MEETING.CIMT.EU | ASSOCIATION FOR CANCER IMMUNOTHERAPY
“The right patient for the right therapy.” CANCER IMMUNOTHERAPY
13th CIMT ANNUAL MEETING ABSTRACTS MAY 11–13, 2015
meeting.cimt.eu cimt.eu ABSTRACTS 2015 13TH ANNUAL MEETING MAY 11–13, 2015 Rheingoldhalle Congress Center Mainz, Germany CIMT Abstract List
Therapeutic Vaccination Abstract List (001 - 020)
No.: Presenter: Short talk: Title:
001 Akers - HLA Class I self-ligandome reveals extreme allelic variability
002 Allen - A novel linear DNA vaccine targeting HPV16 driven malignancies
003 Beckhove yes VXM01, an oral T-cell vaccine targeting VEGFR-2: Further results from a rand- omized, controlled, first-in-man study in pancreatic cancer patients
004 Bialojan - Combined immunotherapy against cancer: Enhanced efficacy of transcutaneous immunization and agonistic CD40 mAb
005 Brown - Local delivery of checkpoint inhibitor antibodies and other therapeutics to tumours by encoding within the oncolytic adenovirus enadenotucirev
006 Buonaguro - Cancer vaccine development for hepatocellular carcinoma – HEPAVAC
007 Cebula - The density of antigen expressing hepatocytes determines the efficacy of thera- peutic vaccination
008 Cerullo - The abstract is withdrawn
009 Chaurasiya - Transcriptionally targeted interleukin-2 gene therapy for breast cancer using mammoglobin promoter
010 Daemen yes A complete reversal of the immunosuppressed tumor environment into an antitu- mor environment upon rationally designed multimodal cancer immunotherapy
011 Deppisch - The combination of trifunctional bispecific antibodies with a CTLA-4-blocking antibody impacts the induction of an immunologic memory in a preclinical mouse model
012 Dilthey - Accurate HLA typing from next-generation sequencing data
013 Frenzel - The GAPVAC consortium: Moving a novel concept of actively personalized can- cer immunotherapy into the clinic
014 Frøsig - Checkpoint inhibitors in cancer immunotherapy: Cross reactivity of a CTLA-4 antibody and IDO-inhibitor L-1MT in pigs
015 Gerer - Immunotherapy of Merkel Cell Carcinoma by therapeutic DC vaccination against the viral oncogenic driver large T antigen
016 Grunwitz - Preclinical evaluation of an mRNA-based immunotherapy against HPV16+ Head and neck squamous cell carcinoma
017 Gustafsson - A novel peptide-conjugate vaccine strategy that targets antigen-presenting cells via endogenous antibodies
018 Harden - Investigation of T-cell responses after DNA vaccination in patients with chronic myeloid leukaemia
019 Heesch - The Mutanome Engineered RNA Immuno-Therapy (MERIT) project: Introducing individualized medicine for the treatment of TNBC
020 Hempel - Vaccinia virus E3-mNRA boosts humoral immune response to self-replicating RNA encoding HA CIMT Abstract List
Therapeutic Vaccination Abstract List (021 - 039)
No.: Presenter: Short talk: Title:
021 Hinkkanen - Neuron-specific microRNA target repeats engineered in alphavirus RNA genome abrogate lethal phenotype but retain viral ability to replicate in the presence of IFN-I in syngeneic CT-2A glioma model
022 Hirayama - Identification of oncofetal antigen (IMP-3)-derived long peptides encompassing both CTL epitopes and multiple HLA-class II-restricted Th cell epitopes
023 Hobernik - CCL22 regulated plasmid DNA as a promising perspective in DC-specific DNA vaccination
024 Höchst yes Controversial effect of atRA in treatment of non-responders in therapeutical vaccination
025 Høgset - Photochemical internalization - an innovative technology giving strong en- hancement of cytotoxic T-cell responses to vaccination
026 Hoppe - Identification of T cell epitopes for a therapeutic HPV16 vaccine
027 Hoyer - Mimicking of CD4+ T-cell help within DCs by transfection with caIKKRNA to improve DC vaccination
028 Hutzler - Oncolytic measles virus for tumor vaccination
029 Jabulowsky yes Lipo-MERIT: a novel nanoparticular formulated tetravalent RNA cancer vaccine for treatment of patients with advanced melanoma
030 Junco Barranco - Heberprovac, a GnRH based therapeutic vaccine to treat advanced prostate can- cer
031 Kanaseki - Identification of the novel antigenic peptide presented by the cancer stem cells of human colon cancer
032 Kappel - Mannose conjugated HPMA-LMA block copolymer micelles for targeting of anti- gen presenting cells and release of anti-tumor agents
033 Khallouf - Evaluation of different adjuvant systems for an epitope-based therapeutic hu- man papillomavirus vaccine
034 Kloke - IVAC MUTANOME - Evaluation of a new personalized therapeutic modality for the treatment of cancer
035 Kloor - Vaccination of MSI-H colorectal cancer patients with frameshift peptide anti- gens - a phase I/IIa clinical trial
036 Koch - A randomized, double-blind, placebo-controlled, phase I/II Trial of RNActive®- vaccine CV9104 in patients with metastatic castrate-refractory prostate cancer (mCRPC): First results of the phase I part
037 Kowalczyk - Self-adjuvanted RNActive® vaccines provide a promising platform for combina- tion therapies with checkpoint inhibitors
038 Löffler - Constructing a peptide vaccine warehouse for the personalized immunotherapy of colorectal cancer
039 Lövgren - Enhanced IL-12 production and T cell stimulation ability by dendritic cells ma- tured in presence of GMP-grade Toll-like receptor ligands and IFN-γ CIMT Abstract List
Therapeutic Vaccination Abstract List (040 - 058)
No.: Presenter: Short talk: Title:
040 Mensali - Invariant chain as a vaccination vehicle for colorectal cancer
041 Milenova - Immunotherapy with a CD40L/4-1BBL double-armed oncolytic adenovirus drives Th1 immunity and controls tumor progression in a pancreatic cancer model
042 Occhipinti - Mutated variant of HER2 as a target for immunotherapy in breast cancer
043 Ophir yes Increased anti-tumor immunity that correlates with clinical benefit and induc- tion of neoantigens reactivity following autologous tumor lysate-pulsed den- dritic cells vaccination in recurrent ovarian cancer
044 Overgaard - Elucidating the T-cell reactivity against porcine IDO and RhoC to establish the pig as an animal model for vaccine development against human cancer
045 Ozimkowski - Phenotypic and functional characterization of monocyte derived dendritic cells (MoDC) generated with CliniMACS Prodigy Instrument
046 Palata - Characterization of antigenic profiles of primary non-small cell lung cancer tumors and lung cancer cell lines for vaccine development
047 Pizzurro - Cytokine-matured DC/Apo-Nec melanoma vaccine presents an improved MMP- 9-dependant migration to lymph nodes that can be modulated by the topical use of Imiquimod
048 Podrazil - Immunological parameters in phase I/II clinical trial of dendritic-cell based immunotherapy (DCVAC/PCa) combined with chemotherapy in patients with metastatic, castration-resistant prostate cancer
049 Préville - Modified Vaccinia Virus Ankara therapeutic efficiency in preclinical models of cancer benefits from the blockade of immune checkpoints
050 Rausch - Combined Immunotherapy: CTLA-4 blockade potentiates anti-tumor response induced by transcutaneous immunization
051 Reuschenbach - p16INK4a as a vaccine target in patients with HPV-associated cancers - results of a phase I/IIa study
052 Rolih - BoHV-4 viral vector as a new approach for ErbB2+ breast cancer immunotherapy
053 Rothe - Enhancing dendritic cell-induced T-cell responses by immunomodulating mol- ecules
054 Rubinsteyn - Spatial and temporal heterogeneity of mutated tumor antigens in a high grade ovarian serous carcinoma
055 Ruiu - TMPRSS4 is a breast cancer stem cell-associated antigen and a putative target for DNA-based vaccination
056 Sayem - Identification of oncofetal antigen Glypican-3-derived long peptides encompass- ing CTL and multiple HLA class II-restricted Th cell epitopes
057 Schnorfeil - Dendritic cell vaccination in postremission therapy of AML: Results of a clinical phase I trial
058 Sedlik - Effective anti-tumor therapy based on a novel anti-Tn antibody conjugated to a cytotoxic drug CIMT Abstract List
Therapeutic Vaccination Abstract List (059 - 075)
No.: Presenter: Short talk: Title:
059 Shraibman - Combining chemotherapy with immunotherapy for the treatment of glioblas- toma
060 Stergiou - Fully synthetic Mucin 1 glycopeptides are effective cancer vaccines and induce high antigen-specific antibody titers
061 Tagliamonte - Novel metronomic chemotherapy and cancer vaccine combinatorial strategy for hepatocellular carcinoma
062 Terhorst - Laser-assisted intradermal delivery of vaccines specific for cross-presenting XCR1+ dendritic cells induces anti-tumoral responses
063 Teulings - Anti-melanoma immunity and local clinical responses of stage III-IV melanoma patients treated with monobenzone and imiquimod; a phase 2a trial
064 Toellner - Vaccines to induce Robo4 and Clec14a specific antibody retard tumour growth
065 Torigoe - Phase I/IIa clinical study of Survivin-2B peptide vaccination in combination with type I interferon for advanced gastrointestinal cancer patients
066 Trautwein - Personalized cancer immunotherapy: patient specific multipeptide vaccination for primary liver cancers
067 Treinies - An RLR/TLR agonist-based combination therapy enhances CTL responses in tumors
068 Tsukahara - Engineering high affinity antibody recognizing naturally presented osteosar- coma antigen PBF peptide
069 Tüttenberg - Sonographic evaluation of lymph node morphology after intranodal vaccination with an RNA-based vaccine in patients with malignant melanoma
070 Uskent - Anti-idiotypic vaccine Racotumomab against NGcGM3 gangloside stabilize disease in rapidly progressing treatment refractory metastatic breast cancer
071 van de Roemer - Individualized tumor immunotherapy-From mutanome to actively personalized RNA based cancer vaccination
072 van de Wall - Development of a next generation Semliki Forest virus-based DNA vaccine against cervical cancer
073 Vormehr - RNA vaccination-induced CD4+ T cells frequently recognize mutated epitopes leading to rejection of aggressively growing tumors
074 Welters - Synthetic long HPV16 E6 and E7 peptides vaccine combined with Aldara as adjuvant to treat HPV16-induced vulvar/vaginal intraepithelial neoplasia
075 Yang - Induction of massive antigen-specific CD8+ T cell cytotoxicity by targeting of antigen into XCR1+ DC combined with potent amplification regimes CIMT Abstract List
New Targets & New Leads Abstract List (076 - 094)
No.: Presenter: Short talk: Title:
076 Bartholomäus - Human tonsil derived T cells show increased responsiveness upon anti-CD28 superagonistic monoclonal antibody treatment compared to blood derived T cells
077 Bassani-Sternberg - In-depth mass spectrometry based immunopeptidomics of melanoma tissues for the development of anti-tumor immunotherapies
078 Boyd - The use of proteomics to analyze whole tumors and identify unique immuno- oncology targets for antibody based therapeutics
079 Burlion - Prevention of Graft-vs-Host-Disease with maintenance of Graft-vs-Leukemia in humanized mice treated with an anti-ICOS monoclonal antibody
080 Charpentier - Meloe, a single polycistronic mRNA translated by various mechanisms to gener- ate multiple potential antigenic peptides in melanoma
081 Chowdhary - A Cancer Research UK Phase I trial of the Anti-CD19 DI-B4 monoclonal anti- body given intravenously, weekly for four weeks in patients with advanced CD19 positive indolent B-cell malignancies
082 Donia - Aberrant expression of MHC Class II in melanoma attracts inflammatory tumor specific CD4+ T cells which dampen CD8+ T cell antitumor activity
083 Ellwanger - EGFRvIII TandAbs are specific and highly potent drug candidates for the treat- ment of solid tumors
084 Goletz yes TrasGEX™, a glyco-optimized anti-HER2 antibody - results from phase I/IIa clinical trials
085 Hassel - Tadalafil in Melanoma (TaMe): a phase 1 trial in patients with metastatic mela- noma (AJCC stage IV or III unresectable) with the PDE-inhibitor Tadalafil
086 Heinz - Zoledronic acid/interleukin-2-expanded γδ T cells augment anti-tumor activity of IMAB362
087 Hemminki - Serum HMGB1 seems as a suitable predictive and prognostic biomarker in pa- tients treated with adenoviral oncolytic immunotherapy
088 Jacobi - A novel synthetic Toll-like-receptor 7 agonist induces lymphoma rejection in an IFNα-dependent manner
089 Jendretzki - A screening approach to identify a lead bispecific antibody targeting CLDN6- positive tumor cells
090 Kamaruzaman - Potential anticancer activity of new β-Carboline derivative on K562 human leukemia cell line
091 Khemlina - Molecular landscape of prostate cancer: Implications for current clinical trials
092 Kowalewski - Immunopeptidome analysis identifies non-mutant epitopes as targets of anti- leukemia T cell responses
093 Kunz - Enabling variant detection in single cell transcriptomes using half cell sequencing
094 Lee - The antitumor effect of the inactivated Sendai virus particles (Hemagglutinat- ing virus of Japan envelope: HVJ-E) on malignant pleural mesothelioma CIMT Abstract List
New Targets & New Leads Abstract List (095 - 114)
No.: Presenter: Short talk: Title:
095 Leon - Rational design of a TGFb1 mutant useful for cancer immunotherapy
096 Marcq - PD-1, PD-L1 and PD-L2 expression in human malignant pleural mesothelioma
097 Michels yes TiMi1 is a novel immune-checkpoint in solid tumors differentially regulating calcium-depending signaling in tumor-infiltrating lymphocytes
098 Peipp - Enhancing natural killer cell-mediated lysis of lymphoma cells by combining therapeutic antibodies with CD20-specific immunoligands engaging NKG2D or NKp30
099 Penny - Phosphopeptides as novel tumour antigens in colorectal cancer
100 Penny - Posttranslationally modified peptides as neoantigens in leukaemia
101 Posselt - RIG-I based immunotherapy of hepatocellular carcinoma (HCC)
102 Reimer - Peptide libraries for comprehensive coverage of the tumor mutanome in im- mune monitoring and immunotherapy
103 Riabov - Stabilin-1 is expressed on tumour-associated macrophages in breast cancer and supports tumour growth in animal model of breast adenocarcinoma by clearance of SPARC
104 Riccardo - Plasma derived exosomes as a reservoir of biomarkers for leukemia patients stratification
105 Schuster - The immunopeptidomic landscape of ovarian cancers - the MUC16/MSLN axis exposes cancer cells to the immune system
106 Seidel - Reduction of minimal residual disease in children with relapsed and refractory ALL by a newly developed, Fc-optimized CD19 antibody
107 Seif - M2 polarization enhances silica nanoparticle uptake by macrophages
108 Sorrentino yes Unraveling the “immune-modulatome” of pancreatic adenocarcinoma (PDAC): a RNAi screening with patient-derived tumor infiltrating lymphocytes (TILs)
109 Souczek - HCMV deletion virus models enable the identification of numerous physiologi- cally relevant T cell epitopes
110 Sun yes Effective in vivo targeting of B cell precursor acute lymphoblastic leukemia by CD70 directed immunotherapy
111 Türeci - Identification of single nucleotide polymorphisms as putative predictive bio- markers for IMAB362 treatment of patients with gastroesophageal cancer
112 Valerius - An engineered IgA antibody against the epidermal growth factor receptor dis- plays improved myeloid effector cell engagement and pharmacokinetics
113 Walter yes Antitumoral activity of IMAB027, the first therapeutic monoclonal antibody targeting the cancer-specific antigen claudin 6 (CLDN6)
114 Zhou - Multiple co-inhibitory pathways contribute to dysfunctionality of intra-tumoral T cells in liver cancer CIMT Abstract List
Improving Immunity Abstract List (115 - 133)
No.: Presenter: Short talk: Title:
115 Barutello - Anti-tumor immunization of mothers delays tumor development in cancer prone offspring
116 Braun yes In vivo silencing of A20 via TLR9-mediated targeted siRNA delivery potentiates antitumor immune response
117 Buonaguro - Laser-assisted epidermal immunogen delivery for optimal targeting of Langher- hans cells in cancer vaccination
118 Creaney - Strong spontaneous tumor neo-antigen responses to tumors induced by a natural human carcinogen, and unmasking of new reactivities with selective treg depletion
119 Dyck - Combination therapy with the TLR7/8 agonist R848 and BAMLET, an α-lactalbumin - oleic acid complex, promotes tumour cell death and anti-tumour immunity
120 Eissler yes Targeting CSF-1R reverses induction of suppressive myeloid cells and controls spontaneous neuroblastoma progression in a murine model
121 Fellermeier - Targeted costimulation with novel single-chain 4-1BBL or OX40L antibody- fusion proteins promotes tumor directed T cell activation
122 Gieseke - Cognate immune response amplifiers - a new RNA based tool for amplifying T- cell responses
123 Gregoire - Measles virus vaccine infects tumor cells and induces antitumor immunogenic response
124 Heidenreich - RNAdjuvant®, a novel, highly-potent RNA-based adjuvant, combines strong im- munostimulatory capacities with a favorable safety profile
125 Hotz - Timing and sequence of pattern-recognition receptor stimulation determines efficacy of cancer immunotherapy with TLR ligands
126 Kapp - EnanDIM®: TLR-9 activating oligodeoxynucleotides stabilized by an evolution- ary concept
127 Lambing - Augmented melanoma immunity by combining irradiation and RIG-I activation
128 Lee yes NK cells expressing Tim-3 and Ceacam1 are functionally impaired
129 Leon - Therapeutic combination of immunetherapies targeting NGcGM3 and EGFR to treat cancer
130 Mottas - Basic evaluation of nanoparticles for cancer immunotherapy: the one plate/one day method
131 Parviainen - Enhancing adoptive T-cell therapy with oncolytic adenovirus encoding CD40- ligand
132 Petrizzo - Functional characterization of biodegradable PLGA and PLGA/PEI nanoparticles as antigen delivery system
133 Rojas Colonelli - RNAi-based silencing of STAT3 during intradermal DNA vaccination promotes antitumor T cell immunity CIMT Abstract List
Improving Immunity Abstract List (134 - 144)
No.: Presenter: Short talk: Title:
134 Rosca - Releasing the cancer immunosuppressing brakes: calling to arms of dendritic cells
135 Rossi yes Delivery of IFNα to PD-L2-expressing cells greatly potentiates chemotherapy efficacy
136 Sanders - N1-methylpseudouridine-modified mRNA outperforms pseudouridine-modified mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice
137 Sanders - Whole cancer cell vaccines based on immunogenically killed cancer cells and dendritic cells
138 Stamova - In-situ activity of tumor specific CTL determines colorectal cancer prognosis
139 Tähtinen - Oncolytic adenovirus improves anti-tumor efficacy of adoptive T-cell therapy by breaking tumor tolerance
140 Temizoz - TLR9 and STING agonists synergistically induce innate and adaptive type-II IFN
141 Thommen - Intratumoral T cell exhaustion increases during cancer progression and limits the activity of T cell-directed immunotherapies
142 Vandenberk - Irradiation of necrotic cancer cells, employed for pulsing dendritic cells (DCs), potentiates DC vaccine-induced antitumor immunity against high-grade glioma
143 Veitonmäki yes ADC-1013, an agonistic CD40 antibody generates immune mediated anti-tumor effects in syngeneic tumor models in hCD40 transgenic mice
144 Wenthe - Gemcitabine reduces the level of TGFβ-1 in plasma from pancreatic cancer pa- tients and potentiates immunotherapy in a model of pancreatic cancer CIMT Abstract List
Cellular Therapy Abstract List (145 - 164)
No.: Presenter: Short talk: Title:
145 Agliardi - Development of EGFRvIII targeted T-cell therapy for brain tumours
146 Amann - HLA.A2-restricted MDM2 (81-88) epitope as a potential tumor-associated anti- gen for TCR gene therapy for multiple myeloma
147 Andersen - Adoptive T cell therapy with intermediate dose interleukin-2 achieves long-last- ing complete responses in heavily pre-treated melanoma patients
148 Bentzen - Peptide-MHC guided stimulation of antigen-specific T cells for immune therapy
149 Bergerhoff - Cellular and molecular events controlling the cytotoxic activity of melanoma- reactive CD4+ T cells
150 Bigalke - Ex-vivo expanded T cells significantly prolong overall survival in stage IV mela- noma patients treated with autologous hTERT/survivin mRNA-loaded dendritic cells
151 Birtel - The importance of costimulation for a novel CAR framework in human T-cells
152 Bonte - Functional evaluation of T cells generated from WT1-TCR transduced human hematopoietic stem cells using the OP9-DL1 coculture system
153 Brabants - Contrasting effects of the anti-cancer MEK 1/2 inhibitor trametinib on human dendritic cell functions
154 Bräunlein - Combining in depth immunopeptidomics of primary melanoma samples and se- lection of high affinity T cells and TCR for the treatment of metastatic melanoma
155 Cappuzzello - Combined therapy of CIK cells and monoclonal antibodies for ovarian cancer: a preclinical study
156 Cartellieri yes A novel modular retargeting platform technology for precise control of CAR T cell reactivity
157 de Witte - Innate transplantation as a low GVHD platform for immune interventions by means of an alpha/beta T-cell depleted allogeneic stem cell transplantation from matched related and unrelated donor grafts
158 Eggers - Expression of PE38-based immunotoxins in T cells introduced by mRNA trans- fection
159 Eiz-Vesper - alloCELL: Selection of suitable T-cell donors and GMP-compliant manufacturing of cells
160 Ellinger - Generation of tumor antigen specific CD4+ and CD8+ T cells by simultaneous MHC-I and -II epitope presentation in vitro and in vivo
161 Enblad - Third generation CAR T-cells targeting CD19 for relapsed and refractory lym- phoma and leukemia - the Swedish experience
162 Farahmand - Targeted delivery of cardiac peptides as anti-cancer agents
163 Foley - T cell receptor modifications to enhance expression, chain pairing, and antigen recognition in T cells for adoptive T cell transfer
164 Genßler - EGFR-targeted CAR NK cells display potent activity against experimental glio- blastoma CIMT Abstract List
Cellular Therapy Abstract List (165 - 184)
No.: Presenter: Short talk: Title:
165 Gross - Messenger RNA encoding constitutively-active TLR4 exerts potent immunostim- ulatory effects on antitumor T cells
166 Guasch - Effect of integrin-mediated cell adhesion, surface stiffness and co-stimulation on CD4+ T cell activation using nanostructures
167 He - Generation of human HCV-specific T cells from hematopoietic progenitor cells using human thymic epithelial cell culture system
168 Hölzel - Modelling genetic heterogeneity as a resistance mechanism to cancer immuno- therapy using CRISPR-Cas9
169 Ikeda - Donor lymphocyte infusion therapy utilizing genetically engineered lympho- cytes that express tumor-specific TCR and siRNA for endogenous TCR enables tumor eradication without GvHD induction
170 Inderberg-Suso - Preclinical validation of a colon cancer specific TCR targeting a neoantigen
171 Karlsson - CAR T cells induce DC maturation via a cell contact-dependent process and enhance their ability to stimulate T cell responses
172 Knippertz - A combined human CD83- and Hsp70B’ promoter system enables transcriptional targeting of dendritic cells in vitro
173 Kobold - Transduction with C-C-chemokine receptor type 4 (CCR4) enhances tumor-spe- cific migration of adoptively transferred T cells in a model of pancreatic cancer
174 Kobold - A new fusion receptor overcomes PD-1-mediated immunosuppression in adop- tive T cell therapy
175 Kobosilova - Expression of immunogenic cell death markers on lung cancer cells
176 Kunert - Engineering and testing of ‘smart’ T cells - counteracting immune evasion by solid tumors
177 Kunert - TCRs specific for MAGE-C2 in combination with epigenetic drugs provide anti- tumor responses toward tumors of multiple histologies
178 Le Poole - Vitiligo T cell receptor SILv44 imparts a Tc17 profile and anti-tumor reactivity on host T cells
179 Liu - Tumor-reactive T-cells from patients with glioblastoma for cellular therapy
180 Lyngaa - Using Merkel cell polyomavirus specific TCR gene therapy for treatment of Mer- kel cell carcinoma
181 Mall - Development of clinically implementable imaging strategies for tracking T cell receptor-transgenic effector T cells
182 Mao - Interleukin-15 potentiates human natural killer cells to resist tumor-induced suppression through mTOR-regulated metabolic control
183 Melief - Immunoregulatory mechanisms in melanoma
184 Meng - Tumor-infiltrating lymphocytes for cellular therapy of patients with pancreatic cancer CIMT Abstract List
Cellular Therapy Abstract List (185 - 205)
No.: Presenter: Short talk: Title:
185 Menger yes TALEN-modified T cells for adoptive cellular therapies: genetic silencing of the glucocorticoid receptor in virus-specific T cells
186 Mesiano - Effective immunotherapy with Cytokine Induced Killer (CIK) cells against au- tologous gastrointestinal stromal tumors
187 Monnot - Enhancing CD8+ CAR T cells antitumour efficiency by miR-155 transgenic expression
188 Moore - Following the fate of genetically modified T cells in melanoma patients
189 Mroz - Targeting Claudin-6 with CAR-engineered T cells for individualized immuno- therapy of ovarian cancer
190 Nishimura - Phase I clinical trial using autologous TIL 1383I TCR transduced T cells
191 Oelsner - Genetically modified cytokine-induced killer (CIK) cells for targeted cancer therapy
192 Klar - A proposal for an individualized immunotherapy of cancer with engineered CAR-/TCR-modified T cells derived from the central memory subset
193 Patasic - Establishment of a non-human primate CAR-T cell model
194 Schaft - Optimised melanoma therapy by generation of T cells expressing two additional T-cell receptors (TETARs)
195 Schütt - Side-by-side comparison of second generation chimeric antigen receptors
196 Simon - In vitro PD-1 blockade to generate optimal effectors for adoptive cell transfer in melanoma
197 Siurala - Enhancement of melanoma adoptive T-cell therapy by adenoviral gene transfer
198 Spear yes TCR-pMHC binding affinity is not the only major influence on functional avidity and polyfunctionality of T cell recognition
199 Straetemans - Untouched GMP-grade purified engineered immune cells
200 Strauss - Myeloid-derived suppressor cell (MDSC) therapy skews the T cell response after bone marrow transplantation towards type 2 immunity and maintains the graft- versus-tumor effect while preventing graft-versus-host disease (GVHD)
201 Temme - Engineering NK cells modified with an EGFRvIII-specific chimeric antigen receptor to overexpress CXCR4 improves immunotherapy of CXCL12/SDF-1α- secreting glioblastoma
202 Tosi - Identification of an HLA-A*0201-restricted immunogenic epitope from the uni- versal tumor antigen DEPDC1
203 Tribble - Fine-tuned T cell receptors for cancer immunotherapy
204 Tubb - Exploring if recurrent somatic mutations can be targeted with TCR gene therapy for cancer
205 Van Caeneghem yes Expression of a chimeric antigen receptor in hematopoietic precursor cells inhibits the generation of a diverse T cell receptor repertoire and induces T Cell Receptor/CD3 negative, CD4 CD8 negative T cells CIMT Abstract List
Cellular Therapy Abstract List (206 - 210)
No.: Presenter: Short talk: Title:
206 Weber - Generation of EBV-specific human CD8+ cytotoxic T lymphocytes with stem cell memory and central memory properties by modulating glycolytic T cell metabolism
207 Weigand - A novel immunotherapy: IMCgp100, a bi-specific TCR-anti-CD3 fusion for potent re-directed killing of melanoma cells
208 William - Optimization of Glioblastoma multiforme in vitro culture conditions for preser- vation of EGFR gene amplification
209 Williams - Gene transfer of an MHC class II-restricted TCR for the treatment of EBV+ Post Transplant Lymphoproliferative Disease
210 Zhang - ErbB2/HER2-targeted CAR NK cells display potent antitumor activity against glioblastoma and enhance survival CIMT Abstract List
Immunomonitoring Abstract List (211 - 228)
No.: Presenter: Short talk: Title:
211 Anastasopoulou - Detection of long lasting memory HER-2/neu specific T cells in vaccinated can- cer patients
212 Attig - Assessment of vaccine-induced immunity following intra lymph node adminis- tration of RIBOLOGICAL® IMMUNOTHERAPY targeting NY-ESO-1 and tyrosi- nase in patients with advanced melanoma
213 Baumeister - The cytokine/chemokine response accompanied with administration of the therapeutic antibody PankoMab-GEX™
214 Baumgaertner - Dissecting the impact of peptide-MHC and TCR-pMHC binding strengths on the functional avidity of tumor-specific CD8 T cells
215 Bode - Differences in NK cell reconstitution after T cell depletion with ATG or Alemtu- zumab in allogeneic hematopoetic stem cell transplantation
216 Challis - CIP NK Proficiency panel 2014: Low inter-lab variation found with NK pheno- typic markers, but high variation in activation and functional markers
217 Chandran.P - Validation of immunomonitoring assays- Multimer staining assay and ELISPOT assay
218 Damuzzo - The abstract is withdrawn
219 de Goeje - Changes in the peripheral blood immune profile upon surgery of early stage lung cancer
220 Dörrie - Electroporation of CD8+ effector T cells with antigen is suitable to test their reactivity to tumor antigens
221 Epp yes Functionally impaired GPI-anchor negative regulatory T cells strongly correlate with acute Graft versus Host Disease after Alemtuzumab-based conditioning regimen
222 Fujii yes Development of a novel “rapid and sensitive” method for detection of cellular immune responses and selection of immunogenic peptides encoded by mutated genes
223 Gustafsson yes Ex vivo circulating whole blood for superior detection of cytokine storms in response to antibody therapies
224 Hawner - Study on the viability, recovery and functionality of PBMCs stored at -80°C versus storage in liquid nitrogen
225 Kini Bailur yes Prognostic impact of circulating plasmacytoid dendritic cells and immunosup- pressive subsets in breast cancer
226 Luiten - Vitiligo-like depigmentation in stage III-IV melanoma patients receiving im- munotherapy and its association with survival: a systematic review and meta- analysis
227 Mansfield - Imaging in cancer immunology: 6-plex immune profiling in situ
228 Melnikov - 10 years immune monitoring of Luminal «B» breast cancer CIMT Abstract List
Immunomonitoring Abstract List (229 - 238)
No.: Presenter: Short talk: Title:
229 Pedersen - Automated analysis of flow cytometry data to reduce inter-lab variation in de- tection of MHC multimer binding T cells
230 Ramskov Andersen yes Identification of novel T cell epitopes in breast cancer
231 Rathinasamy - Emigration of tumor specific regulatory T cells from the bone marrow is trig- gered by S1P1 and correlates with Treg accumulation in breast tumors
232 Santegoets - Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry
233 Tischer - Identification of new immunogenic CD8+ T-cell epitopes from clinically-relevant human Adenovirus serotypes to improve diagnostic and therapeutic options in adoptive immunotherapy
234 Turksma - Antigen specific T cell immunomonitoring; HLA tetramer combinatorial coding for CD8 T cells and CD40L expression on antigen specific CD4 T cells
235 Valous - Visualizing the spatial co-localization of infiltrating immune cells in solid tu- mors using serial tissue sections acquired with whole-slide imaging
236 Vigano - Clinical immunomonitoring strategies defining biomarkers of anti-CD73 mAbs safety and efficacy. The TumAdoR collaborative project
237 Wistuba-Hamprecht - The frequency of circulating Vδ1-positive but not Vδ2-positive γδ T-cells corre- lates negatively with survival in late-stage melanoma and may be increased by ipilimumab treatment
238 Zelba - Correlates of checkpoint inhibitor expression and T cell function in urological tumors CIMT Abstract List
Tumor Biology and Interaction with the Immune System Abstract List (239 - 259)
No.: Presenter: Short talk: Title:
239 Ahmetlić - The role of Foxp3+ regulatory T cells in a murine model of spontaneously de- veloping B-cell lymphoma
240 Baert - ID8-Fluc: a syngeneic mouse model for ovarian cancer
241 Bitzer - Molecular characterization of the Breast Cancer Associated Antigen NY-BR-1
242 Boegel - HLA expression body map
243 Chew - The critical role of immune microenvironment in Hepatocellular carcinoma- the potential of TLR3 ligand as a novel immunotherapy
244 Classen - Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome
245 Coosemans - Immunomodulation in patients with ovarian cancer: a retrospective analysis
246 Das - Establishment of a transplantable, NY-BR-1 expressing breast cancer model in HLA-transgenic mice
247 Duewell yes RIG-I-like helicase-mediated reprogramming of myeloid-derived suppressor cells as novel therapeutic strategy for pancreatic cancer
248 Eichmueller - Generation of murine b2-microglobulin deficient tumor cell lines using the CRISPR/Cas9 system
249 Engel yes Hypoxia-induced reversible dedifferentiation attenuates RIG-I-mediated immu- nity against melanoma
250 Erin yes Activation of neuroimmune pathways decreases tumor-infiltrating myeloid- derived suppressor cells and increases therapeutic effects of radiotherapy on metastatic breast carcinoma
251 Fortis - Intratumoral and systemic immunity as prognostic biomarkers in patients with breast cancer*
252 Greten - CPT1 dependent CD4 T cell death in mice with non-alcoholic steatohepatitis promotes hepatocarcinogenesis
253 Guislain - Activity of tumor-infiltrating lymphocytes can be potentiated by relieving post- transcriptional regulation
254 Havunen - Abscopal effect seen with T-cell therapy enhancing oncolytic adenoviruses armed with TNFa and IL-2
255 Janssen - Do immune features of the tumour have prognostic value in metastatic melanoma?
256 Kaunisto - Local administration of CD40 agonist dynamically modulates the tumor-infil- trating immune cell compartment
257 Knott - Tumor cell-secreted IL-1 mediates CCL22 induction in human PBMC
258 Kober - Characterization of murine GL261 glioma models for oncolytic vaccinia virus therapy
259 Komdeur - CD103-positive T-cells; an immunotherapeutic target for ovarian and endome- trial cancer CIMT Abstract List
Tumor Biology and Interaction with the Immune System Abstract List (260 - 280)
No.: Presenter: Short talk: Title:
260 Korzovatyh - Immune homeostasis of Kidney Cancer
261 Lal - An immunogenomic stratification of colorectal cancer: implications for develop- ment of targeted immunotherapy
262 Lee - The effects of glyco-shortenings Anti-CD52 on PBMC and their anti-inflammato- ry functions
263 Majidzadeh-A - Production of anti-cancer Bi-functional nanobody in bacterial host
264 Maleka - Immunostimulatory AdCD40L gene therapy combined with low-dose cyclophos- phamide in metastatic malignant melanoma patients
265 Mora - N-terminally truncated Interleukin-38 is released from apoptotic tumor cells to limit inflammatory macrophage responses
266 Núñez - Lymph node invasion by tumor cells modifies the distribution and phenotype of dendritic and T cell subsets in breast cancer patients
267 Ochs - Interleukin-22 (IL-22) does not influence methylcholanthren-A-mediated tumori- genesis
268 Okada - Novel immuno-regulatory mechanisms mediated by IDH1 mutations in human gliomas
269 Ozbey - Fluoxetine inhibits tumor-induced IL-6 release from immune cells of draining lymph nodes
270 Ozdemir - The different perspective of tumor immune evasion: Coexistence of cancer stem cells (CSCs) and nonclassical major histocompatibility complex (MHC) class I antigens
271 Parri - Identifying the kinases and phosphatases regulating activated STAT3
272 Parrot - Intra-tumor CD4+CD8+ double-positive T cells: a new target for IL-9
273 Paschen - Monitoring the development of T-cell resistance in malignant melanoma
274 Roskopf - Selective biphenotypic leukemia cell lysis is mediated by dual-targeting triple- body 33-3-19
275 Sanders - Immunological and angiogenic markers during metronomic chemotherapy in spontaneous arising cancers in dogs
276 Sandin - Immunomodulatory kinase inhibitors in tumor immunotherapy
277 Sasada yes Clinical significance of tumor-associated tertiary lymphoid structures in pancre- atic cancer
278 Seida - The potential immunomodulatory role of cortisol on NKG2D activating NK cells receptor in ovarian cancer
279 Soper - Humanized NSGTM mice are a platform for pre-clinical evaluation of immuno- modulatory therapeutics
280 Spille - Macrodissection of human colorectal cancer liver metastases revealed the tumor margin as a precisely separated region with distinct cytokine and chemokine profiles CIMT Abstract List
Tumor Biology and Interaction with the Immune System Abstract List (281 - 292)
No.: Presenter: Short talk: Title:
281 Stanczak - CD33-related siglecs are upregulated on activated and tumor-infiltrating T cells
282 Stoitzner yes Impaired CD8+ T cell responses in the presence of myeloid-derived suppressor cells in a spontaneous mouse melanoma model
283 Taranikanti - Effects of surgical stress on host immunity in breast cancer patients
284 Tognarelli - Characterization of human natural killer cell activity against multiple myeloma
285 Toth - In vitro, in vivo and microscopic analysis of tumor immunotherapy using com- bination of two antibodies against the same ErbB2 target
286 Tuyaerts yes Examination of immune escape mechanisms used by circulating tumour cells (CTC) in gynaecological cancers
287 Vascotto - A novel Toll-Like Receptor 7 agonist with distinct anti-tumoral monotherapeutic effects
288 Vizler - Exploitation of the Thy1-YFP mouse strain for fluorescent imaging of inflamma- tion, wound-healing and experimental tumours
289 Wehner - Accumulation of tolerogenic human 6-sulfo LacNAc+ dendritic cells in renal cell carcinoma is associated with poor prognosis
290 Wouters - Prognostic value of tumor-infiltrating lymphocytes in patients with high grade serous ovarian cancer is dependent on treatment regimen, surgical outcome and T cell differentiation
291 Wulf-Goldenberg - Patient-derived tumor transplant model in humanized mice: a model for investi- gation of immune therapy
292 Xydia - The impact of secondary iTreg clones in the TCR repertoire of tumor patients 001 – 075
Therapeutic Vaccination 001 | THERAPEUTIC VACCINATION
The HLA Class I self-ligandome reveals extreme allelic variability
Akers N.K.1, Finnigan J.P.1, Rubinstein A.1, Hanson R.W.1, Schadt E.E.1, Losic B.1
1Mount Sinai School of Medicine, Genetic and Genomic Sciences, New York, United States
Invoking a T-cell reaction to antigen is dependent of bound peptide varies in magnitude by allele, from on MHC alleles effectively presenting bound antigen negligible differences to >300,000 fewer bound peptide. Predicting immune response is therefore peptides at length 8 compared to length 9 for HLA- reliant on software that calculates the estimated A*02:02. strength of interaction between MHC and peptide. These results indicate a potential for bias away from Many studies have relied on standard single value certain alleles when predicting peptides to illicit cutoffs for binding strength (IC50=500nM) and immune response. It is uncertain if this bias is bio- peptide length (9 amino acids) when making binding logical or technical in origin, but it does draw into predictions. Using the software package NetMHC- question the common tactic of applying a uniform Cons, we have calculated the peptide binding prop- binding threshold for arbitrary alleles and all peptide erties of 2,905 HLA-A, -B, and -C alleles for 48,597 lengths to select p-MHC candidates. The data pre- Ensembl predicted human transcripts at peptide sented offer key insights into this issue, with the lengths 8-12, resulting in 7e8 unique protein allele power of a proteome-sized number of observations. interaction predictions, at a computational cost of ~61 years of CPU time. Preliminary analysis of this dataset reveals signifi- cant differences exist in the number of human pep- tides predicted to be bound by different HLA alleles. Limiting to common alleles, the number of human peptides bound at a cutoff of 500nM ranges 4 orders of magnitude at peptide length 9. HLA-C*07:04 is predicted to bind only 100 human peptides at < =500nM, while HLA-B*15:03 is predicted to bind >1,000,000 peptides using the same threshold. Four alleles are predicted to bind fewer than 1,000 human peptides, and seven are predicted to bind fewer than 10,000 human peptides. Allowing variation in the length of bound peptide shows that in general, the number of peptides pre- dicted to be bound by an allele is maximized at a peptide length of 9. The impact of adjusting length 002 | THERAPEUTIC VACCINATION
A novel linear DNA vaccine targeting HPV16 driven malignancies
Allen A.1, Caproni L.2, Ottensmeier C.1, Savelyeva N.1
1University of Southampton, Cancer Sciences Unit, Southampton, United Kingdom, 2Touchlight Genetics Ltd., c/o Leatherhead Food Research, Surrey, United Kingdom
DNA vaccines delivered by electroporation have seen in wild type, non-tolerant animals. The perfor- performed well in early-stage clinical trials for HPV mance of the doggybone vaccine was comparable to driven malignancies. A novel DNA vaccine design is that of the plasmid vaccine. currently being developed by Touchlight Genetics; it We also found the E6E7 doggybone DNA did not ac- consists of double stranded linear DNA with cova- tivate TLR9 in a reporter gene system, in contrast lently closed ends. In recognition of their visual sim- to the plasmid vaccine which stimulated both the ilarity, the vaccines have been named ‘doggybone’ human and mouse forms of TLR9. However this vaccines (doggybone TM DNA). Using an enzymatic did not appear to affect the in vivo activity of the process the doggybone vaccines can be produced doggybone construct, and both the doggybone and entirely synthetically. This gives them an attractive plasmid vaccines stimulated cytosolic DNA sensing safety profile for early translation into the clinic, as pathways involving STING. there is no requirement for the inclusion of an an- Overall the immunogenicity of the E6E7 doggybone tibiotic resistance gene. Manufacturing can be per- DNA vaccine and its favourable comparison with a formed rapidly and on a large scale. conventional plasmid DNA vaccine, coupled with the Here we have tested the immunogenicity of a dog- manufacturing advantages of a doggybone vaccine gybone DNA vaccine encoding the HPV16 proteins make it a promising new therapeutic option for HPV E6 and E7 in both wild type and tolerant pre-clinical driven cancers. models. The doggybone vaccine was delivered with electroporation, and in wild type mice was able to induce E7 specific CD8+ T cells, detected by an E7 specific tetramer. The E6E7 doggybone vaccine sig- nificantly extended survival of mice in a therapeutic setting, using a transplantable tumour that expresses E6 and E7. Experiments performed in parallel with a plasmid delivered E6E7 vaccination demonstrated that the doggybone based vaccine induced similar numbers of E7 specific CD8+ T cells and comparable extension of survival to the plasmid E6E7 vaccine. In a tolerant setting, using E6E7 transgenic mice, the E6E7 doggybone DNA vaccine was able to induce E7 specific CD8+ T cells, although at lower levels than 003 | THERAPEUTIC VACCINATION
VXM01, an oral T-cell vaccine targeting VEGFR-2: Further results from a randomized, controlled, first-in-man study in pancreatic cancer patients
Schmitz-Winnenthal F.H.1, Hohmann N.2, Haefeli W.E.2, Mikus G.2, Grenacher L.3, Friedrich T.3, Lubenau H.4, Springer M.4, Breiner K.5, Meichle A.4, Weitz J.6, Ulrich A.1, Büchler M.W.1, Knebel P.1, Schmidt T.1, Ge Y.7, Niethammer A.8, Beckhove P.7
1University Clinic of Heidelberg, Clinic for General, Visceral and Transplantation Surgery, Heidelberg, Germany, 2University Clinic of Heidelberg, Clinical Pharmacology and Pharmacoepidemiology, Heidelberg, Germany, 3University Clinic of Heidelberg, Diagnostic and Interventional Radiology, Heidelberg, Germany, 4Vaximm GmbH, Mannheim, Germany, 5Vaximm AG, Basel, Switzerland, 6University Clinic of Dresden, Department of Visceral Surgery, Dresden, Germany, 7German Cancer Research Center, Translational Immunology, Heidelberg, Germany, 8Vaximm AG, Heidelberg, Switzerland ORAL ALK SHORT T 2015
Background: VXM01 is an orally available, bacteri- lymphocyte count decrease preferentially occurred ally transmitted DNA vaccine targeting VEGFR-2. under VXM01, compared to placebo. Increased We here report part II of its first-in-man study that VEGFR-2 specific immune response and expected was designed to evaluate the safety and tolerability changes in biomarkers of antiangiogenic activity in- of VXM01 after monthly boosting. Secondary end- cluding decrease in tumor perfusion, rise in collagen points included VEGFR2 specific T-cell responses, IV and VEGF-A , and mild elevation of blood pres- tumor perfusion changes, and related biomarkers of sure occurred after the initial priming phase similar antiangiogenic or anti tumoral activity. to our previous observations. Monthly single dose Methods: The second part of the trial was a rand- boosting resulted in a target selective and overall omized, double-blind, placebo-controlled study, en- 4 fold enhancement of VEGFR-2 specific T-cell re- rolling 27 patients with advanced pancreatic cancer. sponses. Twelve out of 18 patients (66%) treated with VXM01 or placebo was given on days 1, 3, 5, 7, followed VXM01 had a prime and/or boost specific immune by monthly single doses up to month 6. Twelve patients response, independent of the pre-existence of a de- were treated with VXM01 10(6) CFU, 6 patients with tectable VEGFR2 specific T-cell pool. Among the 18 VXM01 10(7) CFU, and 8 patients with placebo. T-cell VXM01 treated patients, kinetics of CA19-9 levels activity against VEGFR-2 and unrelated control anti- and prolonged overall survival were associated with gens was monitored by IFN-gamma ELISpot before, immune response to VXM01. during and after the vaccination course, including Conclusions: VXM01 showed an acceptable safety boosting. Tumor perfusion, assessed by DCE-MRI, profile without DLTs after boosting. The data confirm biomarkers CA19-9, collagen IV, VEGF-A, and blood that VXM01 induces a VEGFR-2 specific T-cell re- pressure were measured up to 1 month after the last sponse which can be strongly enhanced and main- boosting dose. tained by monthly single dose boosting. Tumor Results: Patients were enrolled from 8/2013 to marker profiles and median overall survival corre- 3/2014. In line with the first part of the trial, adverse lated with immunological response. events such as diarrhea and transient platelet and 004 | THERAPEUTIC VACCINATION
Combined immunotherapy against cancer: Enhanced efficacy of transcutaneous immunization and agonistic CD40 mAb
Bialojan A.D.1, Stein P.2, Rausch J.1, Brühl T.J.3, Probst H.C.3, Schild H.3, Radsak M.P.1
1Third Department of Medicine - Hematology, Oncology, Pneumology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany, 2Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany, 3Institute of Immunology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
Transcutaneous Immunization (TCI) is an innova- in the combinational group, either i.p. or s.c. (d1). 7 tive vaccination strategy offering a simple and nee- days post treatment absolute numbers of SIINFEKL dle-free delivery system using the potent immune specific CD8+ T cells as well as the in vivo cytoly- system of the skin. Therefore it has a promising po- sis of target cells was increased in the group with tential in combating tumors or persistent infectious combinational treatment compared to the group diseases. Our group and others have previously that received only TCI. 30 days after treatment, the shown that TCI with the Toll-like Receptor 7 agonist groups treated with TCI and anti-CD40 still showed imiquimod and a cytotoxic T lymphocyte (CTL) augmented levels of SIINFEKL specific CTL and cyto- epitope induces a specific CTL response. However, lytic responses, whereas in the TCI group no signifi- this effect rapidly decays due to the lack of memory cant response was detected anymore. Subsequently, formation and does not result in an adequate protec- we performed a therapeutic tumor rejection assay by tion against tumors. Furthermore, it is possible to injecting SIINFEKL expressing B16-OVA melanoma circumvent this problem by additional CD40 ligation cells s.c. into the flank of C57BL/6 mice. TCI treat- using an agonistic antibody to amplify the TCI treat- ments were initiated at a tumor size of 3x3mm and ment. Stimulation of CD40 leads to the activation of performed 3 times over three weeks along with either antigen presenting cells such as dentritic cells (DC), 2 or 3 antibody treatments. We observed an en- which are necessary for the initiation of an effective hanced tumor rejection and significantly increased and strong CTL response and are also able to induce overall survival with the combinational treatment. memory CTL formation. We recently established a Longest survival and strongest anti-tumor CTL re- mouse model using a combinational treatment of TCI sponses were achieved with 3 TCI treatments along with imiquimod, a T cell epitope and an agonistic with 3 s.c. antibody treatments. CD40 mAb leading to sufficient tumor protection Taken together, the combination of TCI with imiqui- only in a prophylactic setting. Hence, in the present mod and an adequate costimulation by a CD40 liga- work we evaluated this combinational treatment in a tion may contribute to the development of effective therapeutic setting against B16-OVA melanoma. therapeutical anti-tumor vaccinations. In initial experiments, the CTL response and cyto- toxic potential of CD8+ T cells were evaluated. We performed TCI by applying an imiquimod formula- tion together with the CTL epitope SIINFEKL on the shaved back skin of C57BL/6 mice (d1). In addition, the agonistic CD40 mAb (clone FGK-45) was injected 005 | THERAPEUTIC VACCINATION
Local delivery of checkpoint inhibitor antibodies and other ther- apeutics to tumours by encoding within the oncolytic adenovirus enadenotucirev
Champion B.R.1, Illingworth S.1, Rasiah N.1, Kodialbail P.1, Cochrane D.1, Beadle J.1, Fisher K.1, Brown A.C.N.1
1PsiOxus Therapeutics, Oxford, United Kingdom
We are developing armed versions of the oncolytic human colon carcinoma xenograft models we have adenovirus enadenotucirev (EnAd) for selective shown that the virus activity profile following intra- infection and delivery of immunotherapeutics to tumoural injection is similar to parental EnAd in tumours following systemic dosing. EnAd is a potent, terms of virus replication and gene expression and chimeric Ad11p/Ad3 adenovirus active against a anti-VEGF antibody expression could also be detect- range of epithelial cancer cells. In normal cells, EnAd ed in the tumour tissue as both mRNA and functional is attenuated and shows little or no activity by either antibody. Antibodies were detectable early (within 3 cytotoxicity assay or qPCR. In vivo, EnAd shows ef- days of treatment) and expression was sustained over ficacy in human tumour xenograft models following several weeks. We have now developed an orthotopic intra-tumoural, intravenous and intra-peritoneal in- A549 lung tumour model in which virus activity can jection, and is currently being evaluated clinically be assessed following i.v. dosing. Using this model, for treatment of epithelial cancers. Ongoing clinical a single i.v. dose of NG-135 was able to decrease studies have shown that i.v. dosed EnAd infects and established tumour burden by >90% and increase selectively replicates in tumour cells, producing de- overall survival. Building on this initial exemplifica- tectable levels of viral protein, often with an asso- tion, we have generated and characterized viruses ciated high frequency of intra-tumoral CD8 T cells. encoding immune-modulatory agents including cy- These data indicate that transgene encoded proteins tokines, and checkpoint inhibitor antibodies to PDL1 will also be made in significant amounts by tumours and CTLA4 (IgG1 or ScFv versions). These viruses following i.v. delivery of an armed EnAd virus. produce functional antibodies following tumour cell To develop armed EnAd variants we generated an infection and lead to enhanced T cell activation in in efficient cloning system for rapid engineering of vitro assays using primary human immune cells. In viruses that can produce antibodies and other pay- summary, our data show that EnAd can be modified loads. As an initial exemplification of the platform to produce different antibody payloads following in- we successfully produced EnAd variants encoding fection of human tumour cells in vitro and in vivo. full-length (NG-135) and ScFv (NG-76) forms of anti- Evaluation of the impact of these armed oncolytic human VEGF antibodies. These have similar virus viruses on tumour growth and anti-tumour immune activity profiles to EnAd in cancer cell lines (virus responses are now in progress. replication, gene expression and oncolytic action), but also express and release the respective anti- VEGF antibody forms into the culture supernatant of tumour cells but not non-transformed cells. Using 006 | THERAPEUTIC VACCINATION
Cancer vaccine development for hepatocellular carcinoma – HE PAVAC
Kutscher S.1, Accolla R.2, Ma Y.T.3, Heidenreich R.5, Izzo F.6, Koenigsrainer A.7, Loeffler M.7,8, Mueller P.1, Mayer A.1, Rammensee H.G.8, Sangro B.9, Francque S.10, Valmori D.4, Weinschenk T.1, Singh-Jasuja H.1, Buonaguro L.11
1Immatics Biotechnologies GmbH, Tuebingen, Germany, 2Dept. Surgical and Morphological Sciences, Univ. dell’Insubria, Varese, Italy, 3NIHR Biomedical Research Unit in Liver Disease, School of Immunity and Infection, Univ. of Birmingham, 4INSERM UMR 1102, Saint Herblain, France, 5CUREVAC GmbH, Tuebingen, Germany, 6Liver Surgery Unit, Ist. Naz. Tumori “Pascale”, Napoli, Italy, 7Dept. of Surgery Eberhard Karls Univ., Tuebingen, Germany, 8Dept of Immunology, Eberhard Karls Univ., Tuebingen, Germany, 9Liver Unit, Clinica Universidad de Navarra, and CIBEREHD, Pamplona, Spain, 10Div. of Gastroenterology and Hepatology, Antwerp Univ. Hospital, Edegem, Belgium, 11Mol. Biol and Viral Oncogenesis Unit, Ist. Naz. Tumori “Pascale”, Napoli, Italy
The HEPAVAC Consortium aims to develop a highly different HLA-A*24-restricted TUMAPs have been innovative, novel cancer vaccine approach for hepa- identified from HLA-A*24+ samples. Of these, 33 tocellular carcinoma (HCC). The international project HLA-A*02+ TUMAPS and 33 HLA-A*24+ TUMAPs consortium consists of 9 European Partners from are currently in the process of validation, includ- academia and the biotech industry with complemen- ing peptide synthesis, immunogenicity testing and tary and substantial expertise in developing immu- pharmaceutical evaluation. Most promising TUMAP notherapeutic strategies to treat cancer. The project candidates show selective expression only in HCC has started in September 2013 and is supported by samples and no expression in normal tissues. In par- the European Commission’s 7th Framework Program allel, more than 6600 HLA-DR TUMAPs have been (www.hepavac.eu). identified in Hep3B cells transfected with CIITA as HCC/normal adjacent tissue matched samples have well as an average of 1500 HLA-DR TUMAPs have been collected for HLA immunopeptidome analysis. been identified in HCC samples. Additional HLA-DR 17 HCC samples from HLA-A*02+ patients and 15 TUMAPs derived by distinct CIITA-transfected cell samples from HLA-A*24+ patients have been ana- lines with different HLA-DR haplotypes are under lysed by mass spectrometry (LC-MS/MS). RNA-ex- scrutiny. pression profiles have been established for 12 HCC The discovery phase of the HEPAVAC project is pro- samples. HLA-presentation/expression of peptides ceeding according to the proposed timelines. HCC- and mRNA on primary HCC samples are compared specific epitopes to be included in the vaccine cock- to n>140 normal tissue samples from relevant organs tail will be selected in the coming weeks for GMP (including heart, brain, lung, kidney, liver, nerve, production. In parallel, preclinical studies assessing skin etc.) from Immatics’ database. the formulation and combination of the immunologi- A total of 9051 HLA-A*02-restricted different tumor- cal RNA-based adjuvant (RNAdjuvant®) with peptide associated peptides (TUMAPs) have been identified cocktails are underway. from HLA-A*02+ samples, while a total of 3286 007 | THERAPEUTIC VACCINATION
The density of antigen expressing hepatocytes determines the efficacy of therapeutic vaccination
Cebula M.1, Riehn M.1, Ochel A.2, Hillebrand U.1, Schirmbeck R.3, Hauser H.4, Wirth D.1
1Helmholtz-Zentrum für Infektionsforschung, Modellsysteme für Infektion und Immunität, Braunschweig, Germany, 2Justus-Liebig-University Giessen, FB08-Biology and Chemistry, Giessen, Germany, 3Universitätsklinikum Ulm, Zentrum für Innere Medizin Klinik für Innere Medizin I, Ulm, Germany, 4Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany
Treatment options for cancer patients based on ther- intrahepatic accumulation of antigen specific T cells. apeutic vaccination still do not provide efficacious In agreement with previous observations based on responses capable of eradicating transformed cells adoptive transfer of antigen specific T cells, in mice and controlling the tumor growth. The endogenous with 50% of OVA expressing hepatocytes, neither T cell responses against cancer cells as well as those antigen reduction nor antigen clearance could be induced by therapeutic vaccinations are hampered measured, indicating that in these conditions T cells by a severe exhaustion of specific T cells, mainly the became exhausted and would require additional im- consequence of immunosuppressive tumor environ- munomodulatory treatment or more efficient vacci- ment. In this study we investigated the impact of the nation regime. In contrast T cell effector functions density of antigen expressing hepatocytes on efficacy and complete clearance of ovalbumin hepatocytes of therapeutic vaccination. are observed in mice displaying low antigen density A conditional mouse model RosaOVA X AlbCreERT2 prior to vaccination. was employed to provide hepatocyte specific oval- These data indicate that the density of antigen ex- bumin-antigen expression upon Tamoxifen induced pressing hepatocytes governs the final outcome of activation CreERT2 recombination. A specific feature the therapeutic vaccination and may play critical role of this transgenic model is achieved by stochastic in understanding and establishment of immunother- reversion of the recombination. Thereby, the ovalbu- apy against liver carcinoma. min-antigen expression can be restricted to a frac- tion of hepatocytes resulting in a mosaic pattern of antigen distribution. The degree of mosaicism is ad- justable and depends on tamoxifen dosage. Induction of antigen expression in RosaOVA X AlbCre-ERT2 model do not result in activation of a specific T cell response. Thus, we evaluated if a T cell response induced by peripheral antigen presentation after vaccination would result in a liver specific response. For therapeutic vaccination, a DNA plasmid or ad- enoviral vector encoding the ovalbumin antigen was injected intramuscularly in mice with 10% and 50% of hepatocytes expressing the antigen, respectively. Independently of the antigen density, we observed 008 | THERAPEUTIC VACCINATION
The abstract is withdrawn 009 | THERAPEUTIC VACCINATION
Transcriptionally targeted interleukin-2 gene therapy for breast cancer using mammoglobin promoter
Chaurasiya S.1, Hew P.2, Hergenrother P.J.3, Hitt M.M.4
1University of Alberta, Experimental Oncology, Edmonton, Canada, 2University of Alberta, Oncology, Edmonton, Canada, 3University of Illinois, Biochemistry Department, Urbana-Champaign, United States, 4University of Alberta, Edmonton, Canada
Despite of advances in the screening and treatment have used a breast cancer specific promoter (mam- methods breast cancer remains the most common moglobin promoter) to drive human IL-2 gene. We cancer and leading cause of cancer-related deaths in have found the promoter to be very specific to breast women worldwide. Immuno- therapy may emerge as cancer cells both in vitro, and in vivo in mice. We a better therapeutic approach for breast cancer. Many compared the safety and efficacy of Ad vectors en- types of solid tumors including breast tumors have coding IL-2 under the control of mammoglobin pro- been shown to maintain an immune-suppressive en- moter (Ad-MGB-IL-2) with that of Ad vector encoding vironment. Relieving the immune-suppressive con- IL-2 under the control of CMV promoter (Ad-CMV- dition or activating cytotoxic immune cells could IL-2). While intra-tumoral injection of Ad-CMV- have an anti-tumor effect. Many cytokines including IL-2 was found to be very toxic as determined by interleukin-2 (IL-2), IL-12 and IL-15 have been shown increase in liver enzymes (ALT and AST) and liver to exert anti-tumor effect through activation of cy- histology, Ad-MGB-IL-2 was found to be very safe. totoxic immune cells. Amongst these, IL-2 has been The IL-2 vector treated animals had increased T-cell the most widely studied cytokine for its ant-tumor infiltration in the tumor and an overall increase in effect. IL-2 plays an important role in the activation splenic T cells. However, although the Ad-MGB-IL-2 and proliferation of T- and NK cells. High dose of re- significantly retarded the tumor growth in mice, it combinant IL-2 is used in clinics for the treatment of failed to increase the overall survival of treated mice. melanoma and renal cancer. Systemic use of IL-2 has In order to increase the anti-tumor efficacy of Ad- been shown to result into severe side effects, such as MGB-IL-2 we combined it with PAC-1, a novel pro- vascular leak syndrome, in some patients. The side apoptotic drug, with the idea that apoptosis induced effects associated with systemic use of IL-2 severely by the drug might expose some tumor antigens that limits its use in cancer therapy. One way to minimize would be recognized by T cells and high concen- the side effects of systemic IL-2 therapy could be to tration of IL-2 within the tumor would allow rapid limit the high concentration of IL-2 in the vicinity expansion of the tumor-specific T-cells resulting into of tumor. This could be achieved by delivering IL-2 more killings of cancer cells. However, although the gene to tumor cells through viral vectors. Adenovi- combination treatment was well tolerated in mice rus is one of the most commonly used viral vectors there was no significant increase in the overall sur- in gene therapy. However, even after intra-tumoral vival of treated mice. injection a significant amount of virus escapes tumor and reaches to liver. Therefore, in order to minimize the IL-2 expression in liver and other organs we 010 | THERAPEUTIC VACCINATION
A complete reversal of the immunosuppressed tumor environment into an antitumor environment upon rationally designed multimodal cancer immunotherapy
Draghiciu O.1, Boerma A.1, Hoogeboom B.-N.1, Nijman H.W.1, Daemen T.1
1UMCG, Groningen, Netherlands
ORAL ALK SHORT T 2015
The clinical efficacy of therapeutic cancer vaccines result, the triple treatment strongly enhanced the im- is currently still limited. For effective immunothera- munotherapeutic antitumor effect, blocking tumor peutic responses in cancer patients, multimodal development altogether and leading to 100% tumor- approaches capable of inducing antitumor immune free survival. This study demonstrates that this mul- responses and bypassing tumor-mediated immune timodal approach elicits superior antitumor effects escape seem essential. Here, we report on a combi- and should be considered for clinical applications. nation of sunitinib, single low-dose tumor irradiation and immunization with a therapeutic cancer vaccine based on a Semliki Forest virus vector encoding the oncoproteins E6 and E7 of human papillomavirus References: (SFVeE6,7). We previously demonstrated that low- 1) Oana Draghiciu, Mateusz Walczak, Baukje Nynke Hooge- boom, Kees L.M.C. Franken, Kees J.M. Melief, Hans W. dose irradiation (1) and sunitinib (2) in single com- Nijman andToos Daemen (2014). Therapeutic immuniza- binations with SFVeE6,7 immunizations enhanced tion and local low-dose tumor irradiation, a reinforcing combination. Int. J. of Cancer, 134 (4), 859-872. the intra-tumoral ratio of antitumor effector cells to 2) Oana Draghiciu, Hans W. Nijman, Baukje Nynke Hooge- myeloid-derived suppressor cells (MDSCs). Based on boom, Tjarko Meijerhof and Toos Daemen (2015). Sunitinib depletes myeloid-derived suppressor cells and synergizes these results we designed a regime involving a triple with a cancer vaccine to enhance antigen-specific immune treatment combination. responses and tumor eradication. OncoImmunology DOI:10 .4161/2162402X.2014.989764 The combined treatment of sunitinib, low-dose ir- radiation and SFVeE6,7 immunization resulted in stronger intra-tumoral MDSC depletion than su- nitinib alone. Concomitantly, the highest levels of intra-tumoral E7-specific CD8+ T cells were attained after triple treatment. Approximately 75% of these cells were positive for the early activation marker CD69. The combination of sunitinib, low-dose tumor irradiation and SFVeE6,7 immunization dramatically changed the intra-tumoral immune compartment. Whereas control tumors contained 2 E7-specific CD8+ T cells per 100 MDSCs, triple treatment tumors contained more than 20.000 E7-specific CD8+ T cells per 100 MDSC; a 10,000-fold increased ratio. As a 011 | THERAPEUTIC VACCINATION
The combination of trifunctional bispecific antibodies with a CTLA-4-blocking antibody impacts the induction of an immunologic memory in a preclinical mouse model
Deppisch N.1, Ruf P.2, Eissler N.1, Lindhofer H.2, Mocikat R.1
1Helmholtz-Zentrum München, Institute of Molecular Immunology, Munich, Germany, 2Trion Research GmbH, Martinsried, Germany
Trifunctional bispecific antibodies (trAb) are novel compared to trAb only. Interestingly, the combinato- anticancer drugs that recruit and activate different rial vaccination had strong impact on CD4+ helper T types of immune effector cells at the targeted tumor. cells and led to an increase of CD4+ effector memory Thus, tumor cells are effectively eliminated and a (CD44+CD62L-) and central memory (CD44+C- long-lasting tumor-specific T-cell memory is induced. D62L+) T cells. Additionally, these memory cells ex- In a preclinical mouse model trAb induce T-cell ac- pressed the co-stimulatory molecule CD137, a surface tivation. However, this is followed by induction of marker that is upregulated on T cells following spe- regulatory T cells, which express high levels of the cific TCR activation. Since CD8+ T cells are primar- co-inhibitory molecule CTLA-4. To sustain anti-tu- ily important for direct tumor killing and CD4+ T mor responses and to further enhance the efficien- cells are known to play a major role for the generation cy of trAb, we developed a combinatorial treatment of an immunologic memory, these alterations suggest approach and studied the synergistic effect of trAb an enhanced vaccination effect due to the combina- and a CTLA-4-blocking antibody on direct tumor torial use of trAb and a CTLA-4 blocking antibody. killing as well as on the induction of an immunologic Taken together, the combination of trAb and a CT- memory in an established mouse model. LA-4-blocking antibody showed no additional effect We first examined the direct therapeutic potential of on direct tumor killing. However, the induction of a the combination in vivo, but observed no addition- CD4+ T-cell memory was positively affected. These al survival benefit compared to trAb monotherapy. findings are relevant for the use of trAb in clinics and Since trAb not only mediate direct tumor killing, but provide a promising approach for efficient antitumor also elicit a vaccination effect, we further investigat- vaccination. ed the impact of the combination on the induction of an immune response in vivo. Since cytokines are key players in this context, we analyzed cytokine concentrations in mice sera after immunization. Both treatment groups - trAb only and the combi- nation - showed a comparable Th1 profile. On the cellular level, we observed an enrichment of tumor peptide-specific CD8+ T cells after immunization and in vitro restimulation. However, CD8+ T cells of the combination group showed an impaired effector function in terms of IFN-γ release and cytotoxicity 012 | THERAPEUTIC VACCINATION
Accurate HLA typing from next-generation sequencing data
Dilthey A.1, Kelleher J.1, Iqbal Z.1, Mcvean G.1
1University of Oxford, Oxford, United Kingdom
Classical HLA types describe the structure and 2015, accepted at Nature Genetics; preprint http:// binding affinities of the classical HLA proteins. The dx.doi.org/10.1101/006973). classical HLA proteins are a key mediator of adap- We show that HLA*WGS enables accurate HLA tive immunity: they present short peptide fragments typing at 4-digit resolution from whole-genome Il- from the inside of the cell to the outside, enabling the lumina sequencing data at HLA-A, -B, -C, -DQA1, immune system to scrutinize and react to the cell´s -DQB1, -DRB1. With 250 bp paired-end HiSeq reads internal state. The HLA genes are the most polymor- at 50-60x coverage on a set of 11 samples from the phic loci in the human genome, and it is well-known 1000 Genomes Project, we correctly determine 21 of that they determine the repertoire of presentable 22 validated alleles at HLA-A; 22/22 at HLA-B; 22/22 epitopes. at HLA-C; 22/22 at HLA-DQB1; 16/18 at HLA-DRB1. This has important implications for cancer immu- With 100bp paired-end HiSeq reads for NA12878 (50- notherapy: the set of a tumour´s neo-epitopes is de- 60x), we correctly determine 2/2 alleles for all 6 loci. termined not only by the tumour´s non-synonymous Accuracy on a 1000 Genomes test dataset of whole- mutations, but also by patient HLA types. Whereas exome sequencing data is lower (evaluated at 2-digit non-synonymous mutations are readily accessed by resolution: 95% HLA-A; 91% HLA-B; 95% HLA-C; high-throughput genotyping technology (i.e. whole- 96% HLA-DQA1; 88% HLA-DQB1; 85% HLA-DRB1). genome or whole-exome next-generation sequenc- This is likely driven spatial differences in coverage ing), HLA types (at 4-digit resolution), however, are across the HLA exons, that are likely to be less pro- not - polymorphism in the exons of the HLA genes nounced in current state-of-the-art pulldown panels. makes it difficult to accurately map sequencing reads. By using standard next-generation sequencing data Here we present HLA*WGS, a new algorithm to infer for HLA type inference, HLA*WGS makes neo- HLA types from next-generation sequencing data. epitope discovery more cost-efficient. In addition HLA*WGS is based on an improved read mapping to generating HLA types for the six classical HLA algorithm that utilizes a Population Reference Graph genes, HLA*WGS can also genotype all other HLA (PRG) of the HLA genes, comprising all currently genes (not validated). By improving the mapping of catalogued HLA reference sequences and their reads to HLA genes, HLA*WGS can support the in- allelic variants. The explicit representation of poly- vestigation of cancer-specific alterations in the HLA morphism allows for improved placement of reads components of the antigen presentation pathway, even in regions of population polymorphism (we such as gene loss and nonsynonymous mutations in have recently shown that PRGs also enable improved the HLA genes. genome assembly in the whole MHC; Dilthey et al. 013 | THERAPEUTIC VACCINATION
The GAPVAC consortium: Moving a novel concept of actively personalized cancer immunotherapy into the clinic
Frenzel K.1, Hilf N.2, Heesch S.1, Kuttruff-Coqui S.2, Lindner J.2, Admon A.3, Britten C.M.4, Bukur V.1, van der Burg S.H.4,5, Castle J.6, Diekmann J.1, Dorner S.2, Fritsche J.2, Gouttefangeas C.4,7, Kreiter S.1,6, Kroep J.R.5, Lassen U.8, Lewandrowski P.2, Löwer M.6, Martinez-Ricarte F.9, Maurer D.2, Mendrzyk R.2, Meyer M.2, Müllar S.2, Müller F.1, Okada H.10, Ottensmeier C.11, Paruzynski A.1, Pawlowski N.2, Piro J.12, Ponsati B.12, Poulsen H.S.8, Rössler B.2, Sahuquillo J.9, Al-Salihi O.13, Schoor O.2, Song C.2, Stevanovic S.7, Stevermann L.2, Tabatabai G.14, thor Straten P.8, Wagner C.2, Walter S.2, Weinschenk T.2, Huber C.4, Rammensee H.-G.7, Dietrich P.-Y.15, Wick W.16, Singh-Jasuja H.2, Sahin U.1
1BioNTech Group, Mainz, Germany, 10University of San Francisco, Department of 2immatics biotechnologies GmbH, Tuebingen, Germany, Neurological Surgery, San Francisco, United States, 3TECHNION - Israel Institute of Technology, Haifa, Israel, 11University of Southampton, Southampton, United 4CIMT - Association for Cancer Immunotherapy, Kingdom, Mainz, Germany, 12BCN Peptides S.A, Barcelona, Spain, 5Leiden University Medical Center, Leiden, Netherlands, 13Southampton General Hospital, Southampton, 6TRON -Translational Oncology at the University United Kingdom, Medical Center Mainz, Mainz, Germany, 14University Hospital Tübingen, Interdisciplinary 7Eberhard Karls University Tuebingen, Tuebingen, Division of Neurooncology, Tuebingen, Germany, Germany, 15Université de Genève, Genève, Switzerland, 8Copenhagen University Hospital, Herlev, Denmark, 16University Hospital Heidelberg, Heidelberg, Germany 9Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain,
Introduction: The Glioma Actively Personalized and/or Hiltonol® (poly-ICLC) in addition to standard Vaccine Consortium (GAPVAC) is a European con- chemotherapy and after surgery and initial radio- sortium aiming to introduce a new concept of im- chemotherapy are completed. The clinical trial is ac- munotherapy in a multicenter first-in-human trial companied by an extensive immune monitoring and in patients with glioblastoma (GB). GAPVAC takes biomarker program to assess the mode-of-action of personalization to the next level by engaging mul- the vaccines and to support further clinical develop- tiple independent methodologies to characterize the ment. individual disease and patient’s immune system in The definition of the APVAC vaccines is performed depth and to guide the manufacturing of a unique for each patient individually by comprehensive bio- therapeutic cancer vaccine for every patient informatic and biomarker analyses (e.g. NGS, HLA Methods: Up to 30 patients with newly diagnosed, peptidomics, transcriptomics, immunogenicity fully resectable GB (HLA-A*02/-A*24) will be in- screening). The first vaccine (APVAC1) will consist cluded into the ongoing phase I trial GAPVAC-101. of a tailored selection of 5 to 10 peptides chosen from For every Patient 2 actively personalized vaccines a pre-manufactured GMP warehouse. Peptides con- (termed APVACs) will be subsequently adminis- taining tumor-specific mutations and non-mutated, tered together with the immunomodulators GM-CSF individually over-presented peptides not contained in the warehouse may be selected into the second vaccine (APVAC2) and be produced de novo for every patient. Results: Since start of the project, enormous progress has been made towards the clinical translation. A multi-disciplinary process for the APVAC drug de- velopment was established and its feasibility success- fully tested in multiple test runs. The APVAC1 test compositions varied substantially, thus revealing a high level of personalization. Additionally, mutated and over-presented peptides were identified for the APVAC2 test compositions. In all test runs, the peptide compositions of both APVACs were defined within the predetermined ambitious time of 7 and 14 weeks, respectively. A huge effort was undertaken to establish the logistics of sample and data transfer. In addition, manufacturing of approx. 70 warehouse peptides has been completed. The manufacturing license of the consortium partner University Tuebin- gen to produce APVAC vaccines was received in 2014. In a joint effort, the CTA documents were final- ized and the German national authority approved the start of the GAPVAC-101 study in October 2014. Opening of sites in Spain, Switzerland, Denmark, Netherlands, UK and the USA is ongoing. With the enrollment of the first patients into GAPVAC-101 the novel and ambitious concept of actively personal- ized cancer immunotherapy has been realized in the clinical setting.
GAPVAC is supported by the European Commission’s 7th framework program 2012
014 | THERAPEUTIC VACCINATION
Checkpoint inhibitors in cancer immunotherapy: Cross reactivity of a CTLA-4 antibody and IDO-inhibitor L-1MT in pigs
Al-Shatrawi Z.A.1, Frøsig T.M.1, Jungersen G.1
1Technical University of Denmark, National Veterinary Institute, Department of Immunology and Vaccinology, Frederiksberg, Denmark
Blockade of checkpoint inhibitors has recently specific activation of porcine T cells. This will be shown very convincing results in the treatment of further investigated to provide the basis for in vivo cancer. One key target is CTLA-4, which has been studies investigating checkpoint inhibitor blockade demonstrated to be a potent negative regulator of in combination with other cancer immunotherapies. lymphocyte activation. The treatment with the FDA- Eventually our goal is to establish pigs as an alterna- approved fully human CTLA-4 monoclonal antibody tive large animal model for development and formu- Ipilimumab increases anticancer T-cell reactivity lation of new human cancer vaccines. and overall survival of metastatic cancer patients. Indole-amine 2,3-dioxygenase (IDO) is another checkpoint inhibitor which suppresses T-cell im- munity by the depletion of tryptophan in the T-cell microenvironment, and also inhibition of IDO by L- 1-Methyltryptophan (L-1MT) has shown promising results in clinical phase I/II studies of human cancer such as epithelial ovarian cancer. Pre-clinical immune therapeutic studies are usually performed with mice, but Ipilimumab is not reactive with mouse cells. Recent studies indicate that the pig may be a more suitable animal model for studies of immune reactivity due to higher similarity of the immunome between pig and man. This study is part of the CANVACPIG project “Accelerating develop- ment of vaccines against cancer with pigs as a large animal model” and investigates the reactivity of a fully human monoclonal anti CTLA-4 antibody and L-1MT on porcine immune cells. At the genome level, the homology between human and pig CTLA-4 and IDO is 86% and 73%, respec- tively, while the homology to the mouse is 75% and 63%. Our preliminary in vitro studies indicate that the monoclonal anti CTLA-4 antibody induces a non- 015 | THERAPEUTIC VACCINATION
Immunotherapy of Merkel Cell Carcinoma by therapeutic DC vaccination against the viral oncogenic driver large T antigen
Gerer K.F.1, Dörrie J.1, Schuler G.1, Schaft N.1, Hoyer S.1
1Universitätsklinikum Erlangen, Dermatology, Erlangen, Germany
In about 15% of human cancers a viral involvement pathway (caIKKs), which increased the immunoge- has been discovered. Standard therapies are often nicity of the DCs. not sufficient. Therapeutic vaccines, like dendrit- We co-electroporated the DCs with mRNA coding for ic cell (DC)-based vaccines, represent an option to caIKK and LT antigen, or LT-DCLamp - the latter to generate tumor-specific cytotoxic T cells (CTLs) to permit MHC class II presentation. These transfected destroy tumors. Thereby, the choice of a suitable DCs, expressing the LT and the LT-DCLamp antigens, target antigen is crucial. It is important to use an were used to stimulate autologous CD8+ T cells or a antigen which does not occur in healthy tissues to mixture of CD4+ and CD8+ T cells for several weeks avoid autoimmune reactions. to measure their ability to induce an antigen-specific In this study we examined a viral product which immune response against the LT antigens. After two causes a malignant transformation and still is rounds of stimulation, CD8+ T cells, but more clearly present in an established tumor. The involvement of the combination of CD4+ and CD8+ T cells, stimu- the Merkel cell polyomavirus (MCV) has been asso- lated with the LT-transfected DCs, recognized the ciated with the accruement of Merkel cell carcinoma antigen. The stimulation with LT-DCLamp-transfect- (MCC). Until now, no sufficient therapy is available ed DCs even led to a more effective T-cell induction. for MCC. Hence, we want to resolve this by therapeu- These results show that DCs transfected with tic vaccination with DCs. MCV can integrate into the LT-mRNA were able to present epitopes derived host cell genome and express a truncated form of one thereof. Moreover, these epitopes were immunogen- of its proteins, the large T antigen (LT), which is the ic and efficiently induced T-cell responses, in which oncogenic driver. The LT antigen is a very promising CD4+ T cells seemed to play an important role. antigen, because it is: i) a “foreign” antigen and thus In conclusion, we now possess the technical prereq- not exposed to self-tolerance mechanisms, ii) it is uisites to take this method into the clinic. This ap- similar in various patients, and iii) it is relevant for proach offers a new promising therapy option for a the oncogenic phenotype avoiding the rise of anti- disease without a current approved standard therapy. gen-loss variants of the tumor. Although it is possible to generate, mature, and load autologous DCs with antigen, we observed that these cells need a further activation signal to induce a potent response and a long-lasting immunological memory. This signal was provided by constitutively active mutants of components of the NF-κB signaling 016 | THERAPEUTIC VACCINATION
Preclinical evaluation of an mRNA-based immunotherapy against HPV16+ Head and neck squamous cell carcinoma
Grunwitz C.1,2, Selmi A.3, Schmitt U.1, Byl M.3, Schrörs B.3, Diekmann J.2, Pless B.2, Diken M.3, Kreiter S.3, Vascotto F.3, Türeci Ö.4, Sahin U.1,2,3
1Research Center for Immunotherapy (FZI), Johannes Gutenberg University, Mainz, Germany, 2BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, 3TRON - Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH, Mainz, Germany, 4Ganymed Phamaceuticals AG, Mainz, Germany
The incidence of Head Neck squamous cell carcino- acterize the modulation of the tumour environment
ma (HNSCC) is on the rise and to date treatment is upon RNA[lip] therapy, we analysed the expression of not only associated with facial disfigurement but also receptor/ligand pairs involved in T-cell inhibition. with a high rate of long-term morbidity. Therefore, We found significant up-regulation of co-inhibitory alternative treatment options are needed to improve molecules (e.g. PD1, PD-L1 and CTLA-4) in response
patient outcomes. Multiple lines of evidence linked to RNA[lip], providing a rational approach for combi- the increase in HNSCC to human papillomavirus nation therapies to achieve the ultimate goal of ther- (HPV) infections, primarily associated with HPV-16. apeutic vaccination - sustained and complete tumor HPV+ tumors account for up to 65% of all HNSCC rejection. and up to 80% of squamous cell carcinomas located in the oropharynx. Since expression of the oncopro- teins HPV16 E6 and E7 are required to maintain the proliferative state, they display an excellent target for therapeutic vaccinations. Here we investigated the effect of an RNA-based liposomal formulated vaccine encoding modified sequences of HPV-16 derived E6 and E7. Repetitive immunizations with E6/E7 RNA + [lip] led to substantial antigen specific CD8 T cell ex- pansion, which displayed potent effector function as analysed by IFNγ ELISPOT and intracellular cytokine staining in C57BL/6 and HLA-A2/DR1 transgenic
mice. Assessment of RNA[lip] anti-tumoural activi- ty in established TC-1/luc tumours revealed strong tumour rejection, prolonged survival and profound changes of the tumor-environment. We observed an increased frequency of tumor infiltrating E7-reac- + + tive CD8 T cells, NK cells and beneficial CD8 / Treg ratio as well as polarization of macrophages towards + a CD206- iNOS phenotype. Notably, three RNA [lip] vaccinations resulted in rapid and potent tumour re- jection of well-established tumours. To further char- 017 | THERAPEUTIC VACCINATION
A novel peptide-conjugate vaccine strategy that targets antigen-presenting cells via endogenous antibodies
Gustafsson W.1, Fletcher E.A.K.1,2, van Maren W.3, Cordfunke R.3, Dinkelaar J.4, Castelli R.4, Codee J.D.C.4, van der Marel G.4, Melief C.J.M.3, Drijfhout J.W.3, Ossendorp F.3, Mangsbo S.M.1,2
1Uppsala University, Department of Immunology Genetics and Pathology, Uppsala, Sweden, 2Immuneed Inc., Uppsala, Sweden, 3Leiden University Medical Center, Department of Immunohematology & Blood Transfusion, Leiden, Netherlands, 4Leiden Institute of Chemistry, Department of Bio-organic Synthesis, Leiden, Netherlands
Several cancer immunotherapy strategies, including protein of cytomegalovirus (CMV), strongly reacti- vaccination using long peptides, utilize the ability of vates memory T cells when analyzed in blood from dendritic cells (DCs) to boost an immune response donors with CMV-specific T cells. The CMV specific against antigens. However, water-based peptide vac- T cells produced IFNγ in response to the conjugate cines are rapidly degrades and oil-based delivery illustrating that the uptake of the conjugate leads strategies trap immune cells to unwanted sites. Our to activation of antigen specific T cells. Strikingly novel vaccine (MTTE]3-LSP) consists of three B cell uptake as well as T cell activation occurs at low con- epitopes chemically conjugated to a T cell epitope centrations of the LSP conjugate, superior to a con- composed of a long synthetic peptide (LSP). The LSP jugate lacking the tetanus-based target as well as to can be designed based on the disease of interest. The LSPs alone. Our data show that we have a unique conjugation of a LSP to B cell epitopes facilitates for- delivery system for peptide based vaccines that can mation of immune complexes with circulating anti- aid induction of human T cell responses, which may bodies, enhancing both LPS uptake and activation of ultimately help the patient to mount an immune re- antigen presenting cells such as dendritic cells. The B sponse against for example a tumor. cell epitope MTTE (minimal tetanus toxoid epitope) is a peptide sequence derive from tetanus toxin, which the majority of humans have IgG antibodies against as a result of tetanus vaccination. We have applied our unique whole blood loop system, with intact complement system, to characterize how the vaccine is targeted to human immune cells. The B cell-T cell conjugate ([MTTE]3-LSP) is taken up after 1 hour by human monocytes and blood DCs. The uptake is anti- body-dependent; however, FcγRs appear less import- ant for the internalization of the antigen. The uptake is more likely through the classical pathway of the complement system as it is reduced by blocking C1q (classical pathway), but not when blocking C3 (al- ternative pathway) and at the same time completely
abolished by both EDTA and EGTA. A [MTTE]3-CMV conjugate, containing a T cell epitope from the pp65 018 | THERAPEUTIC VACCINATION
Investigation of T-cell responses after DNA vaccination in patients with chronic myeloid leukaemia
Harden E.1, Cazaly A.1, Thirdborough S.1, Ottensmeier C.1
1University of Southampton, Southampton, United Kingdom
DNA vaccination using the pDOM-epitope design, impaired WT1-specific responses. Ex vivo ELISPOT which fuses domain 1 from tetanus toxin fragment C data revealed that there were indeed T-cell responses protein (DOM) to a tumour-specific peptide of inter- to some of the overlapping peptide pools covering the est, elicits DOM-specific helper T cells and tumour length of DOM. Using intracellular cytokine stain- specific CTL which have the potential to kill tumour ing and an HLA-A2 tetramer we identified a novel cells. This approach is of interest in a setting of DOM-specific epitope which 8/11 patients responded minimal residual disease such as in patients with to. This response often coincided with the presence chronic myeloid leukaemia (CML) who have been of WT1-specific responses rather than occurring in successfully treated with tyrosine kinase inhibitors patients with no WT1-specific T cells. This suggests but are at risk of relapse. that although T-cells against DOM may be slightly During the WIN trial, 12 CML patients were vaccinat- more frequent and numerous, they do not out-com- ed with p.DOM-WT1.37 and p.DOM-WT1.126 DNA pete WT1-specific T cells after DNA vaccination. We vaccines encoding peptide epitopes from Wilm’s believe that we have therefore confirmed the validity tumour antigen (WT1). Blood, leukapheresis and of our approach and our results will inform the plan- bone marrow samples were taken throughout the ning of future clinical trials. vaccination period and beyond to enable immuno- logical monitoring. All patients tested (n=10) had detectable anti-DOM IgG after vaccination, indicat- ing that DNA vaccination was successful. Encourag- ingly, WT1-specific CD8+ T-cell responses were also detected using peptide-specific HLA-A2 tetramers in most evaluable patients; 6/11 for WT1.37 and 2/11 for WT1.126. In addition to the assessment of clinical efficacy, a detailed analysis of the T-cell responses induced is underway. One question we wanted to answer was whether there was any evidence of T-cell competition between CTL directed against the tumour antigen and those that could potentially be induced against the DOM helper sequence. We had hypothesised that patients with strong CTL responses to DOM may have 019 | THERAPEUTIC VACCINATION
The Mutanome Engineered RNA Immuno-Therapy (MERIT) project: Introducing individualized medicine for the treatment of TNBC
Heesch S.1, Britten C.M.1, Bukur V.1, Buck J.1, Castle J.2, Diekmann J.1, Diken M.2, Ewen K.M.1, Frenzel K.1, Kreiter S.2, Kuhn A.N.1, Kuehlcke K.3, Loewer M.2, Haas H.1, Kemmer-Brueck A.1, Kloke B.-P.1, Otte B.1, Paruzynski A.1, Petri S.1, Schwarck D.1, Schmidt M.4, Andre F.5, De Greve J.6, Kuendig T.7, Lindman H.8, Pascolo S.7, Sjoeblom T.8, Thielemans K.6, Zitvogel L.5, Tuereci O.9, Sahin U.1
1BioNTech Group, Mainz, Germany, 6Vrije Universiteit Brussel, Brussel, Belgium, 2TRON -Translational Oncology at the University Medical 7University Hospital of Zurich, Zurich, Switzerland, Center Mainz, Mainz, Germany, 8Uppsala University Hospital, Uppsala, Sweden, 3EUFETS GmbH, Idar-Oberstein, Germany, 9III. Medical department, University Medical Center of the 4University Hospital Mainz, Mainz, Germany, Johannes Gutenberg University, Mainz, Germany 5Gustave Roussy Comprehensive Cancer Center, Paris, France,
The Mutanome Engineered RNA Immuno-Therapy The MERIT project started in June 2013 and by now (MERIT) consortium will clinically and industri- enormous progress has been made towards the clini- ally validate a pioneering RNA-based immunotherapy cal translation of this individualized cancer immu- concept targeting individual tumor antigens and tu- notherapy approach. After discussing the regula- mor-specific mutations in triple negative breast cancer tory challenges with the German national regulatory (TNBC) patients. The treatment of TNBC is hampered agency (PEI), a phase I study is now in preparation. by the apparent lack of established therapeutic targets The trial will start in Q2 2015 in five academic centers like hormonal receptors or HER-2, and therefore chemo- in Europe and will recruit thirty TNBC patients. We therapy and radiotherapy is currently the mainstay of have established a RNA delivery platform as well as a therapy. However, survival rates in TNBC remain poor. MERIT WAREHOUSE containing mRNAs coding for a The MERIT concept attempts to address this unmet selection of TNBC specific antigens. Additionally, we medical need. The personalized treatment consists in have built a multi-disciplinary clinical workflow and (i) injecting vaccines containing “off the shelf” mRNAs trial design tailored to this unique therapeutic concept. selected from a pre-synthesized mRNA vaccine ware- We will describe the therapeutic concept, critical skills, house (MERIT WAREHOUSE) encoding tumor specific and methodologies required for this project, including antigens expressed in the respective patient’s tumor; cancer genomics, NGS, bioinformatics, tumor immu- and (ii) thereafter mRNAs engineered on-demand that nomics, industrial drug development, GMP manufac- encode patient-specific sequence stretches incorporat- turing, clinical immunotherapy, and immunological ing non-synonymous mutations identified by next gen- monitoring. eration sequencing (NGS) and ranked by predicted im- Moreover, an extensive MERIT research program ad- munogenicity (MERIT MUTANOME). The mRNAs are dresses the optimization of algorithms for improved administered intravenously as a nanoparticulate lipo- prediction of immunogenic mutations. Additionally, plex formulation and are selectively delivered to splenic compounds to enhance vaccine efficacy will be devel- APCs. The encoded antigens are translated into proteins oped and improved to support further clinical develop- that are rapidly processed. Subsequent peptide presen- ment. tation on the surface of APCs induces antigen-specific This biomarker-guided, personalized therapy is a col- T cell responses. The central part of the MERIT project, laborative effort of five partners from academia and a multi-center first in-human trial, will assess the fea- industry and is funded by the European Commission’s sibility, safety and biological efficacy of this innovative FP7 and led by BioNTech AG. personalized immunotherapy in TNBC patients. 020 | THERAPEUTIC VACCINATION
Vaccinia virus E3-mNRA boosts humoral immune response to self-replicating RNA encoding HA
Hempel T.1, Beissert T.1, Koste L.2, Erbar S.3, Reuter K.3, Selmi A.1, Diken M.1, Reece S.3, Sahin U.1
1TRON -Translational Oncology at the University Medical Center Mainz, Mainz, Germany, 2Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Mainz, Germany, 3BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany
Self-replicating RNA vectors (replicons) based on We conclude that removal of PKR-mediated blocking alphaviral genomes recently reemerged as power- of translation improves vaccination with replicons. ful vaccine vectors. Immune responses to replicon Intracellular translation of PKR inhibitor mRNA is encoded antigens are driven by an intrinsic self-ad- an advantage of our approach since it might preclude juvanting activity of replicating RNA which relates side-effects potentially related to systemic PKR inhi- to a strong activation of cellular innate immunity by bition with small molecules. Furthermore, the “RNA double stranded RNA (dsRNA) replication intermedi- only” design of our vector platform facilitates formula- ates. However, innate immune response upregulates tion development required for the clinical translation protein kinase R (PKR), which is additionally acti- of our concept and offers a high flexibility, as the E3 vated by dsRNA leading to a global suppression of mRNA can be combined with any antigen-encoding protein translation including the translation of repli- replicon. con vectors. Accordingly PKR inhibitors were shown to enhance translation of replicon RNA, but this ap- proach has not yet been applied to improve vaccina- tion with replicons. Here we demonstrate that PKR inhibition significantly enhances immune response to replicon-encoded antigens. To this aim we combined antigen encoding replicon RNA with non-replicating synthetic mRNA encoding Vaccinia virus PKR inhibi- tors like E3. We observed that co-transfected E3 mRNA increased translation of replicase, an enzyme complex encoded on the 5’ part of replicons that drives rep- lication and subgenomic transcription. Correspond- ingly we also found that E3 boosted RNA replication and the abundance of antigen encoding subgenomic RNA. Furthermore, the translation of subgenomic transcripts raised more than 10-fold upon E3 mRNA cotransfection. In vivo, not only the transgene expres- sion but also humoral immune responses to Influenza HA encoding replicons were enhanced as assessed by a 5-fold increased hemagglutination inhibition titer. 021 | THERAPEUTIC VACCINATION
Neuron-specific microRNA target repeats engineered in alphavirus RNA genome abrogate lethal phenotype but retain viral ability to replicate in the presence of IFN-I in syngeneic CT-2A glioma model
Martikainen M.1, Niittykoski M.1, von und zu Fraunberg M.2, Immonen A.2, Koponen S.2, Vähä-Koskela M.3, Ylösmäki E.4, Saksela K.4, Hinkkanen A.1
1University of Eastern Finland, A. I. Virtanen Institute for Molecular Sciences, Kuopio, Finland, 2Kuopio University Hospital, Neuroscience Center, Kuopio, Finland, 3University of Helsinki, Institute of Biotechnology, Helsinki, Finland, 4University of Helsinki, Haartman Institute, Helsinki, Finland
Malignant glioma is devastating central nervous mice showed response, suggesting that the tumors system disease with no effective treatment avail- are slightly immunogenic. Reminiscent of replica- able. Oncolytic virotherapy with current generation tion, SFV glycoproteins were detected by IHC in viruses has shown poor overall response. This may be SFV4-miRT124 -treated tumors. Neurological symp- partly due to use of nonreplicating vectors or the sen- toms were observed in a fraction (12.5%) of SFV4- sitivity of vectors to tumor type I interferon (IFN-I). miRT124 treated mice and virus antigen was found Neuroattenuated Semliki Forest virus (SFV, alphavi- in the spinal cord and medulla regions. It seems that rus) vector VA7 shows poor oncolytic potency against the nonstructural genome encoding viral replicase syngeneic gliomas due to vector IFN-I sensitivity. VA7 harbors the key elements for engineering of viru- is naturally neuroattenuated whereas SFV4, a proto- lence and cell specific replication. Finally, as com- typic strain, replicates robustly in CNS causing lethal pared to VA7, SFV4-miRT124 was able to replicate encephalitis. In cultured Vero and CT2A glioma cells significantly better in novel human glioblastoma-de- both SFV4 and attenuated SFV4-miRT124, a vector rived cell lines despite IFN-I pretreatment. It seems carrying targets for neuron miRNA124, inhibited that more robust viral replication is required to over- STAT-1 phosphorylation, whereas VA7 did not. In come tumor IFN-I in syngeneic glioma and that strict CT2A tumor mice SFV4-miRT124 was specifically guidance to malignant cells of enhanced oncolytic inhibited in CNS neurons due to multiple inserted vectors might enhance therapy efficacy without com- miRNA target elements recognized by neuronal miR- promising safety. 124. SFV4-miRT124 ignored cellular IFN-I in vitro. The microRNA responsive repeats in viral nonstruc- tural genome were not found to affect viral repli- cation in CT-2A-FLuc mouse glioma cells devoid of miR-124. Similarly to parental SFV4, SFV4-miRT124 replicated in IFN-beta pretreated CT-2A-FLuc cells, whereas VA7 replication was abrogated. A single intraperitoneal dose of SFV4-miRT124 to C57BL/6 mice bearing CT2A tumors resulted in significantly inhibited tumor progression and loss of established gliomas in 50% of mice as analyzed with bioluminescence imaging and MRI. For com- parison, 20% of VA7 treated and 13% of mock-treated 022 | THERAPEUTIC VACCINATION
Identification of oncofetal antigen (IMP-3)-derived long peptides encompassing both CTL epitopes and multiple HLA-class II-restricted Th cell epitopes
Hirayama M.1, Tomita Y.1, Yuno A.2, Sayem M.A.1, Tsukamoto H.1, Irie A.1, Senju S.1, Yoshitake Y.2, Fukuma D.2, Shinohara M.2, Yuba E.3, Kono K.3, Yoshida K.4, Nakamura Y.5, Nakayama H.2, Nishimura Y.1
1Kumamoto University, Immunogenetics, Kumamoto, Japan, 2Kumamoto University, Oral and Maxillofacial Surgery, Kumamoto, Japan, 3Osaka Prefecture University, Department of Applied Chemistry, Graduate School of Engineering, Sakai, Japan, 4OncoTherapy Science Incorporation, Research and Development Division, Tokyo, Japan, 5University of Tokyo, Institute of Medical Science, Laboratory of Molecular Medicine, Human Genome Center, Tokyo, Japan
We recently identified CTL epitopes derived from an could cross-prime IMP-3-SP-specific CTLsin vitro in oncofetal antigen, insulin-like growth factor II mR- human and in vivo in HLA-A2 transgenic mice. The NA-binding protein 3 (IMP-3) which was frequently same LP encapsulated in newly developed pH-sensi- overexpressed in head-and-neck cancers (HNCs) and tive liposome, that can deliver LPs from endosome to lung cancers, but not in many normal adult organs, cytoplasm, was also found to be efficiently cross pre- by using genome-wide cDNA microarray analyses. sented in vitro to stimulate IMP-3-SP-specific human Several phase I/II clinical trials using IMP-3-derived CTLs. Furthermore, we could induce IMP-3-LP-spe- short peptides (SPs) vaccination against several cific Th cells from a HNC patient vaccinated with an types of malignant tumor are ongoing. Recently we IMP3-derived SP. Immune response to these LPs in reported the phase I/II clinical trial of multiple tu- many other HNC patients is now under investigation. mor-associated-antigen (TAA)-derived SPs vaccina- Taken together, IMP-3-derived LPs may be applicable tions including IMP-3-SP for advanced HNC patients. to cancer immunotherapy. In this trial, we observed the vaccinated SPs-specific CTL responses and the prolongation of overall sur- vival of HNC patients without serious adverse effects (Clin Cancer Res 2015; 21:312). In order to further develop peptides-based cancer immunotherapy, we attempted to identify IMP-3-derived long peptides (LPs) containing both CTL and Th cell epitopes in this study. We successfully identified two IMP-3-de- rived LPs recognized by promiscuous HLA-class II- restricted Th cells. One of them encompasses two natural HLA-A2 or A24-restricted CTL epitopes, and these two LPs can stimulate IMP-3-LP-specific Th1 cells restricted by 7 frequent HLA class II mol- ecules. We confirmed that Th cells induced by two LPs responded to autologous dendritic cells (DCs) loaded with IMP-3 protein, suggesting that these IMP-3-derived LPs were possibly naturally processed from IMP-3 protein and presented by DCs. We also demonstrated that one of the IMP-3-derived LPs 023 | THERAPEUTIC VACCINATION
CCL22 regulated plasmid DNA as a promising perspective in DC-specific DNA vaccination
Hobernik D.1, Grabbe S.1, Bros M.1
1University Medical Center of the Johannes Gutenberg University Mainz, Department of Dermatology, Mainz, Germany
For several years DNA vaccination has been gaining ically, a reporter construct which consists of the 250 importance to fight numerous diseases due to the bp proximal CCL22 promoter with a mutated NF-κB possibilities associated with genetically engineered site showed high expression of Luciferase in DC2.4, plasmid DNA. Presently, first clinical trials have whereas no luciferase activity was observed in B16/ started using DNA vaccines in cancer immunothera- OVA-melanoma cells. py. Professional antigen presenting cells like dendrit- Ongoing work will focus on the improvement of DC ic cells (DC), monocytes/macrophages and B cells specificity of promoter activity. Furthermore, the de- play a key role in this therapeutic approach consid- livery of derived DNA vaccines via biodegradable, ering their ability to take up, process and present tu- non-cytotoxic nanoparticles as novel carrier systems mour-associated antigens aimed to induce cytotoxic is tested. anti-tumor immune response. However, uptake of the vaccine by for example myeloid derived suppressor cells can promote tolerance and diminish the overall therapeutic success. Consequently, it is important to develop a delivery system which is specific for DC so that tolerance against tumour-associated antigens can be ruled out. A possible approach for a DC-specific DNA-vaccina- tion strategy is to control expression of the antigen of interest by a promoter which shows predominant activity in DC only. To this end, we chose promoters of genes highly expressed in activated DC like Fascin, CD11c, and CCL22. These promoters were cloned up- stream of a luciferase reporter and transfected into DC2.4, a model DC cell line, and B16melanoma cells as a control. Promoter activities of both full length and deletion promoter test constructs were analysed via photometric luciferase activity measurements. Here we show that transfection of CCL22 promot- er-derived reporter constructs showed a strong pref- erence in DC2.4 compared to B16 cells. More specif- 024 | THERAPEUTIC VACCINATION
Controversial effect of atRA in treatment of non-responders in therapeutical vaccination
Heine A.1,2, Gevensleben H.3, Diehl L.4, Garbi N.2, Brossard P.1, Kurts C.2, Knolle P.5, Höchst B.5
1Medical Clinic III for Oncology, Hematology and Rheumatology, University Hospital, Bonn, Germany, 2Institute of Experimental Immunology, University, Bonn, Germany, 3Institute for Pathology, University Hospital, Bonn, Germany, 4Inst. für Experimentelle Immunologie und Hepatologie Zentrum für Experimentelle Medizin Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany, ORAL 5 ALK Institute of Molecular Immunology, TU München, München, Germany SHORT T 2015
Therapeutic tumor vaccination represents a promis- ing strategy to induce anti-tumor activity in cancer patients. However, usually only a subpopulation of patients benefits from tumor vaccines, whereas others display continuous tumor growth and metastatic spreading. Here we report a similar observation in a B16 melanoma model. Vaccinating these mice with a model tumor antigen and a combination of adjuvants, i.e. TLR-9-ligand CpG and NKT-cell ligand -galactosyl- ceramide, controlled the tumor in most animals (High- Responders), whereas others showed no clear benefit from the treatment (Non-Responders). No differences in numbers of B, NK or T cells were detected between both groups. However, Non-Responders showed sig- nificantly increased numbers of CD11b+Gr-1+myeloid- derived-suppressor-cells (MDSCs). Treatment with all- trans-retinoic-acid (atRA), which has been described to modulate MDSCs, effectively restored the tumor control in Non-responders. Frequencies neither of regulatory T cells nor MDSCs differed after treatment with atRA, but those cells from atRA-treated animals with a pheno- type of monocytic MDSCs (CD11b+Ly6ChighLy6G--cells) had lost their suppressive capacity and displayed up- regulation of MHC-Class-II, indicating acquisition of immune-stimulatory properties. Concomitantly, IFNg production by CD8+ T cells was significantly higher in atRA-treated mice. Taken together, we demonstrate that atRA converts Non-Responders to tumor vaccination to responders, offering a novel opportunity for individualized tumor therapy. 025 | THERAPEUTIC VACCINATION
Photochemical internalization - an innovative technology giving strong enhancement of cytotoxic T-cell responses to vaccination
Høgset A.1, Nedberg A.G.1,2, Edwards V.T.1,3, Håkerud M.1,3, Johansen P.4, Kündig T.M.4, Selbo P.K.1,3
1PCI Biotech AS, Lysaker, Norway, 2Oslo University Hospital – The Norwegian Radium Hospital, Department of Radiation Biology, Oslo, Norway, 3Oslo University Hospital - The Norwegian Radium Hospital, Department of Radiation Biology, Oslo, Norway, 4University Hospital Zürich, Department of Dermatology, Zurich, Switzerland
For many types of vaccination and immunotherapy a mixture of vaccine and TPCS2a intradermally, fol- it is essential to stimulate cytotoxic T-cells (CTLs) to lowed by illumination of the injection site. Used in attack tumour- or infected cells. Activation of CTLs this way PCI strongly enhances CD8 T-cell responses, is typically mediated through MHC Class I antigen as measured by the proliferation of antigen-specific presentation by antigen presenting cells (APCs). This T-cells and intracellular staining for cytokine pro- generally requires cytosolic delivery of the antigen, duction. This effect is achieved both with protein and since the MHC class I presentation machinery is lo- with short and long peptide antigens, and up to a 200 calised in the cytosol. Unfortunately cytosolic de- times enhancement of CD8 T-cell immune responses livery is often difficult to achieve with exogenously has been observed. PCI also gives strong synergistic added peptide or protein antigens, which are pri- effects when used in combination with several com- marily taken up into endocytic vesicles and then monly used vaccine adjuvants. In vivo studies with by default are routed for MHC Class II presentation. therapeutic peptide antigen vaccination in a mouse Thus with current vaccination technologies peptide model for HPV-induced cancer show that the use of and protein vaccines are often ineffective in stimulat- PCI strongly enhances anti-tumour responses to the ing CTL responses. vaccine. Photochemical Internalisation (PCI) is a technology In conclusion, PCI has a completely new mechanism for inducing cytosolic delivery of endocytosed mole- of action as a vaccination enhancement technology, cules, using a photosensitising molecule to make en- representing a new and potent tool for stimulation of
docytic membranes light sensitive, with illumination CTL responses. The TPCS2a photosensitiser is easy inducing permeabilisation of the membranes. Thus, and cheap to produce, withstands autoclavation and PCI has a clear potential for increasing CTL respons- is stable for several years at ambient temperatures.
es by making access for antigens to the MHC Class TPCS2a is currently in clinical development for en- I presentation machinery in the cytosol of APCs, hancement of the effect of cytotoxic anti-cancer
thereby increasing MHC Class I antigen presentation. drugs, and it has been shown that TPCS2a can be Fluorescence microscopy studies show that the pho- administered safely to patients in much higher doses
tosensitiser TPCS2a (disulfonated tetraphenyl chlorin) than what is needed for the use in immunotherapy, co-localises with peptide and protein antigens in en- paving the way for testing the PCI technology in pa- docytic vesicles, and that illumination releases such tients also in the area of cancer vaccination and im- antigens into the cytosol; leading to an enhancement munotherapy. of MHC class I presentation of up to 20 times. In vivo PCI-mediated vaccination is performed by injecting 026 | THERAPEUTIC VACCINATION
Identification of T cell epitopes for a therapeutic HPV16 vaccine
Hoppe S.1,2, Schessner J.P.1, Dressler L.1, Winter J.1, Blatnik R.1,2, Steinbach A.1, Khallouf H.1, Wuehl M.1, Klevenz A.1, Riemer A.B.1,2
1German Cancer Research Center (DKFZ), Immunotherapy and -prevention, Heidelberg, Germany, 2German Center for Infectious Research (DZIF), Molecular Vaccine Design, Heidelberg, Germany
To rationally design therapeutic human papilloma- ously known binding peptides were confirmed and virus (HPV) vaccines, it is crucial to know which 93 novel binding peptides were identified in compe- T cell epitopes are presented on HPV-transformed tition-based binding assays. The presence of binding cells. Due to immune evasion mechanism by the peptides on HPV16-transformed cells is analyzed by virus, not every epitope derived from viral pro- mass spectrometry analysis of immunoprecipitated teins is necessarily presented by Human Leukocyte HLA-peptide complexes. To determine immunoge- Antigen (HLA)-molecules on the cancer cell surface. nicity of binding peptides, peptide-specific short HPV16 has been identified as the causative agent in term T cell lines were generated. To this end, periph- 50% of all cervical cancer cases and in approximate- eral blood mononuclear cells (PBMCs) from buffy ly 95% of all extra-cervical HPV-induced tumors. The coats with known HLA types were stimulated with transforming potential of high-risk HPVs is mediat- previously identified HLA-matching peptides and ed by two consistently expressed viral oncoproteins, cultured for twelve days. Several peptides induced E6 and E7. As the induction and maintenance of the interferon gamma (IFNγ)-responses. Additionally, malignant phenotype depend on these two proteins, peptide-specific long term T cell lines were gener- they represent ideal targets for cancer immunother- ated for the four most promising candidate peptides apy. A therapeutic vaccine that is applicable to ev- from HLA-A24 positive healthy donors. Functional eryone without prior HLA typing needs to contain assays, such as IFNγ-ELISpot and cytotoxicity assays, epitopes for all HLA-types. Definition of epitopes for determined the best vaccine candidates. the five major HLA supertypes allows >95% popu- In conclusion, we identified several new HPV16 E6 lation coverage. and E7 T cell epitopes. Verified epitopes are the basis Up to date, HPV T cell epitopes have mostly been de- of rational therapeutic vaccine design and also im- termined for the most prevalent HLA type, HLA-A2. portant for immunomonitoring purposes. We now aim to identify HPV16 E6 and E7 T cell epi- topes for other common HLA supertypes, HLA-A3, HLA-A11 and HLA-A24. Various in silico prediction algorithms were employed to predict prospective epitopes from the HPV16 E6 and E7 proteins for the mentioned HLA supertypes. Altogether, 64 epitopes, comprised of 8- to 11-mer peptides, were predicted for HLA-A3, 90 epitopes for HLA-A11, and 115 epitopes for HLA-A24. 15 previ- 027 | THERAPEUTIC VACCINATION
Mimicking of CD4+ T-cell help within DCs by transfection with caIKKRNA to improve DC vaccination
Hoyer S.1, Gerer K.F.1, Pfeiffer I.A.1, Wohn C.2, Schuler-Thurner B.1, Schuler G.1, Schaft N.1, Dörrie J.1
1Universitätsklinikum Erlangen, Dermatology, Erlangen, Germany, 2Centre d’Immunologie Marseille-Luminy, Marseille, France
Dendritic cells (DCs) are frequently used for thera- into DCs by mRNA electroporation led to intrinsic peutic vaccination, which aims at the induction of CD40 ligation, without the need of helper cells. Un- an immune response against the tumor. Most com- fortunately, the CD40L-DCs were only capable of re- monly, DCs are generated ex vivo from autologous petitively expanding the CD8+ T cells 4 h after elec- monocytes. These monocyte-derived DCs (moDCs) troporation, but not when used after 24 h. Hence, are matured and loaded with tumor antigen. Com- we directly activated the NF-kB signaling pathway mencing from that base, DC vaccines have been downstream of CD40 within the DCs by the intro- improved in their immunogenic efficiency by equip- duction of constitutively active IKK (caIKK). This ping them with selected features and molecules to also overcame the limited expandability of the CD8+ generate so called designer DCs. We here present an T cells, and like helped DCs, caIKK-DCs induced a approach to improve the DCs’ capability to induce memory-like phenotype of the CD8+ T cells. Most im- more effective and longer lasting immune responses portantly, in contrast to CD40L-DCs, caIKK-DCs were in cancer immunotherapy. functional for a longer time-period, i.e. still had a Standard cytokine-cocktail-matured moDCs were not stimulatory capacity 24 h after electroporation, were able to repetitively stimulate autologous naïve CD8+ capable of secreting cytokines for more than 2 days, T cells in absence of additional activation signals. and surface molecule expression of CD25, CD70, and Because CD4+ T-cell help may rescue this deficiency, OX40L continued to increase over 3 days after caIKK we established a complete human in vitro system to transfection. Thus, caIKK-DCs are a new promising examine the effect of antigen-specific help to DCs type of designer DCs for cancer immunotherapy. on CD8+ T-cell priming and subsequent expansion. The delivered helper signals enabled the DCs to re- petitively stimulate and expand CD8+ T cells. Impor- tantly, the generated T cells were of a memory-like N.S. and J.D. share senior authorship phenotype, which indicates the induction of a longer lasting immunity. We will now assess the nature of these helper signals by utilizing this system. To improve DC vaccination strategies these signals can be emulated to make the presence of specific helper T cells dispensable. One important molecule via which the T-helper cells activate DCs is CD40L. Transfection of this molecule 028 | THERAPEUTIC VACCINATION
Oncolytic measles virus for tumor vaccination
Hutzler S.1, Erbar S.2, Jabulowsky R.2, Beissert T.2, Hanauer J.1, Türeci Ö.3, Mitnacht-Kraus R.3, Kreiter S.4, Diken M.4, Britten C.M.2, Sahin U.2, Mühlebach M.D.1
1Paul-Ehrlich-Institut, Langen, Germany, 2BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, 3GANYMED Pharmaceuticals AG, Mainz, Germany, 4TRON, Mainz, Germany
The concept of tumor vaccination is based on the in- to positional effects of the additional transcription duction of robust immune responses against tumor- units either behind the P (post P) or the H (post H) associated antigens (TAA), which should be selec- gene cassettes of Moraten vaccine strain-derived
tively expressed within the tumor. Whereas in the MVvac2 genome, presented on particles, or not. Based case of foreign antigens an immune response can on these data, viruses with the best expression of be induced quite easily, induction of appropriate im- the different antigen formats were chosen for further munity against self-antigens needs potent antigen characterization. The selected viruses were ampli- presentation to break immune tolerance. fied, and unimpaired growth and genetic stability Tumor vaccines may be further improved by utiliz- of the inserted TAA were demonstrated. To test the ing the properties of oncolytic viruses (OVs), which immunogenic properties, MV-susceptible IFNAR-/- directly kill tumor cells and effectively stimulate -CD46Ge mice are currently immunized with the the immune system. Immunogenic OVs additionally different viruses, and humoral as well as cellular delivering a specific TAA can break immunological immune responses are analyzed. In parallel, an tolerance, induce a specifically redirected immune immune competent, syngeneic tumor model has response, and thereby may increase therapeutic ef- been established. Three different transgenic tumor ficacy. Attenuated measles virus (MV) derived from cell lines expressing the target antigen as well as MV- a vaccine strain is currently tested as OV in clinical receptors cause tumors in MV-susceptible animals. trials. Recombinant MVs additionally reveal excel- Such tumor-bearing animals will be treated by those lent vaccine characteristics per se, inducing potent viruses provoking significant humoral or cellular and long lasting immune responses against endog- immune responses. Thereby, immune responses, enous and foreign antigens, if the latter are addition- tumor growth and overall survival of the animals ally expressed by the recombinant virus. Therefore, will be determined. we develop and aim to validate prototypic replicating MV for oncolysis and simultaneous in situ tumor vac- cination. For that purpose, we use a claudin family member (GNTP01), a recently well characterized tumor-con- fined TAA. Different antigen formats or epitopes of GNTP01 were cloned into the MV genome and re- combinant viruses were generated, which express those epitopes in different amounts as expected due 029 | THERAPEUTIC VACCINATION
Lipo-MERIT: a novel nanoparticular formulated tetravalent RNA cancer vaccine for treatment of patients with advanced melanoma
Jabulowsky R.A.1, Attig S.2, Britten C.M.1, Buch K.1, Buck J.1, Derhovanessian E.3, Diekmann J.1, Esparza I.1, Fritz D.1, Hüsemann Y.1, Jahndel V.1, Kranz L.4, Kühlcke K.5, Laible M.6, Langguth P.1, Luxemburger U.3, Meng M.1, Müller F.3, Reuter K.C.1, Schwarck-Kokarakis D.3, Spieß K.6, Witt M.3, Loquai C.7, Hassel J.C.8,9, Utikal J.10, Kaufmann R.11, Schrott M.12, Kuhn A.N.1, Haas H.1, Diken M.2, Kreiter S.2, Türeci Ö.4, Huber C.3,4, Sahin U.1,2,3
1BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, 10Skin Cancer Unit, German Cancer Research Center 2TRON -Translational Oncology at the University (DKFZ), Heidelberg, Germany and Department of Medical Center Mainz, Mainz, Germany, Dermatology, Venereology and Allergology, Uni- 3BioNTech AG, Mainz, Germany, versity Medical Center Mannheim, Ruprecht-Karl 4III. Medical Department, University Medical Center of University of Heidelberg, Mannheim, Germany, the Johannes Gutenberg-University, Mainz, Germany, 11Department of Dermatology, Venereology and 5EUFETS GmbH, Idar-Oberstein, Germany, Allergology, Johann Wolfgang Goethe University of 6BioNTech Diagnostics GmbH, Mainz, Germany, Frankfurt, Frankfurt, Germany, 7Department of Dermatology, University of Mainz, 12Central-Apotheke, Steinbach/Ts., Mainz, Germany, Germany 8Department of Dermatology, University of Heidel- ORAL T TALK berg, Heidelberg, Germany, SHOR 9National Center for Tumor Diseases (NCT) - Heidel- 2015 berg University Hospital, Heidelberg, Germany,
Immunotherapeutic approaches have evolved as prom- into APCs has further been shown to lead to an en- ising and valid alternatives to available conventional hanced induction of vaccine-induced T-cell responses. cancer treatments. Amongst others, vaccination with Extensive pharmacological characterization of the
tumor antigen-encoding RNAs by local administration RNA(LIP) platform revealed that upon cellular uptake the is currently successfully employed in various clinical encoded antigens will be translated into proteins that trials. Lipo-MERIT (Lipoplex Melanoma RNA Immu- will be rapidly processed into peptide fragments, which notherapy) is a novel RNA immunotherapeutic for sys- after presentation by MHC class I and II molecules on temic application based on a fixed set of four liposome the surface of APCs induce tumour antigen-specific + + complexed RNA drug products (RNA(LIP)) each encod- CD8 and CD4 T-cell responses that spread systemical- ing one shared melanoma-associated antigen. ly. These vaccine-induced T cells have been shown to
Similar to other liposomal drugs, the four RNA(LIP) prod- specifically recognise and kill antigen-positive tumour ucts constituting the investigational medicinal product cells eliciting potent anti-tumoral activity in vivo. In ad- will be prepared individually prior to administration dition, by binding to human Toll-like receptors (TLRs) out of three components, namely solutions containing the administered RNA drug products led to a transient RNA drug product, diluent, and liposomes, that are pro- release of pro-inflammatory cytokines such as IFN-α, vided as a kit. IP-10, and IL-6, enhancing the vaccination effect. The novel lipoplex formulation for the Lipo-MERIT The clinical translation of this pioneering therapeutic vaccine was engineered to protect RNA from degrada- concept is now being realized in a multi-center, first-in- tion by plasma RNases and thus allows for intravenous human phase I trial in patients with malignant melano- administration of RNA products, advancing from local ma to assess safety, tolerability, and immunogenicity of to systemic targeting of antigen-presenting cells (APCs). the Lipo-MERIT immunotherapy.
The improved selective delivery of the RNA(LIP) products 030 | THERAPEUTIC VACCINATION
Heberprovac, a GnRH based therapeutic vaccine to treat advanced prostate cancer
Junco Barranco J.A.1, Fuentes F.1, Calzada L.1, Rodriguez R.2, Baladrón I.3, Pimentel E.4, Basulto R.1, Garay H.5, Reyes O.5, Castro M.1, Porres L.6, González L.2, Valenzuela C.3, Bover E.1, Arteaga N.1, Rodríguez A.7, Bringas R.8, Tudurí H.7, Fernández Y.7, Rodríguez A.9, de Quesada L.9, Guillén G.10
1Center for Genetic Engineeing and Biotechnology, Vaccine, Camaguey, Cuba, 2Hospital Oncológico de Camaguey, Urology, Camaguey, Cuba, 3Center for Genetic Engineeing and Biotechnology, Clinical Trials, Havana, Cuba, 4Center for Genetic Engineeing and Biotechnology, CIGB, Havana, Cuba, 5Center for Genetic Engineeing and Biotechnology, Peptide Synthesis, Havana, Cuba, 6Hospital Oncológico de Camaguey, Clinical Medicine, Camaguey, Cuba, 7Medical University of Camaguey, Pharmacology, Camaguey, Cuba, 8Center for Genetic Engineeing and Biotechnology, Analitic Unit, Havana, Cuba, 9Hospital Oncológico de Camaguey, Clinical Trials, Camaguey, Cuba, 10Center for Genetic Engineeing and Biotechnology, Bioedical Research, Havana, Cuba
GnRH-based vaccines represent a promising an- finished the last immunization and the clinical eval- ti-hormonal treatment alternative in prostate cancer, uation demonstrated the significant reduction of the because they can reduce serum testosterone to cas- primary tumor from grades III/IV to I/II in all the trating levels, avoid the “hot flushes” produced by patients that respond biochemically. GnRH analogues and can be administered in acute Regarding the safety, more adverse effects of the and complicated forms of prostate cancer. The vaccine were those expected according to the mech- present study assesses the application of Heberpro- anism of action of the vaccine. No severe adverse vac, a GnRH based vaccine candidate for patients events were found in any of the patients. Majority of suffering from advanced prostate cancer. them were related with the current vaccination and The main objective of the trial was to evaluate the only the 4.4% of the adverse effects were grade III. As safety and efficacy of this vaccine candidate in 4 conclusion the vaccine proved to be safe and effective levels of dosage. in the 4 dose levels. To this aim, 56 patients affected by advanced pros- tate cancer diagnosed by biopsy, were included. As result, since the first immunizations, the patients exhibited anti GnRH antibodies and in turn, tes- tosterone levels reduction. In concordance with the hormonal and immunological response the patients exhibit a decrease of both; the number of obstruc- tive symptoms as well as the severity of them. There was also a normalization of the prostatic specific antigen (PSA) in the 80% of the patients after they 031 | THERAPEUTIC VACCINATION
Identification of the novel antigenic peptide presented by the cancer stem cells of human colon cancer
Kanaseki T.1, Miyamoto S.1, Takaya A.1, Kochin V.1, Morooka D.1, Hirohashi Y.1, Torigoe T.1, Sato N.1
1Sapporo Meidal University, Pathology, Sapporo, Japan
The cancer stem cells (CSC), a small cell population in cancer cells, are known to be resistant to chemo- therapies and radiotherapies, and therefore respon- sible for relapse and metastasis. Here we enrich a highly tumorigenic CSC population from the colon cancer SW480 line according to the elevated trans- porter activities of ATP-binding cassette, and screen peptides that are presented by HLA-A24 molecules. Large-scale MS/MS sequencing following affinity purification with A24-specific antibody allows us to detect more than 200 natural peptides and we conse- quently identify the one specific to the CSC popula- tion of SW480. The gene encoding the peptides is ex- pressed in a variety of cancer lines but not in a panel of normal tissues. Moreover, the identified peptide is immunogenic to induce CTL responses, from the PBMCs of healthy donors and a cancer patient, to cancer cell lines expressing HLA-A24 and the gene. We consider CSC as a primary target among cells forming a tumor mass, and here report the novel can- didate peptide for CTL-based immunotherapy. 032 | THERAPEUTIC VACCINATION
Mannose conjugated HPMA-LMA block copolymer micelles for targeting of antigen presenting cells and release of anti-tumor agents
Kappel C.A.1, Mohr N.2, Grabbe S.1, Zentel R.2, Bros M.1
1Medical University Mainz, Department of Dermatology, Mainz, Germany, 2Johannes Gutenberg University Mainz, Institute for Organic Chemistry, Mainz, Germany
Mannose-binding receptors such as CD206 (mac- for several agents. One major advantage of this ap- rophage mannose receptor) or CD209 (also known proach is that unintended uptake of nano-carriers as DC-SIGN) are present on the surface of antigen by tumour cells due to the EPR effect may result in presenting cells like monocytes/macrophages and direct tumour cell killing. dendritic cells (DC). They play a major role in the induction and shaping of immunsurface responses including anti-tumour responses. Especially DC have come into the focus of immunotherapeutic strategies aimed to deliver tumour antigens and adjuvants to induce primary tumour-specific immune responses. Here we asked for the potential of mannose-conjugat- ed nanoparticles to target DC as a prerequisite for their use as a reliable carrier system for antigen/adjuvant. In our experiments we used HPMA (N-(2-hydroxypro- pyl)methacrylamid) -LMA (Laurylmethacrylat) based block copolymer micelles with conjugated D-man- nose. These particles were not overly cytotoxic. FACS (fluorescent activated cell sorting) analysis showed binding of the mannose-conjugated blockpolymer to mouse BMDCs (bone marrow derived DC), whereas the non-conjugated control polymer showed almost no binding. Additionally, confocal laser scanning microscopy indicated that these micelles were taken up by BMDC. At the same time, these polymers dis- played no intrinsic immune-modulatory potential. Ongoing experiments are dedicated to test the suit- ability of HPMA-based block copolymers as carriers of antigen/adjuvant. In this regard, we also test the possibility to employ chemotherapeutics that on one hand induce apoptosis in tumour cells, but at the same time promote DC activation as shown by us 033 | THERAPEUTIC VACCINATION
Evaluation of different adjuvant systems for an epitope-based therapeutic human papillomavirus vaccine
Khallouf H.1, Mangei J.1, Klevenz A.1, Blatnik R.1,2, Hoppe S.1,2, Steinbach A.1, Wuehl M.1, Riemer A.B.1,2
1German Cancer Research Center (DKFZ), Immunotherapy and -prevention, Heidelberg, Germany, 2German Center for Infection Research (DZIF), Molecular Vaccine Design, Heidelberg, Germany
High-risk human papillomaviruses (HPVs), such as in vitro. Moreover, we analyzed the adjuvant effects HPV16, cause over 500.000 cervical, anogenital and of MPLA and R848 in vivo using HLA-A2.1-/HLA- oropharyngeal cancer cases annually. The oncopro- DR1-transgenic H-2 class I-/class II-knockout mice.
teins E6 and E7 are indispensable for the induction Mice were subcutaneously vaccinated with E711-19 and maintenance of the malignant phenotype, making + Pan DR T helper epitope (PADRE) emulsified in them ideal targets for therapeutic HPV vaccines. Montanide ISA51 in association with MPLA, R848, E6 and E7 derived epitopes that are present on the or MPLA and R848 in combination. The properties surface of cancer cells are particularly ideal target of TLR ligands as immunostimulatory components structures for an efficient therapeutic HPV vaccine. were investigated by detailed analysis of the induced By using MS3 mass spectrometry, we found that the immune responses 12 days after immunization.
HLA-A2-restricted HPV16 epitope E711-19 is naturally We found that TLR4 and TLR7/8 ligands (alone and in processed and presented in all tested HPV16+ cell combination) were most efficient in activating DCs in
lines. Moreover, E711-19 elicited strong cytotoxic T cell vitro, demonstrated by increased expression of CD80 (CTL) responses in vitro. Therefore, we used this and CD83 on human DCs and CD40 on murine DCs.
epitope as model antigen to determine the optimal In the in vivo experiments, E711-19 induced antigen- vaccine formulation in vivo. specific T cells when coinjected with the adjuvant
Short peptide epitopes (like E711-19) are poorly immu- ISA51. Adding MPLA to ISA51 further significantly nogenic when administered alone without appropriate increased the percentage of antigen-specific T cells. immunostimulatory signals. Additionally, potent and This study validated the use of this complex human- proper activation of antigen presenting cells, mainly ized mouse model to evaluate the immunogenicity of dendritic cells (DCs), is crucial for efficient priming of HLA-A2-retricted HPV epitopes in vivo. Additionally, antigen-specific CTLs. DCs activation can be achieved it demonstrated that adding MPLA to an emulsion- by including appropriate adjuvants. A number of ad- based adjuvant (ISA51) can significantly enhance im- juvants including ISA51 and different TLR agonists munogenicity of epitope-based therapeutic HPV vac-
such as MPLA (TLR4 ligand) and Resiquimod/R-848 cines. The combination of E711-19 + PADRE + MPLA (TLR7/8 ligand) are currently used in the clinic; + ISA51 is currently being evaluated for anti-tumor however, how they influence vaccine immunogenic- efficacy in vivo. ity has not been fully elucidated yet. In this study, we investigated the effects of TLR4 (LPS or MPLA), and TLR7/8 (R848) ligands on the maturation and activation of human and murine DCs 034 | THERAPEUTIC VACCINATION
IVAC MUTANOME - Evaluation of a new personalized therapeutic modality for the treatment of cancer
Kloke B.-P.1, Attig S.2, Bidmon N.3, Bukur V.2, Derhovanessian E.3, Diekmann J.1, Diken M.2, Grabbe S.4, Heesch S.3, Höller C.5, Kühlke K.6, Langer D.3, Löwer M.2, Loquai C.4, Luxemburger U.3, Miller M.1, Müller F.3, Ortseifer I.1, Otte B.1, Paruzynski A.3, Petri S.1, Rae R.2, Seck C.3, Spieß K.3, Tadmor A.D.2, Utikal J.7, Castle J.2, Britten C.M.1, Kemmer-Brück A.3, Vogler I.1, Kuhn A.N.1, Kreiter S.2, Türeci Ö.8, Sahin U.1,2,3
1BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, 2TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany, 3BioNTech AG, Mainz, Germany, 4Department of Dermatology, University of Mainz, Mainz, Germany, 5Division of General Dermatology, Department of Dermatology, Medical University of Vienna, Vienna, Austria, 6EUFETS GmbH, Idar-Oberstein, Germany, 7Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim, Germany, 8III. Medical department, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
Cancer arises from the accumulation of genomic al- With that, the IVAC MUTANOME trial is the first trial terations and epigenetic changes that constitute a in Europe that introduces a fully personalized muta- hallmark of cancer. Owing to the molecular inter-tu- nome RNA vaccine for cancer. The objectives of the mor heterogeneity in cancer, only a minor fraction of clinical trial are to study the feasibility, safety, toler- patients profit from approved therapies. Available tar- ability and immunogenicity of the IVAC MUTANOME geted therapies can only address alterations common approach for malignant melanoma. Feasibility will to a particular type of cancer and induce transient be shown by the proven ability to provide the fully effects due to the generation of resistant sub-clones. In personalized IVAC MUANOME vaccine to patients. contrast, the IVAC MUTANOME project aims to immu- The recruitment of a patient in the trial triggers the nologically target multiple cancer mutations uniquely IVAC MUTANOME process covering (i) the receipt of expressed in a given patient’s tumor. The IVAC MU- tumor and blood sample specimens, (ii) the identifi- TANOME approach should be applicable to the ma- cation, prioritization and confirmation of mutations, jority of patients irrespective of the tumor entity and (iii) testing of pre-existing immunity against private offers the potential to exploit the whole tumor muta- tumor mutations, (iv) the final selection of mutated nome of a given patient using a multi-target approach. sequences, (iv) design, production of a DNA lead The IVAC approach is supported by (i) the availability structure, (v) GMP manufacturing and release of the of technologies that allow fast discovery and valida- patient-specific mRNA, (vi) shipment to the clinical tion of individual mutations based on sequencing trial site, and (vii) the administration of the IMP to of the whole exome and (ii) an innovative vaccine patients. platform based on RNA-technology supporting fast The IVAC MUTANOME recruitment status, manu- manufacturing and release of patient-specific vac- facturing experiences and treatment status of this cines targeting multiple immunogenic mutations first-in-class clinical trial as well as novel data on the within weeks. immune assessment incl. vaccine-induced mutation- A phase I study to test the individualized cancer immu- specific T cell responses of the first six treated pa- notherapeutics for the treatment of malignant melano- tients will be presented. ma was approved and initiated in 2013 (NCT02035956). 035 | THERAPEUTIC VACCINATION
Vaccination of MSI-H colorectal cancer patients with frameshift peptide antigens - a phase I/IIa clinical trial
Kloor M.1, Reuschenbach M.1, Karbach J.2, Rafiyan M.-R.2, Al-Batran S.-E.2, Pauligk C.2, Jäger E.2, von Knebel Doeberitz M.1
1University Hospital, Heidelberg, Germany, 2Krankenhaus Nordwest, Frankfurt, Germany
Background: High level microsatellite instabil- FSP antigens was observed in all patients vaccinated ity (MSI-H) occurs in 15% of colorectal cancers as per protocol. Few patients had stage IV disease and a consequence of DNA mismatch repair deficiency. were evaluable according to RECIST. One heavily MSI-H colorectal cancers are characterized by a pretreated patient with bulky metastases showed a dense immune cell infiltration. They are charac- stable disease and stable CEA levels over 7 months teristic of the inherited HNPCC (hereditary non- under the study treatment. polyposis colorectal cancer) or Lynch syndrome, but Conclusions: Vaccination with FSPs is well toler- can also develop sporadically. DNA mismatch repair ated and leads to the induction of humoral and cel- deficiency causes insertion or deletion mutations at lular immune responses. FSP vaccination represents coding microsatellites, which leads to the generation a promising novel approach for treatment of MSI-H of frameshift peptide (FSP) antigens. FSP antigens colorectal cancer patients and for tumor prevention are attractive targets for vaccination, because they in Lynch syndrome, allowing the evaluation of the are highly immunogenic shared antigens, which di- concept of preventive cancer vaccines in an ideal rectly result from driver mutations in MSI-H cancers. model scenario of a defined high-risk patient popu- To evaluate safety and immunological efficacy of FSP lation. vaccination, we have initiated a clinical phase I/IIa vaccination trial (Micoryx). Methods: The protocol comprised 3 cycles of 4 sub- cutaneous applications of FSP antigens (frameshift variants of the coding microsatellite-containing genes AIM2, HT001, TAF1B) mixed with Montanide ISA-51 VG over a 6 month period. Inclusion criteria were history of colorectal cancer (UICC stage III or IV) who received standard chemotherapy. Phase I of the trial evaluated safety and toxicity as the primary endpoint (6 patients), phase IIa addressed the induc- tion of cellular and humoral immune responses (16 patients). Results: No FSP antigen-associated severe adverse events have been observed. Significant induction of FSP-specific immune responses against one or more 036 | THERAPEUTIC VACCINATION
A randomized, double-blind, placebo-controlled, phase I/II Trial of RNActive®-vaccine CV9104 in patients with metastatic castrate- refractory prostate cancer (mCRPC): First results of the phase I part
Koch S.D.1, Hong H.1, Feyerabend S.2, Retz M.3, Kübler H.3, Heidenreich A.4, van Erps T.4, Schroeder A.1, Scheel B.1, Reus V.1, Kallen K.-J.1, Fotin-Mleczek M.1, Gnad-Vogt U.1, Stenzl A.2
1CureVac GmbH, Tübingen, Germany, 2University Hospital Tübingen, Department of Urology, Tübingen, Germany, 3University Hospital Technical University Munich, Department of Urology, Munich, Germany, 4University Hospital Aachen, Department of Urology, Aachen, Germany
Introduction: CV9104 is a novel mRNA-based thera- Results: Seven patients were enrolled in the phase I peutic vaccine for prostate cancer encoding the pros- part. Six of these patients were evaluable for immune tate cancer-associated antigens PSA, PSMA, PSCA, responses. The independent data monitoring board STEAP, PAP and MUC1 by engineered mRNA mol- recommended continuation into phase II since nature ecules optimized for protein expression and immu- and severity of adverse events were favorable and in nostimulation (RNActive®). CV9103, an mRNA-based line with previous results from CV9103 Recruitment vaccine against 4 of these antigens was previously of the phase II part was finished in 12/2013 with 197 shown to be safe and immunogenic in a phase I/IIA patients randomized. The trial is ongoing. 5 of 6 pa- trial. Immune responses against multiple antigens tients of the phase I part exhibited antigen-specific were associated with improved survival in patients immune responses post vaccination. All CV9104 anti- with mCRPC. gens were immunogenic. Multiple immune respons- Design and Methods: Safety and immunogenicity of es against ≥2 antigens were observed in 2 patients. first human exposure to CV9104 was investigated in the Remarkably, in one patient a substantial, durable phase I part. In the phase II part, chemotherapy-naïve decrease of various MDSC subsets was observed in patients with a- or minimally symptomatic mCRPC addition to phenotypic changes in CD4+ and CD8+ were randomized to CV9104 or placebo. Primary end- T cells consistent with an effector phenotype. point is overall survival. Secondary endpoints include Conclusion: Results from the phase I part suggest progression free survival, quality of life and immuno- that the self-adjuvanted mRNA vaccine CV9104 con- genicity among others. Upon disease or symptom pro- stitutes a well-tolerated approach to induce anti- gression blinded treatment is continued in combination gen-specific cellular and humoral immune responses with the subsequent systemic cancer treatment until against multiple antigens. second progression. Blood samples for immune mon- Trial registration: clinicaltrials.gov NCT01817738 itoring and other biomarkers were taken at baseline, week 6 (1 week post fifth vaccination) and week 24 (6 weeks post ninth vaccination). Cellular and humoral immune responses against all vaccine antigens were analyzed by intracellular cytokine staining and IgM/ IgG ELISA, respectively. Blood leukocyte phenotyp- ing was performed by flow cytometry. Transcriptome analyses in whole blood were performed and serum samples are currently analyzed for miRNAs. 037 | THERAPEUTIC VACCINATION
Self-adjuvanted RNActive® vaccines provide a promising platform for combination therapies with checkpoint inhibitors
Kowalczyk A.1, Huber S.M.2, Heidenreich R.1, Koch S.1, Scheel B.1, Kallen K.-J.1, Gnad-Vogt U.1, Fotin-Mleczek M.1
1CureVac GmbH, Tübingen, Germany, 2University Hospital Tübingen, Department of Radiooncology, Tübingen, Germany
Two-component mRNA vaccines (RNActive®) combine The safety and immunogenicity of RNActive® vac- high antigen expression with strong immune stimu- cines have been already successfully tested in lation. Intradermal injections of RNActive® vaccines humans in two cancer indications: non-small cell induced balanced, potent and long-lasting immune lung cancer (CV9201) and prostate cancer (CV9103). responses which can be boosted via repetitive vac- Data from both trials revealed induction of the cinations. immune responses against all antigens included in Although the strong immune response induced by the vaccination protocol. RNActive® vaccination alone already led to a potent In conclusion, our results demonstrate that intrader- therapeutic anti-tumor response in the E.G7-Oval- mal vaccinations with self-adjuvanting RNActive® bumin tumor model, the combination of RNActive® induce potent anti-tumor immune responses which vaccination with radiation or checkpoint inhibitors can be further enhanced by combination with other elicited strong synergistic effects, resulting in an even therapies such as checkpoint inhibitors or radiation. more effective anti-tumor response. Thus, RNActive® vaccines constitute an attractive Monotherapy with either OVA-RNActive® or radia- vaccination platform for combination therapies in tion was not effective in curing mice bearing large the field of cancer immunotherapy. E.G7-OVA tumors. Also, high dose radiation of tumors induced only transient growth stagnation. In contrast, the combination of RNActive® vaccination and radi- ation dramatically improved anti-tumor efficacy and supported surveillance of large tumors in the treated mice. Combinatorial treatment of mice bearing already es- tablished E.G7-OVA tumors with OVA- RNActive® and checkpoint inhibitors such as anti-CTLA-4 or anti-PD- 1 monoclonal antibody resulted in a strong, syner- gistic anti-tumor effect, yielding a higher frequency of mice with complete tumor rejection compared to treatment with the single components. Remarkably, these complete responders were protected against re-challenge with parental ovalbumin-negative EL-4 tumors, indicating antigen spreading in these groups. 038 | THERAPEUTIC VACCINATION
Constructing a peptide vaccine warehouse for the personalized immunotherapy of colorectal cancer
Löffler M.W.1,2, Kowalewski D.J.1, Dengler F.1,3, Backert L.1,4, Wagner S.2, Kohlbacher O.4, Königsrainer A.2, Kanz L.3, Stevanović S.1,5, Haen S.P.1,3, Rammensee H.-G.1,5
1University of Tübingen, Institute for Cell Biology, Department of Immunology, Tübingen, Germany, 2University Hospital of Tübingen, Department of General, Visceral and Transplant Surgery, Tübingen, Germany, 3University of Tübingen, Internal Medicine, Department for Oncology, Hematology, Immunology, Rheumatology and Pulmonology, Tübingen, Germany, 4University of Tübingen, Applied Bioinformatics, Center for Bioinformatics, Quantitative Biology Center, and Dept. of Computer Science, Tübingen, Germany, 5German Cancer Consortium (DKTK), DKFZ Partner Site Tübingen, Tübingen, Germany
Colorectal cancer (CRC) is still among the world´s of 40 highly specific CRC-associated HLA ligands. leading cancer entities and the second leading These peptides were selected to be restricted by cause for cancer-related death in Germany. In spite one of the eight most frequent HLA-allotypes in the of recent progress made with regard to screening European population allowing for >95% coverage. and early detection, CRC remains an urgent health In order to optimize the applicability of warehouse problem, especially in the advanced stage. With vaccines, we prioritized peptides according to their regard to peptide based immunotherapies, IMA910 frequencies of presentation/detectability on primary a TUMAP-based multi-peptide vaccine for HLA- CRC, including only targets presented on >25% of A*02 positive CRC patients is in advanced clinical allotype-matched tumors. Immunogenicity testing testing. Such an allotype-specific, standardized of these novel vaccine candidates is ongoing. A vaccine however can only cover about 50% of Cau- current outline of a monocentric clinical phase I/II casian patients and does not warrant the flexibility trial in CRC patients envisages the patient-individ- to adapt vaccine composition to the patient-individ- ual analysis of the tumor antigenome, guiding the ual antigenic landscape. Therefore, we are develop- personalized composition of multi-peptide vaccines ing a warehouse-based approach that allows for the from the vaccine warehouse. As primary endpoints personalized composition of multi-peptide vaccines for this clinical trial, we aim at assessing safety and from a range of frequently presented, non-mutated toxicity as well as the feasibility of this novel ap- CRC-associated peptides. Based on a cohort of 30 CRC proach. However, we will further assess the induc- patients, we comparatively and semi-quantitatively tion of peptide-specific immune responses (as surro- mapped the HLA presented antigenome of primary gate endpoint) and progression-free survival in this colorectal tumor samples and paired adjacent benign early clinical setting. The established framework for tissues. Further, cross-evaluation with our in-house the GMP-certified synthesis and formulation of the database of benign tissue antigens guided the as- vaccine cocktails is available in-house at the Univer- sembly of a peptide vaccine warehouse consisting sity of Tübingen GMP center. 039 | THERAPEUTIC VACCINATION
Enhanced IL-12 production and T cell stimulation ability by dendritic cells matured in presence of GMP-grade Toll-like receptor ligands and IFN-γ
Lövgren T.1, Sarhan D.1, Choudhary B.1, Melief J.1, Nyström M.1, Vermeij R.1, Edbäck U.1, Scurti G.2, Nishimura M.2, Lundqvist A.1, Kiessling R.1, Adamson L.1
1Karolinska Institutet, Dept Oncology-Pathology, Stockholm, Sweden, 2Loyola University Chicago, Dept Surgery, Chicago, United States
Although dendritic cell (DC) vaccines induce T cell re- For TLR-7/8 triggering GMP-grade synthetic com- sponses in patients with cancer, long-lasting clinical pound R848 was used throughout the study. For responses are infrequent. There are numerous different TLR-3 triggering poly I:C was used. Poly I:C is a DC maturation protocols but many contain non-GMP synthetic double-stranded RNA (dsRNA) molecule approved reagents and are thus not suitable for the pro- that does not have a fixed length and is heteroge- duction of vaccines to be used in patients. We aimed to neous between producers and batches. Notably, poly generate an optimized DC product using GMP-grade I:C from different companies had high variability in reagents. the ability to activate DC. This was likely to a large Monocyte-derived DC were matured using a two-step extent due to contamination with LPS and possibly maturation protocol. Monocytes were purified and cul- also other substances in non-GMP grade products. tured in CellGro® GMP DC medium for 48 hours in pres- However, heterogeneity in the dsRNA molecule could ence of IL-4 and GM-CSF plus 18 h with different com- also potentially affect the ability to trigger TLR-3 and binations of TNF-α, IFN-γ and Toll-like receptor (TLR) cytosolic RNA-sensors. In the end we decided to use agonists for TLR-3, -4 and -7/8. Following maturation, the GMP-grade, long poly I:C product Hiltonol®. production of IL-12 was evaluated. The ability of the DC to activate allogeneic T cells to Variations between individuals in the ability to respond IFN-γ production was analyzed and correlated well to each maturation stimulus as well as in the overall to the production of IL-12 from the DC. This was also IL-12 producing capacity was observed. Overall, the the case for tyrosinase-peptide loaded DC activation presence of both IFN-γ and TLR-7/8 agonist was essen- of tyrosinase-specific TCR transduced T cells. We are tial for DC to produce high levels of IL-12. Furthermore, now investigating the ability of tumor-lysate loaded the highest induction of IL-12 production was consis- DC to trigger activation of tumor-specific T cells. tently achieved by the combination of IFN-γ, TLR-3, -4 In summary, we have developed a maturation proto- and -7/8 agonist. col to generate GMP-grade DC with excellent ability For TLR-4 triggering, lipopolysacharide (LPS) was used. to produce IL-12 and activate T cells. Using LPS is problematic in a GMP-setting and we tried to exchange LPS to synthetic GMP-grade TLR-4 agonist monophosphoryl lipid A (MPLA). However, addition of MPLA could not substitute for LPS and therefore TLR-4 triggering was excluded. In absence of TLR-4 triggering, the combination including IFN-γ, TLR-3 and TLR-7/8 agonists induced the highest IL-12 levels. 040 | THERAPEUTIC VACCINATION
Invariant chain as a vaccination vehicle for colorectal cancer
Wälchli S.1,2, Inderberg Suso E.M.1, Kucera A.3,4, Mensali N.1,3,4, Fredsvik Gregers T.3, Myhre M.R.1, Gaudernack G.2, Kvalheim G.1, Bakke O.3,4
1OUS-Radium Hospital, Department of Cellular Therapy, Oslo, Norway, 2OUS-IKF, Department of Immunology, Oslo, Norway, 3University of Oslo, Department of Molecular Biosciences, Oslo, Norway, 4Centre for Immune Regulation (CIR), Faculty of Medicine, Oslo, Norway
We and others have previously shown that the HLA- class II scaffolding CLIP peptide of invariant chain (Ii) had the faculty to bind HLA-class I. We further replaced it with an HLA-class I antigenic peptides (Ii-pep) and showed that the loading of this peptide on HLA-class I occurred. When this construct was produced as mRNA and electroporated into dendritic cells (DC), it evoked a specific T-cell response. We here focused our study on a therapeutically relevant peptide derived from a TGFbRII frameshift mutation. This mutation is observed in 76% of colorectal cancer patients with microsatellite instability (MSI). It leads to a change in the protein sequence which gener- ates a peptide presented by the common MHC-class I HLA-A*0201 (-A2). This peptide being a neo-anti- gen is able to induce an efficient immune response as demonstrated in 10/11 patients vaccinated with a similar synthetic peptide. We here show that Ii- TGFbRIIp construct has the ability to load HLA-class I and stimulate specific CD8 T cells. Furthermore, we tested the possibility to expand the HLA coverage by generating Ii constructs with longer peptides derived from the frameshift. We here show that their potency was kept for CD8 T cell activation and that CD4 T cells were also stimulated. This suggests that both HLA-Class I and II were loaded and validates the use of Ii as a vaccination vehicle. 041 | THERAPEUTIC VACCINATION
Immunotherapy with a CD40L/4-1BBL double-armed oncolytic adenovirus drives Th1 immunity and controls tumor progression in a pancreatic cancer model
Svensson E.1, Milenova I.1, Moreno R.2, Ullenhag G.1,3, Alemany R.2, Loskog A.1,4
1Uppsala University, Dept of Immunology, Genetics and Pathology, Uppsala, Sweden, 2IDIBELL Institut Català d’Oncologia, L’Hosptialet de Llobregat, Barcelona, Spain, 3Uppsala University Hospital, Dept of Oncology, Uppsala, Sweden, 4Lokon Pharma AB, Uppsala, Sweden
We hypothesized that an immunostimulatory on- by LOAd703 was as efficient as a similar oncolytic colytic virus (LOAd703) is a potent inducer of an- virus without transgenes demonstrating that the ti-tumor immunity and tested its biological func- double transgene expression did not interfere with tions in models of pancreas cancer. LOAd703 is a viral replication. Repeated (6x) peritumoral injec- double-armed oncolytic adenovirus that introduces tions of LOAd703 in a xenograft Nu/Nu/Panc01 the simultaneous expression of a trimerized mem- model showed efficient tumor cell growth control and brane-bound CD40L and 4-1BBL locally in the tumor sustained complete responses. The effect could be aiming to induce potent Th1-mediated anti-tumor further enhanced by gemcitabine in both xenograft immunity. By utilizing an oncolytic adenovirus se- mice and in a syngeneic immunocompetent C57BL/6/ rotype 5/35 for gene transfer, virally induced tumor Panc02 model. Transduction of human monocyte-de- cell oncolysis will provide a broad release of antigens rived immature DCs resulted in a strong CD40L and at the site of immune activation. Transgene expres- 4-1BBL expression without lysis of the cells. Instead, sion is driven by a separate promoter to allow for the transduced DCs matured as shown by high CD83 efficient expression in both tumor cells and tumor and IL12 expression. The addition of 4-1BBL signifi- stroma while virus replication is restricted to tumor cantly enhanced DC maturation compared to a virus cells by E1A delta24 deletion. CD40 ligand is a potent containing only CD40L, or no transgenes, by express- stimulator of myeloid cells including dendritic cells ing higher levels of CD70, IL12, TNFa, IFNg and IL21. (DCs) that in turn induce robust T cell responses. Further, LOAd703-transduced DCs pulsed with pp65 CD40L can also reduce the levels of myeloid suppres- CMV peptides potently expanded antigen-specific T sor cells and M2 macrophages as well as enhance T cells as well as NK cells. cell infiltration into tumors. Further, CD40-mediated In conclusion, LOAd703 is a novel, double-armed im- signaling combined with TLR stimuli (such as ad- munostimulatory oncolytic gene therapy that initi- enovirus) induces intense Th1 immunity. 4-1BBL is ates robust Th1 immunity, and eradicates pancreatic known to provide expansion and survival signaling cancer in experimental models. A clinical trial using to pre-activated T- and NK cells. LOAd703 for pancreatic cancer is underway. Pancreatic cancer cell lines and healthy exocrine pancreas cells were transduced with LOAd703 which efficiently killed tumor cells while normal cells re- mained intact as evaluated by an MTS assay. CD40L and 4-1BBL were highly expressed by the transduced tumor cells as detected by flow cytometry. Cell lysis 042 | THERAPEUTIC VACCINATION
Mutated variant of HER2 as a target for immunotherapy in breast cancer
Occhipinti S.1, Angelini C.1, Amici A.2, Beano A.3, Bustreo S.3, Marchiò C.1, Novelli F.1, Giovarelli M.1
1University of Turin, Turin, Italy, 2University of Camerino, Camerino, Italy, 3AOU Città della Salute e della Scienza di Torino, Turin, Italy
Introduction: Overexpression of HER2 oncogene sponse. Mutated determinants are potentially immu- in breast tumors has been associated with a more nogenic because they are not controlled by central aggressive disease. Trastuzumab, a HER2-specific tolerance. In fact, Treg stimulated with HER2-DCs monoclonal antibody, has emerged as an important displayed higher suppressive activity on the prolif- intervention for patients with HER2-positive tumors, eration of autologous T cells and higher capacity to but a number of concerns, including resistance, con- degrade ATP compared to control DCs. By contrast, siderable costs and side effects, make active immuno- d16-DCs did not affect the functionality of Treg. therapies a desirable alternative approach. We focus Conclusion: Cancer vaccines based on self antigen our attention on the HER2 oncogenic variant lacking can activate immune tolerance mechanisms hence exon 16 (d16HER2) because of its high frequency in suppress antitumor response. Vaccination with primary and metastatic tumors and its correlation mutated variants of oncoantigen, as in the case of with trastuzumab-resistance. From the immunologic d16HER2, seems to be more promising compared to perspective, mutations can create neoantigens that wild type proteins in eliciting antitumor response in are not subject to central immune tolerance.Due to patients. This finding represent an important point these characteristics, d16HER2 represent a suitable that should be considered to design cancer vaccines target for immunotherapy in breast cancer. in the future. Materials and methods: Dendritic cells (DCs), gener- ated from patients with HER2-overxpressing tumors, were transfected with DNA plasmid coding for wild type HER2 (HER2-DCs) or for d16HER2 (d16-DCs) and used to activate autologous T cells. Activation was evaluated by flow cytometry, ELISpot and cyto- toxic assays. In order to assess the effect of stimula- tion on regulatory T cells (Treg) activation, purified Treg were cultured with transfected DCs. Treg acti- vation was defined as capability to degrade ATP and suppress T cell proliferation. Results and discussion: We observed that HER2-DCs were completely inefficient in eliciting an antitumor response in T cells from breast cancer patients, while d16-DCs were able to mount a specific immune re- 043 | THERAPEUTIC VACCINATION
Increased anti-tumor immunity that correlates with clinical benefit and induction of neoantigens reactivity following autologous tumor lysate-pulsed dendritic cells vaccination in recurrent ovarian cancer
Ophir E.1, Bobisse S.1, Kandalaft L.2,3, Tanyi J.L.4, Genolet R.1, Michel A.1, Baumgartner P.1, Zsiros E.5, Torigian D.A.6, Mick R.7, Harari A.2, Coukos G.1,2,3
1University Hospital of Lausanne, Ludwig center for cancer research, Lausanne, Switzerland, 2University Hospital of Lausanne, Department of Oncology, Lausanne, Switzerland, 3University of Pennsylvania, Ovarian Cancer Research Center, Philadelphia, United States, 4University of Pennsylvania, Division of Gynecologic Oncology, Philadelphia, United States, 5Roswell Park Cancer Institute, Center for Immunotherapy, Buffalo, United States, ORAL 6 ALK University of Pennsylvania, Department of Radiology, Philadelphia, United States, SHORT T 7 University of Pennsylvania, Department of Biostatistics and Epidemiology, Philadelphia, 2015 United States
Autologous whole tumor vaccines encompass the full at EOS and these Pts had significantly longer PFS com- repertoire of patient-specific tumor associated antigens pared to Vx-non-responders (median PFS 14 vs. 4.2 mos). (TAAs). A pilot clinical trial was conducted using autolo- Notably, both CD4 and CD8 T cells responded to the Vx. gous oxidized tumor lysate-pulsed dendritic cell (DCs) Moreover, In accordance with the improved clinical vaccine (Vx) injected intranodally alone, or in combi- outcome, significantly more Pts in cohort 3, treated with nation with bevacizumab (Bev) with or without low- Cy, showed increased immune response toward the Vx dose IV cyclophosphamide (Cy) in patients (Pts) with at EOS (8/10 Vx-responders) compared to Pts in cohort platinum-resistant recurrent ovarian cancer. Pts were 1 and 2, who did not receive Cy (4/12 Vx-responders). vaccinated 5 times every 3 weeks. The Cy was admin- In addition, direct demonstration of increased immune istrated prior to each Vx to deplete regulatory T cells to response toward autologous tumors at EOS was observed enhance the immunological response. Clinical response in 9/13 evaluable Pts (from all cohorts) and these Pts was assessed by RECIST criteria and immune response had significantly longer PFS compared to Pts who did was determined at enrollment and at end of study (EOS) not increase their anti-tumor immune response (median using ELISpot. PFS 11.2 Vs. 3.7 mos). 25 Pts were treated: cohort 1 (Vx only; n=5), cohort 2 The analysis of specific antigens recognized by post-Vx (Vx+Bev; n=10) and cohort 3 (Vx+Bev+Cy; n=10). No T cells showed increased frequency of T cells recogniz- serious adverse events occurred. 16 Pts showed Clini- ing known TAAs (hTert, Mesothelin, WT-1). Moreover, cal benefit (1 partial response, 15 stable disease). Pts by immunological validation of exome-based predicted who received Cy (cohort 3) had improved clinical out- epitopes, we showed for the first time, that vaccination comes with a median progression-free-survival (PFS) of with personalized whole tumor-pulsed DCs could elicit 11.7 months compared to cohorts 1 and 2, where PFS a CD8 T cell response against mutated peptides derived was only 4 months. The median overall survival (OS) of from private non-synonymous somatic tumor mutations. cohorts 1 and 2 was 19.5 mos while for cohort 3 it has In conclusion, the use of oxidized tumor lysate-pulsed not been reached, with median follow-up of 30 mos. The DC vaccine in Pts with recurrent ovarian cancer is safe clinical benefit of the Vx was suggested by comparing to and effective in eliciting anti-tumor activity including a control of 16 Pts from the same institution who only responses against private neoantigens. The addition of received Bev+Cy (without Vx) and had PFS and OS of low dose Cy enhances the immunological and clinical 4.1 and 26.4 mos respectively. responses. Immune monitoring showed that 12/22 evaluable Pts had increased frequency of T cells responding to the Vx 044 | THERAPEUTIC VACCINATION
Elucidating the T-cell reactivity against porcine IDO and RhoC to establish the pig as an animal model for vaccine development against human cancer
Overgaard N.H.1, Frøsig T.M.1, Welner S.1, Rasmussen M.2, Ilsøe M.1, Sørensen M.R.1, Andersen M.H.3, Buus S.2, Jungersen G.1
1Technical University of Denmark, National Veterinary Institute, Department of Immunology and Vaccinology, Frederiksberg, Denmark, 2University of Copenhagen, Department of International Health, Immunology and Microbiology, Copenhagen, Denmark, 3Herlev University Hospital / Haematological Department, Center for Cancer Immune Therapy, Herlev, Denmark
Immune therapy of cancer has recently experienced of 89 stable (t½ ≥ 0.5 hours) peptide-MHC complexes a great breakthrough with prolonged overall survival with SLA-1*04:01, -1*07:02, -2*04:01, -2*05:02 and/or in patients with metastatic disease following the use -3*04:01. For a pilot study, 12 pigs were immunized of checkpoint inhibitors and T cell therapy with ex with overlapping 20-mer peptides spanning the vivo expanded CD8+ cytotoxic T cells (CTLs). In the entire IDO and RhoC sequences formulated in a panel further development of immune therapies against of CTL-inducing adjuvants. Vaccine and adjuvant cancer, vaccine formulations tailored to mount in efficacy will be evaluated through immunological vivo CTL responses towards co-delivered cancer an- assays among others including ex vivo stimulation tigens will be an important hallmark. Recognition of of whole blood with identified stable SLA-binding antigen-derived peptides presented in the context of peptides and quantification of peptide-specific CTLs. major histocompatibility complex (MHC) class I mol- Hence, these data elucidate the potential in using ecules on cancer cells is a requirement for activation pigs as a large animal model for human anti-cancer of CTLs. Previously, the development of therapeu- vaccine development. tic anti-cancer vaccines have largely been based on rodent models, in particular mice; however the ma- jority of these fail to establish a therapeutic response once put into clinical trials. Pigs have the potential of serving as a model superior to rodents as they are more closely related to humans in terms of immu- nology and physiology. Here, we introduce pigs as a supplementary large animal model for human cancer vaccine development via the use of our unique tech- nology for swine leukocyte antigen (SLA) produc- tion. IDO and RhoC, two tumor antigens previously identified as important players in human cancer development and progression, were used as vaccine targets. Using peptide-MHC-I binding predictors we identified IDO-derived and RhoC-derived candidate peptides potentially binding to five different broadly distributed SLA molecules. We measured the pep- tide-SLA complex stability of these and found a total 045 | THERAPEUTIC VACCINATION
Phenotypic and functional characterization of monocyte derived dendritic cells (MoDC) generated with CliniMACS Prodigy Instrument
Ozimkowski T.1, Brüning M.1, Dzionek A.1
1Miltenyi Biotec, Bergisch Gladbach, Germany
Procedures for the preparation of cellular products maturation the expression of receptors involved in for use in man typically comprise several independ- lymph node homing (CCR7) and formation of im- ent processing steps including density gradient cen- munological synapsis between DC and naïve T-cells trifugation, cell separation, cell washing and cell for- (CD80, CD86, CD54, MHC I and MHC II) was strongly mulation. In the particular case of MoDC-vaccines induced. Furthermore, the activation marker CD83 isolated monocytes additionally need to be cultured was uniformly up-regulated on mature MoDCs for several days at controlled temperature and pH. (mMoDCs). mMoDCs were generated with recover- During the cultivation phase manual operations ies of 20% in average as calculated from the number such as media exchange and supplementation of ad- of initially seeded monocytes. ditives are required and generate an additional risk Functional characterization: of contamination. To meet the increasing regulatory imMoDC were very efficient in antigen uptake via requirements for cell-based therapeutics we have in- pinocytosis as indicated by the uptake of FITC-la- tegrated all manufacturing steps in a closed system beled dextran. Cytokine induced maturation led to operated by an automated cell-processing instru- a significant loss of antigen uptake capacity and to ment, the CliniMACS ProdigyTM. down-regulation of the expression of antigen uptake Here we show a summary of the detailed phenotypic receptors CD209 and CD206. This effect was even and functional characterization of MoDC of 6 inde- more pronounced when MoDCs were matured for ad- pendent production batches derived from apheresis ditional 2 days (day 9). In line with the expression samples of healthy donors. of CCR7 and receptors involved in T-cell priming, Recovery and Phenotype: mMoDCs migrated towards CCL19 in a dose-depend- Using the CliniMACS CD14 reagent monocytes were ent manner and induced strong proliferation of allo- routinely enriched to a purity of 97% and a recovery geneic naïve T-cells. Triggering of CD40 on mMoDCs of 81%. Viability of the cells was at 98% in average. using either soluble recombinant CD40L multimer Isolated monocytes were cultured for 6 days in the or CD40L transfected cell line induced production of presence of GM-CSF and IL-4 yielding immature high levels of the bioactive IL-12p70 indicating their MoDCs (imMoDCs) and were subsequently matured capability to induce Th1 polarized T-cell responses. for additional 24 hours in the presence of IL-1b, IL-6, Our data shows the feasibility of the fully automated
TNFa and PGE2. Monocytes completely down-regu- production of MoDC-based vaccines using the Clini- lated the expression of CD14 during the first 6 days MACS ProdigyTM instrument, which is now going to of culture and up-regulated the expression of CD209 be tested in clinical trials. (DC-SIGN) and CD206 (mannose receptor). Upon 046 | THERAPEUTIC VACCINATION
Characterization of antigenic profiles of primary non-small cell lung cancer tumors and lung cancer cell lines for vaccine development
Palata O.1, Hradilová N.1,2, Sadílková L.1, Myšíková D.3, Lischke R.3, Vančurová I.1, Sojka L.1,2, Špíšek R.1,2, Adkins I.1,2
1Sotio a.s., Prague, Czech Republic, 2Department of Immunology, 2nd Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic, 3Thoracic and Lung Transplantation Division, 3rd Department of Surgery, 1st Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic
Cancer cell lines might serve as a universal source of tumor antigens in the development of cancer vaccines. Immunogenic high hydrostatic pressure-killed cancer cell lines are used for dendritic cell-based active cel- lular immunotherapy of prostate and ovarian cancer. We investigated here whether commercially available non-small cell lung cancer (NSCLC) cell lines overlap in selected tumor-specific or tumor-associated anti- gens with primary NSCLC tumors (n=30). A panel of 26 antigens was tested by RT-PCR in se- lected lung cancer adenocarcinoma cell lines A549, H596 and H226, squamous cell carcinoma cell lines H520, SK-MES-1 and H226 and large cell carcinoma cell lines H460 and H661. We found that out of 26 antigens, 17 antigens were differentially expressed in more than one lung cancer cell line. From these, 10 antigens (MAGE-A3, Prame, 5T4, Her2/neu, EpCam, MUC1, STEAP, SOX2, Survivin and WT1) were ex- pressed by at least 4 lung cancer cell lines. The ex- pression of these antigens was significantly enhanced in primary NSCLC cancer tumors in comparison to control non-tumoral lung tissue and peripheral blood mononuclear cells. In addition, their expression was confirmed on a protein level by immunoblotting and flow cytometry. This data showed that selected antigenic profile of lung cancer cell lines overlapped with antigenic profile of primary NSCLC tumors which suggest that lung cancer cell lines would be suitable for generation of dendritic cell-based vaccine for immunotherapy of NSCLC. 047 | THERAPEUTIC VACCINATION
Cytokine-matured DC/Apo-Nec melanoma vaccine presents an improved MMP-9-dependant migration to lymph nodes that can be modulated by the topical use of Imiquimod
Pizzurro G.A.1, Tapia I.J.1, Sganga L.2, Podhajcer O.L.2, Mordoh J.1,2, Barrio M.M.1
1Centro de Investigaciones Oncológicas - FUCA, Buenos Aires, Argentina, 2Fundación Instituto Leloir - Instituto de Investigaciones Bioquímicas de Buenos Aires - CONICET, Buenos Aires, Argentina
Introduction: Dendritic cells (DC) are potent anti- the complete CC. Finally, using the nude mouse gen-presenting cells capable of directing the adaptive model, we analyzed the DC/Apo-Nec vaccine migra- immune response, the foundation for DC-based ther- tion in vivo, in combination with CC and adjuvants. apeutic cancer vaccines. Previously, we have shown In vivo, CC-increased DC/Apo-Nec vaccine migration that gamma-irradiated apoptotic-necrotic melanoma was partially inhibited by the local administration cells (Apo-Nec)-loaded DC efficiently cross-present of MMP-9 Inhibitor I. Forty-eight-hours after vaccine melanoma antigens to specific cytotoxic T lympho- injection in the mouse footpad, human DCs could be cyte clones, generating the DC/Apo-Nec vaccine. detected within the structure of the regional lymph Working on vaccine optimization, we have tested nodes (LNs) and expressed DC differentiation anti- a standard cocktail of pro-inflammatory cytokines gens. Local co-administration of BCG did not affect (CC) and different adjuvants to evaluate phenotypic vaccine LN homing. Vaccine migration was moni- and functional parameters in vitro and in vivo. tored through in vivo imaging, reaching a plateau 3-4 Results: We dissected the contribution of some of the days after injection. This represented around 2.5% CC components, TNF-α, IL-1β, IL-6 and prostaglan- of total injected cells, which remained detectable din E2 (PGE2), regarding vaccine migration in vitro. at least 9 days post-inoculation. Topical use of Im- We found that addition of CC augmented CD40 ex- iquimod cream increased around 15% CC-enhanced pression along with increased IL-12p70 production, vaccine migration, which appeared directly related while PD-L1 expression and IL-10 secretion levels to its daily administration. remained low. This activated phenotype displayed Conclusions: Taken together, the performance of the a proper CD8+ T cell clone stimulation in vitro. An DC/Apo-Nec vaccine is significantly improved when increased expression of maturation markers, such prepared with complete CC, displaying an immu- as CD83, CD86 and MHC class-II altogether, was ob- nostimulatory profile with CD8+ activating capacity. tained with the use of the complete CC. However, Imiquimod improved DC/Apo-Nec vaccine migra- the use of PGE2 alone up-regulated some of these tion, with a low, though persistent, migrating pro- parameters while IL-6 alone could not, unless com- portion localized in regional LNs, which may allow bined with other components. Vaccine migration multiple rounds of T lymphocyte screening. towards CCL19 through Matrigel was significantly improved by the complete CC, as did PGE2 alone. The augmented secretion of MMP-9 is mainly respon- sible for the PGE2 effect, rather than an increase in CCR7 expression that reached its peak when using 048 | THERAPEUTIC VACCINATION
Immunological parameters in phase I/II clinical trial of dendritic-cell based immunotherapy (DCVAC/PCa) combined with chemotherapy in patients with metastatic, castration-resistant prostate cancer
Podrazil M.1, Fucikova J.1,2, Vrabcova P.1, Horvath R.1,3, Becht E.4,5,6, Rozkova D.2, Bilkova P.2, Hromadkova H.1, Kayserova J.1, Vavrova K.1, Lastovicka J.1, Kubackova K.7, Gasova Z.8, Jarolim L.9, Babjuk M.9, Spisek R.1,2, Bartunkova J.1,2
1Department of Immunology, Charles University, 2nd Faculty of Medicine and University Hospital Motol, Prague, Czech Republic, 2Sotio, Prague, Czech Republic, 3Department of Pediatric and Adult Rheumatology, University Hospital Motol, Prague, Czech Republic, 4Institut National de la Santé et de la Recherche Médicale (INSERM), UMRS872, Centre de Recherche des Cordeliers, Paris, France, 5Université Pierre et Marie Curie-Paris 6, UMRS 872, Paris, France, 6Université Paris Descartes, UMRS 872, Paris, France, 7Department of Oncology, Charles University, 2nd Faculty of Medicine and University Hospital Motol, Prague, Czech Republic, 8Institute of Hematology and Blood Transfusion, Prague, Czech Republic, 9Department of Urology, Charles University, 2nd Faculty of Medicine and University Hospital Motol, Prague, Czech Republic
Background: We have performed an open label, single years, median PSA 186 ng/ml and 88% patients had GS arm phase I/II clinical trial in 25 patients with meta- ≥7. No serious DCVAC/PCa-related adverse events have static castrate resistant prostate cancer (mCRPC) eligi- been reported. Therapeutic vaccination with DCVAC/ ble for 1st or 2nd line of docetaxel treatment using autolo- PCa led to a significant improvement in overall survival gous mature dendritic cells pulsed with killed LNCap of patients when compared to nomograms’ predictions. prostate cancer cell line, DCVAC/PCa. With a median overall survival of 19 months, DCVAC/ Methods: DCVAC/PCa treatment consisted of, on PCa treatment resulted in a 7.2 and 6 months improve- average twelve doses of 1x107 dendritic cells injected ment in median overall survival when compared to s.c. Treatment comprised of initial 7d administration Halabi or MSKCC nomograms, respectively. We ob- of metronomic cyclophosphamide and subsequent 2 served no significant changes in T cell subsets during doses of DCVAC/PCa. Patients then started docetaxel the course of the trial. Conversely, the percentages of (75 mg/m2) and prednisone (5 mg twice daily) treat- activated CD3+HLADR+ T cells and of cytotoxic CD8+ ment administered every 3-weeks and DCVAC/PCa T cells were significantly increased. Additionally, a sig- was given every 6 weeks up to a maximum number of nificant decrease in the frequencies of regulatory T cells doses manufactured from one leukapheresis. Immune was observed in the peripheral blood. The long-term monitoring including measurement of humoral and T administration of DCVAC/PCa led to the induction and cell response (serum levels of imunoglobulin G, A, M, maintenance of stable levels of T cells specific against serum autoantibodies, lymphocyte subsets, Tregs, de- multiple tumor antigens, including PSA, MAGE-A1 and tection of antigen-specific T cells against PSA, MAGE- MAGE-A3. There was no obvious correlation between A1, MAGE-A3 and detection of tumor antigen-specific IgG and the CTL response against either PSA or MAGE- antibodies against PSA and MAGE-A3) was performed A3. We did not identify any immunological parameter before the 1st dose of DCVAC/PCa and after the 12th that significantly correlated with better OS. dose or after last dose if less than 10 doses were manu- Conclusions: In patients with mCRPC, the alternat- factured from 1 leukapheresis. The primary endpoint ing administration of DCVAC/PCa cancer immuno- was safety, the secondary endpoint immune responses. therapy and docetaxel was safe and resulted in longer Overall survival (OS) was compared to the predicted OS than expected survival. Concomitant chemotherapy according to Halabi and MSKCC nomograms. did not preclude the induction of specific anti-tumor Results: Data from twenty-five patients were evaluated. cytotoxic T cells. Clinical trial information: EudraCT The median age at the start of immunotherapy was 73 2009-017259-24. 049 | THERAPEUTIC VACCINATION
Modified Vaccinia Virus Ankara therapeutic efficiency in preclinical models of cancer benefits from the blockade of immune checkpoints
Remy-Ziller C.1, Hortelano J.1, Farine I.1, De Meyer M.1, Nourtier V.1, Gantzer M.1, Thioudellet C.1, Slos P.1, Rittner K.1, Préville X.1
1TRANSGENE, Onco-immunology, Illkirch-Graffenstaden Cedex, France
The immunotherapeutic TG4010 has demonstrated clinical benefit for advanced NSCLC patients in com- bination with standard of care chemotherapy in two consecutive phase 2 randomized and controlled clini- cal trials (NCT00415818 and NCT1383148). TG4010 is a Modified Vaccinia virus Ankara (MVA) encoding the human mucin1 (MUC1) tumor associated antigen and human interleukin 2.The blockade of PD-1 pathway with monoclonal antibodies has also demonstrated ef- ficacy for NSCLC patients in phase 2 trials. Hence, we implemented an experimental primary tumor and lung metastasis models to evaluate the rationale of combin- ing both approaches at the preclinical level. We used two different CT26 cell lines expressing b-galactosidase or MUC1 to evaluate the impact on overall survival of the combination of MVA-based and immune block- ade immunotherapies. Synergistic increase in overall survival was observed in the therapeutic CT26-CL25 primary tumor and lung metastasis models upon treat- ment of mice with the combination of MVA-bgal and anti CTLA4 or anti PD-1 in comparison with either treatment alone. We provide evidence that TG4010 synergized with immune checkpoint inhibitors to in- crease overall survival in the therapeutic CT26-MUC1 tumor models in comparison with either treatment ad- ministrated independently. These observations were associated with an increase in the frequency and the quality of antigen-specific tumor infiltrating CD8+ T cells. These studies pave the way for the evaluation of combinatorial therapies including TG4010 and immune checkpoint blockers in the clinic. 050 | THERAPEUTIC VACCINATION
Combined Immunotherapy: CTLA-4 blockade potentiates anti-tumor response induced by transcutaneous immunization
Rausch J.1, Stein P.2, Bialojan A.1, Probst H.C.3, Schild H.3, Radsak M.P.1
1Third Department of Medicine - Hematology, Oncology, Pneumology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany, 2Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany, 3Institute of Immunology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
Immunologic approaches to combat cancer aim at performed an in vivo cytotoxicity assay with CFSE the induction of tumor-reactive immune responses to stained splenocytes. achieve long-term protection. In this context, we es- The results clearly demonstrate, that the combination tablished a Transcutaneous Immunization (TCI) pro- of anti-CTLA-4 and TCI induces superior cytotoxic tocol based on the application of the Toll Like Receptor T-cell priming, leading to a sufficient memory forma- 7 agonist imiquimod onto the skin, mediating peptide- tion. The subcutaneous injection of 300µg CTLA-4 specific T-cell activation via antigen presenting cells. antibody on Day 1 mediates the strongest effects and Our non-invasive vaccination strategy minimizes the was determined as standard treatment for following risk of infection, but nontheless provokes a systemic experiments. reaction by targeting the highly developed dermal We then explored vaccination capacity in a therapeu- immune system. However, induced specific T-cell tic tumor experiment. We inoculated C57BL/6 mice activation and proliferation does not lead to memory with 2x105 B16-OVA melanoma cells and started the formation and only shows partial tumor protection. treatment after a tumor size of >25mm2. The animals As another immunologic option, an antagonistic an- were treated weekly for three consecutive weeks with tibody blocking the immune checkpoint CTLA-4 has TCI and received one antibody application on Day 1. demonstrated great benefit in the clinical treatment of Control groups either received no treatment, only TCI stage IV melanoma. CTLA-4 naturally limits immune or only the antibody. The survival of double-treated reactions permitting self-tolerance. Its antibody most mice was significantly higher compared to mice im- likely acts by blocking inhibitory signals in activated munized with TCI alone. Remarkably, an isolated T cells or by directly inactivating regulatory T- cells. antibody treatment had no effect on tumor growth, Combining TCI with this immunogenic therapy might however was eligible to maximize the potency of the therefore be a promising new approach. answer after initiation. This might be an important We first evaluated the optimal dose, time and route of aspect for non-responders in the clinical application antibody application. Therefore, 50mg of an 5% Im- of anti-CTLA. iquimod-formulation and 100µg of the T-cell epitope Conclusively, double-targeting the immune system po- SIINFEKL were consecutively applied onto the intact tentiates our innate anti-tumor competences leading skin of shaved C57BL/6 mice on Day 1. The CTLA-4 to long-term protection. Combining different immuno- antibody was then injected either intravenously or logic approaches might be the entrance to a new era in subcutaneously on various days and in an altering the combat of cancer by fortifying our own defensive dosage. On Day 7, we analysed the amount of H2-Kb capacities. (SIINFEKL) positive CD8+ cells in the blood and 051 | THERAPEUTIC VACCINATION p16INK4a as a vaccine target in patients with HPV-associated cancers - results of a phase I/IIa study
Reuschenbach M.1, Rafiyan R.2, Pauligk C.2, Karbach J.2, Kloor M.1, Prigge E.-S.1, Sauer M.1, Al Batran S.-E.2, Jäger E.2, von Knebel Doeberitz M.1
1University Hospital Heidelberg, Heidelberg, Germany, 2Krankenhaus Nordwest, Frankfurt, Germany
Background: More than 5% of all cancers are attrib- tion of standard treatment. The protocol comprised a utable to human papillomavirus (HPV) infection, total of 12 subcutaneous applications of a p16 peptide including cervical cancer, other anogenital cancers (P16_37-63) mixed with Montanide® ISA-51 VG over a and cancers of the head and neck region. Despite six months period. After an initial interim safety as- the well-defined carcinogensis of HPV-associated sessment after the first 10 patients the primary end- cancers, still no routine treatment targets specific point was the induction of a cellular and/or humoral molecular alterations. Therapeutic vaccines for pa- immune response against peptide P16_37-63. Sec- tients with HPV-associated neoplasia are considered ondary endpoints were safety of immunization with conceivable strategies to improve outcome. The viral P16_37-63 and tumor response according to RECIST. E6 and E7 oncoproteins have been targets in various Results: 20 patients received at least 4 applications immunotherapeutic, however with limited success. and were evaluable for induction of an immune re- This may in part be due to the not ideal antigenicity sponse. CD4 T cells were induced in 11/20 patients, of the target antigens. E6 and E7 are expressed at low CD8 T cells in 4/20 and antibodies in 14/20 patients. levels even in tumor cells, although they are required No SUSAR or SAR was documented. All reported SAEs to maintain the neoplastic growth of HPV-induced were expectable and not related to study therapy. No cancers. It may thus very well be that cellular an- patients discontinued trial participation due to un- tigens expressed in HPV-transformed cells poten- acceptable toxicities and no dose limiting toxicities tially represent better targets. In particular antigens occurred. The best tumor response was classified as with high expression levels in the respective target stable disease in 10 patients during the treatment and cells have been suggested as ideal candidates. It is a 3 remained stable until end of follow-up. hallmark of HPV-associated cancers that as a direct Conclusions: Vaccination with P16_37-63 induces consequence of HPV oncogene activity the cellular cellular and humoral immune responses and does protein p16INK4a (p16) is strongly overexpressed. With not cause toxicities. Further studies including pa- at the same time absent or very low expression in tients with a defined disease entity and disease normal tissues this expression pattern renders p16 stage will have to evaluate clinical efficacy of this an interesting novel vaccine target. We performed approach. Since p16 is overexpressed already at pre- a phase I/IIa first-in-man trial to evaluate the safety cancer stages, p16-based vaccination has wide pos- and immunogenicity of vaccination with p16. sible indications. Methods: 26 patients with advanced p16-overex- pressing, HPV DNA-positive cancer (anogenital region, head and neck) were included after comple- 052 | THERAPEUTIC VACCINATION
BoHV-4 viral vector as a new approach for ErbB2+ breast cancer immunotherapy
Rolih V.1, Franceschi V.2, Jacca S.2, Bolli E.1, Donofrio G.2, Cavallo F.1, Quaglino E.1
1University of Turin, Turin, Italy, 2University of Parma, Parma, Italy
Indeed, the first issue in the development of an effec- was found in the sera of vaccinated mice and this tive anti-tumor vaccine is to identify an appropriate response correlated to an in vivo delay in the tumor target antigen and an appropriate vaccine formula- appearance. tion. Several reports have demonstrated that cancer Moreover BoVH-4 virus, besides being a good antigen control through active immunotherapy is an attain- delivery system, has also oncolytic properties. There- able goal only when the vaccine targets an oncoanti- fore, the oncolytic viruses provide a number of po- gen (OA). The most important (OA) identified so far tential advantages as cancer vaccines over conven- for breast cancer is ErbB2. Vaccination of rat ErbB2 tional therapies. Firstly, cancer cell death induced transgenic mice with DNA plasmids coding for the by the oncolytic virus provides a natural repertoire extracellular (EC) and transmebrane (TM) domain of of tumor associated antigens (TAAs) in conjunction the rat ErbB2 protein is an effective way to induce a with danger signals, thus contributing to the induc- strong anti-rat immune response able to significantly tion of a potent anti-tumor immunity. Secondly, the delay the onset of autochthonous mammary cancer virus exerts another important function: it efficient- developed by rat ErbB2 transgenic (BALB-neuT) mice. ly present/release TAAs to DCs and danger signals However, while the effectiveness of DNA vaccination which function as adjuvant. For this reason, we used in mice bearing atypical mammary hyperplasia tested in vivo the oncolytic capacity of BoHV-4 viral is clear, its effectiveness results lower when applied vector on a transplantable mouse model. In particu- mice with invasive breast cancer. Bovine Herpesvi- lar, BALB/c mice were inoculated with TUBO cells rus 4 (BoHV-4) has been proposed as a vaccine vector (ErbB2+ murine tumor cell line), then treated intra- because of its minimal pathogenicity and its unlikely tumor with BoHV-4 viral vector. We have observed oncogenicity. For these reason we tested in vivo its an increased antibody and cellular response and a efficacy in BALB-neuT mice. First we tested the ca- complete tumor regression in treated mice. pacity of a recombinant BoHV-4 encoding the EC-TM Taking together, these results demonstrate the effec- of a chimeric rat/human ErbB2 protein to induce an tiveness of BoHV-4 both as a vaccine viral vector and immune response in BALB/c mice. We have observed either as oncolytic virus, opening new perceptive- a strong antibody and cellular-mediated response in ness in the management of ErbB2+ breast cancer. vaccinated mice. Therefore we used this vector to vaccinate 10-week old BALB-neuT mice bearing in their mammary glands in situ lesions. A recombinant BoHV-4 coding for an unrelated protein was used as control. A strong anti-rat ErbB2 immune response 053 | THERAPEUTIC VACCINATION
Enhancing dendritic cell-induced T-cell responses by immunomodulating molecules
Rothe M.D.C.1,2, Schnorfeil F.M.1,2, Lichtenegger F.S.1,2, Schlüter M.1,2, Neitz J.1,2, Hiddemann W.1, Subklewe M.1,2
1Klinikum der Universität München, Department of Internal Medicine III, Munich, Germany, 2Helmholtz Zentrum München, Clinical Cooperation Group Immunotherapy, Munich, Germany
Combinatorial approaches utilizing vaccine strate- upregulated on T cells (Δ% positive CD4+/CD8+: gies together with immune modulators are a prom- PD-1 22.0/7.9, LAG-3 3.6/7.5). The impact of immu- ising treatment in cancer immunotherapy. We have nomodulators on T-cell responses was examined by initiated a proof-of concept phase I/II trial for pos- adding immune checkpoint blocking antibodies to tremission therapy in AML using RNA-transfected the coculture. Addition of the immunomodulatory 3-day, TLR-matured DCs (TLR-3-DCs). Besides thera- drug lenalidomide was also tested. T-cell activation peutic vaccination, immune checkpoint modulation was enhanced by all 4 agents: Blockade of PD-L1, represents another strategy to enhance anti-tumor PD-1 and LAG-3 resulted in a 1.4- (n=11), 2.0- (n=14), immune responses. Here we analyzed the impact of and 5.9- (n=6) fold increase in IFN-γ secretion, re- immune checkpoint modulation on T-cell activation spectively. Combination of anti-LAG-3 and anti- by TLR-3-DCs. PD-1 antibodies resulted even in a 6.9-fold increase Monocyte-derived DCs were generated in 3 days (n=6). And a 5.4-fold increase in IFN-γ release was using a TLR7/8 agonist containing maturation cock- induced by lenalidomide (n=8). All 4 molecules also tail. Expression profile of immune checkpoint mole- enhanced T-cell proliferation. Notably, presence of cules on TLR-3-DCs was analyzed by multiparameter anti-LAG-3 antibody in the coculture further upregu- flow cytometry and median fluorescence intensity lated PD-1 on T cells compared to cocultures without ratio (MFI) calculation. Analysis of basic surface any antibody. markers (CD14, CD83, CCR-7, HLA-DR=LAG3 recep- Our data suggests that the efficacy of DC vaccination tor) showed that mature DCs were generated. Positive can be dramatically enhanced by combination with costimulatory molecules were expressed at a high immunomodulating molecules. Clinical trials will be level (MFI: CD80 32.8; CD86 32.1, n=7) but inhibitory needed to analyze the relevance of those findings. molecules were also expressed to a significant extent (MFI: HVEM 2.0, n=10; ILT-3 2.5, n=7; PD-L1 6.2, n=10). To assess the expression of associated ligands on T cells, before and after activation by DCs, an appropriate coculture system was established: TLR- 3-DCs were pulsed with a CEFT peptide pool and co- cultivated with autologous T cells for 4 days. A strong T-cell activation was observed by peptide pulsed DCs in T-cell proliferation assays (CFSE) and in IFN-y se- cretion (CBA). Upon activation, PD-1 and LAG-3 were 054 | THERAPEUTIC VACCINATION
Spatial and temporal heterogeneity of mutated tumor antigens in a high grade ovarian serous carcinoma
Rubinsteyn A.1, O’Donnell T.1, Ahuja A.1, Martignetti J.1, Pereira E.2, Dottino P.2, Sebra R.1, Hammerbacher J.1, Schadt E.1
1Mount Sinai School of Medicine, Genetics and Genomic Sciences, New York, United States, 2Mount Sinai Hospital, Obstetrics, New York, United States
Background: Mutated tumor antigens seem to play idate variant calling and alignment parameters and a central role in the adaptive immune system´s iden- detect sub-clones of low allelic fraction). Mutated tification and clearance of cancer cells. There has protein sequences are being inferred from somatic recently been significant interest in detecting mu- coding mutations (including frameshifts), and pep- tation-derived tumor specific antigens using high tides overlapping each mutation are being evaluated throughput sequencing and MHC binding predic- for their predicted MHC binding affinity using Net- tion. The neoepitope content of a cancer may serve MHCcons. as a prognostic marker in predicting response to im- munomodulatory therapies such as anti-CTLA4 and anti-PD1 checkpoint blockade. Furthermore, several early stage clinical trials are currently investigating the efficacy of predicted neoepitopes for personal- ized therapeutic cancer vaccination. Questions: 1) To what degree do the detected epitopes vary within a spatially heterogenous tumor? 2) How stable are the detected epitopes over the course of a cancer´s lifetime? 3) Is the variability in detected neoepitopes due to significant differences between tumor samples or an artifact of computational tools and their pa- rameterization? Methods: We are performing exome and RNA se- quencing on nine tumor samples from a single patient´s ovarian carcinoma, collected over a two year period during the course of her treatment. Thus far we have finished RNA sequencing for all the samples and exome sequencing on two tumor samples, along with normal blood. We will soon receive the exome data from the remaining 7 tumor samples, one of which is being sequenced to >1800x coverage (to val- 055 | THERAPEUTIC VACCINATION
TMPRSS4 is a breast cancer stem cell-associated antigen and a putative target for DNA-based vaccination
Ruiu R.1, Lanzardo S.1, Conti L.1, Calogero R.A.1, Cavallo F.1
1Molecular Biotechnology Center, University of Torino, Department of Molecular Biotechnology and Health Sciences, Torino, Italy
Cancer stem cells (CSCs) are a sub-population of high percentage of TMPRSS4-expressing cells were stem-like cells endowed with self-renewal and tumor also positive for Sca-1, thus strengthening the link initiating ability that are thought to be responsible with stem-cell phenotype. We therefore investigated for disease recurrence, metastasis formation and the role of TMPRSS4 in breast CSC biology, and found treatment failures, as they display resistance to tra- that TMPRSS4 downregulation in TUBO cells signifi- ditional cancer therapies. Identification of appropri- cantly reduced their tumorsphere forming ability, in- ate vaccination targets expressed by the tumor bulk dicating CSC self-renewal impairment. Given these in and over-expressed by CSCs is thus essential for the vitro results and the presence of TMPRSS4 expression development of effective cancer immunotherapies. also in primary breast tumors and lung metastases To find possible CSC-associated targets, we performed in BALB-NeuT mice biopsies, we assumed TMPRSS4 a transcriptome microarray analysis to explore genes as an interesting candidate for anti-tumor immu- differentially expressed between a murine ErbB2+ notherapy. Hence, we developed a DNA-based anti- breast cancer cell line (i.e. TUBO cells) cultured as TMPRSS4 vaccine inserting either murine or human an epithelial monolayer and its derived CSC-enriched TMPRSS4 open reading frame in the FDA approved tumorspheres, cultured in non-adherent conditions pVAX1 eukaryotic expression vector. Unlike IgGs as floating spheroids. Genes upregulated in CSCs from BALB/c mice vaccinated with empty pVAX1, were then assessed through meta-analysis of normal IgGs from mice vaccinated with TMPRSS4-pVAX1 and cancerous human breast transcription profiling were able to stain the cell membrane of TMPRSS4- data, ranking them on the basis of their low expres- expressing cells. These results suggest that our DNA sion on normal tissues and of the clinical outcome. vaccine against TMPRSS4 induces the generation of This analysis led us to the identification of TMPRSS4, specific IgGs, encouraging us to test its efficacy on tu- a type II transmembrane serine protease highly ex- morigenesis and metastatic process in mouse models pressed by cancers of diverse origin. TMPRSS4 was in the foreseeable future. recently shown to promote tumor growth, invasion, These preliminary results lay the foundations for a metastasis and epithelial-mesenchymal transition promising study on the role of TMPRSS4 in breast (EMT), though the molecular mechanisms underly- cancer cell stemness, tumorigeneicity and metastatic ing these processes are not fully understood. ability and more importantly for the design of im- We validated TMPRSS4 upregulation in tumor- mune-therapeutic strategies targeting breast CSCs. spheres at both mRNA and protein level. The com- bined staining of TMPRSS4 and the murine CSC marker Stem Cell Antigen 1 (Sca-1) revealed that a 056 | THERAPEUTIC VACCINATION
Identification of oncofetal antigen Glypican-3-derived long peptides encompassing CTL and multiple HLA class II-restricted Th cell epitopes
Sayem M.A.1, Tomita Y.1, Yuno A.1, Hirayama M.1, Yuba E.2, Kono K.2, Yoshikawa T.3, Nakatsura T.3, Nishimura Y.1
1Kumamoto University, Immunogenetics, Kumamoto, Japan, 2Osaka Prefecture University, Applied Chemistry, Sakai, Japan, 3National Cancer Centre, Division of Cancer Immunotherapy, Kashiwa, Japan
Glypican-3 (GPC3), a human oncofetal antigen, is with GPC3-LPs encapsulated in a novel pH-sensi- specifically overexpressed in hepatocellular carcino- tive liposome that can deliver LPs from endosome ma (HCC) and melanoma cells. GPC3-derived short to cytoplasm. HLA-A2 transgenic mice immunized peptides (SPs) vaccine carrying CTL epitopes were with the same LP showed increased GPC3-SP-specific safe, and induced measurable immune and some and HLA-A2-restricted CTL response as compared to clinical responses in a phase I clinical trial for treat- mice immunized with GPC3-SP alone. A part of this ment of HCC (Clin Cancer Res 18:3686, 2012). In this augmented response may be attributed to the help study we aim to identify GPC3-derived long peptides of CD4+ T cell, because this LP stimulated specific (LPs) carrying multiple HLA class II-restricted T mouse I-Ab-restricted CD4+ T cell response in vivo. helper type 1 (Th1) cell epitopes. We predicted five GPC3-LPs-specific and HLA-Class II-restricted T cell candidate LPs using computer algorithm and proved responses were observed in 14 of 20 HCC patients antigenicity of all 5 GPC3-LPs to induce specific vaccinated with GPC3-SPs. Repeated vaccinations CD4+ T-cells by IFN-γ ELISPOT assays. To determine with GPC3-SPs enhanced GPC3-LPs-specific respons- the restriction HLA class II molecules involved in es in 8 of 13 HCC patients. These GPC3-LPs may be antigen presentation, blocking of antigen-induced applicable to immunotherapy of HCC. IFN-γ production was investigated by adding anti- HLA-class II monoclonal antibodies. Determination of specific restriction HLA class II molecules was carried out using L cells transfected with single pair of HLA class II α and β genes or allogeneic PBMC with shared HLA-class II molecule, and found that these Th cells were restricted by seven frequent HLA class II molecules. Majority of these Th cell lines secreted Th1-type cytokines in response to autolo- gous PBMCs pulsed with the cognate LPs. Th cells induced by four GPC3-LPs responded to autologous dendritic cells (DCs) pre-loaded with recombinant human GPC3 suggesting a possible natural process- ing of these LPs from GPC3 protein. One of the LPs was well cross-presented to GPC3-SP-specific CTLs when they were cocultured with DCs preloaded 057 | THERAPEUTIC VACCINATION
Dendritic cell vaccination in postremission therapy of AML: Results of a clinical phase I trial
Schnorfeil F.M.1,2, Lichtenegger F.S.1,2, Brüggemann M.3, Moosmann A.4, Köhnke T.2, Bücklein V.2, Altmann T.2, Geiger C.5, Wagner B.6, Henschler R.6, Hiddemann W.2, Bigalke I.7, Kvalheim G.7, Schendel D.5, Subklewe M.2,4
1Helmholtz Zentrum München, Clinical Cooperation Group Immunotherapy, Munich, Germany, 2Klinikum der Universität München, Department of Internal Medicine III, Munich, Germany, 3University Hospital Schleswig-Holstein, Department of Hematology, Kiel, Germany, 4Helmholtz Zentrum München, Clinical Cooperation Group Immunooncology, Munich, Germany, 5Helmholtz Zentrum München, Institute of Molecular Immunology, Munich, Germany, 6Klinikum der Universität München, Department of Transfusion Medicine, Cellular Therapeutics and Hemostaseology, Munich, Germany, 7The Norwegian Radium Hospital, Oslo University Hospital, Department of Cellular Therapy, Oslo, Norway
Therapeutic vaccination with autologous tumor anti- antigen-specific T cells in vitro. Three patients have gen-loaded dendritic cells (DCs) is a promising treat- completed the vaccination schedule. We observed ment strategy to eradicate residual chemorefractory delayed-type hypersensitivity (DTH) responses at leukemic cells in AML patients. We are currently con- the vaccination site in 4/4 patients, accompanied ducting a proof-of-concept phase I/II (6+14 patients) by slight erythema and indurations at the injection clinical trial using next-generation dendritic cells for site, but no grade III/IV toxicities. Multimer analy- postremission therapy of AML (NCT01734304). En- ses revealed the induction of antigen-specific T cell rollment criteria include a non-favorable risk profile, responses in 2/2 patients: a 10-fold increase of WT1- achievement of complete remission after intensive specific and a 6-fold increase of CMVpp65-specific T induction chemotherapy as well as ineligibility for cells in a CMV-seronegative patient were detected. allogeneic stem cell transplantation. TCR repertoire analyses by next-generation sequenc- Next-generation DCs are generated from patients´ ing revealed an enrichment of particular clonotypes monocytes in a GMP-compliant 3-day protocol that at DTH sites, supporting the observed induction of includes a TLR7/8 agonist containing maturation antigen-specific immune responses. One patient with cocktail. DCs are loaded with ivt-RNA encoding an impending relapse during the vaccination sched- WT1 and PRAME as leukemia-associated antigens ule was treated with a combination of DC vaccination and CMVpp65 as an adjuvant and surrogate antigen. and 5-azacytidine, resulting in MRD conversion. Patients are vaccinated intradermally with 5x106 DCs We thus conclude that vaccination with next-genera- of each antigen up to 10 times within 26 weeks in tion DCs in AML is not only feasible but also induces weekly to monthly intervals. The primary endpoint tumor-specific immune responses in vivo. of the trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control, with a particular focus on MRD conversion. Eight patients have been enrolled so far. In 6/6 cases, leukapheresis yielded sufficient monocytes to gen- erate DCs for clinical application. DCs showed a positive costimulatory profile, secreted IL-12p70, migrated, expressed all three antigens and activated 058 | THERAPEUTIC VACCINATION
Effective anti-tumor therapy based on a novel anti-Tn antibody conjugated to a cytotoxic drug
Sedlik C.1,2,3, Heitzman A.1,2, Ait Sarkouh R.4, Batisse C.1,2, Schmidt F.4, Osinaga E.5, Hubert P.1,2, Amigorena S.1,2,3, Piaggio E.1,2,3
1Institut Curie - Centre de Recherche, Paris, France, 2INSERM U932, Paris, France, 3Centre d’Investigation Clinique Biothérapie CICBT 507, Paris, France, 4Institut Curie - Centre de Recherche, UMR3666/ Unité 1143, Paris, France, 5Institut Pasteur de Montevideo, Montevideo, Uruguay
The antibody-drug conjugate (ADC) technology, combining the specificity of tumor recognition by monoclonal antibodies (mAb) and the potent cy- totoxicity of anti-cancer drugs, is currently under growing interest and development. Tn is a glyco-pep- tidic tumor-associated antigen expressed in many human carcinomas, and poorly detected in normal tissues. Chi-Tn is a mouse/human chimeric mAb that specifically binds to Tn determinants expressed on tumor, but not on normal epithelial cells. We now show, using flow cytometry and deconvolution mi- croscopy, that the Chi-Tn mAb is rapidly internalized and targeted to early and recycling endosomes. The Chi-Tn mAb conjugated to two different cytotoxic drugs (saporin or monomethyl auristatin F (MMAF)) inhibits the growth of Tn-expressing tumor cells in vitro, while leaving the Tn-negative tumor cell prolif- eration unaffected. Therefore, efficient endocytosis and targeting to early/recycling endosomes through Tn targeting is sufficient to induce tumor cell death in vitro. Notably, in a preclinical model to treat mice with established tumor, Chi-Tn mAb conjugated to MMAF shows a potent antitumor efficacy with a highly delayed tumor growth. Thus, the Chi-Tn mAb fulfills the requirements of ADC anti-cancer agents, and is a promising therapeutic vector to deliver cy- totoxic drugs specifically to Tn-expressing tumors. 059 | THERAPEUTIC VACCINATION
Combining chemotherapy with immunotherapy for the treatment of glioblastoma
Shraibman B.1, Barnea E.1, Admon A.1
1Technion-Israel Institute of Technology, Haifa, Israel
Glioblastoma multiforme (GBM) is a deadly type of the clinic. Our preliminary results show significant brain cancer. Most Glioblastoma patients face grim and consistent changes in the HLA peptidome of the prognosis, therefore, better targeted therapeutic cancer cells’ HLA peptidomes after the treatment treatments, such as immunotherapy are needed. with this chemotherapeutic and include identifica- Potentially useful sources of tumor antigens for de- tion of many HLA peptides that are derived from velopment of Glioblastoma immunotherapy are the proteins that are not normally expressed in healthy pools of HLA-bound peptides (the HLA peptidome tissues of the body. or immunopeptidome). One group of antigens that can be used for immunotherapy is tumor antigens that are expressed only in tumor cells, embryonal and immune-privileged tissues, such as the testis, placenta and ovaries. Many of these cancer anti- gens and their derived HLA peptides can be epi- genetically up-regulated in tumors’ cells, follow- ing exposure to DNA demethylating chemotherapy agents, such as 5-aza-2´-deoxycytidine (Decitabine). This project aims to study the HLA peptidomes of human Glioblastoma tumors and cultured cells to identify tumor specific peptides for development of Glioblastoma immunotherapy. We are focus on iden- tifying an up-regulated or newly synthesized HLA peptides that are presented by Glioblastoma cells fol- lowing treatment with Decitabine chemotherapy. We specifically search for antigens that are derived from tumor-testis genes, aiming to combine such chemo- therapy with a cancer immunotherapy composed of the HLA peptides that are induced by the chemo- therapy treatment. We hope that such treatment will enhance killing of tumor cells by T cells directed against the cancer-testis antigens. Such strategy can be rapidly implemented for treating patients in 060 | THERAPEUTIC VACCINATION
Fully synthetic Mucin 1 glycopeptides are effective cancer vaccines and induce high antigen-specific antibody titers
Stergiou N.1, Palitzsch B.2, Schmitt E.1, Kunz H.2
1University Medical Center Mainz, Institute of Immunology, Mainz, Germany, 2Johannes Gutenberg University Mainz, Organic Chemistry, Mainz, Germany
Tumor immunotherapy is considered to be very assays showed that these antibodies bind with promising for cancer treatment. Choosing selec- high affinity selectively to tumor cell-associat- tive tumor antigens is essential for the devel- ed MUC1, demonstrating the diagnostic value of opment of effective anti-tumor vaccine. Among these antibodies. Hence, our developed cancer these, tumor-associated membrane glycopro- vaccines might be used therapeutically to treat teins represent important antigens for the de- MUC1-expressing cancers (breast cancer, pros- velopment of immunotherapies. Many epitheli- tate cancer and pancreatic cancer) by stimulat- al tumor tissue differ from healthy tissue in an ing the body’s natural immune system against altered glycosylation pattern of the cell mem- the tumor-associated glycosylation of MUC1. brane mucin (MUC) 1. However, cancer can be considered as altered self and thus is protect- ed by peripheral tolerance mechanisms of the immune system. To overcome this, cancer vac- cines are designed to induce effective anti-can- cer immune responses. Solid phase glycopep- tide synthesis is now able to produce complex glycopeptide antigens with specific glycosyla- tion patterns. In addition, these structures can be linked to immune stimulants (e.g. tetanus toxoid) which will lead to an activation of B cells and effector T cells thereby leading to an- tigen-specific killing of tumor cells. Especially in personalized medicine the flexible synthesis of these vaccines could become necessary if T helper cell epitopes are required that match dif- ferent human leukocyte antigen types (HLAs). Here we demonstrate that fully synthetic MUC1-glycopeptides can be utilized for cancer vaccination. Immunization studies in mice re- vealed that these vaccines are able to elicit high titers of protective IgG-antibodies. Cell-based 061 | THERAPEUTIC VACCINATION
Novel metronomic chemotherapy and cancer vaccine combinatorial strategy for hepatocellular carcinoma
Tagliamonte M.1, Petrizzo A.1, Napolitano M.1, Luciano A.1, Arra C.1, Maiolino P.1, Tornesello M.L.1, Aurisicchio L.2, Ciliberto G.1, Buonaguro F.M.1, Buonaguro L.1
1Istituto Nazionale Tumori ‘Pascale’, Naples, Italy, 2Takis, S.r.l., Rome, Italy
Hepatocellular carcinoma (HCC) is the most frequent The present study describes a combination of novel primary liver cancer and represents the third and multi-peptide formulation specific for HCV-related the fifth leading cause of cancer-related death world- HCC with a novel multi-drug daily metronomic wide in men and women, respectively. Hepatitis B chemotherapy. (HBV) and hepatitis C (HCV) virus chronic infec- tions account for pathogenesis of more than 80% of primary HCC. Prognosis of HCC is generally poor because of the low effectiveness of available treat- ments and the overall 5-year survival rate is approxi- mately 5-6%. Immunotherapeutic interventions, including cancer vaccines, may represent a novel and effective strat- egy. However, only few immunotherapy trials for HCC have been conducted so far with contrasting results. This is probably due to the strong intrinsic immune suppressive microenvironment in the liver which may represent a major impediment to an ef- fective anti-tumor activity elicited by a therapeutic cancer vaccine. In the present study a novel combinatorial strategy, based on metronomic chemotherapy plus vaccine, is evaluated in a mouse model. The chemotherapy is a multi-drug cocktail including taxanes and alkylating agents, which is administered in a daily metronom- ic fashion. The vaccine is a multi-peptide cocktail including HCV as well as universal tumor antigen hTERT epitopes. The combinatorial strategy de- signed and evaluated in the present study induces an enhanced specific T cell response, when compared to vaccine alone, which correlates to a reduced Treg frequency. 062 | THERAPEUTIC VACCINATION
Laser-assisted intradermal delivery of vaccines specific for cross-presenting XCR1+ dendritic cells induces anti-tumoral responses
Terhorst D.1,2, Fossum E.3, Bogen B.3, Henri S.1, Malissen B.1
1Centre d’Immunologie de Marseille-Luminy, UM2 Aix-Marseille, INSERM U1104, CNRS UMR7280, Marseille, France, 2Department of Dermatology, Charité University Medicine Berlin, Berlin, Germany, 3Center for Immune Regulation, Institute of Immunology, University of Oslo, Oslo, Norway
The development of vaccines inducing efficient CD8+ T cell responses is the focus of intense research. Den- dritic cells (DCs) expressing the XCR1 chemokine re- ceptor excel in presentation of extracellular antigens to CD8+ T cells. Studies aiming at harnessing such cross-presentation potential relied on intravenous in- jection of antigens conjugated to XCL1 - the ligand of XCR1 - or to antibodies specific for XCR1+ DCs. Due to its high DC content, including XCR1+ DCs, the skin dermis is an attractive site for vaccine administra- tion. By creating laser-generated micropores through the epidermis, a model antigen fused to XCL1 was targeted to dermal XCR1+ DCs and triggered antigen- specific CD8+ and CD4+ T cells. It required XCR1+ DC emigration to draining lymph nodes and oc- curred irrespective of Toll Like Receptors. Moreover, a single intradermal immunization protected mice against melanoma tumor growth in prophylactic and therapeutic, adjuvant-free settings. The existence in human of functionally equivalent XCR1+ dermal DCs should permit the translation of topic, needle-free in- tradermal delivery of antigens targeting XCR1+ DCs to human cutaneous vaccinations. 063 | THERAPEUTIC VACCINATION
Anti-melanoma immunity and local clinical responses of stage III-IV melanoma patients treated with monobenzone and imiquimod; a phase 2a trial
Teulings H.-E.1,2, Tjin E.P.M.1, Willemsen K.J.1, van der Kleij S.2, ter Meulen S.2, Kemp E.H.3, Nieweg O.E.2, van der Hage J.A.2, van der Veen J.P.W.1,2, Relyveld G.N.2, Luiten R.M.1
1Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands, 2Antoni van Leeuwenhoek Netherlands Cancer Institute, Amsterdam, Netherlands, 3University of Sheffield, Dept of Human Metabolism, Sheffield, United Kingdom
Purpose: Recent developments indicate immuno- tistical endpoint of treatment efficacy. Continued therapy as a promising approach to treat advanced treatment in responding patients improved the clini- melanoma. Development of vitiligo-like depigmenta- cal responses. Seven patients developed vitiligo-like tion in melanoma patients during immunotherapy is depigmentation. Melanoma-specific CD8+T cell re- a favourable prognostic sign and indicates breakage sponses were induced in 17 patients of 19 patients of tolerance against melanoma antigens. We have tested and melanoma specific antibody responses in developed monobenzone-based therapy that induces 8 patients within 12 weeks. specific immunity against melanocytes and mela- Conclusions: These results indicate that targeting noma cells and inhibits the growth and recurrence melanocytes directly by local skin application of of B16 murine melanoma in vivo. This approach is low-cost and broadly applicable depigmenting agent based on the vitiligo-inducing ability of monoben- monobenzone combined with imiquimod effectively zone combined with immune stimulating adjuvants, induces systemic anti-melanoma immunity (both imiquimod and/or CpG. This phase 2a study evalu- CD8+T-cell and antibody responses) leading to re- ated the local clinical and systemic immunological gression of cutaneous metastases in 35% of patients. effects of topically applied monobenzone combined Prolonged treatment and the combination with CpG with imiquimod (MI) in melanoma patients with cu- may enhance these effects, to achieve abscopal an- taneous metastases. titumor effects. Methods: Patients with stage III or IV melanoma with multiple non-resectable cutaneous melanoma metas- tases were treated with daily monobenzone cream and three times a week imiquimod applied locally to the cutaneous metastases and adjacent skin. After 12 weeks, local tumor response based on RECIST criteria for cutaneous metastases was assessed and the induction of local and anti-melanoma immunity following treatment was evaluated. Results: 21 patients of 25 enrolled patients were evaluable for clinical assessment at 12 weeks. MI therapy was well tolerated. Partial regression of cu- taneous metastases was observed in 8 patients and stable disease in 1 patient, which reached the sta- 064 | THERAPEUTIC VACCINATION
Vaccines to induce Robo4 and Clec14a specific antibody retard tumour growth
Toellner K.-M.1, Cook C.1, Zhuang X.2, Zhang Y.1, Ahmed F.1, Heath V.L.1, Bicknell R.1
1University of Birmingham, Immunity and Infection, Birmingham, United Kingdom, 2University of Birmingham, Cancer Studies, Birmingham, United Kingdom
Tumor endothelial specific expression of Robo4 or Clec14a in adults proteins as anti-cancer targets for immunotherapeutic approaches. We describe how vaccination against the autoantigens Robo4 or Clec14a inhibits angiogenesis and tumor growth. In order to break tolerance, mice were immunised with the Robo4 or CLEC14a fused to foreign carrier pro- teins. Vaccinated mice show a strong antibody re- sponse, with no obvious adverse effects on health. Robo4 vaccinated mice showed impaired fibrovas- cular invasion and angiogenesis in a rodent sponge implantation assay, as well as a reduced growth of implanted syngeneic Lewis lung carcinoma. The an- ti-tumor effect of Robo4 vaccination was present in CD8 deficient mice but absent in B cell or IgG1 knock- out mice, suggesting antibody dependent cell medi- ated cytotoxicity as the anti-vascular/anti-tumour mechanism. Adjuvant free soluble Robo4-carrier and Clec14a conjugates can retard tumour growth in carrier primed mice. These results point to appropri- ate conjugates as potential anti-angiogenic vaccines for cancer patients. Preliminary data from conjugates that may be useful for human vaccination will be presented. 065 | THERAPEUTIC VACCINATION
Phase I/IIa clinical study of Survivin-2B peptide vaccination in combination with type I interferon for advanced gastrointestinal cancer patients
Torigoe T.1, Shima H.2, Mizuguchi T.2, Kutomi G.2, Satomi F.2, Kimura Y.2, Hirohashi Y.1, Tamura Y.1, Kanaseki T.1, Tsukahara T.1, Takahashi A.1, Asanuma H.1, Ito Y.M.3, Hayashi H.4, Sugita O.4, Sato N.1, Hirata K.2
1Sapporo Medical University, Dept. of Pathology, Sapporo, Japan, 2Sapporo Medical University, Dept. of Surgery, Surgical Oncology and Science, Sapporo, Japan, 3Hokkaido University Graduate School of Medicine, Dept. of Biostatistics, Sapporo, Japan, 4Hokkaido University, Center for Translational Research, Sapporo, Japan
Survivin is a member of the inhibitor of apoptosis correlated with anti-tumor responses. In addition, protein (IAP) family. It is expressed in fetal tissues it was indicated that tetramer+ CTL frequency in but not in normal adult tissues. Since Survivin is PBLs before vaccination might become a predictive overexpressed in various types of tumor tissues as biomarker. The present clinical study indicates that well as tumor cell lines, it is considered to be suit- SVN-2B peptide vaccination is safe and can be con- able as a target antigen for cancer vaccine therapy. sidered a potent immunotherapy for HLA-A24-posi- We identified an HLA-A24-restricted antigenic tive gastrointestinal cancer patients. peptide, SVN-2B (AYACNTSTL), derived from a splic- ing variant of Survivin-2B. In the present study, we carried out a phase I/IIa clinical study assessing the safety and efficacy of vaccination with the peptide in patients having advanced gastrointestinal cancer. Vaccinations with 0.1mg, 1.0mg, or 3.0mg doses of the SVN-2B peptide were given subcutaneously four times at 14-day intervals. In 20 patients who received at least one vaccination, grade 1 and grade 2 treatment-related adverse events were observed, including injection site extravasation (grade 2), in- jection site reaction (grade 1), skin induration (grade 1) and fever (grade 1). No severe adverse event was observed in any patient. Based on tumor size evalu- ated by computed tomography, eight of the 15 pa- tients who completed the vaccination schedule were considered to have stable disease as assessed by the RECIST criteria. The patients continued the SVN-2B peptide vaccination in combination with type I in- terferon (IFN) after the initial evaluation. Analy- sis of peripheral blood lymphocytes (PBLs) using HLA-A24/peptide tetramers revealed the significant increase of SVN-2B-specific cytotoxic T lymphocyte frequency in the IFN combination group, which was 066 | THERAPEUTIC VACCINATION
Personalized cancer immunotherapy: patient specific multipeptide vaccination for primary liver cancers
Trautwein N.1, Löffler M.1, Walzer M.1, Nadalin S.2, Schroeder C.3, Bonin M.3, Bauer P.3, Riess O.3, Kohlbacher O.4, Königsrainer A.2, Stevanovic S.1, Rammensee H.-G.1
1Universitaetsklinikum Tuebingen, Immunology, Tübingen, Germany, 2Universitaetsklinikum Tuebingen, General, Visceral- and Transplant Surgery, Tübingen, Germany, 3Universitaetsklinikum Tuebingen, Medical Genetics, Institute of Human Genetics, Tübingen, Germany, 4Universitaetsklinikum Tuebingen, Computer Science, Tübingen, Germany
Primary liver cancers, mainly hepatocellular car- jacent benign tissue. In order to identify the muta- cinomas (HCC), are among the five most common tion derived HLA ligands next generation sequencing cancers globally and a leading cause of cancer (NGS) of the tumor and benign tissue is carried out. related death. More than half a million patients Based on NGS data differences between tumor and are diagnosed with HCC every year. Especially in germline exomes are determined and used to compile Europe and the USA incidence numbers have been a patient specific database. This database contains rising over the last decades. With chemotherapies all new protein sequences resulting from identified and other adjuvant strategies showing only limited mutations. Furthermore the SYFPEITHI algorithm benefit, available therapeutic options are scarce. is used to predict mutated naturally processed and Therefore primary malignancies of the liver are a presented HLA ligands to enable targeted MS. Ana- promising entity for targeted immune intervention lyzing samples from 15 patients (10 HCC/ 5 CCC), we after surgery. Personalized approaches, particularly were able to identify over 5000 different HLA ligands immunotherapeutic ones, may represent promising naturally presented on patients´ tumors and over alternatives with regard to effectiveness and safety 5000 on benign liver tissue respectively. Using this profile. Here we demonstrate an approach to identify strategy we are able to recommend individually tai- real tumor specific antigens with potential for future lored multi-peptide vaccines for each patient. Having clinical application. Our efforts to provide a novel, a GMP peptide production facility available, we plan effective and targeted immunotherapeutic approach on applying this workflow for future clinical trials. focuses on naturally processed and presented HLA We should be able to vaccinate patients with such a ligands directly eluted from patient tumor tissue. cocktail within three months after tumor resection, On the one hand, we are interested in HLA ligands aiming at prevention of metastasis and/or relapse in derived from somatic tumor specific mutations, rep- an adjuvant setting. Once established, this approach resenting neo-epitopes. These peptides should lack should be applicable in many different malignancies. central tolerance and therefore be highly immuno- genic. On the other hand, we have been identifying tumor associated peptides (TUMAPS) for many years which are overrepresented on malignant tissue. TUMAPS, although being possibly presented also on normal tissue, can be used to elicit an anti-tumor T-cell response in cancer patients. To identify these peptides we analyze both tumor and autologous ad- 067 | THERAPEUTIC VACCINATION
An RLR/TLR agonist-based combination therapy enhances CTL responses in tumors
Treinies M.1, Perdicchio M.1, Spinetti T.1, Mottas I.1, Spagnuolo L.1, Bourquin C.1, Hotz C.1
1University of Fribourg, Pharmacology, Fribourg, Switzerland
Cytotoxic T cells are a major component of anti-tu- moral immunity. In order to improve dendritic cell- mediated cytotoxic T lymphocyte (CTL) activation for better anti-tumor responses, we have developed a pattern-recognition receptor (PRR)-based therapy using a combination of RIG-I-like receptor (RLR) and Toll-like receptor (TLR) agonists. The sequential combination of these PRR agonists triggering differ- ent signaling pathways avoids the development of TLR tolerance, a hyporesponsive state induced by repetitive single ligand treatment. It further not only circumvents tolerance but also enhances immune activation. The sequential stimulation leads to in- creased secretion of pro-inflammatory cytokines in mice and to increased numbers of IFNgamma-pro- ducing cytotoxic T cells. Additionally we could show that PRR-based treatment enhances the infiltration of cytotoxic T cells in subcutaneous and autochtho- nous tumors. We are currently further analyzing the mechanisms leading to the enhanced T-cell recruit- ment and investigating the impact of enhanced acti- vation of T cells in tumor-bearing mice. The results of this study may provide an important contribution to the development of complementary immunothera- peutic strategies to target tumors. 068 | THERAPEUTIC VACCINATION
Engineering high affinity antibody recognizing naturally presented osteosarcoma antigen PBF peptide
Tsukahara T.1, Torigoe T.1, Sato N.1
1Sapporo Medical University, Pathology, Sapporo, Japan
Peptide vaccine-based immunotherapy targeting tumor-associated antigens could elicit CTL response against HLA/peptide complex. Therefore, expres- sion of target HLA/peptide complex on tumor cells is prerequisite. In this study, toward the characteri- zation of HLA/peptide complex on tumor cells, we generated artificial antibody reacting with HLA/ peptide complex of an osteosarcoma antigen PBF. We performed biopanning of sFv phage display library derived from naive donors using biotinylated mono- mers (HLA-A2/PBFA2.2 peptide) as an antigen. After the three-round biopanning, we screened 94 soluble scFv clones from output phage using ELISA and iso- lated positive clones reacting with specific monomers but not with irrelevant monomers. As the results, we obtained the scFv clone D12 highly reacting -9 with HLA-A2/PBFA2.2 monomers (KD=1. 53x 10 M). PBFA2.2 peptide-pulsed T2 cells were specifically stained by D12 scFv-hIgG1 and D12 scFv multimer by FACS. Furthermore, D12 scFv multimer could react with HLA-A2+PBF+ osteosarcoma cell lines and si- multaneously demonstrated that the HLA/peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based im- munotherapy and develop antibody-based immuno- therapy. 069 | THERAPEUTIC VACCINATION
Sonographic evaluation of lymph node morphology after intranodal vaccination with an RNA-based vaccine in patients with malignant melanoma
Marg K.R.1, Loquai C.1, Müller-Brenne T.1, Rudolph B.1, Wilden S.1, Schmücker P.1, Kloke B.-P.2, Kemmer-Brück A.3, Attig S.4, Derhovanessian E.3, Pless B.2, Britten C.M.2, Kuhn A.N.2, Kreiter S.4, Türeci Ö.5, Grabbe S.1, Sahin U.2,3,4, Tüttenberg A.1
1University Medicine Mainz, Dept. of Dermatology, Mainz, Germany, 2BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, 3BioNTech AG, Mainz, Germany, 4TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany, 5III. Medical Department, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
In the past, intranodal vaccination of patients with sion of the injected lymph nodes were documented malignant melanoma has been proven to be a safe and analyzed before and directly after vaccination and efficient method for the application of vaccines for a total of 8 vaccinations per patient. A first evalu- based on plasmids, peptides or antigen-loaded den- ation showed a significant increase in lymph node dritic cells. Several clinical trials demonstrated the size directly after vaccination in most patients. Fur- efficient de novo induction as well as an expansion thermore, intraindividual longitudinal analysis in- of already existing effector T cell responses in mela- dicated that most of the patients (8/11) displayed a noma patients at different stages of disease. permanent increase of lymph node volume over the In the present study, the ultrasound guided adminis- course of vaccinations. These findings are currently tration of tumor-antigen encoding RNA in draining correlated to results of immune monitoring as well lymph nodes should lead to a fast and well-directed as clinical response uptake, translation and presentation of the antigen In conclusion, the clinical study confirms a safe and through professional antigen-presenting cells and feasible intranodal vaccination, leading to changes thus to the induction of an antigen-specific immune in lymph node morphology comparable to post-vac- response towards the tumor-antigens NY-ESO-1 and cination lymphadenopathy, indicating the presence Tyrosinase. The intranodal vaccination has the ad- of immunological responses in treated melanoma vantage that antigen expression is limited to the pro- patients. fessional antigen presenting cells in immunocom- petent lymph node and systemic antigen expression is prevented. This is important regarding potential tolerance induction following parenteral applica- tion of antigen alone in the absence of costimulatory signals. As lymph nodes play an important role in the immune system, morphological changes may give a first hint as to the response in patients following intranodal vaccination. Therefore, size, morphology and perfu- 070 | THERAPEUTIC VACCINATION
Anti-idiotypic vaccine Racotumomab against NGcGM3 gangloside stabilize disease in rapidly progressing treatment refractory metastatic breast cancer
Uskent N.1, Sezer M.1, Berberoglu K.1
1Anadolu Health Science Center, Department of Medical Oncology, Kocaeli, Turkey
N-glycolyated gangliosides,especially NGM3 is given intradermaly every 2 weeks for 5 courses,and highly expressed in the cell membrane of certain after than 4 weekly. The pace of the disease pro- human tumors like non-small cell lung cancer gression stabilized at 3 months. The patient clini- and breast cancer ,and not exist in normal cells. cally became almost asymptomatic with minor Racotumomab(Vaxira) is an anti-idiotypic vaccine pain(VAS:1-2) in the legs.Her Performence status against nue-GcGM3 antigen.Racotumomab mimics also improved to ECOG:0. In the seventh month after NGcGM3 antigen and induce an immune response starting Racotumomab PET-CT revealed diminished against the ganglioside.High serumIgM/IgG Abs hipermetabolic activity at all metastatic sites.Tumor against NeuG3GM3 corrolates with survival.A 60 markers(Ca-15-3 and Ca-125) found to be lower from Year-old woman with a known diagnosis of metastat- the pre-treatment high levels. She remains clinically ic breast cancer,involving multiple sites of bones,liver asymptomatic. Our finding suggest that Racotumum- and lungs who had been heavily pretreated and ab vaccine may be tried in the patients who fails from subsequently became unresponsive to 3rd. line multiple lines of chemotherapy and/or endocrine chemotherapies and several attempts of endocrine therapy.HER 2 negative patients may be preferred for treatment, was offered immunotherapy with Racotu- lack of an concrete therapeutic target. mumab plus metronomic treatment along with Cyclo- phosphamide 50 mg po daily and Methotraxate 2.5 mg po bid. Initial pathology and staging of the tumor was invasive ductal carcinoma grade III, Ki-67>%40, ER positive, PR negative, HER2 negative,Luminal B, T4N0M0,for which she underwent neoadjuvant FEC followed by mastectomy and another 6 courses of Docetaxel adjuvant chemotherapy. However , after one year of adjuvant chemotherapy she developed distant metastases in the liver, lung and bones while she was on Letrozol maintanence.3 Lines of chemo- therapy including Carboplatin plus Paclitaxel, Vino- ralbin and Capecitabine+Docetaxel failed for any responce. The disease progressed despite switched endocrine treatments with Fulvestran and Aroma- sine.As an alternative option ,Racatumomab was 071 | THERAPEUTIC VACCINATION
Individualized tumor immunotherapy-From mutanome to actively personalized RNA based cancer vaccination
van de Roemer N.1, Kreiter S.2, Vormehr M.1, Loewer M.2, Selmi A.2, Diekmann J.3, Diken M.2, Vascotto F.2, Brkic M.1, Kind S.3, Schmitt U.1, Türeci Ö.4, Sahin U.1,2,3
1Research Center for Immunotherapy (FZI), Johannes Gutenberg University, Mainz, Germany, 2TRON - Translational Oncology at the University Medical Center of Johannes Gutenberg University, Mainz, Germany, 3Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany, 4Ganymed Phamaceuticals AG, Mainz, Germany
Non-synonymous tumor-specific mutations are an at- as well as the therapeutic setting for a substantial tractive target pool for cancer immunotherapy as they number of mutated epitopes. Surprisingly, we identi- lack expression in healthy tissues and can potentially fied one MHC class II restricted epitope (Mut-30) that be recognized as neo-antigens by the mature T-cell confers tumor control beyond the efficacy of known repertoire. In principle any genetic alteration has the immunodominant tumor-associated antigens like potential to generate mutated peptides that are pre- Trp2 or gp100. To further examine the mechanism sented by either surface MHC class-I or -II molecules behind the anti-tumoral potency of this class II re- and may represent an ideal target for neo-epitope stricted mutated epitope we investigated the active tumor vaccination. As human cancers carry 100-300 role of CD4+, CD8+ and NK1.1 T cells in early and non-synonymous mutations on average which are late stage of tumor development and therapy. Deple- not subject to central immune tolerance, these muta- tion studies demonstrate that CD4+ but not CD8+ T tions can be optimal candidates for vaccine develop- cells are crucial for the observed antitumoral activ- ment. To characterize the immunogenicity in a given ity of Mut-30 RNA vaccination in B16-F10. Mutation- tumor, we implemented a streamlined approach for specific IFN-g expressing CD4+ T cells were shown the prediction and validation of such neo-epitopes to be increased in the tumor tissue after Mut-30 RNA derived from B16-F10 murine melanoma. We iden- vaccination. Finally we showed the direct impact of tified more than 500 non-synonymous point muta- IFN-g on B16-F10 MHC molecules after vaccination tions by whole exome sequencing (1). After selection with Mut-30 RNA. The Data suggest that CD4+ T cell of expressed genes and good potential MHC binders derived IFN-g exerts direct antiproliverative effects of the respective mutated epitopes, 50 mutations on B16-F10 cells in addition to facilitating lytic inter- were chosen and validated by Sanger sequencing. actions of other effectors with tumor targets. Conductive to define the immunogenicity of the mutation-coding sequences, we designed 27-mer peptides (comprising all potential MHC class I and II epitopes of 8 to 14 amino acids in length) carrying (1) Castle JC, et al.: Exploiting the mutanome for tumor vac- cination. Cancer Res 2012, 72:1081-1091 the mutation incorporating either the mutated or the wild-type amino acid to immunize C57BL/6 mice. Anti-tumor potency of all immunogenic epitopes was investigated in a transplantable B16-F10 melanoma model where mice immunized with mutation-encod- ing IVT-RNA revealed tumor control in the protective 072 | THERAPEUTIC VACCINATION
Development of a next generation Semliki Forest virus-based DNA vaccine against cervical cancer
van de Wall S.1, Ljungberg K.2, Ip P.P.1, Boerma A.1, Nijman H.W.3, Liljeström P.2, Daemen T.1
1University of Groningen, University Medical Center Groningen, Department of Medical Microbiology, Groningen, Netherlands, 2Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Stockholm, Sweden, 3University of Groningen, University Medical Center Groningen, Department of Obstetrics & Gynecology, Groningen, Netherlands
Cervical cancer is the second most prevalent cancer among women worldwide. The disease develops as a result of infection with high-risk human papilloma- virus (HPV) through persistent expression of early proteins E6 and E7 with transforming capacities in cervical epithelial cells. Our group pioneered the ap- plication of a replication-defective recombinant viral vector system based on Semliki Forest virus (SFV) for vaccination against cervical cancer. In preclini- cal studies, we demonstrated that recombinant SFV (rSFV) encoding HPV E6 and E7 (rSFVeE6,7) induces robust HPV-specific cellular immune and memory responses upon intramuscular administration in mice resulting in excellent therapeutic anti-tumour efficacy. Despite the clear potency of the SFV vector system, there are a number of inherent challenges. These include manufacturing costs, shelf-life and anti-vector responses. DNA vaccination is an alterna- tive method with the potential to be inexpensive and safe. In this project, the drawbacks associated with both SFV-based vaccines and DNA vaccines are cir- cumvented with the development of a DNA-launched SFV replicon (DREP) strategy. Using intradermal de- livery followed by electroporation, we demonstrate that prime-boost with DREP encoding for E6,7 is as comparable with rSFV in inducing a high frequency of E7-specific cytotoxic T cells. This coincided with effective immunological memory formed with 85% of mice being tumor-free till day 100. This study points towards a potential vaccine platform for clini- cal assessment. 073 | THERAPEUTIC VACCINATION
RNA vaccination-induced CD4+ T cells frequently recognize mutated epitopes leading to rejection of aggressively growing tumors
Vormehr M.1, Kreiter S.2, van de Roemer N.1, Diken M.2, Löwer M.2, Vascotto F.2, Diekmann J.3, Bögel S.2, Schrörs B.2, Schmitt U.2, Holzmann M.2, König A.3, Brikic M.1, Witzel S.2, Kind S.3, Otte B.3, Tadmor A.2, Türeci Ö.2, Sahin U.1,2,3
1Research Center for Immunotherapy (FZI), Johannes Gutenberg University, Mainz, Germany, 2TRON - Translational Oncology at the University Medical Center of Johannes Gutenberg University, Mainz, Germany, 3Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany
Mutations are regarded as ideal targets for cancer tion based MHC class II restricted epitopes are attrac- immunotherapy. As neo-epitopes with strict lack of tive vaccination targets and provide the preclinical expression in any healthy tissue, they are expected to proof of concept for an integrated process from tumor be safe and could bypass the central tolerance mech- sample to a cancer vaccine customized to the unique anisms. Recent advances in nucleic acid sequencing repertoire of each patient`s tumor. technologies have revolutionized the field of genom- ics, allowing the readily targeting of mutated neo- antigens for personalized cancer vaccination. We demonstrated in three independent murine tumor models that a considerable fraction of non-synony- mous cancer mutations is immunogenic and that un- expectedly the immunogenic mutanome is pre-dom- inantly recognized by CD4+ T cells. RNA vaccination with such MHC class II restricted immunogenic mu- tations leads to infiltration of CD4+ and CD8+ T cells into the tumor, reduces intratumoral regulatory T cells and ultimately confers strong anti-tumor activ- ity. Encouraged by these findings we set up a process comprising mutation detection by exome sequenc- ing, selection of vaccine targets by solely bioinfor- matical prioritization of mutated epitopes predicted to be abundantly expressed and presented on MHC class II molecules. Synthetic mRNA vaccines encod- ing multiple of these prioritized mutated epitopes induce potent tumor control and complete rejection of established aggressively growing tumors in mice. Moreover, we demonstrate that CD4+ T cell neo- epitope vaccination primes CTL responses against an independent immunodominant antigen in tumor bearing mice indicating orchestration of antigen spread. Our findings demonstrate that cancer muta- 074 | THERAPEUTIC VACCINATION
Synthetic long HPV16 E6 and E7 peptides vaccine combined with Aldara as adjuvant to treat HPV16-induced vulvar/vaginal intraepithelial neoplasia
Welters M.J.1, van Poelgeest M.I.2, Vermeij R.3, Kenter G.G.4, Nijman H.W.3, Melief C.J.5, van der Burg S.H.1
1Leiden University Medical Center, Clinical Oncology, Leiden, Netherlands, 2Leiden University Medical Center, Gynecology, Leiden, Netherlands, 3University Medical Center Groningen, Gynecology, Groningen, Netherlands, 4Academic Medical Center, Gynecology, Amsterdam, Netherlands, 5ISA Pharmaceuticals, Leiden, Netherlands
High-risk human papillomavirus (HPV) types, such as vaccine. However, in all patients a vaccine-induced HPV16, can cause chronic pre-malignant lesions and T-cell response was observed. Also the clinical out- subsequently cancer of the anogenital tract as well as comes were similar in the two treatment groups. At 3 head and neck region. Previously, we have demon- and 12 months after the last vaccination comparable strated that patients with HPV16-induced high-grade number of patients had a partial (≥50% reduction) or vulvar intraepithelial neoplasia (VIN) can be treated complete disappearance of the lesion as was observed with a vaccine consisting of synthetic long peptides in our previous study in high-grade VIN patients. (SLP) from the HPV16 viral oncoproteins E6 and Moreover, in the current trial we could confirm that E7, resulting in clinical responses associated with a patients with a complete clinical response displayed a strong immune response against these viral proteins. stronger HPV16-specific T-cell response. However, the CD8+ T-cell frequency elicited by the In conclusion, this new independent clinical trial in HPV16 SLP vaccine warrants still improvement. HPV16-induced high-grade VIN/VaIN demonstrates In a preclinical model for melanoma, the application of that the HPV16 SLP is clinical effective and that this the agonist for Toll-like receptor 7 (imiquimod; Aldara clinical outcome is related to the strength of the vac- cream) on top of the SLP vaccination site strongly en- cine-induced T-cell response, which makes this ap- hanced the CD8+ T-cell response against the vaccine proach an effective therapy for this group of patients. peptides, and was accompanied by tumor regression. Therefore, a randomized, open label, phase I/II clini- cal trial in patients with HPV16 positive high-grade VIN or vaginal intraepithelial neoplasia (VaIN) was * the first two authors contributed equally conducted. The predefined endpoints of immune and clinical responses as well as safety of the HPV16 SLP vaccine ISA101 with and without topical application of Aldara on the vaccine site were studied. Patients received 4 vaccinations with a 3-week interval as was given in the previous trial. In total 43 women were enrolled and the intention to treat as well as well as per protocol patient populations were defined. The results of a set of complementary T-cell assays showed that Aldara cream on the vaccine injection site did not improve the immune responses to the 075 | THERAPEUTIC VACCINATION
Induction of massive antigen-specific CD8+ T cell cytotoxicity by targeting of antigen into XCR1+ DC combined with potent amplification regimes
Yang Y.1, Hartung E.1, Jäkel A.1, Gurka S.1, Mages H.W.1, Kroczek R.A.1
1Robert Koch-Institute, Molecular Immunology, Berlin, Germany
Current immunization schemes do not induce potent CD8 memory T cells which can again be quickly re- CD8+ T cell cytotoxicity unless combined with viral amplified to around 50% using the ADAS procedure boost regimes, which are not easily applicable in the (OVA-model). Further, we could observe that the am- human. We have recently shown that the chemokine plification achieved by complexed IL2 can also be receptor XCR1 is selectively expressed on antigen seen with complexed IL15. Currently, experiments cross-presenting dendritic cells (DC), the key players with the B16F10 melanoma lung metastasis model in the induction of CD8+ T cell cytotoxicity. Based are under way and will be presented. We are about on this fact, we have developed strategies to target to immunize Rhesus macaques with the GAG protein protein or peptide antigens into these XCR1+ DC of simian immunodeficiency virus in order to test the by employing an antibody to XCR1 or by utilizing efficacy and safety of the procedure in a non-human the specific chemokine ligand XCL1 together with primate model. If these tests are successful, we will suitable adjuvants (Hartung et al., J Immunol 194 transfer the system into the human. (2015) 1069). This (published) basic immunization can be further strongly amplified using a non-viral amplification regime (ADAS), which raises the level of antigen-specific CD8+ T cells to around 20% of all T cells within few days (OVA or influenza NP as model antigens). Another level of amplification (up to 70% of all CD8+ T cells) can be achieved with further administration of complexed IL2. These ex- panded CD8+ T cells (highly activated, granzyme B and perforin positive), are biologically active in vivo in that they efficiently eradicate established trans- planted tumors. We have now extended our studies to other antigens and modes of amplification and further optimized the procedure. Immunization of the CD8+ T cell compartment with the nucleopro- tein (NP) of influenza, followed by the amplification steps described, fully protected mice from a 10xLD50 infection with the PR8 influenza strain. We have observed that our immunization protocol induces 076 – 114
New Targets & New Leads 076 | NEW TARGETS & NEW LEADS
Human tonsil derived T cells show increased responsiveness upon anti-CD28 superagonistic monoclonal antibody treatment compared to blood derived T cells
Bartholomäus P.1, Kallendrusch S.2, Semmler L.1, Grabski E.1, Buschjäger D.1, Hünig T.3, Schraven B.4, Bechmann I.2, Kalinke U.1
1Twincore, Centre of Experimental and Clinical Infection Research, Institute for Experimental Infection Research, Hannover, Germany, 2University of Leipzig, Institute of Anatomy, Leipzig, Germany, 3Julius-Maximillians University Würzburg, Institute for Virology and Immunobiology, Würzburg, Germany, 4Otto-von-Guericke-University, Institute for Molecular and Clinical Immunology, Magdeburg, Germany
Superagonistic anti-CD28 monoclonal antibodies organotypic tonsil slice cultures mounted cytokine (CD28SA) hold promise as therapeutic agents against responses reminiscent of those detected in the study autoimmunity and cancer. However, in a first clini- participants. Histological analyses revealed that pro- cal trial of the CD28SA TGN1412 a life-threatening liferating T cells were located in B cell rich lymphoid cytokine release syndrome was induced in 6 study nodules. Our results imply that tonsils or second- participants. Only recently it was discovered that ary lymphoid organs might constitute an attractive TGN1412 mediated T cell activation was dependent source of immune cells for preclinical assays to more on tonic pre-activation of T cells and interaction comprehensively appreciate the functional potential with cells expressing Fcγ receptors and/or costim- of new biologics such as CD28SA. ulatory molecules. Thus, conventional screens were not suitable to predict the whole activity spectrum of TGN1412. As known from earlier observations, T cells derived from primary and/or secondary ly- mophoid tissues show a tonic pre-activation status. Furthermore, interaction between TGN1412 dec- orated T cells and FcγR expressing cells is needed to boost the activation. Therefore, we hypothesize that TGN1412 crosslinking and activation of T cells takes place mainly in secondary lymphoid organs, where a high cell density is observed. To test this, tonsil mononuclear cells (TMC) as well as peripher- al blood mononuclear cells (PBMC) from the same donor were isolated, treated with TGN1412 and early cytokine induction and T cell proliferation was mea- sured. We found that TGN1412 treated TMC derived T cells showed significantly stronger responsiveness than PBMC derived T cells. To study whether the complete microarchitecture of a secondary lymphoid tissue was needed, tonsil slice cultures were treated with soluble TGN1412. Of note, within 24h after treatment with soluble TGN1412 freshly prepared 077 | NEW TARGETS & NEW LEADS
In-depth mass spectrometry based immunopeptidomics of melanoma tissues for the development of anti-tumor immunotherapies
Bassani-Sternberg M.1, Bräunlein E.2, Klar R.2, Sinitcyn P.1, Slotta-Huspenina J.3, Werner A.4, Martignoni M.E.4, Hein R.5, Peschel C.2, Busch D.H.6, Cox J.1, Krackhardt A.M.2, Mann M.1
1Max Planck Institute for Biochemistry, Proteomics and Signal Transduction, Martinsried, Germany, 2Medizinische Klinik III, Technical University Munich, Klinikum rechts der Isar, Munich, Germany, 3Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar, Munich, Germany, 4Institute of Surgery, Klinikum rechts der Isar, Technical University Munich, Munich, Germany, 5Department of Dermatology and Allergology, Technical University Munich, Munich, Germany, 6Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technical University Munich, Munich, Germany
Introduction: Metastatic malignant melanoma is a for peptide identification ensured high confidence rapidly increasing and aggressive disease. It is known identifications. to be highly immunogenic; therefore anti-cancer vac- Preliminary data: We accurately identified the most cines and other specific immune intervention strate- comprehensive immune-peptidome from human gies are currently being tested in the clinic. However, melanoma tissues, comprising more than 76,000 little is known about the tumor-specific targets and HLA class I peptides and more than 18,800 HLA the tumor-reactive effector cells that are involved. An class II binding peptides. Among them, we identi- in-depth characterization of the immunopeptidome, fied more than 300 class I and class II epitopes from comprising on the Human Leukocyte Antigen (HLA) known melanoma-specific antigens such as PRAME, class I and class II peptidomes, isolated from mela- Tyrosinase, MAGEA3, PMEL, CTAG1A; most of these noma primary tissues would provide new insights are novel and have never been identified in patient into the immunogenic potential of these tumors. This tissues. We further extended our search to post-trans- is a promising approach for the identification of new lationally modified peptides and mutated peptides targets for the development of immunotherapies, that potentially serve as neo-antigens. In addition, such as peptide vaccine, adoptive T-cell therapies the identification of novel patient specific mutated and for immune-monitoring. epitopes using their exome sequencing information Methods: HLA class I and class II complexes were for the database search is still ongoing. In order to immunoaffinity purified from twenty five primary test the immunogenic potential of selected epitopes melanoma tumors using a recently developed work- in-vitro, we performed T-cell stimulation assays. We flow (Bassani-Sternberg et al. PMID 25576301). The detected elevated levels of peptide-specific T-cells resulting HLA binding peptides were purified from that displayed functional responses against peptide- the HLA molecules, separated by a nanoflow HPLC pulsed target cells as well as tumor reactivity, con- and sprayed directly into a Q Exactive HF mass firming their potential therapeutic value. spectrometer. We used the MaxQuant computational Novel aspect: We established a comprehensive re- environment to identify the HLA peptides, apply- source of the melanoma in-vivo presented immu- ing a database search with no protease specificity nopeptidome with hundreds of novel targets for (i.e. ‘unspecific’ search) and in combination with further development of immunotherapies. de-novo sequencing. A stringent 1% FDR threshold 078 | NEW TARGETS & NEW LEADS
The use of proteomics to analyze whole tumors and identify unique immuno-oncology targets for antibody based therapeutics
Ackroyd J.1, Bisht A.2, Allen J.1, Paris B.1, Barnes M.1, Hudson L.1, Rohlff C.1, Wilson K.2, Boyd R.1, Aud D.2
1Oxford BioTherapeutics Ltd, Abingdon, United Kingdom, 2Oxford BioTherapeutics Inc, San Jose, United States
The recent clinical success of the mAb therapeu- has been used to identify novel oncology therapeutic tics targeting immune checkpoint inhibitor proteins targets for both ADC and BiTE-like approaches. Uti- (PD-1/PD-L1, CTLA-4) has led to an increased ap- lizing OGAP, membrane proteins present in tumors preciation of the potential of utilizing the immune from five cancer indications (Pancreatic, Lung, system in oncology. There are two major strategies Breast, Colorectal and Esophageal cancer) and mul- to elicit either a novel immune anti-tumor response tiple normal tissues or cells were analyzed. Validated or to reactivate a pre-existing anti-tumor response: immune cell markers (such as CD8, OX40, CD79B, by releasing a checkpoint inhibitory pathway via TLR1, TLR2, TLR4, TLR7, CD56, CD204 and CD207) cell surface receptors (such as PD-1/PD-L1, CTLA-4) were profiled across different normal and tumor or by activation of co-stimulatory receptors (such proteomic data sets. This analysis demonstrated as CD40, OX40, or GITR). Both of these strategies we detect key immune cell markers in tumors and of immune modulation utilize cell surface receptors, that different immune cell populations are found and the targeting of antibody therapeutics with the in tumors from the same or different cancer indica- appropriate functional activity to those receptors, to tions. The proteomic data sets were next analyzed for modify immune cell responses and allow for anti- the presence of validated immuno-oncology targets tumor activity. The identification of novel immune- such as inhibitory T cell receptors (ICOS, LAG3,TIM3 modulatory receptors with the potential to be im- and BTLA) or known ligands (PDL-1, PDL-2, B7-H3, muno-oncology therapeutic targets could be of high B7-H4, B7-H5 and HVEM. The expression patterns of value to this anti-tumor approach. these immuno-oncology targets were analyzed to try OGAP is a unique proteomic database that integrates and identify a unique protein signature. Using this information at the tissue, disease and protein isoform protein expression profiling approach we identified level across diseases, indications, and normal tissues most known immuno-oncology targets, validating to clarify membrane protein expression levels and this as an approach to identify a pool of candidate profiles. Specifically, it currently holds information novel immuno-oncology targets. To further develop on ~16,000 human proteins sequenced, ~7,000 our approach we also used sequence based homology membrane proteins, ~35 tissues/organs, and ~17 searching of uncharacterized membrane proteins to cancers. OGAP is fed by a proprietary sample prepa- improve the quality of the pool of candidate novel ration and processing workflow that relies on state- immuno-oncology targets. Several potential novel of-the-art high-throughput mass spectrometry and immuno-oncology targets will be presented. data processing to provide quantitative information on over 4,000 membrane-enriched proteins. OGAP 079 | NEW TARGETS & NEW LEADS
Prevention of Graft-vs-Host-Disease with maintenance of Graft- vs-Leukemia in humanized mice treated with an anti-ICOS monoclonal antibody
Burlion A.1, Petit N.1, Olive D.2, Marodon G.1
1CIMI-PARIS (Immunology and Infectious Diseases Research Center), INSERM U 1135, CNRS ERL 8255, Sorbonne University, Paris, France, 2Paoli-Calmettes Institute, Cancerology Research Center, Marseille, France
Graft-vs-Host-Disease (GVHD) is a major compli- P815 mastocytoma, mimicking an efficient GVL in cation of allogenic bone marrow transplantation humans. Thus, ICOS represents a promising target (BMT) in patients treated for various malignant he- in the management of BMT, preventing GVHD while mopathies. However, GVHD is often necessary to preserving GVL. eliminate residual cancer cells, a process referred to as Graft-vs-Leukemia (GVL). Thus, the conundrum for efficient BMT resides on the control of a low grade GVHD to preserve GVL. Novel therapeutic strategies based on monoclonal antibodies (mAbs) specific for co-stimulatory molecules have proven their efficacy by reaching out effector T cells in the tumor and preventing immunosuppression. Here, we assessed whether a new mAb to ICOS was able to antagonize the co stimulatory signal delivered by ICOS-L in vivo, that would lead to lower T cell activation, preventing GVHD. To test this hypoth- esis, we used a xenogeneic model of human GVHD where human PBMCs are adoptively transferred in immunocompromised NOD.SCID.gc-null mice (NSG). In this model, mice invariably lose weight and ultimately die by day 50. In contrast, 70% of the mice receiving a single injection of the anti- ICOS mAb survived long-term. A strong reduction in perivascular infiltrates was noticed in liver and lungs of treated animals compared to controls. This was associated to a strong decrease in frequencies and numbers of human T cells in the blood and lym- phoid organs, showing that the the mAb prevented T cell expansion in this setting. However, mice were not lymphopenic after treatment since GVHD-pro- tected mice remains competent for rejection of the 080 | NEW TARGETS & NEW LEADS
Meloe, a single polycistronic mRNA translated by various mechanisms to generate multiple potential antigenic peptides in melanoma
Charpentier M.1, Carbonnelle D.1, Florenceau L.1,2, Fortun A.1, Labarriere N.1, Lang F.1
1UMR INSERM U892 - CNRS U6299 - University of Nantes, Nantes, France, 2Nantes Hospital, Nantes, France
Metastatic melanoma is the major cause of death by cu- Since MELOE-4 translation is cap-dependent it should taneous cancers, due to its resistance to classical thera- also be expressed in normal melanocytes and may thus pies such as radio or chemotherapy. For several years, represent the physiological product of meloe mRNA. We our team has worked on the development of new immu- are currently generating a MELOE-4 specific monoclonal notherapies, especially adoptive T cell transfer (ACT). In antibody to directly explore MELOE-4 expression in both ACT, the choice of targeted antigens is of utmost impor- tumor and healthy tissues. To explore the immunogenic- tance and in this regard, we previously identified two ity of MELOE-4, we performed multiple sensitizations of new melanoma antigens involved in immunosurveil- PBMCs from healthy donors or melanoma patients with lance. MELOE-4 and could only obtain one single CD4+ spe- Those antigens named MELOE-1 and MELOE-2 are cific T cell clone and no CD8+ clone, suggesting that translated from distinct ORFs of a polycistronic meloe MELOE-4 specific T cells are scarce. This result is in mRNA and we recently demonstrated that their transla- marked contrast with our previously published observa- tion relied on IRES sequences. Although meloe mRNA tions of a broad and frequent T cell repertoire against is present in normal melanocytes, the translation of MELOE-1. This would be consistent with an immune MELOE-1 and 2 seemed restricted to melanoma cells, tolerance towards an antigen physiologically expressed suggesting that those IRES sequences are specifically on normal melanocytes. activated in tumor cells. In conclusion, our data document that (1) multiple ORFs In the present study, we wondered whether other MELOE from the polycistronic meloe mRNA can be expressed antigens could be produced from this mRNA, whether within cells of the melanocytic lineage through various those proteins would be expressed in healthy melano- translation mechanisms, (2) that cap-dependent transla- cytes and finally if they would be immunogenic. We tion produces a poorly immunogenic protein MELOE-4 focused on two ORFs located at the 5’ end of the meloe that may have a physiological role in melanocytes (3) mRNA. Using transfected GFP constructs, we confirmed that IRES-dependent ORFs of meloe mRNA may gener- that these two ORFs could be translated in melanoma ate the most immunogenic antigens thus prompting us cells thus generating two new antigens, MELOE-3 and to investigate the expression of other ORFs from meloe 4. We observed that the expression of MELOE-4 (closest mRNA. In cancer, IRES-dependent antigens such as to the 5’ end) was much higher than that of MELOE-1, 2 MELOE-1 and 2 could represent a new class of tumor and 3. We then documented that MELOE-4 is translated antigens in that they are both lineage and tumor specific. by the classical cap-dependent pathway while MELOE-3, whose ORF overlaps that of MELOE-4, is translated by defective ribosome scanning. 081 | NEW TARGETS & NEW LEADS
A Cancer Research UK Phase I trial of the Anti-CD19 DI-B4 monoclonal antibody given intravenously, weekly for four weeks in patients with advanced CD19 positive indolent B-cell malignancies
Chowdhary A.1, Challis R.1, Williams A.1, Radford J.2, Collins G.3, Pettitt A.4, Davies A.1
1University of Southampton, Southampton, United Kingdom, 2The Christie NHS Foundation Trust, Manchester, United Kingdom, 3Oxford University Hospitals NHS Trust, Oxford, United Kingdom, 4Royal Liverpool University Hospital, Liverpool, United Kingdom
This Phase I study aims to establish the toxicity sion has been completed successfully. A review after profile, biological effects and maximum tolerated cohort four (500mg dose cohort) demonstrated no dose (MTD) of the low-fucosylated anti-CD19 IgG1 serious adverse events which would prevent dose es- monoclonal antibody DI-B4, in a target patient group calation to 1000mg. Patients 10 -12 have received their including B-cell non-Hodgkin lymphoma (NHL) and first 1000mg drug infusion without serious adverse chronic lymphocytic leukaemia (CLL) in patients re- events. CD20+ B cell depletion was observed in the fractory to conventional treatment. CD19 is widely 250mg dose cohort occurring in week 1 following expressed on malignant B-cells and the low-fuco- the first drug infusion in 2/3 patients in this cohort. sylation should result in enhanced antibody depend- Early cohorts have received DI-B4 well with minimal ent cell mediated cytotoxicity (ADCC) via improved side effects, and CD20+ B cell depletion evident in binding to FCɣ receptors on NK cells and monocytes. patients treated at 4 x 250mg dose cohort. 500mg Further to anti-tumour activity and clinical trial end- doses have also been well tolerated. B cell deple- points on PK and PD effects, exploratory research tion in this cohort was less evident due to clinical assays are also to be performed to investigate pe- B cell depletion prior to enrollment on trial. Dose ripheral blood mononuclear cells (PBMC) and bone infusions have been escalated to 4 weekly infusions marrow depletion using FACs analysis. of 1000mg. The B cell depletions observed at early DI-B4 is intravenously administered once a week for doses support the further investigation of this agent a total of up to four weeks. The initial dose for the in these disease settings. first cohort was 0.02mg with initial intra-patient dose escalation. Fixed dose infusion cohorts at 4 x 250mg, 500mg and 1000mg have been administered. Validat- ed ELISA’s are used to quantify DI-B4 in serum and test for HAHA responses. PBMC are frozen from fresh blood collected over the course of DI-B4 infusions and at end of therapy 8 weeks after the last infusion. CD20+ B cell depletion has been measured on fresh blood over the course of the treatment and the cryo- preserved PBMC will be used to assess B cell subset enumeration and other immune cell modulation. The trial has currently recruited 12 patients. Drug administration for doses up to 500mg per drug infu- 082 | NEW TARGETS & NEW LEADS
Aberrant expression of MHC Class II in melanoma attracts inflammatory tumor specific CD4+ T cells which dampen CD8+ T cell antitumor activity
Donia M.1,2, Andersen R.1,2, Kjeldsen J.W.1, Fagone P.3, Munir S.1, Nicoletti F.3, Andersen M.H.1, Thor Straten P.1, Svane I.M.1,2
1Herlev University Hospital, Center for Cancer Immune Therapy, Herlev, Denmark, 2Herlev University Hospital, Department of Oncology, Herlev, Denmark, 3University of Catania, Department of Bio-Medical Sciences, Catania, Italy
In the absence of a local inflammatory response, the expression of MHC Class II molecules is mainly re- stricted to hematopoietic cells and thymus epithe- lium. However, certain tumors such as melanoma may acquire aberrant constitutive expression of MHC class II. By using a panel of 38 individual short-term cultured melanoma cell lines and corresponding ex- panded autologous tumor infiltrating lymphocytes (TILs), we showed that aberrant constitutive expres- sion of MHC class II on a subset of human melanoma is associated with accumulation of strong MHC class II restricted tumor specific CD4+ T cell responses. Of note, multifunctional characterization revealed that tumor-specific CD4+ T cell responses were dom- inated by TNF production. TNF significantly reduced CD8+ T cell activation in a IFN-γ rich environment, such as at the tumor site. On the other hand, direct CD4+ T cell responses did not significantly influence tumor cell proliferation or viability. In conclusion, we have characterized a novel poten- tial immune escape mechanism activated by mela- nomas through aberrant expression of MHC class II molecules. This attracts melanoma specific CD4+ T cells which generate a local inflammatory response dominated by TNF that in turn inhibits cytotoxic CD8+ T cell responses. 083 | NEW TARGETS & NEW LEADS
EGFRvIII TandAbs are specific and highly potent drug candidates for the treatment of solid tumors
Ellwanger K.1, Reusch U.1, Fucek I.1, Weichel M.1, Herbrecht C.1, Knackmuss S.1, Molkenthin V.2, Treder M.1
1Affimed GmbH, Heidelberg, Germany, 2AbCheck s.r.o., Plzen, Czech Republic
To harness the cytotoxic capacity of immune effector FFPE samples using a high affinity EGFRvIII-binding cells for the treatment of several types of solid tumors, bivalent Diabody. This provides an opportunity to we developed tetravalent, bifunctional antibodies develop cytotoxic antibodies that solely target cancer, that recognize EGFRvIII, the deletion variant III of sparing normal tissues. EGFRvIII/CD3 TandAbs with EGFR. Their second functionality binds with high high affinity for EGFRvIII were most potent in killing affinity to CD3 or CD16A, thereby directing T-cells assays, displaying cytotoxicity towards EGFRvIII- or NK-cells to eliminate EGFRvIII+ cancer cells. expressing F98 glioma, CHO or human DKMG cells
Using phage display, we identified scFv antibodies with EC50 in the range of 1 pM - 10 pM. No cytotoxic- that selectively bind to EGFRvIII but not to the native ity was observed on EGFR+ cells or EGFRvIII-negative form of EGFR. These highly EGFRvIII-specific scFv cells demonstrating the high selectivity of EGFRvIII/ antibodies were substantially improved, employing CD3 TandAbs for the tumor-specific EGFRvIII. High
affinity maturation techniques achieving KDs in the affinity binding to CD3 was necessary for efficacious 100 pM range or lower and were used to construct T cell recruitment as shown by the correlation of a set of bispecific EGFRvIII-targeting TandAbs with CD3-binding and cytotoxic potency of EGFRvIII/CD3 a broad range of binding and cytotoxic properties. TandAbs. Importantly, in the absence of EGFRvIII+ Mono- and bivalent binding constants, specificity for target cells in vitro, TandAbs did not elicit T cell ac- both EGFRvIII and CD3 or CD16A, cytotoxic activity, tivation, as demonstrated by their lack of prolifera- and target-dependent effector-cell activation were tion: this specificity contributed to a good preclini- characterized in a panel of in vitro assays. cal safety profile. Biophysical and pharmacological EGFRvIII-targeting TandAbs exhibited exquisite characterization of several candidates is currently specificity towards the EGFRvIII antigen in Western ongoing, whereby a first non-optimized EGFRvIII/ Blot, SPR, ELISA, and FACS assays of EGFRvIII+ cells. CD3 TandAb demonstrated a robust dose-dependent No binding was observed to recombinant EGFR growth retardation in a proof-of-concept EGFRvIII+ antigen or to EGFR-expressing cells. Apparent af- subcutaneous xenograft tumor model. finities to EGFRvIII in the TandAb format were up to In summary, EGFRvIII/CD3 and EGFRvIII/CD16A 25fold improved relative to the monovalently binding TandAbs are specific and highly potent drug candi- + scFvs, and achieved a KD of 11 pM for the best binding dates for the treatment of EGFRvIII malignancies. EGFRvIII/CD3 TandAb. The expression of EGFRvIII on various solid tumor types, and its absence from healthy tissues was sug- gested by IHC and is examined in more detail on 084 | NEW TARGETS & NEW LEADS
TrasGEX™, a glyco-optimized anti-HER2 antibody - results from phase I/IIa clinical trials
Goletz S.1, Dietrich B.1, Danielczyk A.1, Habel B.1, Flechner A.1, Baumeister H.1
1Glycotope GmbH, Berlin, Germany
ORAL ALK SHORT T 2015
Background: The human epidermal growth factor (IRR) were the most frequently observed drug-relat- receptor 2 (HER2) is a validated target in breast ed AEs (51.4%) all but two of grade 1 or 2. Premedica- cancer and gastric cancer, but also overexpressed tion with paracetamol and steroids reduced the fre- in a variety of other cancers. TrasGEX is a glycoop- quency and intensity of IRRs. The pharmacokinetic timized version of the anti-HER2 antibody trastu- properties were dose-dependent with a maximal zumab. TrasGEX was engineered using the Glyco- half-life of 263 h ± 99 h at 720 mg. One patient with Express™ technology which offers the production of HER2+++ salivary duct tumor developed a CR, two fully human glycosylated biotherapeutics thereby HER2+++ patients reached strong PR (breast: 240 avoiding immunogenicity in patients due to non-hu- mg, FcɣRIIIa allotype: FF, prior trastuzumab non-re- man glycan structures. In addition, TrasGEX is gly- sponder; colon: 480 mg, FV allotype) and 12 (32.4%) cooptimized with respect to manifold improvement showed SD (HER2+ = 4; HER2++ = 3; HER2+++ of anticancer activity, optimization of bioavailability = 5), of which 40% had been exposed to trastuzum- and broadening of the patient and indication cover- ab at earlier therapies. age. In vitro studies showed an approximate 10 to Conclusions: TrasGEX was shown to be safe and 140 fold improvement of ADCC-mediated anti-tumor well tolerated. The pharmacokinetic analysis dem- activity for TrasGEX compared to the non-glycoopti- onstrated that TrasGEX had a very long half life sup- mized trastuzumab depending on the level of HER2 porting a q3w infusion scheme. Evidence of activity expression and of the FcɣRIIIa allotype. was seen in 40.6% of patients, including one CR and Methods: This is a phase I/IIa, dose-escalation trial 2 PRs in patients with progressive disease at study of TrasGEX 12 to 720 mg IV flat-dose q3w in patients entry. Strong responses and clinical benefit was seen progressive with advanced or metastatic cancer in patients who had no benefit or were even progres- for whom no effective standard treatment is avail- sive under prior therapy with the non-glycootimized able and ErbB2 (HER2) positivity (at least 1+). The trastuzumab. In many cases these patients carried primary objective of this first-in-human study was to the FcɣRIIIa F allotype and received low dosages of determine the optimal dose and regimen of TrasGEX the glycooptimized antibody. Clinical benefit was ob- in the study population. served in both, typical and non-typical indications Results: A total of 37 patients were treated with up for trastuzumab. All these facts demonstrate the to 720 mg TrasGEX IV flat-dose q3w in 5 cohorts of advantages of glycooptimization for cancer therapy 3 to 6 patients each and an extension group of 16 using the GlycoExpress™ technology. patients who received 720 mg. No DLT was observed, the MTD was not reached. Infusion-related reactions 085 | NEW TARGETS & NEW LEADS
Tadalafil in Melanoma (TaMe): a phase 1 trial in patients with metastatic melanoma (AJCC stage IV or III unresectable) with the PDE-inhibitor Tadalafil
Hassel J.C.1, Jiang H.2, Halama N.3, Utikal J.2, Enk A.1, Umansky V.2
1University Hospital Heidelberg, Department of Dermatology and National Center for Tumor Diseases, Heidelberg, Germany, 2German Cancer Research Center (DKFZ), Heidelberg and University Hospital Mannheim, Skin Cancer Unit, Department of Dermatology Venereology and Allergology, Mannheim, Germany, 3University Hospital Heidelberg, Department of Medical Oncology, National Center for Tumor Diseases, Heidelberg, Germany
Till the approval of the immune checkpoint blocker patients with metastasized melanoma. The target ipilimumab, which blocks CTLA-4 on activated T population was patients with unresectable stage III lymphocytes, all treatment efforts in metastatic mel- or IV melanoma, who had at least one prior systemic anoma were without great success. Immunosuppres- therapy for metastatic disease and accessible me- sive cells like regulatory T-cells (Treg) and myeloid tastases (Eudract-Nr. 2011-003273-28). In a dose de- derived suppressor cells (MDSCs) were found to escalating trial design 12 patients were treated with inhibit T cell-mediated anti-tumor responses. One tadalafil with a maximum dose of 40mg p.o. once a of the major features of MDSC-induced impairment day for 8 weeks, followed by tumor staging to evalu- of T cell functions is associated with a pronounced ate clinical efficacy. Before start of treatment and down-regulation of the T cell receptor (TCR) z chain 4 weeks later a biopsy was taken to investigate the expression, which plays a key role in coupling the tissue for infiltrating lymphocytes by FACS analysis. TCR-mediated antigen recognition to diverse signal In addition, T cell activation was measured by meas- transduction pathways. Accumulating reports de- uring the TCR z chain expression. Clinically 9 of 12 scribe the relationship between the number of patients revealed a progressive disease. 2 patients MDSCs and the survival rate of tumor-bearing hosts. were stable and continued on tadalafil treatment, one Thus, depletion of the cells or abrogation of their of them being stable on treatment for 5 months now. suppressive function may be a promising tool for This patient with extensive intransit metastases had therapy. MDSCs suppress the anti-tumor immune re- shown progressive disease under ipilimumab treat- sponse in the tumor microenvironment by different ment before. Translational investigations showed mechanisms, involving cyclooxygenase (COX)-2 and uniformly an upregulation of the TCR z chain on T phosphodiesterase (PDE)-5. Recently, PDE-5 inhibi- lymphocytes. Measurement of infiltrating T lympho- tors sildenafil (Viagra®) and tadalafil (Cialis®), which cytes by FACS analysis depended much on the size of are widely used for the treatment of erectile dysfunc- the metastases and was difficult to interpret. Hence, tion, pulmonary hypertension and cardiac hypertro- computer-assisted immunohistochemical analysis of phy, have been shown to exert anti-tumor effects in TILs will be performed. various transplantation mouse models by abrogating MDSC immunosuppressive functions. In a ret trans- genic mouse model, an administration of sildenafil with drinking water led to a significant increase in overall survival of tumor-bearing mice. This encour- aged us to conduct a phase 1 trial with tadalafil in 086 | NEW TARGETS & NEW LEADS
Zoledronic acid/interleukin-2-expanded γδ T cells augment anti-tumor activity of IMAB362
Heinz C.1, Utsch M.1, Posevitz V.1, Attig S.2, Stadler C.3, Sahin U.2,3, Türeci Ö.1
1Ganymed Pharmaceuticals AG, Mainz, Germany, 2TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany, 3BioNTech, Mainz, Germany
IMAB362 is the first therapeutic monoclonal anti- γδ T cells was higher compared to control (IL-2 alone) body specifically targeting the tight junction mole- with a mean maximum lysis of 77±20% vs. 67±23%
cule CLDN18.2. Its anti-tumor activity is mediated via and a mean IMAB362 EC50 of 1.6±1.3 µg/mL vs. 3.6±5.5 e.g. antibody-dependent cellular cytotoxicity (ADCC) µg/mL, respectively. exerted by Fcγ receptor IIIA (CD16) expressing immune To study the in vivo anti-tumor effect of these cells, effector cells such as natural killer and γδ T cells. In- NOD/SCID/γc-/- mice lacking B, T, and NK cells were i.v. creasing the anti-tumor activity of an antibody drug by injected with PBMC containing in vitro expanded γδ T improving its immunomodulatory potential emerges as cells and concurrently s.c. engrafted with CLDN18.2+ promising new concept. The immune modulators zole- tumors cells. All mice received 2.5 µg ZA (i.v. weekly) dronic acid (ZA) and interleukin-2 (IL-2) lead to expan- and 5000 IU IL-2 (alternating i.v./i.p. semi-weekly) to sion and activation of CD16+ γδ T cells. The aim of this maintain and enrich the γδ-T-cell population in vivo. study was, therefore, to investigate the effect of ZA/ Treatment with 200 µg/mL IMAB362 (alternating IL-2 on phenotype and IMAB362-mediated anti-tumor i.v./i.p., semi-weekly) was initiated upon xenograft es- activity of γδ T cell subsets. tablishment. Treatment with MAB362 plus γδ T-cell-en- Treatment with 1 µM ZA and 300 U/mL IL-2 for 14 days riched PBMC abrogated tumor growth in 6/9 mice, increased the frequency of γδ T cells within lympho- whereas γδ T-cell-enriched PBMC alone (tumor growth cytes (CD3+Vγ9+Vδ2+) from 2.1±2.4% to 26.9±10.1% in in 9/9 mice) or IMAB362 alone (5/6 mice) had limited in vitro cultures of human peripheral blood mononu- effect on tumor growth. clear cells (PBMC; data are mean±SD, n=10 donors). In conclusion, ZA/IL-2 activated and expanded immune The frequency of CD16+ γδ T cells within lympho- effector cells are able to augment IMAB362’s anti-tumor cytes increased from 0.5±0.9% to 9.3±7.2% (n=10). activity. These data support the concept of combining The developing γδ T cells were highly differentiated immunomodulation with antibody-based therapeutics, and 96.8±3.3% thereof represent effector memory T such as IMAB362 and provide a preclinical rationale for cells (CD45RA-CCR7-). The frequency of CD57+ γδ T its clinical investigation. Currently, exploratory clinical cells within lymphocytes increased from 0.9±1.6% evaluation of IMAB362 in combination with ZA/IL-2 in to 4.7±4.7%, associated with cytolytic potential. patients with advanced gastroesophageal cancer in the To investigate the effect on IMAB362-mediated ADCC, Phase I PILOT trial is ongoing γδ T cells from ZA/IL-2-enriched PBMC cultures were added to CLDN18.2+ target cells (E:T 40:1) and incubat- ed with increasing concentrations of IMAB362 for 24 h (n=21 donors). The ADCC activity of ZA/IL-2-expanded (ClinicalTrials.gov ID: NCT01671774). 087 | NEW TARGETS & NEW LEADS
Serum HMGB1 seems as a suitable predictive and prognostic biomarker in patients treated with adenoviral oncolytic immunotherapy
Liikanen I.1, Koski A.1, Merisalo-Soikkeli M.1, Hemminki O.1,2, Oksanen M.1, Kairemo K.3, Joensuu T.3, Kanerva A.1,2, Hemminki A.1,4
1University of Helsinki, Helsinki, Finland, 2HUCH, Helsinki, Finland, 3Docrates Cancer Center, Helsinki, Finland, 4TILT Biotherapeutics Ltd, Helsinki, Finland
Effective immunotherapeutics harbor the potential marker for oncolytic immunotherapy with adeno- for toxicity and are expensive to use, thus biomark- viruses. Prospective clinical studies are needed to ers are urgently needed for identification of cancer confirm the results. patients who respond to treatment. 202 cancer patients were treated in this clinical-ep- idemiological study with oncolytic adenoviruses. The value of serum high-mobility group box 1 (HMGB1) protein as a biomarker was addressed. As primary endpoints overall survival and imaging responses were studied and adjusted for confound- ing factors in two multivariate analyses (Cox and logistic regression). Mechanistic studies included assessment of virus replication by quantitative PCR, circulating tumor-specific T-cells by ELISPOT and inflammatory cytokines by cytometric bead array. Patients with low HMGB1 baseline levels (below median concentration) showed significantly im- proved survival (P = 0.008, Log-Rank test) and ra- diological disease control rate (49.2% versus 30.0%, P = 0.038, χ2 test) as compared to high-baseline patients. In multivariate analyses, the low HMGB1 baseline status was a strong prognostic (HR 0.638, 95% CI 0.462-0.881) and the best predictive factor for disease control (OR 2.618, 95% CI 1.004-6.827). Indicative of an immune-mediated mechanism, antitumor T-cell activity in blood and response to immunogenic-transgene coding viruses associ- ated with improved outcome only in HMGB1-low patients. Our results suggest that serum HMGB1 baseline is a useful prognostic and predictive bio- 088 | NEW TARGETS & NEW LEADS
A novel synthetic Toll-like-receptor 7 agonist induces lymphoma rejection in an IFNα-dependent manner
Jacobi S.1, Wiedemann G.1, Chaloupka M.1, Hamm S.2, Strobl S.2, Baumgartner R.3, Endres S.1, Kobold S.1
1Division of Clinical Pharmacology, Department of Medicine IV, Ludwig-Maximilians-Universität München, Munich, Germany, 24SC Discovery GmbH, Martinsried, Germany, 34SC AG, Martinsried, Germany
Toll-like-receptor 7 (TLR7) is located in the endo- IFNαR-/- mice suggesting a TLR7-dependent mode some and is physiologically activated upon binding of action, mainly promoted by Interferon-α. of ssRNA. TLR7 activation results in the release The TLR7-agonist SC1 is a TLR7-specific immune of proinflammatory cytokines and culminates in activator and is effective in the treatment of an NK the activation of cells of the innate and adaptive cell-sensitive murine lymphoma model. SC1 may be immune system. Such rapid immune activation may a promising candidate for use in anti-tumoral im- be used for anti-tumoral immunotherapy. Current- munotherapy. ly, clinical application of synthetic TLR7-agonists is limited to topical treatment of skin tumors. Because of dose limiting toxicities, systemic application of TLR7-agonists is currently not feasible and novel compounds are required to exploit their full poten- tial. All experiments were performed using a novel syn- thetic small molecule predicted to be a TLR7-ago- nist (SC1). We found SC1 to induce IL-6, IL-12p70, MCP-1, MIP-1β, Interferon-α and IP-10 in vivo. Cyto- kine induction by SC1 was TLR7-specific as it was not observed in TLR7-/- splenocytes. In vivo, treat- ment of mice with SC1 specifically enhanced killing
of β2-microglobulin deficient target cells by NK cells
as opposed to β2-microglobulin competent target cells (96,8% vs 0 % specific lysis). Lysis is entirely TLR7-dependent, as shown in TLR7-/- mice. Finally, treatment of RMA-S tumor-bearing mice with SC1 significantly reduced tumor growth as compared to vehicle-treated mice (mean tumor size [mm2] at day 32: 0 vs. 218, respectively, n = 6) and result- ed in prolonged tumor-free survival of SC1-treated mice (median survival was not reached vs. 33 days). Anti-tumoral efficacy was abrogated in TLR7-/- and 089 | NEW TARGETS & NEW LEADS
A screening approach to identify a lead bispecific antibody targeting CLDN6-positive tumor cells
Jendretzki A.1, Stadler C.R.1, Celik L.1, Bähr-Mahmud H.1, Fiedler M.1, Fregin A.2, Kreuzberg M.2, Le Gall F.2, Türeci Ö.2, Sahin U.1
1BioNTech AG, Mainz, Germany, 2Ganymed Phamaceuticals AG, Mainz, Germany
The aim of this work was to construct a bispecific T-cell engager molecule which recruits and directly activates cytotoxic T cells in the proximity of CLDN6- positive tumor cells. The expression of this antigen is highly tumor-specific, except for the expression in human placenta and during embryogenesis. CLDN6 positive tumor entities comprise ovarian, bladder, lung, gastric, pancreatic, breast, hepatic and other solid cancers. The chosen construct format is the well
described bi-(scFv)2 format whose pioneer compound blinatumomab was recently approved by the FDA. In order to receive antibody derivatives which are highly monomeric and not prone to aggregation over a wide range of concentrations, a screening approach was performed. Besides shuffling the positions of the various variable regions, we also modified the con- necting linker length and introduced cysteine resi- dues at strategic positions to allow for the formation of stabilizing intra-domain disulfide bonds. A total of 28 variants were screened regarding producibil- ity, the presence of high molecular weight species (SDS-PAGE and SE-HPLC) and the biological activity (cytotoxicity assay). After this first screening round, four pre-selected candidates were produced in larger scale from stable cell pools for a more thorough analysis. Finally, we selected a single candidate that does not show concentration-dependent aggregation and from which the pure monomeric species could be enriched, without losing the biological activity. 090 | NEW TARGETS & NEW LEADS
Potential anticancer activity of new β-Carboline derivative on K562 human leukemia cell line
Kamaruzaman N.A.1, Mohideen M.1, Lai C.S.1, Mordi M.N.1, Mansor S.M.1
1Centre for Drug Research, Universiti Sains Malaysia, Penang, Malaysia
The role of β-carboline in anticancer has recently further evaluated for potential anticancer treatment. been under discussion. Current anticancer thera- More studies on the mechanism of action of CDR 007 peutics, though are in great numbers, are still in- should be done to fully understand its activity. sufficient due to low efficacy, low selectivity and severe side effects. Thus this study investigated po- tential anticancer activity of β-carboline derivatives in vitro. β-carboline derivatives were synthesized in the laboratory using various substitutions. These derivatives were tested using 3-(4,5-Dimethylthi- azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for potential anticancer activity and selectivity index against four cancer (human leukemia, colon, cervical and liver) and two non-cancerous (human foreskin and mouse embryo fibroblasts) cell lines re- spectively. Mode of death of selected lead compound was evaluated using acridine orange and ethidium bromide (AO/EB) fluorescence staining and caspases
3/7 assay. Based on the IC50 values of the primary and secondary screenings, one lead compound, CDR 007 was selected for its best and most selective inhibitory activity against K562 human leukemia cell line. The derivative was found to act in a concentration-depen- dent manner, with the potential to reach maximal (100%) inhibitory concentration at 9.1 µM, and its inhibitory activity was quick-onset. In addition, CDR 007 caused cell death as both cytostatic and cytocid- al agents, however with a more profound effect as a cytostatic agent. Evaluation of the mode of cell death promoted by CDR 007 has found that the derivative acted through caspases-independent apoptosis. Data from this study supported that CDR 007 should be 091 | NEW TARGETS & NEW LEADS
Molecular landscape of prostate cancer: Implications for current clinical trials
Ikeda S.1, Kurzrock R.1, Khemlina G.2
1UCSD, Hematology-Oncology, La Jolla, United States, 2UCSD, Geriatrics, La Jolla, United States
Castration-resistant prostate cancer (CRPC) is a lethal disease, and improvement with androgen- deprivation therapy has plateaued. Next-generation sequencing studies have led to significant advances in our understanding of genomic alterations in pros- tate cancer. The most common genomic aberrations in this malignancy are the TMPRSS2-ETS transcrip- tion factor fusion, and mutations in TP53, AR, RB1 and PTEN/PIK3CA. In the era of tumor profiling, targeting molecular alterations may provide an op- portunity for new therapeutic approaches. Although there are promising new agents to prosecute a variety of genomic signal abnormalities, biomarker-matched therapy (other than for androgens) were utilized in only 2.0 % of clinical trials (Sept 2011 through Sep- tember 2014; clinicaltrials.gov) for prostate cancer. Although, there are well-known genomic alterna- tions in prostate cancer, molecular-targeted therapy for this malignancy has not been extensively studied in the clinic. We reviewed the genomic landscape of prostate cancer and implications for clinical research in this disease. In conclusion, enhanced efforts to define subsets of patients with prostate cancer based on their molecu- lar anomalies, and match them with cognate thera- pies warrant investigation. 092 | NEW TARGETS & NEW LEADS
Immunopeptidome analysis identifies non-mutant epitopes as targets of anti-leukemia T cell responses
Kowalewski D.J.1, Schuster H.1, Backert L.1,2, Berlin C.3, Kahn S.1, Kohlbacher O.2, Kanz L.3, Salih H.R.3,4, Rammensee H.-G.1,5, Stevanovic S.1,5, Stickel J.S.3
1University of Tübingen / Interfaculty Institute for Cell Biology, Department of Immunology, Tübingen, Germany, 2University of Tübingen, Department of Computer Science, Tübingen, Germany, 3University of Tübingen, Department of Hematology and Oncology, Tübingen, Germany, 4Clinical Collaboration Unit, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany, 5German Cancer Consortium (DKTK), DKFZ partner site Tübingen, Tübingen, Germany
Effective antigen-specific cancer immunotherapy re- sively in CLL patients. These immune responses were quires exact knowledge of tumor-associated epitopes further verified to be strictly leukemia-directed and which can act as rejection antigens. While the mediated by functional CLL patient derived T cells, current paradigm views mutation-derived neo-anti- thus indicating tumor-dependent priming of T cells gens as the most promising targets, we have recently specific for non-mutated tumor epitopes in vivo in CLL demonstrated that leukemia-specific T cell responses patients. A direct correlation between the frequency that are associated with survival benefit in CLL pa- of presentation of these epitopes in CLL patient li- tients target a panel of non-mutated tumor-associated gandomes and immune recognition by CLL patient T antigens. Based on cohorts of 30 CLL patients and cells was observed. Strikingly, retrospective survival 30 healthy individuals we identified a panel of 49 analysis in a cohort of 45 CLL patients - dichotomized CLL-associated antigens represented exclusively on according to their number of CLL antigen specific malignant cells in >20% of analyzed patients. To T cell responses - revealed significantly improved ensure broad clinical applicability, these novel an- overall survival for the patient group recognizing tigens were validated to be broadly and frequently more than one antigen. Together, this suggests that presented across different stages and mutational sub- disease control in CLL might be mediated - at least types of CLL and found to be robustly represented in part- by spontaneous T cell responses targeting in HLA ligandomes of patients undergoing standard non-mutated self-antigens. chemo-immunotherapy. Notably, functional annota- tion clustering and gene expression analysis of these novel antigens did not identify any comprehensive unifying characteristics which might have enabled the development of computational approaches for the prediction of such TAAs. This indicates the unique character of the HLA ligandome and underscores the importance of defining T cell antigens by direct HLA ligandome profiling. Surprisingly, for 14/15 of these novel antigens, functional characterization revealed spontaneous, pre-existing immune responses exclu- 093 | NEW TARGETS & NEW LEADS
Enabling variant detection in single cell transcriptomes using half cell sequencing
Kunz D.1, de Graaf J.2, Albrecht C.2, Löwer M.2, Sorn P.2, Tadmor A.2, Sahin U.1,2
1University Medical Center Mainz, Experimental & Translational Oncology, Mainz, Germany, 2TRON – Translational Oncology at the University Medical Center of Johannes Gutenberg University, Mainz, Germany
Next Generation Sequencing (NGS) is changing the scription in single cells. Using biological replicates way we understand cancer by providing deep in- for mutation validation in CTCs assumes a model sights into the tumors genome, development and that all cells have the same mutations. This might be progression. We can, among other things, monitor true for mutations which occurred early. Mutations expression of shared tumor antigens and identify im- gained later are missed in this process. However to munogenic mutations for individualized therapeutic circumvent this we decided to make technical rep- cancer vaccines (IVACs). licates from single cells, the half-cell approach. By For applications ranging from deciphering tumor het- splitting up the first strand prior to amplification we erogeneity and evolution to circulating tumor cells keep technical errors stochastic distributed. Varia- (CTCs), we would like to profile gene expression and tions which are detected in both cells are unlikely to identify mutations in single cells. However, single be false positives. This might be crucial for individu- cell analysis is complicated by the extremely small alized medication based on CTC samples. amounts of nucleic acids present in single cells and Noise reduction is a key part of mutation detection. the high costs of sample profiling, particularly of Using sophisticated algorithms and filtering strate- exome profiling. gies we are able to separate false positives from true Here, we present a novel method to determine gene positives. Using the extensive knowledge gained from expression and identify mutations in single cells. The the CT26 cell line through exome-seq and RNA-Seq method is robust, reproducible, and costs less than mutations detected using half cell sequencing could existing methods. We isolate RNA from single cells; be confirmed reliably. Aside from high confidence use a modified reverse transcription with 5’ template mutations, up to 99 % of the variants found in the switching; and use tagmentation-based library prep- coding sequences of mRNA of single cells could be aration methods to enable amplification from pico- confirmed either by Bulk RNA-Seq or exome seq. gram amounts of input RNA, reduced hands-on time This shows that the half cell sequencing approach and lower costs. is capable of shedding light on the mutational land- Single cell sequencing is a challenging application scape of single cells. with a small signal to noise ratio. This leads to an un- The method will provide the analytic back-end of certainty whether variants which have been detected newly developed microfluidic device for directly are real or noise coming from reverse transcription, capturing CTCs from patient’s blood. The device is amplification and finally sequencing. A simple ap- in development in an interdisciplinary project of the proach would be to take replicates into account. Bio- CI3 cluster. logical replicates are difficult by the nature of tran- 094 | NEW TARGETS & NEW LEADS
The antitumor effect of the inactivated Sendai virus particles (Hemagglutinating virus of Japan envelope: HVJ-E) on malignant pleural mesothelioma
Lee C.1, Saito A.1, Nakajima T.2, Kaneda Y.3
1Osaka University Hospital, Osaka University, Department of Medical Innovation, Suita, Japan, 2GenomIdea Inc., Ikeda, Japan, 3Osaka University Graduate School of Medicine, Division of Gene Therapy Science, Department of Molecular Therapeutics, Suita, Japan
Hemagglutinaing virus of Japan; Sendai virus was bearing mouse: we examined the antitumor effect first discovered in Japan. It is mouse parainfluenza of HVJ-E by use of orthotopic implantation models. virus, and is not human pathogen. Hemagglutinating the survival rate of HVJ-E group was significantly virus of Japan envelope (HVJ-E) was developed from prolonged compared with control group. We exam- Sendai virus by ultraviolet irradiation. HVJ-E con- ined the mechanism of antitumor effect of HVJ-E on tains negative strand RNA inside its envelope, but it human MPM tumor-bearing mice, and examined the is not human pathogen. There are two glycoproteins, synergistic effects of HVJ-E and antitumor reagents. fusion protein and hemagglutinating neuraminidase We have a plan to do the clinical trial for MPM pa- protein on the its envelope. We found that HVJ-E tients. Therefore, HVJ-E will be a novel promising induced antitumor immunity through the activation tool for cancer treatment as immuno-stimulant and of CTL, the inhibition of regulatory T cell-mediated antitumor drug. immuno-suppression, and the infiltration of NK cells into the tumors through the maturation of dendritic cells. HVJ-E also induces apoptosis in human cancer cells through the retinoic acid inducible protein-I (RIG-I) mediated apoptosis. Therefore, HVJ-E pos- sesses the properties of both immunotherapeutic reagent and antitumor drug. Previously, we revealed that antitumor effects of HVJ-E on malignant mela- noma, and castration resistant prostate cancer, and we did the phase clinical trilas. So we examined anti- tumor effect of HVJ-E on malignant pleural mesothe- lioma by following procedures.The affinity of HVJ-E to MPM cells, and the expression of the receptors of HVJ-E on MPM cells: The same level of the affinity of HVJ-E to human MPM cells was indicated compared with prostate cancer cells as a positive control, and there were large amounts of the receptors of HVJ-E on MPM cells. The cytotoxicity of HVJ-E to each MPM cell: HVJ-E inhibited the cell growth of human MPM cells. The antitumor effect of HVJ-E to MPM tumor- 095 | NEW TARGETS & NEW LEADS
Rational design of a TGFb1 mutant useful for cancer immunotherapy
Corria Osorio J.1, Perez S.1, Garcia C.1, Carmenate T.1, Leon K.1
1Center of Molecular Immunology, Habana, Cuba
Transforming growth factor β (TGFβ) is a pleiotro- pic cytokine that affects tumor growth, metastasis, stroma, and immune response. There are three iso- forms of this cytokine: TGFβ1, TGFβ2 and TGFβ3. They interacts on the cell surface with the TβRII receptor, TβRIII receptor and several of the type I receptors that have been described (ALK5, ALK1, ALK2, ALK3); although ALK5 is the classic type I re- ceptor for these ligands. It has been proposed that the TGFβ ligands bind first to TβRIII, which then trans- fers the cytokine to the TβRII receptor on the cell membrane. TGFβ1 and TGFβ3 isoforms bind to TβRII receptor with high affinity (5-30pM). TβRI (ALK5) is then recruited and activated in a highly coopera- tive manner by the TβRII/TGFβcomplex, forming a heterotrimeric signaling complex. The ALK5 medi- ated signaling plays a pivotal role in regulating the pathogenesis of a wide variety of disorders including cancer. In this work we described the rational design of a TGFβ1 mutant that behave as an antagonist of the TGFβ signaling and has antitumor effects in vitro. The mutant was designed using several bio- informatics tools to predict the mutations that could abrogate the interaction with ALK5 without affecting the binding to the TβRII and TβRIII. The mutant was fused to a human IgG1 Fc region and was obtained using a lentiviral transduction protocol in CHO-K1 cells. To our knowledge this is the first antagonist mutant of TGFb reported in the literature. Our data provide a foundation to support the use of this TGFB1 mutant as a therapeutic agent to treat Cancer: 096 | NEW TARGETS & NEW LEADS
PD-1, PD-L1 and PD-L2 expression in human malignant pleural mesothelioma
Marcq E.1, De Waele J.1, Van Audenaerde J.1, Zwaenepoel K.2, Baas P.3, Pauwels P.2, van Meerbeeck J.P.1,4, Smits E.L.J.1,5
1University of Antwerp, Centre for Oncological Research, Antwerp, Belgium, 2Antwerp University Hospital, Department of Pathology, Antwerp, Belgium, 3Netherlands Cancer Institute, Thoracic Oncology, Amsterdam, Netherlands, 4Antwerp University Hospital, Thoracic Oncology/MOCA, Antwerp, Belgium, 5University of Antwerp, Laboratory of Experimental Hematology, Antwerp, Belgium
Background: The discovery of immune checkpoint Results: PD-1 surface expression was found on T receptors as cytotoxic T lymphocyte antigen-4 cells and not on MPM tumor cells, corresponding to (CTLA-4) and more recently programmed death-1 literature showing that PD-1 is only expressed on T (PD-1) introduced a new era in cancer immunothera- cells, B cells and macrophages. Different expression py. Immune checkpoints are responsible for control- patterns were observed regarding PD-L1 and PD-L2. ling and inactivating the immune system in order Flow cytometry showed significant basal PD-L1 and to avoid autoimmunity and prevent tissue damage. PD-L2 expression on the sarcomatoid VAMT-1 cells PD-1 is expressed primarily on activated effector T as well as low expression levels of PD-L1 on the epi- lymphocytes. Its natural ligands are programmed theloid M28 cell line. Following IFNg stimulation, death ligand-1 (PD-L1) and programmed death PD-L1 expression was induced on the mixed NKI04 ligand-2 (PD-L2). cells and upregulated on the VAMT-1 and M28 cells. Expression of PD-L1/PD-L2 on tumor cells or in Primary MPM cells showed variable expression of stroma impairs effector T lymphocyte activity within PD-L1. IHC data for PD-1 and PD-L1 expression cor- the tumor microenvironment. Trials with antibodies respond to the flow cytometry results. that block the ligand-immune checkpoint interac- Conclusions: Taken together, these data on PD-1, tion have shown promising results in several cancer PD-L1 and PD-L2 expression on human MPM cells types. Until now, they have not been investigated and T cells support further investigation of the ex- in malignant pleural mesothelioma (MPM), a very pression profile of the immune checkpoint PD-1 and aggressive tumor. We investigated PD-1, PD-L1 and its ligands in MPM patients samples. We are current- PD-L2 expression in MPM. Furthermore the effect of ly performing this using multicolor flow cytometry interferon-gamma (IFNg), an important cytokine for and IHC. immune-mediated tumor control, on their expression pattern was analyzed. Material and methods: Flow cytometry and immu- nohistochemistry (IHC) were used for the expression of PD-1, PD-L1 and PD-L2 on human primary MPM and T cells and on MPM cell lines that cover the histological spectrum of MPM, i.e. M28 (epitheloid), VAMT-1 (sarcomatoid) and NKI04 (mixed) mesothe- lioma cells. The effect of stimulation with IFNg on expression of PD-1 and its ligands was measured. 097 | NEW TARGETS & NEW LEADS
TiMi1 is a novel immune-checkpoint in solid tumors differentially regulating calcium-depending signaling in tumor-infiltrating lymphocytes
Michels T.1, Hartl C.A.1, Khandelwal N.1, Breinig M.2, Sorrentino A.1, Mäder C.1, Umansky L.1, Poschke I.3, Offringa R.3, Boutros M.2, Eisenberg G.4, Lotem M.4, Beckhove P.1
1Deutsches Krebsforschungszentrum, Translational Immunology, Heidelberg, Germany, 2Deutsches Krebsforschungszentrum, Signaling and Functional Genomics, Heidelberg, Germany, 3Deutsches Krebsforschungszentrum, Molecular Oncology of Gastrointestinal Tumors, Heidelberg, Germany, 4Hadassah Medical Organization, Sharett Institute of Oncology, Jerusalem, Israel
ORAL ALK SHORT T 2015
Over the last years cancer immunotherapy has seen affecting their viability, increased TIL activity mea- major advances in understanding the role of the sured by production of type 1-associated cytokines immune system in tumor development and treat- (e.g. IFNγ and TNF-α), reduced TC apoptosis and ment of poorly immunogenic cancers. However, increased markers associated with raised activity many patients are still not able to benefit from these and cytotoxicity (4-1BB and CD107a). We verified the advances as tumors employ a plethora of suppressive immune checkpoint function in melanoma patients mechanisms to evade immune destruction that are using an autologous set of melanoma cells and TILs. far more diverse than our current arsenal of avail- Furthermore, we validated the role of TiMi1 in vivo able immunotherapy-based treatments. In this study, using a xenograft mouse model in combination with we established and utilized a novel high throughput adoptive cell transfer. TiMi1-mediated inhibition is RNAi screening to identify new immune checkpoint based on altering the calcium-dependent signaling molecules in melanoma using antigen-specific pa- inside T cells as found by phosphoplex analysis and tient-derived tumor-infiltrating lymphocytes (TILs) next generation RNA-Sequencing. Preliminary ex- in conjunction with primary HLA-matched melano- periments suggest that TiMi1 inhibits TIL-mediated ma cells. Using this approach, we screened a siRNA anti-tumor immune responses in pancreatic (PDAC) library targeting more than 2800 surface receptors and colorectal (CRC) cancers as well. and kinases to explore novel targets for immunother- In summary, we established a novel antigen-specif- apy. The screening resulted in a hit list of 73 can- ic screening approach for immune checkpoints ex- didates that negatively regulated CTL cytotoxicity. pressed in melanoma and were able to identify TiMi1 To streamline the discovery process for large scale as a promising candidate. TiMi1 inhibits T cell re- libraries, we established a secondary screen assaying sponses in melanoma in vitro and in vivo by differen- multiple T cell activation markers, including effector tial regulation of calcium-depending signaling inside cytokines. TILs. TiMi1 might act as an immune checkpoint in One of the strongest candidates from our primary other solid tumors as well. Our novel high-through- and secondary screening is TiMi1 (name altered), a put screening offers a systematic platform to uncover cell surface receptor belonging to the class of GPCRs. the “immune-modulatome” of cancer and subse- We found that knock-down of TiMi1 resulted in in- quently discover novel targets for immunotherapy. creased TIL-mediated killing of M579-A2-luc without 098 | NEW TARGETS & NEW LEADS
Enhancing natural killer cell-mediated lysis of lymphoma cells by combining therapeutic antibodies with CD20-specific immunoligands engaging NKG2D or NKp30
Kellner C.1, Humpe A.1, Repp R.1, Klausz K.1, Derer S.1, Valerius T.1, van de Winkel J.G.J.2, Parren P.W.H.I.2,3, Gramatzki M.1, Peipp M.1
1Christian-Albrechts-University Kiel, Division of Stem Cell Transplantation and Immunotherapy, Kiel, Germany, 2Genmab, Utrecht, Netherlands, 3Dept. of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark
Antibody-dependent cell-mediated cytotoxicity H6:7D8 produced synergistic effects, no significant (ADCC) represents a major effector function of many improvements were obtained by combining the three tumor targeting therapeutic antibodies. Thus, en- agents rituximab, B7-H6:7D8 and ULBP2:7D8. En- hancing ADCC is a promising approach to further hancement of ADCC by the immunoligands was also improve antibody therapy. Here, the CD20-specific achieved when NK cells from lymphoma or leukemia immunoligands ULBP2:7D8 and B7-H6:7D8, which patients were analyzed as effector cells. ULBP2:7D8 engage the stimulatory NK cell receptors natural in particular increased lysis not only of allogeneic killer group 2 member D (NKG2D) and NKp30, re- but also of autologous tumor cells. spectively, were compared for their abilities to boost In summary, co-targeting of NKG2D was more ef- ADCC in an attempt to design an effective antibody fective in promoting NK cell-mediated ADCC than combination strategy. The immunoligands are de- co-ligation of NKp30 and may represent a promising signed as single chain molecules, with a single chain approach to further enhance the efficacy of thera- fragment variable (scFv) of the CD20 antibody 7D8 peutic antibodies. Based on these results we propose fused to UL16-binding protein (ULBP) 2 or B7 homo- a ‘dual-dual-targeting’ concept by co-targeting of logue 6 (B7-H6), which are ligands of the activating two surface antigens on tumor cells and concomi- NK cell receptors NKG2D and NKp30, respectively. tant engagement of two different activating NK cell By binding to lymphoma cells the immunoligands receptors. designated as ULBP2:7D8 and B7-H6:7D8 mimicked an induced self phenotype and thereby triggered NK cells to kill lymphoma and leukemia cells. Both im- munoligands augmented ADCC by NK cells synergis- tically when combined with the lymphoma-directed antibodies rituximab or daratumumab recognizing CD20 and CD38, respectively. Antibody combina- tions with ULBP2:7D8 resulted in higher cytotoxicity (up to 10-fold lower EC50-values) in comparison to combinations with B7-H6:7D8, which in individual experiments failed to boost ADCC. Thus, NK cells were triggered more efficiently when NKG2D rather than NKp30 was co-ligated together with FcγRII- IA. Although a combination of ULBP2:7D8 and B7- 099 | NEW TARGETS & NEW LEADS
Phosphopeptides as novel tumour antigens in colorectal cancer
Penny S.A.1, Abelin J.G.2, Saeed A.Z.1, Malaker S.A.2, Trantham P.D.2, Shabanowitz J.2, Ward S.T.1, Hunt D.F.2,3, Cobbold M.1
1University of Birmingham, School of Immunity and Infection, Birmingham, United Kingdom, 2University of Virginia, Department of Chemistry, Charlottesville, United States, 3University of Virginia, Departments of Biochemistry and Molecular Genetics and Pathology, Charlottesville, United States
Background: There is a pressing need for novel im- Results: We have identified 198 tumour-associated munotherapeutic targets in colorectal cancer (CRC). MHC class-I phosphopeptides from CRC, with dif- Memory CD8 T cell infiltration is now well established ferent HLA-restrictions. There were, on average, 3.1 as a key prognostic indicator in CRC, and it is known times more different phosphopeptides identified on that these tumour infiltrating lymphocytes (TILs) cancer than healthy tissues, at 6.9-fold higher levels. are specifically targeting and killing tumour cells. 24% of these can be attributed to signalling events However, the antigens that these TILs target have not in well-defined cancer pathways and are therefore previously been determined. This has limited the use markers of malignancy. Through analysis of TIL’s cy- of immunotherapies in CRC, despite their efficacy in tokine responses to these phosphopeptides, we have other cancer types. Recently, phosphopeptides have established that they are playing a key role in tu- emerged as strong candidates for tumour-specific an- mour-resident immunity. There were multifunctional tigens, since dysregulation of signalling in cancers TILs present in primary and metastatic tumours that leads to aberrant protein phosphorylation. Here, we recognised and killed in response to these phospho- identify CRC-associated phosphopeptides and assess peptides. Up to 0.7% of expanded TILs targeted each the tumour-resident immunity against these novel phosphopeptide, comparable with responses seen to posttranslationally modified tumour neoantigens. viral epitopes. Immunity to these tumour-associated Methods: We compared tumour and healthy tissue phosphopeptides represents a biological strategy for from CRC patients, to identify tumour-specific MHC distinguishing tumour from healthy tissue. class-I associated phosphopeptides. The tissues were Furthermore, we have shown that healthy donors lysed, the MHC class-I complexes affinity purified, have pre-existing, memory T cell responses to many and the bound peptides eluted. Phosphopeptides (58%) of these CRC-associated phosphopeptides. were enriched using immobilised metal affinity chro- These phosphopeptide-specific T cells are readily matography, and characterised using mass spectrom- expanded ex vivo and kill CRC cell lines. Thus, MHC etry. TILs, from the same tumours, were extracted class-I associated phosphopeptides are ideal im- and expanded, and their responses to the phospho- munotherapeutic targets, as immunity must spare peptides assessed using multiplexed intracellular cy- healthy tissue. tokine staining. Cytolytic activity was observed by Conclusion: The identification of this novel class of staining for surface mobilisation of CD107a. Healthy MHC class-I antigens in CRC offers new hope for the donor responses were quantified using interferon-γ future of immunotherapy in this malignancy. ELISpot and functionality assessed using a europium release killing assay. 100 | NEW TARGETS & NEW LEADS
Posttranslationally modified peptides as neoantigens in leukaemia
Penny S.A.1, Malaker S.A.2, Steadman L.G.1, Trantham P.D.2, Bai D.L.2, Shabanowitz J.2, Cobbold M.1, Hunt D.F.2,3
1University of Birmingham, School of Immunity and Infection, Birmingham, United Kingdom, 2University of Virginia, Department of Chemistry, Charlottesville, United States, 3University of Virginia, Departments of Biochemistry and Molecular Genetics and Pathology, Charlottesville, United States
Background: Immunotherapies are increasingly 56% (5/9) of the O-GlcNAcylated neoantigens tested being utilised in the fight against cancers. However, were immunogenic, with 66% (4/6) of healthy donors advances in immunotherapeutics have been ham- having multifunctional memory CD8 T cell respons- pered by a lack of understanding of how the immune es to them. Cells targeting these neoantigens were response specifically targets cancerous cells. Can- also shown to degranulate. This multifuntionality cerous cells differ from their healthy equivalents, and degranulation in response to antigen indicated producing cancer-specific antigens due to muta- that O-GlcNAc-specific T cells may kill. Indeed, an tions, over- or ectopically expressed proteins, and O-GlcNAc-specific T cell line was grown and these also modified proteins. Cancer-specific antigens cells specifically killed autologous cells pulsed with may derive from the posttranslational modifications the modified peptide, but not the equivalent unmod- (PTMs) associated with the aberrant signalling in ified peptide (p=0.015). We are also investigating cancer. O-linked β-N-acetylglucosamine (O-GlcNAc) CLL patients’ responses to these immunogenic O-Glc- is a PTM that modulates cellular functions through NAcylated neoantigens. extensive cross-talk with the signalling cascades also Conclusions: O-GlcNAcylated neoantigens derive regulated by phosphorylation. Thus, O-GlcNAcylated from aberrations in key cancer pathways, are shared peptides may represent cancer-specific neoantigens. across patients and are immunogenic. CD8 T cells Methods: We eluted MHC class-I associated peptides targeting these O-GlcNAcylated neoantigens specifi- from leukaemic cells to identify O-GlcNAcylated anti- cally recognise and kill only the PTM antigen. There- gens, using enrichment coupled with high-resolution fore, these O-GlcNAcylated neoantigens provide mass spectrometry. Healthy donor immune respons- logical targets for cancer immunotherapy. es were assessed using IFNγ ELISpot and multiplexed intracellular cytokine staining. Functionality was as- sessed using a europium-release killing assay. Results: We have identified 34 MHC class I associat- ed O-GlcNAc neoantigens from primary leukaemia samples, the first tumour antigens containing this PTM. A subset of these neoantigens is linked to key cancer pathways, including the mitogen activated protein kinase (MAPK) and retinoblastoma (RB1) pathways, and these peptides were shared across all of the patient samples tested. 101 | NEW TARGETS & NEW LEADS
RIG-I based immunotherapy of hepatocellular carcinoma (HCC)
Posselt L.1, Lazic I.1, Boehmer D.1, Hoffmann S.1, Duewell P.1, Koenig L.1, Endres S.1, Rothenfusser S.1, Schnurr M.1
1Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Abteilung für Klinische Pharmakologie, München, Germany
Background: Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, leads to a median survival of less than one year, necessitating the search for new therapies. The cytosolic helicase RIG-I senses viral 5’-triphosphate-RNA (ppp-RNA) and induces type I IFN-driven immune responses that combat both viral infections as well as tumors. RIG-I may thus serve as a new target for immunotherapy of HCC. Methods: RIG-I expression in HCC was analyzed by IHC and WB. RIG-I functional activity was assessed by transfection of HCC cells with ppp-RNA. Thera- peutic efficacy was evaluated in vitro and in vivo in an orthotopic HCC mouse model. Seven days after tumor induction ppp-RNA was injected i.v. and survival was monitored. Tumor tissue was analyzed via IHC and qRT-PCR. Results: Treatment of human and murine HCC cells with ppp-RNA induced phosphorylation of IRF-3, pro- duction of IFN-b and CXCL10, indicative of intact RIG-I signaling. In addition, RIG-I activation led to profound tumor cell death in vitro and in vivo. Treatment of mice with orthotopic HCC with ppp-RNA reduced tumor size and significantly prolonged survival. CD8+ T cells as well as NK cells mediated tumor rejection. Conclusion: Our data demonstrate that RIG-I is func- tional in HCC. RIG-I activation leads to systemic and intratumoral type I IFN production accompanied by massive tumor cell death in vitro and in vivo. RIG-I is thus a promising novel target for HCC therapy deserv- ing further evaluation. 102 | NEW TARGETS & NEW LEADS
Peptide libraries for comprehensive coverage of the tumor mutanome in immune monitoring and immunotherapy
Reimer U.1, Eckey M.1, von Hoegen P.1, Knaute T.1, Wenschuh H.1
1JPT Peptide Technologies, Berlin, Germany
Many cancers are associated with functional muta- many challenges. Exemplary, for the ENV protein an tions which can be readily identified using new DNA increase in sequence coverage from 10 % to 34 % sequencing methods. Sequence information is used could be achieved. for the generation of personalized cancer vaccines In order to keep the number of individual peptides with promising clinical benefit (1). Intelligent ways to within peptide pools of tumor antigens at a control- select the relevant, tumor-specific protein-coding mu- lable level a complex filtering of sequence data has to tations are applied to filter the large number of iden- be applied to enrich the library with peptides carry- tified mutations to a manageable number. Besides, ing different tumor-specific and ideally functionally there is an increasing data pool of both germline and relevant mutations. somatic mutations, for the latter frequently even with Our aim is to generate peptide libraries for specific attached information on the tissue and histology. antigens which represent a generic cancer mutanome Although the use of a patient-specific antigen se- applicable to a large number of individual patients quence represents a very promising basis for treat- based on available databases as the catalogue of ment and immune monitoring its identification and somatic mutations in cancer (COSMIC) and epitope translation to corresponding reagents is still slow, information from the immune epitope database laborious and expensive. (IEDB) as well as prediction algorithms. Intelligently designed peptide libraries that cover A library type can contain the full length sequence sequence variability based on known antigen muta- of the antigen enriched by peptides representing tions may serve as easily accessible tools with broad overlapping scans for relevant mutations. Alterna- exertion across patient cohorts in cases where the tively, known or predicted epitopes can be selected individual sequence is not readily available. and the mutated epitopes added. However, mutations Pools of antigen spanning peptides are increasing- can lead to the creation of new epitopes and mutated ly used for T-cell stimulation in T-cell assays and epitopes can escape from MHC-binding. Epitope pre- even for immunotherapeutic treatment. Underly- diction can help to account for these effects. ing peptide libraries are well suited for presenting We have built a pipeline for the efficient generation of sequence diversity. However, the currently used li- such libraries which will be presented here. braries consist of overlapping peptides of wild-type antigen sequences. We recently introduced a concept for diversity enhancement to the design of peptide pools covering immune relevant HIV antigens for 1. Boisguérin et al., (2014) Br. J. Cancer. 111, 1469-75. which diversity between individual viruses poses 103 | NEW TARGETS & NEW LEADS
Stabilin-1 is expressed on tumour-associated macrophages in breast cancer and supports tumour growth in animal model of breast adenocarcinoma by clearance of SPARC
Yin S.1, Riabov V.2
1Anhui Medical University, Hefei, China, 2Heidelberg University, Mannheim, Germany
Tumour-associated macrophages (TAM) are crucial of two cohorts of female patients with breast car- participants in malignant progression. Acquisition cinoma of different stages demonstrated that sta- of M2 (alternatively activated) phenotype by TAM bilin-1 is expressed on significant part of tumour during tumour progression enhances the immuno- associated macrophages. Three types of TAM were suppressive and tumour-supportive properties of identified by co-staining with anti-stabilin-1 RS1 TAM resulting in tumour invasion, metastasis and antibody and anti-CD68 antibody: CD68+stabilin-1-, angiogenesis. Previously we have found that stabi- CD68+stabilin-1+ and CD68-stabilin-1+. The highest lin-1, a multifunctional scavenger/sorting receptor, levels of stabilin-1 expression and highest amount of is a marker of M2 macrophages that is expressed by stabilin-1+ TAM were found on stages I and IIa, sug- TAM in several murine tumour models. However, its gesting that stabilin-1 is required for tumour growth role in tumour progression was not defined. In order at early stages of tumour progression. Our data indi- to identify the role of stabilin-1 in tumour progression, cate that stabilin-1 expression on TAM is necessary on the model of mammary adenocarcinoma (TS/A) was the early stages of tumour growth in human cancer. established in BALB/c mice with stabilin-1 knock- Genetic knockout of stabilin-1 results in decrease in out. The growth of TS/A mammary adenocarcinoma tumour growth in mouse adenocarcinoma model. In in stabilin-1 knockout (ko) mice was suppressed by addition, tumour-associated macrophages deficient 36%. To identify the role of stabilin-1 in TAM biology in stabilin-1 expression have significantly decreased and reveal functions of stabilin-1 that are important ability for the endocytic clearance of SPARC. for tumour progression, isolation of high purity TAM from TS/A murine adenocarcinoma was established and optimized. Flow cytometry analysis revealed that adhesion/internalisation of extracellular SPARC was decreased in the isolated stabilin-1 ko TAM com- pared to wt TAM. Immunofluorescent/confocal mi- croscopy showed that transport of SPARC into the endocytic pathway was significantly impaired in the stabilin-1 ko TAM. Since SPARC is known to inhibit the development of solid tumours including breast cancer we hypothesize that knockout of stabilin-1 in TAM induces accumulation of extracellular SPARC resulting in suppression of tumour growth. Analysis 104 | NEW TARGETS & NEW LEADS
Plasma derived exosomes as a reservoir of biomarkers for leukemia patients stratification
*Riccardo F.1, *Arigoni M.1, Rodriguez-Vicente A.E.2, Delledonne M.3, Hernández-Rivas J.M.2, Zolezzi F.4, Calogero R.A.1
1University of Torino, Torino, Italy, 2University of Salamanca, Salamanca, Spain, 3University of Verona, Verona, Italy, 4Singapore Immunology Network (SIgN), A*STAR, Biopolis, Singapore, Singapore
Hematological diseases account for approximate- intensive follow-up. The analysis allowed the identi- ly 9.5% of the new diagnosed cancers every year. fication of three genes (WDR74, RPPH1, TPT1) able Despite the huge advances in clinical treatment of to discriminate between subsets of MBL and CLL leukemias, their exact etiology and related diagnos- patients. Interestingly, a subset of CLL patients is tic, prognostic and follow up methods remain mainly characterized by an up-modulation of TPT1, which unidentified. As a consequence, early-diagnosis, to- is associated to progression in several tumors and it gether with specifically tailored approaches to leu- is also a predictor of poor prognosis in breast cancer. kemias, still represents a key point in the process Moreover, some CLL patients of the above mentioned of clinical decision making. Thank to the amazing subsets are characterized also by a reduced expres- advances of next-generation sequencing (NGS) tech- sion of 4 miRNAs (miR-146a-5p, miR-151a-3p, miR-22- nologies, a comprehensive exploitation of diagnostic 3p, miR-584-5p), which were shown to act as tumor and prognostic markers in patients affected by he- suppressors in various solid cancers. We have also matological disease could lead to an accurate strati- investigated and characterized other non-coding fication and to the selection of the most appropriate RNAs (snoRNA, snRNA, lncRNA) for their ability to therapy for each leukemia patient, throwing medical refine the stratification of MBL and CLL patients. In sciences into the era of personalized medicine. particular, using a signature based on miR-6813 and Exosomes are cell-derived vesicles that can be con- two small non coding (sn) RNA U2 pseudogenes we sidered as a reservoir of coding and non-coding RNAs managed to stratify CLL patients in 4 groups, which to be used as biomarkers for the diagnosis and prog- are independent by the patient karyotype, while MBL nosis of malignant tumors. Therefore we performed patients grouped all in a separate cluster. Our results a deep characterization of leukemia patients’ plas- indicate that trascriptome analysis of plasma-derived ma-derived exosomes to discover novel signatures exosome has interesting potential to successfully involved in disease prognosis, development and/or identify clinically relevant leukemic biomarkers. We impacting clinical outcome. are applying the same NGS analysis on plasma-de- Firstly, we analyzed by RNA-seq the exosome tran- rived exosomes from Acute Myeloid Leukemia (AML) scriptome of Monoclonal B-cell Lymphocytosis patients to identify a signature predicting the early (MBL) and Chronic Lymphocytic Leukemia (CLL) onset of the disease and/or the risk of recurrence as patients, characterized by similar karyotypes, in well as therapy resistance. order to identify a signature able to discriminate effi- ciently between the two conditions and to differenti- ate non-progressive and progressive cases requiring 105 | NEW TARGETS & NEW LEADS
The immunopeptidomic landscape of ovarian cancers - the MUC16/MSLN axis exposes cancer cells to the immune system
Schuster H.1, Peper J.1, Bösmüller H.2, Backert L.1, Ney B.2, Stevanovic S.1, Rammensee H.-G.1, Staebler A.2, Wagner P.3
1University Tübingen, Interfaculty Institute for Cell Biology, Department of Immunology, Tübingen, Germany, 2University Hospital Tübingen, Institute of Pathology, Tübingen, Germany, 3University Hospital Tübingen, Department of Obstetrics and Gynecology, Tübingen, Germany
Epithelial ovarian cancer (EOC) remains the most ly, ligands derived from mucin 16 and mesothelin, a lethal gynecological disease owing to late diagnosis molecular axis of prognostic importance in EOC, are and frequent resistance to platinum based chemo- immunogenic and frequently presented in a majority therapy. Despite recent improvements, the overall of patients. HLA ligands naturally and exclusively prognosis of patients suffering from ovarian cancer presented by EOC will be integrated into a ware- remains poor and curative treatment is only possi- house for off-the-shelf and personalized vaccination ble by surgical resection at an early non-metastatic of ovarian cancer patients. For the first time we were stage. The lack of treatment options and the high im- able to identify biomarkers, which are able to predict munogenicity of ovarian cancer have long demanded ligand presentation of selected antigens. Thereby, we the use of an immunotherapeutic approach. envisage to further facilitate individualized antigen Immunotherapies are starting a revolution in cancer selection for immunotherapy, directly addressing the therapy with the introduction of checkpoint inhibi- antigenic profile of a patient’s tumor. tors finally unleashing the power of the immune system. Knowledge about which antigens are pre- sented by cancer cells capable of exposing them to immune cells is in contrast still sporadic. This infor- mation is however critically needed for the design of targeted immunotherapies, in order to redirect a liberated immune response to its envisaged target. Large scale HLA ligandome analysis has enabled us to exhaustively characterize the immunopeptidomic landscape of epithelial ovarian cancers. Additional comparative profiling with the immunopeptidome of a variety of benign sources has unveiled a multitude of ovarian cancer antigens (MUC16, MSLN, IDO1, KLK10, FOLR1….) to be presented by HLA class I and class II molecules exclusively on ovarian tumor tissue. Furthermore, isolation of ovarian cancer cells from fresh tumor specimen and subsequent immun- opeptidome analysis confirmed the cancer cell spe- cific origin of HLA presented peptides. Most striking- 106 | NEW TARGETS & NEW LEADS
Reduction of minimal residual disease in children with relapsed and refractory ALL by a newly developed, Fc-optimized CD19 antibody
Seidel U.J.E.1, Grosse-Hovest L.2,3, Schlegel P.1, Hofmann M.2,3, Vogt F.3,4, Schuster F.R.5, Meisel R.5, Witte K.-E.1, Aulwurm S.2,3, Teltschik R.1, Pyz E.3, Rammensee H.-G.3,4, Jung G.3,4, Handgretinger R.1,4, Lang P.1,4
1University Children’s Hospital Tübingen, Department of General Paediatrics, Oncology/Haematology, Tübingen, Germany, 2SYNIMMUNE GmbH, Tübingen, Germany, 3University of Tübingen, Interfaculty Institute for Cell Biology, Department of Immunology, Tübingen, Germany, 4German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Tübingen, Tübingen, Germany, 5Heinrich Heine University, Department of Paediatric Oncology, Haematology and Immunology, Düsseldorf, Germany
B-lineage acute lymphoblastic leukemia (ALL) is the most in sustained remission for 264 - 1115 days (median fol- common childhood cancer. Although this disease can low-up 720 days). be successfully treated in 80% of patients, prognosis for Cytotoxicity assays using patient PBMC on autologous primary refractory or relapsed disease is very poor. Even blasts confirmed sustained functionality of patient effec- after allogeneic stem cell transplantation (SCT), relapse tor cells over the course of 4G7SDIE treatment. Cytotox- rates are considerable and correlate significantly with icity assays were performed using PBMC from transplant- persistent minimal residual disease (MRD) prior to and ed patients obtained at different time points of 4G7SDIE after SCT. MRD constellations represent favorable effec- treatment. Lysis of autologous ALL blasts was increased tor-target ratios and thus might be optimally suited for when 4G7SDIE or autologous patient serum taken after immunotherapeutic intervention with therapeutic anti- antibody infusion was added. After infusion of 20 mg/ bodies. m2 - 40 mg/m2 4G7SDIE serum half-life was 20 h - 43 We developed an Fc-optimized CD19 antibody (4G7SDIE) h. Serum levels of 4G7SDIE remained above saturating
and produced it in pharmaceutical quality. 4G7SDIE me- concentrations of ≥ 700 ng/ml (EC50=65 ng/ml) until the diates markedly enhanced antibodydependent cellular following application in the bi-weekly treatment cycle. cytotoxicity (ADCC) through its improved capability to Notably, in 2/2 analyzed patients under 4G7SDIE therapy, recruit FcγRIIIa bearing effector cells including NK cells a downmodulation of CD19 surface expression on the leu- and γδ T cells. 4G7SDIE significantly increased lysis of kemic blasts was observed. In vitro antigenic shift assays ALL blasts by PBMC in vitro. on patient blasts showed considerable but very hetero- 4G7SDIE was applied on compassionate use to MRD-pos- geneous shift of CD19 surface expression. Furthermore, itive pediatric patients with relapsed or refractory ALL a positive correlation between CD19 surface expression (CR1 n=3, ≥ CR2, n=11). Side effects such as headache levels and 4G7SDIE mediated lysis was observed. These and fever were negligible. In all patients complete CD20+ observations hint at in vivo tumor escape mechanisms B-cell depletion was observed during therapy. After dis- and moreover indicate selective pressure exerted by im- continuation of 4G7SDIE therapy B cell counts recov- munotherapy with 4G7SDIE, underlining its therapeutic ered rapidly to normal levels. In 9/14 patients MRD was potential, but also delineating possible limitations. reduced by ≥ 1 log or fell below MRD-detection thresh- Promising anti-leukemic effects of the 4G7SDIE antibody old of 10-4 over the course of treatment. 2/9 responders have been observed in vitro and in vivo. We are currently were receiving additional treatment. 6 patients have been preparing a clinical phase I/II trial. 107 | NEW TARGETS & NEW LEADS
M2 polarization enhances silica nanoparticle uptake by macrophages
Seif M.1, Hoppstädter J.2, Dembek A.2, Cavelius C.3, Huwer H.4, Kraegeloh A.3, Kiemer A.K.2
1Korea Institute of Science and Technology Europe, Magnetics group, Saarbrücken, Germany, 2Pharmaceutical Biology, Saarland University, Department of Pharmacy, Saarbrücken, Germany, 3INM - Leibniz Institute for New Materials, Nano Cell Interactions Group, Saarbrücken, Germany, 4Voelklingen Heart Centre, Department of Cardiothoracic Surgery, Voelklingen, Germany
Silica nanoparticles (NPs) have been investigated as IL-10 in M2 and TNF-α in M1. In the second model, promising candidates for biomedical applications, PMA (phorbol 12-myristate 13-acetate) differentiat- such as gene transfection and drug delivery. When ed THP-1 cells were polarized towards an M1 or M2 injected into the body NPs are mainly cleared by type by LPS/IFNɣ or IL-10, respectively. In the last macrophages. Macrophages can be divided into at model, M2-like primary human TAMs were isolated least two different cell populations having distinct from lung tumors and M1-like alveolar macrophages functions. Classically activated macrophages (M1) from the surrounding lung tissue. exhibit pro-inflammatory functions and alternatively NPs were added to the macrophages at a concentra- activated macrophages (M2) have anti-inflammatory tion of 50 µg/ml. The cell viability was not affected functions. In a solid tumor, tumor-associated macro- as assessed by MTT assay. Nanoparticle uptake was phages (TAMs) are mostly M2-like macrophages and evaluated by flow cytometry and fluorescence mi- are known to promote cancer progression. Therefore, croscopy. We observed enhanced uptake in M2-po- TAM-targeted strategies represent an interesting ap- larized primary human monocyte-derived macro- proach for cancer therapy. phages compared to M1 cells. M2 polarization was Aim of this study was to determine the influence of also associated with increased nanoparticle uptake macrophage polarization on NP uptake. We therefore in THP- macrophages. In accordance, in vivo polar- examined NP uptake in three models of differentially ized M2-like primary human tumor-associated mac- polarized human macrophages. rophages took up more nanoparticles than M1-like In the first model, peripheral blood monocytes were alveolar macrophages. isolated from buffy coats and differentiated with Our data indicate that the M2 polarization of mac- either macrophage colony-stimulating factor (M-CSF) rophages promotes nanoparticle internalization. or granulocyte macrophage colony-stimulating factor Therefore, drug-loaded silica nanoparticles might (GM-CSF). To generate M1 macrophages, GM-CSF serve to specifically target M2 polarized macro- differentiated macrophages were further activated phages like TAMs. with LPS and IFNɣ. To generate M2 macrophages, M-CSF differentiated macrophages were incubated with IL-10. Macrophage polarization was assessed by flow cytometry and Q-PCR. M1 were identified by an increased CD80 and HLA-II surface expression. M2 displayed a high expression level of CD14 and CD163. Cytokine profiles showed increased mRNA levels of 108 | NEW TARGETS & NEW LEADS
Unraveling the “immune-modulatome” of pancreatic adenocarcinoma (PDAC): a RNAi screening with patient- derived tumor infiltrating lymphocytes (TILs)
Sorrentino A.1, Menevse A.N.1, Michels T.1, Khandelwal N.1, Breining M.2, Poschke I.3, Offring R.3, Boutros M.2, Beckhove P.1
1German Cancer Research Center (DKFZ), Translational Immunology, Heidelberg, Germany, 2German Cancer Research Center (DKFZ), Signaling and Functional Genomics, Heidelberg, Germany, 3 ORAL German Cancer Research Center (DKFZ), Molecular Oncology of Gastrointestinal Tumors, ALK Heidelberg, Germany SHORT T 2015
Background: Pancreatic ductal adenocarcinoma (PDAC) Results: Our screen revealed 155 candidate genes whose accounts for 95% of all pancreatic cancer types. Due to knock-down enhances TIL-mediated killing more effi- the resistance of PDAC towards chemotherapy and radio- ciently than PD-L1 down-regulation. 35% of these genes therapy, traditional treatment strategies are unsuccess- are surface molecules and are most likely to directly ful. Though PDAC was considered to be poorly immuno- mediate tumor immune evasion. Beside novel unde- genic,recent evidence of tumor infiltrating lymphocytes scribed immune checkpoints, our list contains well char- (TILs) in PDAC biopsies shed more light on the involve- acterized immune modulators, supporting the reliability ment of the immune system in this malignancy. Block- of our approach. Of note 13 of our hits were also found ade of immune checkpoints, such as PD-L1 and CTLA-4 in a related melanoma screen and might play a role in have proven clinical success in many cancer entities, but the regulation of immune surveillance of different tumor did not show clinical benefit in PDAC patients, empha- entities. Among our candidates, TONI1 was one of the sizing the need to identify more key players that could most prominent hits. So far, we confirmed the role of radically improve immunotherapy. TONI1 in inhibiting TIL-mediated killing both in chro- Aim: We aim to unravel the whole arsenal of immune mium release and luciferase based cytotoxicity assays. modulators on tumor cells by performing a high-through- Additionally we detected increased T-cell activity upon put RNAi screen and subsequently identifying novel TONI1 down-regulation, as measured with interferon-γ therapeutic targets that could potentially enhance an- ELISPOT and TNF-α and granzyme B ELISA. Since the ti-tumor immune response in PDAC patients. presented work is considered for patent protection, some Methods: We generated a luciferase-expressing PANC-1 gene targets are masked. cell line and knocked-down 2514 genes using a siRNA Conclusion: We set up a robust annd systematic method library. Our library included G-protein coupled recep- to identify novel immune checkpoints for pancreatic tors, protein kinases and 1117surface proteins. We cancer. Further functional validation of our candidate co-cultured HLA-A201+-matched tumor infiltrating lym- genes will prove their potential to be used as relevant phocytes (TILs) derived from a PDAC patient with the therapeutic targets in the clinic. transfected tumor cells. We then measured the remain- ing luciferase intensity of the tumor cells as an estima- tion of TIL-mediated cytotoxicity. In order to exclude genes whose knock-down affected cell viability per se, we cultivated tumor cells with the siRNA library in the absence of TILs. We analyzed the data with the cellHTS2 R package. 109 | NEW TARGETS & NEW LEADS
HCMV deletion virus models enable the identification of numerous physiologically relevant T cell epitopes
Souczek S.1, Kowalewski D.2, Zimmermann C.3, Hengel H.3, Halenius A.3, Stevanovic S.2
1German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Institute of Cell Biology, Tübingen, Germany, 2University Tübingen, Institute of Cell Biology, Tübingen, Germany, 3University Freiburg, Institute of Virology, Freiburg, Germany
Human cytomegalovirus (HCMV) is a large ds DNA viral proteins), only five of which have previously virus with a ~230 kb genome that belongs to the been described. Strikingly, functional characteriza- group of ß-herpesviruses. Whereas most healthy tion by IFNγ-ELISpot and flow cytometry revealed individuals control persistent HCMV infection well, ~40% of novel peptides to be targeted by pep- HCMV can cause severe and sometimes lethal infec- tide-specific and multifunctional T-cell responses. tions in immunocompromised persons. CD8+ T-cell clones specific for the most immunogen- Besides others, one strategy of the virus to evade ic peptides were generated and will be checked for recognition by the host immune system is to inhibit cytolytic activity using HCMV-infected MRC-5 cells the presentation of peptides on MHC molecules by as targets. Taken together, the artificial knockout of the products of some of the viral unique short (US) HCMV immunoevasins enabled the direct identifica- genes. As a result of this down-regulation the iso- tion of HCMV-derived HLA ligands by LC-MS/MS. A lation of naturally presented peptides of HCMV has substantial proportion of these peptides were found not been very successful so far and to our knowl- to be recognized by (memory) T cells of CMV-posi- edge all known HCMV derived T-cell epitopes are tive individuals, underscoring the physiological rele- based on prediction. Surprisingly, despite the strong vance of the identified HLA ligands. Our comprehen- down-regulation of HLA-peptide complexes there is sive analysis of the HCMV immunopeptidome will an efficient control of the virus by healthy individ- provide a rational basis for further improvement of uals. Additionally, HCMV-specific memory T cells intervention approaches to HCMV disease, including could be detected in numerous studies indicating adoptive T-cell therapy and vaccine design. priming of peptide-specific naïve T cells in any phase of HCMV infection. In this study, MRC-5 cells (HLA-A*02:01, -A*29:02, -B*07:02, -B*44:02, -C*05:01, -C*07:02) were infected with HCMV (strain AD169VarL) immunoevasin-de- letion mutants (ΔUS2-US11 or ΔUS2-US6+ΔUS11). HLA ligands were isolated and characterized by liq- uid-chromatography-mass spectometry (LC-MS/MS). In comparison to the WT virus, the number of isolat- ed peptides from the ΔUS2-US11 or ΔUS2-US6+ΔUS11 HCMV infected cells was drastically increased. We were able to identify 217 peptides (derived from >110 110 | NEW TARGETS & NEW LEADS
Effective in vivo targeting of B cell precursor acute lymphoblastic leukemia by CD70 directed immunotherapy
Sun Q.1, Debatin K.-M.1, Meyer L.-H.1
1Universitätsklinikum Ulm, Department of Pediatrics and Adolescent Medicine, Ulm, Germany
ORAL ALK SHORT T 2015
Acute lymphoblastic leukemia (ALL) is the most fre- 8, TTLlong n=20) revealed a higher expression of CD70 quent malignant disorder in children and adolescents. on ALL cells with a TTLshort/early relapse phenotype Despite successful treatment, relapse of the disease compared to TTLlong samples and remission controls remains a major problem associated with poor prog- (n=4). To take advantage of increased CD70 expres- nosis. This emphasizes the need for novel treatment sion in BCP-ALL, the efficacy of monoclonal antibody strategies to be applied in addition to established based CD70-directed immunotherapy was evaluated chemotherapy regimens. Previously, we described a in NOD/SCID mice. A marked reduction of leukemia strong association of leukemia cell engraftment of load in peripheral blood, bone marrow and spleens primary patient B cell precursor (BCP) ALL samples of the animals was detected in anti-CD70 treated transplanted in a NOD/SCID/huALL mouse model CD70hi primograft ALL cells. This effect could be ab- and patient outcome. Rapid onset of leukemia related rogated both by NK-cell depletion in the NOD/SCID morbidity (time to leukemia, TTLshort) is indicative recipients and by using NK-cell deprived NSG mice for patient relapse and characterized by a specific as recipients, indicating that decreased in vivo leu- gene expression profile. Among the top differentially kemia growth upon anti-CD70 treatment is mediated regulated genes, the gene coding for CD70 was identi- by NK-cell induced cytotoxicity of CD70 positive ALL fied to be significantly up-regulated in TTLshort/high cells. Consistently, CD70 antibody-dependent cell- risk ALL. CD70 is a member of the tumor necrosis mediated cytotoxicity (ADCC) was observed in vitro. factor (TNF) family expressed on activated B- and Taken together, we identified significantly up-reg- T-lymphocytes and dendritic cells. Binding of CD70 ulated CD70 expression in BCP-ALL as detected by to its receptor CD27 is involved in regulation of B cell flowcytometry. Targeting of CD70 by directed immu- priming and generation of memory T cells. The re- notherapy with anti-CD70 antibodies led to efficient stricted expression pattern of CD70 in normal hemat- NK-cell dependent lysis of leukemia cells in vitro and opoiesis in contrast to up-regulation in hematological decreased BCP-ALL growth in vivo. Thus, CD70 pro- malignancies makes it an attractive antibody-based vides a novel target for directed immunotherapy of therapeutic target. In this study, we addressed ex- BCP-ALL. pression of CD70 in pediatric patient-derived primo- graft leukemia samples and further evaluated CD70 as a therapeutic target for directed immunotherapy in vitro and in our BCP-ALL xenograft system in vivo. Flowcytometric analyses of CD70 surface expression in 28 patient-derived xenograft samples (TTLshort n= 111 | NEW TARGETS & NEW LEADS
Identification of single nucleotide polymorphisms as putative predictive biomarkers for IMAB362 treatment of patients with gastroesophageal cancer
Türeci Ö.1, Schuler M.2,3, Zvirbule Z.4, Lordick F.5, Krilova A.6, Helbig U.7, Schulze-Bergkamen H.8, Thuß-Patience P.9, von Wichert G.10, Adam B.11, Bauer S.12, Al-Batran S.-E.13, Huber C.14, Bukur V.14,15, Maurus D.1, Sahin U.14,15
1Ganymed Pharmaceuticals AG, Mainz, Germany, 10Schön Klinik Hamburg Eilbek, Hamburg, Germany, 2West German Cancer Center - University Duisburg-Essen, 11University Hospital Knappschaftskrankenhaus Bochum - Essen, Germany, Ruhr-University Bochum, Bochum, Germany, 3German Cancer Consortium (DKTK), Heidelberg, Germany, 12Gemeinschaftspraxis for Hämatologie und Onkologie, Lebach, 4Riga Eastern Clinical University Hospital, Riga, Latvia, Germany, 5University Cancer Center Leipzig (UCCL) - University Medicine 13UCT - University Cancer Center Frankfurt, Krankenhaus Nord- Leipzig, Leipzig, Germany, west, Frankfurt, Germany, 6Piejuras Hospital - Oncology Clinic, Liepaja, Latvia, 14TRON-Translational Oncology at the University Medical Center 7Clinical Center Braunschweig, Braunschweig, Germany, of the Johannes Gutenberg University Mainz, Mainz, Germany, 8National Center for Tumor Diseases (NCT) - Heidelberg 15BioNTech, Mainz, Germany University Hospital, Heidelberg, Germany, 9Campus Virchow-Klinikum, Charité - University Medicine, Berlin, Germany,
IMAB362 is a monoclonal antibody specifically target- patients with advanced GEC included in the interna- ing the tight junction molecule CLDN18.2, which is ex- tional, multicenter, open-label, Phase II MONO study pressed by various solid cancers, e.g. gastroesophage- investigating efficacy and safety of multiple doses of al cancer (GEC). Currently, IMAB362 is under Phase II IMAB362 (ClinicalTrials.gov ID: NCT01197885). Cor- clinical investigation for the treatment of patients with relation was analyzed for the full analysis set (FAS, advanced GEC. The identification of potentially pre- n = 39) and per protocol (PP, n = 21) set. For the dictive biomarker candidates may help to determine correlation of SNP genotypes with clinical benefit, a patient population that would benefit the most from both patient populations were stratified into respond- treatment with IMAB362. Single nucleotide polymor- ers (R, n = 12 for FAS and n = 10 for PP) and non- phisms (SNPs) in several genes have been associated responders (NR, n = 27 for FAS and n = 11 for PP) with clinical outcome of antibody therapy. and statistically analyzed with a Pearson’s Chi2 test Therefore, we evaluated the presence as well as the (or in two cases with a Fisher’s exact test). Correla- correlation with clinical outcome parameters (pro- tion of SNP genotypes with PFS was analyzed with a gression-free survival [PFS] and clinical response) of log-rank test based on Kaplan-Meier estimates. 51 SNP genotypes, including SNPs in genes related Forty-nine of 51 SNP genotypes have a variant allele to immune response or to GEC susceptibility, in 53 pattern in this patient population (n = 53). The fre- quency of 7/48 SNP genotypes studied in this patient population shifted compared to a Caucasian control population (P < 0.05). Three SNPs significantly correlated with clinical benefit in both populations (FAS, P < 0.001-0.05; PP, P < 0.01-0.05). Two of these 3 SNPs significantly correlated with prolonged PFS in both sets (FAS, P < 0.01-0.05; PP, P < 0.001). Ad- ditional 4 SNPs correlated with clinical benefit in either the FAS or the PP (P < 0.05 for 2 SNPs each) population and an additional 2 SNPs correlated with prolonged PFS of patients in the PP set (P < 0.001 for both SNPs). In conclusion, shifts in SNP genotypes in patients with GEC may serve as putative biomarker candi- dates. Moreover, three SNPs (including two immune- related SNPs) correlated with clinical benefit and prolonged PFS in patients treated with IMAB362 and may be potential biomarker candidates with predic- tive or prognostic potential. However, whether these putative biomarker candidates are of a predictive vs. prognostic nature needs further investigation in controlled Phase II and III clinical trials. The FAST clinical trial (ClinicalTrials.gov ID: NCT01630083) is a controlled Phase II trial comparing chemotherapy (EOX) with or without IMAB362 and includes an extensive biomarker program to support the results presented here. 112 | NEW TARGETS & NEW LEADS
An engineered IgA antibody against the epidermal growth factor receptor displays improved myeloid effector cell engagement and pharmacokinetics
Kretschmer A.1, Lohse S.1, Meyer S.2, Meulenbroek L.A.P.2, Jansen J.H.M.2, Nederend M.2, Klausz K.1, Möginger U.3, Kellner C.1, Derer S.1, Sondermann P.4, Schewe D.5, Peipp M.1, Kolarich D.3, Leusen J.H.W.2, Valerius T.1
1Section for Stem Cell Transplantation & Immunotherapy, 2nd Department of Medicine, Kiel, Germany, 2Laboratory for Translational Immunology, University Medical Center, Utrecht, Germany, 3Department of Biomolecular Systems, MPI for Colloids and Interfaces, Potsdam, Germany, 4SuppreMol GmbH, Martinsried, Germany, 5Department of General Pediatrics, Kiel, Germany
Introduction: Antibodies of IgA isotype play an im- tions, but with significantly lower levels of terminal portant role in bridging adaptive and innate immuni- galactose. This molecule demonstrated lower asia- ty. FcαRI (CD89)- dependent engagement of myeloid loglycoprotein-receptor (ASGPR) binding and subse- cells appears to be crucial to activate effector mech- quently improved pharmacokinetics in mice. Com- anisms like phagocytosis or antibody-dependent pared to wild type IgA, this novel molecule displayed cell-mediated cytotoxicity (ADCC). Recently, there enhanced therapeutic efficacy against A431 tumor has been increasing evidence that myeloid cells con- cells in vivo, which required human FcαRI-depen- stitute important effector cells in cancer and cancer dent myeloid effector cell engagement. immunotherapy. Thus, we decided to develop pro- Conclusions: These results demonstrate that an Fc duction and purification technologies for recombi- engineered IgA antibody against EGFR displayed nant IgA antibodies, which demonstrated in vivo improved immunotherapeutic efficacy, which may efficacy in syngeneic and xenogeneic tumor models. overcome some of the limitations (e.g. stability, Here, we describe an Fc engineering approach to pharmacokinetics, in vivo efficacy) of wild type IgA further improve their immunotherapeutic potential. antibodies. Thus, these results promote the concept Methods: Recombinant IgA antibodies against the of FcαRI- dependent engagement of myeloid effector epidermal growth factor receptor (EGFR) were pro- cells as a promising approach for antibody-based duced by co-transfecting CHO-K1 cells with vectors tumor immunotherapy. encoding the 225 variable and Ig alpha heavy and kappa light chain constant regions, respectively. An Fc engineered IgA2m(1) antibody was generated by mutating two N-glycosylation sites (166 and 337) and by removing two free cysteines (311 and 471). The re- sulting antibody variant was compared to wild type IgA2 regarding biochemical characteristics as well as Fab and Fc- mediated effector functions. Additional- ly, serum half-life and in vivo efficacy in a xenogene- ic FcaRI- transgenic tumor model were evaluated. Results: Rational engineering of the constant regions of an IgA2m(1) antibody resulted in monomeric IgA molecules with improved biochemical characteris- tics, identical Fab- and Fc- mediated effector func- 113 | NEW TARGETS & NEW LEADS
Antitumoral activity of IMAB027, the first therapeutic monoclonal antibody targeting the cancer-specific antigen claudin 6 (CLDN6)
Walter K.1, Kreuzberg M.1, Jacobs S.1, Schmitt R.1, Kühnle M.-C.1, Wöll S.1, Rohde C.1, Sahin U.2,3, Türeci Ö.1
1Ganymed Pharmaceuticals AG, Mainz, Germany, 2TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany, 3BioNTech, Mainz, Germany ORAL ALK SHORT T 2015
Monocloncal antibodies (mAB) have emerged as prom- from 64%-96% and a mean IMAB027 EC50 ≤ 0.86 µg/ ising treatment options in solid tumors. Nevertheless, mL. ADCC-mediated mean maximum lysis of TC cells
mAB can be associated with side effects due to the ex- ranged from 81%-87% with mean IMAB027 EC50 ≤ 0.03 pression of their target in healthy organs. CLDN6 is a µg/mL. cancer-cell specific embryonic tight junction protein In vivo, IMAB027 (105 mg/kg per week) significantly absent from healthy tissues in the human adult body. reduced tumor growth in mice bearing subcutaneous This together with its pantumoral expression in solid CLDN6-positive human OC and TC xenografts. Tumor cancers, e.g. approx. 50% of ovarian cancers (OC) and growth inhibition of OC xenografts was 64% (P = 0.01 90% of testicular cancers (TC), make it a prime target for vs vehicle control) and 99% (P < 0.0001 vs vehicle the development of a therapeutic mAB. IMAB027 is the control) of TC xenografts. This translated into a survival first-in-class mAB targeting CLDN6. This study aimed benefit for IMAB027-treated compared to vehicle-treated to investigate the in vitro and in vivo anti-tumor activity mice bearing OC xenografts with a median survival of of IMAB027 in preclinical OC and TC tumor models. 59 days for IMAB027-treated vs 41 days for vehicle-treat- In order to test IMAB027’s anti-tumor activity we de- ed mice (P = 0.005). 73% of mice bearing TC xenografts veloped preclinical tumor models based on human OC were tumor-free at the end of observation time (P < and TC cell lines with either endogenous or exogenous 0.0001 vs vehicle control on day 122). IMAB027 did not expression of CLDN6. IMAB027 specifically bound to bind to healthy tissues in tissue-cross reactivity studies CLDN6 expressed in a range of human OC and TC cell in mouse and monkey. Hence, IMAB027 was well toler- lines. Complement-dependent and antibody-dependent ated in preclinical safety studies in both species. cellular cytotoxicity (CDC and ADCC) are the two major In conclusion, the mAB IMAB027 specifically binds to modes of action of mAB. In vitro, IMAB027 mediated CLDN6 and has in vitro and in vivo anti-tumor activity in specific and efficient CDC and ADCC against CLDN6- preclinical OC and TC tumor models. Together with the positive human OC as well as TC cell lines in the pres- preclinical safety data, these data provide a strong ra- ence of human serum as complement source or human tionale for the clinical development of IMAB027 in both peripheral blood mononuclear cells as source of human OC and TC. Currently, a phase I/II study investigates effector cells, respectively. Mean maximum lysis of IMAB027 in patients with recurrent, advanced OC OC cells by CDC ranged from 63%-73% with mean
IMAB027 EC50 of ≤ 0.27 µg/mL. The mean maximum lysis of OC cells by ADCC ranged from 56%-97% of OC
with mean IMAB027 EC50 ≤ 0.03 µg/mL. TC cells were (ClinicalTrials.gov ID: NCT02054351). killed by CDC with a mean maximum lysis ranging 114 | NEW TARGETS & NEW LEADS
Multiple co-inhibitory pathways contribute to dysfunctionality of intra-tumoral T cells in liver cancer
Zhou G.1, Sprengers D.1, Boor P.1, Polak W.2, de Jonge J.2, Ijzermans J.N.M.2, Grünhagen D.2, Verhoef C.2, Doukas M.3, Bruno M.1, Kwekkeboom J.1
1Erasmus MC-University Medical Center, Gastroenterology and Hepatology, Rotterdam, Netherlands, 2Erasmus MC-University Medical Center, Surgery, Rotterdam, Netherlands, 3Erasmus MC-University Medical Center, Pathology, Rotterdam, Netherlands
Introduction: We recently found that CD4+CD25- T PD-L1, CTLA-4 or isotype control mAb (10ug/ml). cells and CD8+ T cells from tumor tissue of pa- CD4+ and CD8+ T cell proliferation and cytokine tients with liver cancer are functionally compro- production were measured by flow cytometry. mised compared to circulating T cells after depletion Results: Expression of LAG-3, Tim-3, PD-1 and of CD4+CD25+ regulatory T cells. Recent studies CTLA-4 was up-regulated on tumor-infiltrating showed that PD-1/PD-L1 interaction mediates sup- CD4+Foxp3- and CD8+ T cells as compared to those pression of intra-tumoral CD8+ T cells, whereas the in TFL and blood. Their ligands galectin-9, PD-L1, Tim-3/galectin-9 signaling pathway mediates CD4+ CD80 and CD86 were expressed on myeloid dendritic T cell dysfunction in hepatocellular carcinoma cells, monocytes and B cells in the tumors. Interest- (HCC). However, it is unknown whether other co-in- ingly, tumor-derived CD4+Foxp3- and CD8+ T cells hibitory pathways contribute to T-cell dysfunctional- expressing those co-inhibitory receptors displayed ity in liver tumors. Here, we analyzed the expression more proliferative activity (Ki-67) and a more activat- and functional relevance of 7 different co-inhibitory ed status (HLA-DR, CD69) as compared with the cells pathways in patients with HCC or liver metastases without co-inhibitory receptor expression. However, from colorectal cancer (LM-CRC). irrespective of co-inhibitory receptor expression, all Methods: The expression of co-inhibitory receptors intra-tumoral CD8+ T cells showed granzyme B de- LAG-3, Tim-3, PD-1, CTLA-4, BTLA, CD244 and CD160 ficiency compared to those in TFL and blood. Func- was analyzed on paired samples of tumor-infiltrat- tional studies demonstrated that blockade of CTLA-4, ing lymphocytes (TIL), lymphocytes from adjacent PD-L1, LAG-3 or Tim-3 increased the functionality of tumor-free liver tissue (TFL), and peripheral blood tumor-derived CD4+ and CD8+ T cells as shown by mononuclear cells, freshly isolated from patients increased T-cell proliferation and effector cytokine with HCC or LM-CRC undergoing tumor resection. production. In addition, surface stainings of HLA-DR and CD69 Conclusions: In liver tumors, the co-inhibitory recep- and intracellular stainings of Ki-67 and granzyme tors LAG-3, Tim-3, PD-1 and CTLA-4 are selectively B were performed. Expression of the corresponding up-regulated on activated and replicating T cells, ligands was determined on antigen-presenting cells which are probably tumor-reactive T cells. Our func- in the same samples. For functional assays carboxy- tional data suggest that these co-inhibitory pathways fluorescein diacetate succinimidyl ester-labelled TIL restrain the functionality of these T cells, and may were stimulated with CD3/CD28-coated dynabeads be attractive immunotherapeutic targets in patients for 4 days, in the presence or absence of blocking with HCC or LM-CRC. monoclonal antibody(mAb) against LAG-3, Tim-3, 115 – 144
Improving Immunity 115 | IMPROVING IMMUNITY
Anti-tumor immunization of mothers delays tumor development in cancer prone offspring
Barutello G.1, Curcio C.2, Spadaro M.3, Arigoni M.3, Trovato R.3, Ria F.4, Bolli E.3, Quaglino E.3, Riccardo F.3, Holmgren L.5, Forni G.3, Cavallo F.3
1University of Turin, Molecular Biotechnology and Health Sciences, Torino, Italy, 2University of Chieti, Chieti, Italy, 3University of Turin, Torino, Italy, 4University of Rome, Sacro Cuore, Roma, Italy, 5Karolinska Institutet, Stockholm, Sweden
Maternal immunization is successfully applied against some life-threatening infectious diseases as it can protect the mother and her offspring through the passive transfer of maternal antibodies. Here we sought to evaluate whether the concept of maternal immunization could also be applied to cancer immu- noprevention. We have previously shown that anti- bodies induced by DNA vaccination against rat Her2 (neu) protect heterozygous neu-transgenic female (BALB-neuT) mice from autochthonous mammary tumor development. We herein seek to evaluate whether a similar, maternal, immunization can confer anti-tumor protection to BALB-neuT offspring. Signifi- cantly extended tumor-free survival was observed in BALB-neuT offspring born and fed by mothers vacci- nated against neu, as compared to controls. Maternal- ly derived anti-neu IgG were successfully transferred from mothers to newborns and were responsible for the protective effect. Vaccinated mother offspring also developed active immunity against neu as re- vealed by the presence of T-cell-mediated cytotoxicity against the neu immunodominant peptide. This active response was due to the milk transfer of immune-com- plexes that were formed between the neu extracellular domain, shed from vaccine-transfected muscle cells, and the anti-neu IgG induced by the vaccine. These findings show that maternal immunization has the potential to hamper mammary cancer in genetically predestinated offspring and to develop into applica- tions against lethal neonatal cancer diseases for which therapeutic options are currently unavailable. 116 | IMPROVING IMMUNITY
In vivo silencing of A20 via TLR9-mediated targeted siRNA delivery potentiates antitumor immune response
Braun F.C.M.1, van den Brandt J.2, Thomas S.1, Schrank J.1, Bude C.1, Przybylski G.K.1,3, Schmoeckel K.4, Bröker B.M.4, Schmidt C.A.1, Grabarczyk P.1
1University Medicine Greifswald, Molecular Hematology and Oncology, Greifswald, Germany, 2University Medicine Greifswald, Central Core & Research Facility of Laboratory Animals, Greifswald, Germany, 3Polish Academy of Sciences, Institute of Human Genetics, Poznan, Poland, 4University of Greifswald, Institute of Immunology and Transfusion Medicine, Greifswald, Germany ORAL ALK SHORT T 2015
A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling path- ways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of den- dritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of proin- flammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infil- trating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucle- otide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse mela- noma tumor model, silencing of A20 enhances the CpG-triggered induction of NF-κB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimu- latory potency of DCs and provide a strategy for si- multaneous A20 silencing and TLR activation in vivo. 117 | IMPROVING IMMUNITY
Laser-assisted epidermal immunogen delivery for optimal targeting of Langherhans cells in cancer vaccination
Buonaguro L.1, Tagliamonte M.1, Petrizzo A.1, Tornesello M.L.1, Circelli L.1, Luciano A.1, Rea D.1, Barbieri A.1, Arra C.1, Coscia C.2, Buonaguro F.M.1
1Istituto Nazionale Tumori ‘Pascale’, Naples, Italy, 2Pantec Biosolutions AG, Ruggell, Liechtenstein
Efficacy of vaccines is highly dependent on the ef- ficient delivery to professional antigen-presenting cells (APCs), such as dendritic cells (DCs). In par- ticular, Langerhans cells (LC) are members of the dendritic cells family residing in the basal and supra- basal layers of the epidermis. LC have strong immu- nogenic properties, encounter and uptake antigens in peripheral tissues, transport them to regional lymph nodes, present to naive T cells and initiate adaptive immune response. Laser-assisted epidermal immunogen delivery by P.L.E.A.S.E.Ò Professional (Pantec Biosolutions) provides a great opportunity to efficiently deliver vaccine antigens to LC in the epidermis. Indeed, it creates evenly distributed micropores through to the epidermis dramatically increasing skin permeability and enabling topically applied antigens to penetrate much faster and more deeply into the targeted skin layer. Preliminary results in different experimental settings indicate a dramatic enhancement of immune response elicited by laser-assisted epidermal immu- nogen delivery. Taking advantage of such device, we have evaluated in C57BL/6 mice the immunogenicity of candidate peptide cancer vaccine for liver cancer delivered in the epidermis in a side-by-side comparison with standard sub-cutaneous delivery. Immunogenicity results will be described. 118 | IMPROVING IMMUNITY
Strong spontaneous tumor neo-antigen responses to tumors induced by a natural human carcinogen, and unmasking of new reactivities with selective treg depletion
Creaney J.1,2, Ma S.1, Sneddon S.A.1, Tourigny M.R.1, Dick I.M.1, Leon J.S.1, Khong A.1, Fisher S.A.1, Lake R.A.1, Lesterhuis W.J.1, Nowak A.K.1, Leary S.3, Watson M.W.3, Robinson B.W.1
1University of Western Australia, NCARD, Perth, Australia, 2Sir Charles Gairdner Hospital, Perth, Australia, 3Murdoch University, Perth, Australia
A key to improving cancer immunotherapy will be We then determined if neo-antigens could be ‘un- the identification of tumor-specific ‘neo-antigens’ masked’ by therapy. Treg depletion using Foxp3- that arise from mutations. A key to this will be DTR mice, which express diphtheria toxin re- detecting spontaneous responses plus those induc- ceptor under the control of the Foxp3 promoter, ible with therapy, such as Treg depletion, check- unmasked a second neo-antigen, GANAB. GANAB point blockade or immunogenic chemotherapy in a is an alpha glucosidase which cleaves the two in- murine model that mimics its human counterpart. nermost alpha-1,3-linked glucose residues from the In this study we identified single nucleotide vari- Glc(2)Man(9)GlcNAc(2) oligosaccharide precursor ants (SNV) from RNA sequencing of the asbestos of immature glycoproteins. This observation sup- induced murine tumors, AB1 and AB1-HA, which ports the theory that removing Treg cells may are homologues of their human counterpart. broaden the immune response to a greater number Using the NetMHCpan 2.8 algorithm the theoreti- of neo-antigens, a response presumably otherwise cal binding affinity of predicted peptides arising restrained by Treg supression. In contrast, Gem- from high confidence, exonic, non-synonymous citabine, an immunogenic chemotherapy, boosted SNVs was determined for these BALB/c lines. The the Uqcrc2 response but did not unmask GANAB. immunoreactivity to twenty candidate mutation- This work confirms that the approach taken, carrying peptides of increased affinity plus cor- RNAseq plus peptide prediction and ELISPOT responding wild-type peptides was determined testing, before and after therapy, is sufficient to using interferon-γ ELISPOT assays and lymphoid identify patterns of response to relevant natural organs of non-manipulated tumor bearing mice. A tumor neo-antigens. strong endogenous immune response was demon- strated to Uqcrc2, a component of the respiratory chain protein ubiquinol cytochrome complex. This response to Uqcrc2 was greater in the draining lymph node and spleen, as was the response to the model tumor neo-antigen HA, suggesting that the dLN might generally be a better location to look for neo-antigenic reactivities. The magnitude of the response to the Uqcrc2 neo-antigen was similar to that of the strong influenza hemagglutinin antigen transfected as a model tumor neo-antigen. 119 | IMPROVING IMMUNITY
Combination therapy with the TLR7/8 agonist R848 and BAMLET, an α-lactalbumin - oleic acid complex, promotes tumour cell death and anti-tumour immunity
Dyck L.1, Harte N.1, Mok K.H.1, Mills K.H.G.1
1Trinity College Dublin, School of Biochemistry and Immunology, Dublin, Ireland
Tumours have evolved efficient mechanisms to over- tion of BAMLET and R848 conferred a high level of come recognition and elimination by the immune protection, with 57% of mice completely rejecting the system. On the one hand, tumour cells are highly re- tumour. We found a higher frequency of DCs and sistant to cell death by the up-regulation of anti-apop- IFNγ-secreting CD4 and CD8 T cells in the tumour totic proteins and low expression of MHC on their draining lymph nodes and spleens of mice treated cell surface. On the other hand, tumours evade the with the combination of BAMLET and R848, when immune system by creating an immunosuppressive compared with untreated mice or mice treated with environment. In our study, we investigated if a com- either BAMLET or R848 alone. bination therapy targeting both the tumour cells and Our data are consistent with a role for BAMLET in the immune system could overcome tumour resis- directly killing tumour cells, which release antigens tance and immune subversion in a murine model for that are taken up by DCs, but also demonstrate that melanoma. BAMLET, a bovine α-lactalbumin - oleic BAMLET can promote DC maturation, especially acid complex, is a novel and promising candidate when co-activated through a TLR agonist. These for the treatment of cancer because of its capacity to activated DCs promote induction of IFNγ-secreting specifically kill tumour cells. While recent studies T cells that have anti-tumour activity. Our findings focused on direct tumouricidal effects of BAMLET, suggest that BAMLET and R848 are a promising com- we investigated the potential of BAMLET to promote bination therapy against cancer that targets tumour anti-tumour immune responses, when administered cells directly through killing mechanisms and in- alone or in combination with the TLR7/8 agonist directly through activation of anti-tumour immune R848, which also has anti-tumour activity by virtue responses. of its ability to non-specifically stimulate innate and adaptive immunity. Consistent with previous studies, BAMLET induced cell death in B16 melanoma cells in vitro. Surpris- ingly, BAMLET also enhanced MHC class II and co-stimulatory molecule expression on dendritic cells (DCs) and production of IL-12 in a TLR4 inde- pendent manner. Treatment of tumour-bearing mice with either BAMLET or R848 s.c. in the area of the tumour (days 3, 7, 10 and 14) induced some protec- tion against tumour growth. However, the combina- 120 | IMPROVING IMMUNITY
Targeting CSF-1R reverses induction of suppressive myeloid cells and controls spontaneous neuroblastoma progression in a murine model
Eissler N.1, Mao Y.2, Johnsen J.I.1, Kiessling R.2, Kogner P.1
1Karolinska Institutet, Childhood Cancer Research Unit, Department of Women´s and Children´s Health, Stockholm, Sweden, 2Karolinska Institutet, Cancer Centrum Karolinska, Department of Oncology-Pathology, Stockholm, Sweden ORAL ALK SHORT T 2015
Neuroblastoma is the most common extra cranial of established tumors (p< 0.01). Our results dem- solid tumor and the most frequently diagnosed onstrate the essential role of the M-CSF/CSF-1R-axis cancer type among infants. Despite aggressive mul- during the induction of suppressive myeloid cells timodal therapy, high-risk metastatic disease can and emphasize the clinical potential of targeting this induce severe impairments of anti-tumor immunity pathway in patients with neuroblastoma. and has poor clinical outcome. Here, we report that M-CSF (CSF-1) is produced at high levels by neuro- blastoma tumors and infiltration of CSF-1R+ myeloid cells is associated with poor survival in neuroblasto- ma patients. In vitro, neuroblastoma-derived factors hamper myelopoiesis of human CD34+ progenitor cells derived from cord blood, and induce suppres- sive myeloid cells from primary human monocytes and naive murine bone marrow cells through M-CSF/ CSF-1R interaction. In a spontaneous murine model resembling high-risk human neuroblastoma (TH- MYCN model), we observe significant accumulation of myeloid-derived suppressor cells of the granulo- cytic (p< 0.01, grMDSCs, Ly6G+Ly6Clow) and mono- cytic (p< 0.0001, moMDSCs, Ly6GnegLy6Chigh) lineage, as well as F4/80+ macrophages (p< 0.01). Sorted from spleens of tumor-bearing mice, Gr1+ cells conduct potent inhibition of T cells through production of indolamine-2,3-dioxygenase (IDO, p< 0.04) and inducible nitric oxide synthase (iNOS, p< 0.04). Of note, antagonizing CSF-1R with a selective chemical inhibitor (BLZ945; Novartis) abolishes the induction of human and murine MDSCs and TAMs in vitro, and overcomes their induction in vivo. Striking- ly, treatment with BLZ945 elicits robust anti-tumor immune responses and efficiently limits progression 121 | IMPROVING IMMUNITY
Targeted costimulation with novel single-chain 4-1BBL or OX40L antibody-fusion proteins promotes tumor directed T cell activation
Fellermeier S.1, Meyer J.-E.1, Bader S.1, Bondarieva A.1, Türeci Ö.2, Pfizenmaier K.1, Kontermann R.1, Müller D.1
1Universität Stuttgart, Institut für Zellbiologie und Immunologie, Stuttgart, Germany, 2GANYMED Pharmaceuticals AG, Mainz, Germany
Cytokine-mediated costimulation of T cells is an important aspect addressed by novel strategies of cancer immunotherapy. In order to circumvent po- tential side effects caused by systemic activation, we focus on tumor-directed antibody-cytokine fusion proteins, targeting the cytokine to the tumor site and presenting it there in a membrane bound form. The costimulatory activity of tumor-targeted trimeric antibody-fusion proteins comprising the extracellu- lar domain of the TNF-superfamily members 4-1BBL or OX40L has already been demonstrated. Here, we evaluated a new format, generating a set of fusion pro- teins composed of a single-chain antibody fragment (scFv) targeting various tumor-associated antigens and novel single-chain variants of 4-1BBL or OX40L, respectively. Tumor targeting and cytokine recep- tor binding as well as activation was verified using antigen specific tumor cells and HT1080 cells trans- fected to express human 4-1BB or OX40, respectively. Efficient T cell costimulation was demonstrated by enhancement of anti-CD3 mAb induced interferon-g release and proliferation of peripheral blood mono- nuclear cells for all tumor-targeted fusion proteins. Accordingly, the single-chain formats of tumor-tar- geted 4-1BBL or OX40L are promising candidates for translational approaches in cancer immunotherapy. 122 | IMPROVING IMMUNITY
Cognate immune response amplifiers - a new RNA based tool for amplifying T-cell responses
Gieseke F.1, Backer R.1, Grunwitz C.1,2, Sänger B.1, Vascotto F.3, Selmi A.3, Witzel S.3, Schmitt U.2, Diken M.3, Kreiter S.3, Türeci Ö.4, Sahin U.1,2,3
1BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, 2University Medical Center of the Johannes Gutenberg University Mainz, III., Medical Clinic Group of Prof. Dr. U. Sahin Translational Oncology, Mainz, Germany, 3TRON - Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz gGmbH, Mainz, Germany, 4Ganymed Pharmaceuticals AG, Mainz, Germany
Efficient co-stimulation is crucial for T-cell responses. upon engagement of both receptors during cell-cell Therefore, modulation of co-stimulatory signals has interaction TNF-ligand fusion proteins induced activa- become a promising approach in cancer immunother- tion of both receptors efficiently. In order to analyze apy. Targeting individual members of the Tumor ne- effects of TNF ligand fusion proteins on APC : T-cell crosis factor (TNF) superfamily, which play a central interaction, we set up a T-cell proliferation assay. T cells role in T-cell co-stimulation, has already shown promis- were transduced with a T-cell receptor, while autolo- ing antitumor potential. We asked whether the usage gous monocyte derived dendritic cells (DCs) were elec- of TNF receptor ligand fusion proteins could allow us troporated with the corresponding antigen RNA and to boost antigen specific immune responses utilizing the IVT-RNA encoding for a TNF-ligand fusion protein. the spatial proximity of T-cell and APC as well as the RNA encoded fusion proteins with trans-activating up-regulation of TNF receptors on T-cells after antigen function referred to DC : T-cell interaction enhanced contact. Therefore, we aimed to provide co-stimulation T-cell proliferation in an antigen-specific but not in an by RNA encoded fusion proteins consisting of the TNF antigen-unspecific setting. Further also cis-activating receptor ligands CD40L, CD27L, OX40L and 4-1BBL, re- constructs targeting two TNF-receptors both expressed spectively. Single chain constructs encoding for fusion on T cells enhanced antigen-specific T-cell proliferation proteins were cloned and IVT-RNA was produced. Elec- efficiently. troporation of the IVT-RNA encoding fusion constructs In conclusion, we developed a RNA-based tool am- into mammalian cells resulted in production and secre- plifying T-cell responses by targeting the essential tion of fusion proteins which were traceable by flow TNF receptor ligand interactions. These TNF ligand cytometry. In order to test functionality of the secreted fusion constructs acted as co-stimulatory factors only fusion proteins, cell-based reporter assays measuring in an antigen-specific APC : T-cell interaction and did NF-kappa-B signaling upon TNF receptor activation not induce unspecific T-cell responses. Such cognate were set up. IVT-RNAs encoding for only one soluble immune response amplifiers are therefore promising TNF-ligand mediated only limited or even no activa- candidates for cancer immunotherapy. Further in vivo tion of the corresponding receptor, whereas IVT-RNA studies in murine model systems are underway to char- encoded fusion proteins induced efficient receptor ac- acterize the potency and dissect the mode of action of tivation upon cell-mediated cross-presentation, which the different cis- and trans-activating fusion proteins. was achieved by co-incubation of cells expressing the second TNF receptor. Thus, we could show that only 123 | IMPROVING IMMUNITY
Measles virus vaccine infects tumor cells and induces antitumor immunogenic response
Gregoire M.1,2, Boisgerault N.1, Guillerme J.-B.1, Achard C.1, Roulois D.1, Tangy F.3, Fonteneau J.-F.1
1INSERM 892 - CNRS 6299, Nantes cedex, France, 2INSERM - University of Nantes, Nantes, France, 3CNRS URA-3015, Institut Pasteur, Paris, France
Dendritic Cells (DC) are antigen presenting cells specialized in inducing immune responses. Measles Virus vaccine (MV) was recently proposed as anti-tu- mor agent to target and kill specifically tumor cells, without infecting healthy cells. We demonstrat- ed that myeloid dendritic cells (mDC) co-cultured with MV infected tumor cells, actively matured and cross-presented tumor antigen. Recently, we also investigated the effects of MV tumor infected cells on phenotype and functions of plasmacytoid DC. We studied maturation, cytokine production and tumor antigen cross-presentation by mDC and pDC exposed either to the virus alone, MV infected or UV irradi- ated tumor cells. We found that MV infected cells induce DC matu- ration with a strong cytokine production, notably IFN-α, whereas UV irradiated tumor cells and the MV alone did not. We also observed that MV infected and UV irradiated cells were similarly phagocytosed by DC, although this up-take was less important than in myeloïd DC. Interestingly, we observed cross-pre- sentation of tumor antigen to a specific CD8+ T cell clone only when DC are co-cultured with MV infect- ed tumor cells. Altogether, our results suggest that the use of MV, as anti-tumor virotherapy, may induce immunogenic tumor cell death allowing DC to cross-present tumor antigen. 124 | IMPROVING IMMUNITY
RNAdjuvant®, a novel, highly-potent RNA-based adjuvant, combines strong immunostimulatory capacities with a favorable safety profile
Heidenreich R.1, Lutz J.1, Kowalczyk A.1, Baumhof P.1, Scheel B.1, Voss S.1, Kallen K.-J.1, Fotin-Mleczek M.1
1CureVac GmbH, Tübingen, Germany
Purified recombinant proteins and peptides, which sensors at the injection site, avoiding any system- are currently under development in various anti-can- ic cytokine release. These changes are followed by cer vaccination approaches, lack sufficient immuno- activation of different subsets of immune cells in genicity. Therefore, potent adjuvants are needed to the draining lymph nodes. In repeated dose toxici- induce strong and persistent anti-tumor immunity. ty studies carried out in mice and pigs no toxicity However, currently only few adjuvants are licensed, events were observed. most of which primarily enhance antibody, but not In summary, our data suggest that RNAdjuvant® rep- T cell responses. resents a novel, highly efficacious adjuvant candi- In our study, we demonstrate that the novel, well date that enhances anti-cancer responses in vaccina- defined, and thoroughly characterized RNA-based tion regimen by minimizing risk of inducing severe adjuvant, RNAdjuvant®, significantly enhances an- adverse effects, as observed with many other sys- ti-tumor immunity, and that even complete tumor temically acting immunomodulators. Phase I safety rejection can be achieved. We found that in the syn- studies in humans is currently running. Taking into geneic TC-1 tumor, a murine model of human HPV-in- account, that progress in the realm of cancer immu- duced cervical cancer, RNAdjuvant® in combination notherapy is currently severely obstructed by the with E7 long peptide was able to induce functional lack of appropriate adjuvants, we believe that RNAd- memory responses that mediated complete tumor re- juvant® has the potential to revolutionize the field of mission. This observation was striking, as adjuvan- tumor immunotherapies. tation with the commonly used immune enhancer poly(I:C) did not induce protective immunity. Our adjuvant mediates balanced and long-lasting immune responses including strongly enhanced T cell responses, and exhibits an excellent preclinical safety profile. It is composed of a single-stranded, non-coding and non-capped RNA of a defined se- quence, complexed with a specific cationic peptide, is optimized for strong immunostimulatory proper- ties and stabilized against degradation. Our adjuvant acts strictly locally, promoting strong but transient up-regulation of anti-viral and pro-inflammatory cytokines, CXCR3-ligands and cytoplasmic RNA 125 | IMPROVING IMMUNITY
Timing and sequence of pattern-recognition receptor stimulation determines efficacy of cancer immunotherapy with TLR ligands
Hotz C.1, Roetzer L.2, Huber T.2, Sailer A.2, Oberson A.1, Treinies M.1, Mottas I.1, Herbst T.1, Endres S.2, Bourquin C.1
1University of Fribourg, Department of Medicine/Chair of Pharmacology, Fribourg, Switzerland, 2Ludwig-Maximilian University Munich, Division of Clinical Pharmacology, Munich, Germany
Immunotherapy represents an emerging field in the naling pathways but not the receptor expression itself treatment of cancer, aiming to restore effective anti- to enhance subsequent heterologous PRR stimulation tumor immune responses in patients. To this end, the but blunting responses to similar ligands. stimulation of the innate immune system via pattern Our findings may have direct implications for clini- recognition receptors (PRR) such as the Toll-like re- cal studies employing innate immune stimulation for ceptor (TLR) family is in clinical use for the topical cancer immunotherapy. treatment of skin tumors (imiquimod; Aldara®). Further TLR agonists are currently under develop- ment for clinical use. We have demonstrated earlier that TLR-targeting therapy can be limited by the occurrence of TLR tol- erance, a hyporesponsive state induced by repetitive systemic stimulation with single ligands. We estab- lished a protocol employing a synthetic TLR7 ligand that efficiently inhibits the growth of model tumors by taking advantage of the kinetics of receptor sen- sitivity (Bourquin*, Hotz* et al. Cancer Res 2011). In addition to the precise timing of TLR stimulation, the triggering of different receptors in a defined sequence may improve outcome. We therefore investigated the immunological effects of sequential stimulation of different PRR pathways. We found that sequential stimulation of heterologous receptor pathways not only circumvented tolerance but led to enhanced immune response compared to single stimulations. More precisely, we observed higher cytokine secretion as well as increased acti- vation of cytotoxic T lymphocytes and differentiation of type-I T helper cells, which are both implicated in anti-tumoral immunity. We demonstrate that condi- tioning with a PRR ligand rewires intracellular sig- 126 | IMPROVING IMMUNITY
EnanDIM®: TLR-9 activating oligodeoxynucleotides stabilized by an evolutionary concept
Kapp K.1, Kleuss C.1, Schroff M.1, Wittig B.2
1Mologen AG, Berlin, Germany, 2Foundation Institute Molecular Biology and Bioinformatics, Freie Universitaet, Berlin, Germany
Introduction: DNA-based TLR-9 agonists are potent ac- tection were optimized for immunomodulatory activ- tivators of the immune system. Preclinical and ongoing ity resulting in EnanDIM® (Enantiomeric DNA-based clinical studies support the use of TLR-9 agonists as ImmunoModulator). The best EnanDIM® identified, immunomodulators, and proof their anti-tumor effect EnanDIM581, induces a strong activation of cells of by enhancing both the cellular and humoral responses. both the innate and adaptive immune system in the So far two principally different classes of TLR-9 ag- context of peripheral blood mononuclear cells (PBMC). onists are established: The single-stranded oligode- EnanDIM581 leads to activation of plasmacytoid and oxynucleotides of the CpG-ODN type and the cova- myeloid dendritic cells, monocytes, NK cells, NKT lently-closed, dumbbell-shaped DNA molecules of cells, T cells, B cells, and also induces specific cytotox- dSLIM® type. Members of both classes present DNA icity against target cells. Cytokine secretion and activa- with cytosin not methylated in CG-motifs as the main tion of immune cells by EnanDIM581 depend on type I principle of TLR-9 activation. The dSLIM class agonists interferon secreted by plasmacytoid dendritic cells. The are protected against nucleolytic degradation through activation of the IFN-alpha pathway by EnanDIM581 their covalently-closed, natural phosphodiester back- is as efficient as by linear PTO-modified type A and C bone. In contrast, CpG-ODN achieve metabolic stability CpG-ODN. However, no off-target effects on immune by a variety of chemical modifications and/or non-nat- cells are detectable, even at high concentrations of ural nucleotide linkages in their backbone, of which EnanDIM®. the most commonly used are internucleotide phospho- Conclusions: After dSLIM, EnanDIM® comprises a rothioates (PTO). These, however produce off-target second group of TLR-9 agonists with conformation-me- effects in immune cell populations and their toxicity diated nuclease-resistance that significantly activates leads to severe adverse events in clinical trials. endogenous IFN-alpha release from key modulatory Results: To avoid such off-target effects and toxicity we immune cells. The broad immune activation potential used L(+)-deoxyribose containing nucleotides at the with strict CG-motif dependence and the absence of 3’-end of linear oligodeoxynucleotides. These nucleo- off-target effects favors EnanDIM® as TLR-9 agonist for tides are enantiomer to D(-)-deoxyribose containing nu- clinical development in the treatment of cancer, either cleotides. Due to the dominance of D(-)-(deoxy)ribose in as monotherapy, in combination with check point in- nucleotides of present life on earth, the co-evolved nu- hibitors, or other immunotherapeutics. cleases are blind for L(+)-deoxyribose thereby leaving L-protected oligodeoxynucleotides intact. Based on sequence variations around three central CG-motifs, oligonucleotides with 3’-terminal L-pro- 127 | IMPROVING IMMUNITY
Augmented melanoma immunity by combining irradiation and RIG-I activation
Lambing S.1, Holdenrieder S.1, Hagen C.1, Trimpop N.1, Müdder T.2, Garbe S.2, Hartmann G.1, van den Boorn J.1
1University Hospital Bonn, Institute of Clinical Chemistry and Clinical Pharmacology, Bonn, Germany, 2University of Bonn, Department of Radiology, Bonn, Germany
The efficacy of antitumor radiotherapy is often ham- Our data warrant further exploration and develop- pered by tumor radioresistance. Approaches to thera- ment of this combinatorial immunotherapy regimen, peutically enhance radiotherapy are limited. However, to the benefit of future cancer immunotherapy. combinations between radiotherapy and immuno- therapy are largely unexplored. Here, we demonstrate that activation of the retinoic acid inducible gene I (RIG-I) receptor within murine melanoma cells syner- gizes with simultaneous therapeutic irradiation. This approach can embody a new effective combinatorial immunotherapy regimen. Murine B16.F10 melanoma cells were irradiated in vitro and in vivo with a therapeutic dose of 2 Gy under controlled conditions (Mevatron MD linear accelera- tor, Siemens) while simultaneously RIG-I was activat- ed by 5’-triphosphorylated RNA (3pRNA) transfection or intratumoral injection. In comparison to control conditions, melanoma cell apoptosis, MHC class-I up- regulation and IFN type-I, IL6 and CXCL10 cytokine secretion, as well as HMGB1 release and calreticulin exposure, were enhanced by simultaneous irradia- tion and RIG-I activation. The ability of the treated tumor cells to activate melanocyte antigen-specific T cells in vitro and recruit- and activate an immune response in vivo were maximized by the combinato- rial treatment. The ionizing radiation sensitized the tumor cells for 3pRNA-induced cell death via the mito- chondrial apoptosis pathway and elevated cellular IFN responsiveness. In summary, activating RIG-I under simultaneous therapeutic 2 Gy irradiation showed an enhanced effect in both immunogenic cell death and RIG-I-induced immune activation. 128 | IMPROVING IMMUNITY
NK cells expressing Tim-3 and Ceacam1 are functionally impaired
Lee Y.1, Sunwoo J.B.1
1Stanford University School of Medicine, Stanford Cancer Institute; Stanford Institute for Stem Cell Biology and Regenerative Medicine and Program in Immunology, Stanford, United States
ORAL ALK SHORT T 2015
Immune exhaustion arises from cell dysfunction we found Tim-3+Ceacam1+ NK cells to have the during chronic exposure to stimulation, resulting in most impaired cytotoxicity. Importantly, blockade of poor effector functions and proliferative capabilities, Ceacam1 by itself restored cytotoxicity of Tim-3+Cea- which can be detrimental during disease progres- cam1+ NK cells. These findings introduce Tim-3 and sion. Although natural killer (NK) cell exhaustion Ceacam1 as markers of exhaustion in murine NK is poorly understood, there is growing evidence that cells and provide a framework by which to monitor their development of exhaustion may be analogous to NK cell exhaustion following activation. In addition, that of CD8+ T-cells, where there is a progressive loss our results introduce Ceacam1 an important target of effector functions with accumulation of inhibitory for the modulation and enhancement of NK cell ac- receptor expression. In this study, we describe the tivity in the context of chronic stimulation, such as surface receptor T cell immunoglobulin and mucin the tumor microenvironment. domain-3 (Tim-3) and the ITIM-containing Carci- noembryonic antigen-related cell adhesion molecule 1 (Ceacam1) as markers of exhausted murine NK cells. To determine the phenotype of tumor-infiltrating NK cells, we injected B6 mice with RMA-S, RMA-Rae- 1, and YAC-1 cells, and found tumor-infiltrating NK cells expressing varying levels of Ceacam1, Tim-3, and KLRG1. We observed that IL-2 stimulation of Tim-3-KLRG1- NK cells leads to early upregulation of KLRG1, which is then followed by upregulation of Tim-3. Further cytokine stimulation leads to upreg- ulation of Ceacam1 on Tim-3+ NK cells. Tim-3+ NK cells have reduced effector functions, as determined by cytotoxicity assays, CD107a degranulation, and IFNγ secretion; reduced expression of key receptors involved in target cell recognition such as NKG2D; and reduced proliferative capacity, as compared to Tim-3- NK cells. Tim-3+ NK cells can be further sep- arated into Ceacam1- and Ceacam1+ NK cells, where 129 | IMPROVING IMMUNITY
Therapeutic combination of immunetherapies targeting NGcGM3 and EGFR to treat cancer
Gonzalez A.1, Cars A.1, Leon K.1
1Center of Molecular Immunology, Habana, Cuba
At the center of molecular immunology (CIM) several cancer vaccines, immune-modulatory treatments and oncospecific antibodies have been developed over the last 20 years. In particular, CIM vaccines targeting the ganglioside NGcGM3 and the monoclo- nal antibody Nimotuzumab targeting the EGFr are in relatively advanced stages of clinical development. Interestingly several reports in the literature have suggested a functional relationship between these two molecules (NGcGM3 and EGFr) at the tumor cell membrane, providing a rational for exploring the therapeutic combinations of immunetherapies targeting them. In this work we explore, for the first time, such combination in a preclinical setting. We select two murine models: Lewis lung carcinoma (3LL-D122) and mammary adenocarcinoma breast cancer (4T1) where the co-expression of these mol- ecules was initially documented. We observed that indeed the anti-metastatic effect of the NGcGM3 vaccine is synergistically increased by the concomi- tant use of a passive anti-EGFR therapy. We show that such synergisms depends on the mobilization of both CD8+ and NKT+ cells. Moreover, we show that combination treatment trigger a stronger inhibition of the signaling cascades related to these molecules at the tumor cell membrane. Overall this result sup- ports the potential combination of NGcGM3 vaccines and Nimotuzumab in future CIM clinical trials. 130 | IMPROVING IMMUNITY
Basic evaluation of nanoparticles for cancer immunotherapy: the one plate/one day method
Mottas I.1, Milosevic A.2, Thauvin C.3, Delie F.3, Allémann E.3, Fink A.2, Rothen-Rutishauser B.2, Bourquin C.1
1University of Fribourg, Chair of Pharmacology, Fribourg, Switzerland, 2Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland, 3School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland
The main advantages of using nanoparticles as a only one 96-well plate. This results in a rational use drug carriers for cancer immunotherapy are: 1) the of nanoparticles and assays. Moreover, the protocol control over the timing and distribution of drugs is highly reproducible and allows easy comparison 2) the ability to solubilize or stabilize the drug and between different types of nanoparticles. protect it inside the organism, and 3) the possibility This project is part of the recently established Na- to target specific cell types. Additionally, the nano- tional Center of Competence in Research for Bio- particles may accumulate in the tumor microenvi- inspired Stimuli-Responsive Materials (www.bioin- ronment du to enhanced permeation and retention spired-materials.ch). (EPR) effect induced by an inefficient lymphatic drainage coupled with leaky vessels. Because nanoparticles are promising drug delivery system, we are investigating their use for cancer im- munotherapy. Whereas many types of highly diverse nanomaterials are well characterized, we and others have shown that their characteristics in cell culture are difficult to predict. Therefore, we have estab- lished a new standardized operating protocol (SOP) to evaluate the potential of nanoparticles for biologi- cal use. Three parameters are tested in parallel: cy- totoxicity is measured by an MTT assay and flow cytometry, immunogenicity is quantified by surface activation markers and by cytokine secretion, and cellular uptake is determined by flow cytometry and confocal microscopy. To become a potential drug de- livery system, nanoparticles must have low cytotox- icity and must be taken up by immune cells. In case of failure, discussions with chemists and physicists are necessary to design different or adapted nanopar- ticles suitable for cancer immunotherapy. The real impact of this SOP is the fact that all these data are obtained in one experimental day using 131 | IMPROVING IMMUNITY
Enhancing adoptive T-cell therapy with oncolytic adenovirus encoding CD40-ligand
Parviainen S.1,2, Bramante S.1, Hemminki O.1, Diaconu I.1, Hemminki A.1,2
1University of Helsinki, Cancer Gene Therapy Group, Department of Pathology and Transplantation laboratory, Haartman institute, Helsinki, Finland, 2TILT Biotherapeutics Ltd, Helsinki, Finland
T-cell therapy has resulted in remarkable recent clini- isting immunity against serotype 5 adenovirus. Also, cal breakthroughs in the treatment of melanoma and the ability of Ad3 to open intercellular tight junc- CD19+ leukemia, with most patients responding and tions could be a benefit when combined with T-cell many possibly completely cured. However, despite therapy. Human data has shown the ability of Ad3 to these impressive examples in small patient groups, reach tumors through the intravenous route. in most trials benefits have been limited. Several To deeply dissect how CD40L-encoding adenovirus immune evasion mechanisms have been shown to affects the immune system, we generated a murine limit the efficacy of infiltrating T-cells, eventually version of the virus (Ad5/3-CMV-mCD40L). With leading to tumor tolerance. Oncolytic adenoviruses these two viruses we are able to study the effects cause biological effects which could be utilized to of the transgene on tumor-infiltrating lymphocytes, reverse the immunosuppression. In addition to direct the T-cell graft and the composition of tumor stroma anti-tumoral effect, oncolysis can trigger danger from different aspects by studying e.g. the tumor signals at the tumor site and enhance the release of growth, apoptosis and the infiltration of tumor-spe- tumor-specific antigens, which can be taken up by cific T-cells, B cells, NK cells and ultimately dendritic antigen-presenting cells and presented to effector cells. These findings support development of clinical T-cells. trials where T-cell therapy is enhanced with onco- To achieve optimal activation of the T-cell graft, we lytic adenovirus. armed viruses with CD40L, which can trigger several immune mechanisms resulting in reduction in tumor immunosuppression. One of these is a T-helper type 1 (Th1) response which leads to activation of cyto- toxic T-cells and reduction of immune suppression. Another is direct activation of tumor resident den- dritic cells. Therefore, we constructed selectively oncolytic serotype 3 adenovirus (Ad3) featuring human telomerase (hTERT) promoter and human CD40L (Ad3-hTERT-CMV-hCD40L) which could be combined with adoptive T-cell therapy. One issue with oncolytic adenoviruses is suboptimal systemic delivery, which can be solved by using a fully Ad3 platform since most patients have with high pre-ex- 132 | IMPROVING IMMUNITY
Functional characterization of biodegradable PLGA and PLGA/PEI nanoparticles as antigen delivery system
Petrizzo A.1, Conte C.2, Tagliamonte M.1, Bifulco K.1, Carriero V.1, De Stradis A.3, Tornesello M.L.1, Buonaguro F.M.1, Quaglia F.2, Buonaguro L.1
1Istituto Nazionale Tumori ‘Pascale’, Naples, Italy, 2University of Naples ‘Federico II’, Dept. of Pharmacy, Naples, Italy, 3Natl. Res. Council - Inst. Sustainable Plant Protection, Bari, Italy
Efficacy of vaccines based on peptide may suffer from limited stability and inefficient delivery to pro- fessional antigen-presenting cells (APCs), such as dendritic cells (DCs). In order to overcome such limi- tations, several biodegradable nanoparticles (NPs) as delivery system have been and are developed. The present study describes the extensive biological characterization of NPs based on poly (D,L-lactic-co- glycolic acid - PLGA) alone or in association with polyethylenimine (PLGA/PEI) for subsequent ex- ploitation as antigen delivery system in therapeutic cancer vaccine development. Both PLGA and PLGA/PEI NPs don’t show any toxic effects when loaded on either mouse hepatoma cell line or human PBMCs. Flow cytometry as well as confocal laser scanning microscopy (CLSM) show that PLGA/PEI NPs are more readily taken up than PLGA NPs by both CD14+ monocytes and mouse Hepa 1-6. Intracellular localization of both NPs has been assessed in sequential image acquisition by TEM. Finally, OVA antigen loaded on either of the two NPs induces an activation of autologous naïve CD4+ T cells in an ex vivo priming experiment more efficiently than free antigen. In conclusion, both PLGA and PLGA/PEI NPs are an efficient antigen delivery system since they are non-toxic and enhance antigen presentation to pro- fessional APCs, suggesting their potential efficient exploitation in therapeutic cancer vaccine develop- ment. 133 | IMPROVING IMMUNITY
RNAi-based silencing of STAT3 during intradermal DNA vaccination promotes antitumor T cell immunity
Rojas Colonelli N.1, Galvez Cancino F.1, Flores C.1, Ligtenberg M.2, Kiessling R.2, Lladser A.1
1Fundación Ciencia & Vida, Laboratory of Gene Immunotherapy, Santiago, Chile, 2Karolinska Institutet, Immune and Gene Therapy Laboratory, Stockholm, Sweden
The signal transducer and activator of transcription and flow cytometry analysis. Mice immunized with (STAT) 3 has been identified as an intrinsic negative pTRP2 showed TRP2-specific CD8+ T cell responses regulator of dendritic cell function and the induction that were increased with the co-administration of of antitumor T cell immunity. Accordingly, develop- shSTAT3 as compared with control shRNA. Vaccinat- ing an RNAi-based adjuvant targeting STAT3 repre- ed mice were then intravenously and subcutaneously sents an interesting strategy to improve the potency challenged with B16F10 melanoma cells. Animals of DNA vaccines encoding tumor antigens. treated with pTRP2 and shSTAT3 showed enhanced A short hairpin RNA (shRNA) against STAT3 protection against melanoma as noted by decreased (shSTAT3) and the melanoma antigen tyrosinase- lung metastasis formation and subcutaneous tumor related protein 2 (pTRP2) plasmids were generated. growth, respectively. In a therapeutic setting, we ob- STAT3 knock-down was determined in vitro by RT- served a delay in tumor growth and an increased in qPCR, western blot and flow cytometry in transfected TRP2-specific CD8+ T cell response in spleen when cells lines and in vivo by RT-qPCR in mouse skin elec- the animals were treated with pTRP2 and shSTAT3. troporated with plasmid DNA. STAT3 expression was In conclusion, we showed that silencing STAT3 increased in tissue treated with either empty plasmid during intradermal DNA electroporation promotes or control shRNA. In contrast, shSTAT3 was able to maturation of dendritic cells and antitumor CTL re- decrease STAT3 expression in mouse skin and trans- sponses in a mouse melanoma model. fected cells. Also, we evaluated the maturation status Acknowledgement: CONICYT PFB-16, CONICYT- of skin-derived dendritic cells in draining lymph 791100038, FONDECYT-11110525, CONICYT Fellow- nodes by flow cytometry. Intradermal DNA elec- ship, CORFO INNOVA 12IDL2-13348. troporation induced maturation of dermal dendritic cells, which was further promoted by co-adminis- tration of shSTAT3, as compared to control shRNA or empty plasmid. Similar results were observed when human monocyte-derived dendritic cells were electroporated and shSTAT3 further increased their ability to promote proliferation of T cells in a mixed lymphocyte reaction. Antigen-specific CD8+ T cell responses were eval- uated in mice after two vaccinations by in vitro peptide stimulation followed by cytokine staining 134 | IMPROVING IMMUNITY
Releasing the cancer immunosuppressing brakes: calling to arms of dendritic cells
Wingham B.J.1, Greenman J.1, Rosca E.V.1
1University of Hull, School of Biological Sciences, Hull, United Kingdom
Phase III clinical trials in active cancer immu- potentially lead to the different activation of effector notherapy have risen sharply in the last 5 years, T cells and perhaps even of T regulatory cells. with hundreds of ongoing trials, however only one Detailed characterisation in the changes of cy- therapy is currently approved, indicating a strong tokines (IL2, IL6, and IL10) along with changes in T need for better optimisation of this approach, and cell effector activity subsequent to this activation, especially for its translation in vivo rather than the are ongoing. Further studies will convert the multi- current ex vivo. Active immunotherapy is unique as valent targeting to thermally responsive liposomes it relies on reciprocal learning; while the immune which will enable further activation of the immune system teaches researchers about its intricacies, system using focused ultrasound, an emerging non- the researchers teach the immune system how to invasive cancer treatment modality. overcome the breaks imposed by the tumour and become a much more effective weapon of destruc- tion. Our research focuses on encouraging the re-activa- tion of the immune system through specific target- ing of dendritic cells. We have developed a multi- valent nanoplex capable of inducing the activation of immature dendritic cells present in the tumour microenvironment. Engaging the main activation receptor, CD40 via a specific peptide, we can modu- late the activation of these cells leading to further stimulation of the effector T cells. Taking advantage of multivalency binding, and thus enhanced avidity, specific targeting becomes possible even in the pres- ence of low expression of activating receptors. The nanoplex, assembled using the Biotin-AMSYEG-
SWRAWVMWGGCG-NH 2 peptide and streptavidin, has demonstrated enhanced binding and activation over the single targeting peptide. This activation translates in changes of the expression levels of re- ceptors such as CD80, CD86, CD 11a, b, c which will 135 | IMPROVING IMMUNITY
Delivery of IFNα to PD-L2-expressing cells greatly potentiates chemotherapy efficacy
Rossi A.1, Macchia I.1, La Sorsa V.1, Sestili P.1, Garcin G.2, Bordat Y.2, Uzé G.2, Belardelli F.1, Proietti E.1, Bracci L.1
1Istituto Superiore di Sanità, Rome, Italy, 2University of Montpellier II, Montpellier, France
ORAL ALK SHORT T 2015
Cancers develop in complex tissue environments a longer survival time, as compared to CTX alone. which they depend on for sustained growth, inva- The therapeutic effect observed in mice treated with sion and metastasis[1]. It’s now clear that certain CTX and PD-L2-IFN-Nb was comparable to that of chemotherapeutic drugs, including the alkylating CTX and a 100-fold higher dose of soluble IFN-I and agent cyclophosphamide (CTX), exert their activi- stronger than that of a comparable dose of soluble ty not only by directly killing tumor cells, but also IFN-I. Of note, PD-L2-IFN-Nb or soluble IFN, without by subverting the immunosuppressive equilibrium prior CTX administration, had no therapeutic effect. within the tumor microenvironment [2]. We ob- Preliminary data from mice bearing PD-L2- tumors, served that in CTX-treated animals, the recruitment showed no synergistic therapeutic effect between of activated cross-presenting DC is paralleled by the CTX and IFN-I treatment, independently of IFN-tar- accrual of macrophages and of granulocytic my- geting. Our data so far suggest that targeted delivery eloid-derived suppressor cells expressing high levels of IFN-I to specific components of tumor microen- of the immunosuppressive surface molecule pro- vironment, including tumor cells, greatly increases grammed death-ligand 2 (PD-L2). We previously re- chemotherapy therapeutic effect and represents an ported a strong synergistic antitumor effect between attractive strategy to circumvent IFN-related toxicity. CTX and type I interferon (IFN-I) administration in mice bearing implantable tumors[3]. Indeed, these cytokines have considerable clinical potential as anticancer agents due to their antiproliferative and 1. Quail and Joyce. Nat Med, 2013 2. Bracci L et al. Cell Death Differ, 2013 immunomodulatory effects. However, their clinical 3. Schiavoni G et al. Cancer Res, 2011 use has been severely hampered by their intrinsic, 4. Bracarda S et al. Eur J Cancer, 2010 5. Garcin G et al. Nat Commun, 2014 systemic toxicity mainly due to the heterogeneity of cell subsets they act on[4]. In the present study we aimed at evaluating the therapeutic efficacy of a nanobody (Nb) selectively targeting the interfer- on-α (IFNα) molecule to PD-L2-expressing cells[5] when administered three days after CTX treatment i.e., at the peak of PD-L2+ cells recruitment within the tumor mass. In mice bearing PD-L2+ tumors the injection of PD-L2-IFN-Nb after CTX administra- tion induced a bigger reduction of tumor size and 136 | IMPROVING IMMUNITY
N1-methylpseudouridine-modified mRNA outperforms pseudouri- dine-modified mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice
Andries O.1, De Smedt S.2, Weiss R.3, Kitada T.3, Sanders N.1
1Ghent University, Laboratory of Gene Therapy, Merelbeke, Belgium, 2Ghent University, Ghent Research Group on Nanomedicine, Faculty of Pharmaceutical Sciences, Ghent, Belgium, 3Massachusetts Institute of Technology, Department of Biological Engineering, Cambridge, United States
In this work we demonstrated that the incorporation of N1-methylpseudouridine (mΨ) in mRNA during in vitro transcription drastically improved the trans- lational capacity of mRNA compared to non-modi- fied and pseudouridine Ψ( )-modified mRNA. The superiority of mΨ-modified mRNA overΨ -modified mRNA was demonstrated after lipofection in human lung epithelial cells, human foreskin fibroblasts, murine myoblast cells, human cervix epithelial cells, and human primary keratinocytes from neo- natal foreskin. We also showed in these cell lines that mΨ-modified mRNA was less toxic than non-modi- fied or e.g. Ψ-modified mRNA. In line with this obser- vation mΨ-modified mRNA did not induce cytokines, while an extensive and moderate cytokine induction was observed with respectively non-modified and Ψ-modified mRNA. Furthermore, we showed that the superiority of mΨ-modified mRNA overΨ -modified mRNA may be due to its improved ability to evade TLR3 activation. Finally, mΨ-modified mRNA result- ed also in a much higher luciferase expression after intradermal and intramuscular electroporation or li- pofection in mice than non-modified or Ψ-modified mRNA. 137 | IMPROVING IMMUNITY
Whole cancer cell vaccines based on immunogenically killed cancer cells and dendritic cells
Cicchelero L.1, Sofie Denies S.1, de Rooster H.2, Sanders N.1
1Ghent University, Laboratory of Gene Therapy, Merelbeke, Belgium, 2Ghent University, Department of Medicine and Clinical Biology of Small Animals, Merelbeke, Belgium
Immunogenic cell death offers interesting opportuni- ties for the manufacture of cancer cell vaccines, as it increases the immunogenicity of the dead cancer cells. Furthermore, fusion of cancer cells with den- dritic cells is considered a superior method for gen- erating whole cancer cell vaccines. Therefore, in this work, we determined in naïve mice whether immu- nogenic killed cancer cells elicit a stronger immune and anti-tumor response after fusion or co-incuba- tion with dendritic cells. After tumor inoculation, the type of immune response in the prophylactically vac- cinated mice differed between the groups. In more detail, fusion vaccines elicited a humoral anticancer response, whereas the co-incubation and cancer cell vaccine mainly induced a cellular response. Despite these differences, all three approaches offered pro- phylactic protection against tumor development in a murine mammary carcinoma model. In summary, it can be concluded that whole cancer cell vaccines based on immunogenically killed cancer cells may not necessarily require association with dendritic cells to elicit a protective anticancer immune re- sponse. If this finding can be endorsed in other cancer models, the manufacture of cancer cell vac- cines would greatly benefit from this new insight, as production of dendritic cell-based vaccines is labori- ous, time-consuming and expensive. 138 | IMPROVING IMMUNITY
In-situ activity of tumor specific CTL determines colorectal cancer prognosis
Stamova S.1, Reissfelder C.2, Gossmann C.1, Braun M.1, Bonertz A.3, Walliczek U.4, Koch M.2, Benner A.1, Jäger D.5, Halama N.5, Khazaie K.6, Weitz J.2, Beckhove P.1
1Deutsches Krebsforschungszentrum, Heidelberg, Germany, 2University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany, 3Paul Ehrlich Institute, Langen, Germany, 4Ruprecht-Karls University, Heidelberg, Germany, 5National Center of Tumor Diseases, Heidelberg, Germany, 6Mayo Clinic College of Medicine and Graduate School, Rochester, United States
T cell infiltrates are a major prognostic factor in colorec- tal cancer (CRC) but their potential functional role in prolonged patient survival remains unclear. We there- fore studied in altogether 190 CRC patients the presence of functionally active tumor infiltrating lymphocytes (TIL), their tumor specificity and their correlation to long term survival. Using intracytoplasmic cytokine staining in conjunc- tion with tumor peptide loaded human leukocyte antigen (HLA)-multimers and antigen specific cytokine secretion assays we demonstrate that tumor necrosis factor alpha (TNFα) expression delineates a population of tumor antigen (TA) specific, in situ active cytotoxic T cells (CTL). Increased TNFα expression in TIL strong- ly correlated to increased total amount of intratumoral TNFα and thus indicated tumor specific CTL activity in situ. As such, increased TNFα concentration was an in- dependent prognostic factor in a retrospective multivar- iate analysis of 102 CRC patients involving a.o. immune parameters such as infiltration by CD4+ and CD8+ T conventional cells, regulatory T cells or mast cells. Thus, the prognostic impact of T cell infiltrates in CRC maybe largely based on subpopulations of in situ active TA specific T cells suggesting their causal implication in patient survival. Intratumoral TNFα resembles a T cell function-associated prognostic parameter for CRC, which may be more accurate than mere T cell infiltra- tion. 139 | IMPROVING IMMUNITY
Oncolytic adenovirus improves anti-tumor efficacy of adoptive T-cell therapy by breaking tumor tolerance
Tähtinen S.1, Grönberg-Vähä-Koskela S.1, Lumen D.2, Merisalo-Soikkeli M.1, Siurala M.1,3, Airaksinen A.2, Vähä-Koskela M.1, Hemminki A.1,3
1University of Helsinki, Cancer Gene Therapy Group, Department of Pathology and Transplantation Laboratory, Haartman Institute, Helsinki, Finland, 2University of Helsinki, Laboratory of Radiochemistry, Department of Chemistry, Helsinki, Finland, 3TILT Biotherapeutics Ltd, Helsinki, Finland
Background: Despite the fast growing rate in the de- lymphocytes and F4/80+ macrophages was seen ad- velopment of novel adoptive T-cell therapies (ACT), enovirus-treated tumors suggesting enhanced tumor the clinical benefit in established solid tumors has immunogenicity. Pro-inflammatory tumor microen- remained modest. Roadblocks to effective immune vironment mediated by adenovirus infection led to attack by infiltrating tumor-specific T-cells include expression of co-stimulatory signals on CD11c+ den- several immune evasion mechanisms, which con- dritic cells and subsequent activation of T-cells, thus tribute to tumor tolerance. Oncolytic virotherapy is breaking the tumor-induced peripheral tolerance of the use of cancer cell specific, conditionally repli- T-cells. Moreover, epitope spreading in the form of cative viruses in the treatment of cancer. Oncolytic endogenous anti-melanoma T-cells was detected in virotherapy can enhance epitope spreading by direct the combination treated mice indicating that the dual oncolysis of tumor cells, leading to enhanced antigen approach can lead to systemic anti-tumor immunity. presentation by dendritic cells and increased immu- Conclusions: Treatment with oncolytic adenovirus nogenicity of the established tumor. Here, we studied can overcome resistance of B16.OVA murine mela- the combination of these two established forms of noma tumors to T-cell therapy by recruitment and immunotherapy in a highly resistant and poorly im- stimulation of tumor-infiltrating immune cells, thus munogenic B16.OVA mouse melanoma model. resulting in improved efficacy of adoptive T-cell Methods: Immunocompetent B16.OVA bearing therapy. In our model, adenovirus was required to C57BL/6 female mice were adoptively transferred unleash the power of ACT for therapeutic effects. with CD8a+ enriched, OVA-specific OT-I lymphocytes Importantly, these two modalities are not merely an and treated with intratumoral injections 5/3-fiber attractive combination, but could represent a way to chimeric adenovirus (diluted in saline) on six con- achieve “CD19-like” results in the treatment of solid secutive days. Tumor growth of mice was monitored tumors. every 2-3 days by using electronic calipers. Mice were sacrificed on day 14 post-transfer and immune composition of harvested tumors was analyzed by flow cytometry. Results: Following adoptive transfer of OT-I lympho- cytes, superior tumor growth control was observed in adenovirus-treated mice compared to control mice, even in the absence of active oncolysis. Significant increase in infiltration of CD45+ leukocytes, CD8+ 140 | IMPROVING IMMUNITY
TLR9 and STING agonists synergistically induce innate and adaptive type-II IFN
Temizoz B.1, Kuroda E.1, Ohata K.1, Jounai N.2, Ozasa K.2, Kobiyama K.2, Aoshi T.2, Ishii K.J.1,2
1Osaka University, Immunology Frontier Research Center, Laboratory of Vaccine Science, Osaka, Japan, 2National Institute of Biomedical Innovation, Laboratory of Adjuvant Innovation, Osaka, Japan
TLR9 ligand, CpG oligodeoxynucleotide (ODN), immunization. Finally, by using the syngenic mouse is one of the clinically-tested adjuvants that can tumor models, we show that intra-tumoral injection induce type-1 immunity. Recently, cyclic dinucleo- of CpG ODN and cGAMP has reduced the tumor tides, known as bacterial sencond messengers, have size significanly better than those treated alone. In been demonstrated to be potent agonistic ligands conclusion, combination of CpG ODN and cGAMP for STING to induce type-I IFN production. Cyclic is better than the singular use, as not only a potent GMP-AMP (cGAMP), one of the recently identified type-1 adjuvant for vaccines that require strong cel- mammalian cyclic dinucleotides, has been reported lular immune responses, but also an antigen-free an- to act as an adjuvant. Several concerns, however, are ti-tumor agent, by activating not only mouse, but also raised that currently available CpG ODN (K-type) is human cells towards synergistic IFNγ production. a weak inducer of type-I and type-II IFNs, and that Thus, further mechanistic and optimization studies STING-ligands are shown to induce type-2 immune are required for clarifying the therapeutic potential responses, rather than type-1 immunity, thereby of the combination. limiting their potential therapeutic applications. In this study, we found synergistic activity of TLR9 and STING agonists in innate and adaptive type-II IFN (IFNγ) induction, resulting in a potent type-1 vaccine adjuvant and an immuno-therapeutic agent in ex- planted tumor models. Our in vitro studies in human and mouse PBMC suggest that the synergistic effect between CpG ODN (K3) and cGAMP, culminated in IFNγ production by NK cells, was partly due to the
Th1-inducing cytokines, including IL-12 produced by both cDCs and pDCs. As a result, intramuscular im- munization of mice with the combination of protein antigen, CpG ODN and cGAMP resulted in the in-
duction of significanly higher antigen specific Th1 and CD8+ T cell responses, than those immunized with CpG ODN alone. Futhermore, type-2 immune responses that are induced by cGAMP immuniza- tion, are significantly suppressed in the combination 141 | IMPROVING IMMUNITY
Intratumoral T cell exhaustion increases during cancer progression and limits the activity of T cell-directed immunotherapies
Thommen D.S.1,2, Schreiner J.2, Herzig P.2, Müller P.2, Savic Prince S.3, Lardinois D.4, Zippelius A.1,2
1University Hospital Basel, Medical Oncology, Basel, Switzerland, 2Laboratory of Cancer Immunology, Department of Biomedicine, Basel, Switzerland, 3University Hospital Basel, Institute of Pathology, Basel, Switzerland, 4University Hospital Basel, Department of Surgery, Switzerland
Introduction: Dysfunctional T cells present in ma- correlated with poor response to PD-1 blockade. Of lignant lesions are characterized by a sustained and note, PD-1hi expression marked a particularly dys- highly diverse expression of inhibitory receptors, also functional T cell subset with high levels of additional referred to as immune checkpoints. Yet, their rela- inhibitory receptors and, thus, may assist in identify- tive functional significance in different cancer types ing patients likely to respond to inhibitory receptor remains incompletely understood. Immunotherapeu- specific antibodies. tic compounds targeting intratumoral immune cells Conclusions: In summary, our data suggest that can- represent a promising clinical approach to harness cer-associated T cell dysfunction is largely reflected T cells to fight against cancer. However, it is cur- by cumulative expression of inhibitory receptors and rently unknown whether the dysfunctional state of abundance of PD-1hi T cells. Ongoing experimen- T cells embedded into the tumor microenvironment tal work, which will be presented in detail in the imprints on the therapeutic activity of T cell-directed meeting, indicates that T cell dysfunction consider- therapies. ably alters the therapeutic efficacy of T cell-directed Methods: We performed a comprehensive charac- therapies and requires combinatorial immunothera- terization of the diversity and expression patterns of py approaches to unleash the full armamentarium of inhibitory receptors on tumor-infiltrating T cells in T cell effector functions. different tumor types as well as of their activation upon polyclonal stimulation. Results: We noted a large heterogeneity in the ex- pression levels of PD-1, Tim-3, CTLA-4, LAG-3 and BTLA on intratumoral CD8+ T cells from patients with different cancer types. A clear correlation was established between increased expression of these inhibitory co-receptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Co-expression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T cell function only in a subset of patients. A high frequency of PD-1hi T cells 142 | IMPROVING IMMUNITY
Irradiation of necrotic cancer cells, employed for pulsing dendritic cells (DCs), potentiates DC vaccine-induced antitumor immunity against high-grade glioma
Vandenberk L.1, Garg A.D.2, Verschuere T.1, Koks C.1, Belmans J.1, De Vleeschouwer S.3, Agostinis P.2, Van Gool S.W.1
1KU Leuven, Laboratory of Pediatric Immunology, Leuven, Belgium, 2KU Leuven, Laboratory of Cell Death Research and Therapy, Leuven, Belgium, 3KU Leuven, Experimental Neurosurgery and Neuroanatomy, Leuven, Belgium
Despite trimodal standard-of-care therapy, the prog- of DCs. Further analysis showed that, the overall nosis of patients diagnosed with high-grade glioma content of carbonylated proteins - a surrogate of im- (HGG) remains particularly dismal. Dendritic cell munogenic oxidation-associated molecular patterns (DC)-based immunotherapy has yielded promising (OAMPs) - in the FT-necrotic lysate, was increased results with objective responses reported in 15 % by the irradiation treatment. Moreover, we found a of HGG patients. The efficacy of DC vaccinations is striking correlation between the amount of protein however abated by the profound HGG-induced im- carbonylation in tumor lysates and the DC vaccine- munosuppression and, in part, a lack of attention mediated tumor rejection capacity (such that the towards immunogenicity of the tumor lysate. Our latter was partially but not significantly abrogated by literature analysis of DC vaccination clinical trials in addition of bona fide anti-oxidants). Together these HGG patients showed that the two most frequently data strongly advocate the use of protein oxidation- used methods for preparing tumor lysate are: freeze- inducing modalities like irradiation for increasing thaw (FT)-induced necrosis or FT-necrosis followed the immunogenicity of tumor lysate used for pulsing by high-dose X-ray irradiation. However, from the DC vaccines. available clinical evidence, it is not clear which of the above methodologies have a superior immunogenic potential. In view of these findings, we made use of the GL261 glioma murine model to directly compare the immunogenicity of FT-necrotic and irradiated FT-necrotic tumor lysates. Interestingly, we observed that pulsing of DCs with irradiated FT-necrotic (com- pared to FT-necrotic only) tumor lysate prolonged overall survival and increased tumor rejection in glioma-challenged mice. This was associated with an increase in brain-infiltrating effector T cells and an increase in the CD8+ T cell to Treg ratio, paral- leled by a reduced accumulation of regulatory T cells (Tregs), tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Pulsing of DCs with irradiated FT-necrotic tumor lysate did not influence phenotypic and functional maturation 143 | IMPROVING IMMUNITY
ADC-1013, an agonistic CD40 antibody generates immune mediated anti-tumor effects in syngeneic tumor models in hCD40 transgenic mice
Veitonmäki N.1, Mangsbo S.2, Gustafsson E.2, Furebring C.1, Norlen P.1, Enell-Smith K.1, Tötterman T.2, Ellmark P.1,3
1Alligator Bioscience, Lund, Sweden, 2Uppsala University, Immunology, Genetics and Pathology, IGP, Uppsala, Sweden, 3Lund University, Department of Immunotechnology, Lund, Sweden ORAL ALK SHORT T 2015
Local administration of immune activating anti- bodies may increase the efficacy and reduce the immune-related adverse events associated with sys- temic immunotherapy of cancer. Here we report the development of a fully human agonistic CD40 anti- body (IgG1), ADC-1013, which has been optimized by increased affinity, potency and tumor retention. Syngeneic tumor models, both negative and positive for human CD40, were used to demonstrate anti- tumor effects in a human CD40 transgenic mouse (hCD40tg). ADC-1013 induced significant anti-tumor responses and long term tumor immunity in a CD40 negative bladder cancer model (MB49). The anti-tu- mor immunity was shown to be T-cell dependent and naïve mice were protected from tumor challenge by transplantation of splenocytes from cured hCD40tg mice. Local treatment induced systemic anti-tumor effects as peritumoral administration of ADC-1013 in one tumor prevented the tumor growth in a distal un- treated tumor. Further, significant anti-tumor effects were demonstrated in two aggressive subcutaneous melanoma models, positive and negative for human CD40, respectively. ADC-1013 is a fully human and highly potent agonis- tic CD40 immunomodulatory antibody developed for immunotherapy of cancer. ADC-1013 will enter first in human clinical trials in patients with advanced solid tumors in early 2015. 144 | IMPROVING IMMUNITY
Gemcitabine reduces the level of TGFβ-1 in plasma from pancreatic cancer patients and potentiates immunotherapy in a model of pancreatic cancer
Svensson E.1, Wenthe J.1, Irenaeus S.1,2, Ullenhag G.1,2, Loskog A.1,3
1Uppsala University, Dept of Immunology, Genetics and Pathology, Uppsala, Sweden, 2Uppsala University Hospital, Dept of Oncology, Uppsala, Sweden, 3Lokon Pharma AB, Uppsala, Sweden
Immunotherapy can be potentiated by conditioning granulocytic MDSCs were similar between HCs and regimens that reduce suppressive immune cells prior patients. Overall MDSCs were not affected by gemcit- or during treatment. Commonly, chemotherapeutics abine treatment at these time points. Normal mono- with lymphodepleting ability, such as cyclophospha- cytes were affected by gemcitabine as shown by a mide, can reduce the levels of T regulatory cells (Tregs) transient decrease of the activation markers CD86 and in cancer patients and the tyrosine kinase inhibitor HLA-DR. Tregs were increased in patients compared sunitinib decreases myeloid derived suppressor cells to HCs and while gemcitabine treatment initially de- (MDSCs) in renal cell carcinoma. We hypothesized creased Tregs the levels were increased after one week that gemcitabine, standard treatment for pancreatic of resting. Likewise, immunosuppressive TGFβ-1 was cancer, may be used as a metronomic conditioning for significantly reduced after treatment but the levels these patients. Hence, the immune profile of pancre- were restored at day 28. The levels of natural killer atic cancer patients was determined at different time cells (NKs) and T lymphocytes in the patients were points post gemcitabine initiation, and the combina- equal to the levels in HCs but the T lymphocytes had tion of gemcitabine with immunotherapy was evalu- a lower expression of CD107a than HCs. Nevertheless, ated in an experimental model of pancreatic cancer. gemcitabine treatment did not affect the lymphocyte Patients with pancreatic cancer received standard proliferation capacity to OKT-3 and IL-2 at any time cycles of gemcitabine consisting of weekly doses for point. In mice, mLOAd703 adenovirus therapy can three weeks followed by one resting week prior to the reduce tumor growth. However, gemcitabine given next cycle. Peripheral blood mononuclear cells and metronomic during this immunotherapy significantly plasma were collected at the start of the first, second enhanced the efficacy of mLOAd703. and third treatment of gemcitabine (day 0, 7, 14) and In conclusion, gemcitabine regulates the immune after the first week of resting (day 28). The samples system in patients with pancreatic cancer including were analyzed by flow cytometry, Meso Scale multi- molecules such as TGFβ-1 but do not hamper the plex assay, ELISA and Alamar Blue proliferation kit. ability of lymphocytes to expand to stimuli. In a model Murine pancreatic cancer cells (Panc02) were im- of murine pancreatic cancer, gemcitabine potentiated planted into C57BL6 mice and the tumors were treated the effect of the immunotherapy mLOAd703. These with gemcitabine weekly, with adenovirus immuno- results hold promise for combining gemcitabine with stimulatory gene therapy (murine LOAd703) biweekly, immunotherapy in clinical trials. or the combination of both treatments. Pancreatic cancer patients had a higher level of mono- cytic MDSCs compared to healthy controls (HC) while 145 – 210
Cellular Therapy 145 | CELLULAR THERAPY
Development of EGFRvIII targeted T-cell therapy for brain tumours
Agliardi G.1, Kiru L.2, Chen D.Y.1, Ramasawmy R.2, Siow B.2, Lythgoe M.2, Anderson J.1, Pule M.3, Badar A.2, Straathof K.1
1Universty College London, Institute of Child Health, London, United Kingdom, 2UCL, Centre for Advanced Biomedical Imaging, London, United Kingdom, 3UCL Cancer Institute, London, United Kingdom
High grade gliomas are aggressive brain tumours. EGFRvIII-specific second generation murine CAR, The main challenges in the treatment of this disease Firefly Luciferase and a CD34-based marker gene are its location within the central nervous system from a single retroviral vector. Antigen-specific lysis (CNS) and its capability to spread within the brain of GL216_EGFRvIII cells by EGFRvIII-CAR expressing parenchyma. These features make recurrence fol- splenocytes was demonstrated in a chromium release lowing current standard therapy including surgery, assay. EGFRvIII_GL261 were stereotactically inject- radiotherapy and temozolomide common. Therefore ed into the right striatum of syngeneic C57Bl6 mice. there is an urgent need for new treatment strategies. Mice with established tumours as determined by Chimeric antigen receptor (CAR)-based immuno- MRI (ICON system, Bruker), received 5 Gy total body therapy has shown promising results in haemato- irradiation followed by intravenous injection of 5x106 logical cancers. Treating solid cancers with the same EGFRvIII-CAR or irrelevant CAR transduced spleno- approach, however, represents a bigger challenge, cytes. Migration of CAR-transduced splenocytes into as modified T-cells have to actively migrate to the the brain was monitored by serial bioluminescence tumour and will likely encounter a highly immu- imaging. Effect on tumour growth is determined by nosuppressive environment. So far, the majority of serial MRI imaging and immunohistopathological studies on CAR immunotherapy in brain tumours examination of brain tissue upon sacrifice. Data to have used xenograft models in which human-derived date demonstrate that 72 hours after administration glioma cells have been injected in immunocompro- EGFRvIII-CAR expressing splenocytes are present at mised mice. These models can recapitulate the fea- the tumour site and persist over time in an antigen- tures of the human cancer, but provide limited infor- dependent manner. Efficacy data are currently being mation regarding the optimal setting and efficacy for acquired. cell-based immunotherapeutics. In summary, we have demonstrated in vitro func- Here we used tumour antigen Epidermal Growth tion and specificity of a murine second generation Factor Receptor variant III (EGFRvIII) to develop EGFRvIII-specific CAR and, importantly, capabil- an immunocompetent murine model to study cell ity of these CAR T-cells to systemically engraft and therapy approaches for brain tumours. EGFRvIII, migrate from the periphery to accumulate in the a variant form of EGFR lacking exons 2 to 7, is a CNS after antigen recognition. This immunocompe- mutation present in approximately 30% of gliomas tent murine model provides an informative tool to while absent in normal tissue. Murine glioma cell develop and evaluate immunotherapy approaches for line GL261 was transduced to express EGFRvIII. brain tumours. Activated splenocytes were modified to express an 146 | CELLULAR THERAPY
HLA.A2-restricted MDM2 (81-88) epitope as a potential tumor- associated antigen for TCR gene therapy for multiple myeloma
Amann E.1, Antunes E.1, Jacobi B.1, Theobald M.1,2,3, Echchannaoui H.1,2
1University Medical Center of the Johannes Gutenberg University Mainz, Third Department of Medicine, Mainz, Germany, 2German Cancer Research Center, German Cancer Consortium, Frankfurt/Mainz, Germany, 3Johannes Gutenberg University Mainz, Research Center Immunology, Mainz, Germany
Background: Adoptive transfer of T cells retrovirally screened MM cell lines for MDM2 protein expression transduced with a tumor-associated antigen (TAA) via Western Blot and determined cell surface expres- -specific T cell receptor (TCR) has shown promise in sion of HLA.A2 by flow cytometry. the treatment of cancer patients. The human homolo- In our xenograft mouse model the MM cell line NCI- gous of the murine double-minute 2 protein (MDM2) H929 cells was injected subcutaneously in NOD-scid TAA is overexpressed in a variety of human tumors, gamma (NSG) mice and human T cells transduced including soft tissue sarcoma, multiple myeloma with the cys.opt. MDM2 TCR were injected intrave- (MM), glioblastoma and melanoma. MDM2 protein nously when the tumor were palpable. overexpression is particularly observed in invasive Results: TCR expression in human T cells transduc- and metastatic melanoma and is associated with en- ed with the cys.op. MDM2 TCR was higher compared hanced survival of MM cells. For our purpose we to T cells transduced with the wt TCR whereas the have generated and optimized an HLA.A2-restricted expression levels in transduced Jurkat 76 were equal. CD8-dependent MDM2 (81-88)-specific TCR isolated The MM cell line NCI-H929 showed, after transfec- from a high-avidity murine CTL clone derived from tion with HLA.A2 molecule, one of the highest ex- CD8 x A2Kb transgenic mice. Our aims is to genetical- pression levels of this molecule and the strongest ly equip human T cells with MDM2 TCR for targeting MDM2 expression. This cell line showed also a very cancers overexpressing MDM2 in adoptive immuno- good recognition by the cys.opt. MDM2 TCR in vitro therapy. in a cytolytic assay. We could verify this data in vivo Methods: We modified the wild-type (wt) MDM2- in a xenograft mouse model where we could observe TCR-construct to increase the expression level of in- a prolonged overall survival in mice which received troduced TCR and to reduce potential autoreactivity. the MDM2-specific TCR transduced T cells compared The nucleotide sequence was codon-optimized (opt.) to the Mock-treated group and we detected homing of and TCR chains cloned in a bicistronic retroviral transduced T cells to the tumor. vector containing the self-cleaving 2A virus-derived Conclusion: These results demonstrate that the cys. peptide. To enhance the safety of introduced TCR we opt. MDM2-specific TCR shows an improved safety also introduced an additional inter-chain disulfide compared to the wt TCR and MM can be targeted by bond (cys.) between TCR α and β constant domains this TCR in vitro and in vivo. In addition we are cur- to prevent mixed heterodimers formation with poten- rently establishing a melanoma mouse model. Using tial autoreactivity. Human T cells were retrovirally MDM2-specific TCR in adoptive T cell immunothera- transduced with the cys.opt. MDM2 TCR and TCR py may be a promising approach for targeting cancer expression level was analysed by flow cytometry. We cells overexpressing MDM2. 147 | CELLULAR THERAPY
Adoptive T cell therapy with intermediate dose interleukin-2 achieves long-lasting complete responses in heavily pre-treated melanoma patients
Andersen R.1, Donia M.1, Ellebæk E.1, Holz Borch T.1, Kongsted P.1, Zeeberg Iversen T.1, Met Ö.2, Hald Andersen M.2, Thor Straten P.2, Svane I.M.1
1Herlev University Hospital, Center for Cancer Immune Therapy, Department of Hematology and Department of Oncology, Herlev, Denmark, 2Herlev University Hospital, Center for Cancer Immune Therapy, Department of Hematology, Herlev, Denmark
Background: Adoptive cell therapy (ACT) with infused and importantly, in most responding pa- tumor infiltrating lymphocytes (TILs) achieved tients we observed induction and persistence of impressive clinical results in several single insti- anti-tumor T-cell responses in the peripheral blood. tution phase I/II clinical trials performed outside Conclusion: As the first European institution we Europe, and holds the promise to enter the main- show that TIL-based ACT is reliable, logistically stream of standard melanoma care in the near feasible and clinically effective. Importantly, a high future. However, although transient, the toxicities response rate including long-lasting complete re- associated with high-dose bolus interleukin-2 (IL-2) sponses can be induced after TIL infusion followed classically administered together with TILs are by a reduced regimen of IL-2, which considerably severe and recent results have questioned its use. reduced the occurrence of severe side effects. Effec- To further scrutinize IL-2 dosing, we conducted a tive TIL therapy is associated with induction and clinical trial administering TILs after classical lym- long-term persistence in the blood of T cells produc- phodepletion but followed by an attenuated regimen ing in vitro anticancer responses. of IL-2. Materials and methods: A total of 25 patients with progressive metastatic melanoma, PS ≤ 1, age < 70 and at least one resectable metastasis were included in this phase II trial (NCT00937625). TIL infusion was preceded by standard lymphodepleting chemo- therapy, with cyclophosphamide and fludarabine, but followed by an intermediate dose IL-2 admin- istered in an intravenous, continuous decrescendo regimen. Results: 25 patients were treated and the reduced dose of IL-2 considerably reduced the toxicity of the treatment. Imaging evaluations showed three com- plete responses (38+, 24+ and 14+ months) and seven partial responses (36+, 27+, 20+, 12, 12, 9+ and 8 months) with most responses still ongoing (response rate 40%). Clinical responses were asso- ciated with high numbers of tumor reactive T-cells 148 | CELLULAR THERAPY
Peptide-MHC guided stimulation of antigen-specific T cells for immune therapy
Bentzen A.K.1, Rasmussen V.M.1, Pedersen N.W.1, Jakobsen S.N.2, Hadrup S.R.1
1Technical University of Denmark, Section for Immunology and Vaccinology, Frederiksberg C, Denmark, 2Immudex, Copenhagen, Denmark
The capacity of T cells to specifically eliminate MHC responsive T cells of choice in a heterogeneous tumors have repeatedly been demonstrated in clini- mixture of lymphocytes. cal studies of adoptively transferred tumor infil- We have set out to investigate the optimal composi- trating lymphocytes in malignant melanoma, with tion of the MHC klickmer reagents that will foster a promising response rates of 20-50%. The treatment persistent phenotype and functional characteristics strategy has the major advantage that patients obtain of T cells ideal for target cell killing and antigen- an immunological memory directed against their specific memory. We have confirmed that klickmer own tumor. Still a great proportion of patients do not reagents generated with peptide-MHC molecules of respond and in this regard it seems that the speci- choice bind with sufficient avidity to cognate anti- ficity and quality of the applied cells are of crucial gen-specific T cells. Importantly we have verified importance for tumor-cell killing and development of that the interaction with antigen-specific T cells is tumor-specific memory. Nevertheless, current meas- retained when these reagents carry costimulatory ures for rapid expansion of T cells from tumor mate- molecules, IL-15 and CD86, in different ratios along rial does not offer any way of discriminating cancer- with a given peptide-MHC molecule. specific T cells from those of irrelevant specificities Preliminary data after culturing of peripheral blood or even inhibitory functionalities. Moreover, there mononuclear cells from healthy donors already sug- is a screwing towards preferential growth of virus- gests a preferential expansion of those antigen-spe- specific T cell populations as well as an increased cific T cells that are specifically targeted with MHC exhaustion phenotype of T cells after culturing. klickmer reagents carrying IL-15 and CD86. Cur- To reverse these phenomena we are developing ar- rently, various combinations of cytokines are being tificial antigen presenting scaffolds using the MHC investigated to optimize the MHC directed culturing klickmer system from Immudex - an MHC multimer system. composed of a dextran polysaccharide backbone with peptide-MHC and a number of free streptavi- din sites for attachment of biotinylated molecules of choice. These are utilized to attach co-stimulatory molecules and cytokines to provide stimulation in an antigen-specific manner. Such reagents can efficient- ly multiply and functionally redirect cancer-specific T cells through peptide-MHC directed manipulation, which allows efficient expansion only of the peptide- 149 | CELLULAR THERAPY
Cellular and molecular events controlling the cytotoxic activity of melanoma-reactive CD4+ T cells
Bergerhoff K.F.1, Sledzinska A.1, Veliça P.2, Arce F.1, Briesemeister D.1, Ono M.3, Stauss H.2, Chakraverty R.4, Peggs K.S.1, Quezada S.A.1
1UCL Cancer Institute, University College London, Research Department of Haematology, Cancer Immunology Unit, London, United Kingdom, 2Royal Free Hospital, University College London, Institute of Immunity & Transplantation, London, United Kingdom, 3Institute of Child Health, University College London, Immunobiology Unit, London, United Kingdom, 4University College London, Department of Haematology, Division of Cancer Studies, London, United Kingdom
While there is an abundance of studies on CD8+ CTLs cytotoxic phenotype while preserving the production in cancer therapy, the potential of tumour-reactive of inflammatory cytokines. This study underlines the CD4+ T cells remains understudied. Recently, we importance of mTORC1 signalling for the cytotoxic underscored the significance of melanoma-specif- activity of highly plastic, tumour specific CD4+Trp1 ic CD4+ T cells (CD4+Trp1) by demonstrating their T cells. capacity to acquire cytotoxic activity in vivo and to promote complete eradication of established lesions resulting in long-term survival and protection. In order to determine the molecular and cellular mechanisms underpinning CD4+ cytotoxicity we compared CD4+Trp1 cells differentiated in vivo into helper and cytotoxic lineages. Microarray and FACS analysis of tumour infiltrating CD4+Trp1 illustrated a highly plastic phenotype: Th1 and Th2 specific tran- scription factors Gata3 and T-bet were co-expressed and inflammatory cytokines IFNγ, TNFα and IL-2 were secreted. Additionally, CD8+ lineage specific transcription factor Runx3 expression was elevat- ed on cytotoxic CD4+Trp1 and correlated with high levels of GzmB expression. However, and in contrast to classical CD8+ CTLs, cytotoxic CD4+ T cells lack expression of CD8+ memory associated transcription factor Eomes. Further microarray analysis revealed a high correlation of tumour infiltrating CD4+Trp1 cells with a full effector CD8+ T cell gene signature rather than a CD4+ or CD8+ memory phenotype. Futher- more, due to the important role of mTOR signaling in CD8+ effector differentiation, inhibition of mTORC1 via administration of Rapamycin and genetic engi- neering of CD4+Trp1 cells was performed. Disruption of mTORC1 signaling prevented the acquisition of a 150 | CELLULAR THERAPY
Ex-vivo expanded T cells significantly prolong overall survival in stage IV melanoma patients treated with autologous hTERT/ survivin mRNA-loaded dendritic cells
Bigalke I.1, Brinchmann-Torhaug S.2, Lundby M.1, Mollatt C.1, Suso-Inderberg E.-M.1, Gaudernack G.3, Rasmussen A.-M.3, Aamdal S.2, Kvalheim G.1
1Oslo University Hospital - The Norwegian Radium Hospital, Dept. of Cellular Therapy, Oslo, Norway, 2Oslo University Hospital - The Norwegian Radium Hospital, Dept. of Clinical Cancer Research, Oslo, Norway, 3Oslo University Hospital - The Norwegian Radium Hospital, Dept. of Immunology, Oslo, Norway
In previous trials using autologous dendritic cells bine and Cyclophosphamide and 3x1010 T cells were (DCs) loaded with mRNA encoding tumor specific transfused fresh after removal of the CD3/CD28 antigens to treat malignant melanoma we found that beads. DC vaccination was continued the day after immune responses occurred in some patients but T cell transfer. only for a limited time. Following these results we de- Three patients with immune responses have been signed a new clinical trial to investigate, whether ad- treated with this DC/T cell combination. Patients one ditional transfusion of ex-vivo expanded autologous T and two had a sustained response against hTERT pep- cells could prolong the effect in immune responders. tides after treatment with T cells. In patient one this Melanoma stage IV patients were included after response dropped 20 months after start of DC-vacci- having received standard treatment. nation correlating with progression of the disease. DCs were generated by a standard protocol using a In patient two the response dropped 29 months after maturation cocktail as described by Jonuleit et al. start of DC-vaccination and was combined with a and transfected with hTERT and survivin encoding progression of disease from week 31 on. In patient mRNA. Patients were vaccinated 4x with 5 - 10 x three the response against hTERT peptides vanished 106 DCs per antigen with weekly intervals and then at the time of T cell transfer and was combined with boosted every month. a strong response against survivin peptides. The Blood samples were taken before start of DC vaccina- disease progressed after 11 months. tion and at defined time points during vaccination. Patients who did not mount any immune response PBMC were isolated and immune responses were had a PFS of 3 to 10 months while patients who determined by measuring Thymidine incorporation showed an immune response against hTERT alone after stimulation with different peptide pools for had a PFS of 8 to 13 months. hTERT and survivin. Tregs did not play any significant role in the clinical Patients who showed immune responses against outcome. hTERT and/or survivin during DC vaccination were Our data show that positive immune responses offered additional T cell treatment. against hTERT after initial DC therapy correlate to a T cells were enriched from leukapheresis by elutri- better clinical outcome and additional T cell transfer ation. Prior to T cell expansion regulatory T cells results in a significantly longer PFS. (Tregs) were depleted with CD25 Dynabeads. There- after T cells were expanded in the WAVE bioreactor by adding CD3/CD28 Dynabeads and IL-2. Patients were non-myeloablative conditioned with Fludara- 151 | CELLULAR THERAPY
The importance of costimulation for a novel CAR framework in human T-cells
Birtel M.1, Voss R.-H.2, Yildiz O.3, Breitkreuz A.3, Mroz K.3, Simon P.3, Sahin U.1,2,3
1TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany, 2Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Mainz, Germany, 3BioNTech, Mainz, Germany
Chimeric antigen receptor (CAR) design is based on classical structure is able to homodimerize via its the signaling machinery of the T-cell receptor (TCR). CD28, CD3ζ and Ig domains. Hence, in an attempt First generation CARs consist of a tumor associated to increase valency and function of our receptor, a antigen specific scFv-fragment of an antibody, which scFv fragment was cloned also to the Cα molecule. is hooked onto the signaling moiety CD3ζ. For second Antigen binding could be improved, and a better and third generation CARs, either one or two co-stim- T-cell response was observed in proliferation assays ulatory signaling domains (like CD28 and 41BB) and IFNγ ELISAs. were incorporated into the CAR structure, improv- Pursuing the idea of costimulation in the classical ing overall proliferation and survival of T-cells. The CAR concept, we further investigated an alternative high affinity of antibodies to their cognate antigen, approach of auto-costimulation of T-cells on func- the incorporation of signaling moieties and thus, the tion. The costimulatory ligands CD80 and 41BBL, uncoupling from normal T-cell signaling, poses the normally only present on APCs, were expressed risk of on-target/off-tumor effects. Hence, for the on T-cells to enhance T-cell responses in cis upon sake of the patient’s health alternative CAR formats CD28- and 41BB-receptor binding. This mechanism are under investigation. showed a superior antigen specific effect in cell pro- We designed a tumor reactive scCAR molecule liferation and IFNγ secretion. However, intracellular specific for the tight junction protein Claudin 6. It (ic) IFNγ production on a T-cell clonal level was not consists of a scFv fragment chimerized to the Cα increased, leaving us to speculate that costimulation domain of the mouse TCR molecule (VH-linker-VL- in cis merely affects bulk proliferation of T-cells. In Cα), coexpressed with a membrane resident TCRα contrast to this observation, costimulation in trans derived Cα-domain. This CAR mimics the natural by providing the myelogenous leukemia K562 with situation in a T-cell, since it relies on the recruit- these ligands, thereby producing a so-called artificial ment of endogenous CD3 components, in particular APC (aAPC), led to a profound upregulation of both, CD3ζ, and co-stimulatory proteins essential for T-cell IFNγ secretion and ic IFNγ production. activation. For CAR functional studies, we took ad- The costimulatory assisted scCAR + Cα design may vantage of in vitro transcribed RNA electroporation provide an alternative approach more strictly relying into human CD8+ T-cells. So far, comparison of the on native T-cell signaling and thus, a better concord classical CAR with the new scCAR + Cα model elic- with safety aspects in immunotherapy of cancer. ited a lower proliferation and IFNγ production with an at the same time almost equal cytotoxic effector function. In contrast to the novel CAR design, the 152 | CELLULAR THERAPY
Functional evaluation of T cells generated from WT1-TCR transduced human hematopoietic stem cells using the OP9-DL1 coculture system
Bonte S.1, Snauwaert S.2, Goetgeluk G.3, Heemskerk M.H.M.4, Stauss H.5, Stripecke R.6, Vandekerckhove B.3, Kerre T.1,2
1Ghent University, Internal Medicine, Ghent, Belgium, 2Ghent University Hospital, Hematology, Ghent, Belgium, 3Ghent University, Clinical Chemistry, Microbiology and Immunology, Ghent, Belgium, 4Leiden University Medical Center, Hematology, Leiden, Netherlands, 5University College London, Immunology, London, United Kingdom, 6Hannover Medical School, Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany
Background: Acute myeloid leukemia (AML) remains cell line (K562-A2-WT1-fLuc, R. Stripecke), or lucifer- a therapeutic challenge as many patients relapse after ase-transduced, WT1+, HLA-A2+ primary AML cells chemotherapy. Allogeneic stem cell transplantation (cultured short-term with cytokines or expanded is in most of these patients the only option for cure, long-term on MS-5 in the presence of cytokines), and but carries a high risk of morbidity and mortality and 24 h later, with 5x106 or 107 WT1- or CMV-TCR (neg- a suitable donor may be lacking. Therefore, we have ative control) T cells. Mice were evaluated using the developed a novel immunotherapeutic treatment for IVIS bioluminescence assay. this population. We generate in vitro, starting from Results: Both 51Cr release assay and ELISA showed hematopoietic precursor cells (HPC), T cells that rec- that, upon activation, the in vitro generated WT1-TCR ognize WT1, a tumor antigen that is overexpressed T cells showed specific cytokine production and ef- on 70% of the AMLs. In this study we have evaluated ficient killing of WT1+ HLA-A2+ tumor cells and not the functionality and specificity of these WT1-direct- control cells. ed T cells both in vitro and in vivo. In vivo, we observed that the K562-A2-WT1-fLuc Methods: Cord blood-isolated CD34+ HPC were cul- cell line homed to ovaria and brain (female) or liver, tured on OP9-DL1 in the presence of the cytokines testes and brain (male) and these are largely sanctu- IL-7, FLT3L and SCF for 2 weeks, until T cell com- ary sites, not reached by the T cells, therefore result- mitment. Subsequently, they were transduced with ing in low efficiency. a WT1 (H. Stauss) or a CMV (M. Heemskerk) TCR Currently, experiments are ongoing evaluating the and again co-cultured until CD4+CD8+ double pos- efficacy of the T cells against luciferase-transduced itive cells were abundantly present (generally after primary AML cells, as these cells are expected to another 2 weeks). At that point, the agonist peptide home to the bone marrow and blood of the mice, was added to the culture together with IL-7, and 5 reflecting more the physiological situation, and can days later cells were harvested and expanded in the be more easily reached by T cells. Results of these presence of IL-2. experiments will be presented at CIMT. T cells were evaluated using a 51Chromium release Conclusions: We have shown that, using the OP9-DL1 assay, for cytotoxicity against WT1 and HLA-A2 pos- model and agonist selection, we were able to generate itive and negative targets. Also, upon activation, pro- large numbers of tumor-specific, naive and resting duction of IFNγ was evaluated using ELISA. T cells. After expansion and activation, these cells Immunodeficient NSG mice were irradiated (200 show specificity and functionality in vitro and are cGy), and 24 h later injected intravenously with either currently being evaluated in an in vivo immunodefi- a luciferase-positive, WT1, HLA-A2 transduced K562 cient mouse model. 153 | CELLULAR THERAPY
Contrasting effects of the anti-cancer MEK 1/2 inhibitor trametinib on human dendritic cell functions
Brabants E.1, Heyns K.1, Vermaelen K.1
1Ghent University, Department of Pulmonary Medicine, Ghent, Belgium
Introduction: Evidence is emerging that pharma- Doses above 10 µM led to excessive DC toxicity. cological inhibition in the oncogenic Ras-Raf-MEK- Preconditioning DCs with 0.1µM Trametinib led to ERK/MAPK cascade can trigger immunological col- higher expression levels of the maturation markers lateral effects in tumors. CD40, CD70, CD83, CD86, CCR7 and HLA-DR, but MEK inhibitors are a promising targeted therapy in impaired the expression of the co-inhibitory mole- cancers driven by oncogenic KRAS mutations, in- cule PD-L1. Furthermore, trametinib strongly para- cluding a subset of lung adenocarcinomas. While lyzed the IL-10 secretion capacity of TLR-ligand-ma- direct anti-tumor responses of MEK inhibitors have tured DCs, while preserving and even upregulating been well described, only a limited number of IL12p70 release. studies have examined on-target/off-tumor effects At the functional level, trametinib-preconditioned of selective MEK inhibitors on immune cells. DCs loaded with an HLA-A2-restricted MART-1 Dendritic cells (DCs), when properly activated, are peptide suppressed IL-10 production capacity of critical in initiating anti-tumor immune responses. CD8+ T-cells and induced a higher percentage DC activation can be achieved by triggering toll- of MART1-tetramer+ and IFN-gamma+ CD8+ like receptors, which stimulate MAPK such as p38 T-cells. In sharp contrast, exposing DC-T-cell cocul- MAPK and ERK. Still, controversy exists about the tures to trametinib negatively affected the percent- impact of MEK/ERK inhibition on DC biology. In age divided IFN-gamma+ CD4+ T-cells and CD8+ this study we sought to examine effects of the MEK T-cells. inhibitor trametinib, currently under clinical de- Conclusion: In vitro exposure of monocyte-derived, velopment for lung cancer, on DC phenotype and TLR-ligand-matured DCs to low doses of the selec- function. tive MEK 1/2 inhibitor trametinib enhanced DC Results: We confirmed the already described bio- immunogenic features while preventing the up- logical response to trametinib exposure using the regulation of immunosuppressive factors. However, mouse and human lung cancer cell lines LLC respec- trametinib showed detrimental effects on T-cell tively A549, with a clear dose-dependent growth in- functions. Together, these results warrant investi- hibition, as well as cytotoxicity at higher dose. gation on the impact of MEK inhibition on tumor- Effects on DC functions were assessed using infiltrating immune cells in vivo. In addition, these trametinib-preconditioned, TLR-ligand-matured data suggest the use of clinical grade MEK inhibi- human monocyte-derived DCs. Biological effects tors as a preconditioning regimen for the production were examined in a trametinib dose range of 0µM - of DCs for cancer immunotherapy. 0.1µM - 1µM - 10µM - 20µM. 154 | CELLULAR THERAPY
Combining in depth immunopeptidomics of primary melanoma samples and selection of high affinity T cells and TCR for the treatment of metastatic melanoma
Bräunlein E.1, Bassani-Sternberg M.2, Klar R.1, Audehm S.1, Koch T.1, Rämisch S.1, Slotta-Huspenina J.3, Schlitter A.M.3, Specht K.3, Hein R.4, Werner A.5, Martignoni M.E.5, Busch D.H.6, Peschel C.1, Mann M.2, Krackhardt A.M.1
1III. Medizinische Klinik, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany, 2Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Munich, Germany, 3Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar, Munich, Germany, 4Department of Dermatology and Allergology, Technische Universität München, Munich, Germany, 5Institute of Surgery, Klinikum rechts der Isar, Technische Universität München, Munich, Germany, 6Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
Malignant Melanoma (MM) is one of the most rapidly were stimulated with peptide-pulsed dendritic cells in progressing tumor entities with poor prognosis and a single HLA-mismatched setting. After screening for limited therapy options. Recent advances in the field peptide-specific T cells by peptide/HLA multimer stain- of immunotherapy, especially concerning checkpoint ing, we were able to detect enrichment for 3 different inhibitors, showed encouraging results in this disease epitopes derived from the CTA preferentially expressed entity by unleashing endogenous tumor reactivities. antigen of melanoma (PRAME) and from paired box 3 However, as not all treated patients respond to these (PAX3). Multimer-based sorting and cloning by limiting novel therapies, alternative options are badly needed. dilution resulted in the identification of peptide-specific An alternative highly promising immunotherapeutic T-cell clones, which also recognize cell lines with en- approach is the adoptive transfer of T-cells transgenic dogenous target antigen expression, underscoring the for T-cell receptors (TCR) with defined peptide specifici- functional relevance of isolated TCR. For confirmation ty. Since knowledge of human leukocyte antigen (HLA) of target-dependent reactivity, cell lines are transduced ligands naturally presented on primary melanoma cells with a retroviral vector coding for PRAME or PAX3 at the is limited, we used the immunopeptidomic approach for moment. Specific recognition of these transgenic targets the identification of suitable targets for adoptive immu- by expanded T-cell clones will be assessed as well as notherapy. By HLA class I and II immunoprecipitation functional avidity against T2 cells pulsed with titrated of primary tumor material from 25 melanoma patients peptide concentrations. A further detailed characteriza- and mass spectrometric analysis of eluted peptides, we tion of the reactivity pattern and potential cross-reactivi- identified over 70 000 HLA class I ligands and proceeded ties of these T-cell clones and their TCR is under current with a systematic data analysis for the identification of investigation. most promising target candidates. Selection criteria com- This approach emphasizes the great potential of sys- prised differentiation antigens, cancer-testis antigens tematic epitope identification for efficient isolation of (CTA) as well as candidates with previously described highly tumor-reactive TCR by the immunopeptidomic tumor-associated overexpression. This selection process approach. Our results might improve immunotherapeu- lead to a preliminary list of 24 highly attractive candidate tic treatment options for MM such as the development of peptides restricted to common HLA alleles, e.g. HLA- a TCR library and combination of checkpoint inhibitors A*01:01, -A*02:01, -B*0702. In order to isolate peptide spe- with peptide vaccinations. cific high affinity TCR, naïve T cells from healthy donors 155 | CELLULAR THERAPY
Combined therapy of CIK cells and monoclonal antibodies for ovarian cancer: a preclinical study
Cappuzzello E.1, Sommaggio R.1, Tosi A.1, Zanovello P.1, Rosato A.1
1University of Padova, Padova, Italy
Cytokine-Induced Killer (CIK) cells are NK-like T cells mab during the cytotoxic assay increased CIK cell capable of recognizing target tumor cells without the antitumor activity, according to the expression level need of antigen-specific priming. CIK cell cultures of ligands. This combined treatment was also evalu- are composed by CD3+CD56− T cells, CD3−CD56+ NK ated in vivo in NOD/SCID common γ chain knockout cells and the CD3+CD56+ subpopulation, which is (NSG) mice bearing intraperitoneal ovarian cancers responsible for antitumor activity. For their distinc- induced by either an established cell line (SKOV-3), tive features, CIK cells are an attractive approach for or primary samples. We observed a delay in tumor cellular immunotherapy. In this study we obtained growth and an increase in survival in mice treated CIK cells from healthy donor PBMCs stimulated in with the therapy combining CIK cells and mAbs, vitro with IFNγ, OKT3 and IL-2 for 28 days. At this compared to those receiving CIK cells or mAbs alone. timepoint, about 40% of the culture was composed Overall, these results demonstrate that CIK cells are by CD3+CD56+ double positive cells expressing high able to kill target cells also through ADCC, and that levels of CD8, NKG2D, CD11, and TCR α/β. Different- their activity can be enhanced by the co-administra- ly from previously reported data, we also observed tion of monoclonal antibodies. Our study provide a that they maintained good levels of CD16 expression preclinical proof-of-concept for an effective strategy (mean 17%, range 9-29%), although a certain donor- to treat ovarian cancer, which implies the assessment dependency was noticed. Based on this observation, of Her2 and EGFR expression on each patient tumor we hypothesized the possibility of increasing the for a prior estimation of the efficacy of the treatment. antitumor activity through the antibody-dependent Overall, our findings not only support a CIK-based cell-mediated cytotoxicity (ADCC) accountable to the therapy for ovarian cancer treatment, but also envis- CD16 + fraction of CD3+CD56+ population, by combin- age that a combined approach can virtually improve ing CIK-based treatment with the administration all CIK-based immunotheraphy protocols. of monoclonal antibodies (mAbs) already routinely used in therapies, such as trastuzumab (anti-Her2 mAb) or cetuximab (anti-EGFR mAb). Therefore, CIK cells were challenged against both established cell lines and primary samples of ovarian cancer, and displayed a high, albeit variable, capability to kill different samples. After flow cytometry assessment of Her2 and EGFR expression on target cells, we dem- onstrated that the addition of trastuzumab or cetuxi- 156 | CELLULAR THERAPY
A novel modular retargeting platform technology for precise control of CAR T cell reactivity
Cartellieri M.1,2, Loff S.1,3, von Bonin M.4, Bejestani E.P.4, Ehninger A.3, Feldmann A.1, Koristka S.1, Arndt C.1, Ehninger G.4, Bachmann M.1,5
1TU Dresden, University Cancer Center (UCC), Medical Faculty Carl Gustav Carus, Tumorimmunology, Dresden, Germany, 2Cellex Patient Treatment GmbH, Dresden, Germany, 3GEMoaB Monoclonals GmbH, Dresden, Germany, 4TU Dresden, Medical Faculty Carl Gustav Carus, Medical Clinic and Policlinic I, Dresden, Germany, ORAL 5Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer T TALK Research, Dresden, Germany SHOR 2015
The adoptive transfer of T cells engineered with are engineered to express a universal CAR (UniCAR), chimeric antigen receptors (CARs) is currently con- which has specificity for a short peptide motif of 10 sidered as a highly promising therapeutic option for amino acids derived from a human nuclear protein. treatment of otherwise incurable malignant diseas- Thus, T cells engineered to express UniCAR remain es. CARs combine the cellular and humoral arm of inactivated after re-infusion, since the UniCAR target the immune response by assembling a single-chain is not available for binding under physiological con- fragment variable (scFv) as binding moiety which ditions. The ultimate antigenspecificity of the system provides the antigen-specificity and an activating is provided separately by targeting modules (TMs) immune receptor. It has been demonstrated both in comprising a binding domain e.g., a tumorantigen vitro and in vivo, that CAR engrafted effector T cells specific scFv, fused to the nuclear antigen motif rec- mediate long-lasting anti-tumor responses. Despite ognized by the UniCAR binding domain. Here we encouraging clinical efficacy targeting CD19 in provide first in vitro and in vivo prove of concept for recent clinical trials, the appearance of potentially this new approach. Antigen-specific redirection of T life-threatening adverse reactions and the lack of cells armed with the universal CAR in the presence of control mechanisms once initiated, prevent more different targeting modules against various antigens widespread application of the CAR technology. (PSCA, PSMA, CD33, CD123, CD19) was effective at To overcome limitations of conventional CAR T cells, femtomolar concentrations of the targeting module. a unique chimeric antigen receptor (UniCAR) tech- Taken together, the modular nature of UniCAR tech- nology was developed which allows precise control nology will allow retargeting of autologous, patient- of CAR T cell reactivity, thus lowering the risk of derived T cells to several antigens under controlled side effects while preserving efficacy. Moreover, pharmacological conditions and has the potential to the UniCAR technology enables the retargeting of become a highly effective treatment option for late engrafted T cells against more than one antigen stage cancer patients with reduced risks for side simultaneously or subsequently, thus reducing the effects. risk for development of antigen-loss tumor variants under treatment. The UniCAR technology splits the signaling and antigen-binding aspects of conven- tional CAR into two individual components. T cells 157 | CELLULAR THERAPY
Innate transplantation as a low GVHD platform for immune interventions by means of an alpha/beta T-cell depleted allogeneic stem cell transplantation from matched related and unrelated donor grafts de Witte M.A.1, Fleurke G.2, van de Wagen L.2, Slaper I.3, Kuball J.1,2
1University Medical Center Utrecht, Hematology, Utrecht, Netherlands, 2University Medical Center Utrecht, Laboratory of Translational Immunology, Utrecht, Netherlands, 3University Medical Center Utrecht, Cell Therapy Facility, Utrecht, Netherlands
Introduction: The outcome of allo-SCT in patients covery within 3 weeks. Primary engraftment (chi- with poor risk leukemia is still hampered by infec- merism > 95%) was observed in all patients (n=22). tions, graft versus host disease (GVHD) and relapse. Immune reconstitution primarily consisted of NK The innate immune system has been reported to con- cells, which contract around 4 months post SCT. In ad- tribute to control of infected or transformed cells, with dition, γδT cells were present at normal numbers the lower incidence of GVHD. Specific depletion ofαβ T- first half year post SCT, whereas the adaptive immune cells - key players in the development of GVHD - will repertoire shows a delayed reconstitution. Under this render NK cells and γδT cells within the allograft. Re- “innate control”, no increase in CMV or EBV reactiva- cently reported results have shown the great promise tions has been observed so far. Up to date, none of the of this approach in haploindentical transplantations. patients developed aGVHD > grade II. Within this study, we aim to extend αβT- cell depleted Conclusion: “Innate-Tx” is a low toxicity platform, allo-SCT to patients with a MRD or MUD. resulting in a swift reconstitution of innate cells (NK Methods: All patients with hematological malignan- cells and γδ T cells) the first 6 months post transplan- cies and an indication for allo-SCT were eligible. Either tation, followed by a subsequent reconstitution of the HLA matched siblings (MRD) or HLA matched (9 or adaptive immune repertoire. The diversity and func- 10/10) unrelated donors (MUD) were eligible. αβT-cell tionality of the reconstituted innate cells remains to reduction was performed by negative selection with be addressed. However, the low incidence of aGVHD anti-αβTCR antibodies in combination with magnetic and sufficient control of infections in this protocol microbeads, using the automated CliniMACS device suggest that this transplantation strategy can serve (Miltenyi Biotec, Bergisch Gladbach, Germany). The as a platform for subsequent immunological inter- maximal contamination with αβT-cells for all dose ventions such as a pre-emptive DLI or transfer of ge- levels was 5x105/kg. The conditioning regimen con- netically modified T cells. sisted of: ATG (Genzyme®) 4 or 6 mg/m2 + fludara- bine 120 mg/m2 + busilvex AUC=90 followed by αβT- cell depleted grafts from matched related or unrelated donors. No additional immune suppression was given post allo-SCT. Results: A ~4 log depletion of αβT-cells has been observed in the product with a recovery of ~75% of CD34+ cells. The combination of ATG/fludarabine/ busilvex was well tolerated with a hematological re- 158 | CELLULAR THERAPY
Expression of PE38-based immunotoxins in T cells introduced by mRNA transfection
Eggers R.1, Philippi A.1, Breinig F.2, Schmitt M.J.2
1Korea Institute of Science and Technology Europe Forschungsgesellschaft mbH, Magnetics Group, Saarbrücken, Germany, 2Universität des Saarlandes, Molekular- und Zellbiologie, Saarbrücken, Germany
The long-term goal of the presented project is to use T cells and HEK293 cells, and expression levels de- T cells for targeted delivery of an anti-cancer-agent termined by Western analyses correlated with the to a tumor. In order to achieve this goal via T cell amount of mRNA used for transfection. Although a mediated immunotherapy, autologous T cells are decrease in cell viability was detected in IT express- transfected with mRNA coding for a bacterial A/B ing T cells, impaired T cells still retained residual class protein toxin engineered to specifically attack effector function mediated by the bispecific OKT3x- cancer cells. The recombinant toxin secreted by T HEA125 antibody. Genetic modification of the cata- cells should result in an increased efficiency in adop- lytic center of the toxin domain of vegf-PE38 restored tive T cell therapy. cell viability of IT synthesizing T cells, indicating Here we transfected primary, ex vivo activated T cells that attenuated immunotoxin variants are a suitable by electroporation with in vitro synthesized mRNA. tool to avoid self-killing of IT expressing T cells. Transfection was established with eGFP as model protein, and PE38-based immunotoxins (IT) were chosen as anti-cancer-agent. As immune component, single chain Fv of anti-Her2/neu antibody (e23) or
vascular endothelial growth factor165 (VEGF) were used. While immunotoxin e23-PE38 targets Her2/ neu positive tumors like breast cancer, vegf-PE38 targets the tumor neovasculature. In order to achieve IT secretion and prevent self-killing, an endoplasmic reticulum (ER) signal sequence was inserted into the expression vector to direct ER import of each IT. Transfection efficiency and subsequent protein trans- location into the ER of T cells and HEK293 cells was analyzed by using an eGFP reporter containing an N-terminal signal peptide. Transfection efficiencies reached 80 % for T cells and 90 % for HEK293 cells without impairing cell viability. Fluorescence mi- croscopy of the transfected cells indicated that eGFP was mainly localized in ER/Golgi-like structures. Both immunotoxins were successfully expressed in 159 | CELLULAR THERAPY alloCELL: Selection of suitable T-cell donors and GMP-compliant manufacturing of cells
Tischer S.1,2, Priesner C.3, Arseniev L.3, Martens J.1, Goudeva L.1, Heuft H.-G.1, Figueiredo C.1, Köhl U.2,3, Blasczyk R.1,2, Maecker-Kolhoff B.2,4, Eiz-Vesper B.1,2
1Hannover Medical School, Institute for Transfusion Medicine, Hannover, Germany, 2Hannover Medical School, Integrated Research and Treatment Center (IFB-Tx), Hannover, Germany, 3Hannover Medical School, Institute for Celltherapeutics, Hannover, Germany, 4Hannover Medical School, Department of Pediatric Hematology and Oncology, Hannover, Germany
Patients after transplantation are rendered suscep- from clinically active viral disease (>6 months). tible to infections and reactivations caused by cy- These data underline the need for (i) accurate mon- tomegalovirus (CMV), Epstein-Barr virus (EBV), itoring of viral load and antiviral T-cell frequencies human herpesvirus 6 (HHV6), adenovirus (ADV), in patients, (ii) early selection of suitable T-cell and polyoma virus BK (BKV). The shortcomings of donors and (iii) support data about safe application conventional therapies have increased the interest in of third-party antiviral T-cell products. adoptively transferred antiviral T cells. The alloCELL lab and registry established a compre- hensive protocol to consider clinical requirements of patients at high risk or with failed conventional antiviral therapy and obtained the manufacturing license for generating clinical-grade CMV-, ADV-, EBV- and multivirus-specific T-cell products. T-cell donors are defined as eligible if ≥0.03% virus-specif- ic IFN-γ+ T cells are detectable. A related or at least 3/6 HLA-A/B/DR-matched alloCELL donor (www. allocell.com) is chosen, if the stem cell donor is not eligible. Determination of antiviral T-cell frequencies in donors and patients was done routinely by IFN-γ EliSpot and multimer staining. Thirteen out of 45 monitored patients failed to respond to antiviral treatment and were assigned to receive adoptive T-cell transfer. 20 family, 2 stem cell and 11 allo- CELL donors were tested. Clinical-grade CMV-, EBV-, ADV- and CMV/ADV-specific T cells were generated from family and alloCELL donors. Patients receiving T cells at Hannover Medical School (n = 2) were continuously monitored to determine frequency and chimerism. None of the patients showed significant infusion-related side effects and both remained free 160 | CELLULAR THERAPY
Generation of tumor antigen specific CD4+ and CD8+ T cells by simultaneous MHC-I and -II epitope presentation in vitro and in vivo
Ellinger C.1,2, Wehner C.1,2, Raffegerst S.1,2, Wilde S.1,2, Weis M.1,2, Longinotti G.1,2, Sailer N.1,2, Schendel D.J.1,2, Milošević S.1,2
1Medigene Immunotherapies GmbH, Martinsried, Germany, 2Helmholtz Center Munich, Institute of Molecular Immunology, Munich, Germany
In the past few years, activation of the immune system marker CD154 on CD4+ T cells or CD137 on CD8+ T to effectively target the patient’s own tumors was cells. In this manner, we were able to induce and demonstrated to be a promising alternative strategy identify TAA-specific CD4+ and CD8+ T cells in the for the treatment of cancer patients. Dendritic cells very same approach. (DCs) are the immune systems most potent antigen Furthermore, efficiency of DCs loaded with ILS- presenting cells (APC) and can present antigens on TAA-ivtRNA compared to conventional TAA-ivtRNA MHC-I and -II molecules, leading to the activation to prime antigen specific T cells in-vivo was investi- of CD8+ as well as CD4+ T cells. Stimulated CD4+ gated by using the humanized NOD/scid IL2Rgnull T cells act as helper cells for cytotoxic CD8+ T cells (NSG) mouse model. Therefore, NSG mice were en- to kill tumor cells by inducing T cell proliferation grafted with human peripheral blood mononuclear and antigen presentation by DCs. CD4+ T cells also cells (PBMC) and vaccinated twice with DCs either have substantial cytotoxic potential and can directly transfected with conventional TAA-ivtRNA or with destroy tumor cells bearing MHC-II molecules. As ILS-TAA-ivtRNA. Re-isolated PBMC of mice vacci- an efficient anti-tumor response strongly depends on nated with ILS-TAA-ivtRNA-transfected DCs showed the interaction between DCs, CD4+ and CD8+ T cells, higher amounts of antigen-specific CD8+ T cells and these cell types are considered to be essential for suc- stronger cytotoxic activity against tumor cells. cessful immunotherapeutic strategies. These in vitro and in vivo data illustrate the benefits In most of the clinical trials using DCs, the APCs of loading DCs with ILS-linked target antigens as were either endogenously or exogenously loaded CD8+ and CD4+ T cells can be induced side by side with tumor antigens for the presentation on MHC-I allowing interactions and T cell help. rather than MHC-II. Due to the differences concern- ing the antigen presentation pathways, simultaneous loading of MHC-I and -II was hardly successful so far. To overcome this obstacle, we used internal cell lo- calization signals (ILS) to force MHC-II cross-presen- tation of tumor-associated antigens (TAAs) encoded by in vitro transcribed (ivt) RNA. Thus, peripheral blood lymphocytes were primed by DCs expressing TAAs on MHC-I and -II, facilitating the activation and interaction of CD4+ and CD8+ T cells. TAA-specific T cells were isolated either by the activation induced 161 | CELLULAR THERAPY
Third generation CAR T-cells targeting CD19 for relapsed and refractory lymphoma and leukemia - the Swedish experience
Enblad G.1,2, Karlsson H.1, Wikström K.I.3, Essand M.1, Savaldo B.4, Brenner M.K.4, Dotti G.4, Höglund M.5,6, Hagberg H.1,2, Loskog A.1
1Uppsala University, Immunology, Genetics and Pathology, Uppsala, Sweden, 2Uppsala University Hospital, Oncology, Uppsala, Sweden, 3Karolinska University Hospital, Vecura, Stockholm, Sweden, 4Baylor College of Medicine, Center for Cell and Gene therapy, Houston, United States, 5Uppsala University, Medical Sciences, Uppsala, Sweden, 6Uppsala University Hospital, Hematology, Uppsala, Sweden
Chimeric antigen receptor (CAR) T-cells have Patient 1 (DLBCL) had a mild cytokine release syn- shown promising results in patients with B-cell drome (CRS), after four weeks (never requiring treat- malignancy. In preclinical studies we showed that ment) followed by a complete response of his lym- CD19-directed third generation CAR T-cells contain- phoma. A relapse and a second CRS occurred after ing signaling domains from both CD28 and 4-1BB as six weeks and he was treated with prednisone with co-stimulatory molecules have a higher activation good symptomatic effect and reduction of tumour status and greater proliferation in response to anti- size. The patient progressed after three months. Pa- gens. We here report the first results from a phase tients #2-4 (CLL, MCL, MCL) all progressed after I/IIa study (NCT:02132624) using these 3G CAR 2,1, and 3 months, respectively. Patient #5 (DLBCL) T-cells targeting CD19. was in remission at the time of CAR T cell infusion Patients with relapsed or refractory CD19+ B-cell and remains in complete remission (3+ months) malignancy were eligible provided there was no and patient 6 progressed after 1 month. Patients no other curative treatment available. Of the first eight 7 and 8 are too early to evaluate. No patients had patients reported here seven had lymphoma and major toxicity of CNS or other organs. However, all one patient acute lymphoblastic leukemia (ALL). lymphoma patients had received rituximab as part We manufactured autologous CAR T cells using of their salvage treatment prior to CAR T-cell infu- a gamma retrovirus encoding the CAR. The con- sion that might explain the lack of CRS. ditioning regimen was dependent on the type of Eight patients have been treated with increasing lymphoma or leukemia and the previous treatment, doses of CAR T-cells in Sweden. All treatments were with the intent to both reduce tumor burden and given as out-patients. The conditioning has been achieve lymphodepletion. Two patients (DLBCL, relatively mild as compared to previous published MCL) received pegylated doxorubicin and gemcit- studies. One patient entered complete remission abine and one patient (MCL) received rituximab and after treatment (DLBCL) and one was in remission bendamustin as the final treatment prior to CAR at the time of treatment and is still in CR; 4 have T-cell infusion. The remaining five patients (CLL, progressed; and 2 are too early to evaluate. Too few DLBCL, FL transformed, DLBCL and ALL) received patients have been treated to reach definitive con- cyclophosphamide, mainly 500 mg/m2 d 1 and clusions about efficacy but more intensive condi- fludarabine 25 mg /m2 d 1-3. The patients received tioning or repeat dosage may be required to achieve one infusion of CAR T cells starting at a dose of long-term remission in B-lymphoma patients. The 2x10e7 cells/m2 (patients 1 and 2), 1x10e8 (patients transgene PCR levels for expansion or persistence 3,4,6) and 2x10e8 (patients 5,7,8). are now being analyzed. 162 | CELLULAR THERAPY
Targeted delivery of cardiac peptides as anti-cancer agents
Farahmand L.1, Majidzadeh-A K.1,2, Salehi M.1, Esmaeili R.1
1Cancer Genetics Department, Breast Cancer Research Center, ACECR, Tehran, Iran, Islamic Republic of, 2Tasnim Biotechnology Research Center (TBRC), school of medicine, AJA University of Medical Sciences, Tehran, Iran, Islamic Republic of
Introduction: The pitfalls in the current cancer ther- Considering that the Ribosome Binding Site (RBS) apies have already encouraged scholars to conduct was put at the beginning of each gene, the possibility comprehensive researches to finding new treatment for individual and simultaneous expression of three approaches in this area. A recent focus of such re- peptides as well as the GFP inside a bacterial host search is to study the application of bacteria in target- was provided. The leader sequence led to the secre- ed therapy of cancer through the direct expression of tion of peptides outside the cells. The expression of anti-cancer compounds in tumor tissue by using the GFP inside the bacterial host facilitated the detection intrinsic anti-tumor properties of the bacteria. Fur- of cells that produce peptides. thermore, studies have shown that the peptides in Conclusions: The designed construct can be applied which produced in heart (atrial natriuretic peptide) for bacterial drug delivery or through increasing the have anti-cancer properties that help to reduce the potency of bacterial anti-cancer effects (such as Sal- size of tumor under in vitro and in vivo conditions. monella typhimurium, Listeria, E.Coli, etc.). Also, it Materials and methods: For the purposes of the is possible to deliver other genes of anti-cancer pro- present research, a construct was designed to express teins and peptides in a direct and targeted manner three atrial peptides in conditions similar to those of to the tumor tissue and accordingly produce the anaerobic conditions of tumors that have the most desired proteins and peptides inside the area where effect on breast cancer cells. The NADH-dependent the tumor is located. nitrite reductase (nirB) promoter of E.coli genome was used. The construct used in the research was designed based on the separate cistronic model. Ac- cordingly, the abovementioned peptides were cloned under the control of the one promoter as well as the Green Fluorescent Protein (GFP) which itself was under the control of another promoter as a reporter gene. In order to secretion the peptides to outside the bacterial cell, an IgK leader sequence was inserted at the beginning of each peptide sequences. After the cloning, the E.coli bacteria were cultured under an- aerobic conditions. Results: The bacterial construct was successfully designed by applying the separate cistronic model. 163 | CELLULAR THERAPY
T cell receptor modifications to enhance expression, chain pairing, and antigen recognition in T cells for adoptive T cell transfer
Foley K.1, Spear T.1, Nagato K.2, Murray D.1, Garrett-Mayer E.3, Nishimura M.I.1
1Loyola University Chicago, Department of Surgery, Maywood, United States, 2Chiba University, Department of General Thoracic Surgery, Chiba, Japan, 3Medical University of South Carolina, Department of Public Health Services, Charleston, United States
T cell receptor (TCR) gene modified T cells for adop- due to the 1:1 stoichiometric ratio between CD34t and tive T cell transfer therapy have been shown to have the TCR proteins. This allows us to directly compare clinical success in treating melanoma and other ma- the TCR expression and T cell function of each TCR lignancies by redirecting the specificity of peripheral modification using this internal reference standard. blood lymphocytes (PBL) to recognize tumor and/ Our results revealed that the murinized Cβ2 TCR or viral associated antigens of choice. One of the and the leucine zipper TCR have the highest levels of challenges in using TCR gene modified T cells for cell surface expression per transduced T cells when adoptive transfer include proper expression of the in- compared to the wild type TCR. It is also evident troduced TCR and function. Mispairing between en- in this study that although some modifications have dogenous and introduced alpha and beta TCR chains higher levels of TCR cell surface expression, this does allows for the potential of unanticipated off-target not always result in increased T cell function. Our reactivity or autoimmunity. Additionally, chain mi- studies have given us a better understanding of how spairing reduces TCR expression which can result these TCR modifications can impact TCR expression in impaired therapeutic efficacy against targeted and T cell function that may allow for optimization antigens. One approach to improve TCR expression of adoptive T cell transfer to treat viral infections and and pairing on the cell surface and to enhance T cell malignancies. function involves the modification of the introduced TCR genes to promote proper pairing. Some of these modifications to augment proper TCR pairing include introducing a disulfide bridge in the alpha/beta con- stant regions, substituting human with murine con- stant regions, codon optimization to enhance protein synthesis, TCR chain leucine zipper fusions, and a single chain TCR. We have previously identified and cloned a novel TCR from HLA-A2 restricted, HCV NS3:1406-1415-reactive T cell clone and successful- ly redirected antigen specificity of normal PBL-de- rived T cells using a recombinant retroviral vector encoding for the TCR genes. We have also developed a unique surface transduction marker, CD34t, that allows for the exact measurement of TCR expression 164 | CELLULAR THERAPY
EGFR-targeted CAR NK cells display potent activity against experimental glioblastoma
Genßler S.1, Burger M.2,3, Zhang C.1, Hattingen E.4, Steinbach J.P.2,3, Wels W.S.1,3
1Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany, 2Institute for Neurooncology, Goethe University, Frankfurt am Main, Germany, 3German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany, 4Institute of Neuroradiology, Goethe University, Frankfurt am Main, Germany
Glioblastoma (GBM) is the most common primary human GBM cells, which was dependent on EGFR malignant brain tumor and is currently incurable. levels and the duration of co-culture. EGFRvIII-tar- Epidermal growth factor receptor (EGFR) and its geted effector cells only lysed EGFRvIII-positive GBM mutant EGFRvIII are overexpressed in a large pro- cells, while dual-specific CAR NK cells were active portion of glioblastomas and contribute to malignant against both types of tumor cells. In NOD-SCID IL2R transformation and aggressiveness. However, strat- γnull (NSG) mice carrying intracranial GBM xenografts egies to interfere with EGFR/EGFRvIII signaling or either expressing wildtype EGFR, EGFRvIII or both vaccination approaches against EGFRvIII showed receptors, intratumoral treatment with dual-specific only limited clinical success. Adoptive transfer of NK cells was superior to treatment with the corre- ex vivo activated lymphocytes such as natural killer sponding monospecific effector cells resulting in a (NK) cells may represent a promising alternative marked extension of symptom-free survival. immunotherapeutic approach. NK cells can respond rapidly to transformed and stressed cells, and have the intrinsic potential to reach their targets in almost all body tissues. In addition to primary NK cells, also continuously expanding cytotoxic cell lines such as NK-92 are being considered for adoptive cancer im- munotherapy, and general safety of infusion of NK-92 cells has been established in phase I clinical trials. The therapeutic utility of NK-92 cells can be further enhanced by genetic modification with chimeric antigen receptors (CARs), which recognize antigens differentially expressed on the surface of tumor cells. To target NK-92 cells towards GBM, we developed optimized CARs that employ scFv antibody frag- ments either recognizing epitopes restricted to EGFR or EGFRvIII, or an epitope common to both target receptors. For signaling these CARs carry a compos- ite CD28-CD3 zeta domain. Functional analysis in vitro revealed high and specific cytotoxicity of EG- FR-targeted NK cells against established and primary 165 | CELLULAR THERAPY
Messenger RNA encoding constitutively-active TLR4 exerts potent immunostimulatory effects on antitumor T cells
Gross G.1,2, Pato A.3, Eisenberg G.3, Machlenkin A.3, Margalit A.1,2, Frankenburg S.3, Merims S.3, Peretz T.3, Lotem M.3
1MIGAL Galilee Research Institute, Immunology, Kiryat Shmona, Israel, 2Tel-Hai College, Upper Galilee, Israel, 3Hadassah Hebrew University Hospital, Sharett Institute of Oncology, Jerusalem, Israel
A growing number of clinical trials evaluating dif- polypeptide product. We then found that the mere ferent approaches for adoptive cell therapy (ACT) expression of caTLR4 mRNA in polyclonal CD4 and of cancer show encouraging results. Many of these CD8 T cells, bulk anti-melanoma TIL cultures and approaches utilize T cells which are either isolated TIL-derived CD8 T cell clones induced massive pro- directly from the tumor (tumor infiltrating lympho- duction of IFN-γ and that this effect could be clearly cytes, TILs) or genetically redirected by virtue of manifested with as little as 1 mg mRNA. Furthermore, tumor-specific TCRs or chimeric antigen receptors caTLR4 increased surface expression of CD25, CD69 (CARs). To further improve the clinical efficacy of and CD137 (4-1BB) in all primary T cells and TILs therapeutic T cells new strategies are required for tested. Expression of caTLR4 in a melanoma-specific counteracting a variety of cell-extrinsic and intrinsic CD8 T cell clone triggered the secretion of a panel of factors which limit their actual antitumor activity in cytokines and chemokines. Importantly, caTLR4 en- vivo. hanced the anti-melanoma response of bulk TILs for The ability of toll-like receptor (TLR) ligands to at least 4 days post-transfection, but only in the pres- act directly on practically all T cell subsets is now ence of autologous and not HLA-I-mismatched target well-established. TLR engagement on human effec- melanoma cells, as judged by CD107a expression and tor CD8 and CD4 T cells has been shown to augment secretion of IFN-γ, TNF-α and GM-CSF. survival, proliferation, production of pro-inflamma- Our results point to caTRL4 as a potent T cell adju- tory cytokines, target cell killing and resistance to vant and to mRNA as a safe and effective means for suppression by Treg cells. Removal of the extracel- delivering this gene to different human T cell popu- lular domain of TLRs mimics ligand binding and lations employed in cancer ACT. results in constitutive signaling. Hence, the use of truncated TLRs offers a new tool for reprogramming T cells so as to enhance their functional properties and potentially improve the clinical outcome of ACT. To test this hypothesis we generated a constitutive- ly-active derivative of TLR4 (caTLR4). To prevent in- cessant activation of signaling circuits we examined the effects of this construct on human T cells through the delivery of in-vitro-transcribed mRNA by electro- poration. We first validated transfection efficacy, du- ration of the transfected mRNA and expression of the 166 | CELLULAR THERAPY
Effect of integrin-mediated cell adhesion, surface stiffness and co-stimulation on CD4+ T cell activation using nanostructures
Guasch J.1,2, Belz C.A.1,2, Spatz J.P.1,2
1Max Planck Institute for Intelligent Systems, Department of New Materials and Biosystems, Stuttgart, Germany, 2University of Heidelberg, Department of Biophysical Chemistry, Heidelberg, Germany
The encouraging results of adoptive cell thera- pies demand more efficient cell culture systems to achieve large amounts of autologous T cells. Glass surfaces decorated with quasi-hexagonal nanoarrays of gold nanoparticles (AuNPs) functionalized with antiCD3 have been demonstrated to be more efficient than continuous functionalized Au substrates due to their capacity of presenting molecules and proteins to cells in a relevant orientation and spacing.[1] Build- ing on these findings, we transferred the ordered nanostructures prepared on glass to poly(ethylene glycol) (PEG) hydrogels, which were cross-linked with small fibronectin-derived peptide ligands such as RGD and LDV. Thus, we studied the influence of adhesive backgrounds in combination with the an- tiCD3-functionalized AuNPs, with different separa- tions ranging from 20 to 150 nm, on the activation of primary human CD4+ T cells. Early activation markers, cytokine secretion, proliferation, and differ- entiation were analyzed demonstrating the influence of both interparticle distances and integrin-mediated adhesion. Furthermore, we analyzed the influence of the substrate stiffness on T cell activation in the range between 10 kPa to 6 MPa as well as different co-stimulation methods to combine with our nano- structured surfaces with immobilized antiCD3.
[1] J. Matic, J. Deeg, A. Scheffold, I. Goldstein, J. P. Spatz, Nano Lett. 2013, 13, 5090. 167 | CELLULAR THERAPY
Generation of human HCV-specific T cells from hematopoietic progenitor cells using human thymic epithelial cell culture system
He E.1, Wang X.2, Zhang S.3, Moxley K.2, Scurti G.2, Nishimura M.2, Le P.3
1Loyola University Medical Center, Department of Surgery, Maywood, United States, 2Loyola University Chicago, Cardinal Bernardin Cancer Center, Maywood, United States, 3Loyola University Chicago, Department of Microbiology and Immunology, Maywood, United States
Adoptive cell transfer using tumor-infiltrating lym- 133pos cells with lymphoid potential; 2) promotion of phocytes (TIL) to eradicate tumors has shown signifi- these cells to commit to T lineage and to develop into cant progress. The current standard is to introduce mature T cells. Our co-culture system with TEC and genes encoding T cell receptors (TCR) with known TEC-DL4 has successfully generated T lineage CD7pos tumor antigen specificity into mature autologous T cells, the immature double positive CD4+CD8+ and cells. These are then infused back into the patient. the mature single positive CD4+ and CD8+ cells that This approach has several limitations. Firstly, trans- also express CD3. Results with the TEC-A2 and TEC- genic T cells have limited survival time when infused A2-DL4 and the TCR1406 are forthcoming. into the patient resulting in transient anti-tumor ac- tivity. Additionally, the specificity and efficacy of transduced TCR is reduced resulting from the mis- pairing of transduced TCRs with endogenous TCR chains - new TCRs could be generated that would potentially be reactive against self-antigens resulting in autoimmunity. To overcome these issues, we have devised a novel system where peripherally mobilized hematopoietic progenitor cells (HPCs) are co-cultured on human thymic epithelial cells (TEC) that have been engi- neered to express the human Notch ligand Delta-like 4 (TEC-DL4). We have determined the cytokine con- ditions and the duration of Notch signaling required to generate mature CD4+ and CD8+ T cells from HPCs. To adapt the co-culture system to generate T cells with TCR1406 which is specific for Hepati- tis C Virus (HCV), we have generated a new line of TEC that express HLA-A2 (TEC-A2), the restrictive element of the TCR1406 as well as TEC-A2 that over express DL4 (TEC-A2DL4). Our strategy to generate functional mature T cells includes: 1) expansion conditions for CD34pos CD- 168 | CELLULAR THERAPY
Modelling genetic heterogeneity as a resistance mechanism to cancer immunotherapy using CRISPR-Cas9
Glodde N.1, Bald T.1, van den Boorn-Konijnenberg D.2, Schmid-Burgk J.3, Hornung V.3, Tüting T.1, Hölzel M.2
1University Hospital Bonn, Dermatology, Bonn, Germany, 2University Hospital, Institute for Clinical Chemistry and Clinical Pharmacology, Bonn, Germany, 3University Hospital Bonn, Institute of Molecular Medicine, Bonn, Germany
Phenotypic adaptation and genetic selection are dis- tinct evolutionary mechanisms of resistance to cancer therapy. Here we used a multimodal antigen-specific T-cell immunotherapy in a transplantable syngeneic mouse melanoma model to simultaneously compare how adaptive antigen down-regulation and selection of genetic antigen-loss variants contribute to therapy resistance. We generated genetic heterogeneity of an endogenous lineage antigen using the CRISPR-Cas9 genome engineering technology and scrutinized monoclonal versus polyclonal genome editing strate- gies. Regional genetic heterogeneity was visualized using fluorescent cell labelling to enable tracing of clonal evolutions as well as reciprocal tumour-im- mune cell interactions in a therapy and genotype dependent manner. Our results show that wild-type melanoma cells adaptively suppressed target antigen expression at recurrence, but genetic antigen-loss variants were also strongly enriched. This demon- strates that total antigen levels are critical immuno- therapeutic determinants and emphasizes the need to assess both the regulation and the genetic hetero- geneity of target antigens for personalized cancer im- munotherapies. 169 | CELLULAR THERAPY
Donor lymphocyte infusion therapy utilizing genetically engineered lymphocytes that express tumor-specific TCR and siRNA for endogenous TCR enables tumor eradication without GvHD induction
Ikeda H.1, Ueno H.1, Tawara I.2, Kawamura A.3, Okamori K.1, Imai N.4, Okamoto S.3, Mineno J.3, Takesako K.3, Katayama N.2, Shiku H.1
1Mie University Graduate School of Medicine, Department of Immuno-Gene Therapy, Tsu, Japan, 2Mie University Graduate School of Medicine, Department of Hematology/Oncology, Tsu, Japan, 3Takara Bio Inc., Ohtsu, Japan, 4Icahn School of Medicine at Mt. Sinai, New York, United States
The patients with relapsed hematological malignan- destruction. The development of GvHD was depend- cy after allogeneic hematopoetic stem cell transplan- ent on both the CD4+ and CD8+ T cells suggest- tation (HSCT) can be treated by donor lymphocyte ing that the simple purification of CD8+ T cells was infusion (DLI). However, the efficacy is often limited not a sufficient enough strategy to prevent GvHD. In in accosiation with the development of Graft-versus- contrast, adoptive transfer of NY-ESO-1-specific TCR Host Disease (GvHD) as a serious adverse event. By gene-transduced T cells with siTCR vector induced the use of the novel retrovirus vector that specifically complete tumor regression without the development silences endogenous TCR in gene-engineered T cells, of GvHD. we intended to develop the DLI with lymphocytes en- The results here suggest that the allogeneic T cells gineered to express tumor-specific TCR that exhibit transduced with a tumor-specific TCR by siTCR increased tumor-specificity in combination with de- vector showed increased tumor-reactivity with di- creased GvHD-inducing potential. minished GvHD potential. These T cells will be ap- Human PBMC were transduced with a high affin- plicable to the donor lymphocytes infusion therapy ity TCR specific to a cancer/testis antigen, NY-ESO- after allogeneic stem cell transplantation for the 1, by the retrovirus vector with siRNA specific to treatment of hematological malignacy. Currently we the endogenous TCR (siTCR vector). Resulting TCR plan a clinical trial of DLI with siTCR technology for gene-transduced T cells were examined for the ex- the patients with relapsed NY-ESO-1 positive adult T pression of their endogenous TCR by flow cytometry cell leukemia/lymphoma after HSCT. and for their reactivity to allogeneic LCL by 3H-up- take proliferation assay. The genetically engineered human lymphocytes showed antigen specific cyto- toxic activity and significantly reduced expression of endogenous TCR associated with dramatically diminished reactivity to allogeneic lymphocytes. When adoptively transferred into immunodeficient NOG mice that were innoculated with a NY-ESO-1 ex- pressing human tumor cell line NW-MEL-38, control non-engineered T cells failed to control the growth of human tumor cells despite the fact that the lym- phocytes were allogeneic to the tumor cells and de- veloped lethal GvHD including severe bone-marrow 170 | CELLULAR THERAPY
Preclinical validation of a colon cancer specific TCR targeting a neoantigen
Inderberg-Suso E.M.1, Wälchli S.1, Myhre M.R.1, Trachsel S.1, Olweus J.2, Lislerud K.1, Almåsbak H.1, Kvalheim G.1, Gaudernack G.2
1Oslo University Hospital - The Norwegian Radium Hospital, Dept. of Cellular Therapy, Oslo, Norway, 2Oslo University Hospital - The Norwegian Radium Hospital, Section for Immunology, Oslo, Norway
T-cell receptor (TCR) transfer is an attractive strategy from CD4pos T helper clones. These HLA-DR restrict- to increase the number of cancer-specific T cells in ed TCRs were shown to be functional in both CD8pos adoptive cell therapy. However, recent clinical and and CD4pos T cells upon transient redirection. The pre-clinical findings indicate that careful consider- redirected T cells specifically recognized antigen ation of the target antigen is required to avoid on-tar- presenting cells loaded with exogenous peptide or get, off-site toxicity. A safer alternative to targeting transfected with mRNA encoding the epitope. tumour-associated self-antigens may be to direct Finally, T cells transduced with the HLA-A2-restrict- engineered T cells against mutated proteins such as ed TGFβRIImut-specific TCR were demonstrated to sig- frequently occurring frameshift mutations. Further- nificantly reduce the growth of colorectal cancer and more, such frameshift mutations result in novel poly- enhance survival in a NOD/SCID xenograft mouse peptides allowing selection of TCRs from the non-tol- model. erant T-cell repertoire circumventing the problem of low affinity TCRs due to central tolerance. The transforming growth factor β Receptor II frameshift mutation (TGFβRIImut) is found in Lynch syndrome cancer patients and in around 15% of sporadic col- orectal and gastric cancers displaying microsatellite instability. The -1A mutation within a stretch of 10 adenine bases (nucleotides 709-718) of the TGFβRII gene gives rise to immunogenic peptides previously used for vaccination of MSI+ colorectal cancer pa- tients in a Phase I clinical trial. From one clinically responding patients we isolat- ed a CD8neg/CD4neg CTL clone from which we cloned an HLA-A2-restricted TGFβRIImut-specific TCR. We showed that both CD8pos and CD4pos T cells transient- ly redirected with this TGFβRIImut-TCR recognised colon carcinoma cell lines harbouring the frameshift mutation, indicating CD8 co-receptor independency. From two other vaccinated patients we isolated and cloned HLA-DR restricted TGFβRIImut-specific TCRs 171 | CELLULAR THERAPY
CAR T cells induce DC maturation via a cell contact-dependent process and enhance their ability to stimulate T cell responses
Karlsson H.1, Gustafsson W.1, Olsson-Strömberg U.2,3, Savoldo B.4, Dotti G.4, Loskog A.1
1Uppsala University, Department of Immunology, Genetics and Pathology, Uppsala, Sweden, 2Uppsala University, Department of Medical Sciences, Uppsala, Sweden, 3Uppsala University Hospital, Section of Hematology, Uppsala, Sweden, 4Baylor College of Medicine, Center for Cell and Gene Therapy, Houston, United States
Gene engineered T cells expressing a chimeric antigen with untransduced (59%) or Mock (50%) T cells ex- receptor (CAR) to target tumor cells are currently in pressed less CD83. DCs cultured alone maintained clinical investigation. The CAR receptor targets a tu- their immature phenotype and lacked CD83 while mor-associated antigen via a single-chain antibody positive control DCs were strongly CD83 positive extracellular domain and provides activation stimuli (93%). Co-cultures were also performed in a tran- via signaling domains in the intracellular portion. swell system where the cells share medium and can CAR T cells are activated upon antigen stimulation communicate via cytokines but cannot physically via the CAR. Activated T cells can up-regulate mole- interact. In this system, no maturation of the imma- cules such as CD40 ligand (CD40L) that is an import- ture DCs was seen, indicating that (in this setting) ant stimulator of dendritic cells (DCs). We hypothe- cell-cell contact is crucial for the maturation process size that CAR T cells may express CD40L as well as since cytokines alone couldn’t induce maturation. other molecules involved in the activation of anti- Furthermore, in the co-culture supernatants, cy- gen-presenting cells such as dendritic cells (DCs) and tokines indicative of DC maturation, such as IL-12, thereby participate in bystander immune responses IL-1β, IL-6, IFNγ and TNFα were present. The same to kill the tumor. cytokines were detected at lower levels or not detect- To perform proof of concept, human DCs were pre- ed at all in the transwell cultures. pared from CD14+ monocytes and co-cultured with Hence, CAR T cells may directly kill tumor cells but autologous CAR T cells, control T cells or with TNFα may also participate to drive activation of bystander (positive control) for 48 hours. The cell co-cultures tumor immunity via activation of DCs to broaden the were analyzed by multicolor flow cytometry to de- immune reaction against the tumors. termine the activation status of the DCs and the T cell phenotype. The DCs were then used in an assay to activate and expand CMV+ T cells to demonstrate functional capacity. Prior to co-culture, the CAR T cells expressed higher levels of CD40L, CD70 and CD27 than Mock T cells or untransduced T cells. CD40L decreased post co-cul- ture in all groups while CD70 increased in the CAR T cell group. CD27 expression was stable. The majority (91%) of DCs co-cultured with CAR T cells expressed the maturation marker CD83, while DCs co-cultured 172 | CELLULAR THERAPY
A combined human CD83- and Hsp70B’ promoter system enables transcriptional targeting of dendritic cells in vitro
Knippertz I.1, Deinzer A.1, Nettelbeck D.M.2, Steinkasserer A.1
1Universitätsklinikum Erlangen, Dept. of Immune Modulation at the Dept. of Dermatology, Erlangen, Germany, 2Heidelberg University, Helmholtz-University Group Oncolytic Adenoviruses at the DKFZ and Dept. of Dermatology, Heidelberg, Germany
Dendritic cells (DCs) play an important role in the to the short heat shock response element Hsp70B’ induction of immune responses towards tumors. In encoded on a second vector driving the expression order to improve their capacity to activate tumor- of the transgenes MelanA and IL-12p70. To show specific cytotoxic T cells, one strategy in DC-immu- “proof of principle” we used the murine DC-like cell notherapy is the concurrent overexpression of tumor line XS52, where we demonstrated a specific expres- antigens and immune-modifying mediators, such as sion of MelanA and IL-12p70 after co-transfection of MelanA and IL-12p70, respectively. Thus, we used respective plasmid-vectors. Next, we generated cor- the newly characterized human CD83 promoter in responding adenoviral vectors for the transcriptional combination with the heat shock protein (Hsp)- 70B’ targeting of mDCs. Here, we could show a highly promoter to transcriptionally target DCs in vitro, to specific expression of MelanA and IL-12p70 only in express different therapeutic transgenes. We first immunogenic mature DCs, but not in tolerogenic im- identified a 216bp short Hsp70B’ core promoter mature DCs, which was mediated by transcriptional element induced by a mutated constitutive active control of the human CD83 promoter. heat shock factor (mHSF)-1 to strongly drive gene Taken together, the newly generated MS-system expression of therapeutic transgenes MelanA and allows specific and simultaneous expression of dif- IL-12p70 in HeLa cells as well as in human mature ferent therapeutic transgenes in mature DCs in vitro, monocyte-derived dendritic cells (mDCs). Further- representing a promising tool to improve future DC- more, to overexpress mHSF-1 in DCs we also used based immunotherapies in vivo. the adenoviral vectors (Ad5mHSF1). Therefore, DCs were mock-treated or transduced with Ad5mHSF1 or control virus Ad5Luc1 and expression of various heat shock proteins was analyzed by Western Blot. Furthermore, we examined the surface expression of maturation markers like CD25, CD80, CD83, CD86, HLA-ABC and HLA-DR on transduced and control DCs as well as their cytokine profile (IL-1ß, IL-6, IL-8, IL-10, IL-12p70, TNF-a) in cell culture supernatants. Finally, we generated a two vector-based system - the “multiple switch (MS)” system - where the expression of mHSF-1 is controlled by the mDC-specific human CD83 promoter. Subsequently, mHSF-1 then binds 173 | CELLULAR THERAPY
Transduction with C-C-chemokine receptor type 4 (CCR4) enhances tumor-specific migration of adoptively transferred T cells in a model of pancreatic cancer
Rapp M.1, Grassmann S.1, Rataj F.1, Endres S.1, Anz D.1, Kobold S.1
1Klinikum der Ludwig-Maximilians-Universität, Abteilung für Klinische Pharmakologie, München, Germany
Introduction: Regulatory T cells selectively express C-C-chemokine receptor type 4 (CCR4) and are at- tracted by intratumoral CCL22 while CCR4 is absent from most cytotoxic T cells. We hypothesized that inducing forced expression of CCR4 on adoptively transferred cytotoxic T cells could enhance thera- peutic efficacy. Methods: CCR4 and a new non-functional CCR4-de- letion mutant were cloned into the retroviral vector pMP71 and transduced into murine ovalbumin-spe- cific T cells (OT-1). T cell migration and function in vitro were assessed using transwell assays. Results: In vitro, CCR4- but not CCR4del-transduced OT-1 T cells specifically migrated towards recombi- nant CCL22 and tumor-induced CCL22 (25-fold in- crease). In vivo, CCR4-transduced OT-1 T cells cured 50 % of mice bearing established Panc-OVA tumors compared to 12 % in the CCR4del- or untransduced OT-1 T cell- treated mice (n = 8 per group, p < 0.0001). Cured mice were protected from rechallenge, indicating the induction of anti-tumor immunity. Enhanced numbers of CCR4-transduced OT-1 T cells compared to CCR4del-transduced cells were found in the tumor, suggesting enhanced homing to the tumor site as the mode of action of CCR4-transduced T cells. Conclusions: The transduction of cytotoxic T cells with CCR4 represents a new therapeutic approach for effectively guiding adoptively transferred T cells into pancreatic tumors and thereby inducing a potent antitumor immune response. 174 | CELLULAR THERAPY
A new fusion receptor overcomes PD-1-mediated immunosuppression in adoptive T cell therapy
Kobold S.1, Grassmann S.1, Rataj F.1, Kraus F.1, Chaloupka M.1, Lampert C.1, Rothenfusser S.1, Schnurr M.1, Endres S.1
1Klinikum der Ludwig-Maximilians-Universität, Abteilung für Klinische Pharmakologie, München, Germany
Background: Recent evidence suggests that the co-inhibitory PD-1-PD-L1 axis plays a major role in adoptive T cell therapy failure. We hypothesized that a new fusion receptor reverting PD-1-mediated inhi- bition into CD28-costimulation may brake peripheral tolerance. Methods: Different PD-1-CD28 fusion receptor con- structs were created and retrovirally transduced into primary, ovalbumine specific CD8+ T cells (OT-1). Cytokine release, proliferation, cytotoxicity and tumor recognition were analyzed in vitro. Anti-tumor efficacy and mode of action were investigated in mice bearing subcutaneous Panc-OVA tumors. Results: Transduction of the PD-1-CD28 receptor constructs mediated enhanced cytokine-release, pronounced T cell proliferation and T cell-induced lysis of target tumor cells upon PD-L1 engagement. The PD-1-CD28 receptor function was dependent on two of the CD28-signaling motifs and IFN-γ release. Treatment of mice with established Panc-OVA tumors with fusion receptor-transduced OT-1 T cells mediat- ed complete tumor regression in 83% of mice. Mice rejecting the tumor were protected upon subsequent rechallenge with either ovalbumin positive or neg- ative tumors, indicative of a memory response and epitope spreading. Treatment efficacy was associated with accumulation of IFN-g producing T cells in the tumors. Conclusions: Transduction of T cells with this new receptor has the potential of braking the PD-1-PD-L1 immunosuppressive axis in ACT. 175 | CELLULAR THERAPY
Expression of immunogenic cell death markers on lung cancer cells
Kobosilova L.1,2, Hradilova N.1,2, Adkins I.1,2, Fucikova J.1,2, Spisek R.1,2, Kusova Moserova I.1,2
1Sotio, Prague, Czech Republic, 2Charles University, 2nd Faculty of Medicine, Prague, Czech Republic
Immunogenic cell death (ICD) is a type of cell death cytosis of HHP-treated tumor cells and they induced induced by various chemotherapeutics such as bor- lower numbers of regulatory T cells compared to im- tezomib, oxaliplatin and anthracyclines or physical mature DCs. modalities such as photodynamic therapy (PDT), ra- In conclusion, using a wide range of methods we diotherapy and high hydrostatic pressure (HHP). ICD confirmed that HHP is reliable and potent inducer of is characterized by presence of specific molecules in- ICD in lung cancer cell lines H520 and H522. Pheno- cluding surface exposed calreticulin (CRT) and the typic and functional characteristics of DCs were de- heat shock proteins HSP70 and HSP90. Release of scribed as well as decreased induction of regulatory ATP and high-mobility group box protein 1 (HMGB1) T-lymphocytes by pulsed DCs. These properties are belongs to other typical characteristics. important for possible use of autologous DC vaccine. We used HHP for induction of ICD in lung cancer cell This allows for the testing of HHP killed immunogen- lines H520 and H522 in comparison with non-immu- ic tumor cells in cancer immunotherapy trials. nogenic UV-B treatment. The kinetics of cell death and exposure of ICD markers was analyzed by flow cytometry, western blotting and confocal micros- copy. We also pulsed dendritic cells (DCs) with HHP-treated tumor cells and observed phagocytosis and maturation markers of DCs. Treatment by HHP induced expression of immuno- genic markers CRT, HSP70 and HSP90 on the cell surface of lung cancer cell lines. HHP also induced secretion of ATP to the extracellular milieu. More- over, activation of caspases (-8, -9, -3) and other pro- teins (phosphorylation of eIF2α) which are crucial in ER-stress mediated apoptotic pathway, was observed after HHP treatment. DCs pulsed with HHP-treated tumor cells showed phenotypic maturation char- acterized by upregulation of maturation molecule CD83, costimulation molecules CD80 and CD86, chemokine receptor CCR7 and MHC class II molecule HLA-DR. Pulsed DCs had also higher rate of phago- 176 | CELLULAR THERAPY
Engineering and testing of ‘smart’ T cells - counteracting immune evasion by solid tumors
Kunert A.1, Chmielewski M.2, Berrevoets C.1, Wijers R.1, Abken H.2, Debets R.1
1Erasmus MC, Medical Oncology, Rotterdam, Netherlands, 2Department I of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
Adoptive therapy with tumor-targeted, engineered T We observed that simultaneous transduction of the cells constitutes a promising new treatment approach TCR and induced cytokine transgenes, in combina- for patients with malignant disease. Treatment with T tion with a delayed start of T cell selection was most cells generally results in significant tumor regression, optimal with respect to numbers of T cells expressing however, it is mostly followed by tumor recurrence both constructs. We were further able to show that and incomplete responses. The tumor micro-environ- combination of the TCR with these induced mediator ment may direct immune evasion and result in insuf- constructs restricts the release of IL-12 and IL-18 to ficient numbers and function of intra-tumor T cells T cells exposed to target-antigen-positive cell lines. and monocytes (Straetemans, Mol Ther, 2015). In the Notably, production of IL-12 and IL-18 in these cul- present study, we propose a strategy that relies on tures increased the production of IFNγ by T cells 5-fold smart T cells (in extension to Chmielewski, Cancer and 7-fold, respectively. When co-culturing tumor cell Res, 2011) that locally produce Interleukin (IL) -12 and lines with a combination of IL-12- and IL-18-transduc- IL-18. Both of these cytokines are known stimulators ed T cells, we observed a stronger enhancement of of immune cell activation, differentiation and Interfer- IFNγ production than for either cytokine alone. on gamma (IFNγ) production by T cells - all of which Currently we are conducting an in vivo experiment, are crucial factors contributing to tumor clearance. using B57/Bl6 mice to investigate the effect the local- In this study, we engineered T cells with a TCR di- ized production of IL-12 and IL-18 has on the overall rected against a melanoma target antigen and addi- tumor growth and local IFNγ production, but also on tionally equipped them with a construct that allows the numbers and activation of intra-tumoral T cells inducible production of either murine IL-12 or murine and monocytes. IL-18. Cytokine production is under the control of a nuclear-factor of the activated T-cell (NFAT)-derived minimal promoter, ensuring that release of the trans- genic product is only initiated upon target-specific ac- tivation of the T cell via its TCR. We optimized the sequence of transductions, the concentration of select- ing antibiotics, as well as the timing and duration of T cell selection following transduction. Target specific release of IL-12 and/or IL-18 was confirmed via co-cul- tures of transduced T cells with antigen-positive and antigen-negative tumor cell lines. 177 | CELLULAR THERAPY
TCRs specific for MAGE-C2 in combination with epigenetic drugs provide anti-tumor responses toward tumors of multiple histologies
Kunert A.1, Van Steenbergen-Langeveld S.1, Van Brakel M.1, Oresta B.1, da Silva M.1, Coulie P.2, Debets R.1
1Erasmus MC, Medical Oncology, Rotterdam, Netherlands, 2Laboratory of Human Tumor Immunology, de Duve Institute for Cellular Pathology, Brussels, Belgium
T cells gene-engineered with T cell receptors (TCR) Comparative analysis of T cell functions toward ti- have shown significant clinical successes for pa- trated amounts of peptide enabled us to group TCRs, tients with advanced melanoma and other tumors. in particular HLA-A2-restricted TCRs, according to These trials, however, point out that further devel- their binding strength for MC2. Remarkably, mobili- opment of TCR gene therapy requires better choices zation of CD107a and production of IFNγ in response of target antigens to limit T cell-mediated toxici- to MC2-positive tumor cells were highest when ties in patients. In the present study, we assessed using TCRs with an intermediate affinity. In addi- whether and under which conditions MAGE-C2 tion, pre-treatment of tumor cells with the epigene- (MC2) provides an effective target antigen for T cell tic drugs Azacytidine, Valproate (which are already therapy. The rationale for MC2 include: its suppres- used in clinical studies) and the immune-stimulat- sive role in p53-dependent apoptosis in tumor cells; ing cytokine IFNγ further enhanced T cell respon- its epigenetically silenced expression in healthy siveness. With these conditions, TCR-transduced T adult tissues; its ability to induce an anti-tumor T cells showed distinctive responses toward multiple cell response in patients without signs of toxicity; MC2-positive cell lines from melanoma, head-and- and its presence in multiple tumor types, such as neck cancer, bladder cancer and triple-negative breast melanoma, head-and-neck cancer, bladder cancer, cancers, but not MC2-negative tumor cell lines. breast cancer, non-small cell lung cancer, and mul- In conclusion, we have selected an intermediate af- tiple myeloma. finity MC2-specific TCR as well as pre-treatment con- Here, we have isolated 10 sets of TCRα and TCRβ se- ditions that are now translated to a clinical phase I/II quences from melanoma patients who showed clin- study to treat patients with advanced melanoma and ical responses upon vaccination, accompanied by head-and-neck cancer. significant frequencies of MC2-specific CD8 T cells. These TCRs are directed against 4 different MC2 epitopes presented by Human Leukocyte-associated Antigens (HLA-)A2 (ALK, LLF), HLA-B44 (SES) or HLA-B52 (ASS). TCR genes were codon-optimized, cloned into a clinical pMP71 vector using a TCRb- 2A-TCRa configuration, and introduced into T cells from healthy donors. TCR T cells were subsequently enriched to ensure high and equal surface expres- sion of MC2 TCRs. 178 | CELLULAR THERAPY
Vitiligo T cell receptor SILv44 imparts a Tc17 profile and anti-tumor reactivity on host T cells
Eby J.M.1, Klarquist J.1, Li M.2, Wainwright D.A.3, Watkins S.K.1, Paulos C.M.2, Westerhof W.4, Mehrotra S.2, Garrett-Mayer E.2, Luiten R.M.4, Nishimura M.I.2, Le Poole C.1
1Loyola University Chicago, Maywood, United States, 2Medical University of South Carolina, Charleston, United States, 3Northwestern University, Chicago, United States, 4University of Amsterdam, Amsterdam, Netherlands
We questioned the role of T cell receptors (TCRs) in defining functional differences among autoimmune and tumor reactive T cells. We thus probed the reac- tivity of TCRs reactive with the same antigenic peptide from vitiligo and melanoma patients, after expression within the same host cells. For these studies, we prop- agated T cells from depigmenting skin, identified a gp100-reactive TCR named SILv44 for ‘skin infiltrating lymphocytes from vitiligo’, and cloned it into a ret- roviral vector to allow a comparison to TCRs R6C12 and T4H2 from circulating melanoma T cells upon T cell transduction. Although melanoma patients were
peptide gp100209-2M vaccinated to drive high avidity T cell responses, greatest IL-2 secretion was observed by SILv44 expressing CD8+ Jurkats. In transduced CD8+ primary human T cells, SILv44 expressing CTL responded to 888A2+ melanoma cells by enhanced CD107A surface expression and IL-17A secretion; the latter was also observed in response to melanocytes. Importantly, cytokine expression by TCR transduced T cells shifted from IFN-γ to IL-17A by limiting peptide availability to physiologic concentrations. Superior an- ti-tumor efficacy of SILv44 transduced T cells was ob- served in vivo, where IL-17A expressing T cells limited the growth of 888A2+ tumors in SCID/beige mice. This comparison provides surprising cues about TCR con- tributions to T cell plasticity, supporting a role for TCR variable regions in defining T cell cytokine secretion patterns and function. The data further suggest that vitiligo patient skin can provide a valuable source of T cell receptors with therapeutic potential in melanoma. 179 | CELLULAR THERAPY
Tumor-reactive T-cells from patients with glioblastoma for cellular therapy
Liu Z.1, Meng Q.1, Poiret T.1, Rane L.1, Ambati A.1, Dodoo E.2, Maeurer M.3
1Karolinska Institutet, Division of Therapeutic Immunology, Department of Laboratory Medicine, Stockholm, Sweden, 2Karolinska Institutet, Department of Laboratory Medicine, Department of Neurosurgery, Karolinska Hospital, Stockholm, Sweden, 3Karolinska Institutet, Division of Therapeutic Immunology, Department of Laboratory Medicine, CAST, Karolinska University Hospital, Stockholm, Sweden
Background: Tumor-infiltrating T-cells (TILs) may 4.70%, in CD4+ TILs: median: 0.83%, mean: 1.50%), represent a viable source of T-cells for the biologi- CTLA-4 (in CD8+ TILs: median: 2.69%, mean: 5.20%, cal treatment of patients with tumors of the central in CD4+ TILs: median: 0.76%, mean: 1.06%) and nervous system. We established a rapid TIL expan- LAG-3 (in CD8+ TILs: median: 2.79%, mean: 4.30%, sion protocol for patients with glioblastoma, and in CD4+ TILs: median: 1.52%, mean: 2.05%). TIL cul- tested the recognition of autologous tumor cell lines tures exhibited preferential usage of Vβ families (i.e defined by cytokine production and cytotoxicity. up to 99.60% Vβ2 or 97.00% Vβ5.1 in CD4+ or CD8+ Material and methods: Glioma tumor tissue was ob- TIL). TIL from individual patients exhibited NY-ESO- tained from 16 patients with glioblastoma, tumor cell 1 specificity up to 2% in CD4 and CD8+ T-cells, yet up lines were established, TILs could successfully ex- to 25% TNFα production directed against autologous panded in 16/16 cases. Intracellular cytokine stain- tumor cells, defined by ICS. TILs showed strong cy- ing (ICS) was used to detect antigen-specific immune tolytic functions directed against autologous tumor responses. Autologous tumor cells or TAAs (NY-ESO- cells, i.e. up to 70% specific lysis at an effector/target 1, Survivin and EGFRvIII peptides) were co-cultured ratio of 25/1. with TILs in the presence of brefeldin A as well as Conclusion: TILs from glioma samples can be reli- in medium (negative control), or PMA+Ionomy- ably and successfully expanded, they show prefer- cin (positive control). CD3, CD4 and CD8 markers ential expansion of VB families, exhibit a Th1-cy- were combined with either IL-2, IL-17, TNFα, IFNγ tokine production pattern and recognize autologous production or 4-1BB expression. Vβ family composi- tumor cells. Tumor samples are currently sequenced tion, exhaustion/activation as well as differentiation to detect the nominal targets. Glioma - reactive TIL markers were tested by flow cytometry, cytotoxic represents a viable source the cellular therapy for pa- T-cell responses were measured using a Cr51 release tients with glioblastoma, a phase I safety trial for the assay. treatment of patients with glioblastoma is prepared Results: 16/16 TILs could successfully expanded. The at Karolinska Hospital. majority of TIL exhibited a mixture of CD4+, CD8+, as well as CD3+ (TCRα/β) CD4-CD8- T-cells with an memory cell phenotype (CD45RA-CCR7+or CD45RA- CCR7-). TILs showed low frequencies of exhaustion markers, i.e. PD-1 (in CD8+ TILs: median 1.53%, mean: 23.13%, in CD4+ TILs: median: 8.95%, mean: 10.30%), TIM-3 (in CD8+ TILs: median: 1.61%, mean: 180 | CELLULAR THERAPY
Using Merkel cell polyomavirus specific TCR gene therapy for treatment of Merkel cell carcinoma
Lyngaa R.1, Pedersen N.W.2, Linnemann C.3, Schrama D.S.4, Ibrani D.5, Met Ö.6, Thor Straten P.2, Nghiem P.7, Becker J.8, Hadrup S.R.1
1Veterinary Institute, Technical University of Denmark, Section of Immunology and Vaccinology, Fredriksberg C, Denmark, 2Herlev Hospital, Center for Cancer Immune Therapy, Herlev, Denmark, 3Netherlands Cancer Institute, Dept. of Immunology, Amsterdam, Netherlands, 4Medical University of Graz, General Dermatology, Graz, Austria, 5University of Washington, Departments of Medicine/Dermatology, Seattle, United States, 6Herlev Hospital, Center for Cancer Immunotherapy and Department of Oncology, Herlev, Denmark, 7University of Washington, General Dermatology, Seattle, United States, 8Medical University of Graz, Departments of Medicine/Dermatology, Graz, Austria
T cell receptor gene-therapy provides an ideal teins Large T antigen and small T antigen were ex- concept for an off-the-shelves immunotherapeutic clusively present in peripheral blood of MCC patients, strategy. This strategy has no requirement for a pre- but not in healthy donors. We further demonstrate existing anti-tumor immune response in tumor infil- both the processing and presentation of the oncopro- trating lymphocytes. T cell receptor gene therapy has tein-derived epitopes, as well as the lytic activity of entered the clinic and shown potential for successful oncoprotein-specific T cells towards MHC-matched cancer treatment. However, the clinical evaluation- MCC cells. Demonstrating the presence of oncopro- has also highlighted the need for selection of truly tein-specific T cells among tumor infiltrating lym- cancer-specific targets. Since the gene-modified T phocytes ex vivo further substantiated the relevance cells are very potent in cell killing, these may exert of the identified epitopes. The viral epitopes repre- dramatic side effects if their target is recognized on sents specific targets and should be ideal for TCR- other tissues than the tumor cells. Merkel cell car- gene therapy approaches. We are establishing an in cinoma (MCC) is a highly aggressive skin cancer vivo system for testing the ability of MCPyV specific that mainly affects the elderly and immunosup- TCR gene-modified T-cells to repress tumor growth. pressed population and is associated with Merkel We have isolated and sequenced T cell receptors spe- cell polyomavirus (MCPyV). Due to the clear viral cific for the MCPyV oncogenic proteins restricted to correlation we speculated that CD8+ T cells specific HLA-A2, B7 and A24 and inserted the receptor into for viral epitopes would be present in MCC patients a retroviral vector. Currently we are testing in vitro and some might also be specific for MCC patients. transduction systems with the purpose of introduc- We have identified MCPyV specific T cells using a ing the TCRs into human PBMC, injecting them into high-throughput platform for T-cell enrichment and immune deficient NOG mice carrying HLA matched combinatorial encoding of major histocompatibility MCPyV positive tumor to investigate the tumor rejec- complex (MHC) class I multimers. Strikingly, T-cell tion capacity of these gene-modified T cells. responses against the two oncogenic MCPyV pro- 181 | CELLULAR THERAPY
Development of clinically implementable imaging strategies for tracking T cell receptor-transgenic effector T cells
Mall S.1, Yusufi N.2, Klar R.1, Bianchi H.1, Laitinen I.2, Steiger K.3, Aichler M.4, Feuchtinger A.4, Walch A.4, Schwaiger M.2, Peschel C.1, D´Alessandria C.2, Krackhardt A.1
1Technical University Munich, Klinikum rechts der Isar, Munich, Germany, 2Technical University Munich, Nuklearmedizinische Klinik und Poliklinik, Munich, Germany, 3Technical University Munich, Institut für Allgemeine Pathologie und Pathologische Anatomie, Munich, Germany, 4Helmholtz Center Munich, Research Unit Analytical Pathology, Munich, Germany
Transfer of T lymphocytes that have been genetically ML2. After tumor inoculation, TCR-transduced human modified to express T-cell receptors (TCR) specific for TCM were transferred intratumorally and PET/CT tumor associated antigens, is an upcoming therapy for imaging was performed at different time points post diverse cancers. However, first clinical trials revealed intravenous injection of 89Zr-TCRmu-mAb. A clear risks regarding safety as well as limited efficacy using signal was detected in tumors injected with TCR trans- this approach. In order to understand T-cell traffick- genic T cells, but also high background signal was ing, expansion and functionality in vivo, the develop- observed in tumors injected with nontransduced TCM ment of non-invasive and highly sensitive cell-track- or PBS. In vitro analysis revealed high expression of ing technologies would be of high value. Moreover, Fc-receptors on the surface of tumor cells, which may clinical translation of such technologies would further account for the binding of the TCRmu-mAb. As this provide possibilities to improve this therapeutic ap- occurrence might be relevant as well in the human proach in humans. We therefore aimed to establish a system, we produced a F(ab)´2 fragment of the anti- non-invasive, clinically translatable imaging approach body. In vitro characterization of 89Zr-TCRmu-F(ab)´2 for in vivo monitoring of adoptively transferred T cells showed similar binding affinities to TCM compared to engineered with tumor-specific TCRs. the full antibody. Functionality of T cells after label- We have previously isolated several TCR specifically ling showed no differences compared to 89Zr-TCRmu- recognizing peptides derived from diverse tumor as- mAb labelled or unlabelled cells. In vivo application sociated antigens and selected one TCR which demon- demonstrated a strong signal on the tumor site where strated strong anti-leukemic efficacy by recognizing TCR-transgenic T cells were injected, but no signal the hematopoietic differentiation antigen myeloper- when nontransduced TCM or PBS were used. These oxidase. results correspond to ex vivo biodistribution and au- Central memory T-cells (TCM) were retrovirally trans- toradiography data. duced with the selected TCR. To monitor TCR-trans- In summary, we developed a non-invasive imaging duced T cells in vivo, we used Zirconium-89 labelled model for tracking specifically human TCR-engi- anti-murine TCR antibody (TCRmu-mAb), which neered lymphocytes in vivo. This TCRmu-F(ab)´2- binds to the murinized part of the introduced TCR. fragment based PET imaging modality may be useful Specific 89Zr-TCRmu-mAb binding to TCR-transduced to monitor adoptively transferred TCR-transgenic T TCM was observed while functionality of the cells re- cells in murine models in order to optimize safety and mained unaffected. efficacy of such therapies. A xenogenic mouse model was established using NSG mice injected subcutaneously with the AML-cell line 182 | CELLULAR THERAPY
Interleukin-15 potentiates human natural killer cells to resist tumor- induced suppression through mTOR-regulated metabolic control
Mao Y.1, Zhang X.2, Wennerberg E.2, vo Hoef V.2, Larsson O.2, Linder S.2, Kiessling R.2, Lundqvist A.2
1Karolinska Institutet, Department of Oncology-Pathology, Stockholm, Sweden, 2Karolinska Institutet, Stockholm, Sweden
In cancer patients, anti-tumor functions of NK cells duced suppression. It provides evidence that imple- are severely impaired by a variety of immunosup- mentation of IL-15 may greatly improve the clinical pressive mechanisms. Interleukin (IL)-2 and -15 are efficacy of adoptive NK cell therapy for the treatment two essential cytokines regulating the development of human cancers. and function of human natural killer (NK) cells. Here, we compared the role of IL-2 and IL-15 to render resis- tance of human NK cells to tumor-induced suppres- sion. We found that early-passage melanoma tumor cells strongly inhibited functions of IL-2 activated NK cells through production of prostaglandin E2 (PGE2). Under the same condition, IL-15 activated NK cells could significantly retain the ability to proliferate (p< 0.05) and lyse target cells (p< 0.05). Further- more, melanoma patient-derived NK cells stimulated by IL-15 had significantly improved production of IFNg (p< 0.05) and cytotoxicity against melanoma targets (p< 0.01) compared with IL-2 activation. In further analysis, we found that IL-15 enhanced surface anti-oxidant, retention of membrane-bound IL-15 and elicited stronger anti-apoptotic signaling through Bcl-2 in primary human NK cells. Analyzed in real-time, IL-15 elicited superior basal (p< 0.05) and maximal (p< 0.05) mitochondrial as well as glycolytic potential (p< 0.05) through mTOR-gov- erned machineries. Even though IL-15 did not gen- erate higher numbers of NK cells after expansion (14 days), we observed up-regulated surface expression of CD25 (IL2Rα) and enhanced in vitro durability, in comparison to IL-2-expanded cells. Altogether, our study uncovers distinct properties between IL-2 and IL-15 on primary human NK cells under tumor-in- 183 | CELLULAR THERAPY
Immunoregulatory mechanisms in melanoma
Melief S.1, Visser M.1, van der Burg S.1, Verdegaal E.1
1Leiden University Medical Center, Clinical Oncology, Leiden, Netherlands
In our ongoing clinical trial we apply adoptive Other factors that might hamper the generation of T-cell transfer to treat malignant melanoma. In tumor-reactive T cells in MLTC are immunosuppres- this protocol, patients are infused with peripheral sive molecules that are expressed by the tumor cells. blood-derived tumor-specific autologous T cells that Indoleamine 2,3-dioxygenase (IDO) is such a factor, are generated in a mixed lymphocyte tumor culture it inhibits the proliferation of T cells by degrading (MLTC). tryptophan and is also described to induce Tregs. T cells that were transfused to non-responder pa- Our tumor cell lines showed differential expression tients showed a lower expansion rate in vitro than of IDO mRNA and all lines showed upregulation the T cells that were generated for responder pa- of IDO expression after stimulation with IFNγ. The tients. The aim of the current project is to gain more IDO inhibitor 1-methyl-D-tryptophan (1-MT-D) was insight in the underlying mechanisms of the gen- added to the MLTC 3 times per week and this in- eration of T cell batches, with the goal to improve creased the proliferation of T cells for most tumor the expansion of melanoma-specific T cells and to lines. In addition, the number of tumor-reactive T increase the frequency of responder patients. cells, measured by expression of CD137, CD154 and The difference in expansion rates might be explained the cytokines IL-2 and IFNγ, was increased when by the high frequency of CD4+CD25hiFoxP3+ regula- 1-MT-D was added to the MLTC. tory T cells (Tregs) in T cell batches from non-re- We conclude that depletion of CD25+ cells from the sponder patients. Therefore, CD25+/ h i cells were de- MLTC does not consistently lead to improved T-cell pleted during in vitro expansion. For some patients expansion. Although CD4 depletion from PBMC this had a beneficial effect on the expansion, but this improves the expansion for some patients and it result was not consistent when repeated for multiple increases the frequency of tumor-specific CD8 T patients. CD25 depletion by magnetic activated cell cells in all T cell batches, this procedure is not pre- sorting (MACS) lacked could not separate between ferred because all CD4 tumor-reactive T cells are CD25hi Tregs and CD25+ activated T cells. Since lost. Addition of the IDO-inhibitor 1-MT-D results Tregs accumulate in the CD4 fraction of the T cells, in better proliferation rates and increased numbers we continued by depleting the CD4 population from of tumor-reactive T cells. Therefore, adding 1-MT-D the PBMC at the start of the MLTC. This resulted in to our clinical MLTC protocol might increase the increased proliferation for some T cell batches and efficiency of generating tumor-reactive T cells and all T cell batches contained a higher frequency of thereby enhance the response to adoptive T cell CD8+CD137+ tumor-reactive T-cells, which might be transfer in malignant melanoma patients. explained by the absence of Tregs in these cultures. 184 | CELLULAR THERAPY
Tumor-infiltrating lymphocytes for cellular therapy of patients with pancreatic cancer
Meng Q.1, Rangelova E.2, Liu Z.1, Ambati A.1, Poiret T.1, Rane L.1, Xie S.3, Verbeke C.4, Jiri B.5, Dodoo E.5, Löhr M.2, Segersvärd R.2, Maeurer M.6
1Karolinska Institutet, Division of Therapeutic Immunology, Department of Laboratory Medicine, Stockholm, Sweden, 2Karolinska Institutet, Department of Clinical Science Intervention and Technology, Karolinska Hospital, Stockholm, Sweden, 3Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Stockholm, Sweden, 4Karolinska Institutet, Division of Pathology, Department of Laboratory Medicine, Stockholm, Sweden, 5Karolinska Institutet, Department of Neurosurgery, Stockholm, Sweden, 6Karolinska Institutet, Division of Therapeutic Immunology, Department of Laboratory Medicine, CAST, Karolinska University Hospital, Stockholm, Sweden
Purpose: The generation of T lymphocytes with Results: We could reliably expand TILs, that resided specific reactivity against autologous tumor cells is predominantly in the central memory population a prerequisite for effective adoptive transfer thera- (CD45RA-CCR7+), from 17/17 patients. TIL showed pies. We established a protocol for tumor infiltrating reactivity to common TAAs, i.e. to mesothelin (16 lymphocytes (TILs) cultures from biopsies obtained /17), survivin (12 / 17) or to NY-ESO-1(11 / 17) defined from patients with pancreatic ductal adenocarcino- by intracellular cytokine staining. Some TIL exhib- ma (PDAC). ited strong cytotoxicity directed against the autolo- Methods: Tumor biopsy specimens obtained from 17 gous tumor cell line. 12/17 of CD8+ TILs are either patients with PDAC were cultured with TIL medium, oligo- or monoclonal (e.g. some CD8+ TIL showed containing IL-2, IL-15 and IL-21. After 10 days of 99.3% Vb13.2, 77% Vb1, 68.7% Vb22, or 64% ex- culture, TILs were stimulated with anti-CD3 (OKT3) pansion of the Vb14 family). An Vb13,2+ CD8+ TIL and rapidly expanded. The specific activity of TILs clone strongly recognizes autologous tumor cells, towards shared tumor associated antigens (TAAs, defined by INF-γ production, and could be blocked mesothelin, survivin and NY-ESO-1) was detected by by the anti-HLA class 1 antibody (W6/32). We were Intracellular staining (ICS) by flow cytometry, cyto- able to expand about 1.5 x 10e9 TIL from small PDAC toxicity was measured using a Chromium-51 release biopsies. assay and the specific activity of TILs against au- Conclusion: We have optimized methods for gener- tologous tumor was measured by INF-γ production. ating pancreatic cancer specific TIL cultures from TCRVb analysis was investigated by a multiplex an- small resected tumor specimens. Tumor specimens tibody TCR Vb specific panel. The TCR clonality was are currently sequenced to detect potential targets evaluated a by CDR3 region PCR analysis and subse- for anti-PDAC directed T-cell clones. A phase I study quent sequencing. The T-cell phenotype, as well as is prepared at Karolinska for the cellular therapy of activation/exhaustion marker profile was tested by patients with pancreatic cancer. flow cytometry. 185 | CELLULAR THERAPY
TALEN-modified T cells for adoptive cellular therapies: genetic silencing of the glucocorticoid receptor in virus-specific T cells
Menger L.1, Gouble A.2, Smith J.2, Quezada S.A.1, Peggs K.S.1
1UCL Cancer Institute, London, United Kingdom, 2Cellectis S.A, Paris, France
ORAL ALK SHORT T 2015
Cytomegalovirus (CMV) remains an important cause provide engineered antigen specific (viral- or tu- of morbidity and mortality following allogeneic mour-reactive) T cell immunotherapy. haematopoietic stem cell transplantation (alloSCT) during the treatment of haematological malignan- cies. Accelerated reconstitution of anti-viral immu- nity is now clinically achievable through adoptive transfer of HLA-Streptamer®-selected CMV-specific CD8+ T cells, as shown by the results of a recent ran- domized trial (CMV-ASPECT). However, the highest clinical risk group, patients with active graft-versus- host disease (GVHD) receiving systemic corticoste- roid therapy is excluded, due to suppression of T cell function and induction of apoptosis, entirely depen- dent on the glucocorticoid receptor (GR). In order to obviate the impact of glucocorticoids on CMV-spe- cific immunity, we propose a protocol for adoptive transfer of GR gene inactivated CMV-specific T cells. The recently described transcription activator-like effector nucleases (TALENs) are highly efficient and can specifically silence target genes. Using electro- poration of TALEN mRNA targeting the GR gene, we demonstrate efficient gene inactivation (60%) of Streptamer®-selected human primary CMV-reactive T cells (99.8% purity), conferring resistance to gluco- corticoids in vitro and most importantly in vivo, after transfer into irradiated NOD-scid IL2rγnull (NSG) mice. Finally, GR-KO CMV-specific CD8+ T cells retain spe- cific killing activity against target cells pulsed with the CMV peptide pp65 after treatment with glucocor- ticoids. This proof of concept is the first step towards a clinically translational protocol relevant to rapidly 186 | CELLULAR THERAPY
Effective immunotherapy with Cytokine Induced Killer (CIK) cells against autologous gastrointestinal stromal tumors
Mesiano G.1, Leuci V.1, Gammaitoni L.1, Giraudo L.1, Capellero S.1, D’Ambrosio L.1,2, Rotolo R.2, Fiorino E.2, Melano C.2, Pisacane A.1, Grignani G.1, Aglietta M.1,2, Sangiolo D.1,2
1Candiolo Cancer Institute-FPO IRCCS, Candiolo, Italy, 2University of Torino, Torino, Italy
Background: Cytokine-Induced Killer (CIK) cells are gous GIST targets (n=6) even at minimal effector/ ex vivo expanded T lymphocytes with mixed T-NK target (E/T) ratios. Mean specific killing was 81% phenotype, endowed with MHC-unrestricted antitu- (E/T=40:1), 73% (E/T=20:1), 63% (E/T=10:1), 55% mor activity. We previously reported their preclinical (E/T=5:1), 35% (E/T=1:1) 26% (E/T=1:2); 21% (E/ activity against sarcomas but few data are available T=1:4). Treatment in vitro for 72 hours with imatinib against gastrointestinal stromal tumors (GIST) and (10-15 µM) effectively inhibited viability of GIST cells complementarity with molecular targeted therapy. (>80%). GIST surviving exposure to imatinib were Almost 90% of GIST harbor activating mutations in efficiently killed by autologous CIK cells (n=3). Mean the KIT receptor gene and are treated with small-mol- specific killing was 95% (E/T=40:1, 20:1, 10:1), 83% ecule KIT inhibitor imatinib. Clinical results are (E/T=5:1), 78% (E/T=1:1), 50% (E/T=1:2); 26% (E/ often impressive but transitory and patients almost T=1:4). In selected experiments (n=2) we evaluated invariably relapse. A challenging research issue is vi- the presence of putative gCSC by engineering GIST sualization and killing of putative cancer stem cells cultures with a lentiviral CSC-detector vector encod- (CSC) considered implicated in disease relapse. ing the eGFP transgene under control of stem-gene Main aim: was to explore the preclinical antitumor promoter Oct4. This approach was recently validated activity of CIK cells against autologous GIST. Spe- in our lab and allowed to visualize a subpopulation cific objectives were complementarity with Imatinib of putative GFP+gCSC (mean 5%). We confirmed by and potential to kill putative GIST cancer stem cells flow-cytometry that the antitumor activity of CIK (gCSC). cells, described above, fully involved GFP+gCSC at Experimental design and results: We generated all E/T ratios explored. CIK cells from 6 patients with advanced GIST. Stan- Conclusions: We reported first proof of concept that dard protocol involved culturing of peripheral blood CIK cells can efficiently kill autologous GIST cells, in- mononuclear cells for 3 weeks with timed addition cluding a subset of putative gCSC considered import- of IL2, Ab anti-CD3 (OKT3) and IFN-γ. Primary GIST ant for disease relapse and chemoresistance. Antitu- cultures were generated from surgical biopsies and mor activity was retained at unfavorable E/T ratios, used as autologous targets. GIST were validated by more representative of realistic clinic scenarios. Our pathology revision, expression of DOG1 (discov- findings provide biologic basis for future clinical ered on GIST-1) protein and sensitivity to imatinib. studies, supporting integration of immunotherapy Stress inducible molecule ULBP-2 was the most ex- with imatinib-based molecular targeted approaches. pressed (55%) among known ligands recognized by CIK cells. CIK cells effectively killed all autolo- 187 | CELLULAR THERAPY
Enhancing CD8+ CAR T cells antitumour efficiency by miR-155 transgenic expression
Monnot G.C.1, Santoro S.2, Alatzoglou D.1, Dudda J.1, Coukos G.1,2, Romero P.1
1UNIL, LICR, Epalinges, Switzerland, 2University of Pennsylvania, Philadelphia, United States
Chimeric antigen receptors (CARs) are fusion pro- These results suggest the potential of using microR- teins expressed at the cell surface designed to redi- NAs for the enhancement of CAR antitumor therapy rect autologous patients’ T cells against a tumour and future experiments will be performed to assess antigen. CARs incorporate a single chain with the the in vivo expansion potential, maintenance over antigen binding domains of an antibody fused to the time and therapeutic activity of T cells reprogrammed CD3-zeta intracellular signaling domain, allowing T with this novel CAR design. cell activation upon recognition of a specific target. Adoptive transfer of CAR+ T cells has shown great therapeutic potential in patients with B-cell malig- nancies. Here, we aim to improve further the efficiency of current approaches to CAR+ T cell therapy for solid tumours via the transgenic expression of miR-155. The rationale is based on observations that enforced expression of this microRNA in CD8+ T cell dramati- cally improved the antitumor response in a mouse model of melanoma. We constructed a lentiviral vector expressing a CAR specific for the human pros- tate-specific membrane antigen (PSMA), fused to the CD28 costimulatory signalling domain spliced to the CD3-zeta signalling chain. In addition, our construct includes the expression of miR-155, constitutively or under control of the NFAT-responsive promoter. Our results show that upon activation, miR-155 was up- regulated in CD8+ CAR+ NFAT miR-155. This was sufficient to induce a better proliferation of the cells in vitro, but not an increase of the therapeutic effect in vivo. Moreover, CAR expressing constitutive miR-155 had increased proliferative potential, as well as a better resistance to apoptosis. CAR+ T cells can effi- ciently and specifically kill PSMA+ endothelial cells. 188 | CELLULAR THERAPY
Following the fate of genetically modified T cells in melanoma patients
Moore T.1, Scurti G.1, Moxley K.1, Regan C.1, Hutchens K.2, Godellas C.3, Clark A.L.4, Dropulic B.5, Embree H.5, Shirai K.6, Garrett-Mayer E.7, Li M.8, Eby J.1, Le Poole C.1, Clark J.I.9, Nishimura M.I.1
1Loyola University Chicago, Oncology Research Institute, Maywood, United States, 2Loyola University Chicago, Pathology, Maywood, United States, 3Loyola University Chicago, Surgery, Maywood, United States, 4Loyola University Chicago, Maywood, United States, 5Lentigen Technology, Gaithersburg, United States, 6Medical University of South Carolina, Hematology and Oncology, Charleston, United States, 7Medical University of South Carolina, Biostatistics, Charleston, United States, 8Bluebird Bio, Cambridge, United States, 9Loyola University Chicago, Hematology and Oncology, Maywood, United States
Metastatic melanoma is a devastating disease with few in vitro, transduced T cells lose transgene expression treatment options available. In an open clinical trial for over time, and culturing in the absence of cytokines Stage IV melanoma patients, we are testing a promis- dramatically increases the rate of transgene expression ing new therapy in which the patient’s own T cells are loss. As seen ex vivo, restimulation of in vitro cultured genetically engineered with a T cell receptor (TIL 1383I T cells restores transgene expression. Interestingly, the TCR) targeting the melanoma antigen tyrosinase. In- cells that express the most transgene are those that cluded in the TIL 1383I TCR vector is a nonfunctional are undergoing cell division. These data suggest that CD34 marker that can be readily tracked by flow cy- mitogenic factors causing cell division may promote tometry. These transgenic T cells are infused into the transgene expression. patient with the goals of killing tumor cells and pro- Although we have seen clinical responses to the trans- moting anti-tumor inflammatory responses. ferred transgenic T cells, we hypothesize that both The fate of the transgenic T cells is monitored in fol- loss of transgene expression and increasing levels of low-up samples of patient peripheral blood, to iden- exhaustion may be hindering the efficacy of our trans- tify T cell persistence and function. We find that ferred transgenic T cells. We are therefore looking the transgenic T cells persist at low levels for long into therapies that might be added to our protocol periods of time in the patient. Preliminary data sug- that address these concerns. High-dose IL-2 is cur- gests that many of the transgenic T cells are activated rently FDA-approved for treatment of melanoma and in the patient, indicating that they are responding to is known to induce widespread T cell proliferation. antigen. A subset of these activated cells show signs PD-1 blockade can reduce and/or prevent exhaustion of becoming exhausted after transfer, expressing el- and is currently being used as a therapeutic agent for evated levels of PD-1. Interestingly, we find that the melanoma. We plan to test these therapies and poten- percentage of transgene-expressing T cells in patient tially amend our protocol in the future to reduce the samples is significantly augmented by cytokine stimu- effects of exhaustion and transgene loss on transgenic lation and/or CD3/CD28 stimulation. When cultured anti-tumor T cells. 189 | CELLULAR THERAPY
Targeting Claudin-6 with CAR-engineered T cells for individualized immunotherapy of ovarian cancer
Mroz K.A.1, Simon P.1, Mades A.2, Birtel M.3, Tolliver C.1, Klein O.1, Büchling T.3, Löw R.4, Voss R.-H.2, Hartwig U.2, Külcke K.4, Walter K.5, Türeci Ö.5, Sahin U.2,3
1BioNTech Cell & Gene Therapies GmbH, Mainz, Germany, 2Universal Medical Center of Johannes Gutenberg-University, Department of Hematology and Oncology, Mainz, Germany, 3TRON gGmbH, Translational Oncology at the University Medical Center, Mainz, Germany, 4EUFETS GmbH, Mainz, Germany, 5GANYMED Pharmaceuticals AG, Mainz, Germany
The potential of engineered antigen-specific T cells to final lead candidate was tested in vitro for its ability eradicate tumors provoked the development of novel to direct CAR-transduced T cells to specifically lyse, T cell based immunotherapy approaches. These proliferate and secrete cytokines in response to anti- include the adoptive transfer of autologous T cells gen-expressing target cells. genetically engineered with tumor antigen-specific Multiple producer cell clones were generated and the receptors, such as conventional α/β TCRs or chime- final clone for the generation of a master cell bank ric antigen receptors (CAR). CARs are recombinant under GMP-compliant conditions was selected based receptors that combine HLA independent scFv-me- on best titer and growing properties. diated antigen-binding with T cell signaling. We We further evaluated the in vivo antitumoral activ- designed a second generation CAR with specificity ity of the CLDN6-CAR using an ovarian carcinoma for Claudin-6 (CLDN6), coupled to CD28 and CD3ζ xenograft model. It could be demonstrated that the signaling domains. CLDN6 is an onco-fetal gene that adoptive transfer of CLDN6-CAR transduced T cells belongs to the family of tight junction proteins and resulted in a significantly slowed tumor growth com- is expressed in human stem cells and during early pared to control CAR T cells. stage of epidermal morphogenesis. As it is absent in adult healthy tissues, but overexpressed in different cancers including ovarian cancer, CLDN6 represents an ideal target antigen for immunotherapy based on CAR-engineered T cells. Our lead candidate for preclinical testing was selected among different CAR formats after functional char- acterization in vitro using mRNA transfer to achieve rapid expression of the different CLND6-CAR con- structs in T lymphocytes. Subsequently, we select- ed the self-inactivating (SIN) vector pES12.6 among different retroviral vector backbones for stable gene transfer of the CLDN6-CAR providing the final lead structure for preclinical and clinical testing. The 190 | CELLULAR THERAPY
Phase I clinical trial using autologous TIL 1383I TCR transduced T cells
Nishimura M.I.1, Regan C.2, Scurti G.1, Hutchens K.3, Dropulic B.4, Embree H.4, Shirai K.5, Garrett-Mayer E.6, Godellas C.1, Clark A.L.7, Li M.8, Eby J.3, Moxley K.1, Moore T.1, Le Poole C.3, Clark J.I.2
1Loyola University Chicago, Department of Surgery, Maywood, United States, 2Loyola University Chicago, Department of Medicine, Maywood, United States, 3Loyola University Chicago, Department of Pathology, Maywood, United States, 4Lentigen Corporation, Gaithersburg, United States, 5Medical University of South Carolina, Department of Medicine, Charleston, United States, 6Medical University of South Carolina, Department of Public Health Sciences, Charleston, United States, 7Loyola University Chicago, Cardinal Bernardin Cancer Center, Maywood, United States, 8Medical University of South Carolina, Department of Surgery, Charleston, United States
Adoptive T cell transfer of T cell receptor (TCR) gene full screening process for inclusion in the trial. Of modified T cells have been used to treat patients the six subjects that were fully screened, three were for more than a decade. While objective clinical re- deemed to be eligible for the study and were then sponses have been observed in patients treated with treated at the initial dose. The T cell infusions were TCR gene modified T cells, we have a long way to go well tolerated and no serious adverse events related to make this a safe and effective treatment for cancer to the T cell infusion were observed. All subjects had patients. We have previously reported identifying, mild skin rashes (Grade I or II). One subject had a cloning, and characterizing an HLA-A2 restricted, pancytopenia 4 weeks post infusion that was rapidly tyrosinase reactive TCR. This TCR was cloned from resolved with standard care. There was evidence of a CD4+ T cell meaning it ability to mediate tumor rec- T cell activity in two of the patients with one patient ognition was independent of CD8 co-receptor expres- having a transient partial response and another sion. All of the pre-clinical in vitro results indicate having progressive vitiligo. Despite the low number that TIL 1383I TCR transduced CD4+ and CD8+ T cells of patients, the results strongly suggest that TIL 1383I can recognize both efficiently recognize HLA-A2+ ty- TCR transduced T cells can be delivered safely to rosinase+ cells. In vivo TIL 1383I TCR expressing T patients, the have biologic activity in humans, and cells can mediate rejection of established HLA-A2+ no serious autoimmune has been observed. tyrosinase+ tumors. And while our transgenic mouse data revealed the potential for TIL 1383I TCR express- ing T cells to recognize melanocytes leading to au- toimmunity, no evidence of self reactivity was ever seen in any of our adoptive T cell transfer models. We concluded that the TIL 1383I TCR had all of the desired properties for use in treating patients with TCR gene modified T cells. A phase I clinical trial was initiated where autolo- gous T cells were engineered using a lentiviral vector encoding the TIL 1383I TCR. Of the 46 subjects in the initial screening for the trial, six went through the 191 | CELLULAR THERAPY
Genetically modified cytokine-induced killer (CIK) cells for targeted cancer therapy
Oelsner S.1, Wagner J.2, Friede M.E.1, Pfirrmann V.2, Rettinger E.2, Schubert R.3, Ullrich E.2, Bader P.2, Wels W.S.1
1Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt/Main, Germany, 2Goethe University Frankfurt, Hospital for Children and Adolescents, Dept. Stem Cell Transplantation and Immunology, Frankfurt/Main, Germany, 3Goethe University Frankfurt, Hospital for Children and Adolescents, Dept. Allergology, Pneumology and Cystic Fibrosis, Frankfurt/Main, Germany
Pre-emptive immunotherapy after HSC transplanta- Effects of exposure to lentiviral vector particles on tion based on minimal residual disease (MRD) status the development of CIK cell subpopulations were with donor lymphocyte infusions (DLI) using cyto- monitored over four weeks of continuous culture. kine-induced killer (CIK) cells may be beneficial to In in vitro cytotoxicity assays we could demonstrate prevent relapse without causing graft-versus-host-dis- potent and selective cytotoxicity of retargeted CIK ease (GvHD). CIK cells are a heterogeneous effector cells towards established cancer cell lines expressing cell population including T cells (CD3+CD56-), natural CD19 and primary pre-B-ALL blasts. killer (NK) cells (CD3-CD56+) and natural killer T Furthermore, anti-leukemic activity of retargeted CIK (NKT) cells (CD3+CD56+), that exhibit non-MHC-re- cells against established primary pre-B-ALL blasts stricted cytotoxic activity and are generated by ex was demonstrated in vivo in a NOD/SCID common vivo expansion of peripheral blood mononuclear cells gamma chain knockout (NSG) mouse model using (PBMC) through the addition of interferon (IFN)-γ, bioluminescence imaging. anti-CD3 antibody, IL-2 and IL-15. While CIK cells have shown potent in vivo activity against various cancer types such as lymphomas or colorectal cancer, their cytotoxicity against B-ALL, characterized by the expression of CD19, has been limited. Hence, retargeting of CIK cells using chimeric antigen receptors (CARs) to facilitate selective target cell recognition and enhance specific cytotoxicity rep- resents a promising approach. CAR comprise an extra- cellular scFv antibody fragment as an antigen-bind- ing domain, linked via a flexible hinge region and a transmembrane domain to an intracellular signaling moiety such as CD3 zeta chain (first generation CAR), or zeta chain fused to a co-stimulatory protein domain such as CD28 (second generation CAR). We established an optimized protocol for transduc- tion of CIK cells with CD19-specific lentiviral CAR constructs, and characterized cells for expression of an EGFP marker gene and CAR surface expression. 192 | CELLULAR THERAPY
A proposal for an individualized immunotherapy of cancer with engineered CAR-/TCR-modified T cells derived from the central memory subset
On behalf of the Invictus Consortium: Klar R.1, Busch D.2, Edinger M.3, Einsele H.4, Germeroth L.5, Hauser A.3, Herr W.3, Hildebrandt M.6, Hudecek M.4, Krackhardt A.1, Middeler G.7, Nerreter T.4, Perisic T.6, Peschel C.1, Riddell S.8, Slobodianski A.6
1Medizinische Klinik III, Klinikum rechts der Isar, TU München, München, Germany, 2Institut für medizinsiche Mikrobiologie, Immunologie und Hygiene, TU München, München, Germany, 3Klinik und Poliklinik für Innere Medizin III, Universitätsklinikum Regensburg, Regensburg, Germany, 4Department of Medicine II, Clinic of the University of Würzburg (UKW), Würzburg, Germany, 5Stage cell therapeutics GmbH, Göttingen, Germany, 6TUMCells, TUM School of Medicine, München, Germany, 7Diapharm GmbH & Co. KG, Lübeck, Germany, 8Fred Hutchinson Cancer Research Center (FHCRC), Seattle, United States
Adoptive immunotherapy with T cells that were redi- (INVICTUS 2). This receptor serves as a prototype for rected by genetic engineering to express a T cell recep- a library of leukemia-reactive TCR that will allow per- tor (TCR) or chimeric antigen receptor (CAR) to target sonalized therapy of a broader patient population in the tumor cells is emerging as an effective strategy to treat future. patients with a variety of life threatening malignancies. During the concept development phase, several pre- We have shown that the central memory T cell (TCM) requisites for realization of clinical translation have subset is endowed with features of stemness, and exper- been fulfilled and a full proposal is under review at the iments in preclinical models have shown a superior en- moment. Scientific advice has been obtained from Paul graftment capability of TCM compared to other memory Ehrlich Institut and the framework for pivotal preclini- T cell subsets. Thus, we anticipate that TCR- and CAR- cal studies and subsequent clinical trials with ROR1- modified T-cells derived from this subset may exert a CAR and MPO-TCR modified TCM been discussed. long-lasting therapeutic effect, and we have developed The extensive safety data obtained for the TCR in vitro a clinical concept as a proposal to the Federal Ministry of were considered sufficient to compensate the lack of a Education and Research (BMBF) initiative “Innovationen suitable animal model. Preliminary safety testing of für die individualisierte Medizin”. ROR1-CAR modified T cells has been performed in a We have recently upscaled the isolation of TCM under non-human primate model and will be completed in IN- GMP-conditions at TUMCells using reversible Fab- VICTUS 1. We established a risk management concept Streptamer technology. This process will serve as a with defined milestones to control progress and success common trunk for subsequent transduction of retroviral of the preclinical and clinical work, to ensure maximum vectors encoding a tumor-reactive TCR or CAR. The CAR safety of the treated patients. - developed at FHCRC and UKW - recognizes the tumor The consortium has claimed a patent for the TCR, holds antigen ROR1 in an HLA-independent fashion and will patents in CAR technology and cell selection reagents be used to target the two most common solid tumors, and freedom to operate until marketing authorization breast and lung cancer (INVICTUS 1). The TCR - devel- has been ascertained for all reagents necessary for the oped at TUM - is specific for an HLA-B*07:02-restricted manufacturing process. In summary, the INVICTUS epitope derived from myeloperoxidase (MPO) and will consortium is developing a modular concept of endow- be used to target acute myeloid leukemia (AML) in the ing tumor reactivity to TCM by genetic engineering with context of allogenic stem cell transplantations, where tumor-targeting receptors to provide novel therapies for the patient but not the donor is HLA-B*07:02 positive patients in need. 193 | CELLULAR THERAPY
Establishment of a non-human primate CAR-T cell model
Patasic L.1, Seifried J.1, Schneider I.2, Tondera C.1, Schmitz H.1, Hombach A.3, Abken H.3, König R.1,4, Cichutek K.5
1Paul-Ehrlich-Institut, Host-Pathogen Interactions, Langen, Germany, 2Paul-Ehrlich-Institut, Molecular Biotechnology and Gene Therapy, Langen, Germany, 3Center for Molecular Medicine Cologne, Clinic I for Internal Medicine, Köln, Germany, 4Sanford-Burnham Medical Research Institute, Infectious & Inflammatory Disease Center, La Jolla, United States, 5Paul-Ehrlich-Institut, Langen, Germany
Genetically modified T cells expressingC himeric activation and functionality of the CAR-T cell, com- Antigen Receptors (CAR), are transgenic for artifi- parable to humans with respect to both, target cell cial antigen receptors consisting of a binding domain killing and potentially occurring side effects. attached to signaling domains derived from the in- tracellular part of the T cell receptor complex. The extracellular part is composed of a targeting entity which specifically binds to the desired antigen on a respective target cell. The intracellular part converts antigen binding into activation of the CAR-T cell, leading to profoundly specific killing of the bound target cell. Another great advantage over other T cell based therapy approaches is the MHC independent binding and activation. CAR-T cells are a promising therapeutic option. Efficacy against leukemia, cer- ebral tumors and CAR-T cells targeting HIV envelope has been demonstrated in numerous clinical studies. Nevertheless, severe side effects and even death as a result of cytokine storms during treatment have been reported. To further investigate function, effi- cacy and long term survival of CAR-T cells in vivo, appropriate non-human primate models are needed. So far, very few data are available for generation and efficacy of CAR-T cells in rhesus macaques. However, even if killing might be comparable, nothing is known about analogy of signaling pathways leading to these severe side effects, between human and non- human primates. Therefore, we aim to investigate the functionality of species-specific CAR activation domains in non-human primates. We pose the ques- tion, if adaption of the intracellular CAR signaling domain to the respective species leads to optimized 194 | CELLULAR THERAPY
Optimised melanoma therapy by generation of T cells expressing two additional T-cell receptors (TETARs)
Höfflin S.1,2, Prommersberger S.1,2, Uslu U.1, Schuler G.1, Schmidt C.W.3, Lennerz V.4, Dörrie J.1, Schaft N.1
1Universitätsklinikum Erlangen, Dermatology, Erlangen, Germany, 2Friedrich-Alexander-Universität Erlangen-Nürnberg, Genetics, Erlangen, Germany, 3QIMR Berghofer Medical Research Institute, Cancer Immunotherapy Laboratory, Brisbane, Australia, 4Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Internal Medicine III, Mainz, Germany
Adoptive T-cell therapy of cancer often fails due noma-antigen and an individually mutated antigen to the tumour cells’ immune escape mechanisms, for the use in personalised adoptive T-cell therapy of like antigen loss or down-regulation and defects in melanoma. Additionally, the possibility to combine the antigen processing and MHC presentation ma- normal TCRs and CARs in TETARs opens up new chinery. To anticipate immune escape by loss of a avenues in the treatment of cancer. single antigen, it would be advantageous to equip T cells with multiple specificities. To study the pos- sible interference of two exogenous T-cell receptors (TCRs) in one cell, and to examine how to counter- (S.H. and S.P. share first authorship; J.D. and N.S. share senior authorship) act competing effects, we generated TETARs, CD8+ T cells expressing two additional T-cell receptors by simultaneous transfection with two TCRs using RNA electroporation. The TETARs were equipped with one TCR specific for the common melanoma antigen gp100 and one TCR recognising a patient-specific, individual mutation of CCT6A (chaperonin contain- ing TCP1, subunit 6A) termed “CCT6Am TCR”. These CD8+ T cells proved functional in cytokine secretion and lytic activity upon stimulation with each of their cognate antigens, although some reciprocal inhibi- tion was observed. Murinisation of the CCT6Am TCR increased and prolonged its expression and increased the lytic capacity of the dual-specific T cells. To partially overcome MHC-restriction of the gener- ated TETARs, we further investigated the possibility to combine normal full-length TCRs with chimeric antigen receptors (CARs), which recognise native antigens expressed on the cell surface of the tumour cells. Taken together, we generated functional, dual-spe- cific CD8+ T cells directed against a common mela- 195 | CELLULAR THERAPY
Side-by-side comparison of second generation chimeric antigen receptors
Schütt A.1, Poxon J.1, Gilham D.1
1University of Manchester, Clinical and Experimental Immunotherapy Group, Manchester, United Kingdom
Personalized medicine, especially in the field of im- The objective of this research is a side-by-side compar- munotherapy has made tremendous progress in the ison of neuroblastoma specific CARs targeting neural past 10 years. It is now well understood that patients cell adhesion molecule (NCAM), which differ in their often exhibit anti-cancer immune responses, however co-stimulatory domains. In particular we focused on effector functions are often silenced or suppressed. CD28 and variants, which seem to be promising can- Adoptive T-cell therapy (ACT) utilises patient derived didates. Furthermore we investigated the impact of tumour tissue and activates existing tumour reactive CD2, CD6, CD134 (OX40) and CD137 (4-1BB) signalling T-cells or genetically engineering T-cells from patient domains on CAR T-cell activation. T-cells of healthy blood to express anti-cancer receptors. First genera- donors have been genetically engineered using retro- tion chimeric antigen receptors (CARs) are artificial viruses to express NCAM-CARs and tested in co-cul- receptors comprising an antibody derived antigen ture with NCAM positive cell lines of neuroblastoma binding domain fused to the T-cell receptor signal- origin. To measure CAR T-cell activation, production ling domain CD3 zeta. Second generation receptors of interferon gamma and interleukin-2 were assessed, include a second signalling domain derived from as well as degranulation (CD107a) and killing func- well know co-stimulatory molecules in T-cell acti- tion (WST-1). We could clearly see that CAR constructs vation. Adding co-stimulatory signalling domains is containing CD28 lead to a strong T-cell activation, necessary for long-time survival and persistence of however severe side effects are reported when using T cells, which is crucial for treating cancer patients. full length CD28 co-stimulatory domains in vivo. Trun- The antigen binding domain can descend from any cated versions of CD28 showed less toxicity but whilst target-specific monoclonal antibody which opens the exerting high levels of activation and could provide a possibility to treat tumours of diverse origins with the viable alternative to full length CD28 in CAR settings. same method. However, different kinds of tumours Our data also shows that CD2 is the most promising (solid or blood borne cancer) require different strate- alternative to CD28 for CAR T-cell co-stimulation. To gies, largely due to target antigen origin which may prove the universal applicability of these CAR settings be from tumour tissue, malignantly altered cells or we are currently exploring a range of further targets, tumour stroma. Clinical trials using second genera- including carcinoembryonic antigen (CEA) and Dis- tion CARs have shown promising results, however, ialoganglioside (GD2). variations in CAR design, recruitment criteria, treat- ment schedules and supporting therapies make results difficult to compare. As a consequence optimal condi- tions for CAR therapy remain largely undetermined. 196 | CELLULAR THERAPY
In vitro PD-1 blockade to generate optimal effectors for adoptive cell transfer in melanoma
Simon S.1,2, Vignard V.1,2,3, Florenceau L.1,2,3, Dreno B.1,2,3, Khammari A.1,2,3, Labarriere N.1,2,3
1Nantes University, Inserm UMR892-CNRS 6299, Nantes, France, 2LabEx IGO “Immunotherapy, Graft, Oncology”, Nantes, France, 3CHU, Nantes, France
We developed a clinical-grade production process of T affinity specific T cell clones negative for PD-1 -ex lymphocytes specific for melanoma antigens, usable pression are not or rarely present in peripheral blood, for adoptive cell transfer in melanoma patients. These as they are probably eliminated by negative selec- specific T cells are produced from HLA-A2 patient’s tion, due to a too high reactivity. Thus, the selection PBMC by a peptide stimulation step, followed by a of PD-1neg T cell clones would not be a good option sorting/amplification step. Sorted T lymphocytes are for adoptive cell transfer. Ideal specific T cells should polyclonal, fully specific of targeted antigens and re- combine a high avidity for the targeted antigen and active against melanoma cells. the lowest PD-1 expression. An extensive analysis of these specific T cells showed We thus modified the production procedure of spe- that a fraction of these T lymphocytes constitutively cific T cells, adding a PD-1 blocking antibody during expressed the PD-1 molecule that could potentially each step. We showed that the absolute number of compromise their in vivo functions, while another antigen specific T cells increased upon addition of fraction remained unable to express this molecule. this Ab during the peptide stimulation step, and that This result prompted us to characterize the mecha- the recovered specific T cell repertoire was biased nisms leading to PD-1 expression on melanoma spe- towards a higher avidity T cell repertoire. Moreover, cific T lymphocytes, and to define culture conditions we documented a strong decrease of PD-1 expression for amplifying specific T cells lacking this molecule. on specific T cells generated with the anti-PD-1 block- We isolated Melan-A and MELOE-1 specific T cell ing Ab, partially explained by a progressive remeth- clones stably expressing different levels of PD-1 at ylation of PD-1 promoter in these T cells. Consistently rest. We showed that PD-1neg T cell clones remained with these results, reactivity of specific T cells gener- unable to express this molecule, even after activa- ated with anti-PD-1 Ab is not or much less affected tion. The regulation of PD-1 expression occurs at the by the presence of PD-L1 on melanoma target cells. transcriptional level, with major differences in the Therefore, we reckon that the use of anti-PD-1 block- methylation pattern of the PD-1 promoter. From a ing Ab during the production process of melanoma functional point of view, activation of PD-1pos T cell specific T cells will ensure recovery of high affinity clones was strongly inhibited in response to PD-L1 polyclonal T cells insensitive to the functional inhi- expressing melanoma cells, whereas no inhibition bition mediated by PD-1/PD-1 ligand signalization. was detected for PD-1neg T cell clones. More surpris- ingly, we observed that PD-1neg T cell clones were altogether of lower avidity than PD-1pos ones. These results suggest that in physiological conditions, high 197 | CELLULAR THERAPY
Enhancement of melanoma adoptive T-cell therapy by adenoviral gene transfer
Siurala M.1,2, Havunen R.1, Bramante S.1, Saha D.1, Tähtinen S.1, Vähä-Koskela M.1, Parviainen S.1,2, Hemminki A.1,2
1Cancer Gene Therapy Group, Department of Pathology and Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland, 2TILT Biotherapeutics Ltd, Helsinki, Finland
Adoptive cell transfer (ACT) has shown promising p=0.001; triple combination vs. Ad5-CMV-mTNFa + results in clinical trials for melanoma with response OT-1, p=0.049). In summary, these results support rates up to 50%. We aimed to enhance the efficacy of the further development of cytokine-armed adenovi- ACT by coupling it with adenoviruses coding for im- ruses for enhancing adoptive T-cell therapy. munostimulatory cytokines. The non-replicating ad- enoviruses that were constructed (Ad5-CMV-mIFNg, Ad5-CMV-mIL2, Ad5-CMV-mIFNb and Ad5-CMV- mTNFa) were confirmed to produce biologically active murine cytokines in vitro. Additionally, in vivo studies confirmed that virus-encoded cytokines were produced locally when injected into B16-OVA tumors whereas serum levels were low. These viruses were then used in combination with adoptive transfer of OT-1 TCR transgenic T-cells to treat C57BL/6 mice bearing B16-OVA melanoma tumors. The animals were administered intraperitoneally with 1.5 x 106 CD8+-enriched OT-1 cells with or without intratu- moral injections of cytokine-coding adenoviruses (1 x 109 viral particles/tumor). Combination treatment with Ad5-CMV-mIL2 and OT-1 resulted in statisti- cally significant antitumor efficacy when compared with either monotherapy and untreated control (com- bination vs. virus, p=0.026; combination vs. OT1, p=0.003; combination vs. mock, p< 0.001). Similar results were obtained with other cytokine-coding virus and OT-1 combinations, except for mIFNb. In further experiments a triple combination of Ad5- CMV-mIL2 + Ad5-CMV-mTNFa (1:1 ratio) and OT-1 T-cells was used to treat B16-OVA; improved anti- tumor efficacy was observed over dual agent thera- pies (triple combination vs. Ad5-CMV-mIL2 + OT-1, 198 | CELLULAR THERAPY
TCR-pMHC binding affinity is not the only major influence on functional avidity and polyfunctionality of T cell recognition
Spear T.1, Simms P.2, Wang Y.3, Hellman L.3, Baker B.M.3, Rosen H.R.4, Nishimura M.I.1
1Loyola University Chicago, Cardinal Bernardin Cancer Center, Maywood, United States, 2Loyola University Chicago, Flow Cytometry Core Facility, Maywood, United States, 3University of Notre Dame, Department of Chemistry & Biochemistry, Notre Dame, United States, 4 ORAL University of Colorado, Division of Gastroenterology & Hepatology, Denver, United States ALK SHORT T 2015
Affinity between a TCR and its pMHC ligand is component for full recognition. Furthermore, differ- thought to be the most important factor governing ential recognition patterns between peptide-loaded T cell recognition. Many approaches to improve targets and tumor targets expressing NS3 variants genetically-modified T cells for adoptive transfer suggest that antigen density also plays a role in rec- include TCR affinity-enhancement, often by generat- ognition potential. Moreover, transduced Jurkat76 ing random alterations through phage display. This cells which lack an endogenous TCR can now recog- method of enhancing affinity, however, may not be nize previously thought CD8-dependent variants in the best answer, as it can cause off-tumor effects the absence of CD8, but some require the lck-binding against low-level antigen presence, elicit cross-re- domain to achieve full functional output. Immuno- activity against unpredicted epitopes, and can limit fluorsecent analysis of 6 intracellular cytokines and cross-reactivity potential against epitope variants lytic marker CD107a (yielding 128 possible function- that may arise in diseases with high rates of antigen al combinations) after stimulation with our panel of mutation. Here, we demonstrate that while affinity peptides shows distinct polyfunctional profiles, even plays a role in TCR-pMHC recognition, it is not the between peptides with the same or similar measured
only important factor in achieving full polyfunction- KD (determined by surface plasmon resonance). Ulti- al potential of a T cell. mately, our crystal structure data of this TCR-pMHC We have previously identified a novel HLA-A2-re- give us the unique ability to predict structural in- stricted CD8-independent TCR reactive to hepatitis teractions, how each mutant epitope may influence C virus (HCV) NS3:1406-1415. However, we have binding and ultimately functional recognition, and observed distinct recognition patterns by cytokine how changes in affinity may be compensated for by release against a panel of 8 naturally occurring the presence and function of CD8 and by altering mutant epitopes in both a Jurkat cell line and PBL-de- TCR and antigen densities. rived T cells transduced with retroviral vectors to Together, these data suggest a TCR’s engagement express this promiscuous TCR. However, Jurkat cells with pMHC and ultimately its functional recogni- engineered to express either full length CD8αβ or a tion is not necessarily defined by binding affinity. A truncated version lacking intracellular lck-binding better understanding of other biophysical influences domains, recognize mutants in patterns distinct from on polyfunctional recognition of variant ligands may each other and from unmodified Jurkats, demonstrat- allow for a more informative rational design of TCRs ing that while affinity enhancement by CD8 may be to improve adoptive cell transfer. required for recognition of certain mutant epitopes, others may also require the functional signaling 199 | CELLULAR THERAPY
Untouched GMP-grade purified engineered immune cells
Straetemans T.1, Grunder C.1, Heijhuurs S.1, Hol S.1, Slaper-Cortenbach I.2, Bonig H.3, Sebestyen Z.1, Kuball J.4
1UMC Utrecht, Laboratory of Translational Immunology, Utrecht, Netherlands, 2UMC Utrecht, Cell Therapy Facility, Utrecht, Netherlands, 3Institute for Transfusion Medicine and Immunohematology, Frankfurt, Germany, 4UMC Utrecht, Hematology / Laboratory of Translational Immunology, Utrecht, Netherlands
Engineering T cells with receptors to re-direct the immune system against cancer has most recent- ly been described as one of the scientific break- throughs. However, a main challenge remains the GMP-grade purification of immune cells selectively expressing the introduced receptor in order to reduce potential side effects due to poorly or non-engineered immune cells. By taking advantage of an optimized expression system, a model γδT cell receptor (TCR) naturally in- terfering with endogenous αβTCR chains, and GMP- grade anti-αβTCR beads we propose a novel strategy to isolate engineered immune cells. This untouched enrichment of engineered immune cells translated into highly purified receptor engineered cells with strong anti-tumor reactivity both in vitro but also in vivo in two different humanized mouse models. In addition, this approach eliminated residual allo-reac- tivity of engineered immune cells, which remained absent even with long-term suboptimal interference with endogenous TCR chains such as in resting cells, while tumor control was preserved. Importantly, we demonstrate that the enrichment process can be ex- tended to any αβTCR that, by design, will compete for endogenous αβTCR expression. All together, we present a novel GMP-grade enrich- ment method of untouched engineered immune cells, which is applicable to all receptor-modified cells and suitable for an autologous but also allogeneic clinical scenario. 200 | CELLULAR THERAPY
Myeloid-derived suppressor cell (MDSC) therapy skews the T cell response after bone marrow transplantation towards type 2 immunity and maintains the graft-versus-tumor effect while preventing graft-versus-host disease (GVHD)
Messmann J.J.1, Leithäuser F.2, Lutz M.B.3, Debatin K.-M.1, Strauss G.1
1University Medical Center Ulm, Department of Pediatrics and Adolescent Medicine, Ulm, Germany, 2University of Ulm, Institute of Pathology, Ulm, Germany, 3Julius-Maximillians University Würzburg, Institute of Virology and Immunobiology, Würzburg, Germany
Allogeneic bone marrow transplantation (BMT) is a GVHD and attenuated histological GVHD. MDSCs curative treatment modality for hematopoietic ma- expanded in vivo and invaded lymphoid and GVHD lignancies such as acute and chronic leukemias and target organs. MDSC-mediated GVHD suppression lymphomas. Mature donor T cells in the allograft was antigen-independent since transplantation of support engraftment, promote early T cell immu- MHC class I deficient MDSCs prevented GVHD devel- nity of the recipient and most importantly mediate opment comparable to wild type MDSCs. Inhibition the graft-versus-tumor (GVT) effect. However, these of GVHD required the presence of MDSCs during T donor-derived T cells are also responsible for the cell priming because transplantation of MDSCs one induction of graft-versus-host disease (GVHD). My- week after BMT was ineffective in GVHD prevention. eloid-derived suppressor cells (MDSCs) are a hetero- Interestingly, MDSC treatment did not significantly geneous population of myeloid precursors suppress- reduce allogeneic T cell numbers in lymphoid and ing versatile T cell functions and might therefore be GVHD target organs or change their homing be- used in GVHD prophylaxis. However, MDSC therapy havior or phenotype. However, MDSCs skewed al- is only reasonable if MDSCs do not abrogate the GVT logeneic T cells towards type 2 T cells up-regulating effect. Th2-specific transcription factors and cytokines. Therefore, we tested whether in vitro-generated Most importantly, co-transplantation of MDSCs and MDSCs suppress GVHD development without inter- development of Tc2 cells did not abrogate the GVT fering with the GVT effect. effect, since alloantigen-specific T cells efficiently MDSCs were generated in vitro by culturing BM cells eradicated syngeneic tumor cell lines. in the presence of GM-CSF and G-CSF. After 4 days In summary, MDSC-mediated Th2 skewing of the more than 90% of the cells exhibited the CD11b+Gr-1+ immune response might be used as a cellular treat- MDSC phenotype and were then co-transplanted ment strategy after allogeneic BMT to prevent GVHD with allogeneic BM and spleen cells into lethally ir- without abolishing anti-tumor cytotoxicity. radiated recipient mice. By co-injecting syngeneic tumor cells together with the transplant, the impact of MDSC transplantation on the GVT effect was ana- lyzed. In-vitro generated MDSCs efficiently suppressed al- loantigen-specific T cell proliferation in vitro. Trans- plantation of 1x107 MDSCs together with allogeneic BM and spleen cells efficiently prevented clinical 201 | CELLULAR THERAPY
Engineering NK cells modified with an EGFRvIII-specific chimeric antigen receptor to overexpress CXCR4 improves immunotherapy of CXCL12/SDF-1α-secreting glioblastoma
Temme A.1, Müller N.1, Michen S.1, Tietze S.1, Schmitz M.2, Schulte A.3, Lamszus K.3, Töpfer K.1, Pastan I.4, Schackert G.5
1TU Dresden, University Cancer Center (UCC), Medical Faculty Carl Gustav Carus, Department of Neurosurgery, Section Exp. Neurosurgery / Tumor Immunology, Dresden, Germany, 2TU Dresden, Medical Faculty Carl Gustav Carus, Institute of Immunology, Dresden, Germany, 3University Medical Center Hamburg-Eppendorf, Laboratory for Brain Tumor Biology, Department of Neurosurgery, Hamburg, Germany, 4National Cancer Institute, National Institutes of Health, Laboratory of Molecular Biology, Center for Cancer Research, Bethesda, United States, 5TU Dresden, Department of Neurosurgery, Dresden, Germany
NK cells are promising effector cells for adjuvant conferred a specific chemotaxis to CXCL12/SDF-1α immunotherapy of cancer. So far, several preclini- secreting U87-MG glioblastoma cells. Moreover the cal studies have shown the feasibility of gene-engi- administration of such NK cells resulted in a sig- neered NK cells, which upon expression of chimeric nificantly increased survival of U87-MG xenografted antigen receptors (CARs) are redirected to otherwise mice when compared to the treatment with NK cells NK-cell resistant tumors. Yet, we reasoned that the expressing only the EGFRvIII-specific CAR. We con- efficiency of an immunotherapy using CAR-modified clude that chemokine receptor engineered NK cells NK cells critically relies on efficient migration to the with concomitant expression of a tumor-specific CAR tumor site and might be improved by the engraft- are a promising tool to improve adoptive tumor im- ment of a receptor specific for a chemokine released munotherapy. by the tumor. Based on the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell recep- tors, we constructed an EGFRvIII-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII+ glioblastoma cells in vitro and to established subcutaneous U87-MGEGFRvIII tumor xenografts. Notably, infused NK cells with expres- sion of MR1.1-DAP12 caused significantly delayed tumor growth and increased median survival time when compared to NK cells transduced with an ITAM-defective CAR or with DAP12 only. Yet, the further genetic engineering of these EGFRvIII-spe- cific NK cells with the chemokine receptor CXCR4 202 | CELLULAR THERAPY
Identification of an HLA-A*0201-restricted immunogenic epitope from the universal tumor antigen DEPDC1
Tosi A.1, Sommaggio R.1, Cappuzzello E.1, Zanovello P.1, Rosato A.1
1University of Padova, Padova, Italy
With the discovery of tumor-specific and tumor-as- in response not only to peptide-loaded cells but also sociated antigens, cancer vaccine strategies as well to HLA-A*0201-positive tumor targets. Taken togeth- as adoptive cell therapy with antigen-specific T cells er, these findings indicate that this HLA-A*0201-re- have become a promising therapeutic approach. Re- stricted DEPDC1-derived peptide is a putative tumor cently, DEPDC1 has been described to play an im- antigen that could be exploited for vaccination portant function in cancer cell growth/survival, as its against many different tumors that overexpress the siRNA-mediated knock down suppresses tumor cell DEPDC1 protein. growth and increases the number of apoptotic cells, while its overexpression is linked to a bad prognosis in patients with different types of tumors. These data suggest an important involvement of this protein in tumor progression, and therefore its immunological targeting could represent an important strategy to counteract tumor growth and metastasis. A deep search in the Oncomine database confirmed the wide overexpression of DEPDC1 in tumors but its almost complete absence in normal tissues, thus indicating that it can be regarded as a universal tumor antigen and might represent a potential and safe target for immunotherapy. Thereafter, protein expression was assessed in a large set of tumor cell lines, and several HLA-A*0201-restricted candidate peptides were identified by the epitope prediction programs BIMAS, NetMHC and NetCTL, with the final aim to assess their capacity to induce peptide-specific cy- totoxic T lymphocytes (CTLs) from peripheral blood mononuclear cells of HLA-A*0201-positive healthy donors. Among ten different candidates tested, one immunodominant epitope was identified that was capable of inducing CTL populations producing in- terferon-γ and exerting a relevant cytotoxic activity 203 | CELLULAR THERAPY
Fine-tuned T cell receptors for cancer immunotherapy
Tribble N.1, Jakobsen B.1, Pumphrey N.1
1Adaptimmune Ltd, Abingdon, United Kingdom
Adoptive T cell therapy (ACT) with gene-modified T cell receptors (TCR) with optimal affinity for a T cells is emerging as a promising strategy for the given tumour antigen to address the problem of low treatment of many forms of cancer. The success of T cell antigen affinity imposed by thymic selection. ACT depends on comprehensive and robust safety Fine-tuning each antigen-specific TCR is essential to testing to mitigate the risks posed by target presen- ensure the appropriate balance between functionali- tation on healthy tissue and off target cross-reactiv- ty and specificity. A TCR has been developed to target ity to a homologous peptide. The use of engineered the HLA-A2 restricted NY-ESO-1 cancer-testis antigen TCRs for ACT has potential advantages over the that is overexpressed in a wide range of cancer types. more clinically advanced approach using chimeric Preliminary results from a pilot clinical trial in syno- antigen receptors (CAR). Despite some encouraging vial sarcoma patients indicate responses in four of early results with CARs, their wider applicability five patients, including one complete response. is restricted by a lack of tumour-specific antibody targets. Moreover, CARs have proved very promis- ing for hematologic malignancies but have so far had less success against solid tumors. TCRs have access to the larger pool of intracellular antigens presented on the cell surface as short peptides bound to major histocompatibility complex (MHC). A larger choice permits the selection of antigens with little or no ex- pression in normal tissues, thereby reducing the risk of off target effects. TCRs also represent the natural antigen receptor for T cells, thus allowing for correct immune synapse formation and T cell activation via native signalling pathways. Furthermore, TCRs have a much lower affinity than antibodies for their anti- gens and require less than ten interactions to activate a T cell. The combination of low affinity and exqui- site functional sensitivity could prevent T cells from getting stuck at the periphery of cancerous tissues, allowing good penetration of solid tumours. We have developed methods to produce precision-engineered 204 | CELLULAR THERAPY
Exploring if recurrent somatic mutations can be targeted with TCR gene therapy for cancer
Tubb V.M.1, Bendle G.M.1
1University of Birmingham, School of Cancer Science, Birmingham, United Kingdom
The transfer of tumour-specific T cell receptor (TCR) derived from recurrent somatic mutations. These TCR α and β genes can efficiently redirect T cell specificity genes can be used as tools to assess whether puta- to engineer tumour-reactive T cells for use in cancer tive peptide epitopes derived from recurrent somatic immunotherapy. The clinical testing of this approach mutations are naturally processed and presented on has demonstrated its therapeutic potential, but it has tumour cells and may potentially be utilized for TCR also revealed that identifying suitable target anti- gene therapy of cancer. gens is a critical issue, as life-threatening toxicities can occur if the target antigen is expressed in vital normal tissues. Neo-antigens generated by mutations in tumour cells are a class of tumour antigens that are likely to rep- resent safe targets for TCR gene therapy. However, most neo-antigens are encoded by patient specific mutations and therefore will be difficult to target with this approach. Nevertheless, some somatic mu- tations occur recurrently between cancer patients and neo-antigens derived from these would represent very attractive targets. Therefore, we set out to assess if recurrent mutations encode immunogenic peptide epitopes presented by common HLA class I alleles and isolate TCR genes specific for such neo-antigens. In this project we have generated a panel of predicted neo-antigen peptide epitopes derived from recurrent somatic mutations and explored if an experimental protocol that combines peptide/MHC class I tetram- er technology, cell sorting technologies and our TCR gene capture technology, can be used to isolate TCR genes specific for these putative neo-antigens from T cells of healthy donor PBMCs. Our results show that this approach can be used to isolate TCR genes specific for predicted neo-antigens 205 | CELLULAR THERAPY
Expression of a chimeric antigen receptor in hematopoietic precursor cells inhibits the generation of a diverse T cell receptor repertoire and induces T Cell Receptor/CD3 negative, CD4 CD8 negative T cells
Van Caeneghem Y.1, Goetgeluk G.1, Weening K.1, Verstichel G.1, Vanhee S.1, Bonte S.1, Taghon T.1, Leclercq G.1, Abken H.2, Kerre T.1, Vandekerckhove B.1
1Ghent University, Ghent, Belgium, 2University of Cologne, Cologne, Germany ORAL ALK SHORT T 2015
CD19-specific chimeric antigen receptor (CAR) T cell tor-α and IL2, when compared to TCR-TD cells. Cy- therapy was recently shown to induce complete re- tokine production is specific since only CEA+ tumor mission in patient with relapsed acute and chronic lines can induce this production. We subsequently lymphoblastic leukemia. It is generally believed that assessed whether the CAR-TD cells were capable of cure is dependent on the long term persistence of specific cell lysis of a CEA+ tumor line. The lytic ac- these CAR+ T cells. To improve the persistence of tivity was in the same range as the TCR-TD controle CAR+ T cells, it has been suggested to transduce and was CEA specific. These results show that the hematopoietic precursor cells (HPC) rather than cells have indeed functional activity of T cells mature T cells in the context of an allogeneic or au- We believe therefore that care should be taken that tologous transplantation. We here studied the effect the infusion in patients of CAR-TD HPC in the setting of transgenic CAR expression in HPCs on T cell dif- of an autologous or allogeneic transplantation does ferentiation in the OP9-DL1 system. Human HPC not compromize the development of a diverse T cell were retrovirally transduced with express a carci- receptor repertoire. In vitro generated CD3- CAR+ T no-embryonic antigen (CEA)-specific, second gener- cells are of major interest since they do not carry ation CAR (CAR-CD3ζ-CD28). As a control, HPC were (alloreactive) TCRs and may be ideally suited as “of transduced with the α- and β-chain of a regular T cell the shelf” third party T cells for adoptive cell therapy. receptor (TCR). The transduced (TD) cells were sub- sequently cultured on OP9-DL1 cells in the presence of IL7, stem cell factor and Flt3-ligand. We found that the CAR-TD cells differentiate to CD4 CD8 double positive (DP) immature precursor T cells. However, few cells pass through the proliferative DP pathway but rather go towards the double negative (DN) path- ways with full maturation towards CD1-CD27+ cells. These mature CAR-TD cells were TCR and CD3 nega- tive, suggesting that the expression of a CAR in early T cell precursors shuts down rearrangements of the endogenous TCR chains. To further document their T cell nature, cytokine production was tested after spe- cific antigen stimulation. The CAR-TD cells produced higher amounts of interferon-γ, tumor necrosis fac- 206 | CELLULAR THERAPY
Generation of EBV-specific human CD8+ cytotoxic T lymphocytes with stem cell memory and central memory properties by modulating glycolytic T cell metabolism
Weber I.1, Khan S.1, Theobald M.1, Hartwig U.F.1
1University Medical Center Mainz, III. Dept. of Internal Medicine - Hematology, Internal Oncology & Pneumology, Mainz, Germany
Adoptive transfer of virus-specific and TCR- or CAR-re- tion. OXPHOS and aerobic glycolysis was assessed by programmed T cells has advanced as a valuable cellu- measuring oxygen-consumption rate (OCR) and ex- lar therapy for opportunistic viral infections, EBV-me- tracellular acidification rate (ECAR) using a Seahorse diated lymphoma and leukemia relapse. However, Analyzer. durable clinical responses are often hampered by Upon repetitive restimulation of naive T cells we limited capability of terminally differentiated, high obtained strong expansion of EBV-reactive CTL ex- + + - + + + avidity effector T cells (TEFF) to establish sustained an- pressing a CD8 CD45RA CD45RO CD95 CD27 CD28 + + + - + tileukemic immunity whereas less differentiated stem CD62L CCR7 TSCM and CD8 CD45RA CD45RO C- + + + + + cell memory (TSCM) and central memory (TCM) T cells D95 CD27 CD28 CD62L CCR7 TCM phenotype in the could be shown to elicit potent antitumor responses, presence of low glucose, glutamine and 2-DG when
prolonged survival and memory. Moreover, TSCM and compared to untreated CTL. This effect was also seen + TCM depend less on glucose consumption to drive ox- in total CD8 T cells although less pronounced. In con- idative phosphorylation (OXPHOS) as their primary trast, supply of galactose or oleic-acid to glucose free
source of ATP whereas TEFF undergo additional aerobic (but glutamine containing) medium did not result in
glycolysis to generate the ATP needed. In the current reduced TEFF differentiation, suggesting OXPHOS by study, we therefore investigated means of modulating galactose converted to glucose and fatty acid oxida-
T cell metabolism to generate EBV-specific TSCM and tion. Moreover, 2-DG treated TSCM and TCM as well as + TCM to be used for adoptive transfer or to be addition- total CD8 CTL showed less lactate production and ally reprogrammed by e.g. TCR-transfer. ECAR as compared to untreated controls, suggesting + + Naïve CD8 CD45RA T cells isolated from PBMC of that differentiation to TEFF requires aerobic glycolyt- healthy HLA-A2+ donors by MACS® and total CD8+ ic ATP production. Functional assays in vitro did not
T cells were primed by autologous DCs loaded with only reveal cytolytic activity of 2-DG treated EBV-TSCM
EBV-peptides and restimulated with peptide loaded and TCM comparable to controls but elicited superior autologous PBMC in the presence of low glucose (1 migration properties of these cells when tested to un- mM), glutamine, an optimized cytokine cocktail treated naïve or total CD8+ T cells in a transwell mi- and 1 mM of the non-metabolizable glucose analog gration assay. 2-deoxy-glucose (2-DG). Moreover, either galactose or In summary, these studies demonstrate that modulat- 9-oleic acid was added to glucose deprived cultures. ing T cell metabolism may be a promising approach to
In addition to phenotypic and functional in vitro generate TSCM/CM in vitro for improved adoptive cellular analyses by FACS, ELISPOT and 51Cr-release assays, therapy. we determined glucose uptake and lactate produc- 207 | CELLULAR THERAPY
A novel immunotherapy: IMCgp100, a bi-specific TCR-anti-CD3 fusion for potent re-directed killing of melanoma cells
Weigand L.U.1, Bossi G.1, Harper J.1, Dukes J.1, Liddy N.1, Paston S.1, Mc Grath Y.1, Mahon T.1, Molloy P.1, Sami M.1, Baston E.1, Cameron B.1, Johnson A.1, Vuidepot A.1, Hassan N.1, Jakobsen B.K.1
1Immunocore Ltd, Abingdon, United Kingdom
T cells may recognise malignant cells by binding regimen, for which the maximal tolerated dose tumour-associated peptides in the context of Human (MTD) has been reached at 600 ng/kg (or 50 ug total Leukocyte Antigen (HLA). Such tumour-specific T dose) and is now in the dose expansion phase; arm 2 cells have been shown to be present in cancer pa- involves 4 daily doses every three weeks and is cur- tients albeit with low affinities. Since most tumour- rently in dose escalation. The drug is well-tolerated; associated antigens are auto-antigens, potential high toxicities of IMCgp100 were consistent with the mode affinity T cells may have been depleted during thymic of action of the drug i.e. IMCgp100 mediated T cell selection. Due to their low affinity, HLA down regu- mobilisation, activation and tumour killing. So far, lation and the immunosuppressive tumour micro- objective durable responses have been achieved. environment, these T cells are insufficient to allow tumour eradication. To overcome these limitations, we have developed novel, bi-functional soluble mol- ecules termed ImmTACs (Immune mobilising mono- clonal TCR Against Cancer) which re-direct T cells to target and destroy tumour cells with a high degree of potency and specificity. An ImmTAC comprises a high affinity ‘monoclonal’ T cell Receptor (mTCR) targeting a cancer-associated HLA-peptide complex, fused to an anti-CD3 scFv domain which activates an anti-tumour T cell response. IMCgp100 is our first clinical candidate molecule which is currently in a Phase I/IIa clinical study in patients with melanoma. The engineered TCR portion of the drug targets the gp100 peptide 280-288 in the context of HLA-A2, the presentation of which is markedly elevated on the surface of melanoma cells compared to melanocytes. In vitro, IMCgp100 showed high potency against tumour cells, and at anticipated clinical concentrations no significant reactivity was observed against gp100 negative targets. The clinical study has two arms: arm 1 follows a weekly dosing 208 | CELLULAR THERAPY
Optimization of Glioblastoma multiforme in vitro culture conditions for preservation of EGFR gene amplification
Mokri P.1, William D.1, Classen C.F.1
1University Childrens Hospital Rostock, Brain Tumor Vaccine Group, Rostock, Germany
Glioblastoma multiforme (GBM) is a brain tumor copy number, albeit lower than the egfr copy number with desperate prognosis both in adults and children. of the original xenograft tumor. Furthermore, we de- Numerous efforts are made to investigate cellular termined the amount of EGFR protein in the differ- mechanisms in GBM tumor cells and to develop ent cultures using immunofluorescence analysis. We new, more effective therapies. Hence, optimal in found that cells cultured under serum free conditions vitro models of GBM are essential, generated from have higher amounts of EGFR than cells cultured individual patients and analysed at early passages to under standard conditions, with lower EGFR upon avoid culture artefacts. higher EGF supplementation in the medium. Different studies demonstrated that amplification of In summary, we demonstrated that an elevated egfr the egfr gene occurs in many cases of GBM, leading to copy number can be maintained over several pas- enhanced activation of the EGFR signalling pathway sages in vitro. However, egfr copy number still de- and thus driving tumor growth. Although mutations creased initially under serum free conditions. Hence, are well maintained in in vitro models of GBM, one culture conditions are subject to further optimiza- major cell culture artefact is the rapid loss of ampli- tion to maintain egfr copy numbers that are closer fication of the egfr gene during cell culture (DMEM/ to the original tumor. Nevertheless, in vitro cultures Ham’s F12, 10% FCS). of GBM cells with elevated egfr copy number are a In this project we aim at maintaining egfr gene valuable tool to gain better understanding of EGFR amplification duringin vitro culture by optimizing driven tumors, which contributes to the improve- culture conditions. Therefore we established cell cul- ment of therapeutic strategies - conventional and im- tures from a GBM xenograft tumor with a high copy munological - to overcome this deadly disease. number of egfr under serum free conditions with varying concentrations of EGF. We could show that cells cultured under these conditions grow preferably as 3-dimensional spheroids, whereas cells cultured under standard conditions (+10% FCS) grow as ad- herent monolayer. We determined the copy number of egfr in tumor cells from several passages. As ex- pected, the cells cultured under standard conditions (+10% FCS) lost amplification of egfr with increas- ing number of passages. However, the cells cultured under serum free conditions maintained an elevated 209 | CELLULAR THERAPY
Gene transfer of an MHC class II-restricted TCR for the treatment of EBV+ Post Transplant Lymphoproliferative Disease
Williams A.1, Long H.M.1, Lee S.P.1
1University of Birmingham, College of Medical and Dental Sciences, Birmingham, United Kingdom
CD4+ T-cells play a pivotal role within the immune lated from a high avidity clone, and sequence opti- response, and multiple studies have highlighted their mised in vitro for efficient gene translation and TCR importance in anti-tumour immunity. In addition to chain pairing. their helper functions, CD4+ T cells can act as direct We have transduced healthy donor PBMCs by retro- effectors. For virus-infections or cancers arising in viral transfer and demonstrated the specificity and MHC II positive cells, this opens an exciting thera- function of the transduced cells. We have shown that peutic opportunity. the TCR-transduced T cells specifically recognise Our interest is in Post-Transplant Lymphoprolifera- the PRS peptide with a high avidity. Interestingly, tive Disease (PTLD), a life threatening complication the TCR is functional in both the CD4+ and CD8+ of solid organ and hematopoietic transplantation. subsets of the transduced T cells, proliferating and PTLD is often associated with Epstein Barr Virus - a producing multiple cytokines in response to physi- persistent B cell tropic virus that is under the control ological levels of EBNA2 processed and presented of T cell immunity. The disease arises in immuno- by EBV-infected B cells. Furthermore, both CD4+ deficient patients, where the lack of immune control and CD8+ transduced T-cells have direct cytotoxic leads to EBV driven B-cell proliferations. Important- effects against EBV-infected cells, as measured by ly, the outgrowing B cells of PTLD are constitutively CD107a expression, chromium release assays and MHC II-positive, and express a range of viral antigens outgrowth assays. We have also demonstrated that that are targets of the CD4+ T cell response. Further- the transduced cells retain helper functions, as they more, we have previously demonstrated that isolated are able to induce maturation of dendritic cells. virus-specific CD4+ T cell clones are able to directly Results from this study highlight that TCR gene trans- recognise EBV-infected B cells in vitro, suggesting fer with EBV-specific MHC II-restricted TCRs can therapeutic promise. generate polyclonal T cells with functional capacity Therefore, in this study we are exploring the anti- against virus-infected cells, which may be useful in tumour potential of EBV-specific, MHC II-restricted rapid and reliable generation of T cells for treatment T cells, generated by gene transfer of a virus-specific of PTLD. Given the importance of CD4 T cells for T cell receptor (TCR). The TCR is specific for the PRS anti-tumour responses this study also highlights the epitope (PRSPTVFYNIPPMPLPPSQL) from the EBV potential for using TCR gene transfer to target such protein EBNA2 expressed in PTLD. This epitope was cells towards other MHC class II-positive tumours. selected as it is efficiently presented on the surface of virus-infected B cells and restricted through the common MHC II allele DRB3*02. The TCR was iso- 210 | CELLULAR THERAPY
ErbB2/HER2-targeted CAR NK cells display potent antitumor activity against glioblastoma and enhance survival
Zhang C.1,2, Burger M.2,3, Jennewein L.4, Genßler S.1, Schönfeld K.1, Hattingen E.5, Mittelbronn M.4, Tonn T.6, Steinbach J.P.2,3, Wels W.S.1,2
1Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany, 2German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany, 3Institute for Neurooncology, Goethe University, Frankfurt am Main, Germany, 4Edinger Institute, Goethe University, Frankfurt am Main, Germany, 5Institute of Neuroradiology, Goethe University, Frankfurt am Main, Germany, 6Institute for Transfusion Medicine, German Red Cross Blood Donation Service North-East and Medical Faculty Carl Gustav Carus, TU Dresden, Dresden, Germany
Significant progress has been made over the last adoptive immunotherapy of ErbB2-positive glioblas- decade towards realizing the potential of natural toma. We evaluated the activity of NK-92/5.28.z cells killer (NK) cells for cancer immunotherapy. NK cells against a panel of glioblastoma cell lines and primary can respond rapidly to transformed and stressed glioblastoma cultures and demonstrated selective in cells, and have the intrinsic potential to extravasate vitro cell killing that was dependent on the level of and reach their targets in almost all body tissues. ErbB2 expression by the target cells and the time of In addition to donor-derived primary NK cells, also their exposure to the NK cells. Antigen specificity continuously expanding cytotoxic cell lines such as and selective cytotoxicity of NK-92/5.28.z cells were NK-92 are being considered for adoptive cancer im- retained in vivo, resulting in antitumoral activity munotherapy. High cytotoxicity of NK-92 has pre- against orthotopic glioblastoma xenografts in NSG viously been shown against malignant cells of he- mice. Further proliferation of NK-92/5.28.z cells was matologic origin in preclinical studies, and general prevented after irradiation with 5 or 10 Gy included safety of infusion of NK-92 cells has been established as a safety measure, while the in vivo antitumor ac- in phase I clinical trials. To enhance their thera- tivity of the irradiated cells was not impaired. Our peutic utility, here we genetically modified NK-92 results suggest adoptive transfer of ErbB2-specific cells to express a chimeric antigen receptor (CAR), NK-92/5.28.z cells as a promising new immunother- consisting of an ErbB2-specific scFv antibody frag- apy approach for ErbB2-positive glioblastoma. ment fused via a linker to a composite CD28-CD3 zeta signaling domain. GMP-compliant protocols for vector production, lentiviral transduction and ex- pansion of a genetically modified NK-92 single cell clone (NK-92/5.28.z) were established. Functional analysis of NK-92/5.28.z cells revealed high and stable CAR expression, selective cytotoxicity against ErbB2-expressing but otherwise NK-resistant tumor cells of different origins in vitro, as well as homing to ErbB2-expressing tumors in vivo. Ongoing work now focuses on the development of these cells for 211 – 238
Immuno- monitoring 211 | IMMUNOMONITORING
Detection of long lasting memory HER-2/neu specific T cells in vaccinated cancer patients
Anastasopoulou E.A.1, Voutsas I.F.1, Perez S.A.1, Baxevanis C.N.1
1Cancer Immunology and Immunotherapy Center,Saint Savas Cancer Hospital, Athens, Greece
Antitumor T cell-mediated immunity is a key bio- Methods: Peripheral blood mononuclear cells marker for most vaccines along with immunother- (PBMCs) were thawed and stimulated in vitro apies and involves the activity of specialized cells using the AE37 peptide (10µg/ml) in the presence including antigen specific cytotoxic T lymphocytes of IL-2 (20IU/ml) for 10-15 days. We used samples (CTLs) and CD4+ helper T lymphocytes. In this from DRB1*11+ (DR11) patients at different time- project we investigated HER-2/neu (HER2) specific points post vaccination based on their availability. T cells in patients vaccinated with a 15-mer from AE37-specific CD4+ T cells were assessed using te-
the intracellular domain of HER2 (AE36 peptide: tramer DR11/AE37(LRMKGVGSPYVSRLLGICL). Further pheno-
HER2 776-790), which was linked to a 4-mer (LRMK typic characterization and cytokine profile (intra- or Ii-key) from the invariant chain of MHC class cellular staining after 24hrs challenge with AE37 II molecules (LRMK/GVGSPYVSRLLGICL or AE37 loaded DCs) were performed with multicolor flow hybrid peptide), in a phase I clinical trial. Published cytometry (BDFacs ARIA III-up to 10 parameters). results from this trial showed that the vaccine is Results: Antigen-specific CD4+ T cells were detected safe and induces HER2-specific immunity in a het- in all analyzed vaccinated patients. Further anal- erogeneous population of HER2 + prostate cancer ysis demonstrated that these cells have a memory patients. Because AE37 vaccine is a multiepitope phenotype and, most important, are not regulatory vaccine containing overlapping CD4+T-helper and T cells. Additionally, we found that AE37-responsive CD8+ CTL epitopes, it would be important to monitor CD4+ T cells in their vast majority are polyfunction- the presence of both CD4+ and CD8+ antigen specific al, producing mainly IFN-γ, IL-2, IL-4 and TNF-a T-cell populations in our vaccinated patient popula- with a very low percentage expressing IL-10 and tion. Investigating vaccine specific CD4+T-cell pop- I L -17. ulations can be really a challenge due to the high Conclusion: The AE37 vaccine specifically induces polymorphism of MHC class II molecules, especially memory CD4+ T cells with a polyfunctional, mainly, those encoded by the DRB1 locus, and the reduced Th1 profile. binding affinity of antigenic peptides derived from tumor and self-antigens compared to peptides derived from pathogens. In the present study, we aimed to detect HER2 specific CD4+ T cells in vacci- nated patients and further characterize them based on their regulatory phenotype, memory status and cytokine profile. 212 | IMMUNOMONITORING
Assessment of vaccine-induced immunity following intra lymph node administration of RIBOLOGICAL® IMMUNOTHERAPY targeting NY-ESO-1 and tyrosinase in patients with advanced melanoma
Attig S.1, Derhovanessian E.2, Bidmon N.2, Rae R.1, Schröder H.2, Kloke B.-P.3, Diekman J.3, Kemmer-Brueck A.2, Luxemburger U.2, Diken M.1, Pless B.3, Loquai C.4, Schadendorf D.5, Höller C.6, Utikal J.7, Britten C.M.3, Kuhn A.N.3, Kreiter S.1, Türeci Ö.8, Sahin U.1,2,3
1TRON - Translationale Onkologie an der Universitätsmedizin Mainz gGmbH, Mainz , Germany, 2BioNTech AG, Mainz, Germany, 3BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, 4Department of Dermatology, University of Mainz, Mainz, Germany, 5Department of Dermatology, University Hospital Essen, Essen, Germany, 6Department of Dermatology, Division of General Dermatology and Medical University Vienna, Vienna, Austria, 7Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim, Germany, 8III. Medical Department, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
There are about 160,000 new cases of melanoma to 1000µg RNA per injection. All 5 dose levels were worldwide each year. Despite advances in the treat- well tolerated and no dose limiting toxicities were ment of early-stage melanoma, a significant fraction observed. Pharmacodynamic activity of RIBOLOGI- of patients will eventually develop metastatic mela- CAL® IMMUNOTHERAPY was assessed by intrader- noma with a poor prognosis. Immunotherapy may mal injections of RNA to induce DTH reactions, flow address the unmet medical need in patients with ad- cytometric analysis of skin infiltrating lymphocytes, vanced melanoma. as well as IFN-gamma ELISPOT assays in peripheral BioNTech RNA Pharmaceuticals GmbH has devel- blood mononuclear cells (PBMC). In addition detailed oped an innovative RNA-based immunotherapy immune phenotyping data were generated on the fre- platform called RIBOLOGICAL® harboring propri- quency of circulating Tregs, MDSCs and the activa- etary structural features and sequence elements tion status of different immune subsets. that increase its bioavailability, immunogenicity and We will present results from the immunological immunomodulatory properties. Based on an exten- readouts that have been made indicating a high rate sive preclinical program and a favorable risk benefit of vaccine-induced immunity. Immune responses analysis the company initiated a first in human trial against a microbial marker antigen and the cancer to test the safety, tolerability and pharmacodynam- germline antigen NY-ESO-1 were more frequently ics activity of RIBOLOGICAL® IMMUNOTHERAPY observed than immune responses against the differ- targeting the known tumor-associated antigens NY- entiation antigen tyrosinase. ESO-1 and tyrosinase (NCT01684241). So far 28 patients with stage III and IV malignant mel- anoma were recruited to this phase I trial that includs 5 dose escalation cohorts ranging from 50µg RNA up 213 | IMMUNOMONITORING
The cytokine/chemokine response accompanied with administration of the therapeutic antibody PankoMab-GEX™
Baumeister H.1, Stahn R.1, Karsten U.1, Goletz S.1
1Glycotope GmbH, Berlin, Germany
PankoMab-GEX™ is a humanized and glyco-opti- 4 h and 24 h after the start of first infusion and an- mized IgG1 mAb recognizing the novel carbohy- alyzed for cytokine/chemokine release. Except for drate-induced conformational TA-MUC1 epitope and very few measurements no or only very weak in- is currently evaluated in a phase II clinical trial. The flammatory responses of IL-1β, IL-8 and TNF-α were epitope comprises a tumor specific carbohydrate observed within 24h after 1st administration of Pan- antigen together with the immunodominant peptide koMab-GEX™. Weak stimulations of IFN-γ observed region of MUC1 and is virtually only expressed on 3-6 h after start of 1st infusion may indicate some tumor cells in a wide variety of cancers with high stimulation of NK cells after infusion of PankoM- incidences. The glycosylation of PankoMab-GEX™ ab-GEX™. Analysis of the complement factor C3a and was optimized with the GlycoExpress™ system the eosinophil cationic protein (ECP) in a subset of using human glycoengineered production cell lines 27 patients revealed the absence of complement or leading to improved anti-tumor efficacy and reduced allergic reactions. immunogenicity due to a lack of non-human glycan Finally, a 30 cytokine/chemokine array analysis was structures. PankoMab-GEX™’s anti-tumor activity is performed with five samples per patient (between 0 mediated at least by antibody dependent cellular cy- and 24 h after infusion) from 15 patients. The array totoxicity (ADCC) and phagocytosis. analysis confirmed the absence of the above men- In a phase I clinical trial 74 patients received Pan- tioned pro-inflammatory response, and detected only koMab-GEX™ infusions weekly (Q1W), every two very weak responses. Only in case of the macrophage weeks (Q2W) or every three weeks (Q3W) in doses inflammatory proteins (MIP) α1 and especially 1β a of 1 mg to 2200 mg. A clinical benefit that was more >10fold peak stimulation was found 3 h after start of frequent at higher doses was experienced by a third infusion in 8 of the 15 patients. The response is dose of evaluable patients, all with advanced progressive independent and does not correlate with the occur- disease at study entry and a broad variety of adeno- rence of IRR. The chemokines MIP-1α and MIP-1β are carcinomas. No MTD was reached. The commonly known to be secreted by a number of immune cells, with therapeutic antibodies observed infusion-relat- such as macrophages, NK cells and monocytes. The ed reactions (IRRs) were mild to moderate (Grade 1 / potential role of MIP-1α and MIP-1β stimulation after 2) and occurred in 53 % of the patients mostly with PankoMab-GEX™ infusion is discussed. the 1st infusion. To better understand the systemic activity of Pank- oMab-GEX™, serum samples were taken before start of first infusion, at the end of infusion and between 214 | IMMUNOMONITORING
Dissecting the impact of peptide-MHC and TCR-pMHC binding strengths on the functional avidity of tumor-specific CD8 T cells
Baumgaertner P.1, Hebeisen M.1, Costa-Nunes C.M.1, Bordry N.1, Schmidt J.1,2, Guillaume P.2, Luescher I.F.1,2, Rufer N.1,3, Speiser D.E.1,3
1Ludwig Center for Cancer Research/ University of Lausanne, Department of Oncology, Lausanne, Switzerland, 2TCMetrix, Lausanne, Switzerland, 3University Hospital Center (CHUV), Department of Oncology, Lausanne, Switzerland
CD8 T cell responses are regulated via the specific different Melan-A variants. Interestingly, native pep- interactions between TCR and immunogenic pep- tide-derived T cell showed higher functional avidi- tides presented by MHC. We investigated the func- ties when recognizing weak MHC binding peptides. tional impact of peptide:MHC binding strength using In turn, analog peptide-derived T cell clones required mutated peptides derived from TAA Melan-A/Mart- strong MHC binding peptides to show high function-
126-35 and NY-ESO-1157-165 and of TCR:pMHC binding al avidity. using mutated TCRs with increasing affinities for the This study demonstrates and quantifies the impact of pMHC complex. peptide:MHC binding strength on the tri-molecular We selected 17 Melan-A and 21 NY-ESO-1 peptide TCR:pMHC affinity. This knowledge is fundamental variants according to their binding strength to the for the choice of peptides used for assessing T cell MHC determined by in silico prediction. The capacity responses, and for the development of vaccination of the peptide variants to form stable peptide:MHC against infectious and malignant diseases. complexes was first determined by soluble refolding assays in a cell-free system. We developed further a cell-based competition assay to evaluate the strength of peptide:MHC binding. A selection of eight NY-ESO-1 variants with various binding affinities for MHC was tested in functional killing assays in combination with a panel of NY- ESO-1-specific CD8 T cells expressing TCRs of various affinities. We found that peptides attaching strongly to the MHC were able to compensate for the loss of functional avidity of low affinity TCR, but never to a level comparable to the wild-type or affinity-opti- mized TCRs. A selection of twelve Melan-A variants was used in functional killing assays on Melan-A specific T cell clones from melanoma patients vaccinated with either the native (low) or the analog peptide (10x increased binding strength). We determined the functional avidity of 34 T cell clones using these 215 | IMMUNOMONITORING
Differences in NK cell reconstitution after T cell depletion with ATG or Alemtuzumab in allogeneic hematopoetic stem cell transplantation
Bode S.1, Lay A.N.1, Wolff D.1, Theobald M.1, Meyer R.G.1, Ullrich E.2, Wagner E.M.1
1University Medical Center Mainz, Hematology, Oncology and Pneumology, Mainz, Germany, 2Goethe University Frankfurt, Childrens Hospital, Hematology and Oncology, Cellular Immunology, Frankfurt, Germany
Introduction: Antithymocyteglobulin (ATG) and CD52neg NK cells at d+60. CD69 is present on CD52pos/ the monoclonal anti-CD52-antibody Alemtuzumab neg NK cells (R2), whereas CD62L is expressed exclu- are frequently used for T cell depletion (TCD) in the sively on CD52pos cells (R2). Treatment with ATG does context of allogeneic hematopoietic stem cell trans- not alter these additional markers. plantation (HSCT). We already showed long-term R2 showed the highest density of CD52 in healthy persistence of functionally impaired CD52-/Glyko- controls, both TCD-antibodies induced downregula- sylphosphatidyl-Inositol (GPI)-negative effector and tion of CD52 and GPI-anchors on NK cells and altered regulatory T cells following Alemtuzumab-based the distribution of activating or inhibitory markers. conditioning. In this study, we analyze the pheno- Conclusion: The reconstitution of functionally im- type on natural killer (NK) cells of patients under- paired GPIneg T cells may be partly responsible for the going HSCT with different antibodies used for TCD. viral complications after Alemtuzumab-based con- Methods: We examined peripheral blood samples of ditioning. In this context, NK cells play a major role 10 patients following conditioning with Alemtuzmab especially early after HSCT. Here we provide data on and 10 patients with ATG on d+60 and d+365 after a relevant percentage of CD52neg/GPIneg NK cells and HSCT. By using multicolor FACS-analysis, we phe- their specific subset distribution. Depended on the notypically distinguished the NK cell subpopula- time after HSCT and conditioning, expression of GPI- tions CD56brightCD16dim (R1), CD56dimCD16neg/CD56dim- anchors, natural cytotoxicity receptors, maturation- CD16int (R2) and CD56dimCD16bright (R3). CD52neg NK and activation markers varies. This may impact the cell subsets were characterized and compared to NK cell mediated immune responses. Further inves- healthy donors regarding their expression of surface tigations will help to identify patients with a need for antigens involved in cytotoxicity (NKp44, NKp46, cellular therapies, for example NK-cell derived DLI. NKG2A, NKG2D), maturation, activation (CD62L, CD69) and GPI-anchor expression. Results: R2 and R3 are the major NK cell popula- tions early after HSCT, CD52neg NK cells are present in most patients, but predominantly after Alemtu- zumab treatment (55-90%, decreasing over time). GPI-anchors are downregulated in CD52neg NK cells, a complete loss of GPI-anchors was only found in R2 of Alemtuzumab-treated patients. After Alemtuzumab, NKG2A/NKG2D expression is increased mainly for 216 | IMMUNOMONITORING
CIP NK Proficiency panel 2014: Low inter-lab variation found with NK phenotypic markers, but high variation in activation and functional markers
Challis R.J.1, Chudley L.1, Coleman A.1, Gao Y.1, Khakoo S.2, Williams A.1, Ottensmeier C.1
1University of Southampton, NIHR/CRUK Experimental Cancer Medicine Centre, Southampton, United Kingdom, 2University of Southampton, Clinical Experimental Sciences, Southampton, United Kingdom
Aims and methods: An NK proficiency panel was or- ulations. This variation was increased, although not ganized in association with the CIMT Immunoguid- significantly, following stimulation in CD56+ cells; ing Program (CIP) in 2014 to assess variations in the CD56bright/CD16- cells; and NKp46+ cell popula- phenotyping of human NK cells by flow cytometry. tions. In the CD56lowCD16+ cells, we observed dif- 21 participants from Europe and the USA registered ferential effects on the CD16 expression, with some an interest in participating in the experimental phase stimulation protocols causing a marked down regula- and were sent PBMC samples derived from the buffy tion of CD16, leading to a marked increase in the vari- coats of 3 UK national blood service donors. Partici- ation within this population following stimulation pants were asked to quantify predefined NK cell phe- (p≤0.05). In general variation across the activation notypes in the following way: live cells by inclusion and functional markers was also higher, particularly of a live/dead marker and CD3/CD19- and CD56+ in the case of CD69 and NKG2D, where there was cells; CD56lowCD16+ cells; CD56bright/CD16- cells less clear definition between the positive and nega- and the alternative NK marker NKp46+ cell popula- tive populations. In these cases, the fold-change in tions. Additional markers of NK cell activation (CD69 the populations following stimulation was more in- and NKG2D) and functional assessment (IFNg and formative. Additionally we observed that different CD107a) were also requested. Stimulated and un- types of stimulation had differential effects on the stimulated cells were analysed, although labs were activation/functional markers. free to choose their own staining panel configuration Conclusions: Good consensus on the definition of and stimulation protocols. the basic phenotypic markers was observed in un- Results: 20 laboratories performed the experimen- stimulated cells with low inter-laboratory variation. tal stainings, and returned their data to the central However the variation in the activation markers was laboratory. This data was collated centrally in South- high in both stimulated and unstimulated cells, as ampton, and a written report returned to each of the was the observed variation in the activation and participating labs informing them of their perfor- functional markers following stimulation. mance relative to the group as a whole, and of the inter-lab variation observed between the requested populations. In general the inter-lab variation of the main NK phenotypes (CD56+ cells; CD56lowCD16+ cells; CD56bright/CD16- cells and NKp46+ cell pop- ulations) was low in unstimulated cells, % cv range approximately 9 - 65 % amongst the donors and pop- 217 | IMMUNOMONITORING
Validation of immunomonitoring assays- Multimer staining assay and ELISPOT assay
Chandran.P A.1, Rusch E.1, Backert L.2, Laske K.3, Rammensee H.-G.1, Gouttefangeas C.1
1University of Tübingen, Interfaculty Institute for Cell Biology, Department of Immunology, Tübingen, Germany, 2University of Tubingen, Center of Bioinformatics Tubingen (ZBIT), Tübingen, Germany, 3Immatics Biotechnologies GmbH, Tübingen, Germany
Our group monitors T cell responses to vaccinated lyzing spot counts after coating unspecific proteins, peptides in cancer patients by probing the presence response of MHC mismatched cells, etc. Precision of peptide specific T cells in the peripheral blood col- and variability among technical replicates within an lected during multiple vaccination time points. Rou- assay, assays performed on several days by the same tinely used assays, to detect peptide specific cells as operator or by several operators were also quanti- well as to assess their functionality, are multimer fied. Linear relationship of the assay read out to the (oligomeric MHCs loaded with vaccinated peptides) actual amount of target cells was checked as well. A staining assay (MSA) and Interferon-gamma (IFN-g) common issue during immunomonitoring of patient ELISPOT assay (IEA). However, in order to use these material is scarcity of sample material. We evaluated assays for such a purpose, the assays have to qualify the influence of the no. of cells used for each assay certain acceptance criterion. Assay validation essen- on the final read out. tially qualifies an assay to be applied in a particular Definition of a truly positive response in both assays investigation. Validation is the last step the before the has been quite problematic and largely subjective application of an assay, following assay adaptation, with several groups adopting multiple empirical cri- establishment, standardization and optimization. teria to define a positive response. In the multimer Since the assay will be applied to monitor responses assay, we were able to successfully validate a lower to several different peptides, we decided to validate limit of quantitation of a multimer(+) population. In the underlying assay rather than the multimers or the ELISPOT assay, where tests are often performed peptides. We used viral peptide loaded MHC-multim- in triplicates or duplicates, we adopted a distribution- ers and viral peptides to assess viral antigen specific free resampling-based statistical method which was T cells for the MSA and IEA respectively. During vali- closest to our in-lab empirical method. We did this dation, various parameters of the assay like speci- by calculating the distances between the empirical ficity, repeatability (precision), linearity, sensitivity statistical methods through simulated spot counts and robustness were assessed for both assays. for negative control and samples. Specificity of the MSA was determined by assess- Together, in addition to qualifying the assays for our ing the percentage of multimer(+) CD8(+) T cells intended purpose, validation of both the assays have binding unspecifically to multimers under several further informed us of their strengths and limita- conditions. These included binding of multimers to tions. MHC-mismatched PBMCs, binding of self-antigenic multimers and binding of 2 different multimers to the same cell. Specificity of IEA was tested by ana- 218 | IMMUNOMONITORING
The abstract is withdrawn 219 | IMMUNOMONITORING
Changes in the peripheral blood immune profile upon surgery of early stage lung cancer
de Goeje P.L.1, Hegmans J.P.J.J.1, Waasdorp C.1, Senan S.2, Smit E.F.3, Aerts J.G.J.V.1,4
1Erasmus MC, Pulmonary Medicine, Rotterdam, Netherlands, 2VU Medical Center, Radiotherapy, Amsterdam, Netherlands, 3Antoni van Leeuwenhoek Netherlands Cancer Institute, Thorax Oncology, Amsterdam, Netherlands, 4Amphia Hospital, Breda, Netherlands
The immune system plays an important role in the again to baseline level. Myeloid-derived suppressor initiation and progression of cancer. Novel immu- cells are known for their capacity to suppress the notherapies show promising results, and also the T cell response and increased levels of these cells patient’s individual immune profile shows to have correlate with reduced survival. We were therefore prognostic and predictive value. Recently, the ‘im- interested in the kinetics and activation of T cells in munoscore’ has been proposed as an addition to the these patients, and designed a staining that includes classical TNM staging, to improve survival predic- multiple activation markers (such as CD69 and CD25) tion. However, biopsies in which the immune status and inhibitory markers (such as PD-1 and CTLA-4), can be reliably evaluated are often not available. for various T cell subsets, including regulatory T Also, monitoring the immune status of patients in cells. Moreover, we stimulated the patients’ cells in time is not possible from a single biopsy. Therefore, vitro to study IFNγ and granzyme B expression as we study the immune profile in peripheral blood of an indirect measure of cytotoxic capacity. We expect lung cancer patients to monitor the immune status in to report the results on the T cells at the conference time, before and during treatment. as well. Both the baseline immune profile of the pa- Early stage non-small cell lung cancer (NSCLC) is tients and the changes over time will be correlated usually treated by surgical removal of the tumor. Al- with recurrence and survival. The importance of though it is not an immune therapy, surgery is known the immune contexture in cancer and the effects of to influence the immune system. To get more insight non-immunotherepeutic treatments on the immune in the effect of regular treatment on the immune system are increasingly being recognized. However, system, we aimed to examine the immune status of detailed knowledge about the immune profile as a the patient and the kinetics as a consequence of this whole in peripheral blood is lacking. Our results will treatment. Blood was collected from NSCLC patients give insight into the kinetics of the immune status before and during 6 weeks after surgery and the iso- will provide after surgery, which may have prognos- lated mononuclear fraction was analyzed with flow tic or predictive value. Moreover, the results might cytometry. We analyzed immune regulatory popula- provide insight into the optimal time frame for addi- tions such as regulatory T cells and myeloid-derived tional immunotherapeutic treatments that modulate suppressor cells, and focused on activation and ex- certain (anti-cancer) immune responses. haustion markers on CD4+ and CD8+ and γδ T cells. In the myeloid cell population, we found that myeloid-de- rived suppressor cells numbers were increased in the first weeks after treatment, but thereafter reclined 220 | IMMUNOMONITORING
Electroporation of CD8+ effector T cells with antigen is suitable to test their reactivity to tumor antigens
Prommersberger S.1,2, Höfflin S.1,2, Schuler-Thurner B.1, Schuler G.1, Schaft N.1, Dörrie J.1
1Universitätsklinikum Erlangen, Hautklinik, Erlangen, Germany, 2Friedrich-Alexander-Universität Erlangen-Nürnberg, Genetics, Erlangen, Germany
The identification and quantification of tumor-anti- of high and intermediate affinity could be detected gen-specific CD8+ T cells is a challenge often faced in with our new screening method. immunological research. Immunomonitoring during Next, we analyzed the immunogenicity of the fre- therapeutic vaccination and the evaluation of new quently occurring tumor driver mutations BRAFV600E target antigens are just two occasions. To detect and and NRASQ61K in healthy donors. Besides a massive monitor such T cells without requirement for char- donor variance, we observed that not only the acterized T-cell epitopes and independent from HLA mutated but also the wild-type versions of the an- haplotypes or additional target cells, we expressed tigens could induce immune responses, and that the antigen in the T cells themselves, taking advan- the induced T cells also displayed cross-reactivity tage of their capability to present antigens on their between both versions. The tumor-associated antigen own MHC molecules. We achieved this by electropo- Wilms’ Tumor protein 1 (WT1) was also examined in ration with antigen-encoding mRNA. our system, and we could show that WT1 RNA-elec- The reliability of this read-out system was verified troporated DC were capable to expand specific CD8+ using the melanoma antigen MelanA. This antigen T cells from both HLA-A02- and HLA-A24-positive contains a highly immunodominant HLA-A2-re- healthy donor material. stricted epitope and healthy donors of this haplo- In conclusion, we showed that this read-out system is type display measurable numbers of specific CD8+ quick and easy to perform, independent of the donors T cells, which can easily be expanded in vitro. By HLA type, and circumvents the need for additional repetitive stimulation with MelanA-expressing au- cells as targets. It can be used in pre-clinical research tologous dendritic cells (DC), we generated pools to test new antigens for their immunogenic potential of bulk T cells, containing MelanA-specific cells in and for immunomonitoring in cancer patients. various percentages. These T cells were electropo- rated with MelanA-encoding RNA, resulting in a transfection efficiency of over 80%. They were also able to process and present the antigen and to stimu- (S.P. and S.H. contributed equally, J.D. and N.S. share senior authorship) late each other, shown by cytokine-secretion-based assays. The numbers of cytokine-producing T cells after mRNA electroporation highly correlated with those observed after peptide pulsing, when interme- diate peptide concentrations were used (R2 = 0.80 by Pearson-Test). These results indicate that T cells 221 | IMMUNOMONITORING
Functionally impaired GPI-anchor negative regulatory T cells strongly correlate with acute Graft versus Host Disease after Alemtuzumab-based conditioning regimen
Epp K.1, Schäfer L.1, Theobald M.1, Bopp T.2, Meyer R.1, Wagner E.M.1
1Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Department of Heamatology, Medical Oncology, and Pneumology, Mainz, Germany, 2Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Department of Immunology, Mainz, Germany ORAL ALK SHORT T 2015
Introduction: Alemtuzumab is a monoclonal CD52- Results: Patients with aGvHD showed significantly el- antibody, which is administered for T-cell depletion evated percentages of CD52 negative Treg: mean 55.3% (TCD) in the context of allogeneic hematopoietic (range 34.4-79.7%) in comparison to only 10.1% (range stem cell transplantation to prevent graft versus host 1-21.3%) in patients with chronic our without GvHD. disease (GvHD). Nevertheless, half of our patients By FLAER-staining we confirmed that loss of CD52 treated with Alemtuzumab developed acute graft correlates with the absence of GPI-anchors on the cell versus host disease (aGvHD), mainly overall Grade surface. I-II°. Since especially regulatory T cells (Treg) play a Patients who overcame aGvHD reconstituted CD52 pos- major role in controlling GvHD, we hypothesized that itive Treg, whereas CD52 negative Treg remained the they might be functionally impaired and not able to dominant population in patients with ongoing aGvHD. prevent patients of GvHD. The fraction of activated GARP positive Treg was Methods: We analyzed peripheral blood samples mainly detected among the CD52 positive Treg popula- of 20 patients with aGvHD, 10 patients with tion. All other markers showed a heterogeneous expres- chronic GvHD and 12 patients who never devel- sion profile with a tendency towards lower expression oped GvHD after Alemtuzumab-mediated TCD. on CD52 negative Treg. Treg were identified as CD3+CD4+CD25+CD127- or Suppression-assays showed a higher suppressive capac- CD3+CD4+CD25+FoxP3+ and subsets described by ity of CD52 positive ex vivo Treg. expression of CD52. Since CD52 is linked to the mem- Discussion: CD52 negative/ GPI-anchor negative Treg brane by a glycosylphosphatidylinositol (GPI)-anchor, reconstituted in patients after Alemtuzumab mediated we used FLAER to stain for GPI-anchors themselves. TCD and mainly persisted in patients with aGvHD. To further investigate Treg activation, we stained ad- These Treg were less likely to express the activation ditional markers: CD39, CD44, GITR, CXCR3, CCR5, marker GARP. Functional assays demonstrated that CTLA-4, GARP and Granzyme. CD52 negative Treg were functionally impaired- in To observe Treg reconstitution after HSCT, we ana- patients developing aGVHD, these impaired Treg rep- lysed samples from patients with aGvHD at different resented the major Treg-population. Our preliminary time points and correlated our findings with the clini- data suggest that CD52 negative Treg, like other CD52 cal course of GvHD. Treg function was evaluated in negative T-cell subpopulations (previously shown), are CFSE-suppression-assays: patient derived ex vivo Treg functionally altered. We hypothesize that this might were FACS-sorted according to their CD52 expression promote aGvHD. These CD52 negative Treg could be and incubated with proliferating CFSE-stained CD4- useful to diagnose and guide immunosuppressive effector T cells from healthy donors. therapy in patients with acute GVHD. 222 | IMMUNOMONITORING
Development of a novel “rapid and sensitive” method for detection of cellular immune responses and selection of immunogenic peptides encoded by mutated genes
Fujii K.1, Miyahara Y.1, Shiku H.1
1Mie University Graduate School of Medicine, Tsu, Japan
ORAL ALK SHORT T 2015
Various types of cancer vaccines have been con- (9mer) or even addition of long-peptides (20mer). ducted and evaluated for their clinical efficacies. This phenomenon was equally observed when we An appropriate assessment of vaccine-induced cel- investigated specific immune responses against NY- lular responses is critically important for evaluation ESO-1 by the use of PBMCs from patients previously of vaccine itself, however, we are often faced with vaccinated with NY-ESO-1 protein. We confirmed problems. For instance, the IFN-g ELISPOT and te- that these chemokines’ mRNA was mainly produced tramer assays, “gold standard” immune-monitoring in CD14+ cells and caused by minute IFN-g secreted methods, are known to be difficult to be standard- by antigen-specific T cells in PBMCs. It should be em- ized between facilities due to diverse protocols, es- phasized that amplified reactions driven by IFN-g/ pecially when used with long term cultured T cells. IFN-g receptor axis make this novel method more This defect of these conventional assays cause less sensitive than conventional assays. We proved this reproducibility and partly disturbed the precise eval- kinetics was same in murine immune systems as uation of vaccine so far. To overcome this obstacle, well. In particular, taking advantage of high sensitiv- we explored the possibility of utilizing quantitative ity of this method, we successfully detected specific RT-PCR (qPCR) method for evaluation of cellular immune responses against mutated antigens-derived immune responses with minimum cultivation in epitopes in tumor-bearing mice, even without long- vitro. Firstly, we tried to investigate which cytokines/ term in vitro cultivation. Collectively, this method chemokines increased at protein levels in the culture could be not only superior to conventional methods medium after overnight incubation of tumor cells in respect of reproducibility and sensitivity, but also with tumor-specific human T cells. After screening of could be a useful tool for selection of immunogenic forty-eight different human cytokines/chemokines, peptides for individualized mutanome-vaccines. we selected candidate cytokines/chemokines in- creased quantitatively in an antigen recognition dependent manner. Then, by utilizing Epstein-Barr virus specific immune responses, we investigated which gene’s transcript increased, especially within a few hours, after antigen recognition. Among of candidate genes, we observed that mRNA of three different chemokines (CXCL9, 10 and 11), which are ligands for CXCR3, rapidly increased in amount within short periods after addition of short-peptides 223 | IMMUNOMONITORING
Ex vivo circulating whole blood for superior detection of cytokine storms in response to antibody therapies
Gustafsson W.1, Fletcher E.A.K.1,2, Gustafsson J.1, Mangsbo S.M.1,2
1Uppsala University, Department of Immunology Genetics and Pathology, Uppsala, Sweden, 2Immuneed Inc., Uppsala, Sweden
ORAL ALK SHORT T 2015
The strong need for assays that can provide adequate ment reduces the need of soluble anticoagulant (i.e. information on immunotoxicity and immune effica- low or no soluble anticoagulant added), and thereby cy came into light after a disastrous clinical trial in preserves an intact complement system. The unique 2006, in where injection of the CD28 superagonist features of our whole blood loop system make it a TGN1412, led to life-threatening conditions for six new promising method for safety testing of novel im- out of six healthy trail participants. Current in vitro munotherapies/biologicals. assays often fail to predict cytokine storm responses in humans. Published assays display high inter-in- dividual variability, low specificity and cytokine release only after 24hours, not in line with a rapid 1. Suntharalingam, G. et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. The New in vivo cytokine storm. Using our proprietary whole England journal of medicine 355, 1018-1028 (2006). blood loop system we show that a cytokine storm can be detected as early as after 4 hour incubation. All donors tested (n=10) respond with release of IFNγ to anti-CD28 (ANC28.1/5D10) and to anti-CD3 (OKT3) stimulation, while no donors respond in a standard cytokine release assay (4 hours) and only 1/10 (anti- CD28) and 7/10 (OKT3) respond after a 24 hour stim- ulation. The stimulation also gives rise to the release of TNFα in a similar manner. IL-2 was the key cyto- kine inducing the severe cytokine storm in the disas- trous clinical trial in 2006. In our blood loop system, a greater and quicker IL-2 release than in other whole blood in vitro assays is observed. In addition, there is also a release of other proinflammatory cytokines (TNFα, IFNγ and IL-6) in response to TGN1412 when using our loop system (mimicking the in vivo clini- cal trial findings1), also indicative of a true cytokine storm. Our ex vivo loop system uniquely consists of fresh whole blood kept in circulation using a surface heparinized tubing system. The circulatory environ- 224 | IMMUNOMONITORING
Study on the viability, recovery and functionality of PBMCs stored at -80°C versus storage in liquid nitrogen
Hawner C.1, Goldberg P.2, Maas M.1, Seck C.1, Bidmon N.2, Vogler I.3, Godehardt E.3, Rösler T.2, Attig S.2, Tran H.Q.1, Mueller F.1, Sahin U.2,4,5
1BioNTech AG, Mainz, Germany, 2TRON - Translational Oncology at the University Medical Center of Johannes Gutenberg University, Mainz, Germany, 3BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, 4BioNTech Group, Mainz, Germany, 5University Medical Center of Johannes Gutenberg-University, Mainz, Germany
Background: The goal of this study was to find out stored at -80°C was 10% lower than that of samples whether PBMCs could be stored at -80°C without loss stored at LN2. of recovery, viability or functionality of viable cells Regarding the functional read-out assay, reduced re- or whether storage in the gas phase of liquid nitrogen coveries of viable cells were detected directly after (LN2) is required to assure satisfying assay perfor- thawing and after overnight resting for samples mance. stored at -80°C. Furthermore, reduced recoveries Study Design: PBMCs were isolated from blood of 13 and viabilities were detected after antigen-specific in healthy donors and divided into several aliquots. Half vitro expansion for cells stored at -80°C. The cells of of the aliquots of each donor was stored at -80°C and these samples also resulted in a lower expansion rate the other half in LN2. Over a period of 3 months cell of model antigen-specific T cells leading to a reduced aliquots of each donor and storage condition were -sensitivity to detect an existing response. Based on subsequently thawed every two weeks and tested for these observations and transferring the conclusions the recovery of viable cells and viability. to a situation where low frequented and neoepitope- Additionally, the functionality of antigen-specific specific T cells are analyzed storage of samples in T cells from aliquots stored at -80°C or LN2 for a LN2 is recommended to reduce the difficulty of de- period of two months was tested in a peptide-based tection. IFN-y ELISPOT before and after in vitro stimulation. Conclusion: Short term storage (2 months) at -80°C For this purpose, PBMCs from 3 donors were thawed affects PBMC quality negatively. In order to achieve and rested overnight. In an in vitro culture, the an- good viability and acceptable recovery of viable cells tigen-specific T cells were expanded based on FluM1 as well as high functionality of antigen-specific T (GILGFVFTL) and CMVpp65 (NLVPMVATV)-specific cells, storage in the gas phase of LN2 is required. peptide stimulation. Results: Over the course of 3 months, the viability of cells stored at -80°C decreased significantly from 89% to 84 % while the viability of samples stored in LN2 remained constant (≈ 92 %). With regards to the recovery no difference could be detected after 3 weeks, however as of week 5, the recovery of samples 225 | IMMUNOMONITORING
Prognostic impact of circulating plasmacytoid dendritic cells and immunosuppressive subsets in breast cancer
Kini Bailur J.1, Gueckel B.2, Derhovanessian E.1, Pawelec G.1
1University Hospital Tübingen, Centre for medical research, Internal Medicine II, Tübingen, Germany, 2University of Tübingen, Radiology Clinic, Tübingen, Germany
ORAL ALK SHORT T 2015
Identifying immune signatures in blood that are in- association between 5-year survival and >median formative for breast cancer patient survival would levels of circulating pDCs (p=0.03) in non-metastatic not only be useful for prognosis but might also patients. We found that 5-year survival also correlat- provide mechanistic insights into processes facil- ed positively with the presence of patients´ CD8+ but itating survival. Dendritic cells (DCs) are import- not CD4+ T-cell responses to Her-2 peptides in vitro ant antigen presenting cells, of which there are two (p=0.04). Patients who had a CD8+ T-cell response main subtypes, myeloid dendritic cells (mDCs) and to Her-2 together with a low ratio of MDSCs to pDCs plasmacytoid dendritic cells (pDCs). Recently, pDCs had 100% 5-year survival in non-metastatic (p=0.04) have gained attention as increased levels of pDCs in and all (non-metastatic and metastatic) patients the tumor microenvironment have been negatively (p=0.009). High levels of pDCs and the presence of correlated with survival in several different cancers. a CD8+ T-cell response to Her-2 were independent However, the prognostic relevance, if any, of circulat- positive survival indicators according to multivariate ing pDCs has not been explored in detail. As well as Cox analysis. pDCs in the tumor, high levels of circulating MDSCs Our results suggest that circulating pDCs could be and Tregs also have a negative impact on survival a positive prognostic indicator in breast cancer, to- in different cancers. The present study focuses on gether with CD8+ T-cell reactivity to Her-2 antigens. investigating the prognostic relevance of circulating These two prognostic indicators were independent total DCs, mDCs and pDCs separately. The frequen- and emphasize the important role of immunity in en- cies of peripheral DCs, MDSCs and Tregs were as- suring breast cancer patient survival, even in those sessed in relation to in vitro T-cell responses to Her-2 not undergoing immunotherapy. antigens in 75 untreated breast cancer patients 28-87 years of age at diagnosis. The T-cell response to Her-2 was analyzed after a 12-day in vitro expansion of memory cells and simultaneous intracytoplasmic staining of TNF, IFN-γ, IL-2, IL-5, IL-10 and IL-17. We observed that patients with later stage tumors had lower levels of circulating pDCs(Lin-CD14-HLA- DR+CD123+) (p=0.008), and that ratios of MDSCs (Lin-CD14+HLA-DR-) to pDCs(p=0.03) and Tregs (CD4+FoxP3+CD25+CD127lo) to pDCs (p=0.02) were higher in later stage tumors.There was a positive 226 | IMMUNOMONITORING
Vitiligo-like depigmentation in stage III-IV melanoma patients receiving immunotherapy and its association with survival: a systematic review and meta-analysis
Teulings H.-E.1, Limpens J.1, Jansen S.M.1, Zwinderman A.H.1, Reitsma J.B.2, Spuls P.I.1, Luiten R.M.1
1Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands, 2University Medical Center Utrecht, Utrecht, Netherlands
Background: Vitiligo-like depigmentation in mela- Conclusions: Although vitiligo occurs only in a low noma patients may be associated with more favorable percentage of melanoma patients treated with immu- clinical outcome. We conducted a systematic review notherapy, our findings suggest clear survival benefit of stage III-IV melanoma patients treated with immu- in these patients. Awareness of vitiligo induction in notherapy to determine the cumulative incidence of melanoma patients is important as indicator of robust vitiligo-like depigmentation and the prognostic value anti-melanoma immunity and associated improved of vitiligo development on survival. survival. Methods: We systemically searched and selected all studies on melanoma immunotherapy which report- ed on autoimmune toxicity and/or vitiligo between 1995 and 2013. Methodological quality of each study was appraised using adapted criteria for systematic reviews in prognostic studies. Random-effect models were used to calculate summary estimates of the cumulative incidence of vitiligo-like depigmentation across studies. The prognostic value of vitiligo-like de- pigmentation on survival outcome was assessed using random-effects Cox regression survival analyses. Results: Hundred thirty seven studies were identified comprising 139 treatment arms (11 general immune stimulation, 84 vaccine, 28 antibody-based, 16 adop- tive transfer) including a total of 5737 patients. The overall cumulative incidence of vitiligo was 3.4% (95% CI: 2.5%-4.5%). In 27 studies reporting indi- vidual patient data, vitiligo development was signifi- cantly associated with both progression-free-survival, hazard ratio (HR) 0.51 (95% CI 0.32-0.82; p< 0.005) and overall survival HR 0.25 (CI 0.10-0.61;p< 0.003), indicating that these patients have 2 to 4 times less risk of disease progression/death, as compared to pa- tients without vitiligo development. 227 | IMMUNOMONITORING
Imaging in cancer immunology: 6-plex immune profiling in situ
Mansfield J.1, Wendik B.1, Hoyt C.1, Stack E.1, Feldman M.2, Bifulco C.3, Fox B.3
1PerkinElmer, Hopkinton, United States, 2University of Pennsylvania, Philadelphia, United States, 3Providence Cancer Center, Portland, United States
There has been a rapid grown in the field of tumor FOXP3 and DAPI in head and neck squamous cell immunobiology in recent years as a result of recent carcinoma. Each example will show the application successes in cancer immunotherapies, and it is be- of the multiplexed staining, per-cell quantitation and coming clear that immune cells play many some- cellular phenotyping from multispectral images of times conflicting roles in the tumor microenviron- FFPE tissue sections, as well as methods to explore ment. However, obtaining phenotypic information the spatial distributions of the phenotyped cells in about the various immune cells that play these roles and around the tumor. in and around the tumor has been a challenge. Exist- ing methods can either deliver phenotypic informa- tion on homogenous samples (e.g., flow cytometry or PCR) or morphologic information on single immu- nomarkers (standard IHC). We present here a meth- odology for delivering quantitative per-cell marker expression and phenotyping, analogous to that ob- tained from flow cytometry, but from cells imaged in situ in FFPE tissue sections. This methodology combines: the sequential multi- marker labeling of up to 8 antigens using antibod- ies all of the same species in a single section; au- tomated multispectral imaging (MSI) to remove the typically problematic FFPE tissue autofluorescence and correct cross-talk between fluorescent channels; and an automated analysis that can quantitate the per-cell marker expression, determine the cellular phenotype, count these cells separately in the tumor compartment and in the stroma and provide high- resolution images of their distributions. We present here examples of this new methodol- ogy: the simultaneous labeling, analysis and valida- tion of CD4, CD8, CD20, PD-L1, Foxp3, cytokeratin and DAPI in breast cancer; and CD8, CD34, PD-L1, 228 | IMMUNOMONITORING
10 years immune monitoring of Luminal «B» breast cancer
Melnikov D.Y.1, Pushina I.V.2, Malyshev V.2, Baykin Y.B.3, Melnikova O.V.3, Lagereva Y.G.3, Ivanova I.Y.4, Schirshova V.V.1, Korzovatih E.A.1
1Ural State Medical University, Oncology, Yekaterinburg, Russian Federation, 2Hospital ‘Zdorovie 365’, Oncology, Yekaterinburg, Russian Federation, 3Diagnostic of City Center, Laboratory Diagnostic, Yekaterinburg, Russian Federation, 4Hospital 31, Oncology, Novouralsk, Russian Federation
Recently the treatments of oncology diseases have CD19,20/0,15±0,11*109l (CI 0,07±0,22*109l). The he- taken the fourth equivalent method - the immunol- moglobin was 122,2±19,9g/l (CI 108,2±136,2g/l)- and ogy. Our knowledge about immunity and immune thrombocytosis - 190,7±31,7*109l (CI 168,4±213,0*109l). homeostasis (IH) has changes our clinical overview Indexes the Group II were high level CD4+/0,61±0,14*109l about malignant growth. But at the same time we (CI 0,46±0,75*109l, in 47,1%, n=8) p≤0,5; 35,3% Pts don’t understand changing of IH and breast cancer (n=6) had increase or decrease as CD8+/0,55±0,12*109l (BC) biology which changing disease from latent to (CI 0,48±0,63*109l) as 0,21±0,03*109l (CI 0,16±0,25*109l, aggressive forms. in 17,6% pts (n=2), p≤0,5); 29,4% (n=5) had increase The aim of our research is to reveal immune indexes CD19,20/ 0,29±0,12*109l (CI 0,16±0,41*109l) p≤0,5. The progression of Luminal B (LB) BC patients for 10 indexes of Hb - 141,7±5,6g/l (CI 137,6±144,4g/l; in years of monitoring. 35,3%, n=6) and of thrombocyte - 221,80±40,2*109l(CI We included 27 patients with LB BC T1-4dN0-2M0 195,1±248,5*10 9l; in 70,6%, n=12) also increased the stages, 32-75 years old, from the base date 2004-2014 average indicators of metastasis group p≤0,5. Changes at the department of oncology in USMU, Yekaterin- of CD16 and parameters of cytokines did not have any burg. The patients (Pts) of group I had metastasis of accurate differences. diseases (n=10, immune examine (IE) n=10; ratio Conclusion: LB of BC has the highest variability of 51,9±11,6 age, follow up 3,2±2,1 years). The Pts of IH during ten years of monitoring after of treatment. group II had no metastasis (n=17, IE n=127, medium LB BC has characteristic indexes of immunity that is 7,5±3,7, ratio 50,3±9,9 age, follow up 6,5±3,7 years). typical for metastasis diseases. Stability IH of LB BC The treatment of BC was given according to San-Gal- without metastasis has a high index of CD4+ in 47,1 len 2003 with correction of next years. Monitoring % (n=8) of cases, a high index of CD19,20 in 29,4% of IH was done annually on hemogramma indexes: (n=5) of cases and in 52,9% (n=9) - increased or hemoglobin and thrombocytosis: as such indexes IE decreased indexes of CD8+(p≤0,5). Disorganization - CD4+, CD8+, CD16, CD19,20; and indexes of cyto- of immune indexes can be seen as a risk of selective kines - TNF-α, IL-2, IFN-γ. The Pts of group I had IE metastasis or a new cancer. Compensation of IH must before 3-6 months of metastasis. Statistical analysis be the aim for immune rehabilitation and control of was performed using the chi-square test, t, p and metastasis. estimating the corresponding hazard ratio with 95% confidential interval (CI). Indexes IE of the Group I were CD4+/0,45±0,09*109l (CI 0,39-0,52*109l); CD8+/0,35±0,15*109l (CI 0,22±0,45*109l); 229 | IMMUNOMONITORING
Automated analysis of flow cytometry data to reduce inter-lab variation in detection of MHC multimer binding T cells
Pedersen N.W.1, Hoff M.1, Petersen N.1, Qian M.2, Stanton R.2, Scheuerman R.2, Halgreen C.3, Jacobsen K.3, Hadrup S.R.1
1Technical University of Denmark, Department of Immunology and Vaccinology, Frederiksberg, Denmark, 2J. Craig Venter Institute, La Jolla, United States, 3Immudex, Copenhagen, Denmark
In an effort to improve quality in assessment of sponses against Epstein-Barr virus (EBV)- and Influ- antigen responsive T cells, the organizations CIMT enza virus (FLU)-derived T-cell epitopes in the range and CIC has conducted numbers of proficiency of 0.04 - 5.33% of CD8 T-cells and additionally MHC panels to enhance quality and promote assay har- multimers with an irrelevant peptide were included monization. This has successfully improved the as a negative control. ability to uniformly identify MHC multimer binding Preliminary data shows the feasibility for detection T-cell populations following flow cytometry analysis. of MHC multimer reactive T-cell populations using Yet, the gating strategies and subjective gate-border FLOCK (n=31) and SWIFT (n=10). Especially when decisions taken by the individual labs may provide attempting to identify the large and intermediate variation to the assessment of antigen specific T-cells populations, both software tools performed quite when comparing data across laboratories, but also well and correlated with what was found by manual over time-courses in individual labs. Therefore strat- gating (r=0.92 for SWIFT, r=0.88 for FLOCK). When egies to provide automated analysis of MHC multim- it came to the low frequency populations, both soft- er binding T-cells form an ideal solution to decrease ware tools - FLOCK a bit more than SWIFT - did subjectivity and variation. The challenge of auto- however have some difficulties as there were several matic analysis of MHC multimer responsive T-cell cases where no cells were assigned to the specific populations is that these are often of low frequency populations. A disadvantage of both solutions was and low fluorescence intensity. that they both required extensive manual pre-filter- We sat out to asses, using a recent MHC multimer ing and/or gating of the data alongside with the com- proficiency panel dataset, if MHC multimer binding putational analysis. Thus to allow a fully automated T-cells could be analyzed with the computational so- approach, we did an automated pre-filtering step lution currently available and if such analyses would using a not yet publicly available algorithm. Compar- reduce the variation across different laboratories. ison of the automatically pre-filtered data with the We have at first instance used FLOCK, SWIFT and manually pre-gated data is currently ongoing. Fur- ReFlow (ungoing). The MHC multimer proficiency thermore, a central manual gating of all labs is also panel included 51 labs, from which 31 had provided being conducted in order to evaluate whether this in datasets suitable for our analyses. Each lab received itself would reduce variance between different labs. the same donor material and same MHC multimers, but used their own antibodies, staining protocols and gating strategies, which varied significantly from lab to lab. The two donors used held T-cell re- 230 | IMMUNOMONITORING
Identification of novel T cell epitopes in breast cancer
Ramskov Andersen S.1, Sick Andersen R.2, Mørch Frøsig T.1, Thor Straten P.2, Reker Hadrup S.1
1Technical University of Denmark / National Veterinary Institute, Section for Immunology and Vaccinology, Frederiksberg C, Denmark, 2Herlev University Hospital / Haematological Department, Center for Cancer Immune Therapy, Herlev, Denmark ORAL ALK SHORT T 2015
Immune therapy has established itself as the fourth fluorescence activated cell sorting and expanded in column of cancer treatment with major break- culture. Functional testing of these cultures in in- throughs in recent years, as highlighted by the ap- tracellular cytokine staining and 51Cr release assays proval of immune checkpoint blockade antibodies. demonstrated tumor-killing capacity of the T cells, Even in immunologically silent cancers as breast indicating that aromatase might be a novel T cell cancer, preliminary results have encouraged further target. However, final confirmation of the peptides studies. However little is still known as to what spe- as true T cell epitopes still needs to be done through cific targets are important in eradicating the cancer demonstration of intracellular processing and pres- cells, and identification of novel T cell targets is entation at the cell surface. therefore of paramount interest. If true T cell epitopes are found in these proteins, In this study four breast cancer associated proteins; these might be valuable targets for new immune- aromatase, prolactin, NEK3 and PIAS3 were investi- mediated treatment strategies and could potentially gated as potential novel T cell targets. In an initial improve current treatment options for breast cancer. screen of the protein sequences by use of web-based prediction databases, 415 peptides were predicted as HLA-A2 and HLA-B7 binding peptides. Subsequent experimental binding analysis in a high-throughput MHC ELISA platform confirmed sufficient binding for 147 of the 415 predicted binders. These selected peptides were evaluated as targets for T cell reactiv- ity in peripheral blood from breast cancer patients and healthy donors by use of combinatorial encod- ing of MHC multimers and multicolor flow cytom- etry. Peptide specific cytotoxic T cell responses were observed in 8 out of 18 patients, whereas responses were found in only 2 of 13 healthy donors. Thus, the selected proteins; aromatase, prolactin, Nek3 and PIAS3, are indeed targets for T cell reactivity. Two aromatase (ARO9(305) and ARO10(304)) specific T cell populations, found in peripheral blood from breast cancer patient CM P53-016, were isolated by 231 | IMMUNOMONITORING
Emigration of tumor specific regulatory T cells from the bone marrow is triggered by S1P1 and correlates with Treg accumulation in breast tumors
Rathinasamy A.1, Dettling S.2, Ge Y.1, Umansky L.1, Herold-Mende C.2, Domschke C.3, Schütz F.3, Beckhove P.1
1German Cancer Research Center, Translational Immunology, Heidelberg, Germany, 2University Hospital Heidelberg, Division of Experimental Neurosurgery, Heidelberg, Germany, 3University Hospital Heidelberg, Department of Gynecology and Obstetrics, Heidelberg, Germany
High regulatory T cell (Treg) infiltration in breast tumors is associated with reduced survival. However, the source of tumor infiltrating Treg and signals underlying their migration from lymphoid organs to the tumor tissue remain elusive. We here dem- onstrate that pronounced Treg infiltration in human breast tumors correlates with a selective reduction of tumor antigen specifc Treg from the bone marrow. Using MHC-II tumor peptide tetramers we further- more show that tumor specific bone marrow Treg selectively express Sphingosine-1-phosphate recep- tor 1 (S1P1), the receptor that mediates cell egress. S1P1 was upregulated in Treg upon TCR stimulation mediated by bone marrow resident antigen present- ing cells and triggered selective Treg but not Tcon cell migration in response to S1P gradients between bone marrow and blood which we found to be sig- nificantly increased in breast cancer patients. Taken together, our data suggest a crucial role for bone marrow antigen presenting cells in regulating the surface expression of S1P1 on tumor specific Treg in the bone marrow which may represent an important source of tumor infiltrating Treg. 232 | IMMUNOMONITORING
Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry
Santegoets S.J.1, Dijkgraaf E.M.1, Kroep J.R.1, Welters M.J.1, van der Burg S.H.1
1Leiden University Medical Center, Clinical Oncology, Leiden, Netherlands
Regulatory T cell (Treg)-mediated immunosuppres- CD3, CD4, CD25, CD127, Foxp3, Ki67 and CD45RA sion is considered a major obstacle for successful and a corresponding robust and undisputable gating cancer immunotherapy. Given their profound effect strategy for the context-dependent analysis of Treg on the outcome of immunotherapy trials, Tregs are by flow cytometry. The proposed essential marker being studied extensively in clinical trials. Unfortu- set will be used to launch proficiency panels and har- nately, no consensus has been reached about a) the monize the phenotypic analysis of Tregs among the (minimal number of) markers and b) the gating strat- CIMT Immunoguiding Program (CIP) participating egy required to define human Tregs, making it dif- laboratories. ficult to draw final conclusions. Therefore, we had or- ganized a workshop on the detection and functional testing of (antigen-specific) Tregs with 40 participants discussing different approaches of analyses and the importance of different markers and context in which Tregs were analyzed. Based on the outcome of this workshop and subsequent discussions, we generated a rationally based ranking list of “Treg markers”. In this study, the proposed Treg markers were tested to get insight in the overlap and differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. We here show that the CD3, CD4, CD25, CD127 and FoxP3 markers are the minimally required markers to define human Tregs. Furthermore, we highly rec- ommend the use of Ki67 and CD45RA as they provide additional information on their activation status. We have validated the use of this essential marker set in a series of PBMC from healthy donors and cancer patients, as well as in tumor draining lymph nodes and in tumor-infiltrating lymphocytes from freshly dispersed tumors. In conclusion, we have formulat- ed an essential marker set comprising antibodies to 233 | IMMUNOMONITORING
Identification of new immunogenic CD8+ T-cell epitopes from clinically-relevant human Adenovirus serotypes to improve diagnostic and therapeutic options in adoptive immunotherapy
Tischer S.1,2, Bunse C.1, Lahrberg J.1, Kwoczek J.1, Heim A.3, Geyeregger R.4, Blasczyk R.1,2, Maecker-Kolhoff B.2,5, Eiz-Vesper B.1,2
1Hannover Medical School, Institute for Transfusion Medicine, Hannover, Germany, 2Hannover Medical School, Integrated Research and Treatment Center (IFB-Tx), Hannover, Germany, 3Hannover Medical School, Institute for Virology, Hannover, Germany, 4St. Anna Kinderkrebsforschung e.V., Children’s Cancer Research Institute, Vienna, Austria, 5Hannover Medical School, Department of Pediatric Hematology and Oncology, Hannover, Germany
Infection with human Adenovirus (ADV) constitutes peptide epitopes derived from ADV proteins hexon a major cause of morbidity and mortality in paediat- (n=2) and penton (n=4) were identified as potential ric patients after allogeneic haematopoietic stem cell immunogenic. No T-cell responses were detectable transplantation (HSCT). T cells as most potent effec- for candidate peptides derived from ADV proteins tors of the human immune system are crucial for the fiber and E3. ADV-specific T cells against these six control and clearance of ADV. Adoptive transfer of peptides were further analysed for T-cell pheno- ADV-specific T cells offers an effective and non-toxic type, cytotoxicity and proliferation capacity. Pep-
immunotherapeutic strategy to safely and effective- t ides A*02 _HexonTLL and A*01_PentonSTD elicited the ly reduce or prevent clinical manifestation of ADV strongest IFN-γ secretion in donors’ and patients’ T + in HSCT recipients. Only few CD8 T-cell epitopes cells. 68% of donors showed A*02_HexonTLL-positive derived from the major capsid protein hexon have memory T cells, while in 66% of donors A*01_Pen-
been identified as immunodominant so far and their tonSTD-specific T cells were found. The immunoge- clinical relevance has remained largely unknown. nicity of the epitopes was confirmed in ADV-infected These facts make diagnosis and therapeutic treat- patients. Interestingly, we found higher frequencies
ment very complex. Identification of new immu- of A*01_PentonSTD-specific T cells in patients in the nogenic epitopes is of great importance to improve early stage of infection, whereas frequencies of
adoptive immunotherapy by broadening the number A*02 _HexonTLL-specific T cells increased in the late of target antigens. stages of ADV infection. In this study we applied the strategy of reverse im- In conclusion, we identified 6/27 candidate epitopes munology to map cytotoxic CD8+ T-cell epitopes as potential immunogenic antigens to induce func- from ADV proteins hexon, penton, fiber and E3 of tional active ADV-specific effector T cells. 4/6 identi- clinical-relevant serotypes 1, 2, 5 and 31 in frequent fied epitopes derived from the penton protein are the HLA alleles (A*01, 02, 03 and B*08). Established algo- first described that are presented by MHC class I and rithms (SYFPEITHI, Bimas, NetMHC) were applied to not derived from the hexon protein. Newly identi- predict candidate epitopes from the primary protein fied immunogenic ADV epitopes allow improved risk sequences. 27 epitopes were identified and the respec- assessment in HSCT recipients by accurate monitor- tive nonamer peptides were synthesized to monitor ing of ADV-specific T-cells and represent promising ADV-specific T-cell responses in in healthy donors (n candidates for further immunotherapy, making this = 64) and ADV-infected patients after HSCT (n = approach applicable to a broader patient cohort. 26). The candidate peptides were verified by IFN-γ EliSpot assay and flow cytometry, in which 6/27 234 | IMMUNOMONITORING
Antigen specific T cell immunomonitoring; HLA tetramer combinatorial coding for CD8 T cells and CD40L expression on antigen specific CD4 T cells
Turksma A.W.1, Mok J.Y.2, Nguyen M.2, Brasser G.3, Hombrink P.3, van Esch W.2, van Ham M.1, ten Brinke A.1
1Sanquin, Blood Supply Foundation, Immunopathology, Amsterdam, Netherlands, 2Sanquin Blood Supply Foundation, Reagents, Amsterdam, Netherlands, 3Sanquin Blood Supply Foundation, Hematopoiesis, Amsterdam, Netherlands
Immunomonitoring of T cell based immune re- ground staining. This technique was successfully sponses becomes increasingly important for the combined with phenotypic markers to define the development of a wide range of therapeutic applica- memory status of the antigen specific T cells. The tions covering cancer, infectious and autoimmune detection of antigen specific CD4+ T cells by CD40L diseases and vaccine design. Since their discovery detection was highly sensitive with background fluorescently labelled HLA-tetramer complexes have levels of around 0.01%. Antigen specificity could be become a cornerstone for monitoring T cell specifici- combined with cytokine production such as IFN-y ties and frequencies. The availability of HLA-tetram- and IL-2. er combinatorial coding (HTCC) technology, using These antigen specific analysis of CD4+ and CD8+ dual-colour encoded HLA-tetramer complexes, now T cells are in particular suitable for the analysis of enables the simultaneous detection of up to 28 differ- the breath of T cell responses induced by the tumor, ent antigen-specific T cell responses in a single bio- cancer therapy or immune modulating therapies. logical sample. In this way, comprehensive epitope discovery screens can be performed using patient material for which the amount of material is limited. Using class II tetramers to determine CD4+ T cell specificities is less robust than class I tetramer detec- tion. Therefore, a different approach may be useful to determine antigen specific CD4+ T cells. After antigen specific TCR stimulation CD4+ T cells up regulate CD40L. Detection of CD40L by flow cytom- etry can be combined with functional analysis such as cytokine production. Peripheral blood mononuclear cells derived from healthy donors have been used to standardize and validate the HTCC technology and CD40L assay, using antigens such as CMV, EBV and tetanus toxoid. The detection of antigen specific CD8+ T cells by dual coding (n=26) was found to be as efficient as detection with conventionally fluorescently-labeled HLA multimers (n=8) with an even lower back- 235 | IMMUNOMONITORING
Visualizing the spatial co-localization of infiltrating immune cells in solid tumors using serial tissue sections acquired with whole-slide imaging
Valous N.A.1, Halama N.2, Zörnig I.2, Klupp F.3, Ulrich A.3, Kahlert C.3, Jäger D.2
1National Center for Tumor Diseases, German Cancer Research Center, Heidelberg, Germany, 2National Center for Tumor Diseases, Heidelberg University Hospital, Heidelberg, Germany, 3Department of General, Visceral and Transplantation Surgery, Heidelberg University Hospital, Heidelberg, Germany
Characterization of infiltrating immune cells in solid using standard immunohistochemistry methods, the tumors has moved into scientific and clinical focus distribution and visualization of multiple biomarkers for prognostic and therapeutic stratification. Spatial can be realized in a single virtual section. Moreo- profiling using immunohistochemistry is the current ver, the method is transferrable to other biomarker state-of-the-art. However, simultaneous evaluation of studies aiming to detect co-localization of multiple surface markers and cytokine productions of cells is proteins. difficult. Multiplex-staining technologies are limited by the availability of primary antibodies from dif- ferent species; directly labelled antibodies often still require an exclusive tissue preparation procedure. To address these problems and open-up more oppor- tunities in characterizing the immunological tumor microenvironment, a multistage image processing pipeline in thin sequential tissue sections - stained for specific biomarkers and then computationally rendered into a single image - was developed. The au- tomated workflow is based on deformable image reg- istration exploiting the Markov random field formu- lation and discrete optimization algorithms, removal of histological noise through quaternionic operations and 2D histogram variance thresholding, colour de- convolution for stain separation, color/brightness/ saturation geometric transformations in a Clifford algebra framework, and post-processing operations. Here, we present an example of spatial co-localiza- tion visualization in colorectal liver metastases for CD8, CCL5, and CD4 staining (plus hemalaun coun- terstaining) using clinical formalin-fixed paraffin- embedded serial tissue sections (thickness ~ 5 µm) acquired with a whole-slide imaging system. This in silico approach has merits and demonstrates that 236 | IMMUNOMONITORING
Clinical immunomonitoring strategies defining biomarkers of anti-CD73 mAbs safety and efficacy. The TumAdoR collaborative project
Vigano S.1,2, Jandus C.2, Harari A.1, Gourdin N.3, Menetrier-Caux C.3, Caux C.3, Romero P.2
1Center of Experimental Therapeutics, University of Lausanne, Oncology, Lausanne, Switzerland, 2Ludwig Center for Cancer Research/ University of Lausanne, Lausanne, Switzerland, 3Center Leon Bérard, Lyon, France
The natural progression of tumors is usually asso- get effects of neutralizing CD73 Abs. In particular, ciated with, and even sustained by, tumor-driven we developed and validated fluorescently labeled immune-modulating mechanisms. Among these antibody panels to evaluate the differentiation and the over-expression of the membrane-bound nu- polarization of T cells, B cells, NK cells, DCs, mono- cleotidase CD73 by various cell types in the tumor cytes and their activation, exhaustion, cytotoxic and microenvironment (ecto-5’-nucleotidase) increases cytokine profiles by flow cytometry. Immuno-moni- the concentration of extracellular adenosine (Ado), toring is crucial in the evaluation of immunotherapy which suppresses both innate and adaptive immune interventions to identify relevant predictive biomark- responses and through multiple activities contrib- ers of efficacy and safety. utes to tumor progression. The conversion of ATP into Ado is tightly regulated by cell membrane ec- toenzymes. However, while the conversion of ATP into AMP, predominantly catalyzed by CD39, is re- versible, the CD73 mediated catalysis of AMP into Ado is virtually irreversible, thus representing a crucial checkpoint in conversion of pro-inflamma- tory ATP into immunosuppressive Ado. Recent suc- cesses of anti-CTLA-4, anti-PD-1 and PD-L1 mAbs open the door to a highly promising novel class of therapeutics targeting tumor-driven immunosup- pressive pathways. In this context the TumAdoR is a collaborative EU-supported consortium that aims at bringing blocking anti-CD73 mAb candidates to a first in-human clinical trial. Such antibodies would be applicable to a wide range of cancers. Among the tasks shared by the partners, specific diagnostic tools are in development to assess target expression and to monitor relevant immune parameters in patients before and during the treatment. Antibody panels for immuno-monitoring have been developed to assess by multicolor flow cytometry on-target and off-tar- 237 | IMMUNOMONITORING
The frequency of circulating Vδ1-positive but not Vδ2-positive γδ T-cells correlates negatively with survival in late-stage melanoma and may be increased by ipilimumab treatment
Wistuba-Hamprecht K.1, Martens A.1, Weide B.1, Pawelec G.1
1Universitaetsklinikum Tuebingen, Tuebingen, Germany
Recently, interest has resurged in the possibility of increase of peripheral Vδ1+ T-cells (p=0.0391, Wil- using γδ-T cells in cancer immunotherapy, because coxon) but not of Vδ2+ T-cells (p=0.3750, Wilcoxon). these cells can kill melanoma cells in vitro, and IL-2 We are currently analyzing data from a larger pro- and zoledronate treatment can expand some human spective study of ipilimumab treatment with over 100 γδ cells in vitro and in animal models in vivo. The patients, to confirm or refute these results. presence of the main subsets of γδ-T cells in late- stage melanoma patients and relationship with sur- vival has not been extensively explored, although relatively lower percentages of Vδ2-positive cells has been reported in melanoma. Here, we present pre- liminary data on associations between the major γδ-T cell subsets and survival in melanoma patients, and whether baseline data are informative for predict- ing responses to ipilimumab treatment. We used a carefully validated set of monoclonal antibodies to assess γδ-phenotypes, because we found that certain commercial reagents were not behaving as specified (Wistuba-Hamprecht et al. OMIP-020: phenotypic characterization of human γδ T-cells by multicolor flow cytometry. Cytometry A. 2014; 85:522-4). Com- paring a total of 39 stage IV melanoma patients with 27 controls, we found no overall differences in Vδ1 or Vδ2 T-cell frequencies, or in proportions of naïve (CD27, CD28-double positive)-vs-late-differentiated (CD27,CD28-double negative) γδ cells. However, Ka- plan-Meier plots stratifying individual patients into groups with Vδ1 or Vδ2 frequencies above or below the median showed that the presence of Vδ1 but not Vδ2 cells negatively correlated with longer survival (p=0.033 log-rank; p=0.939 log-rank). In a subgroup of 9 patients, ipilimumab treatment resulted in an 238 | IMMUNOMONITORING
Correlates of checkpoint inhibitor expression and T cell function in urological tumors
Zelba H.1, Hennenlotter J.2, Zettl M.3, Bedke J.2, Stenzl A.2, Rammensee H.-G.1, Gouttefangeas C.1
1University of Tübingen, Department of Immunology, Tübingen, Germany, 2University of Tübingen, Department of Urology, Tübingen, Germany, 3Boehringer Ingelheim, Vienna, Austria
Purpose: Three immune checkpoint inhibitors target- Results: While the percentage of iR+ T cells was low ing CTLA4 and PD1 have been recently approved for in PBMCs (except BTLA), both CD4+ and CD8+ TILs the treatment of metastatic melanoma. CTLA-4 and showed high expression levels of iR, with LAG3 and PD-1 are expressed by activated CD4+ and CD8+ T PD1 being most often expressed. However, iR expres- cells and transduce an inhibitory signal when bound sion and co-expression was highly heterogeneous to their respective ligands, thereby downregulating T among patients. cell functions. In some cancer types, these receptors Within CD3+ T cells, we observed that the percentage are upregulated on TILs and the increased receptor-li- of LAG3+, PD1+ and CTLA4+ cells (both CD4+ and gand interactions might contribute to inhibition of an- CD8+ T cells) is increased in PBMCs from cancer pa- ti-cancer immune responses. The blockade of CTLA4 tients (CP) compared to that of HD. When CP PBMCs and/or PD1 leads to a prolonged survival and signifi- were compared with autologous TILs, we saw in- cant tumor shrinkage in a fraction of patients with ad- creased frequency of LAG3+, PD1+ as well as TIM3+ vanced melanoma. Further checkpoint receptors that T cells in TILs compared to PBMCs. BTLA+ T cells could be targeted by immunotherapy approaches are were decreased, while the percentage of CTLA4+ T intensively examined at the moment. Here, we inves- cells did not differ between PBMCs and TILs. Inter- tigated the expression pattern of 5 different inhibitory estingly, only a minor fraction of CD4+ and CD8+ receptors (iR) on PBMCs and autologous TILs obtained T cells in both PBMCs and TILs co-expressed more from patients with renal cell carcinoma or prostate than one iR. cancer. After in vitro stimulation using several stimuli, TILs Experimental Design: TILs were isolated by a com- showed decreased functionality compared to autolo- bined mechanical and enzymatic dissociation from gous PBMCs. At the same time, we observed that CP fresh primary tumor tissues. Autologous PBMCs were PBMCs (both CD4+ and CD8+ T cells) showed an isolated from whole blood by density gradient centrif- increased frequency of cytokine-producing cells (IL2, ugation. PBMCs from healthy blood bank donors (HD) TNF and IFNg) compared to HD PBMCs. were used as control samples. Cells were stained for Conclusion: We observed that all five investigated iRs multiparametric flow cytometry in order to determine are highly expressed on CD4+ and CD8+ TILs. The the co-expression of iRs on various cell populations, heterogeneity of iR expression and co-expression of as well as the percentage and expression levels of iR receptors suggests that a personalized therapy might on various T cell subsets. In addition, functional at- be the most promising approach using checkpoint tributes of both PBMCs and TILs were assessed by in- blockade. tracellular cytokine staining after in vitro stimulation. 239 – 292
Tumor Biology and Interaction with the Immune System 239 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
The role of Foxp3+ regulatory T cells in a murine model of spontaneously developing B-cell lymphoma
Ahmetlić F.1, Riedel T.1, Sparwasser T.2, Röcken M.3, Mocikat R.1
1Helmholtz-Zentrum München, Institut für Molekulare Immunologie, Munich, Germany, 2TWINCORE GmbH, Institut für Infektionsimmunologie, Hannover, Germany, 3Eberhard-Karls-Universität, Universitäts-Hautklinik, Tübingen, Germany
As transplantable tumor models do not reflect the in vitro cocultures in which Treg showed strong pro- clinical situation, we investigated the interactions liferation (Ki-67) with the c-MYC lymphoma-derived between tumor cells and the immune system with cell line 291 suggest a specific recognition of lym- a c-MYC transgenic mouse model of endogenously phoma antigens. arising B-cell lymphoma. In many tumors, a perma- Elevated amounts of the cytokine IL-10 were found in nent immune activation leads to T-cell suppression the tumor microenvironment upon in vitro stimula- because of T cell exhaustion or induction of regula- tion pointing to an immunosuppressive tumor micro- tory T cells (Treg). Therefore, the function of tumor- environment. This was confirmed by the presence of reactive T lymphocytes and the mechanisms of their coinhibitory receptors on intratumoral T cells, like exhaustion as well as the induction of Treg are to be CTLA-4, which was only increased on Foxp3+ Treg examined. whereas Foxp3- Teff and CD8+ CTL showed very low An augmented fraction of CD4+ Foxp3+ Treg was CTLA-4 levels. found in spleens and lymph nodes of tumor-bearing Furthermore, in vitro suppression assays showed c-MYC mice. Both the transcription factor Helios and that Treg from c-MYC as well as wt mice potently the transmembrane protein Neuropilin-1 (Nrp-1) are and dose-dependently suppress the proliferation of markers for thymus-derived natural Treg (nTreg). CD4+ Teff. Considering the highly increased amount These Nrp-1+ Helios+ nTreg were predominantly of Treg cells in c-MYC lymphomas, we suggest that found in c-MYC lymphomas whereas Nrp1- Helios- the suppression of CD4+ Teff cells in tumor-bearing iTreg that develop by conversion of naïve CD4+ mice is stronger. Foxp3- T cells in the periphery, were present in low The depletion of Foxp3+ GFP+ Tregs in DEREG/c- numbers. MYC mice by i.p. injection of diphtheria toxin im- In comparison to wildtype (wt) mice, T-cell subpop- proved the survival slightly. Therefore, Treg deple- ulations (CD4+ Teff, CD8+ CTL and Treg) in the tion alone might not be sufficient for controlling tumor had an activated phenotype as evidenced by tumor development. upregulation of CD69 and downregulation of CD62L. In conclusion, the Treg population, together with In parts, they showed increased IFN-g production, the exhausted T cells in tumour-bearing mice reflect proliferation in vivo and were able to degranulate an immunosuppressive tumour microenvironment, (CD8+ CTL) in vitro. which favours tumour growth. The high expression of the costimulatory molecule CD137 by Treg compared to CD4+ Teff and CD8+ CTL indicates a TCR-specific activation. Additionally, 240 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
ID8-Fluc: a syngeneic mouse model for ovarian cancer
Baert T.1,2, Verschuere T.3, Van Hoylandt A.2, Gijsbers R.4, Van Gool S.5, Vergote I.1,2, Coosemans A.1,2
1UZ Leuven, Department of Gynaecology and Obstetrics, Leuven, Belgium, 2KU Leuven, Department of Oncology, Laboratory of Gynaecologic Oncology, Leuven, Belgium, 3KU Leuven, Department of Neuroscience, Laboratory of Experimental Neurosurgery, Leuven, Belgium, 4KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Laboratory of Molecular Virology and Gene Therapy, Leuven, Belgium, 5KU Leuven, Department of Microbiology and Immunology, Laboratory of Pediatric Immunology, Leuven, Belgium
Introduction: Mouse models are widely used in the three weeks after tumor challenge. At the beginning study of anticancer treatments. However, the clinical of the fourth week, a rapid increase in tumor load relevance of these models is crucial. For epithelial could be observed (up to 4,11 x 109 p/s). Five mice ovarian cancer the development of the ID8/MOSEC were sacrificed at day 58 to confirm BLI scanning cell-line by Katherine Roby meant a breakthrough. results with microscopic tumor examination and five The peritoneal spread of the disease correlates with mice were kept to determine overall survival. One FIGO III-IV ovarian cancer and the histology corre- mouse became cachectic with intestinal obstruction, lates to a high-grade serous ovarian cancer. Our goal one mouse developed signs of anemia and dyspnea, is to evaluate the clinical relevance of the model and and three mice showed progressive ascites and were possibilities for reliable monitoring of tumor growth. sacrificed at 32g Materials and methods: Six to eight weeks old At sacrification of all mice, macroscopic peritoneal female C57BL/6J-Tyrc-2J/J (Charles River Laborato- metastatic load was present in all and confirmed at ries, MO, USA) mice were inoculated (n=10) intra- microscopic examination. peritoneal with 10 x 106 ID8-Fluc cells. ID8 cells were Conclusion: The ID8-Fluc mouse model reflects the stably transfected with the Firefly Luciferase gene different clinical presentations of metastatic high- using lentiviral transfection. Mice were weighed grade serous ovarian cancer in humans, ranging minimally 3 times a week. Tumor growth was evalu- from bowel-obstruction with cachexia to the devel- ated weekly, using bioluminescence imaging (BLI). opment of massive ascites. Tumor growth can reli- For this procedure mice were anesthetized with 2% ably be followed through BLI scanning. This model Isoflurane (ISO-VET, Piramal Healthcare, UK) and opens new perspectives in the development of new injected intraperitoneal with 126 mg/kg Luciferine therapies in ovarian cancer. (Promega, WI, USA). Mice were sacrificed according to European directives on animal welfare. Tumor bi- opsies were stained for hematoxylin and eosin. Results: Weight curves in all mice showed the devel- opment of progressive ascites starting from day 55 after tumor inoculation. BLI scanning showed tumor establishment in all mice one week after tumor in- oculation with an average signal of 3,74 x 108 p/s. Tumor load remained relatively stable over the first three weeks, with a mean BLI signal of 5,73 x 108 p/s 241 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Molecular characterization of the Breast Cancer Associated Antigen NY-BR-1
Bitzer J.1, Kosaloglu Z.2, Halama N.1, Ziegelmeier C.1, Lerchl T.2, Spille A.2, Huang Z.3, Zapatka M.3, Lichter P.3, Pudenz M.4, Köllensperger E.5, Eichmüller S.6, Schneeweiss A.7, Marmé F.7, Zörnig I.1, Jäger D.1,2
1National Center for Tumor Diseases (NCT) and Heidelberg University Hospital, Department of Medical Oncology, Heidelberg, Germany, 2National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Clinical Cooperation Unit ‘Applied Tumor Immunity’, Heidelberg, Germany, 3German Cancer Research Center (DKFZ), Division of Molecular Genetics, Heidelberg, Germany, 4German Cancer Research Center (DKFZ), Division Epigenomics and Cancer Risk Factors, Heidelberg, Germany, 5Heidelberg University Hospital-Ethianum, Clinic for Plastic and Reconstructive Surgery, Aesthetic and Preventive Medicine, Heidelberg, Germany, 6German Cancer Research Center (DKFZ), Department of Translational Immunology, Heidelberg, Germany, 7National Center for Tumor Diseases (NCT) and Heidelberg University Hospital, Department of Gynecology and Obstetrics, Heidelberg, Germany
Breast cancer is one of the most common malignan- transcriptional regulation of NY-BR-1. For in silico cies with increasing incidence and it is a leading analyses, we used data from publicly available re- cause of death among women. Although early stage sources such as dbSNP, ICGC, TCGA, ENCODE, and breast cancer can be effectively treated, there are NCBI SRA. Additionally, we analyzed the methylation limited numbers of treatment options available for status of various fragments in the promoter region patients with advanced and metastatic disease. Thus, of NY-BR-1 using the EpiTYPER - MassARRAY tech- there is a clear need to define new targets for the de- nology. Here, we present preliminary results of our velopment of novel therapeutic strategies.The breast analysis and evaluate their implication for further cancer associated antigen NY-BR-1 was identified by investigation of NY-BR-1. a serological screening strategy (SEREX). NY-BR-1 is expressed in the majority (>70%) of breast tumors as well as metastases, in normal breast tissue, in testis, and occasionally in prostate tissue. NY-BR-1 was con- firmed as an immunogenic antigen as we could detect NY-BR-1 specific spontaneous humoral and cellular immune responses in several breast cancer patients. Due to its tissue restricted expression pattern and immunogenicity, NY-BR-1 represents a promising target antigen for immunotherapeutic interventions. However, the biological function, regulatory mecha- nisms, and interaction partners of NY-BR-1 are still unknown.We combined integrative functional anal- yses involving genomics, transcriptomics and epig- enomics as well as wet lab techniques to explore the 242 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
HLA expression body map
Boegel S.1,2, Löwer M.2, Bukur T.1,2, Sorn P.2, Scholtalbers J.2, Bukur V.2, Castle J.C.2, Sahin U.1,2
1University Medical Center of the Johannes Gutenberg-University Mainz, AG Sahin, Mainz, Germany, 2TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany
The Human Leukocyte Antigen (HLA) molecules as baseline when examining HLA downregulation display peptide antigens that are derived from in- in tumors or choosing targets for cancer immuno- tracellular (HLA Class I) and extracellular (Class II) therapy. In addition, we examine the comparability proteins on the surface of nucleated cells. They play of RNA-Seq experiments from different projects. a central role in the adaptive immunity by presenting self, abnormal self (neo-epitopes arising from muta- tions) and foreign (pathogenic) peptides to T lympho- cytes, which subsequently are capable of eliminating those cells. There is increasing evidence that diseased cells down-regulate or even loose HLA expression to escape T cell mediated killing. Due to their exten- sive genetic heterogeneity, high-throughput profiling of HLA expression is not possible with standard NGS RNA-Seq processing algorithms. Using our recently developed algorithm seq2HLA, we re-analyze over 3000 public RNA-Seq NGS datasets (Genotype Tissue Expression project, Human Protein Atlas, Blueprint, NCBI Sequence Read Archive) comprising more than 30 normal human tissues and cell types (including a variety of immune cells) to create a HLA class I (clas- sical and non classical) and II normal tissue expres- sion map. We find high HLA expression in spleen and bone marrow and low expression in brain, muscle and testis. Interestingly, we find high HLA class I and high HLA class II expression in lung and colon (compared to spleen). In addition, we do not find massive imbalance in locus specific HLA Class 1 ex- pression between the different tissues. This is - to our knowledge- the first HLA expression body map providing a reference catalog of HLA expression in normal human tissues and cells, which can be used 243 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
The critical role of immune microenvironment in Hepatocellular carcinoma- the potential of TLR3 ligand as a novel immunotherapy
Chew V.1,2, Chen J.M.2, Bard-Chapeau E.3, Oh H.C.4, Heikenwalder M.5, Ng I.6, Abastado J.-P.2
1Singhealth Translational Immunology and Inflammation Centre, Singapore, Singapore, 2Singapore Immunology Network (SIgN), A*STAR, Singapore, Singapore, 3Institute of Molecular and Cell Biology (IMCB), Singapore, Singapore, 4National Cancer Centre, Singapore, Singapore, 5Institute of Virology, Technical University München/Helmholtz Zentrum München, Munich, Germany, 6The University of Hong Kong, Department of Pathology, Hong Kong, Hong Kong
Hepatocellular carcinoma (HCC) is an aggressive intratumoral chemokines expression, T- and NK-cells cancer that is linked to chronically dysregulated activation and tumor infiltration as well as enhanced liver inflammation. However, appropriate immune apoptosis and reduced proliferation of the tumor pa- responses can control HCC progression. renchyma cells. We previously identified an 14 immune-gene signa- Hence, tumor immune microenvironment is critical ture that predicts patient survival in a total of 155 for HCC progression and TLR3 is shown to be an HCC patients from Singapore, Hong Kong and Zurich important modulator of tumor progression and a po- irrespective of patient ethnicity and disease aetiol- tential target for immunotherapy. ogy. Importantly, it predicts the survival of patients with early disease (stages I and II), for whom classi- cal clinical parameters provide limited information. This signature includes the chemokine genes CXCL10 and CCL5, markers of T helper 1 (Th1), CD8+ T and natural killer (NK) cells, inflammatory cytokines (tumour necrosis factor a, interferon g) and Toll-like receptor-3 (TL3). We further investigated the underlying mechanism TLR3 in HCC. TLR3 expression was also associated with longer survival in HCC patients (HR = 2.1, P = 0.002, n=172). TLR3 expression by both tumor parenchyma and tumor-infiltrating NK cells asso- ciates with patient survival. In-vitro, TLR3 activa- tion increased cell death and enhanced chemokines (CXCL10 and CCL5) expression in TLR3+ SNU182 HCC cell line as well as promoted NK cell activation and its cytotoxicity against HCC cells. In vivo, using both a transplanted and spontaneous mouse models of liver tumor, treatment with TLR3 ligand increased 244 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome
Classen C.F.1
1University Childrens Hospital Rostock, Pediatric Oncology Hematology, Rostock, Germany
Macrophage Activation Syndrome (MAS) is a well- that even in cases appearing as reactive MAS, some known disorder, characterized by hyperinflamma- variants of genes regulating cytotoxic cell function tion and multiorgan infiltration by activated mac- were found. In conclusion, MAS/HLH is a group of rophages, which is a rare complication of juvenile disorders in which both pathogenetic concept and rheumatoid arthritis and other autoimmune disor- therapeutic approach will have to be redefined, based ders. Hemophagocytic Lymphohistiocytosis (HLH) on a new understanding of numerous genetic and ac- is a similar hyperinflammation, developing both in quired factors that require a new and individualized certain genetically defined disorders like perforin algorithm of diagnostic and therapeutic procedures. deficiency, or secondarily in malignant diseases or immunodeficiency, triggered e.g. by infections. In fact, both disorders appear to be just two variants within the same spectrum of disease; characterized to different degrees by cytopenias, hepatospleno- megaly and lymph node enlargement, hyperferri- tinemia, hypertriglyceridemia, hypofibrinogenemia, increase of soluble IL-2 receptor, hemophagocytosis, and impaired cellular cytotoxicity. A similar pattern of reaction may even be triggered by infections like leishmaniosis or metabolic diseases like Wolman’s disease. While in HLH, a protocol of dexametha- sone, cyclosporine A and etoposide, combined with intrathecal methotrexate induces remission in most cases, enabling bone marrow transplantation in cases of congenital disorder, and leading to a main- tenance therapy that may than be tapered, in cases of acquired disease, or, alternatively, antithymocyte globulin, in MAS traditionally steroids were given alone with addition of cyclosporine A in cases of in- sufficient response. Since meanwhile, the two syn- dromes seem to be very much overlapping, numer- ous genetic analyses have been performed, showing 245 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Immunomodulation in patients with ovarian cancer: a retrospective analysis
Coosemans A.1,2, Decoene J.2, Baert T.1,2, Laenen A.3, Kasran A.4, Verschuere T.5, Seys S.4, Van Gool S.W.4, Vergote I.1,6
1KU Leuven, Department of Oncology, Leuven, Belgium, 2UZ Leuven, Gynaecology and Obstetrics, Leuven, Belgium, 3KU Leuven, Biostatistics and Statistical Bioinformatics Centre of Leuven, Leuven, Belgium, 4KU Leuven, Department of Microbiology and Immunology, Leuven, Belgium, 5KU Leuven, Department of Neuroscience, Leuven, Belgium, 6UZ Leuven, Gynaecologic Oncology, Leuven, Belgium
Study goal: In general, it has become clear that the growth factor) for Treg, M2 and MDSC. Moreover, we immune system controls the development of cancer. determined the presence of Galectin-1, involved in However, if the immune system fails, malignant cells angiogenesis and tumor-mediated immune evasion. start proliferation and the tumor becomes clinically Metabolites were measured using Enzyme-Linked apparent. Regulatory T cells (Treg), myeloid-derived Immuno Sorbent Assay (ELISA) or Cytometric Bead suppressor cells (MDSC) and tumor-associated mac- Array (CBA). rophages (TAM) are crucial in the regulation of this Results: The mean age was 61,9 years. 90% had immune escape. Since this process has barely been serous ovarian carcinoma (65,25% FIGO stage III; studied in ovarian cancer, we explored the changes 33,75% stage IV). We found significant lower levels in the immune system at diagnosis, after administra- of IL-10, VEGF, TGF-β and arginase and higher levels tion of chemotherapy and after (interval) debulking of galectin-1 after three cycles of paclitaxel-carbopla- surgery. tin compared to diagnosis. After debulking surgery, Material and methods: Retrospectively, we collected a decrease in IL-10 reached significance. In a multi- 135 serum samples from 80 ovarian cancer patients, variate analysis, including the FIGO stage, galectin-1 available from a historical tumor bank. Samples and CCL2 appeared independent prognostic factors, were gathered at diagnosis (n=50), after primary reducing the progression-free and overall survival. debulking surgery (n=15), after chemotherapy (pa- Conclusions: This study was able to demonstrate clitaxel-carboplatin) (n=40) and interval debulking changes in the immune system of ovarian cancer pa- surgery (n=19). Of 40 patients, two or more consecu- tients due to chemotherapy and surgery. We defined tive samples were available. Furthermore, ten serum two new prognostic markers. These results can be samples from an age-matched control group were useful to determine the best treatment at the most collected. To estimate the presence of Treg, MDSC suitable moment, in order to further modulate the and TAM (more specifically M2) in serum, we ana- immune system in a positive way. lyzed the presence of metabolites that influence their behavior: IL-4 (interleukin), IL-13 and arginase spe- cifically for M2, IL-10 and VEGF (vascular endothe- lial growth factor) for MDSC and M2, CCL2 (chemo- kine (C-C) motif ligand 2) and TGF-β (transforming 246 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Establishment of a transplantable, NY-BR-1 expressing breast cancer model in HLA-transgenic mice
Das K.1, Gardyan A.1, Vormehr M.2, Mueller-Decker K.3, Zoernig I.4, Jaeger D.4, Osen W.1, Eichmueller S.B.1
1DKFZ, Translational Immunology, Heidelberg, Germany, 2Johannes Gutenberg University Medical Center, Mainz, Germany, 3DKFZ, Core Facility Tumor Models, Heidelberg, Germany, 4University Hospital Heidelberg, Department of Medical Oncology, NCT, Heidelberg, Germany
Breast cancer is one of the leading causes of cancer gate the effect of adoptive co-transfer of the afore- related deaths in women worldwide and current mentioned CTL line and HLA-DR3- and HLA-DR4- standard therapies show limited efficacy. However, restricted NY-BR-1-specifc CD4+ T cell lines into immunotherapeutic approaches like adoptive T cell tumor bearing HLA-transgenic mice on the tumor transfer might represent an attractive option for the growth, survival and the composition of the tumor treatment of breast carcinoma. The differentiation infiltrating immune cells are currently ongoing. The antigen NY-BR-1 appears as a suitable target for T cell results presented here demonstrate the first NY-BR-1 immunotherapy against breast cancer as it is over- expressing mouse tumor model, allowing the investi- expressed in 60% of breast carcinomas compared to gation of NY-BR-1-specific immune responsesin vivo. healthy breast. The aim of this project is to establish a NY-BR-1-expressing, transplantable tumor model in HLA transgenic mice that would allow to investigate the functional role of NY-BR-1-specific HLA-restrict- ed CD4+ T cells in vivo with respect to their capacity to sustain cytotoxic T lymphocytes (CTL)-mediated tumor attack. The effect of interaction of NY-BR- 1-specific CD4+ T cells and tumor-associated mac- rophages (TAMs) on the polarization of TAMs will be investigated as well. Stable transfectant clones of the C57BL/6 derived lymphoma cell line EL4 and of the mammary adenocarcinoma cell line EO771 express- ing NY-BR-1 were established which gave rise to sub- cutaneous tumors in HLA-DR4 transgenic mice. Flow cytometric analysis revealed that CD11b+F4/80+ macrophages constituted a significant proportion of the tumor infiltrating immune cells. An H2-Db-re- stricted, NY-BR-1-specific CTL epitope was identified and immunization of C57BL/6 mice with recombi- nant adenovirus encoding NY-BR-1 resulted in the generation of NY-BR-1 specific, H2-Db-restricted CTL line reactive to the same epitope. Studies to investi- 247 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
RIG-I-like helicase-mediated reprogramming of myeloid-derived suppressor cells as novel therapeutic strategy for pancreatic cancer
Duewell P.1, Kirchleitner S.V.1, Endres S.1, Schnurr M.1
1Klinikum der Universität München, Department of Medicine, Munich, Germany
ORAL ALK SHORT T 2015
Background: Treatment of patients with advanced profiling of MDSC revealed that tumor MDSC express pancreatic cancer is mostly limited to chemothera- an immunosuppressive M2/G2 phenotype with high py or irradiation. The tumor is extremely apoptosis levels of TGF-β, arginase, VEGF-α, STAT3, Nos2 and resistant and creates a potent immunosuppressive NOX2. Treatment with RLH ligands resulted in a phe- network with dense infiltration of regulatory T cells notype switch towards M1/G1, with elevated levels of and cells of the myeloid lineage, such as macrophages TNF-α, CCL5 and IFN-β. and myeloid-derived suppressor cells (MDSC). MDSC Conclusion: RLH ligands induce an immunogenic are versatile undifferentiated cells of monocytic form of tumor cell death and prolong survival in a (M-MDSC) and granulocytic (G-MDSC) origin that murine model of pancreatic cancer. This correlates exhibit multiple immunosuppressive mechanisms with a reduced number and less suppressive function and promote tumor growth. We could recently iden- of MDSC as well as enhanced effector T cell function tify the RIG-I-like helicases (RLH) to induce an im- in tumors. RLH ligands could be exploited to induce munogenic form of tumor cell death and bypass im- a phenotype switch of MDSC towards M1/G1 and to munosuppressive mechanisms. The synthetic RLH unleash the potential of tumor-resident CTL for effec- ligand polyinosinic:polycytidylic acid (poly(I:C)) tive immune control of pancreatic cancer. induces a type I IFN-driven immune response with the release of danger signals (calreticulin, CXCL10, HMGB1) and upregulation of MHC-I as well as CD95 (Fas) on tumor cells. This involved DC activation with enhanced antigen cross-presentation to CD8+ T cells, linking innate with adaptive immunity. Results: Survival of poly(I:C)-PEI treated mice with orthotopic pancreatic tumors was significantly pro- longed. Analysis of tumor tissue show an increased infiltration with activated CD8+ T cells expressing CD69 and IFN-γ, as well as markers associated with lytic function, such as FasL and perforin. This was paralleled by a reduced number of G-MDSC in tumors. Furthermore, tumor-resident MDSC of treated mice lost their suppressive function on CD8+ T cell prolif- eration in vitro, compared to PBS-treated mice. RNA 248 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Generation of murine b2-microglobulin deficient tumor cell lines using the CRISPR/Cas9 system
Nicolaus C.1, Das K.1, Goyal A.2, Diederichs S.2, Dickes E.1, Osen W.1, Eichmueller S.B.1
1DKFZ, Translational Immunology, Heidelberg, Germany, 2DKFZ, Division of RNA Biology and Cancer, Heidelberg, Germany
Stable surface expression of the MHC class I complex class I negative murine cell lines represent a valuable crucially depends on the association of the MHC class tool that could be used as controls when analyzing
I heavy chain with β2 microglobulin (b2m). In order class I restricted T cell responses or investigating NK to generate MHC class I negative murine cell lines, cell activity. The CRISPR/Cas9 system has proven to we have used the CRISPR/Cas9 system to knock out be a straight forward and very effective technique for
the b2m gene in the melanoma cell line B16F10 and in producing MHC knock out cell lines. a transfectant clone derived from the murine breast cancer cell line EO771 stably expressing NY-BR-1, called EO-NY (see poster K. Das et al.). Expression of
a b2m-specific gRNA-Cas9 complex in these cell lines resulted in the outgrowth of completely MHC class I negative cells within 25 days post transfection. Upon FACS sorting and limiting dilution of the transfect- ed bulk culture individual knockout clones could be obtained. Validation by FACS confirmed absent
surface expression of b2m and the MHC I molecules H2-Db and H2-Kb. Importantly, both cell lines lost their susceptibility for recognition by cognate CTL lines, even when pulsed with high peptide concen-
trations. Sequencing results of the b2m gene of one knockout cell line showed insertions and deletions of variable lengths at the genomic target site, all but one resulting in a frameshift and thereby destroying the reading frame. One six-nucleotide deletion did not induce a frameshift but destroyed the start codon,
thereby causing the knockout of the b2m gene. The B16F10 knock out lines contained four different ab- errations, which is in line with the polyploid karyo- type of this tumor cell line. In the EO-NY knock out line we found three different aberrations arguing for three copies of chromosome #2 in this cell line. MHC 249 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Hypoxia-induced reversible dedifferentiation attenuates RIG-I-mediated immunity against melanoma
Engel C.1, Hagen C.1, Horváth D.1, Drijfhout J.W.2, Wenzel D.3, Tüting T.4, Hartmann G.1, van den Boorn J.G.1
1University Hospital Bonn, Institute for clinical chemistry and clinical pharmacology, Bonn, Germany, 2Leiden University Medical Center, Department of Immunohematology and Bloodtransfusion, Leiden, Netherlands, 3University of Bonn, Institute of Physiology I, Bonn, Germany, 4University Hospital Bonn, Department for Dermatology and Allergy, Bonn, Germany ORAL ALK SHORT T 2015
Classic cancer therapies like chemotherapy and irra- resulted in an augmented intratumoral CXCL10 pro- diation are often hampered by solid-tumor hypoxia. duction, enhanced anti-tumor efficacy and prolonged Whether intratumoral immune-activation, in particu- survival. lar by intracellular innate immune-receptors like reti- Taken together, our results establish the impairing noic acid-inducible gene-I (RIG-I), is likewise impaired effects of hypoxia-induced tumor cell dedifferentia- remains unknown. We here establish RIG-I function tion on intratumoral RIG-I functionality in malignant to be disadvantageously modulated in hypoxic murine cells, and highlight measures to counteract these sup- melanoma cells, attributable to oxidative stress-in- pressive effects. duced cellular dedifferentiation and spontaneous NFκB activation. In a panel of melanoma cells 5’-triphosphate RNA (3pR- NA)-induced RIG-I protein-upregulation and CXCL10
(IP10)-secretion was blunted by hypoxia (pO2=2%), an effect absent in non-malignant control cells. Ac- cordingly, in vitro co-cultures of 3pRNA-transfected hypoxic melanoma cells with Pmel-1 splenocytes re- vealed suppressed CD8+ T cell activation in response to hypoxic melanoma cells, indicated by a reduced induction of CD69 and perforin expression. Likewise, NK cell responsiveness was reduced. Hypoxia medi- ated a time-dependent increase in cellular oxidative stress and induced NFκB activation. Melanoma cells responded by cellular dedifferentiation and loss of gp100 differentiation-antigen expression. ROS-scav- enging or NFκB inhibition, could rescue RIG-I-respon- siveness and differentiation antigen-expression under hypoxia. In vivo ROS-scavenging by systemic vitamin C during intratumoral 3pRNA-based immunotherapy 250 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Activation of neuroimmune pathways decreases tumor-infiltrating myeloid-derived suppressor cells and increases therapeutic effects of radiotherapy on metastatic breast carcinoma
Erin N.1, Korcum A.1, Tanrıöver G.1, Kale Ş.1, Demir N.1, Köksoy S.1
1Akdeniz University, Antalya, Turkey
ORAL ALK SHORT T 2015
Recent studies document the importance of neuro- ing mice. These original findings demonstrate that nal dysfunction in cancer development and metas- SP through neuroimmune modulation can prevent tasis. We reported previously that both depletion of formation of immune suppression in the tumor mi- neuropeptides in capsaicin-sensitive sensory nerve croenvironment, enhance cytotoxic immunity in the endings and vagotomy increases metastasis of triple presence of RT and prevent metastatic growth. negative breast carcinoma. Of the sensory neuro- peptides, Substance P (SP) is distributed widely for regulation of immune functions. We therefore exam- ined the affects of continuous exposure to low doses of SP on brain metastatic cells of the mouse breast carcinoma (4TBM) in the presence of radiotherapy (RT) thought to increase antigenicity of cancer cells. 4TBM cells have a cancer stem cell phenotype and induce extensive visceral metastasis after orthotopic inoculation into the mammary pad. Results demon- strated that SP treatment decreases the number of tumor-infiltrating myeloid-derived suppressor cells as well as the TNF-a response to LPS challenge. SP also increased CD4+CD25b cells in draining lymph nodes of tumor-bearing animals and IFN-γ secretion from leukocyte culture prepared from lymph nodes and spleens of tumor-bearing animals. SP also pre- vented tumor-induced degeneration of sensory nerve endings and altered release of angiogenic factors from cancer-associated fibroblasts (CAF) and tumor ex- plants. In accordance with these observed immuno- logical effects, combination treatment of continuous SP with a single dose of RT induced complete tumor regression and significantly reduced or prevented metastasis in 50% of the animals while suppressing primary tumor growth and metastasis in the remain- 251 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Intratumoral and systemic immunity as prognostic biomarkers in patients with breast cancer*
Fortis S.P.1, Sotiriadou N.N.2, Sofopoulos M.2, Haritos C.1, Arnogiannaki N.2, Anastasopoulos N.1, Pawelec G.3, Papamichail M.1, Perez S.A.1, Baxevanis C.N.1
1Cancer Immunology and Immunotherapy Center,Saint Savas Cancer Hospital, Athens, Greece, 2Saint Savas Cancer Hospital, Pathology Department, Athens, Greece, 3Center for Medical Research, Eberhard-Karls-Universität, Tübingen, Germany
It is already established that the immune system plays affin-embedded tissues. The density (i.e. the number a crucial role in cancer elimination, dormancy and of positive cells per mm2 tissue surface area) and the progression. Correlating immune parameters, either relative ratios among the different subpopulations, in in circulation or in the tumor microenvironment, with the different locations, were analyzed. As indicators established prognostic clinicopathological variables of peripheral immunity several cytokines/chemokines and disease outcome is currently at the forefront of bio- (i.e. ΤGF-β, IL-1β, ΙL-1Ra, IL-2, IL-4, IL-5, IL-8, IL-9, marker research. IL-10, IL-12, IL-15, IL-17, IFN-γ, ΤNF-α, GM-CSF, CCL5) Treatment of breast cancer (BC) is becoming increas- were determined in the serum of BC patients with ingly individualized and is guided by many clinicopath- LUMINEX platform technology. From the circulating ological variables, such as tumour staging, histological cytokines/chemokines statistically significant differ- type and grade, hormonal receptors status and HER-2/ ences between BC patients (n=49) and normal indi- neu gene amplification. Retrospective analyses eval- viduals (n=12) were found for IL-9 (increased in BC, uating the presence and density of intratumoral and p=0.0004), IL-1Ra and CCL5 (decreased in BC patients, stromal tumor-infiltrating lymphocytes (TILs) in BC p=0.0283 and p=0.0059, respectively). IL-1Ra was sig- have revealed a correlation with overall survival (OS) nificantly lower in HER-2/neu+ patients (p=0.0202). in different subtypes of the disease. However, there are IL-9 and IL-10 were found to be increased in advanced no studies correlating the prognostic impact of TIL lo- disease stage (stage 3 vs 1,2 p=0.0064 and p=0.0227, cation within distinct tumor regions (e.g. tumor center respectively) while the CD4/CD8 ratio in the tumor and invasive margin), a parameter that gained major center was higher in early stage disease (stages 1,2 vs importance for the establishment of the immunoscore 3: p=0.0404). Interestingly, there was a direct correla- (IS) in colon cancer. tion between IL-9 and IL-10 levels (p=0.0015) and an In the present study, we sought to correlate density and inverse correlation between IL-9 and IL-10 with the localization of TILs and levels of circulating cytokines/ CD4/CD8 ratio (p=0.0177 and p=0.0527, respectively). chemokines with established prognostic clinicopatho- Further analyses are ongoing and will be reported at logical parameters (stage, HER-2/neu amplification and the meeting. hormone receptor expression status), in BC patients with invasive ductal carcinoma without neoadjuvant therapy. Serum from patients was obtained one day before surgery and compared with normal donors. * This study is sponsored by a grant from the bilateral Re- search and Technology Greek-German collaboration The analysis of immune cell infiltrates (CD4+, CD8+, FoxP3+, and CD163+) was performed by IHC in par- 252 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
CPT1 dependent CD4 T cell death in mice with non-alcoholic steatohepatitis promotes hepatocarcinogenesis
Ma C.1, Felsher D.2, Greten T.1
1National Cancer Institute, National Institutes of Health, Bethesda, United States, 2Stanford University School of Medicine, Stanford, United States
Non-alcoholic steatohepatitis (NASH), a common that N-acetyl cysteine reversed the NASH-induced condition in obese patients, is an important risk hepatic CD4 T cell decrease and delayed NASH-pro- factor for HCC. However, only little information moted tumor development. Our results suggest the exists about the immunological mechanisms of how critical role of CD4 T cell in the disease progression NASH promotes hepatocarcinogenesis. Methionine of NASH to HCC, and provide a new link between choline deficient (MCD) diet is widely used to induce lipid metabolism dysfunction with impaired anti-tu- NASH. In this study we fed Lap-tTA-myc mice, which mor surveillance. develop HCC, MCD diet. MCD diet accelerated tumor development in MYC on mice. A robust decrease of hepatic CD4 but not CD8 T cells was observed in NASH mice, which was exacerbated by MYC trans- gene expression. Choline-deficient and amino ac- id-defined (CDAA) diet and high fat (HF) diet treat- ment also induced a liver specific CD4 T cell decrease confirming it is a general phenomena in NASH. As expected higher level of cell death was detected in hepatic CD4 T cells after NASH. Phenotypic analysis demonstrated that these CD4 T cells had a CD44high- CD62Llow effector memory phenotype and produced more IFNγ. Selective CD4 T cell depletion further ac- celerated HCC development suggesting an important role of these cells for tumor development. Next, we studied the cause of CD4 T cells. In co-culture exper- iments we demonstrated that lipid-laden hepatocytes from mice on MCD diet induced selective CD4 but not CD8 T cells death through a contact-indepen- dent mechanism. FFAs were found from hepatocyte culture medium to be responsible for T cell cytotox- icity, and linoleic acid recapitulates the selective tox- icity to CD4 T cells. Blocking ROS abolished linoleic acid-induced CD4 T cell death. In vivo study showed 253 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Activity of tumor-infiltrating lymphocytes can be potentiated by relieving post-transcriptional regulation
Guislain A.1, Salerno F.1, Nicolet B.1, Young H.A.2, Wolkers M.C.1
1Sanquin Blood Supply Foundation, Amsterdam, Netherlands, 2National Cancer Institute, National Institutes of Health, Frederick, United States
Cytotoxic T cells are crucial for tumor immune surveil- lance. However, tumor-infiltrating lymphocytes (TILs) are often rendered inert by the tumor environment and fail to produce effector molecules required to kill tumor cells. Despite this failure in cytokine production, TILs express substantial amounts of cytokine mRNA. This finding implies that the production of effector mole- cules is regulated by post-transcriptional events. Here, we investigated whether adoptive T cell therapy could be improved by interfering with post-transcrip- tional regulation of cytokines. We treated B16 melano- ma-bearing mice with T cells containing a germ-line deletion of AU-rich regulatory elements located in the 3’Untranslated Region (ARE-Del). ARE-Del T cells con- tinuously produced IFN-γ within the tumor environ- ment, in contrast to WT T cells that had lost their capac- ity to produce this cytokine. Like WT T cells, ARE-DEL T cells expressed PD-1 and LAG-3, and the production of TNF-α and IL-2 remained undetectable. The compo- sition of immune infiltrates also remained unchanged. Importantly, the increased potential of ARE-Del T cells to produce IFN-γ resulted in a significant delay of tumor growth and a prolonged survival rate when compared to mice that had received WT T cell therapy. This im- proved anti-tumoral response was at least in part a direct effect of IFN-γ on the tumor cells. In conclusion, our data demonstrate that post-transcriptional modula- tors prevent the production of IFN-γ in TILs, and that the efficacy of TIL therapy can be improved by relieving this translational block. 254 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Abscopal effect seen with T-cell therapy enhancing oncolytic adenoviruses armed with TNFa and IL-2
Havunen R.1, Parviainen S.1,2, Siurala M.1,2, Behr M.1, Nettelbeck D.3, Ehrhardt A.4, Hemminki A.1,2
1University of Helsinki, Haartman institute, Medicum, Helsinki, Finland, 2TILT Biotherapeutics Ltd, Helsinki, Finland, 3German Cancer Research Center (DKFZ), Heidelberg, Germany, 4University Witten/Herdecke, Institute for Virology and Microbiology, Witten, Germany
Adenovirus itself can induce immune responses into only one of the two tumors. Tumor size, activa- against tumors, but the antitumor effect increases tion of immune cells and the viral transduction of when the viruses are armed with immune system non-injected tumors were studied. Our data shows stimulating factors such as cytokines. We found that the oncolytic and cytokine production capability of the most promising factors to stimulate the graft Ad5/3-E2F-D24-hTNFa-IRES-hIL2 adenoviruses. In used in adoptive T-cell therapy are interleukin (IL) mice we saw the abscopal effect: also the non-inject- -2 and Tumor Necrosis Factor alpha (TNFa). IL-2 is ed tumors became smaller when viruses were armed a commonly used cytokine in treating malignant with TNFa and IL2. This has important implications melanoma and renal cell carcinoma. Like IL-2, TNFa for human trials, as injection of one tumor achieves activates immune cells, but it is also known for its an- systemic improvement in efficacy of T-cell therapy. ti-tumor properties. These cytokines can, however, lead to severe side effects when administered sys- temically. Previously, we and others have achieved long lasting, high level cytokine expression locally but low level systemically when using armed onco- lytic viruses in vivo. Therefore, we developed replica- tive oncolytic adenoviruses expressing one or both above mentioned human cytokines. In addition, we have non-replicative adenoviruses with murine IL-2 and TNFa which facilitate immunological studies in mice. Several cell lines were infected with variable amount of viral particles to test the functionality of the replicative viruses. The proportion of surviving cells was measured with MTS-assay. The secretion of cytokines was tested with FACS based bead array and their biological activity with indicator cell lines. In mice, we studied if the murine cytokine armed viruses could bring about the abscopal effect. We im- planted two B16-OVA melanoma tumors to the flanks of C57BL/6 mice and treated them with an adoptive T-cell transfer systemically and with virus injections 255 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Do immune features of the tumour have prognostic value in metastatic melanoma?
Janssen N.1, Shipp C.1, Weide B.2, Pawelec G.1
1Universitaetsklinikum Tuebingen, Center for Medical Research, Tuebingen, Germany, 2Universitaetsklinikum Tuebingen, Department of Dermatology, Tuebingen, Germany
In cancer patients, interactions between the immune features of the tumour microenvironment such as the system and the tumour can result in cancer control, local presence of cytokines is not known. Here, we or have a tumour-stimulating effect. Recent evi- have measured levels of myeloid cells and cytokines dence suggests that the immunological characteris- in the tumour mass in order to build a more detailed tics within the tumour are a major prognostic factor, picture of how immunological features of the tumour largely based on studies in primary excised colorectal are related to clinical outcome of the patient. Pre- cancers, where the “immune contexture” had greater liminary results in metastatic melanoma deposits prognostic power than the standard staging system showed that IL-6 and GM-CSF were expressed in the for predicting post-operative survival. Accordingly, a tumour of every patient examined (n=64 and 47, re- new scoring system called the “immune score”, was spectively). Despite this, a large degree of variation established which emphasises the prognostic effect in the expression of these cytokines across patients of T cells in the tumour, thereby underlining the was observed. Analysis of the tumour-infiltrating importance of interactions between immune system cells showed the frequency of myeloid cells to range and the tumour in determining outcome. Studies considerably across patients (n = 75). In particular, supporting this idea have shown a correlation the levels of CD14+CD16+ and CD14+CD16- mono- between high relative levels of tumour-infiltrating cytic cells were different across patients. In addition lymphocytes (TILs) and improved clinical outcome to cellular and soluble components of the tumour mi- in several cancer types. In contrast, pro-tumour croenvironment, we plan to measure levels of senes- effects can be mediated through the suppression of cent and proliferating T-cells, transcription factors, anti-tumour immunity or stimulation of cancer cells fibroblasts and MHC molecules in order to paint a by secreted products. In particular, so-called mye- more detailed picture of the tumour microenviron- loid-derived suppressor cells (MDSCs) of the innate ment. Ongoing evaluation of these results will reveal immune system appear to play a negative role in the degree by which these features relate to patient anti-tumour immunity. This notion is supported by outcome. Our results will shed light on the question several studies suggesting that lower relative levels of of whether the “immune contexture” is predictive of MDSCs in blood are associated with a more favorable survival in metastatic and not only primary cancer, clinical outcome. Whether this association between and whether it is informative in melanoma as well MDSC level and patient prognosis is also seen for as colon cancer. myeloid cells occurring in the tumour mass has not yet been evaluated, with the exception of tumour-in- filtrating macrophages. Similarly, the impact of other 256 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Local administration of CD40 agonist dynamically modulates the tumor-infiltrating immune cell compartment
Kaunisto A.1, Veitonmäki N.1, Ellmark P.1,2
1Alligator Bioscience AB, Lund, Sweden, 2Lund University, Department of Immunotechnology, Lund, Sweden
T cells, dendritic cells and macrophages are known to mediate the anti-tumor effects of CD40 agonists. However, the effects of many myeloid cell populations on the CD40-related anti-tumor response remain in- completely understood. We have analyzed in vivo re- sponses of tumor-infiltrating myeloid cells and lym- phocytes by flow cytometry and elucidated how the murine anti-CD40 agonistic antibody FGK45 affects myeloid cell populations in a syngeneic murine sub- cutaneous cancer model. In addition, we have inves- tigated local anti-CD40 immunotherapy either alone or together with other therapeutic modalities. Combining the CD40 agonist with chemotherapeu- tic agents and checkpoint inhibitors slows down tumor progression and prolongs survival. Moreo- ver, our results suggest that CD40 agonist treatment causes dynamic changes in both tumor-infiltrating myeloid cells and T cells. These results may explain, in part, why combination therapies may be required for optimal anti-tumor responses in certain tumor models. 257 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Tumor cell-secreted IL-1 mediates CCL22 induction in human PBMC
Knott M.1, Wiedemann G.1, Rapp M.1, Endres S.1, Anz D.1
1LMU Munich, Clinical Pharmacology, Munich, Germany
Intratumoral immunosuppression is considered one of the striking mechanisms leading to the immune escape of cancer cells. Tumor-induced immunosuppression can be achieved by an altered expression level of im- munostimulatory or -inhibitory surface molecules such as MHC-I or PD-L1 on the tumor cells, by the secretion of enzymes or cytokines contributing to an immunosup- pressive microenvironment or the attraction of immuno- suppressive cells such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). One mechanism by which Treg are attracted to the tumor is intratumoral production of the chemokine CCL22. Here we show, that not only intratumoral but also systemic CCL22 levels are elevated in tumor-bearing mice and that CCL22 secretion takes place in immune cells and not in the cancer cells themselves. This is in line with our finding, that both murine and humane immune cells secrete CCL22 in vitro when stimulated with tumor-conditioned medium (TCM). The tumor cell- mediated induction of CCL22 in human peripheral blood mononuclear cells (PBMC) is abrogated when anakinra, a recombinant IL-1R1 antagonist, is added to the TCM. As further analysis revealed, IL-1a is expressed by IMIM PC1 pancreatic cancer cells and is able to induce CCL22 in PBMC when applied in its recombinant form. To conclude, we identified tumor cell-derived IL-1 as strong inducer of the Treg-attracting chemokine CCL22 in human PBMC. Blocking the IL-1 pathway could repre- sent a promising strategy for the reduction of Treg infil- tration-mediated immunosuppression in cancer therapy. 258 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Characterization of murine GL261 glioma models for oncolytic vaccinia virus therapy
Kober C.1, Rohn S.1, Weibel S.1, Chen N.G.2, Szalay A.A.1,2,3
1University of Wuerzburg, Department of Biochemistry, Wuerzburg, Germany, 2Genelux Corporation, San Diego, United States, 3Rebecca & John Moores Comprehensive Cancer Center, Department of Radiation Medicine and Applied Sciences, San Diego, United States
Glioblastoma multiforme (GBM) is one of the most were analyzed in detail in cell cultures for VACV malignant forms of brain cancer (WHO IV) and infection in polarization and co culture experi- in spite of extensive research still a fatal disease. ments. Oncolytic virotherapy is a novel treatment option We found that GL261 glioma cells implanted sub- for GBM studied in preclinical and clinical trials. cutaneously or orthotopically into Balb/c athymic, Vaccinia virus (VACV) is one promising candidate C57BL/6 athymic or into C57BL/6 wt mice formed in this field. Oncolytic VACVs were proven as safe individual tumor microenvironments that support- therapeutics in patients with different types of ed oncolytic virotherapy with different outcomes. cancer in clinical trials but the excellent preclini- Surprisingly, only Balb/c athymic mice with sub- cal research findings still need to be demonstrated cutaneous tumors exhibited intratumoral viral in human patients. To develop a successful onco- replication. All other models were non-respond- lytic virotherapy for GBM a more defined under- ers to oncolytic virotherapy with LIVP 1.1.1. We standing of the individual tumor biology and the identified endogenous IFN-γ expression levels, interaction of VACV with the tumor microenviron- that upregulate MHCII expression on GL261 cells ment is required. in C57BL/6 wt mice leading to the non-permissive Comparing tumors at different locations and mice status in these tumor cells. In addition, we demon- with different immunologic and genetic back- strated that the intratumoral tumors were infiltrat- grounds we set out to analyze the replication effi- ed by microglia and astrocytes, independent of cacy and oncolytic potential of the VACV LIVP 1.1.1 virus infection, as a consequence of tumor growth. in murine GL261 glioma models. LIVP 1.1.1 is an In cell culture experiments, using BV-2 microglia attenuated wild type isolate of the Lister strain. We and IMA2.1 astrocytes we showed, that both cell expected to identify potential microenvironmental types clear viral particles efficiently by uptake and differences, parameters or factors which may be inclusion of VACV. responsible for treatment success or failure. Taken together, testing of cancer patients for IFN-γ Virus replication analyses were performed in both, levels in tumor biopsies prior to oncolytic VACV cell culture experiments and in tumor tissues. The therapy may become a useful prognostic marker tumor microenvironments of subcutaneous GL261 for successful therapeutic outcome. gliomas were analyzed by RBM ELISA; Flow cy- tometry and immunohistochemistry. Orthotopic GL261 gliomas were analyzed by immunohisto- chemistry. BV-2 microglia and IMA2.1 astrocytes 259 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
CD103-positive T-cells; an immunotherapeutic target for ovarian and endometrial cancer
Komdeur F.L.1, Workel H.H.1, Wouters M.C.1, Klip H.G.1, Plat A.1, Wisman G.B.1, Duiker E.W.2, Hollema H.2, de Bruyn M.1, Nijman H.W.1
1University of Groningen, University Medical Center Groningen, Obstetrics and Gynecology, Groningen, Netherlands, 2University of Groningen, University Medical Center Groningen, Pathology, Groningen, Netherlands
Introduction: A recent report has demonstrated that tion of CD103-positive T-cells was strongly associ- the presence of CD103-positive T-cells correlates with ated with disease specific survival in high risk EC a favorable prognosis in patients with high grade (p=0.017), and HGSC in patients with less than 1 serous ovarian cancer (HGSC). CD103 is a tissue resi- centimeter macroscopic residual tumor after cytore- dent T-cell marker and was proposed to define the in- ductive surgery (p=0.011). traepithelial population of cytolytic T-cells. As such, Conclusions: CD103 is a marker for intraepithelial CD103-positive T-cells could be a relevant target for T-cells in EC and HGSC. We found a strong associa- adoptive cell transfer-based immunotherapy across tion with favorable prognosis in both high-risk EC epithelial malignancies, including endometrial and HGSC. Therefore CD103 represents a potential cancer (EC). Here, we confirmed these recent find- target population for use in adoptive transfer therapy ings and extended them to EC. in these epithelial malignancies. Future studies to Methods: Cohorts of endometrioid EC- and HGSC- extend these findings to additional epithelial malig- patients were analysed by immunohistochemistry nancies are warrented. for the presence of CD103-positive tumor-infiltrating lymphocytes (TIL) using tissue microarrays. The prognostic significance of CD103 TIL was evaluated by Kaplan-Meier analysis. Immunofluorescence was performed to determine the T-cell localization on full slides. Tumor digests, derived from surgically removed tissue, were analyzed for further charac- terization of the TIL using flow cytometry. Results: We found CD103 to be preferentially ex- pressed on intraepithelial CD8-positive T-cells in EC and HGSC. By contrast, CD103+/CD8+ TIL were not present within healthy endometrial tissue. CD103-positive cells represented ~20% (median) of the total TIL fraction in both EC and HGSC tumor digests. CD103-positive TIL were predominantly of the CD45RO+/CCR7- effector memory phenotype (60%) and to a lesser extent of the CD45RO+/CCR7+ central memory phenotype (40%). Tumor infiltra- 260 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Immune homeostasis of Kidney Cancer
Korzovatyh E.1, Shirshova V.1, Melnikov D.1
1Ural State Medical University, Department of Oncology and Medical Radiology, Ekaterinburg, Russian Federation
The Kidney Cancer (KC) is a disease with poor 1,12) and for group III - 0,29±0,14х10^9 l (CI -0,11- prognosis. The role of immunotherapy in treatment 0,48). The Cr TNF-α group I was 85,9±48,4 (CI 30,3- KC is an established fact. Use cytokines can not 141,7), Cr IL-2 - 53,5±41,1 (CI 6,5-100,6), Cr IFN-γ help because the local tumor growth and systemic - 96,8±119,2 (CI - 40,2-233,8). The Cr TNF-α group II immune responses are not identical. The aim of this was 40,7±22,4 (CI -27,7-109,2), Cr IL-2 69,8±82,4 (CI work is to evaluate the immune homeostasis (IH) and -180,8-320,4) Cr IFN-γ 181,0±199,4 (CI 425,6-787,7). different clinical manifestation of KC. The Cr TNF-α group III was 109,6±119,2 (CI -55,9- 13 patients of the department of oncology of the 275,1), Cr IL-2 - 10177±20148 (CI -17793-38147,9), Cr USMU have been included from 2010 to 2014, the IFN-γ - 8662,7±10067,3 (CI -5312,9-22638,5). mean age is 56,7±6,9 years old. All the patients (pts) We concluded that IH and clinical forms of KC were were divided into three groups: the group I - local different. The group I had normal Cr of inflammation diseases 5 pts (T1N0M0-T2N1MO), the group II - for IL-2 and IFN-γ. The group III had high level of leu- region forms 4 pts (T3N0M0-T2N2M0) and the group cocytes and granulocytes with the highest activity III - metastatic diseases 4 pts (T-anyN-anyM1). The Cr of IL-2 and IFN-γ. The group II had a diverse im- elements of the study were based on the indicators munological parameters that requires high-quality of blood count: the level of leukocytes, lymphocytes, diagnostics of latent metastasis and individual type granulocytes; the indicators of immunograme were: of cytokine therapy. CD3, CD4, CD8, CD16, CD19,20; and inflammatory cytokines. The range of reaction was index «Cr» (Cr=nCD3+stimulation/nCD3+wild where n=count CD3+ that can synthesize cytokines TNF-α, IL-2, IFN-γ in the tests after stimulation and in wild) with 95% confidence interval (CI), index t, p. The group I had indices of leucocytes 5,8±1,7х10^9 l (CI 3,8-7,8), group II - 7,0±2,3х10^9 l (CI 3,8-10,2) and group III - 8,6±0,9 х10^9 l (CI 7,2-9,9). The indices of granulocytes for group I were 3,4±1,5 х10^9 l (CI 1,7- 5,1), for group II - 3,7±1,4 х10^9 l (CI 1,1-6,2; р ≥ .05) and for group III - 6,1±1,03х10^9 l (CI 4,6-7,5; р .02). The indices of CD16+ for group I were 0,32±0,09х10^9 l (CI 0,18-0,44), for group II - 0,5±0,33х10^9 l (CI 0,01- 261 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
An immunogenomic stratification of colorectal cancer: implications for development of targeted immunotherapy
Lal N.1, Beggs A.1, Willcox B.1, Middleton G.1
1University of Birmingham, School of Cancer Sciences, Birmingham, United Kingdom
Although tumour infiltrating lymphocyte (TIL) trast, RAS mutant tumours predict for a relatively density is prognostic and predictive in colorectal poor immune infiltration and low inhibitory mole- cancer, the impact of tumour genetics upon colorectal cule expression. In this setting checkpoint blockade immunobiology is unclear. Identification of genetic may be less efficacious, highlighting a requirement factors that influence the tumour immunophenotype for novel strategies in this patient group. is essential to improve the effectiveness of stratified immunotherapy approaches. We carried out a bio- informatic analysis of colorectal cancer data in The Cancer Genome Atlas (TCGA) involving two-dimen- sional hierarchical clustering to define an immune signature that we used to characterise the immune response across key patient groups. An immune sig- nature termed The Co-ordinate Immune Response Cluster (CIRC) comprising 28 genes was co-ordi- nately regulated across the patient population. Four patient groups were delineated on the basis of cluster expression. Group A, which was heavily enriched for patients with microsatellite instability (MSI-H) and POL mutations, exhibited high CIRC expression, in- cluding the presence of several inhibitory molecules: CTLA4, PDL1, PDL2, LAG3 and TIM3. In contrast, RAS mutation was enriched in patient groups with lower CIRC expression. This work links the genet- ics and immunobiology of colorectal tumourigenesis, with implications for the development of stratified immunotherapeutic approaches. Microsatellite in- stability and POL mutations are linked with high mutational burden and high immune infiltration, but the coordinate expression of inhibitory pathways observed suggests combination checkpoint blockade therapy may be required to improve efficacy. In con- 262 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
The effects of glyco-shortenings Anti-CD52 on PBMC and their anti-inflammatory functions
Yu C.-C.1, Chen C.-Y.1, Lee M.-S.1, Chan M.-C.1, Lee C.-C.1
1Development Center for Biotechnology, Institute of Biologics, New Taipei City, Taiwan, Republic of China
Anti-CD52 (Alemtuzumab) is a drug used in the treat- CD52 decreased the inflammatory cytokines, Inter- ment of chronic lymphocytic leukemia (CLL), cuta- feron gamma and TNF-alpha, which were induced neous T-cell lymphoma (CTCL) and T-cell lymphoma by anti-CD52-treated PBMCs. The anti-inflammatory under the trade names Campath, MabCampath and functions of both glyco-shortening Anti-CD52 were Campath-1H, and in the treatment of multiple sclero- also supported by the increase of regulatory T cell sis as Lemtrada. It is also used in some conditioning (CD4+CD25+, Treg) population and the induction regimens for bone marrow transplantation, kidney of Foxp3 expression in CD4+T cells than Anti-CD52 transplantation and islet cell transplantation. One when co-treated with anti-CD3 on CD4+ T cells. Our of the mechanism of Campath is to induce ADCC, results suggest the glyco-shortening Anti-CD52 may CDC or apoptosis of CD52-expressed T cells, however have the anti-inflammatory functions and may be many investigations also suggest that Campath may used for inflammatory diseases therapy. lead the immune system to induce the regulator T cells (Tregs) to suppress the function of effective T cells. Since many studies shown that the composition of glycan may influence the therapeutic efficacy of antibody drugs, we investigated the effects of Anti- CD52 with glycan-shortenings on PBMC cells. We use PNGase and EndoS to treat Anti-CD52, and most glycan of the Anti-CD52 will be cleaved to an aglycan (AG) and a Anti-CD52-GlcNAc-Fucose (GF), respec- tively. After protein A purification, the composition and the purity of these glyco-shortening Anti-CD52 were identified and confirmed by SDS-PAGE , AAL- Blot and LC/MS/MS analysis. Our investigations showed that either anti-CD52-AG or anti-CD52-GF did decrease the ADCC activity on Jurkat cells than theirs parental Anti-CD52 antibody. Both of these glyco- shortening Anti-CD52 also less-deprived the CD52+ T cells populations and decreased the PBMC apopto- sis level than the full glycan of anti-CD52. However, we found both of these two glyco-shortening Anti- 263 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Production of anti-cancer Bi-functional nanobody in bacterial host
Majidzadeh-A K.1,2, Farahmand L.1, Esmaeili R.1, Salehi M.1, Sanati H.3
1Breast Cancer Genetics Department, Breast Cancer Research Center, ACECR, Tehran, Iran, Islamic Republic of, 2Tasnim Biotechnology Research Center (TBRC), School of Medicine, AJA University of Medical Sciences, Tehran, Iran, Islamic Republic of, 3Cancer Alternative and Complementary Medicine Department, Breast Cancer Research Center, ACECR, Tehran, Iran, Islamic Republic of
Introduction: Cancer therapy based on antibodies ately. Evaluation of protein concentration after puri- and their different classes were caused to appear out- fication indicated high level expression of nanobody standing clinical results in improvement of patients in bacterial host. Besides, producing secretory forms suffering. Nanobodies are the variable single domain was caused to gain active nanobody with no extra of camel antibodies. These smallest antigen binding amino acids. anti MUC1×CD3 nanobody have anti- fragments have unique advantages compared to the proliferative effect on breast cancer cells and have conventional therapeutic antibodies. MUC1 is over- proliferative effect on T-cells. expressed in more than 90% of breast cancers and Discussion: As compared to the other methods of correlates with metastasis and recurrence. Bi-specif- producing antibody which are more expensive and ic Nanobodies, not only bind to antigen of cancer cell time-consuming, bi-specific nanobody against MUC1 but also can stimulate cytotoxic T cells. In addition, & CD3 may be more functional and effective in some it will have a synergistic effect on tumor cell eradica- host with economic cost. According to the individual tion and deals with tumor escape. characteristics of nanobodies, after passing more de- Materials and methods: In order to expression of velopmental steps, it can be used in diagnosis and nanobody in bacterial host, a gene cassette was targeted therapy of cancer patients. designed that include the nanobody sequences of anti-CD3 and anti-MUC1.Two nanobodies were con- juncted by a IgG2 Lama hinge linker of together. The aim of additional signal sequence at the beginning of construct was producing secreted proteins and histidine sequence due to purification was added at the end. After cloning, construct was transform into the bacterial host. Nanobody expression was done in bacteria and it was purified by chromatography. Nanobody function carried out by specific cell line. Results: Bi-specific anti MUC1×CD3 nanobody was designed successfully. All stages of cloning and ex- pression of nanobody in E.coli were done appropri- 264 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Immunostimulatory AdCD40L gene therapy combined with low-dose cyclophosphamide in metastatic malignant melanoma patients
Maleka A.1,2, Lodskog A.1, Mangsbo S.1, Svensson E.1, Lundberg C.3, Nillson A.3, Krause J.3, Agnarsdóttir M.1,4, Ahlström H.3,5, Tötterman T.H.1, Ullenhag G.1,2
1Uppsala University, Department of Immunology, Genetics and Pathology (IGP), Uppsala, Sweden, 2Uppsala University Hospital, Department of Oncology, Uppsala, Sweden, 3Akademiska University Hospital, Division of Radiology, Uppsala, Sweden, 4Akademiska University Hospital, Department of Pathology and Cytology, Uppsala, Sweden, 5Akademiska University Hospital, Department of Surgery, Uppsala, Sweden
Current approaches for treating metastatic malignant Antibodies against adenovirus were measured by the melanoma (MM) are not effective enough and are as- Adenovirus IgG ELISA. Biopsy lysates were generated sociated with serious adverse events. Due to its immu- from frozen tumor biopsies. Peripheral blood mononu- nogenicity, MM is an attractive target for mmunostimu- clear cells were analyzed by flow cytometry. latory gene therapy. AdCD40L treatment was safe with mild transient reac- AdCD40L is a replication-deficient adenovirus carrying tions such as fever, increased liver enzymes and pain the gene for CD40 ligand (CD40L). In order to evaluate at injection site. No OR were recorded in MRI-scans, the feasibility and effectiveness of AdCD40L-treatment however, local and distant responses were seen in PET- and if cyclophosphamide can improve the treatment ef- scans. In group A, two patients experienced reduced ficacy, an open-label phase I/IIa trial with intra-tumor tumor activity in the majority of lesions one patient had AdCD40L injections +/- low dose cyclophosphamide similar activity as before treatment in both post-treat- conditioning was conducted. The patients were moni- ment evaluations. In group B, two patients experienced tored for toxicity, immune modulation and tumor re- reduced tumor activity in all lesions and two patients sponses. All enrolled patients had failed standard treat- reduced tumor activity in the injected metastasis as ments. well as in the majority of lesions. Two patients had the The trial was divided in two parts. In the first part, same activity as before treatment in both post-treatment (group A), six patients received four weekly AdCD40L- evaluations. The median survival in A was 18 weeks intratumoral injections only. In part B, nine patients re- and in B 34 weeks. At six months one patient was still ceived low dose (300 mg/m2) cyclophosphamide condi- alive in A while seven of nine in B. The 6-months sur- tioning prior to the first and fourth AdCD40L injection. vival was significantly different (p=0.0475). However, Only six patients were treated with AdCD40L alone the overall survival (OS) was not significantly different since none of them experienced an objective response (p=0.236). Patients with the best survival developed (OR). Blood was drawn at multiple time points for the highest levels of activated T cells and experienced chemistry, hematology and immunology evaluations. a pronounced decrease of intratumoral IL8. Pretreatment PET/CT and whole-body MRI scans were In this phase I/IIa study of MM, AdCD40L therapy was performed and repeated two and six weeks after the given for the first time to metastatic cancer patients and last AdCD40L-injection. Tumor responses were meas- was well tolerated. The results of local and distant re- ured by assessing RECIST criteria and tumor metabolic sponses along with the better survival in the low dose activity using the SUVmax value. cyclophosphamide group are encouraging. 265 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
N-terminally truncated Interleukin-38 is released from apoptotic tumor cells to limit inflammatory macrophage responses
Mora J.1,2, Schlemmer A.1, Wittig I.1,3, Jung M.1, Frank A.C.1, Weigert A.1, Bruene B.1
1Goethe University Frankfurt am Main, Institute of Biochemistry I, Faculty of Medicine, Frankfurt am Main, Germany, 2University of Costa Rica, Faculty of Micriobiology, San Jose, Costa Rica, 3Goethe University Frankfurt am Main, Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Frankfurt am Main, Germany
Different modes of cell death regulate immunity. Whereas necrotic (necroptotic, pyroptotic) cell death triggers inflammation, apoptosis contributes to its resolution and induces a tolerogenic response. Inter- leukin-1 (IL-1) family cytokines are key players in this interaction. A number of IL-1 family cytokines are produced by necrotic cells to induce sterile inflam- mation. However, release of IL-1 family proteins from apoptotic cells to regulate inflammation was not de- scribed. Here we show that IL-38, a poorly charac- terized IL-1 family cytokine, is produced by human apoptotic tumor cells to limit inflammation. Depletion of IL-38 in apoptotic tumor cells provoked enhanced IL-6 and IL-8 levels and AP1 activation in co-cultured human primary macrophages, subsequently induc- ing Th17 cell expansion at the expense of IL-10 pro- ducing T cells. IL-38 was N-terminally processed in apoptotic cells to generate a mature cytokine with distinct properties. Both full-length and truncated IL-38 bound to X-linked interleukin-1 receptor acces- sory protein-like 1 (IL1RAPL1). However, whereas the IL-38 precursor induced an increase in IL-6 produc- tion by human macrophages, truncated IL-38 reduced IL-6 production by attenuating the JNK/AP1 pathway downstream of IL1RAPL1. In conclusion, we identi- fied a mechanism of apoptotic cell-dependent immune regulation requiring IL-38 processing and secretion, which might be relevant in resolution of inflamma- tion, autoimmunity and cancer, where dying tumor cells produce IL-38 to promote the survival of their siblings through limiting protective immunity. 266 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Lymph node invasion by tumor cells modifies the distribution and phenotype of dendritic and T cell subsets in breast cancer patients
Núñez N.1, Nadan A.T.1, Segura E.1, Pérol L.1, Milder M.1, Viel S.1, de la Rochere P.1, Loirat D.1, Meseure D.1, Amzallag N.1, Sastre-Garau X.1, Sedlik C.1, Amigorena S.1, Piaggio E.1
1Institut Curie, Paris, France
In human breast cancer the invasion of tumor-drain- being the IFN-γ production significantly higher in ing lymph nodes (TDLNs) contributes to disease pro- INV TDLNs (P < 0.05). Overall, we observed that gression and has predictive value. TDLNs dendritic immune cells with tolerogenic potential (Tregs, mac- cells (DCs) present tumor antigens to naïve T cells rophages) and effector CD4+ and CD8+ T cells from and induce their polarization into different function- metastatic TDLNs, both show evidence of high acti- al subsets (Th1, Th2, regulatory T cells (Treg) …) vation. These results potentially explain the power leading to antitumor T cell responses or to tolerance. ofimmunotherapies targeting the former ones that Here, we compared the immune profile of 70 untreat- unleash the anti-tumor activity of the highly activat- ed breast cancer Invaded (INV) versus Non-invaded ed effector T cells. (NI) TDLNs, with the aim to identify immunomod- ulatory mechanisms associated with the presence of the metastatic tumor cells. We studied by multi-col- or flow cytometry the distribution of 6 different DC subpopulations and observed in INV TDLNs a significant decrease in the percentage of BDCA1+ DCs (P < 0.05) and a significant increase in the per- centage of CD11c+HLADR+CD14+ cells (P < 0.01), including macrophages and inflammatory DCs, com- pared to NI TDLNs (P < 0.05). Interestingly, in vitro, these CD14+ myeloid cells induced the expression of FoxP3 in proliferating CD4+ T cells, suggesting a suppressive role. We also found a significant lower frequency of naïve conventional and Treg cells in INVTDLNs (P < 0.05). Both, in NI and INV TDLNs, memory conventional and Treg cells were highly po- larized, mainly to the Th1, but also to the Th2, Th17, Tfh and Th22 phenotypes, as determined by chemo- kine receptor and transcription factor expression. Further functional analysis showed that after ex-vivo PMA/Iono stimulation, the CD4+ and CD8+ T cells, but not the Tregs produced high amounts of IFN-γ, 267 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Interleukin-22 (IL-22) does not influence methylcholanthren-A- mediated tumorigenesis
Ochs C.1, May P.1, Wenk D.1, Endres S.1, Kobold S.1
1Ludwig-Maximilian-Universität, Division of Clinical Pharmacology, München, Germany
Background: Interleukin-22 (IL-22) is a unique cy- Conclusion: Endogenous IL-22 does not appear to tokine expressed by immune cell subtypes such as play a role in MCA-induced sarcoma model in Balb/c T and NK cells and acting exclusively on Interleu- mice. The findings in this inflammation-based tumo- kin-22-receptor-1 (IL-22-R1) positive epithelial cells. rigenesis model are in contrast to published findings Recently, we have demonstrated that expression of on enhancing tumor promoting role of IL-22 in the IL-22 is frequently found in primary human small APC-min colon cancer model. and large cell lung cancer samples. It is also known that IL-22 is found at high levels in the primary tumor, serum and in the malignant pleural effusion of non-small cell lung cancer patients. However, the influence of IL-22 on the development of a spontane- ous experimental tumor remained unaddressed. Methods: To test the influence of IL-22 on tumorigen- esis, we used the murine methylcholantren-A (MCA) model. MCA was injected at different doses (100 µg, 200 µg and 400 µg) subcutaneously in wild type or IL-22-KO Balb/c mice. Tumor onset and growth was monitored. Results: The time of tumor development correlated with the dose of MCA used. In wild type mice, 100, 200 and 400 µg of MCA led to a median onset of tumor growth (first day of palpable lesion) at days 124, 83 and 70 respectively (n = 4 to 6 per group). IL-22- KO mice, had similar intervals until tumor onset of 131, 88 and 70 days, respectively. Tumor growth after development was also similar between MCA dosage groups. No difference in median overall survival was noted in IL-22-KO (173, 161 and 164 days) versus wild type (163, 165 and 165 days) mice. We could not observe any impact of IL-22-KO on the phenotype of mice developing sarcoma (weight or behavior). 268 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Novel immuno-regulatory mechanisms mediated by IDH1 mutations in human gliomas
Kohanbash G.1, Ahn B.2, Shrivastav S.1, Costello J.1, Okada H.1
1University of California San Francisco, San Francisco, United States, 2University of Pittsburgh, Pittsburgh, United States
Mutations of the isocitrate dehydrogenase (IDH) the differential CXCL10 expression levels are not metabolic enzymes IDH1 and IDH2 have been likely to be merely secondary to the environmen- found to be early and frequent genetic alterations tal effects in vivo, but likely mediated by the IDH1 in World Health Organization (WHO) Grades 2 and mutation through intrinsic cellular mechanisms, 3 gliomas. All IDH mutations confer a novel gain- such as IDH-mutation-induced promotor methyla- of-function activity of converting α-ketoglutarate tion. Indeed, analysis of the TCGA Illumina ifinium (αKG) to 2-hydroxyglutarate (2HG), thereby pro- 450K methylation database indicates significantly moting the induction of histone and DNA hyper- higher levels of methylation of the CXCL10 promot- methylation. In the Cancer Genome Atlas (TCGA) er in IDH1-mutated cases compared to non-mutated database, we found that IDH-mutated cases exhibit cases. Treatment of GL261-IDH1-MUT cells with a decreased expression levels of type-1 effector T novel IDH1 inhibitor IDH1-C35, which inhibits 2HG cell response-related genes, including CD8, IFNG, production and induces demethylation of histone OAS2, GMZA, CXCL9 and CXCL10 compared with H3K9me3, restored CXCL10 expression to levels comparable non-mutated cases. On the other hand, observed in GL261-IDH1-WT cells. Our data reveal type-2 and regulatory T cell response genes, such a novel immune-regulatory mechanism mediated as IL5 and TGFb1, and MHC class I-related genes by IDH mutations. Based on our previous studies are not significantly different between IDH-mutat- demonstrating the importance of CXCL10 for accu- ed vs. non-mutated cases. mulation of effector cytotoxic T cells to the brain To address biological mechanisms underlying tumor site, our data imply that reversal of IDH-mu- these observations, we created mouse GL261 tations by a specific inhibitor or de-methylation glioma cells stably transfected with cDNAs for strategies may improve the efficacy of immunother- wild-type (GL261-IDH1-WT) or mutant IDH1 apies in IDH-mutated gliomas. (GL261-IDH1-MUT). Consistent with the TCGA cases, GL261-IDH1-MUT cells, when grown in the brain of syngeneic C57BL/6 mice, demonstrated lower numbers of infiltrating CD8 T-cells as well as lower CXCL10 and IFNG expression levels, com- pared with GL261-IDH1-WT tumors. In vitro culture experiments revealed that GL261-IDH1-MUT tumor cells express decreased levels of CXCL10 compared to GL261-IDH1-WT cells, strongly suggesting that 269 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Fluoxetine inhibits tumor-induced IL-6 release from immune cells of draining lymph nodes
Ozbey G.1, Erin N.1
1Akdeniz University Medical Faculty, Pharmacology, Antalya, Turkey
Fluoxetine (FLU), a widely used selective serotonin inflammatory response may differ in 4TLM bearing reuptake inhibitor (SSRI) for major depression, is mice. Further studies are needed to confirm these in known to alter immune function and inflammatory vivo effects using different doses of FLU on metastat- response. A growing body of findings indicates that ic breast cancer. FLU is able to modulate carcinogenesis and metas- tasis of breast cancer. The results of these studies are controversial which might be due to differential effects of FLU under in-vitro and in-vivo conditions. Because most previous studies were performed under in-vitro conditions using cell cultures we here exam- ined systemic effects of FLU on a syngeneic model of metastatic breast carcinoma. Specifically Balb/c mice were inoculated orthotopically with highly met- astatic breast carcinoma cells which were derived from liver metastasis of 4T1 murine breast carcinoma (4TLM). Mice were treated with daily i.p. injections of saline and FLU (3 mg/kg) for 4 weeks 48 h after injection of cancer cells. Tumor growth, lung metas- tasis and cytokine levels in secretomes of spleen and lymph nodes induced by lipopolysaccharide (LPS), concanavalin-A (ConA) or radiated 4TLM cells were determined. IL-6 release from secretomes of lymph nodes challenged with irradiated 4TLM cells was markedly decreased in FLU treated group. Similarly in FLU-treated group TNF-α secretion were reduced in secretomes of lymph nodes challenged by LPS. On contrary, TNF-α levels of FLU-treated animals were increased in secretomes of spleen challenged by LPS. The tumor growth and lung metastasis did not significantly differ among the groups. These results suggest that the effects of FLU on systemic and local 270 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
The different perspective of tumor immune evasion: Coexistence of cancer stem cells (CSCs) and nonclassical major histocompatibility complex (MHC) class I antigens
Özgul Özdemir R.B.1, Ozdemir A.T.2, Oltulu F.3, Kurt K.4, Yiğittürk G.3, Kırmaz C.5
1Manisa State Hospital, Clinical Immunology and Allergy, Manisa, Turkey, 2Ege University, Institute of Health Sciences, Department of Stem Cell, İzmir, Turkey, 3Ege University, Faculty of Medicine, Department of Histology and Embryology, İzmir, Turkey, 4Merkezefendi State Hospital, Medical Pathology Lab, Manisa, Turkey, 5Celal Bayar University, Faculty of Medicine, Clinical Immunology and Allergy, Manisa, Turkey
Cancer stem cells (CSCs) are unique subset of the CD44, NANOG, Oct3/4, HLA-G and HLA-E were sta- tumor cells and can separate from the other tumor tistically higher than the control group (p< 0.001) cells by the own specific markers. These cells show except CD133 (p=0.9963). These results suggested resistance to both chemotherapy and radiotherapy, an association between nonclassical MHC I antigens also they can escape from the immune system suc- and CSCs. We think the nonclassical MHC I antigens cessfully. They have different and effective cam- are may be important targets or markers for gastro- ouflage for the two important cell of anti-cancer intestinal tract tumors. defense, CD8 T cells (CTL) and Natural Killer (NK) cells. Nonclassical MHC I antigens are one of the immune evasion mechanisms. These molecules have different types of including HLA-G, HLA-E and HLA-F and potent inhibition and apoptosis signals for CTL and NK cells. CSCs and nonclassical MHC I antigens are associated with poor prognosis independently. However, there is not enough article in the current literature exam- ining the relationship between these two situations. Our purpose, examine the relationship between nonclassical MHC I antigens and CSCs in terms of immune system escape mechanisms. In this study, we did immunohistochemical analysis (H-SCORE) of CD133, CD44, NANOG, Oct3/4, HLA-G and HLA-E on collected pathology material from the advanced level of colorectal cancer (n=7), gastric cancer patients (n=7) and control subject of both cancers (n=14). In the gastric cancer samples all markers were statisti- cally higher than the control group (p< 0.001) except CD44 (p=0.9954). In the colorectal cancer samples 271 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Identifying the kinases and phosphatases regulating activated STAT3
Parri E.1, van Adrichem A.1, Kaustio M.1, Turunen L.1, Wennerberg K.1
1University of Helsinki, Institute for Molecular Medicine, FIMM, Helsinki, Finland
The signal transducer and activator of transcription these regulators active STAT3 can be inhibited or 3 (STAT3) protein is linked to inflammatory tumor- the regulators can change the pro-cancer immune igenesis, where it selectively induces and maintains responses. We developed cell lines stably express- the pro-cancer inflammatory microenvironment. ing STAT3(wt) or STAT3(Y640F) and a STAT3 re- STAT3 is persistently activated in cancers either sponsive luciferase reporter element. We screened through upregulation or activating mutations of them against human kinase and phosphatase siRNA upstream signaling pathways. Active STAT3 both library in a high-throughput manner. Monitoring promotes pro-oncogenic inflammatory pathways, STAT3 expression activity and cell viability we such as nuclear factor-κB and interleukin-6 (IL-6)- found several unexpected kinase and phosphatase Janus kinase pathways, and suppresses anti-tumor hit genes that regulate STAT3(Y640F) activity (i.e immune responses through T helper cells. In addi- PAK4, HIPK1, EPHA8, CTDP1). Some of the found tion STAT3 has been linked to increased metastasis regulators (i.e. BMX, CDK8/4, PTK6) were expected and drug resistance. Strikingly, in several types of to have effect on STAT3(Y640F) activity. Based on cancer, including T-cell large granular lymphocytic the pooled and individual siRNA screens we found (T-LGL), CD30+ T-cell lymphomas, chronic lymph- 6 regulators inhibiting the activity of constitutive- oproliferative disorders of NK cells and inflamma- ly active STAT3(Y640F) that had not been described tory hepatocellular adenomas, the STAT3 gene has before and 4 regulators that appear to further activate been shown to be frequently mutated resulting in already hyperactive STAT3(Y640F). We are currently a hyperactive STAT3 protein. To better understand exploring how knockdown of these genes affect the these diseases and how STAT3 regulates anti-cancer expression of STAT3-regulated immunoregulatory immunity and therapeutic resistance in cancers in proteins on the surface of the reporter cells as well general, we are studying these activated variants of as other cell models. STAT3, how they regulate cancer cell biology and how their activity may be modulated. The regulation of active STAT3 mutants is largely unknown and direct targeting of non-enzymat- ic STAT3 is difficult. Since we know that activat- ing phosphorylation of the STAT3 mutants is still required for their activity, we are exploring, how protein kinases and phosphatases regulate wild type and active STAT3 mutants and whether through 272 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Intra-tumor CD4+CD8+ double-positive T cells: a new target for IL-9
Parrot T.1, Allard M.1, Desfrançois-Noël J.2, Oger R.1, Benlalam H.1, Raingeard de la Blétière D.1,3, Pressier L.1, Labarrière N.1, Delneste Y.1, Guardiola P.1,3, Gervois N.1
1Centre de Recherche en Cancérologie Nantes-Angers - INSERM U892/CNRS 6299, Nantes, France, 2Cytometry Facility “CytoCell”, SFR François Bonamy, Nantes, France, 3SNP Transcriptome & Epigenomics Facility, CHU, Angers, France
The study of the tumor infiltrate composition in mel- To conclude, IL-9 would take part in the maintenance anomas enabled us to identify a non-conventional of DP T cells within the tumor infiltrate and would CD4+CD8+ double positive T lymphocyte sub-popula- favor their functional activity. Our results extend the tion (double-positive (DP) T cells) characterized by the pleiotropic effect of IL-9 already described for mast stable co-expression of the CD4 and CD8 molecules. cells, T reg, Th17 and monocytes, to intra-tumor DP DP T cell frequency can reach up to 10% of tumor T cells [2]. infiltrating lymphocytes and increases with advanced cancer stage, which suggests a role of these cells in anti-tumor immune responses [1]. Similarly to the conventional effector CD8+ lympho- References: cytes, DP T cells recognize tumor cells in an HLA-I [1] Desfrancois J, Moreau-Aubry A, Vignard V, Godet Y, Khammari A, et al. (2010) Double positive CD4CD8 alpha- restricted context and in a CD8 dependent way. beta T cells: a new tumor-reactive population in human However, DP T cells exert a poor cytotoxic activity melanomas. PLoS One 5(1): e8437. and differ from CD8 T cells by their strong ability to [2] Noelle RJ, Nowak EC. Cellular sources and immune func- tions of interleukin-9. Nat Rev Immunol. 2010;10:683-687. produce diverse Th1 and Th2 cytokines. In order to delineate the functions of DP T cells, their transcriptome profile was documented and compared with the one of CD8 infiltrating lymphocytes. It ap- peared that DP T cells overexpress the IL-9 recep- tor raising the question of the impact of IL-9 on the biology of these cells. We showed, that IL-9 enhances the survival of DP T cells by protecting them from ap- optosis, and favors their proliferation. In addition, IL-9 increases in a dose-dependent manner the cytokine production of DP T cells. In particular, the amount of IL-13 secreted in presence of IL-9 is liable to exert bio- logical effects on macrophages polarization, dendritic cells maturation or on tumor cells themselves. Finally, IL-9 increases perforine /granzyme B production and degranulation of DP T cells leading to an enhanced cytotoxic capacity against melanoma cell line. 273 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Monitoring the development of T-cell resistance in malignant melanoma
Sucker A.1, Zhao F.1, Real B.1, Horn S.1, Schilling B.1, Lennerz V.2, Kloor M.3, Ferrone S.4, Schadendorf D.1, Griewank K.1, Paschen A.1
1University Hospital Essen, Department of Dermatology, Essen, Germany, 2University Medical Center of the Johannes Gutenberg University Mainz, Medical Oncology, Mainz, Germany, 3University of Heidelberg, Institute of Pathology, Heidelberg, Germany, 4Massachusetts General Hospital, Department of Surgery, Boston, United States
CD8+ T lymphocytes (CTL) can exert potent in vivo cy- totoxicity against autologous melanoma cells as dem- onstrated in clinical trials with immune-modulatory antibodies. However, T-cell activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here we studied the im- munogenicity of melanoma cells from three consecu- tive patient lesions (Ma-Mel-48a, -48b, -48c) obtained within one year of developing stage IV disease. In vitro, stimulation of autologous CD8+ T cells was found to be strong for Ma-Mel-48a cells, significantly lower for Ma-Mel-48b cells and completely abrogated in case of Ma-Mel-48c cells. The latter was due to an ir- reversible HLA class I-negative phenotype of Ma-Mel- 48c cells, verified alsoin situ. An inactivating gene mutation in one allele of B2M, and concomitant loss of the other allele due to a larger deletion on chromo- some 15q were causative for HLA class I loss. Interest- ingly, SNP array analyses showed the loss of chro- mosome 15q was already present in both Ma-Mel-48a and Ma-Mel-48b cells, identifying this alteration as an early predisposing genetic event in the develop- ment of b2m deficiency. The same sequence of genetic alterations, i.e. chromosome 15q aberration followed by B2M gene mutation, was observed also in other melanoma patient models. Our study reveals the genetic evolvement of T-cell re- sistance in melanoma. Based on these data we suggest screening of tumor samples for genetic alterations with potential strong impact on immunogenicity for further improvement of melanoma immunotherapy. 274 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Selective biphenotypic leukemia cell lysis is mediated by dual-targeting triplebody 33-3-19
Roskopf C.C.1, Braciak T.A.1, Fenn N.2, Kobold S.3, Jacob U.4, Fey G.H.4, Hopfner K.-P.2, Oduncu F.S.1
1Klinikum der Universität München, Medizinische Klinik und Poliklinik IV, Hämatologie/Onkologie, Munich, Germany, 2Ludwig-Maximilians Universität München, Department of Biochemistry/Gene Center, Munich, Germany, 3Klinikum der Universität München, Medizinische Klinik und Poliklinik IV, Division of Clinical Pharmacology, Munich, Germany, 4Spectramab GmbH, Munich, Germany
The principle of multispecific targeting was put the SEM target cells. 19-3-19 or a bispecific T cell forward in targeted tumor therapy to prevent immune engager (BiTE) mixture of 19-3 and 33-3 induced the escape and increase selectivity. To this end, antibody lysis of both cell lines equally. Finally, 33-3-19 led to derivatives in the triplebody format were developed. the elimination of > 95 % colony-forming leukemia Triplebodies, which are composed of three linked cells. single chain variable fragments (scFvs), can simul- These results highlight the potential of dual-target- taneously target two different tumor associated anti- ing agents for efficient and selective immune-inter- gens on the same cell (“dual-targeting”), while con- vention in cancer. comitantly recruiting immune effector cells. Triplebody 33-3-19 was designed to test dual target- ing in combination with effector T cell recruitment. It was produced in suspension-adapted 293F cells, puri- fied and tested using standard molecular biology and flow cytometric techniques. 33-3-19 binds specifically to the B lymphoid marker CD19 and the myeloid marker CD33, a combination that only occurs on biphenotypic leukemia cells. Further, it recruits T cells via the CD3 epsilon chain. The triplebody activated T cells as efficiently as the recently described monotargeting triplebody 19-3-19 (Roskopf et al. 2014) and induced the specific lysis of established myeloid (MOLM13, THP-1) as well as B cell lines (SEM, Raji) and of primary patient cells
at low picomolar concentrations. EC50 values ranged from 0,1 to 28 pM. Importantly, 33-3-19 induced the preferential lysis of double- over single-positive leu- kemia cells in a target cell mixture: When both, the CD19 single-positive cell line SEM and the (CD19 plus CD33) double-positive cell line BV173 were present in a cytotoxicity test, the BV173 target cells were elimi- nated more readily in the presence of 33-3-19 than 275 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Immunological and angiogenic markers during metronomic chemotherapy in spontaneous arising cancers in dogs
Denies S.1, Cicchelero L.1, de Rooster H.2, Daminet S.2, Polis I.2, Van de Maele I.2, Sanders N.1
1Ghent University, Laboratory of Gene Therapy, Merelbeke, Belgium, 2Ghent University, Department of Medicine and Clinical Biology of Small Animals, Merelbeke, Belgium
Regulatory T cells (Tregs) suppress the anti-tumor depleting them. Metronomic cyclophosphamide can immune response while thrombospondin-1 and be such a strategy. More research is needed to iden- vascular endothelial growth factor (VEGF) are key tify optimal dosing for clinical efficacy, as none of angiogenic factors implicated in cancer progression. the evaluated treatments had an effect on angiogenic Metronomic chemotherapy stimulates the immune factors, considered biomarkers for efficacy of metro- response via depletion of Tregs and suppresses angi- nomic chemotherapy. ogenesis. Blood was collected in 10 healthy dogs and in 30 dogs with advanced stage cancer before and after four weeks of metronomic cyclophosphamide (12.5mg/m 2), temozolomide (6.6mg/m 2) or cyclo- phosphamide and temozolomide. Tregs were quanti- fied with flow cytometry, for the first time in canine cancer patients with the recently available anti-ca- nine CD25 marker included. Plasma levels of throm- bospondin-1 and VEGF were measured with ELISA. There was a significant difference in percentage Tregs between cancer patients and healthy dogs (4.2 +/- 2.1% vs 2.5 +/-0.7%, p< 0.001) (t-test). A signifi- cant decrease was noted in patients treated with cy- clophosphamide (5.2+/-2.2% vs 3.8+/-1.7%, p=0.02) and the combination (3.7+/-2.0% vs 2.8+/-1.0%, p=0.03) (paired t-test). Treatment with temozolomide had no effect on the percentage Tregs. Thrombos- pondin-1 levels were significantly lower (927.26+/- 783.11 pg/ml vs 121.67+/-90.92 pg/ml, p=0.001) and VEGF significantly higher (57.17 +/-28.49 pg/ml vs 139.27+/-132.66 pg/ml, p= 0.03) (Mann-Whitney-U test) in cancer patients than in healthy dogs but both were not influenced by any metronomic treatment regimen. As Tregs are increased in cancer patients, immunotherapy should include a strategy aimed at 276 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Immunomodulatory kinase inhibitors in tumor immunotherapy
Sandin L.C.1, Schneider J.1, Hahn U.1, Guse J.-H.1, Burnet M.1
1Synovo GmbH, Tuebingen, Germany
The tumor microenvironment in advanced cancer of these mice there was a shift in the mRNA levels patients is characterized by immunosuppression from immunosuppressive (IL10) towards immunoac- induced by tumor- and/or tumor stroma-derived tivation (IL12, IFNg). factors leading to influx of regulatory cell types. SYD003 recently received Orphan Drug Designation These factors and cells inhibit immune activation from both the European Medicines Agency and the and facilitate the progression and metastasis of the US Food and Drug Administration for treatment of tumor leading to treatment failure. Therefore, thera- pancreatic cancer. As SYD003 is non-cytotoxic and peutic approaches targeting the tumor microenvi- well tolerated, it presents a useful potential addition ronment rather than the tumor itself are potentially to standard of care in these patients. useful and synergistic to current standard of care. Mitogen-Activated Protein Kinases (MAPK) are abun- dantly expressed in different cell types. They regu- late important cellular responses such as prolifera- tion, differentiation and apoptosis, which represents signaling pathways often dysregulated in cancer cells. Most approved MAPK inhibitors for cancer therapy aim to target over-expressed kinases in the malignant cells while our lead compound SYD003, a p38alpha MAPK inhibitor, stimulates the produc- tion of IL12. Bone marrow-derived macrophages ac- tivated towards an immunosuppressive phenotype increased the production of IL12 in a dose-dependent manner, whereas, at the same time decreasing their secretion of IL10. A similar pattern was observed for bone marrow-derived macrophages activated towards an immunoactivating phenotype as well as for bone marrow-derived DCs. In vivo, SYD003 has proven anti-tumor activity in various transplantable tumor models (e.g. MB49 bladder cancer) and in a spontaneous tumor model of pancreatic cancer (KPC mice). In the tumor tissue 277 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Clinical significance of tumor-associated tertiary lymphoid structures in pancreatic cancer
Sasada T.1,2, Suekane S.2,3, Reinherz E.L.2
1Kanagawa Cancer Center Research Institute, Cancer Vaccine Center, Yokohama, Japan, 2Dana-Farber Cancer Institute, Cancer Vaccine Center, Boston, United States, 3Kurume University School of Medicine, Department of Urology, Kurume, Japan ORAL ALK SHORT T 2015
Purpose: Limited information has been available significant prognostic factor for better overall sur- on tumor-infiltrating immune cells in pancreatic vival [Hazard ratio = 0.647, P = 0.017; Hazard ratio cancer. Immunohistochemistry (IHC) was performed = 1.059, P = 0.043; respectively]. to characterize tumor-infiltrating cells in pancreatic Discussion: Our findings have provided new infor- tumor tissues. mation on the host immune responses against pan- Materials and methods: IHC with anti-CD20 mAb creatic tumors. Tumor-infiltrating B cells, especially was performed to characterize tumor-infiltrating B tumor-associated TLS, might be associated with cells in pancreatic tumor tissues. For more precise better prognosis in pancreatic cancer. Further elu- assessment of 4 different types of immune cells, in- cidation of target antigens of tumor-infiltrating B cluding CD8+ and CD4+ T cells, FoxP3+ regulatory cells might be helpful for better understanding the T (Treg) cells, and CD20+ B cells, multiplexed IHC anti-tumor immune responses and ultimately accel- with quantum dot nanocrystals (Qdot-IHC) was erating development of novel immunotherapeutic ap- developed. The clinical value of tumor-infiltrating proaches for pancreatic cancer. In addition, of note immune cells was statistically evaluated. is that our novel methodology, Qdot-IHC, enables si- Results: In a cohort of 46 pancreatic cancer patients, multaneous and precise assessment of multiple (up 32 patients (69.6%) showed infiltration of CD20+ B to 4) different types of immune cells, which might cells, which frequently aggregated and formed tu- be quite useful for future studies to characterize in mor-associated tertiary lymphoid structures (TLS), detail the types and spatial locations of tumor-infil- within tumors. The patients with more B-cell infil- trating immune cells. tration showed a significantly better prognosis than those with less B-cell infiltration (median survival, 718 vs. 283 days; P = 0.021). To characterize tumor- infiltrating CD20+ B cells and other immune cells more precisely, Qdot-IHC was performed in tumor tissues from a second cohort of 46 pancreatic cancer patients. Tumor-associated TLS were detected in 22 patients (47.8%), and the presence of TLS was sig- nificantly correlated with more numbers of tumor- infiltrating CD8+ cells (P = 0.011). Statistical analy- sis demonstrated that more numbers of TLS and less numbers of FoxP3+ Treg cells within tumors were a 278 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
The potential immunomodulatory role of cortisol on NKG2D activating NK cells receptor in ovarian cancer
Seida A.A.1
1Cairo University, Microbiology and Immunology Department, Giza, Egypt
Background: Tumour-infiltrating myeloid-derived both cortisol and TGF-β1. Cortisol and TGF-β1 were suppressor cells (MDSC) or tumour-associated mac- further found to act synergistically in downregulat- rophages (TAM) which are abundant in ovarian ing NKG2D. However, RU486 and SD208 were able cancer show a high expression of the enzyme 11Be- to restore the activating NKG2D receptor expression ta-Hydroxysteroid dehydrogenase I (11β-HSD1) which on NK cells. able to convert inactive cortisone into active cortisol which has been detected in serum, ascitic fluid and tumour exudates from ovarian cancer patients. Con- sidering that cortisol has a strong suppressive effect on immune cells, we investigated the effect of corti- sol or TGF-β or both since they do not act separately of each other but modulate each other’s activities on the activating NK cell receptor (NKG2D) expression. This shall now be investigated by pharmacological inhibitors of cortisol (RU486) and TGF-β (SD208). Material and methods: Using immunohistochemis- try, real-time PCR, luminescent immunoassays (LIA), Flow cytometry and immunofluorescent double staining. Results: We found that 11β-HSD1 enzyme is highly expressed in human and murine ovarian cancer tissue via real-time PCR and immunohistochemis- try. Luminescent immunoassays (LIA) showed el- evated cortisol in serum, ascitic fluid and tumour exudates from ovarian cancer patients. Immunoflu- orescent double staining revealed a co-localization of 11β-HSD1 with CD14, CD68, and CD85, but not with EpCAM. Expression of 11b-HSD1 can thus be attributed to TAM or MDSC. Expression of NKG2D on NK cells was analyzed by flow cytometry. The result indicated that NKG2D is downregulated by 279 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Humanized NSGTM mice are a platform for pre-clinical evaluation of immuno-modulatory therapeutics
Soper B.M.1, Wang M.1
1The Jackson Laboratory, Bar Harbor, United States
With the development of the NSGTM mouse (NOD.Cg- samples respond to standard of care drugs (SOC) in Prkdcscid Il2rgtm1Wjl/SzJ), a better small animal model the presence of human immunity, and (2) can the to host human cancer cells as well as human immune human immune cells diminish tumor growth when cells for experimental and pre-clinical testing is treated with clinically relevant immune checkpoint available to researchers. Both applications allow for inhibitors? First, a colon PDX sample was engrafted more specialized, human-specific research questions in hu-CD34 NSGTM mice and treated with vehicle or to be addressed that were not possible previously. the SOC drugs 5-FU or Avastin. Both drugs slowed However, several cancer therapeutics in pre-clinical tumor growth rates compared to the control indicat- development rely on functional human immunity ing response to SOC drugs in the presence of human for therapeutic effect and the current models are immunity. Second, MDA-MB-231 breast cancer cells not independently adequate for this purpose. JAX were transplanted into hu-CD34 NSGTM mice. 98.8% In Vivo Pharmacology Services has combined the of these cells expressed PD-L1, which when bound to CD34+ hematopoietic stem cell engrafted human- PD1 on T cells renders the T cells anergic. When mice ized NSGTM (hu-CD34 NSGTM) with human patient were treated with Keytruda, an antibody that blocks derived xenograft (PDX) to create a platform for pre- PD1/PD-L1 binding, tumor growth was significantly clinical studies in immuno-oncology. Initial experi- diminished. In a similar study, hu-CD34 NSGTM mice ments to establish and validate the model found that were transplanted with a PDX breast tumor in which non-HLA matched PDX tumors grow well in hu-CD34 56.9% of the cells in the tumor expressed PD-L1. This NSGTM (3 different solid tumor PDX tested), despite tumor also showed a dramatic suppression of tumor concerns over transplant rejection. Also, growth growth following treatment with Keytruda. This data of a non-HLA matched human ovarian tumor was demonstrated the human T cells could be induced to relatively unchanged when transplanted either prior respond to either a human cell line or a heterologous to development of immunity or after. Finally, when patient tumor following treatment with the check- human breast, lung, or soft tissue carcinoma PDX point inhibitor Keytruda. Taken together, hu-CD34 tumors were engrafted to either NSGTM or hu-CD34 NSGTM mice support growth of non-HLA matched NSGTM, tumor take was 100% and growth rate was human tumors and serve as a pre-clinical platform similar regardless of host, indicating the human to test new immuno-oncology therapeutics. immune cells did not influence the growth of the tumors. To further test the model, experiments were conducted that addressed questions important for pre-clinical immuno-oncology testing: (1) do the PDX 280 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Macrodissection of human colorectal cancer liver metastases revealed the tumor margin as a precisely separated region with distinct cytokine and chemokine profiles
Spille A.1,2, Zoernig I.2, Kahlert C.3, Klupp F.4, Ulrich A.4, Weitz J.3, Jaeger D.1,2, Halama N.2
1Clinical Cooperation Unit "Applied Tumor Immunity", National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany, 2Department of Medical Oncology, National Center for Tumor Diseases (NCT) and University Hospital Heidelberg, Heidelberg, Germany, 3Department of Surgery, University Hospital Dresden, Dresden, Germany, 4Department of Surgery, University Hospital Heidelberg, Heidelberg, Germany
In the tumor microenvironment (TME) of human the same macrodissected tissues gave an excellent re- colorectal cancer liver metastases (CRC-LM) lo- producibility. Additionally, side by side comparison calization and high densities of tumor infiltrating with laser-assisted microdissected samples validated lymphocytes (TIL) have been associated with good the results of macrodissecton. In conclusion, macro- treatment responses and prognosis. However, little is dissection is a simple and reliable tool to separate known about intratumoral migration of TIL and the adjacent liver, invasive margin and liver metastasis analysis of the unknown processes that either retard in CRC-LM tissues. Furthermore, macrodissection re- or enhance T-cell recruitment, migration and activa- vealed the invasive margin as a precisely separated tion in the TME is a necessary step for the optimiza- region with reproducible as well as distinct cytokine tion of CRC-LM therapies. Especially cytokines and and chemokine profiles. The functional role of the chemokines play an important role in these processes individual cytokine and chemokine profiles in the in- by recruiting and activating an anti-tumor immune vasive margin will be investigated in future studies. response on the one hand and by forming a tumor- promoting and immunosuppressive environment on the other hand. The strong accumulation of TIL at the tumor margin in therapy-responding CRC-LM patients leads to the assumption, that this region ex- hibits a distinct cytokine and chemokine profile com- pared to the tumor and adjacent liver. In addition, the metastasis and adjacent liver in CRC-LM tissues can be simply divided macroscopically leading to the presumption that macrodissection is a conveni- ent tool to dissect CRC-LM tissues. For these reasons, our research group used macrodissection to divide CRC-LM tissues into the three regions liver metas- tasis, invasive margin and adjacent liver for subse- quent quantitative cytokine and chemokine analysis. As assumed, cytokines and chemokines show a dis- tinct pattern in the invasive margin compared to the liver metastasis and the adjacent liver. Comparison between different invasive margin serial sections of 281 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
CD33-related siglecs are upregulated on activated and tumor-infiltrating T cells
Stanczak M.1,2, Thommen D.1,2, Varki A.3, Zippelius A.1,2, Läubli H.1,2
1University of Basel, Department of Biomedicine, Basel, Switzerland, 2University Hospital Basel, Division of Oncology, Department of Medicine, Basel, Switzerland, 3University of California, San Diego, Departments of Medicine and Cellular and Molecular Medicine, La Jolla, United States
Most tumor cells are recognized by the immune cells initially expressed very low amounts of CD33r- system and eliminated before developing into clini- Siglecs predominantly on effector memory T cells. cally detectable tumors. However, during cancer Furthermore, engagement of Siglec-9 during T cell progression tumor cells evade immune-mediated activation inhibited both activation and prolifera- destruction by generating an immunosuppressive tion, additionally supporting a role for CD33rSiglecs tumor microenvironment and upregulation of im- in regulation of T cell activation. Future studies will muno-inhibitory molecules such as PD-L1. Recently, aim to investigate the functional consequences of in- tumor hypersialylation was shown to produce ligands hibition of CD33rSiglecs on CD8 T cells from healthy for inhibitory CD33-related sialic acid binding im- donors and tumor infiltrating T cells. munoglobulin-like lectins (CD33rSiglecs) including Siglec-9. Engagement of Siglec-9 by tumor-associated ligands inhibited neutrophils and NK cells and pro- moted immune evasion of tumor cells. Interestingly, not only cell surface bound ligands for CD33-related Siglecs are upregulated in tumors, but also secreted ligands like the recently identified heavily N-glyco- sylated lectin galactoside-binding soluble 3 binding protein (LGALS3BP). Analysis of CD33rSiglecs on tumor infiltrating leu- kocytes revealed an expression on myeloid cells, but also a significant proportion of tumor infiltrat- ing CD8 T cells expressing inhibitory CD33rSiglecs including Siglec-9 and Siglec-10. Positive correlation with markers of T cell exhaustion such as Tim-3 sug- gests a possible role for Siglec-9 during T cell dys- function in cancer. Further studies on T cell receptor-mediated activa- tion of CD8 T cells from healthy donors showed an upregulated expression of various CD33rSiglecs on proliferating CD8 T cells upon stimulation, while in accordance with previous analyses quiescent CD8 T 282 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Impaired CD8+ T cell responses in the presence of myeloid-derived suppressor cells in a spontaneous mouse melanoma model
Mairhofer D.G.1, Schaffenrath S.1,2, Tripp C.H.1,2, Ortner D.1, Fleming V.3, Heger L.3, Komenda K.1, Reider D.1,2, Duziak D.3, Chen S.4, Becker J.C.5, Stoitzner P.1
1Medical University of Innsbruck, Department of Dermatology & Venereology, Innsbruck, Austria, 2Oncotyrol- Center for Personalized Medicine, Innsbruck, Austria, 3Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Dermatology, Erlangen, Germany, 4Rutgers University, Chemical Biology Lab for Cancer Research, Piscataway, United States, 5University Hospital Essen, German Cancer Consortium, Translational Skin Cancer Research, ORAL Essen, Germany ALK SHORT T 2015
Murine tumor models that closely reflect human of a spontaneous melanoma mouse model that pro- diseases are important tools to investigate car- vides an interesting tool to develop improved im- cinogenesis and tumor immunity. The transgenic munotherapeutical strategies. (tg) mouse strain tg(Grm1)EPv develops spontane- ous melanoma due to ectopic overexpression of the metabotropic glutamate receptor 1 (Grm1) in melanocytes, causing spontaneous melanoma de- velopment. In the present study, we characterized the immune status and functional properties of immune cells in tumor-bearing mice. Melanoma development was accompaigned by a reduction of CD4+ T cells including regulatory T cells (Tregs) in the CD45+ leukocytes present in tumor tissue and draining lymph nodes (LN). In contrast, percent- ages of CD8+ T cells were unchanged and these cells showed an activated phenotype in tumor- bearing mice. Endogenous gp100-specific CD8+ T cells were not deleted during tumor development, as revealed by pentamer staining in skin and LN. However, CD8+ T cells were unresponsive to ex vivo restimulation with the melanoma-associated antigen gp100. Interestingly, immunosuppressive myeloid-derived suppressor cells (MDSC) were re- cruited to tumor tissue with a preferential accumu- lation of granulocytic MDSC (grMDSC) over mono- cytic MDSC (moMDSC). Both subsets suppressed gp100-specific transgenic CD8+ T cell responses in vitro. In this work, we describe the immune status 283 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Effects of surgical stress on host immunity in breast cancer patients
Taranikanti M.1,2, Panda S.1, Behara J.1, Yasmeen N.1, Siddiqui A.R.O.1
1Shadan Institute of Medical Sciences, Physiology, Hyderabad, India, 2Dr B R Ambedkar Open University, Psychology, Hyderabad, India
Background: Surgery is a form of stress that can presses immunity in breast cancer patients follow- have significant effects on immune system, pos- ing surgery should not be used for at least 3 weeks sibly by reducing the ability to fight the disease after surgery. progression and also metastatic spread. Cellular immunity plays an important role in the control of malignancy. The objective of the study was to understand the effects of surgical stress on cell me- diated immunity in breast cancer patients. Methods: 31 women with operable early stage breast cancer, who underwent surgery and were also receiving adjuvant chemotherapy were in- cluded in the study. Blood samples were collected from the patients during the pre-operative period and twice during the post-operative period, after 72 hours and after 21 days. Informed consent was taken from all the participants of the study. Pe- ripheral Blood mononuclear cells were isolated and incubated with FITC / PE conjugated monoclonal antibodies. Samples were subjected to flow cyto- metric analysis to obtain the mean values of lym- phocyte sub-populations as percentage. A ques- tionnaire was also given to all study participants to assess the levels of stress of having breast cancer. Results: The mean values of lymphocyte sub-pop- ulation were found to be significantly lower (p< 0.5) in the early post-operative period in breast cancer patients. However, the values returned to near pre-operative levels after 21 days. A positive correlation existed between psychological stress of having the disease and low host immunity. This indicates that any intervention that further sup- 284 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Characterization of human natural killer cell activity against multiple myeloma
Tognarelli S.1,2, von Metzler I.3, Jacobs B.4,5, Mackensen A.4, Ullrich E.2,3
1Universitätsklinikum Frankfurt, Pediatric Hematology and Oncology, Frankfurt am Main, Germany, 2Johann Wolfgang Goethe University, LOEWE Center for Cell and Gene Therapy, Frankfurt am Main, Germany, 3Universitätsklinikum Frankfurt, Hematology and Oncology, Frankfurt am Main, Germany, 4Universitätsklinikum Erlangen, Hematology and Oncology, Erlangen, Germany, 5Institute for Cancer Research, Cancer Immunology, Oslo, Norway
Multiple myeloma (MM) is a tumor formed by the with focus on activating and chemokine receptors as clonal expansion of malignant plasma cells, compris- well as on their modulation following activation and ing about 10% of all hematologic malignancies and in vitro culturing. Interestingly, we observed that having a 5-years survival rate of just 45%. Therapies various MM cell lines differentially expressed ligands such as Stem Cell Transplantation (SCT), Proteasome for NK receptors that impact NK cell mediated MM Inhibitors (Bortezomib, Carfilzomib, Marizomib) and killing in combination with the HLA-KIR mismatch. Immunomodulatory Drugs (IMiDs) including Tha- We also analyzed the maintenance of migratory ca- lidomide, Lenalidomide and Pomalidomide improve pacity, with particular interest in bone marrow (BM)- patients’ outcome, nevertheless MM mostly remains homing chemokine receptors and strategies to rescue incurable. Natural Killer (NK) cells range from 2 to and/or improve NK cell migration. In addition, we 18% of human peripheral blood lymphocytes and validated the pathways involved in NK cytotoxicity show potent anti-tumor capacities. Remarkably, an using agonists and antagonists of several NKR in increased NK cell number has shown a positive cor- order to dissect their role in MM killing. relation with the MM patients’ outcome and clini- Furthermore, we are currently investigating the pos- cal trials with infusion of activated NK cells in MM sible suppressive influence of specific BM stromal patients are currently under evaluation. However, components, performing phenotypic and functional MM can use multiple tumor immune escape mecha- analysis of NK cells that have been co-cultured with nisms (TIEM) to evade NK cell recognition. Current stromal cell populations. strategies that aim to further improve the efficacy Finally, peripheral blood samples from a cohort of of NK donor lymphocyte infusions (NK-DLI) include newly diagnosed MM patients have already been the use of antibodies directed against NK inhibitory collected and will be analyzed in the context of our receptors (iKIRs) to block NK cell inhibition. experimental design. We wondered if a strategy to increase the activ- In conclusion, our study will shed new light on ity of activating receptors might also be beneficial. NK - BM stroma - MM interactions and will help to Therefore, we aimed to characterize human MM cell improve current NK cell therapy protocols against lines and the different pathways involved in MM - multiple myeloma. NK cell crosstalk. First, we studied the expression of activating receptor- ligands by different human MM cell lines with special interest in NKG2D-, DNAM- 1-, TRAILR- and FAS-ligands. Next, we addressed the expression of several NK cell receptors (NKR) 285 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
In vitro, in vivo and microscopic analysis of tumor immunotherapy using combination of two antibodies against the same ErbB2 target
Toth G.1, Szoor A.1, Simon L.1, Szollosi J.1, Vereb G.1
1University of Debrecen, Department of Biophysics and Cell Biology, Debrecen, Hungary
Trastuzumab (Herceptin®), a humanized anti-ErbB2 diated in vitro ADCC-based killing, while F(ab’)2 antibody is a specific targeted therapy against ErbB2 fragments did not. positive tumors, with a history of both success and a Formation of immunological synapses was verified high rate of therapy resistance. Another humanized by confocal microscopy. NK-92 cells were able to antibody, pertuzumab (Perjeta®) inhibits ErbB2 het- form synapses upon recognition of whole antibod- erodimerization. While these antibodies have been ies by their high affinity FcγRIII, while owed to the developed based on the in vitro direct cellular effect lack of Fc region, synapse formation did not occur
of their mouse parent antibodies, there is the possi- with F(ab’)2-s. bility that their in vivo mechanism of action roots For in vivo ADCC study, JIMT-1 cells were inoculat- rather in antibody dependent cellular cytotoxicity ed s.c. in severe combined immunodeficiency mice. (ADCC) exerted by natural killer (NK) cells. The whole antibodies were able to inhibit tumor
Our goal was to ascertain the extent of contribution growth, but their F(ab’)2 fragments were ineffective. of the direct cellular effect of the antibodies and that of the in vivo evoked ADCC to tumor growth
inhibition. We generated the F(ab’)2 fragments of the antibodies lacking the Fc part, that have the ability to generate direct cellular effects, but lack the ADCC component. Affinity and lack of Fc fragment
on F(ab’)2-s were tested with immunofluorescence
in flow cytometry. In vitro EC50 was assessed with an MTT based assay. The effect on proliferation of in vitro sensitive BT-474 and resistant JIMT-1 cell lines, both ErbB2 positive, was not affected by the
removal of the Fc region. Based on the EC50 values determined as single agents on BT-474 cells, isoboles for a range of combination were also measured for
both the whole antibodies and the F(ab)’2 fragments and no co-operativity was found. Non-radioactive in vitro ADCC assay on JIMT-1cell line was optimized with CD16.176V.NK-92 high af- finity natural killer cell line. Intact antibodies me- 286 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Examination of immune escape mechanisms used by circulating tumour cells (CTC) in gynaecological cancers
Tuyaerts S.1, Euteneuer A.1, Everaert T.1, Amant F.2
1KU Leuven, Department of Oncology, Leuven, Belgium, 2UZ Leuven, Leuven, Belgium
ORAL ALK SHORT T 2015
Introduction: Ovarian cancer is mostly diagnosed in the RosetteSep® CTC isolation kit was used, where advanced stages while for uterine cancer, recurrent unwanted cells are labelled with tetrameric antibody and metastatic disease remain major obstacles. Both complexes which then make rosettes with red blood ovarian cancer and uterine cancer comprise carcino- cells from the blood. The tumour cells are enriched mas and sarcomas. The metastatic process of tumours on a density gradient. Enriched CTC were analysed by is poorly understood but it is accepted that CTC, tumour flow cytometry, PAP staining, fluorescence staining or cells that have entered the circulation, mediate metas- qPCR. The procedure based on OncoQuick Plus® was tasis. In spite of being rare and exposed to immune selected for CTC isolation from patient samples. attack, CTC must be capable to escape the immune Results: CTC enriched by different methods were anal- system. Little is known about the interaction of CTC ysed by flow cytometry (FSC/SSC, CD45, CD15, EpCAM, with the immune system, most likely due to their cytokeratin, viability dye) or diameter measurement by scarcity. CTC analysis in patients with gynaecological microscopy after PAP staining. To assess CTC purity tumours will give us more insight into the immune and recovery, diameter measurement of cells was escape process and metastasis formation. For CTC iso- optimal. However, flow cytometry adds information lation, we focused on antigen-independent approaches, about expression of certain molecules by the isolated because CTC from sarcomas do not express EpCAM cells. Concerning the comparison of the 3 techniques, and for tumour cells to enter the blood, they need to the resulting CTC purities were comparable, with all undergo epithelial-to-mesenchymal transformation methods showing a high variability. CTC recovery was (EMT), by which they downregulate or lose EpCAM also similar between the 3 techniques, with slightly expression. better results for the OncoQuick Plus® based procedure, Material and methods: For optimization of CTC iso- which is now being used to isolate CTC from the blood lation, blood from healthy donors was spiked with of cancer patients, which are compared to tumour cells various amounts of ovarian or uterine cancer cell lines from the original tumour. (OVCAR3, HEC-1A or MES-SA) and processed using Discussion: The OncoQuick Plus® based procedure different techniques. The first approach comprised was selected for CTC isolation from patients with gy- the density-gradient based OncoQuick® procedure naecological cancer. We are analysing several known followed by depletion of contaminating blood cells immune escape mechanisms (downregulation of via CD45-labeled dynabeads. The second protocol HLA, expression of CD47, HLA-E, HLA-G, PD-L1, EMT used OncoQuick Plus® instead of OncoQuick®, an op- markers) on CTC isolated from patient blood. timised separation medium resulting in less depletion of unwanted MNC but higher CTC enrichment. At last, 287 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
A novel Toll-Like Receptor 7 agonist with distinct anti-tumoral monotherapeutic effects
Vascotto F.1, Petschenka J.1, Reuter K.2, Vormehr M.1,3, Roth R.2, König A.2, Worm C.2, Brkic M.3, Krause N.2, Schmitt U.3, Diken M.1, Kreiter S.1, Hamm S.4, Strobl S.4, Türeci Ö.5, Sahin U.1,2,6
1TRON - Translational Oncology at the University Medical Center of the Johannes Gutenberg University gGmbH, Mainz, Germany, 2BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, 3Universal Medical Center of Johannes Gutenberg-University, Mainz, Germany, 44SC Discovery GmbH, Martinsried, Germany, 5Ganymed Phamaceuticals AG, Mainz, Germany, 6Research Center Immunology (FZI), University Medical Center of Johannes Gutenberg University, Mainz, Germany
Toll-like receptor (TLR) ligation activates both the Anti-tumoral SC1 treatment activated splenic pDC, innate and adaptive immune systems and plays an cDC, macrophages as well as monocytes and aug- important role in antiviral and anti-tumoral im- mented the effector immune response by increas- munity. TLR agonists are ranked in the National ing CD8/regulatory T cell ratio. In addition, SC1 Cancer Institute list of immunotherapeutic agents induced polarization of macrophages towards an with the highest potential to treat cancer. Among M1 phenotype in spleen as well as in tumor. By in- them, the TLR7 agonist imiquimod is U.S. Food creasing expression levels of chemokine/cytokine/ and Drug Administration-approved as a topical interleukins and integrins, SC1 promoted a change treatment for premalignant and early skin cancers of the tumor environment favoring a Th1 immune and genital warts. However, it displays disadvanta- response and the recruitment of immune cells. geous toxic effects after systemic delivery. Interestingly, during the in vivo therapeutic treat- Here we describe a preclinical study using SC1, ment using anti-PD-L1 antibody in CT26 tumor a novel small molecule TLR7 agonist, against bearing mice, SC1 improved the anti-tumoral re- several murine tumor models. In the therapeutic sponse and survival of mice. setting, repetitive intravenous injections of SC1 In conclusion, this novel TLR7 agonist, in a non- prevented lung metastasis formation of 4T1 ortho- toxic systemic regiment acts as strong anti-tumoral topic breast tumor and delayed B16 melanomasu- agent modulating tumor environment, potentiat- bcutaneous (s.c.) tumor growth. In the CT26 colon ing immune response via CD8 T cells, indicating s.c. carcinoma model, SC1 conferred a potent anti- promising implications in oncological therapeutic tumoral effect, prolonging survival and increas- treatments. ing the anti-tumoral immune response. We found that SC1 treatment increased frequency of tumor antigen specific (gp70) CD8 T cells detectable in blood, spleen and tumors, which showed effector functions and mediated SC1 anti-tumor therapy. Notably, SC1 treated mice mounted a memory CD8 T cell response, which protected mice against several tumor re-challenges. 288 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Exploitation of the Thy1-YFP mouse strain for fluorescent imaging of inflammation, wound-healing and experimental tumours
Jósvay K.1, Winter Z.2, Katona R.L.3, Pecze L.1, Marton A.1, Buhala A.2, Szakonyi G.2, Oláh Z.2, Vizler C.1
1Biological Research Centre of HAS, Institute of Biochemistry, Szeged, Hungary, 2Institute of Pharmaceutical Analysis, Faculty of Pharmacy, University of Szeged, Szeged, Hungary, 3Biological Research Centre of HAS, Institute of Genetics, Szeged, Hungary
Thy1 (CD90) is a cell surface glycoprotein expressed on several cell types, including neurons and T cells, and is known to be involved in cell adhesion, mi- gration and signal transduction. The B6.Cg-Tg(Thy1- YFP)16Jrs/J transgenic mouse strain was created for visualizing neuronal development and regenera- tion. This mouse strain expresses yellow fluorescent protein (YFP) specifically in the peripheral nerves and the central nervous system under the control of truncated regulatory sequences of the Thy1 gene. We hypothesized that Thy1-activating conditions, as in- flammation, wound healing or tumour growth could also activate the truncated Thy1 regulatory sequenc- es used for creating the transgenic mouse. Local inflammation induced by subcutaneous injection of complete Freund´s adjuvant, as well as the experi- mental wound-healing milieu created by superficial scalpel cuts triggered YFP fluorescence in the B6.Cg- Tg(Thy1-YFP)16Jrs/J mice. We demonstrated that the stroma of both 4T1 or MC26 subcutaneous tumours is readily detectable via the YFP fluorescence in BALB/c(Thy1-YFP) mice established by back-crosses from the original transgenic strain. The yellow-green fluorescent stromal cells were clearly distinguishable from the red fluorescent 4T1 carcinoma cells created by transfection with red fluorescent protein. In con- clusion, we described a mouse model for fluorescent imaging of non-labelled subcutaneous tumours. In addition, the model gives opportunities for studying eventual overlapping pathways of wound-healing, inflammation and tumour growth. 289 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Accumulation of tolerogenic human 6-sulfo LacNAc+ dendritic cells in renal cell carcinoma is associated with poor prognosis
Wehner R.1, Toma M.2, Kloß A.1, Füssel S.3, Seliger B.4, Noessner E.5, Schäkel K.6, Wirth M.3, Baretton G.2, Schmitz M.1
1Institute of Immunology, Medical Faculty, Dresden University of Technology, Dresden, Germany, 2Institute of Pathology, University Hospital of Dresden, Dresden, Germany, 3Department of Urology, University Hospital of Dresden, Dresden, Germany, 4Institute for Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany, 5Institute of Molecular Immunology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany, 6Department of Dermatology, University Hospital, Heidelberg, Germany
Dendritic cells (DCs) play a pivotal role in the induc- ing slanDCs expressed HLA-DR and low densities of tion and maintenance of innate and adaptive antitu- CD86, but CD40, CD80, CD83 and CCR7 could not mor immunity. Previously, we reported that 6-sulfo be detected. Whereas the proinflammatory cytokines LacNAc+ (slan) DCs (formerly termed M-DC8+ DCs), TNF-alpha and IL-12 were not found, a proportion of representing a myeloid human blood DC subset, slanDCs showed expression of the anti-inflammatory produce various proinflammatory cytokines, display cytokine IL-10. Further studies revealed that primary cytotoxic activity and efficiently stimulate natural ccRCC cells efficiently impair the capacity of slanDCs killer (NK) cells and T lymphocytes (Immunity to stimulate CD4+ and CD8+ T-cell proliferation and 2002;17:289-301, Immunity 2006;24:767-777). More Th1 programming. In addition, ccRCC cells signifi- recently, it has been demonstrated that slanDCs ac- cantly inhibited slanDC-mediated NK cell activation. cumulate in metastatic tumor-draining lymph nodes In conclusion, these findings indicate that higher from cancer patients (Nat Commun 2014;5:3029). slanDC numbers in ccRCC tissues are associated These findings indicate that slanDCs may contribute with poor prognosis. The induction of a tolerogenic to antitumor immunity. phenotype in slanDCs leading to an insufficient ac- In the present study, we investigated the character- tivation of innate and adaptive antitumor immunity istics of human slanDCs in clear cell renal cell car- may represent a novel immune escape mechanism cinoma (ccRCC). When evaluating 263 ccRCC and of ccRCC. These observations may have implications 227 tumor-free tissue samples, we found increased for the design of therapeutic strategies that harness frequencies of slanDCs in ccRCC tissues compared tumor-directed functional properties of DCs against to tumor-free tissues. slanDCs were also detectable ccRCC. in the majority of 24 metastatic lymph nodes and 67 distant metastases from ccRCC patients. In addition, we associated the frequency of slanDCs in ccRCC tissues with important clinico-pathological charac- teristics of patients. Remarkably, a higher density of slanDCs was significantly correlated with a reduced progression-free, tumor-specific or overall survival of ccRCC patients. Freshly prepared ccRCC-infiltrat- 290 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Prognostic value of tumor-infiltrating lymphocytes in patients with high grade serous ovarian cancer is dependent on treatment regimen, surgical outcome and T cell differentiation
Wouters M.C.A.1,2, Komdeur F.L.1, Workel H.H.1, Klip H.G.1, Plat A.1, Kooi N.M.3, Wisman G.B.A.1, de Jong S.3, Hollema H.4, Duiker E.W.4, Daemen T.2, de Bruyn M.1, Nijman H.W.1
1University of Groningen, University Medical Center Groningen, Obstetrics and Gynecology, Groningen, Netherlands, 2University of Groningen, University Medical Center Groningen, Medical Microbiology, Groningen, Netherlands, 3University of Groningen, University Medical Center Groningen, Medical Oncology, Groningen, Netherlands, 4University of Groningen, University Medical Center Groningen, Pathology, Groningen, Netherlands
Purpose: Tumor-infiltrating lymphocytes (TILs) are and CD27 TIL showed a similar prognostic benefit in associated with a better prognosis in high grade patients where complete primary surgical debulking serous ovarian cancer (HGSC). The prognostic effect was achieved. By contrast, only CD27 proved to be of TIL in relation to chemotherapeutic treatment a prognostic factor in patients with residual macro- and surgical outcome, however, remains controver- scopic tissue after surgery. In patients treated with sial. In large part, this is likely due to the type of neo-adjuvant chemotherapy, the total number of in- TIL studied (CD3 vs. CD8), and the heterogeneity in filtrating CD8+ and CD27+ cells was not different the studied patient populations. To address this, we from that of patients that had not received chemo- assessed CD8+ TIL infiltration in a homogeneous therapy. Interestingly, neither CD8 or CD27 proved to cohort of advanced stage HGSC. We hypothesized be of prognostic benefit in these patients. that CD27+ TIL (representing a less differentiated Conclusions: Based on our data, CD27 represents an subset of TIL) would be associated with a superior interesting target for agonistic immunotherapy in prognostic benefit to CD8+ in HGSC patients. There- HGSC. Furthermore, our findings indicate the differ- fore, we studied the CD27+ TIL population in HGSC entiation status of TILs, as well as treatment regimen and determined whether expression of this marker and surgical result should be taken into account influenced the prognostic value of T cells in relation when studying immune factors in ovarian cancer, or to residual tumor tissue after surgery. selecting patients for immunotherapy trials. Experimental Design: TILs isolated from fresh tumor tissue were phenotyped by multi-parameter flow cy- tometry. Based on that, Tissue Microarray (TMA) slides with samples of 171 patients treated with cytoreductive surgery combined with adjuvant or neo-adjuvant chemotherapy were analyzed for CD8 and CD27 cell infiltration by immunohistochemistry. Furthermore, co-expression of CD8 and CD27 was determined by immunofluorescencs on full slides. Results: CD27+ TIL were found to predominantly be a (CD8+) CTL subset, expressing CD45RO, CCR7 and PD1. Furthermore, the recently described tumor- reactive CD137+ TIL subset was found predomi- nantly within the CD8+CD27+ subset of TILs. CD8 291 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
Patient-derived tumor transplant model in humanized mice: a model for investigation of immune therapy
Wulf-Goldenberg A.1, Stecklum M.1, Böhmer M.1, Fichtner I.1,2, Hoffmann J.1
1epo GmbH, Berlin, Germany, 2MDC Berlin-Buch, Berlin, Germany
The identification and validation of new targets for mor-bearing humanized mice and found ipilimumab antitumor immune therapy is still a challenge for the could lead to a slight tumor growth delay and an preclinical research as the classical syngeneic tumor increased percentage of T-cells in the blood and in models are of limited translational value and patient the tumor. derived human tumor xenograft models (PDX) are Initially results revealed that our humanized mouse growing on immunodeficient animals. models could enable a more appropriate preclinical To overcome these constraints our aim is the devel- assessment of immune-based therapeutic antitumor opment of PDX models on mice with a functional strategies especially when combining the human- human immune system to improve predictability of ized mouse with patient-derived xenografts. drug efficacy and safety. We reconstituted a human immune system by en- grafting human hematopoietic stem cells isolated from cord blood in immunodeficient mice. Twelve weeks later stable expression of lymphoid cell lin- eages in peripheral blood and secondary lymphoid tissues could be detected by flow cytometry and immunohistochemistry analysis. To evaluate the lineage-specific differentiation of human cells, mice were treated with different cytokines (IL-3, IL-2 and IL-15) after stem cell transplantation. Treatment with cytokines resulted in a decrease of circulating human leukocytes as well as the portion of B cells and an increase of monocytes and NK cells in the peripheral blood and bone marrow. At the time when the human immune system is de- veloped, established patient-derived tumors were transplanted on these humanized mice. Human tumors developed on humanized mice without evi- dence of rejection. We also tested the effect of ipilimumab (anti-CTLA-4 [cytotoxic T-lymphocyte antigen-4] mAB) in these tu- 292 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
The impact of secondary iTreg clones in the TCR repertoire of tumor patients
Xydia M.1, Ruggiero E.2, Mastitskaya S.2, Halama N.2, Schmidt M.2, von Kalle C.2, Beckhove P.1
1German Cancer Research Center (DKFZ), Heidelberg, Germany, 2National Center for Tumor Diseases, Heidelberg, Germany
Regulatory CD4+ T cells (Treg) have a detrimental TCRβ amplification with a multiplex single-cell PCR effect in efficient cancer immunotherapy. Recog- method. The TCR pool of Treg and Tconv was char- nizing the tumor as self, they suppress the function acterized after RNA extraction and high-throughput of effector T cells (Teff) hindering tumor eradica- TCRβ sequencing. Interestingly, the Treg subset in tion. Increased Treg numbers have been reported tumor patients contained a few massively expanded in various tumor entities and correlate with poor clones compared to the highly polyclonal Treg pool patient survival. Animal studies suggest that Treg in healthy individuals. A similar oligoclonal pattern accumulation may be due to the expansion of pre-ex- was also observed within the TA-specific Tconv isting thymic-derived natural Treg but also through subset. Nonetheless, dominant Treg clones were not the conversion of conventional T cells (Tconv) into identical with highly expanded clones within the induced Treg (iTreg) upon suboptimal stimulation TA-specific Teff or the total Tconv population. In under tumor-driven suppressive conditions. However, general, total Treg and total Tconv showed no major little is known about iTreg generation in cancer pa- overlap in peripheral blood of tumor patients and tients. Therefore, we investigated the existence of healthy individuals, arguing against the existence iTreg in tumor patients and their contribution to the of tumor-specific iTreg in the circulation. To investi- total Treg TCR pool in the periphery. To this end, pe- gate whether iTreg exist within the breast tumors of ripheral blood from breast cancer patients was used the same patients, we analyzed the TCR repertoire to characterize the TCR repertoire of total Treg and of tumor-infiltrating CD4+Tconv and CD4+Treg using compare it to the TCR pool of Tumor Antigen (TA)-re- single-cell Laser Microdissection combined with a active Teff or total Tconv for the identification of over- novel gDNA-based TCRβ sequencing method. Within lapping TCRs, representing iTreg. The same analysis the tumor CD4+Tconv and CD4+Treg shared domi- was performed in healthy individuals, as negative nant highly expanded clones representing 10%-65% control. In brief, purified T cells were separated into of the tumor-infiltrating Treg repertoire but with no highly pure Treg (CD4+CD25+CD127-) and Tconv impact on Treg in peripheral blood. In conclusion, (CD4+CD25-CD127+/-) using FACS sorting. Tconv were our data suggest that secondary Treg conversion stimulated with autologous dendritic cells present- from dominant Tconv clones within the tumor may ing either the breast cancer-antigen Mammaglobin have a major impact on the tumor-infiltrating but not (MAM I) or the negative control human Immuno- on the circulating Treg population. globulin (IgG). Antigen-reactive IFNγ+CD4+Teff were isolated by the IFNγ-secretion assay and their TCR repertoire was analyzed using single-cell sorting and
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