eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-01
HPV-Associated Tumor Eradication by Vaccination with Synthetic Short Peptides and Particle-Forming Liposomes X. He1, S. Zhou1, B. Quinn1, D. Jahagirdar2, J. Ortega2, S. Abrams3, J. Lovell1 1University at bufflo, Biomedical engineering, Amherst, United States, 2McGill University, Department of Anatomy and Cell Biology, Montreal, Canada, 3Roswell Park Comprehensive Cancer Center, Department of Immunology, Buffalo, United States
Text Human papilloma virus (HPV)-16 is associated with cervical cancers and induces expression of the E6 and E7 oncogenes. Using a murine cell line that expresses these, the genes were sequenced and 6 predicted major histocompatibility complex (MHC) class I (MHC-I) epitopes were identified. A liposomal vaccine adjuvant based on cobalt-porphyrin-phospholipid (CoPoP) was admixed with synthetic 9-mer epitopes appended with 3 histidine residues, resulting in rapid formation of stable peptide-liposome particles. Synthetic monophosphoryl lipid A and QS-21 were also included in the liposome bilayer. Immunization with multivalent peptides led to protection from tumor challenge. Of the peptides screened, only the previously identified E749-57 epitope was functional. The peptide-liposome particles that formed upon mixing E7HHH49-57 with CoPoP liposomes were stable in serum and avidly taken up by immune cells in vitro. Immunization with
E7HHH49-57 admixed with CoPoP liposomes resulted in robust protection from tumor challenge and re- challenge. Immunization with the synthetic short peptide at a dose of 100 ng protected mice in a therapeutic tumor challenge when admixed with CoPoP liposomes, whereas 200-fold higher peptide doses were ineffective with the Poly(I:C) adjuvant. CoPoP induced strong infiltrating CD8+ T response within the tumor microenvironment with improved functional profile. Vaccine monotherapy using nanogram dosing of the
E7HHH49-57 peptide admixed with CoPoP reversed the growth of large established tumors, permanently eradicating subcutaneous tumors upwards of 100 mm3, and also completely eradicated lung tumors in a metastasis model.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-02
Tuning LNPs to target antigen presenting cells in spleen induces CD8 T-cell responses and tumor regression in mice S. Bevers1, 2, S. A. A. Kooijmans3, E. Van de Velde1, M. J. Evers3, S. Seghers1, J. J. Gitz-François3, L. Hassler4, K. Breckpot2, T. Bastogne4, 5, R. M. Schiffelers3, S. De Koker1 1eTheRNA Immunotherapies, Niel, Belgium, 2Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Brussels, Belgium, 3CDL Research, University Medical Center Utrecht, Utrecht, Netherlands, 4CYBERnano, Villers-lès-Nancy, France, 5CRAN, Université de Lorraine, CNRS, INRIA BIGS, Vandœuvre- lès-Nancy, France
Text Intravenous (i.v.) delivery of vaccines is emerging as an appealing approach to evoke high-quality T-cell responses needed to combat tumors. Messenger RNA (mRNA) holds huge potential to use in vaccination because of its ease of production, intrinsic adjuvant activity and the high versatility in antigen design, but also requires a potent delivery vehicle to enable antigen expression and immune activation. Lipid based nanoparticles (LNP) are currently the clinically most advanced tool to protect mRNA from degradation and efficiently deliver it inside cells. Yet, i.v administered LNPs generally tend to distribute to the liver. We hypothesized LNPs can be altered to target antigen presenting cells in the spleen and hereby orchestrate specific and durable immune responses against cancer antigens. By using a design of experiment methodology we were able to screen LNPs in a cost and time effective manner. LNP compositions that were evaluated solely differed in the molar ratios of ionizable lipid, phospholipid, cholesterol and PEG-lipid and the choice of the PEG-lipid. Bayesian Regression modeling enabled us to identify LNP compositions conferring maximum immunogenicity upon i.v. administration. To link LNP compositions to T cell responses and biodistribution, we prepared the same LNPs packaging either the viral oncoprotein E7 as an antigen or Cy-5 labelled luciferase mRNA. The optimized LNP compositions showed increased localization in the spleen (of mice and non-human primates) and uptake by antigen presenting cells. Furthermore, they induced high magnitude CD8 T cell responses that conferred strong antitumor immunity and prolonged survival of TC-1 tumor bearing mice. Mechanistically, type I interferons and phagocytes were found to be essential for eliciting strong T-cell responses. Unexpectedly, we identified B cells as major mediators of the vaccine- elicited T-cell response, revealing a previously undocumented role of these cells in the immune response to mRNA LNPs. Our data highlight the potential of optimizing LNP compositions by tailoring of the molar ratio of the lipids compromising the LNPs and provide insight in the cell types involved in the mRNA LNP-induced immune response. Collectively, our results illustrate the great promise of intravenous mRNA LNP vaccination for the treatment of cancer.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-03
Rapid prime-boost amplification of T cell responses using heterologous, costimulatory liposome vaccines T. Wirth1, D. Ostroumov1, B. Slütter2 1Medical School Hannover, Gastroenterology, Hepatology and Endocrinology, Hannover, Germany, 2University of Leiden, Division of Biotherapeutics, Leiden, Netherlands
Text Therapeutic vaccinations for cancer therapy require rapid and reliable induction of cancer-specific T cell responses in patients. Current vaccination regimens, however, rely on repeated antigen stimulations over an extensive period of time and still fail to reach the magnitude required for therapeutic effects. Here we describe the establishment of a heterologous vaccination regimen that is able to induce robust CD4 and CD8 T cells responses in less than two weeks in mice. Following a primary immunization with liposomes (LS) containing peptide and the STING agonist cdi-GMP, the resulting immune response is boosted only seven days later with a combination of a costimulatory agonist (Co), antigen in the form of soluble peptide (A) and a TLR3 agonist (T). Using this heterologous LS-CoAT prime-boost vaccination we demonstrate robust expansion of neoantigen-specific CD8 T cells, reaching a magnitude of up to 40% of Adpgk-specific T cells in the total CD8 T cell population after 14 days. LS-CoAT could be used with both short and long peptides and was similarly efficient in inducing CD4 T cell responses or combined CD4/CD8 T cell responses. LS-CoAT therefore represents a novel heterologous prime-boost vaccination approach which induces rapid T cell responses of unprecedented magnitude, thus warranting further evaluation of this vaccination method for cancer treatment.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-04
Developing a therapeutic HPV16 vaccination strategy utilizing a vaginal tumor model in MHC-humanized mice S. Zottnick1, 2, 3, S. Kruse1, 2, M. Bozza4, A. Voß1, 2, A. Klevenz1, 2, R. Yang5, F. Rösl5, R. P. Harbottle4, A. B. Riemer1, 2 1German Cancer Research Center (DKFZ), Immunotherapy and Immunoprevention, Heidelberg, Germany, 2German Center for Infection Research (DZIF), partner site Heidelberg, Molecular Vaccine Design, Heidelberg, Germany, 3Heidelberg University, Faculty of Biosciences, Heidelberg, Germany, 4German Cancer Research Center (DKFZ), DNA Vectors, Heidelberg, Germany, 5German Cancer Research Center (DKFZ), Viral Transformation Mechanisms, Heidelberg, Germany
Text We recently demonstrated the therapeutic efficacy of amphiphilic peptide vaccinations in a subcutaneous HPV16 (human papillomavirus 16) tumor model in MHC-humanized A2.DR1 mice. To better mimic the situation in patients, we established the model at an orthotopic location, namely the vaginal mucosa, as the final goal is to examine tumors located in this region to mimic cervicovaginal cancers. The mucosa is more difficult to access for T cells than the subcutis, therefore the orthotopic location will allow for a more detailed understanding of the requirements for successful therapeutic HPV16 vaccination. A suitable tumor model has to express the vaccination target proteins HPV16 E6 and E7, as well as an activated oncoprotein for tumorigenicity and luciferase for eventual tracking of tumor growth. For this project, we followed two paths: first, we adapted our HPV16-positive, but E6/E7-independent, tumor model PAP-A2 for the orthotopic setting and second, we developed a new tumor model based on E6/E7-dependent lung cells from A2.DR1 mice. Both cell types were transduced with HPV16 E6 and E7, transfected with luciferase and - in the case of the lung cells - with activated H-Ras to render them tumorigenic. Western blot analysis confirmed the expression of E6, E7 and H-Ras, and luminescence measurements proved luciferase expression. After validation of tumorigenicity of subcutaneously injected cells, cells were instilled intravaginally into A2.DR1 mice. Subsequent monitoring of the genital region revealed the growth of tumors in the vaginal cavity. We also determined the minimal cell number needed for reliable tumor formation. In addition to the establishment of the vaginal tumor model in A2.DR1 mice, we examine strategies to induce the migration of HPV16-specific T cells to the mucosa. This can be either achieved by directly inducing tissue-resident T cells on-site or to influence the trafficking of vaccination-induced T cells towards the vaginal mucosa. Vaccine candidates are peptide-coupled or peptide-loaded nanoparticles, peptides encapsulated in liposomes or mRNA vaccines. The trafficking of T cells towards the vagina can be achieved by utilizing immunomodulating substances or by induction of inflammation, for example by irradiation. The induction and attraction of HPV16-specific T cells will then be tested for their capacity to tackle the previously established vaginal tumors. Taken together, the new HPV16 orthotopic tumor model in MHC-humanized A2.DR1 mice will help to develop effective therapeutic HPV16 vaccinations by allowing to study and influence the trafficking of T cells to the mucosal tumor site.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-05
Individualized Multimodal Immunotherapy for cervical cancer: a single insitution experience S. Van Gool, J. Makalowski, V. Schirrmacher, W. Stücker Immun-Onkologisches Zentrum Köln, Köln, Germany
Text In spite of several treatment options today, the overall prognosis for patients with cervical cancer remains poor, and innovative treatments are needed. We retrospectively analyzed patients treated with IO-Vac®, an autologous DC vaccine approved in May 2015 by the German authorities as advanced therapy medicinal product for use in human. IO-Vac® was given as part of individualized multimodal immunotherapy (IMI) consisting of Newcastle Disease Virus injections (NDV) + sessions of modulated electroyhperthermia (mEHT) + IO-Vac® + personalized immunomodulatory strategies. Since then, 11 patients were treated. Median age at diagnosis and at start of IMI was 41y resp 43y. Only one patient had local disease at first diagnosis. IMI was started as part of first line treatment in 3 patients (2 patients surgery + IMI, one patient biopsy + radiochemotherapy + IMI), at first relapse in 3 patients, or later-on in the disease course (3-6 events). Patients received in median 2 (1-3) IO-Vac®, 25 (3-73) sessions of NDV/mEHT and 3 (2-8) total body hyperthermia sessions. Four patients received Ozon treatment, 3 patients received additional Keytruda and 2 patients received Herceptin. Antibody treatments were based on the analyses of target mRNA expression in circulating cancer cells. Six patients are alive with a median follow up of 55 months (16-111) after diagnosis and 30m (2-65) after start of IMI. Four patients died in median 33m (13-52) and 11m (6-27) after diagnosis resp start of IMI. Treatment was given in ambulatory setting, and no major adverse reactions were detected. IMI can contribute to improve OS for patients with cervical cancer. Further investigations on larger groups of patients with longer follow up are needed to demonstrate the additive value of IMI in the treatment of cervical cancer.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-06
The retroviral engineering of a human plasmacytoid dendritic cell-based vaccine allowed the priming and expansion of multispecific viral and tumor antigen-specific T-cells in multiple HLA contexts. K. Lenogue1, A. Walencik2, K. Laulagnier1, J.-P. Molens2, H. Benlalam3, B. Dreno4, P. Coulie5, M. Pule6, L. Chaperot2, J. Plumas1 1PDC*line Pharma, La Tronche, France, 2Etablissement Français du Sang Auvergne Rhône-Alpes, Research and development laboratory, La Tronche, France, 3CRCINA, INSERM, Université de Nantes, Nantes, France, 4CHU Nantes, Onco-dermatology department, Nantes, France, 5De Duve Institute, Université Catholique de Louvain, Brussels, Belgium, 6Cancer Institute, University College London, London, United Kingdom
Text Because dendritic cells are crucial to prime and expand antigen-specific CD8+ T-cells, several strategies are designed to use them in therapeutic vaccines against infectious diseases or cancer. In this context, off-the- shelf allogeneic dendritic cell-based platforms are more attractive than individualized autologous vaccines tailored to each patient. We have previously shown that a unique dendritic cell line (PDC*line) platform of plasmacytoid origin, was able to prime and expand tumor-specific CD8+ T cells in vitro and in vivo in a first- in-human clinical trial with melanoma patients. The aim of the present study was to improve the PDC*line platform using retroviral engineering. The transduced PDC*line cells were cocultured with either Peripheral blood mononuclear cells (PBMCs) or antigen-specific T-cell clones. The antigen presentation efficiency was displayed by the expansion of antigen-specific CD8+ T cells present in PBMCs or the secretion of cytokines by T-cell clones measured using flow cytometry. We demonstrated that the clinical-grade PDC*line transduced with genes encoding whole viral or tumoral proteins efficiently processed the transduced antigens and stably presented the derived peptides to specific CD8+ T cells both in HLA-A*02:01 and HLA- B*07:02 molecules expressed by PDC*line. When PDC*line cells were transduced with retroviral constructs encoding a polyepitope composed of four HLA-A*02:01-restricted peptides from the tumoral or viral antigens, the cells were able to mount a multispecific CD8+ T-cell response against peptides of the polyepitope. We also demonstrated that the addition of a part of the sequence of the Lysosome-associated membrane protein-1 (LAMP-1) to the whole protein or to the polyepitope greatly improved the presentation of some peptides. Lastly, we used retroviral transduction to express a new HLA class I molecule, HLA-B*35:02, in PDC*line. After loading with HLA-matched peptides, this new PDC*line successfully presented the peptides in the endogenous HLA-A*02:01 or in the new HLA-B*35:02 molecules. The HLA-A*24:02 allele was also of interest as the Asian population expresses it more than the Caucasian population. The PDC*line transduced with HLA-A*24:02 and loaded with the HLA-matched peptide from the HIV protein Nef was able to prime and expand specific CD8+ T-cells (unpublished results). The retroviral engineering can thus benefit a broadened population of patients through the easy addition of new HLA class I molecules. The demonstration of the effective retroviral transduction of PDC*line cells strengthens and broadens the scope of the PDC*line antigen presentation platform, which can be used in adoptive or active immunotherapy.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-07
Exploiting the pre-existing immunity to enhance oncolytic cancer immunotherapy S. Feola1, S. Tähtinen1, C. Capasso1, N. Laustio1, C. Groeneveldt2, E. O. Ylösmäki1, L. Ylösmäki3, B. Martins1, M. Fusciello3, M. Medeot4, M. Tagliamonte5, J. Chiaro1, F. Hamdan6, K. Peltonen3, T. Ranki7, L. Buonaguro5, V. Cerullo3 1University of Helsinki, Pharmacy, Helsinki, Finland, 2Faculty of Science, Leiden University, Leiden, Netherlands, 3University of Helsinki, Helsinki, Finland, 4University of Padova, Department of Pharmaceutical and Pharmacological Sciences, Padova, Italy, 5Istituto Nazionale Tumori IRCCS Fondazione Pascale, Napoli, Italy, 6University of Helsinki, Pharmacy, Finland, 7ValoTherapeutics, Helsinki, Finland
Text Due to the high coverage of international vaccine programs, majority of the worldwide population has been vaccinated against common pathogens, thus generating a strong repertoire of memory T cells against pathogens. Following immunization, naïve T cells differentiate into T effector memory (TEM) cells that are rapidly re-called by encountering the antigen, and into T central memory (TCM) cells that are found mainly in lymphoid organs and are not immediately triggered in response to pathogens. Cancer immunotherapy relies on the engagement of CD8+ anti-tumor cytotoxic T lymphocytes (CTLs) that recognize peptides presented on the major histocompatibility complex I (MHC-I). However, it has been demonstrated that the cooperation of CD4+ and CD8+ T cells is required for an efficient anti-tumor immunity to occur. Indeed, CD4+ T cells provide signals that improve the functionality of CD8+ T cells within the tumor microenvironment (TME). Even though the CD4+T cell help is required for an optimally strong anti-tumor immune response to occur, it is still not clear and not proper investigated how to capitalize the crosstalk between CD4+ and CD8+ T cells in cancer treatment regiments. Therefore, we want to investigate whether the pathogen related CD4+T cell memory populations could be re-engaged to support the CTLs, converting a weak primary anti-tumor immune response unto a stronger secondary one. To this end, we used PeptiCRAd; this technology consists of an oncolytic adenovirus coated with MHC-I restricted tumor-specific peptides that we developed it further by introducing pathogen specific MHC-II-restricted peptides. We used tetanus vaccine pre-immunized mice, challenged with B16.OVA tumors and treated with the newly developed hybrid TT-OVA-PeptiCRAd containing both tumor- and tetanus toxoid-specific peptides. The treatment with such hybrid PeptiCRAd showed significantly enhanced anti-tumor efficacy and led to the induction of TT-specific, CD40 ligand expressing CD4+ T helper cells and maturation of antigen presenting cells (APCs). Moreover, the anti-tumor effect was even more prominent when combined with the immune checkpoint inhibitor anti-PD1, strengthening the rationale behind combination therapy with oncolytic viruses. We validated this strategy by targeting different pathogens (e.g. Diphtheria and Pertussis).Consistent with our previous results, engagement of CD4+ T cells by Diphtheria-Pertussis-specific MHC-II-restricted peptides should result in a slower tumor growth in pre-immunized mice.These results indicated that the proposed mechanism of action is not restricted to tetanus, but the principle could be applied to other vaccine formulations.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-08
T-cell responses induced by an individualized neoantigen specific immune therapy in post (neo)adjuvant patients with triple negative breast cancer I. Vogler1, E. Derhovanessian2, T. Omokoko3, S. Attig4, N. Bidmon1, A. Cortini3, S. Newrzela5, J. Grützner2, S. Bolte6, D. Langer6, M. Eichbaum7, H. Lindman8, S. Pascolo9, M. Schmidt10, A. Schneeweiss11, T. Sjöblom12, Ö. Türeci13, U. Sahin13 1BioNTech SE, Immunotherapies and Pre-clinical Research - Immunogenicity Testing, Mainz, Germany, 2BioNTech SE, Biolytics, Mainz, Germany, 3BioNTech Cell & Gene Therapies GmbH, TCR Discovery, Mainz, Germany, 4TRON gGmbH, IDC Immunomonitoring,, Mainz, Germany, 5BioNTech Cell & Gene Therapies GmbH, Immunoreceptor Validation, Mainz, Germany, 6BioNTech SE, Clinical Operations, Mainz, Germany, 7Helios Dr. Horst Schmidt Kliniken, Klinik für Gynäkologie und Gynäkologische Onkologie, Wiesbaden, Germany, 8University Hospital Uppsala Akademiska Sjukhuset, Department of Immunology, Genetics and Pathology, Uppsala, Sweden, 9University Hospital Zurich, Department of Dermatology, Zürich, Switzerland, 10Klinik und Poliklinik für Geburtshilfe und Frauengesundheit Universitätsmedizin der Johannes Gutenberg- Universität Mainz, Konservative und Molekulare Gynäkologische Onkologie, Mainz, Germany, 11University Hospital and German Cancer Research Center, Gynecologic Oncology Division, National Center for Tumor Diseases, Heidelberg, Germany, 12Uppsala University, Department of Immunology, Uppsala, Sweden, 13BioNTech SE, Management Board, Mainz, Germany
Text Triple negative breast cancer (TNBC) presents with an aggressive clinical phenotype lacking expression of conventional drug targets. Overall survival in early- and advanced-stage disease is poor. The objective of the phase I TNBC-MERIT trial (NCT02316457) was to assess the feasibility, safety, and immunogenicity of a liposome-formulated intravenous RNA vaccine encoding different tumor antigen categories in early TNBC following surgery and (neo-) adjuvant chemotherapy. Patients in two arms were vaccinated with a personalized set of pre-manufactured non-mutated shared tumor-associated antigens with or without universal T helper epitopes. In a third arm, patients were vaccinated with IVAC_M_uID, an on-demand manufactured Individualized NeoAntigen Specific Immunotherapy (iNeST) encoding neoepitopes derived from up to 20 cancer mutations determined by next generation sequencing (NGS). This report provides a first data analysis of immune responses in vaccinated patients as analyzed by IFNγ-ELISpot, multimer staining, TCR repertoire profiling and single cell TCR sequencing. Immunogenicity data was generated from all 14 patients treated with the neoantigen vaccine. All analyzed patients had vaccine-induced CD4+ and/or CD8+ T-cell responses against 1 to 10 of the vaccine neoepitopes detected by IFNγ ELISpot, ex vivo or after in vitro stimulation. A substantial number of T-cell responses against individual neoepitopes were induced de novo and of high magnitude (up to 10.3% of peripheral CD8+ T cells). One of the index patients characterized in more detail mounted CD4+ and/or CD8+ T-cell responses against 10 of 20 vaccine neoepitopes. The highly poly-epitopic TCR-clonotype diversified CD8+ T-cell response comprised in aggregate about 30% of total peripheral CD8+ T cells after the last vaccination. This patient was further monitored after the end of trial, and vaccine-specific CD8+ T cells still comprised 8% of all CD8+ T cells 600 days after the last vaccination. Vaccine-induced CD8+ T cells were of effector/memory, PD1+ phenotype, with a high fraction IFNγ/TNFα/Mip-1a&b triple-positive. This first data suggests that the vaccine is highly efficient in inducing strong poly-epitopic T-cell responses in patients with TNBC in the post-(neo) adjuvant setting, justifying further trials to assess the efficacy of the vaccine in this patient population.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-10
Superior T cell responses from DNA vaccines encoding Fc-engineered antibody and Fc-fusion proteins through enhanced intermolecular cooperativity V. Brentville1, M. Vankemmelbeke1, P. Symonds1, K. Cook1, R. Metheringham1, L. Durrant1, 2 1Scancell Ltd, Nottingham, United Kingdom, 2University of Nottingham, Division of Cancer & Stem Cells, Nottingham, United Kingdom
Text Anti-tumour immunity requires strong CD4 responses to overcome the suppressive tumour environment and high avidity CD8 responses to efficiently kill tumour cells. Immunisation of mice with an antibody-encoding DNA vaccine (ImmunoBodyTM) stimulates potent CD4 responses and high avidity CD8 T cell responses compared to standard peptide or DC based vaccination. High avidity responses are generated through direct transfection of antigen presenting cells and cross presentation via Fc-mediated interaction with FcγR1 expressed on activated dendritic cells. These T cells mediate efficient tumour therapy in human clinical trials.1 We have previously shown that Fc-engineering of a human IgG1 antibody to contain residues from murine IgG3 promotes non-covalent association between neighbouring Fc regions resulting in superior binding and function of the antibody.2 Here, we apply this approach across two DNA vaccine modalities, encoding tumour-epitope containing antibody and COVID19 Fc-fusion proteins. We demonstrate that the vaccines containing the modified human IgG1 Fc region stimulate higher frequency and avidity T cell responses and superior anti-tumour responses compared to vaccines containing the unmodified Fc region. This suggests that enhanced avidity for FcγRI results in more efficient antigen presentation and translates into superior tumour therapy. The creation of an improved vaccine, with enhanced immunogenicity, through establishing intermolecular cooperativity binding may lead to superior clinical utility.
1. Patel PM, et al. Targeting gp100 and TRP-2 with a DNA vaccine: Incorporating T cell epitopes with a human IgG1 antibody induces potent T cell responses that are associated with favourable clinical outcome in a phase I/II trial. Oncoimmunology 2018;7(6):e1433516. 2. Vankemmelbeke, et al. Engineering the Human Fc Region Enables Direct Cell Killing by Cancer Glycan- Targeting Antibodies without the Need for Immune Effector Cells or Complement. Cancer research 2020;80(16):3399-412.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-11
Achieving dendritic cell subset-specific targeting in vivo by site-directed conjugation of targeting antibodies to nanocarriers M. Fichter1, 2, M. Brückner2, J. Simon2, G. Kuhn1, J. Schunke1, T. Klaus1, R. da Costa Marques1, S. Grabbe1, K. Landfester2, V. Mailänder1, 2 1University Medical Center of the Johannes Gutenberg University Mainz, Department of Dermatology, Mainz, Germany, 2Max Planck Institute for Polymer Research, Mainz, Germany
Text The major challenge of nanocarrier-based anti-cancer vaccination approaches is their targeted delivery of antigens and immunostimulatory agents to cells of interest, such as specific subtypes of dendritic cells (DCs), in order to induce robust antigen-specific anti-tumor responses. An undirected cell and body distribution of nanocarriers can lead to unwanted delivery to other immune cell types like macrophages reducing the vaccine efficacy. An often-used approach to overcome this issue is the surface functionalization of nanocarriers with targeting moieties, such as antibodies, mediating cell type-specific interaction. Numerous studies could successfully prove the targeting efficiency of antibody-conjugated carrier systems in vitro, however, most of them failed in vivo when targeting DCs that is partly due to cells of the reticuloendothelial system unspecifically clearing nanocarriers from the blood stream via Fc receptor ligation. Therefore, this study shows a surface functionalization strategy to site-specifically attach antibodies in an orientated direction onto the nanocarrier surface. Different DC-targeting antibodies, such as anti-CD11c, anti-CLEC9A, anti-DEC205 and anti-XCR1, were conjugated to the nanocarrier surface at their Fc domains. Anti-mouse CD11c antibody-conjugated nanocarriers specifically accumulated in the targeted organ (spleen) over time. Additionally, antibodies against CD11c and CLEC9A proved to specifically direct nanocarriers to the targeted DC subtype, conventional DCs type 1. In conclusion, site-directed antibody conjugation to nanocarriers is essential in order to avoid unspecific uptake by non-target cells while achieving antibody-specific targeting of DC subsets. This novel conjugation technique paves the way for the development of antibody-functionalized nanocarriers for DC-based vaccination approaches in the field of cancer immunotherapy.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-12
Multicomponent adjuvantation of antigen-based nanocapsules using site-directed click chemistry crosslinking for the treatment of melanoma J. Schunke1, N. Hüppe2, M. Fichter1, 2, D. Paßlick1, S. Grabbe1, K. Landfester2, V. Mailänder1 1University Medical Center of the Johannes Gutenberg University, Dermatology, Mainz, Germany, 2Max Planck Institute for Polymer Research, Mainz, Germany
Text Nanocarrier-based antigen delivery is a promising vaccination approach in the context of tumor therapy. Formulating polymeric nanocapsules (NCs) out of tumor antigens in combination with vaccine adjuvants enables efficient targeting and maturation of dendritic cells (DCs), essential prerequisites for the induction of vigorous cellular immune responses. Aim of the present study was the synthesis of polymeric protein nanocapsules composed exclusively of vaccine antigens and encapsulated with combinations of adjuvants, as well as the evaluation of their potential to induce antigen-specific immune responses. The NCs consisting of either Human Serum Albumin (HSA) or ovalbumin (OVA), were bioorthogonally crosslinked by copper-free azide-alkyne Click-Chemistry using the inverse miniemulsion technique. This method ensures integrity and processability of crosslinked antigens leading to effective epitope presentation by dendritic cells. The inverse miniemulsion approach led to polymeric nanocapsules with a spherical morphology that were efficiently ingested by DCs. In addition, a combination of the vaccine adjuvants Resiquimod (R848), muramyl dipeptide (MDP) and Polyinosinic:Polycytidylic acid (Poly I:C) was encapsulated to efficiently trigger strong DC activation analyzed by costimulatory surface marker expression and the secretion of pro-inflammatory cytokines. The induction of robust antigen-specific T cell proliferation was observed in DC-T cell co-cultures. The high biocompatibility and the suitability of this vaccination platform was shown using the OVA-NC formulation as a therapeutic vaccine in the B16/OVA-Luc melanoma model. In conclusion, multiadjuvant-functionalized protein nanocapsules are a promising delivery system for the combined delivery of antigens and vaccine adjuvants to dendritic cells promoting T cell-based immune responses. This novel anti-tumor vaccination strategy avoids the use of structural compounds, increases the antigen load of DCs and bears the potential to overcome tolerance and to induce vigorous antigen-specific anti-cancer immunity.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-13
Identification of neoantigens features required for effective anti-tumor response of viral vectored vaccines M. De Lucia1, I. Garzia1, L. Avalle2, F. Troise1, L. Nocchi1, F. Langone1, G. Leoni1, G. Cotugno1, V. Bignone1, E. Di Matteo1, V. Poli2, E. Scarselli1, A. M. D'Alise1 1Nouscom Srl, Rome, Italy, 2University of Turin, Department of Molecular Biotechnology and Health Sciences, Turin, Italy
Text Tumor neoantigens (nAgs) are non-self peptides derived from somatic mutations occurring in cancer cells. The lack of expression in normal tissues makes them a promising target for cancer immunotherapy. However, one of the greatest remaining challenges is the successful identification of nAgs that can generate tumor specific T cell responses and have therapeutic effects. Therefore, unravelling the nAgs features crucial for effective anti-tumor response could provide important feedback for improvement of current in silico prediction algorithms. Great Apes-derived adenoviral vectors (GAds) are a potent, clinically validated, vaccine platform that allow the inclusion of multiple nAgs, increasing the chance of targeting relevant T cell epitopes. Exploiting this feature, starting from the colon carcinoma CT26 tumor model, we generated a GAd- based vaccine encoding 31 nAgs, that showed high efficacy in preventing tumor onset and curing large established tumors, in combination with checkpoint blockade. Nevertheless, only 6 out of 31 predicted nAgs were able to induce strong T cell responses, 3 eliciting CD8+ and 3 CD4+ T response. These observations have paved the way to investigate the contribution of each single immunogenic nAg in order to disclose features relevant for anti-tumor efficacy. To that, we generated a series of GAd constructs encoding individual immunogenic reactivities and tested them in prophylactic and therapeutic tumor settings. Results demonstrated that, beyond the capability to induce a specific T cell immune response, other nAgs features have to be taken into account. The ability of tumor-expressed nAgs to elicit spontaneous T cell responses and their expression levels on target tumor cells are indeed further aspects that could contribute to the design optimization of more effective anti-tumor vaccines.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-14
Characterizing the CD8+ T cell effector subtype composition upon treatment with antigen-armed VSV-GP T. Hofer1, 2, D. Berger1, 2, S. Danklmaier1, 2, E. Richter1, 2, D. von Laer1, K. Das1, 2, G. Wollmann1, 2 1Medical University Innsbruck, Institute of Virology, Innsbruck, Austria, 2Medical University Innsbruck, CD- Laboratory for Viral Immunotherapy of Cancer, Innsbruck, Austria
Text VSV-GP (vesicular stomatitis virus pseudo-typed with the glycoprotein of the lymphocytic choriomeningitis virus) is a promising oncolytic virus. The virus has a broad cellular tropism and fast replication cycle. While VSV-GP spread is quickly contained within normal tissue via a potent IFN-I response, tumor cells allow viral replication and lysis as they often exhibit a defective or mutated IFN-I pathway. In addition, VSV-GP shows potential as an anti-cancer vaccine, as it can express transgenes such as tumor- associated antigens (TAA) of up to 3 kb in size. By administrating our VSV-GP in a prime-boost regimen, we were able to increase the frequency of TAA specific cytotoxic T cells. However, high frequency of TAA specific cytotoxic T cells did not always result in a therapeutic effect, confirming previous reports that high frequency of circulating tumor specific cytotoxic T cells does not always correlate with therapeutic benefit. Therefore, we hypothesize that both quantity and quality of TAA specific T cells determine treatment efficacy of our tumor vaccine. We define quality as the surface marker phenotype of CD8+ T cell effector cells. Using flow cytometry of peripheral blood after various vaccine regimen, we assessed the expression of KLRG-1 and/or CD127/IL-7R to discriminate between memory precursor effector cells (MPEC); short-lived effector cells (SLEC), early effector cells (EEC) and double-positive effector cells (DP). We characterized the CD8+ T cell effector phenotype composition of VSV-GP-TAA depending on the dose, the vaccine antigen and the route in non-tumor bearing C57/bl6 mice. In addition, we assessed the CD8+ T cell effector phenotype composition in the blood of tumor-bearing animals. We found that antigen type does not significantly impact the CD8+ T cell effector subtype composition. We did, however, observe a tendency for an increased SLECs portion with increasing vaccine dose, although not statistically significant. Regarding the different routes of application, i.v. and i.m. injections increased the SLEC proportion while decreasing the MPEC proportion compared to s.c. and i.p. vaccinated mice. In regards to tumor-bearing animals, we observed that our VSV-GP-TAA vector displays a SLEC dominant phenotype profile. Our results indicate clear differences in the CD8+ T cell effector subtype composition depending on the route of application. Future studies will focus on the direct comparison of MPECs and SLEC CD8+ T cells regarding their respective roles in treatment efficacy.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-15
Combining intratumoral administration of inflammatory allogeneic DCs with systemic anti-CTLA-4 treatment leads to tumor eradication and is associated with peripheral expansion of CD8+ effector cells with a “tumor-matched” phenotype A. Karlsson-Parra1, A. Iskar2, D. Yu2, C. Jin2 1Immunicum AB, Stockholm, Sweden, 2Uppsala University, Immunology, Genetics and Pathology, Uppsala, Sweden
Text Based on paired single-cell RNA and TCR seq. to detect and characterize circulating CD8+ T cells with TCRs in mice that match to tumor-infiltrating cells, an effector population of “tumor-matched” CD8+ T cells with a unique phenotype signature, including surface protein expression of NKG2D, CD39 and CX3CR1, was recently reported. Notably, non-matching blood CD8+ T cells were generally negative for these markers. The present study was aimed at exploring the frequency of CD8+ T cells expressing these “tumor-matching” markers in the blood of Balb/c mice with CT-26 tumors treated with intratumoral injections of allogeneic inflammatory DCs (aimed at inducing recruitment and activation of endogenous immune cells, including NK cells and DCs) and/or systemic anti-CTLA-4 treatment. BM-derived immature DCs were produced from allogeneic C57BL6 mice and activated ex vivo with a cocktail consisting of polyIC, R848 and IFN-gamma. Balb/c mice with large CT-26 tumors (median volume around 100 mm3 at start of treatment, Day 1) were treated with inflammatory allogeneic DCs intratumorally and/or anti-CTLA-4 (BioXcell clone 9H10) intraperitoneally at Day 1 and Day 7. Monotherapy with allogeneic inflammatory DCs induced a weakly delayed tumor progression while monotherpay with anti-CTLA-4 led to a significant delay in tumor progression, but without complete tumor eradication. In contrast, the combined treatment led to CD8-dependent tumor eradication in 50-80% of treated mice. Mice with complete tumor eradication where found to be protected from tumor growth upon subsequent tumor rechallenge. In a separate study using the same treatment protocols the mice were euthanized at Day 12 when peripheral blood was collected. Monotherapy with anti-CTLA-4 was found to increase the frequency of CD8+ T cells expressing NKG2D, CX3CR1 and CD39 as compared with vehicle-treated control mice, while monotherapy with allogeneic DCs did not substantially affect the frequency of CD8+ T cells expressing these markers. The combined treatment induced an increase of NKG2D and CX3CR1 expressing CD8+ T cells from 2.9% to 7.7% and 5.8 to 8.0%, respectively, as compared to anti-CTLA-4 monotherpay, while the frequency of CD39- expressing CD8+ T cells remained unchanged at 9.5-10%. Taken together, our data indicate that anti-CTLA- 4 treatment leads to expansion of peripheral blood CD8+ T cells with a “tumor-matching” effector phenotype (NKG2D, CXCR1 and CD39 expression), and that addition of intratumorally injected allogeneic inflammatory DCs further enhance the frequency of NKG2D and CXCR1 expressing CD8+ T cells. These data combined with efficacy data in this tumor model suggest an increased activity within central parts of the cancer immunity cycle.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-16
A peptide-based vaccine to induce SARS-CoV-2-specific T cells in cancer patients with disease- or treatment-induced antibody deficiency J. S. Heitmann1, T. Bilich1, 2, 3, C. Tandler1, 2, J. Reusch1, Y. Maringer1, 2, 3, M. Denk2, 4, M. Richter2, 4, A. Nelde1, 2, 3, M. Löffler2, 3, H.-G. Rammensee2, 3, 4, H. R. Salih1, 3, J. S. Walz1, 2, 3, 5 1Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Department of Internal Medicine, University Hospital Tübingen, Tübingen, Germany, 2Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen, Germany, 3Cluster of Excellence iFIT (EXC2180) “Image- Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany, 4German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), partner site Tübingen, Tübingen, Germany, 5Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and Robert Bosch Center for Tumor Diseases (RBCT), Stuttgart, Germany
Text SARS-CoV-2 causing COVID-19 has evolved to a pandemic with dramatic socioeconomic consequences. The recent approval of vaccines provides hope for millions of people at risk for a worse outcome of COVID-19. However, so far largely neglected is the group of patients unable to mount humoral i.e. antibody-mediated SARS- CoV-2 immune responses either in the context of natural infection or upon prophylactic vaccination. This group comprises individuals with congenital B cell deficiency, but notably also cancer patients, in particular after B cell depleting therapy as routinely applied in leukemia and lymphoma. These patients are at high risk for a severe course of COVID-19 and may not receive or will mount only suboptimal immune responses to available vaccines mainly aiming for antibody induction. Besides humoral immunity, T cell immunity is key for the control of viral infections. We developed a peptide-based vaccine termed CoVac-1, which primarily aims for the induction of SARS-CoV-2-specific T cells. CoVac-1 comprises six promiscuous HLA-DR-binding SARS-CoV-2-derived T cell epitopes from various viral proteins, which enables the induction of broad anti-SARS-CoV-2 T cell immunity independent of newly arising mutations. CoVac-1 T cell epitopes are of pathophysiological relevance in the natural course of COVID-19 and also for long term T cell immunity after infection (Nelde et al. Nat Immunol 2021, Bilich et al. Science Transl Med in press). Besides the CoVac-1 peptides, the vaccine contains the novel TLR1/2 ligand XS15 and Montanide ISA 51 VG as adjuvants. CoVac-1 is currently evaluated in a first-in-human clinical trial in healthy adults (P-pVAC-SARS-CoV-2 study, NCT04546841). Preliminary data from the study show a good safety and tolerability profile without relevant systemic adverse events. As intended, all so far vaccinated study subjects (n = 12) developed a granuloma at injection site (Rammensee et al. JIC 2019). We observed T cell induction on day 28 after one single dose of CoVac-1 in 100% of study subjects. Earliest T cell responses were evidenced at day 14 in 92% of cases. Immune responses were directed against multiple of the vaccine peptides (median 5/subject, range 4-6). Moreover, CD8+ T cell responses against HLA class I-presented T cell epitopes, embedded in the HLA-DR-restricted CoVac-1 vaccine peptides, were detected in 75% of study subjects. Thus, CoVac-1 represents a promising vaccine candidate for the induction of broad T cell immunity in the high-risk population of patients with antibody deficiency, which will be evaluated in an upcoming study (B-pVAC-SARS-CoV-2, EudraCT- Nr. 2021-001070-38).
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-17
A modular self-adjuvanting cancer vaccine combined with an oncolytic vaccine induces potent antitumor immunity K. Das1, 2, E. Belnoue3, M. Rossi3, T. Hofer1, 2, S. Danklmaier1, 2, T. Nolden4, L.-M. Schreiber1, 2, K. Angerer1, 2, J. Kimpel2, B. Spiesschaert4, S. Hoegler5, L. Kenner5, 6, D. von Laer2, K. Elbers4, M. Derouazi3, G. Wollmann1, 2 1Medical University Innsbruck, Christian Doppler Laboratory for Viral Immunotherapy of Cancer, Innsbruck, Austria, 2Medical University Innsbruck, Institute for Virology, Innsbruck, Austria, 3AMAL Therapeutics, Geneva, Switzerland, 4Viratherapeutics GmbH, Innsbruck, Austria, 5University of Veterinary Medicine Vienna, Unit of Laboratory Animal Pathology, Vienna, Austria, 6Medical University of Vienna, Department of Experimental Pathology, Vienna, Austria
Text Eliciting functional tumor-specific cytotoxic T cells through therapeutic cancer vaccination has regained attractiveness as it offers together with oncolytic viruses new combination opportunities to address resistance to checkpoint blockade therapy. The self-adjuvanting protein vaccine KISIMATM and the recombinant oncolytic virus VSV-GP-TAA (vesicular stomatitis virus pseudotyped with LCMV GP expressing tumor-associated antigens) are both promising vaccine candidates that when combined in heterologous prime-boost regimen with an optimized schedule and route of administration offer a new cancer vaccination opportunity. Indeed, priming with KISIMA-TAA followed by VSV-GP-TAA boost induced a large pool of polyfunctional and persistent antigen-specific cytotoxic T cells in the periphery as well as within the tumor in several tumor models with varying degrees of susceptibility for viral replication. Additionally, transcriptome analysis of a cold tumor model revealed profound changes in the tumor microenvironment upon heterologous vaccination, including a strong upregulation of gene signatures of several pro-inflammatory cytokines and chemokines required for antitumor immunity along with dendritic and T cell trafficking and activation. This was corroborated by flow-cytometric analysis of tumor-infiltrating leukocytes showing massive CD8+ and CD4+ T cell infiltration as well as repolarization of M2-like macrophages towards M1- phenotype. The presence of the CD8+ T cells within the tumor core was confirmed by immunohistochemistry analysis. Moreover, combining heterologous vaccination with checkpoint blockade further improved its therapeutic efficacy and the number of long-term survivors. Overall, heterologous vaccination with KISIMA- TAA and VSV-GP-TAA could sensitize non-inflamed tumors with few endogenous tumor-reactive T cells to checkpoint blockade therapy.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-18
A novel DC-targeted vaccine harnessing CD4 T cell help to improve cancer vaccination C. Le Gall1, 2, F. Fennemann1, I. Ramos-Tomillero1, 3, Y. Dölen1, 2, L. de Haas1, K. Schouren1, K. van Riessen1, L. Smeding1, S. van der Schoot1, 2, C. Figdor1, 2, 3, M. Verdoes1, 3 1Radboud Institute for Molecular Life Sciences, Tumor Immunology, Nijmegen, Netherlands, 2Oncode Institute, Nijmegen, Netherlands, 3Institute for Chemical Immunology, Nijmegen, Netherlands
Text Due to their ability to take up and present antigens, dendritic cells (DCs) shape immune responses by priming cytotoxic and helper T lymphocytes. Shortly after their discovery, it became evident that DCs were holding major promises in efforts to generate therapeutic antitumour immunity. Administrating tumor antigens to DCs in presence of adjuvants enables de novo T cell priming, and stimulates antitumour immune responses.
The identification of specific surface receptors on DCs has been a breakthrough in the field, as it allows precise vaccine delivery to DCs in vivo by using monoclonal antibodies and antibody fragments. We, and others, have used single-domain antibodies (VHHs) targeting DCs with the goal to generate anti-tumor vaccines. VHHs are praised for their highly versatile applications, and due to their structure, it is notably possible to introduce multiple functional groups without hampering their binding efficiency.
We conjugated epitopes derived from a model antigen to VHHs targeting DCs using an optimized approach, and set out to maximize CD8 T cell activation by harnessing CD4 T cell help. We show in vitro and in vivo that CD8 T cell activation is maximized after priming by DCs concurrently presenting both class I and class II antigens. Importantly, CD8 T cell priming was not maximal when class I and class II epitopes were delivered separately. By analyzing the dynamics of CD4 and CD8 T cell recruitment on DCs, we show that this phenomenon is highly timing dependent, and relying on simultaneous recruitment of antigen-specific CD4 and CD8 T cells onto single DCs.
Our strategy is able to substantially increase cytotoxic T cell activation by DCs, which we believe will be of importance in efforts to improve therapeutic vaccines for cancer.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-19
Post translationally modified peptides containing homocitrulline can be an effective anti-tumour target for multiple HLA- types. K. Cook, V. Brentville, P. Symonds, S. Shah, L. Durrant Scancell Ltd, Biodiscovery Institute, Nottingham, United Kingdom
Text CD4 epitopes from post translationally modified (PTM) proteins allow the generation of cytotoxic CD4 cells capable of targeting tumour. Homocitrulline is an amino acid PTM produced when lysine is carbamylated. We have previously shown that homocitrullinated epitopes can be a target for effective tumour immunity. This mechanism was fully characterised in HLA transgenic mice expressing the human DP4 allele. In this study we aimed to determine whether HLA type would limit the applicability of a homocitrulline based vaccine targeting tumours. Transgenic HLA-DR4, DR1 or DP4 and wild type mice were immunised with a variety of homocitrullinated peptides and the HLA restriction was fully characterised. We limited the study to peptides that produced modification-specific immune responses with minimal wild type cross reactivity. In addition, human donor peripheral blood responses were assessed in both healthy donor and cancer patients using CFSE proliferation assays. TCR analysis of the proliferating cells was also performed. Donor HLA-types were determined. In vivo studies identified a number of homocitrullinated peptides that can induce modification-specific immune responses. All HLA-types tested could recognise at least one modification- specific epitope. However, responses to individually epitopes varied by HLA type. Two examples are a homocitrullinated epitope from vimentin which can induce responses in HLA-DR4 and HLA-DR1 but not HLA-DP4 mice and an epitope from Cytokeratin 8 (Cyk8) which can induce immune responses in HLA-DR1 and HLA-DP4 but not HLA-DR4 mice. In the B16 melanoma tumour model Vimentin 116Hcit was associated with a significant survival advantage in both HLA-DR4 (p=0.0042) and DR1 (p=0.0010) mice and Cyk8 in HLA-DR1 (p=0.0072) and DP4 (p<0.0001). Combining multiple peptides as a single vaccine allowed additional HLA coverage to be achieved. PBMC studies showed that most healthy donors have proliferative responses to at least one of the three homocitrullinated peptides tested. TCR analysis of proliferating cells showed this is a clonal response and that there is some overlap within donors in the TCR clones responding to different homocitrullinated epitopes. Cancer patients also showed responses to homocitrullinated peptides, however, a significantly lower proportion of donors responded than seen in healthy donors. Together this data shows that modification-specific immune responses to homocitrullinated epitopes are not confined to the HLA-DP4 allele. Combination of multiple homocitrullinated epitopes can induce immune responses in most individuals regardless of HLA-type. This suggests that vaccines containing carefully chosen homocitrullinated epitopes could be effective in most patients.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-20
Results of the therapeutic cancer vaccine HepaVac-101 clinical trial in HCC patients. L. Buonaguro1, M. Loeffler2, F. Izzo1, S. Gori3, P. Ascierto1, A. Mayer-Mokler4, A. Königsrainer2, Y. T. Ma5, B. Sangro6, S. Francque7, R. Accolla8, L. Vonghia8, A. Avallone1, J. Ludwig4, D. Alcoba4, K. Aslan4, R. Mendrzyk4, H. Schuster4, D. Valmori9, T. Chaumette9, R. Heidenreich10, C. Gouttefangeas11, G. Forlani8, M. Tagliamonte1, M. Iñarrairaegui6, U. Gnad- Vogt10, C. Reinhardt4, T. Weinschenk4, H.-G. Rammensee11 1Istituto Nazionale Tumori - IRCCS Pascale, Experimental Oncology, Napoli, Italy, 2University Hospital Tübingen, Department of General, Visceral and Transplant Surgery, Tübingen, Germany, 3IRCCS Ospedale Sacro Cuore Don Calabria, Negrar, Italy, 4Immatics Biotechnologies GmbH, Tübingen, Germany, 5University of Birmingham, School of Immunity and Infection, NIHR Biomedical Research Unit in Liver Disease, Birmingham, United Kingdom, 6Clinica Universidad de Navarra and CIBEREHD, Liver Unit, Pamplona, Spain, 7Antwerp Univ. Hospital, Division of Gastroenterology and Hepatology, Edegem, Belgium, 8University dell´Insubria, Department of Surgical and Morphological Sciences, Varese, Italy, 9University of Nantes, Nantes, France, 10CureVac AG, Tübingen, Germany, 11University of Tübingen, Interfaculty Institute for Cell Biology, Department of Immunology, Tübingen, Germany
Text Background and Aims: Hepatocellular carcinoma (HCC) is the third leading cause of death from cancer globally with an extremely variable 5-year survival rate. Immunotherapy strategies for HCC may represent a key therapeutic tool to improve clinical outcome in HCC patients. HepaVac-101 (EudraCT Number: 2015-003389-10; NCT03203005), is a single-arm, first-in- man Phase I/II clinical trial evaluating a therapeutic cancer vaccine in patients affected by HCC. It is a highly innovative, novel approach based on a multi-peptide vaccine (IMA970A) combined with the TLR7/8/RIG I agonist CV8102 as an adjuvant.
Method: The IMA970A off-the-shelf vaccine includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides identified and selected from native human HCC tumor tissue by applying the XPRESIDENT® discovery platform. CV8102 is a novel ribonucleic acid (RNA) based immunostimulatory agent inducing a balanced Th1/Th2 immune response. HLA-A*02 and/or A*24-positive patients with very early, early and intermediate stage HCCs have been enrolled to be treated with 9 intradermal vaccinations consisting of IMA970A plus CV8102 following a single pre-vaccination infusion of low-dose cyclophosphamide acting as an immunomodulator. Patients have completed standard of care treatments at time of enrolment and received study drugs without concomitant anti-tumor therapy with no evidence of disease reactivation. The primary endpoints of the HepaVac-101 clinical trial are safety, tolerability, and antigen specific T cell response. Secondary/exploratory endpoints are additional immunological parameters in blood, infiltrating T-lymphocytes in tumor tissue, biomarkers in blood and tissue, disease-free survival/progression-free survival and overall survival.
Results: Patients were enrolled in 6 centers located in 5 European countries i.e. Italy (Naples and Negrar/Varese), Germany (Tübingen), UK (Birmingham), Spain (Pamplona) and Belgium (Antwerp). The end of trial (EOT) was reached on last December 20, 2019. 82 HCC patients have been screened for suitable HLA haplotypes, 22 patients were put on study treatment (i.e. received at least the pre-treatment with cyclophosphamide). The vaccination showed a good safety profile with no major SAE nor AESI reported during the protocol. Immunogenicity data show immune response against at least 1 vaccine class I TAA and 1 vaccine class II TAA in about 70% and 50% of the vaccinees, respectively. More detailed immunological evaluations will be available at time of the meeting.
Conclusions: The results of the HEPAVAC-101 clinical trial provide the grounding evidences for futher evaluation in subsequent steps.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-21
Considering “self-like” epitopes in pathogen and cancer vaccine design for better efficacy and safety G. Richard1, M. F. Princiotta1, L. Moise2, 3, C. Boyle2, R. F. Sweis4, W. D. Martin2, A. S. De Groot2, 3 1EpiVax Therapeutics, Providence, United States, 2EpiVax, Providence, United States, 3University of Georgia, Athens, United States, 4University of Chicago, Chicago, United States
Text Tools that facilitate the in silico prediction of immune response to cancers, biothreats, and emerging infectious diseases can accelerate the delivery of novel vaccines to the clinic. Epitope-driven vaccines represent perhaps the most stripped-down products of existing vaccine pipelines. Accumulating evidence is showing that these vaccines may eventually prove to be rapid to develop and effective in cancer and emerging infectious diseases. Our group recently discovered that the prediction of epitopes for vaccines is improved when T cell epitopes homologous to self-epitopes are removed, as T cells that recognize these cross-conserved "self-like" epitopes may be tolerant to them or actively tolerogenic. The process of selecting the best epitopes involves a set of tools that were originally developed for a "genome-to-vaccine" toolkit (iVAX) for emerging infectious diseases and have more recently been adapted in a pipeline (Ancer) that designs vaccines for individualized cancer immunotherapy. Traditional vaccine design and manufacturing timelines remain a hurdle for the adoption of therapeutic personalized cancer vaccines. In silico neoantigen pipelines are key to alleviate some of these impediments, if they identify the "correct" neoantigens. Using animal models, our group showcased that influenza subunit vaccines are more immunogenic and protective after removing self-like inhibitory epitopes from antigen sequences. Similar methodologies were utilized to identify immunogenic SARS-CoV-2 spike, membrane, and envelope epitopes tested in naïve and convalescent PBMCs. In addition, we showed in retrospective analyses of bladder cancer mutanomes from The Cancer Genome Atlas (TCGA) that identifying and removing self-like neoepitopes from neoantigen burden calculations improved prediction of patient survival over comparative approaches that use patient tumor mutational burden (TMB) or neoepitopes identified with publicly available T cell epitope prediction tools. The routine screening of self-like sequences combined with accurate T cell epitope prediction tools will allow the rapid design of immunogenic, effective, and safe prophylactic infectious disease vaccines and therapeutic cancer vaccines.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-22
Intratumoral delivery of a self-amplifying RNA expressing interleukin-12 by electroporation induces antitumor effects in a mouse colorectal cancer model N. Silva-Pilipich1, A. Lasarte-Cía2, J. J. Lasarte2, C. Smerdou1 1Cima Universidad de Navarra and Instituto de Investigación Sanitaria de Navarra (IdISNA), Division of Gene Therapy and Regulation of Gene Expression, Pamplona, Spain, 2Cima Universidad de Navarra and Instituto de Investigación Sanitaria de Navarra (IdISNA), Immunology and Immunotherapy Program, Pamplona, Spain
Text Alphavirus vectors based on self-amplifying RNA (saRNA) generate high and transient levels of transgene expression and induce innate immune responses, constituting an interesting tool for antitumor therapy. Although these vectors are usually delivered as viral particles it is also possible to administer them as RNA, although the high instability of this molecule may hinder its therapeutic effect. Here we have evaluated a quick and simple strategy to increase RNA uptake based on in vivo electroporation. We used Semliki Forest Virus (SFV) saRNA for local treatment of murine colorectal MC38 subcutaneous tumors. First, we optimized the conditions for SFV saRNA electroporation in these tumors using a vector coding for luciferase and evaluating the expression of this reporter gene by in vivo bioluminescence imaging. This study showed that optimal in vivo transfection was obtained by giving eight pulses of 1200 V/cm with a duration of 0.1 ms each, after injection of the saRNA into the tumor. Then, we evaluated the therapeutic potential of this approach using a saRNA coding for interleukin-12 (SFV-IL-12), a proinflammatory cytokine that has a potent antitumor effect. The delivery of SFV-IL-12 saRNA by electroporation led to a decrease in tumor growth and to a significantly higher survival compared to the control group that received saline. saRNA administration or electroporation as single therapies did not show significant antitumor effects compared to the control group. Our results suggest that delivery of saRNA by electroporation could represent an attractive strategy for local cancer immunotherapy. This approach could have an easier translation to the clinical practice, especially for tumors that can be easily accessed.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-23
Creating next-generation DC vaccines tailored to immuno-contexture of immunologically “silent” or lymphocytes-depleted tumors. J. Sprooten1, D. Borras1, I. Vanmeerbeek1, S. Krautwald2, B. Van Den Eynde3, 4, 5, J. Murphy6, S. Schlenner7, P. Agostinis1, 8, S. Tejpar9, A. Garg1 1KU Leuven, Cellular and Molecular Medicine, Leuven, Belgium, 2University Hospital SchleswigHolstein, Nephrology and Hypertension, Kiel, Germany, 3UCLouvain, de Duve Institute, Brussels, Belgium, 4Ludwig Institute for Cancer Research, Brussels, Belgium, 5Walloon Excellence in Life Sciences and Biotechnology, Brussels, Belgium, 6University of Melbourne, Medical Biology, Melbourne, Australia, 7KU Leuven, Microbiology, Immunology and Transplantation, Leuven, Belgium, 8VIB, Center for Cancer Biology, Leuven, Belgium, 9University hospital Leuven, Oncology, Leuven, Belgium
Text In the last decade, immunotherapy got a major boost in interest creating multiple anti-cancer immune treatment regimens such as checkpoint blockers. However, in certain immuno-contexts checkpoint blockade has failed to meet expectations resulting in limited treatment response. These immunological quiet or lymphocyte-depleted tumors fail to respond due to a scarcity of tumor infiltrating T-cells and a lack of TCR diversity. We believe that using dendritic cell (DC) vaccines, to evade immunosuppression of these tumor types by allowing exogenously tumor antigens pulsed DCs to prime T cells with cancer antigens and increase TCR diversity, would increase checkpoint blocker efficiency. The aim of this project was to create next-generation DC vaccines tailored to immuno-contexture of immunologically “silent” or lymphocytes-depleted tumors, addressing a critical unmet clinical need. At the outset, we found that such tumors had severe dysregulations in type I interferon (IFN) pathways (especially TLR4 discordance and IFNα/β defects). Remarkably, a trajectory analyses mapping ex vivo-generated human DC vaccines administered to cancer patients, showed that, DC vaccines developed towards highly contrary trajectories in terms of antigen-specific T cell responses and patient prognosis. Interestingly, the DC vaccine trajectory with positive prognosis and highest antigen-specific T cell responses enriched for type I IFN response and immunogenic NFkB signaling. Therefore, our research on next-generation DC vaccines development will focus on: the immunogenic potential of the dying/dead cancer cells as antigen-sources and the maturation factor type I interferon (IFN) for stimulating DCs. We found, by using the murine tumor cell line TC-1, which mimics the immunological quiet tumor type, undergoing either apoptosis or necroptosis as an antigen source, that apoptotic cells stimulate DCs in the most optimal matter. Being that both phagocytosis, maturation, pro-inflammatory cytokine expression and secretion were significantly upregulated, which was even more so combined with type I IFN. In prophylactic and curative in vivo experiments using our DC vaccines in the TC-1 mouse model showed that all our vaccines increased tumor free survival or tumor growth. Unexpectedly, we observed that type I IFN was decreasing the vaccine efficiency for apoptotic cancer cell stimulated DCs. However, DCs pulsed with necroptotic cancer cells showed an increase in vaccine efficiency when IFN was added. We are currently analyzing the regulatory response in our DC vaccines as a sign of overstimulation and hope to unravel their implications for anticancer DC vaccines in the context of immunologically “silent” or lymphocytes-depleted tumors.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-24
Universal HLA-specific peptide scaffold for improved anti-tumor response M. Tagliamonte1, A. Mauriello1, B. Cavalluzzo1, C. Ragone1, C. Manolio1, L. Vitagliano2, M. Ruvo2, A. Barbieri3, G. Botti4, L. Buonaguro1 1Istituto Nazionale per lo Studio e la Cura dei Tumori IRCCS-, Cancer Immunoregulation Unit, Naples, Italy, 2National Research Council, Institute of Biostructures and Bioimaging, Naples, Italy, 3Istituto Nazionale per lo Studio e la Cura dei Tumori IRCCS- "Fondazione Pascale", Animal Facility, Naples, Italy, 4Istituto Nazionale per lo Studio e la Cura dei Tumori IRCCS- "Fondazione Pascale", Scientific Direction, Naples, Italy
Text We have previously shown the efficacy of a multi-peptide vaccine, based on Tumor Associated Antigens (TAAs), in delaying tumor growth and prolonging survival in vivo when combined with low dose metronomic chemotherapy (MCT) and an anti-PD-1 checkpoint inhibitor (CI). However, TAAs are cellular self-antigens mostly overexpressed in cancer cells with low expression in normal cells. For this reason, they may be subject to both central and peripheral tolerance mechanisms leading to an inefficient immune response. In order to potentiate the immunogenicity of TAAs, heteroclitic peptides were designed according to prediction algorithms and specific modifications were introduced in the amino acid residues binding to HLA as well as to TCR. In particular, an HLA-specific scaffold was designed for a general optimization of the antigen presentation to TCR. The efficacy of such heteroclitic peptides was assessed in C57BL/6 mice injected with syngeneic melanoma B16F10 or lung TC1 tumor cell lines, in combination with metronomic chemotherapy and checkpoint inhibitors. The immunogenicity of heteroclitic peptides was significantly stronger than the corresponding wt peptide and the modification involving both HLA and TCR binding residues scored the strongest. Strikingly, Tumor growth in the TC1 model was significantly delayed, in a therapeutic setting, and completely controlled, in a preventive setting. The anti-tumor effect was associated with strong specific T cell immune response and significant reduction of immunosuppressive cell populations. Overall, we demonstrated that modified TAAs show higher immunogenicity compared to wild-type, eliciting cross- reacting CD8+ T cells able to kill tumor cells presenting the wild-type antigen. In particular, we showed that peptides modified in both HLA and TCR binding residues induce the strongest anti-tumor T cell response, supporting the notion that an HLA-specific scaffold can be used for the optimal antigen presentation to TCR.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-25
Preliminary immunogenicity analysis of HB-201 and HB-202, an arenavirus-based cancer immunotherapy, in patients with advanced HPV16+ cancers K. Katchar1, M. Schwendinger1, D. M. DaSilva2, D. Edwards1, X. Qing1, K. Schlienger1, H. Lauterbach1, K. Orlinger1, I. Matushansky1 1HOOKIPA Pharma, New York, United States, 2University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, United States
Text Background: Human Papillomavirus 16 (HPV16) is linked to most HPV-associated cancers such as cervical, head and neck, vaginal and anal cancers. Treatment options are limited for patients with HPV16+ recurrent or metastatic cancers. The generation and maintenance of the HPV16+ malignant state requires the stable expression of HPV16-specific E7 and E6 oncogenes, which therefore constitute attractive targets for immunotherapy. HB-201 and HB-202 are both replicating live-attenuated vectors based on arenaviruses LCMV and PICV, respectively, expressing the non-oncogenic HPV16 E7E6 fusion protein for induction of tumor specific T cell responses. In preclinical models, administration of HB-201 alone and sequential administration of HB-202 followed by HB-201 was safe and demonstrated potent immunogenicity by induction of E7 and E6 -specific CD8+ T cell responses and efficient tumor control of TC-1 tumors. Methods: A first-in-human, Phase 1/2 open-labelled clinical trial of HB-201 single vector therapy and HB-201 & HB-202 two-vector therapy in patients with treatment-refractory HPV16+ cancers is currently ongoing (NCT04180215). Here, we present first immunogenicity results from the dose escalation Phase 1 of this study. The Phase 1 of the trial is designed to evaluate different dose levels and dosing schedules of HB-201 as a single-vector therapy or as an alternating two-vector therapy together with HB-202. Peripheral blood mononuclear cells (PBMCs) were collected before and after treatment from all patients. PBMCs from a subset of patients were examined for HPV16-specific T cell responses measured by IFN-γ enzyme-linked immunospot assay and intracellular cytokine staining. Paired tissue biopsy and serum samples were also collected and being currently evaluated for histology, cytokines, and pharmacokinetics. Results: We demonstrated induction of a HPV16-specific T cell response in PBMCs after a single dose of HB-201 or HB- 202. The magnitude of the T cell response allowed direct detection ex vivo without prior in vitro expansion. Additional exploratory analysis will be available at the time of the meeting. Conclusion: These preliminary data provide the first demonstration of arenavirus vectors ability to induce HPV16-specific T cells in cancer patients. This is achieved following a single injection of HB-201 or HB-202. Arenavirus vectors expressing E7E6 may constitute a new potential therapy for patients with treatment refractory HPV16+ cancers. Clinical data will be presented in an upcoming scientific meeting. Additional schedules, alternating two-vector therapy with HB-201/HB-202, and combination with anti-PD-1 mAbs are being explored in additional cohorts.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-26
Priming and expansion of functional antitumor T-cells in melanoma patients: results of phase I clinical trial with GeniusVac- Mel4, an innovative cancer vaccine based on a plasmacytoid dendritic cell line J. Charles1, 2, L. Chaperot2, 3, D. Hannani4, J. Bruder Costa2, 3, 5, I. Templier1, S. Trabelsi1, H. Gil6, A. Moisan2, 7, V. Persoons7, H. Hegelhofer7, E. Schir8, J.-L. Quesada9, C. Mendoza9, C. Aspord2, 3, O. Manches2, 3, P. Coulie10, A. Khammari11, B. Dreno11, M.-T. Leccia1, 2, J. Plumas2, 3, 12 1CHU Grenoble Alpes, Dermatology Department, Pôle Pluridisciplinaire de Médecine,, Grenoble, France, 2INSERMU1209/UGA, Immunology and immunotherapy in chronic diseases, Grenoble, France, 3EFS- Auvergne-Rhone-Alpes, R&D, Grenoble, France, 4PDC*line Pharma, Grenoble, Grenoble, France, 5CHU Grenoble Alpes, Dermatology Department, Pôle Pluridisciplinaire de Médecine, Grenoble, France, 6CHU Grenoble Alpes, Pathology department, Grenoble, France, 7EFS, Cell Therapy and Engineering Unit, St Ismier, France, 8CHU Grenoble Alpes, DRCI, Grenoble, France, 9CHU Grenoble Alpes, CIC, Grenoble, France, 10Université Catholique de Louvain, de Duve Institute, Brussels, Belgium, 11CHU Nantes,, Onco- dermatology Department, Nantes, France, 12PDC*line Pharma, Grenoble, France
Text Cancer vaccines represent an attractive therapeutic approach that could synergize with immune checkpoint inhibitors to induce protective antitumor immunity. Among dendritic cells, plasmacytoid dendritic cells (PDC) display all the hallmarks of good cancer vaccine candidates, as they are able to prime efficient antitumor specific CD8+ T cell responses. An allogeneic HLA-A*02:01 PDC line was developed, suitable for GMP production, and fully characterized for antigen-presenting function in preclinical studies, in vitro and in vivo, allowing setting-up a clinical trial with this unique tool. We describe here the results of immune and clinical activities observed in the first-in-man phase Ib clinical trial applying this strategy to treat metastatic melanoma patients. The PDC line was loaded with 4 melanoma antigens (Melan-A, gp100, tyrosinase and Mage-A3), irradiated, and frozen. Three weekly subcutaneous injections of the thawed vaccine were performed for each patient, in 3 groups receiving 20, 40 or 60 millions of cells. The safety and tolerability were excellent; the vaccine was well tolerated without vaccine-induced side effects recorded. Regarding clinical activity, four patients remained stable according to IrRC and vitiligo appeared. The frequency of anti-tumor specific T lymphocytes increased in two patients, with a naïve to memory switch observed in these cells, demonstrating vaccine efficacy to boost T cell responses. In one patient, PD-L1+ myeloid and PD1+ antitumor-specific cells were found in the tumor bed, suggesting the presence of checkpoint blockade involved in tumor escape. We also analyzed in vitro, the cytokine secretion and cytotoxic activity of the antitumor specific cells that were restimulated with the vaccine in 14-day cultures. In addition, with melanoma samples of an independent cohort we found that therapeutic anti-PD1 synergized with the PDC line loaded with tumor antigen to induce in vitro antitumor T-cells. This clinical trial demonstrates the feasibility and biological efficacy of this original platform based on a unique PDC line based-vaccine that is able to prime and expand functional antitumor CD8+ responses in cancer patients. Next step should be to evaluate this vaccine strategy in combination with immune checkpoint inhibitors
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-27
Interim results of a phase I neoantigen-directed personalized cancer vaccine trial J. Ingels1, L. De Cock2, R. Mayer2, S. Desmet3, K. Heyns4, N. Lootens3, M. Brusseel3, D. Stevens4, J. Van Dorpe1, F. Impens2, K. Vermaelen4, B. Menten2, B. Vandekerckhove1 1University of Ghent, Diagnostic Sciences, Ghent, Belgium, 2University of Ghent, Biomolecular Science, Ghent, Belgium, 3Ghent University Hospital, GMP Unit Cell Therapy, Ghent, Belgium, 4University of Ghent, Department of Respiratory Medicine, Ghent, Belgium
Text Immune checkpoint blockers (ICB) have revolutionized the treatment of cancer. Clinical efficacy of ICBs have especially been demonstrated in tumors with a high mutational burden, such as lung cancers. However, only a small subset of the patients responds to treatment. As it is demonstrated that the efficacy of ICBs relies on the reactivation of T cells directed to neoantigens, a rational strategy to enhance the response rate is to combine ICBs with an agent that broadens the immune reactivity. Therefore, we developed a neoantigen targeted vaccine which is composed of dendritic cells loaded with messenger RNA (mRNA) encoding a selected set of neoantigens.
The vaccine is now being evaluated in 6 early stage lung cancer patients, who underwent surgical resection, to assess the safety and immunogenicity of the vaccine. A tumor and a healthy blood sample are collected and subjected to DNA and RNA sequencing for neoantigens selection through epitope prediction algorithms. Simultaneously, a larger tumor sample is subjected to mass-spectrometry-based immunopeptidomics, which allows direct identification of neoantigenic peptides bound in the HLA cleft. Subsequently, mRNA encoding up to 6 selected neoantigens is designed, produced and loaded onto dendritic cells manufactured from autologous monocytes. The vaccine is administrated in escalating doses and first results show safety at the highest dose. First immunomonitoring results show neoantigen specific immune responses already after a single dose.
During the presentation, we will present the epitope prediction pipeline and show evidence that 1. the tumor effectively expresses predicted neoepitopes, 2. potent neoantigen directed T cell responses are induced after vaccination.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-28
The oncolytic HSV-1 virus T-VEC induces immunogenic cell death of melanoma cells and efficiently matures monocyte derived as well as BDCA-1+/BDCA-3+ myeloid dendritic cells J. De Munck1, P. Kalus1, H. Smets1, K. Broos2, J. Tijtgat3, I. Van Riet4, I. Dufait3, 5, J. K. Schwarze3, C. Goyvaert2, B. Neyns3, S. Tuyaerts3, 6, K. Breckpot2, J. L. Aerts1 1Vrije Universiteit Brussel, Laboratory for Neuro-Aging and Viro-Immunotherapy, Jette, Belgium, 2Vrije Universiteit Brussel, Laboratory for Molecular and Cellular Therapy, Jette, Belgium, 3Universitair Ziekenhuis Brussel, Departement of Medical Oncology, Jette, Belgium, 4Universitair Ziekenhuis Brussel, Departement of Hematology, Jette, Belgium, 5Vrije Universiteit Brussel, Laboratory of Translational Radiation Oncology, supportive care and Physics, Jette, Belgium, 6Vrije Universiteit Brussel, Laboratory of Medical and Molecular Oncology, Jette, Belgium
Text Oncolytic viruses (OVs) are designed to selectively replicate within cancer cells upon infection and cause cell death leading to the release of mature virions that can subsequently infect neighboring cells. Talimogene laherparepvec (T- VEC), an HSV-1-derived OV, expresses GM-CSF which improves its immunogenicity. In the Optim phase III trial, intralesional injection of T-VEC in melanoma patients induced rejection of treated tumors and distant lesions in a significant proportion of patients. This abscopal effect suggests that antigenic fragments of ruptured tumors presumably activate tumor resident dendritic cells (DCs) and subsequently tumor specific T cell responses. So far, a comprehensive analysis on the immune activating potential of T-VEC is not available. In addition, it is highly likely that the mode of inducing (immunogenic) tumor cell death is an important contributing factor.
We studied the capacity of T-VEC to infect and replicate within human melanoma cell lines (624-mel, 938-mel, MZ2 and CHL-1) and to induce (immunogenic) cell death. Further, we assessed their ability to mature human monocyte-derived DCs (moDCs) as well as human BDCA-1+/BDCA-3+ DCs (see abstract Tijtgat J., De Munck J. et al. for more details) and stimulate cross-presentation of tumor antigens, enabling them to coordinate anti-tumor T cell responses. Experiments were performed in 2D and 3D cultures. In parallel, analogous experiments are currently being done using the mouse melanoma cell line D4M.3A.
Using the IncuCyte® Live Cell Analysis System and ELISA for measuring GM-CSF concentrations, we show that T-VEC is able to infect, replicate, and induce cell death in all studied melanoma cell lines. The type of cell death could be characterized using western blot for different cell death associated proteins (RIPK1, RIPK3, MLKL, caspase-8, FADD and SMAC). Using flow cytometry for the evaluation of ATP release and calreticulin exposure and ELISA for the detection of HMGB-1, it could be documented that the induced type of cell death is immunogenic. Finally, with a flow- cytometry based method, we demonstrated that dying target cells (labeled with Cell Tracker Green®) were taken up by DCs.
In conclusion, T-VEC is able to infect, replicate and induce immunogenic cell death in melanoma cell lines which subsequently leads to tumor fragment uptake by and maturation of DCs. Further research is currently being performed to investigate whether these DCs can cross-present tumor antigens to T cells and whether these T cells are capable to subsequently induce immune-mediated tumor cell killing.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-29
Identification and functional characterization of HLA-A1- and HLA-A2-restricted HPV16 E6- and E7-derived epitopes K. Öhlenschläger1, 2, 3, S. Hoppe1, 2, R. Blatnik1, 2, J. Heinze1, 3, M. Bonsack1, 2, A. Riemer1, 2 1German Cancer Reserach Center (DKFZ), Immunotherapy and Immunoprevention, Heidelberg, Germany, 2German Center for Infection Research (DZIF), Molecular Vaccine Design, Heidelberg, Germany, 3Heidelberg University, Faculty of Biosciences, Heidelberg, Germany
Text For the development of a therapeutic vaccine against human papillomavirus (HPV) it is fundamental to identify viral epitopes that are bona fide presented on the surface of HPV-transformed cells and are able to induce immunological responses. The HPV oncoproteins E6 and E7 are expressed in HPV-transformed cells of all disease stages and are therefore ideal targets for immunotherapies. As the presentation of epitopes is specific to human leukocyte antigen (HLA) molecules, an epitope-specific peptide-based vaccine needs to match a patient’s HLA-type. The number of epitopes that are required to achieve a broad population coverage can be reduced by exploitation of HLA-supertypes, each of which covers HLA-types with similar binding preferences. Within our process of identifying suitable HPV16 E6-/ E7-derived epitopes for therapeutic vaccine design, possible target peptides for five HLA-supertypes were preselected by state-of-the-art in silico HLA-binding predictions, and validated regarding their HLA binding in in vitro competitive cellular binding assays. These resulted in identification of 47 HLA-A2 and 28 HLA-A1 binding peptides, out of which 22 HLA-A2- and 24 HLA-A1-ligands have not been described before. The immunogenicity of these peptides was assessed in interferon-γ (INFγ) ELISpot assays in which peripheral blood mononuclear cells of HLA-matched heathy female donors were tested for T cell memory responses. Twelve HLA-A2+ and ten HLA-A1+ donors were analyzed and 22 HLA-A2 and three HLA-A1 restricted epitopes were found to be recognized by these donors. The INFγ ELISpot assays were supplemented with a flow cytometry staining for T cell surface markers and an intracellular cytokine staining (ICS) for IFNγ, TNFα and granzyme B to further characterize the immune memory response and the responsive cell population. The combination of ELISpot assays with ICS data added an additional dimension of information and the data highlighted the benefit of characterizing a peptide-mediated immune response in more than one assay. The ICS data allowed the identification of peptides that induced reactivity in CD4+ T cells rather than in CD8+ T cells or did e.g. not induce a TNFα response. Therefore, that data is expected to predict if a peptide mediates death of HPV16-transformed target cells. The functional characterization of the identified immunogenic peptides in cytotoxicity assays and assessment of further HLA supertypes is currently ongoing. The definition of validated HPV16 E6-/E7-derived HLA-A2 and HLA-A1 epitopes contributes to an HPV16 epitope repertoire map and thus to the development of a therapeutic HPV vaccine as treatment option for persistent HPV16 infections and HPV16-driven malignancies.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-30
Liver sinusoidal endothelial cells are largely responsible for liver accumulation of antibody-conjugated nanoparticles C. Kappel, C. Medina-Montano University Medical Center Mainz, Dermatology, Mainz, Germany
Text Despite considerable progress in the design of multifunctionalized nanoparticles (NP) aimed to selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell targeting antibodies per NP determines the extent of NP liver accumulation.
We used polysarcosine-based peptobrushes (PB) which show low unspecific cell binding and remain in the circulation for >24h. We conjugated PB with defined numbers of antibodies (2, 6 and 12; strain-promoted azide–alkyne cycloaddition) specific for the dendritic cell (DC) surface receptor, DEC205. The time- dependent biodistribution of PB-antibody conjugates in mice was assessed by in vivo imaging and ex vivo flow cytometric analysis of cells derived from retrieved organs (liver, spleen, lymph nodes).
We demonstrated that liver accumulation of the applied PB-antibody formulations positively correlates with the numbers of antibodies conjugated per the PB and is mostly due to binding of exposed Fc ends to Fc receptors (FcR) expressed on liver sinusoidal endothelial cells (LSEC) as shown by blocking experiments with an anti-FcR antibody. Conjugation of PB with an average of 2 full aDEC205 antibodies as well as higher numbers of F(ab’)2 fragments, lacking the Fc part, largely prevents liver accumulation while still conferring sufficient DC targeting in secondary lymphoid organs. Moreover, we showed that the use of higher antibody amounts leads to the formation of a more pronounced protein corona, mainly consisting of antibodies and complement factors, which in turn may contribute to other unwanted cell binding mechanisms. Since LSEC are potent inducers of antigen-specific tolerance, nano-vaccines intended to induce immunity should aim to minimize uptake by LSEC in order to prevent tolerance induction, which underlines the importance of controlling antibody numbers on NP-based vaccines. However, when receiving suitable stimuli LSEC are also capable to promote immune activation, which could also be exploited by NP immunotherapy.
Cumulatively, our study demonstrates that LSEC play a yet scarcely acknowledged role in liver entrapment of antibody-coated NP, and that low antibody numbers on NP or the use of F(ab’)2 antibody fragments are both necessary to minimize unwanted liver accumulation and sufficient for cell type-specific targeting of secondary lymphoid organs.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-31
Nanoparticles that contain NY-ESO-1 peptides and an iNKT cell agonist induce strong immune responses. Y. Dolen1, U. Gileadi2, J.-L. Chen2, M. Valente3, E. Jager4, J. Creemers1, V. Cerundolo2, M. Diken5, C. Figdor1, J. de Vries1 1Radboud Institute for Molecular Life Sciences, Tumor Immunology, Nijmegen, Netherlands, 2Weatherall Institute of Molecular Medicine, MRC Human Immunology Unit, Oxford, United Kingdom, 3Centre d’Immunologie de Marseille-Luminy, Marseille, France, 4Institute of Macromolecular Chemistry v.v.i, Prague, Czech Republic, 5Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz gGmbH, Mainz, Germany
Text Identification of tumor-specific antigens and the production of personalized cancer vaccines can be time- consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as off-the-shelf immunotherapy. Here, we sought to optimize an immunogenic tumor-associated antigen, NY-ESO-1, and a novel iNKT cell agonist IMM60 for PLGA nanoparticle-mediated vaccine delivery.
Three peptide sequences (85-111, 117-143, and 157-165) were selected based on their immunogenicity and wide HLA coverage. To evaluate antigen processing and presentation by different HLA types, nanoparticles were loaded to moDCs and co-cultured with autologous CD8 and CD4 T cells transfected with mRNAs encoding different NY-ESO-1 TCRs. Additionally, patient-derived CD8 and CD4 T cells were stimulated with immortalized B cells loaded with nanoparticle or soluble peptide formulations. Moreover, wild-type and HLA- A2 transgenic mice were used to determine in vivo immunogenicity of the nanoparticle vaccine.
In vitro, IFN-y production of TCR transfected T cells by particles containing peptides was on par with soluble peptides and much higher than particles loaded with whole NY-ESO-1 protein. These results were confirmed by in vitro re-stimulation of patient-derived T cells, indicating wide HLA coverage and immunogenicity of the nanoparticles containing peptides. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo.
In conclusion, we demonstrated the feasibility to encapsulate three NY-ESO-1-derived peptides together with IMM60 in PLGA nanoparticles. All peptides are efficiently processed and presented by multiple HLA types. Furthermore, iNKT cell help was provided for multiple epitope-specific CD8 and CD4 T cell responses, and a de novo antibody response was observed in NY-ESO-1 positive tumor-bearing mice
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-32
Immunogenicity and HLA binding affinity of MSI-associated frameshift peptide neoantigens A. Hernandez-Sanchez1, 2, G. Litau2, K. Urban2, M. Kloor1, 2, M. von Knebel Doeberitz1, 2 1German Cancer Research Center (DKFZ), Clinical Cooperation Unit Applied Tumor Biology, Heidelberg, Germany, 2Heidelberg University Hospital, Institute of Pathology, Department of Applied Tumor Biology, Heidelberg, Germany
Text Lynch syndrome (LS) is the most common inherited cancer syndrome and caused by germline DNA mismatch repair (MMR) gene variants. LS predisposes patients to the development of microsatellite-unstable (MSI) tumors. Due to MMR deficiency, MMR tumors accumulate high numbers of indel mutations at coding microsatellites (cMS), which can result in the generation of frameshift peptide (FSP) neoantigens. Due to Darwinian evolution, MSI tumors share similar cMS mutations and FSP neoantigens. The shared pool of FSP neoantigens and the ability to identify Lynch syndrome carriers provide an ideal opportunity for the development of a preventive vaccine against MSI tumors. We have successfully concluded a clinical phase I/IIa trial demonstrating the safety and immunological efficacy of a vaccine encompassing 3 FSPs. Recently, we identified promising novel FSP candidates with high predicted immunogenicity and high cMS mutation frequency in MSI cancer.
Here we aimed to describe the immunological properties of these FSPs and their potential for the development of a neoantigen-based vaccine against MSI cancers. First, binding of predicted epitopes to HLA-A2*02:01 was determined in vitro using a competition-based binding assay. Second, to evaluate antigen-specific HLA-A2*02:01-mediated T cell responses, HLA-A2*02:01-transgenic mice were vaccinated with the respective FSP. Interferon gamma (IFNɣ) ELISpot was performed with whole splenocytes; for the most promising candidates, CD4+ and CD8+ T cell responses were assessed separately.
IFNɣ ELISpot demonstrated that 5 out of the 8 candidate FSPs were able to induce FSP-specific T cells, indicating processing and presentation of the FSP-derived epitopes. The competition-based binding assay showed multiple high binder (IC50 < 5 μM) HLA-A2*02:01-restricted epitopes for 3 FSPs. In addition, intermediate (5 μM < IC50 < 15 μM) and low (15 μM < IC50 < 100 μM) binding epitopes were identified for additional FSPs.
Our results confirm the immunogenicity of 5 out of 8 candidate FSPs, 3 of which harbor multiple epitopes that bind with high affinity to HLA-A2*02:01. The high number of confirmed epitopes already for one specific HLA allele underscores the great potential of MSI-induced FSPs as a neoantigen pool for vaccination approaches for prevention and treatment of MSI cancers. Further preclinical research is performed to promote this goal.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-33
A Therapeutic Silica-Nanoparticle Vaccine Against HPV16-Induced Malignancies L. T. Roth1, 2, 3, S. Kruse1, S. Zottnick1, 2, 4, A. Klevenz1, 2, E. Junglas5, E. Feidt5, A. K. Grabowska5, A. Kübelbeck5, A. B. Riemer1, 2 1German Cancer Research Center, Heidelberg, Germany, 2German Center for Infection Research (DZIF), Molecular Vaccine Design, Heidelberg, Germany, 3Heidelberg University, Faculty of Medicine, Heidelberg, Germany, 4Heidelberg University, Faculty of Biosciences, Heidelberg, Germany, 5Life Science Inkubator GmbH, Silvacx, Bonn, Germany
Text High-risk types of human papillomavirus (HPV), including HPV16 and 18, cause more than 600,000 anogenital and oropharyngeal cancer cases worldwide per year. The existing prophylactic vaccinations against HPV prevent infection, but are not able to cure infection or neoplasia. Current therapeutic interventions such as removal of premalignant precursor lesions, or surgery, chemotherapy or radiotherapy against established tumors, are invasive and incur adverse side effects. Development of a HPV-specific therapeutic vaccine would provide a truly tumor-specific therapeutic approach, with the added benefit of induction of immunological memory. Here we present a therapeutic vaccine formulation based on a validated HLA-A2-restricted epitope derived from the HPV16 oncoprotein E7, which is expressed in all stages of HPV-mediated carcinogenesis. It is combined with a novel vaccination vehicle consisting of silica nanoparticles. These safe and biodegradable nanoparticles are loaded with epitopes of interest via a previously optimized surface modification and spacer/linker sequence. Confocal microscopy showed the uptake of fluorescently labeled nanoparticles, and the presentation of the fluorescently labeled H2-Kb model epitope OVA/257-264 (SIINFEKL) on antigen-presenting cells. These findings were corroborated by ImageStream analysis. Presentation of the epitope delivered by nanoparticles was further proven in an assay with a TCR-like antibody, recognizing the SIINFEKL/H2-kb complex.To test the efficacy of a silica nanoparticle-based vaccination formulation in vivo, the naturally presented and immunogenic HLA-A2-restricted HPV16 epitope E7/11-19 was linked to nanoparticles. This vaccine was tested in the MHC-humanized mouse model A2.DR1, with and without syngeneic HPV16 E6/E7-positive PAP-A2 tumors. Vaccinated mice did not only develop a robust epitope-specific T cell response but were also able to successfully eliminate subcutaneous tumors. Taken together, the silica nanoparticle-based epitope-specific vaccine is a promising candidate for therapeutic HPV vaccination development. Ongoing studies assess its efficacy against orthotopic, i.e. genital or oropharyngeal mucosal tumors, as these are the true targets of any HPV therapy.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-34
IVAC-ALL-1: Updated analysis of a phase I/II clinical trial on personalized peptide vaccination based on patient-individual tumor-specific variants in relapsed pediatric acute lymphoblastic leukemia A. Rabsteyn1, 2, C. Mohr3, J. Matthes4, O. Witt2, 5, R. Meisel2, 6, C. Chen-Santel2, 7, T. Feuchtinger2, 8, C. Schroeder4, N. Casadei4, M. Denk2, 9, A. von Stackelberg2, 7, J. Schulte2, 7, T. Milde2, 5, S. Stevanovic2, 9, O. Riess4, O. Kohlbacher3, H.- G. Rammensee2, 9, S. Pfister2, 5, R. Handgretinger1, 2, P. Lang1, 2 1University Children's Hospital, Department of General Pediatrics, Oncology / Hematology, Tübingen, Germany, 2German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Sites Tübingen, Heidelberg, Düsseldorf, Berlin and München, Germany, 3Institute for Applied Bioinformatics, University of Tübingen, Tübingen, Germany, 4Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany, 5Heidelberg University Hospital, Department of Pediatric Hematology and Oncology, Heidelberg, Germany, 6Clinic for Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine University Düsseldorf, Division of Pediatric Stem Cell Therapy, Düsseldorf, Germany, 7Charite University Medicine, Pediatric Oncology and Hematology, Berlin, Germany, 8Dr. von Hauner'sches Children's Hospital LMU Munich, Department of Pediatrics, München, Germany, 9Institute for Cell Biology, University of Tübingen, Department of Immunology, Tübingen, Germany
Text Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Only about 20% of patients develop a relapse after standard therapy, however these patients have a dismal prognosis. Prevention of relapse after first-line chemotherapy or stem cell transplantation is therefore an urgent clinical need. We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. We started clinical translation of this approach by starting a phase I/II clinical trial in 2016 (NCT03559413). Besides feasibility and toxicity assessments, we aim to assess the capability of the peptide vaccination to induce neoantigen-specific T cell responses in high-risk ALL patients to target residual tumor cells and prevent leukemic relapses. Nonsynonymous mutations are identified by a comparative bioinformatic pipeline using whole exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. HLA binding peptides harboring altered amino acids are subsequently predicted in silico by algorithms SYFPEITHI, NetMHC and NetMHCpan. Vaccine cocktails consisting of 3-5 individual peptides and a control wildtype peptide derived from the antigen Survivin are produced and formulated under GMP conditions. The vaccination schedule is 16 vaccinations over 33 weeks using GM-CSF and Imiquimod as adjuvants. Response to the vaccination is monitored by detection of vaccine-induced neoantigen-specific T cells in peripheral blood of patients. Until now, 28 patients were recruited, whole exome sequencing was completed for 21 patients. We found an average of 78.5 mutations per patient on DNA level. Based on these data, an average of 154 HLA binders derived from neoantigens could be predicted per patient. An average expression of 33.6% of mutations was assessed by RNA sequencing. In all cases expressed mutations could be identified and cocktail design was feasible. Until now, 15 patients received vaccinations. The vaccine was generally well tolerated and no or only mild side effects were observed. Immune monitoring revealed sustained T cell responses against vaccinated peptides in 10/11 evaluated patients. Peptide-specific T cell responses were of CD4+ and CD8+ subtype.Concluding, whole exome sequencing of pediatric ALL yields small amounts of expressed, tumor-specific mutations. These few mutations are sufficient to predict HLA-binding peptides that can be used to formulate individualized peptide vaccine cocktails under GMP conditions. Vaccination with such peptide cocktails is able to induce de novo T cell responses against neoantigens in patients.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-35
Modelling Immune Response by Joining Advancements in Machine Learning Technologies and Dataset Improvements M. Lang1, A. N. Okandan1, G. Reuber1, N. L. Carranza2, M. Jedidi2, C. Peikert1, P. Pandey1, R. Hilker1, B. Schrörs3, F. Lang3, M. Löwer3, M. Skwark2, K. Beguir2, U. Sahin1, 3, 4 1BioNTech, Mainz, Germany, 2InstaDeep Ltd., London, United Kingdom, 3TRON, Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz gGmbH, Mainz, Germany, 4Research Center for Immunotherapy, University Medical Center at the Johannes Gutenberg University Mainz, Mainz, Germany
Text The cellular immune response towards an antigen is a result of a complex machinery. It is determined by the abundance of the antigen, the processing and presentation of the antigenic peptide on major histocompatibility complex (MHC) molecules, and finally by the recognition of antigen-specific T cells. Several prediction algorithms for MHC presentation have been developed by training neural networks and other machine learning models on MHC ligands. Newer algorithms show improved performance by using ligands derived from mass spectrometry (MS). These ligands determined by MS are not artificially loaded onto the MHC complex but show naturally occurring events. Thus, their appearance encompasses all cellular processing steps that precede the binding event. However, as the presentation is not the only part that defines the immune response, we used additional features and a large dataset collection of immune response data that enabled us to tackle further determinants of T Cell responses. With this approach, we are able to improve the immune response prediction significantly.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-36
An adaptable antibody-based platform for flexible synthetic peptide-based cargo delivery based on agonistic CD40-directed antibodies M. Eltahir1, 2, I. Olsson3, M. Lord3, A. Chourlia3, L. Dahllund4, 5, A. Olsson4, 5, A. Saleh6, J. Ytterberg6, A. Lindqvist6, O. Andersson4, 5, H. Persson4, 5, S. Mangsbo3 1Uppsala University, Pharmaceutical Bioscience, Science for Life Laboratory, Uppsala, Sweden, 2Uppsala University, Immunology Genetics and Pathology, Uppsala, Sweden, 3Uppsala university, Pharmaceutical Bioscience, Science for Life Laboratory, Uppsala, Sweden, 4SciLifeLab Drug Discovery and Development, Stockholm, Sweden, 5Royal Institute of Technology (KTH), School of Engineering Sciences in Chemistry, Biotechnology and Health, Stockholm, Sweden, 6Uppsala universitet, Department of Pharmacy, Translational PKPD Research Group, Science for Life Laboratory, Uppsala, Sweden
Text Much attention has focused on developing CD40 directed agonistic monoclonal antibody therapy. These antibodies have been profiled for their epitope specificity and isotype in relation to their agonistic potential. Still, clinical impact relies on a well-balanced clinical efficacy versus target-mediated toxicity. As CD40- mediated immune activation must rely on a combination of co-stimulation of antigen-presenting cells alongside antigen-presentation, alternative approaches to improve the therapeutic outcome of CD40- targeting strategies should focus on providing optimal antigen presentation together with CD40 stimulation. Herein we developed a bispecific antibody targeting CD40 as a means to carry in cargo (herein synthetic peptides) into antigen-presenting cells using a high-affinity interaction between the antibody and the cargo peptide, further referred to as the Antibody Drug Affinity Conjugate (ADAC) technology. The bispecific antibody preclinical efficacy was confirmed using in vitro and in vivo T cell activation/proliferation assays. In an in vitro DC-T cell co-culture model using the CD4 epitope OVA 329-337 (OT-II) or the CD8 epitope OVA257-
264 (SIINFEKL), the non-covalent, high-affinity coupling of antigens to the anti-CD40 bispecific antibody via a peptide-Tag (pTag) was shown, in a target-specific manner, to improve both CD4+ and CD8+ T cell expansion in vitro. This strategy, delivering CD40-mediated antigen cargo via affinity coupling, resulted in a 4-6 fold increase in activated T cells compared to uncoupled antigens in vitro. Furthermore, using the hgp100 in vivo vaccination model, a significant enhancement in proliferating antigen-specific CD8+ T cell pool was observed with a 4-fold increase in proliferating hgp100-specific T cells when the ADAC was used. In addition, the strategy dramatically improved the in vitro (human and mouse plasma) and the in vivo (mouse plasma) half-life of the synthetic peptide drug part through the high-affinity drug cargo strategy. Overall, the ADAC technology provides a robust, flexible platform for personalized cancer vaccines. Future applications of the technology involve pandemic preparedness to viral genetic drift as well as neoepitope vaccination strategies where the bispecific antibody is an off-the-shelve product, and synthetic peptide production is synthesized based on next-generation sequencing data mining.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-37
Medical Gas Plasma Jet Technology Targets Murine Melanoma in an Immunogenic Fashion S. Bekeschus1, R. Clemen1, E. Freund1, 2, A. Schmidt1 1Leibniz Institute for Plasma Science and Technology (INP), ZIK plasmatis, Greifswald, Germany, 2Greifswald University Medical Center, Department of General, Visceral, Thoracic, and Vascular Surgery, Greifswald, Germany
Text Medical technologies from physics are imperative in the diagnosis and therapy of many types of diseases. In 2013, a novel cold physical plasma treatment concept was accredited for clinical therapy. This gas plasma jet technology generates large amounts of different reactive oxygen and nitrogen species (ROS). Using a melanoma model, gas plasma technology is tested as a novel anticancer agent. Plasma technology derived ROS diminish tumor growth in vitro and in vivo. Varying the feed gas mixture modifies the composition of ROS. Conditions rich in atomic oxygen correlate with killing activity and elevate intratumoral immune- infiltrates of CD8(+) cytotoxic T-cells and dendritic cells. T-cells from secondary lymphoid organs of these mice stimulated with B16 melanoma cells ex vivo show higher activation levels as well. This correlates with immunogenic cancer cell death and higher calreticulin and heat-shock protein 90 expressions induced by gas plasma treatment in melanoma cells. To test the immunogenicity of gas plasma treated melanoma cells, 50% of mice vaccinated with these cells are protected from tumor growth compared to 1/6 and 5/6 mice negative control (mitomycin C) and positive control (mitoxantrone), respectively. Gas plasma jet technology is concluded to provide immunoprotection against malignant melanoma both in vitro and in vivo.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-38
Gas plasma technology augments ovalbumin immunogenicity and OT-II T cell activation conferring tumor protection in mice R. Clemen1, E. Freund1, 2, L. Miebach1, 2, B. Bröker3, S. Bekeschus1 1Leibniz Institute for Plasma Science and Technology (INP), ZIK plasmatis 'Plasma-Redox-Effects', Greifswald, Germany, 2University Medicine Greifswald, Department of General, Visceral, Thoracic, and Vascular Surgery, Greifswald, Germany, 3University Medicine Greifswald, Department of Immunology, Greifswald, Germany
Text Reactive oxygen species (ROS/RNS) are produced during inflammation and elicit protein modifications, but the immunological consequences are largely unknown. Gas plasma technology capable of generating an unmatched variety of ROS/RNS is deployed to mimic inflammation and study the significance of ROS/RNS modifications using the model protein chicken ovalbumin (Ova vs. oxOva). Dynamic light scattering and circular dichroism spectroscopy revealed structural modifications in oxOva compared to Ova. T cells from Ova-specific OT-II but not from C57BL/6 or SKH-1 wild-type mice presented enhanced activation after Ova addition in vitro. When administered in vivo, OxOva exacerbated this activation, including increased release of interferon-gamma, a known anti-melanoma agent. Wild-type mice that were vaccinated with oxOva followed by inoculation of syngeneic B16F10 Ova-expressing melanoma cells showed enhanced T cell numbers and -activation, decreased tumor burden, and elevated numbers of antigen-presenting cells when compared to their Ova-vaccinated counterparts. Analysis of oxOva using mass spectrometry identified three hot spot regions rich in oxidative modifications that are associated with the increased T cell activation. Using Ova as a model protein, the findings suggest an immunomodulating role of multi-ROS/RNS modifications that may spur novel research lines in inflammation research and for vaccination strategies in oncology.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-39
The effect of liposomes from total lipid of Mycobacterium semgmatis with mice bone marrow- derived dendritic cells R. Kadir1, N. e. Mat Luwi1, S. Ahmad1, R. Mohamud1, A. Acosta2, M. E. Sarmiento2, M. N. Norazmi2 1Universiti Sains Malaysia, Immunology, Kota Bharu, Malaysia, 2Universiti Sains Malaysia, School of Health Sciences, Kota Bharu, Malaysia
Text Liposomes are biocompatible and biodegradable lipid based-nanoparticles in the range of 25 to 2500 nm. Liposomes have one or more concentric bilayers membrane composed of synthetic or naturally derived- phospholipid with mixed lipid chain. It demonstrates advance technology to deliver active molecules to the location of action and currently several formulations in clinical use. It also shows their adjuvant effects to dendritic cells (DCs), with unique capacity of stimulating primary immune responses. Thus, the aim of this study to investigate the effects of liposomes derived from Mycobacterium smegmatis on DCs. Liposomes were synthesized from total lipid of M. smegmatis and mature bone marrow derived- dendritic cells (BMDCs) were generated from Balb/c mice bone marrow. The recognition and internalization of BMDCs were observed and analyzed with FACS analysis, SEM and confocal microscopy after 24 hours exposure with liposomes. The result of SEM images of liposomes showing the spherical structures with average size between 20 nm-80 nm that can be classified as small unilamellar vesicles (SUVs). Flow cytometry analysis demonstrated that the percentage of CD86+, CD86+, CD11c+ and MHCII+ was increase in the BMDCs exposed to liposomes compared to the BMDCs without liposomes and with the inhibitors. SEM and confocal microscopy images show the uptake of liposomes and it was internalized by BMDCs. Besides, the secretion of IFN-g, TNF-α and IL-4 of BMDCs exposed to liposomes was higher compared to other groups. In conclusion, the results obtained in the current study may revealed several promising aspect of liposomes as a future candidate for an effective therapeutic application. These findings need further research before can be integrated into development of future drug delivery agent, vaccine and immunotherapeutic treatments.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-40
T cell receptor discovery for cancer and beyond C. Wagner1, F. Brunk1, A. Moritz1, J. Walz2, 3, 4, A. Nelde2, 3, 4, N. Casadei5, C. Reinhardt1, H. Singh1, H.-G. Rammensee2, 4, D. Maurer1 1immatics biotechnologies, Tuebingen, Germany, 2Institute for Cell Biology, University of Tübingen, Immunology, Tübingen, Germany, 3Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Department of Internal Medicine, University Hospital Tübingen, Tübingen, Germany, 4Cluster of Excellence iFIT (EXC2180) ‘Image-Guided and Functionally Instructed Tumor Therapies’, University of Tübingen,, Tübingen, Germany, 5NGS Competence Center Tübingen, Institute of Medical Genetics and Applied Genomics, University Hospital Tübingen, Tübingen, Germany
Text T cell receptor (TCR)-mediated immunotherapy holds great promise for the treatment of malignant diseases, including solid cancers. A prerequisite for successful therapy is the availability of highly tumor-specific targets in combination with potent and safe TCRs. TCRs naturally recognize human leukocyte antigen (HLA)-bound peptides derived from foreign and endogenous proteins regardless of their extracellular or intracellular location, creating a broad target space for TCR-based approaches. At Immatics, cancer-specific targets are discovered through the target discovery platform XPRESIDENT®, that integrates quantitative mass spectrometry, transcriptomics and bioinformatics. The TCR platform XCEPTOR® generates highly specific TCRs towards these novel tumor targets. We will exemplify how TCRs are identified from human T cells either directly from the natural repertoire or after in vitro pre-expansion, and how they are thoroughly characterized, embedding information from the large XPRESIDENT® collection of healthy tissues and tumor biopsies to investigate on- and off-target specificity of TCR candidates. The best TCRs are developed towards adoptive cell therapy applications such as our ACTengine® trials, where the patient´s own T cells are lentiviral engineered with a tumor-specific TCR. Given the ongoing global COVID-19 pandemic, we demonstrated the universality of the established TCR discovery approach and used the platform to rapidly identify novel SARS-CoV2 specific TCRs against recently identified SARS-CoV-2 epitopes. While T cells have been recognized as major player in effective anti-SARS-CoV-2 immunity, data relating a functional T cell response to individual TCRs is still sparse. We provide insights into the characteristics of individual HLA- A*02:01- and HLA-A*24:02-restricted SARS-CoV-2-reactive TCRs, isolated from convalescent COVID-19 patients. These SARS-CoV-2-reactive TCRs mediated T cell effector functions upon re-expression in primary CD8+ T cells. We also provide an example of long-lasting anti-SARS-CoV-2 memory by re-isolation of one of the retrieved TCRs five months after the initial sampling. Taken together, these findings contribute to the understanding of anti-SARS-CoV-2 T cell immunity and signal the versatility of our XCEPTOR® TCR platform for discovering TCRs for cancer and possibly beyond.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-41
An in-depth computational investigation of cell populations with prognostic significance at diagnosis of acute myeloid leukemia S. Bonte1, 2, A. Couckuyt3, 4, V. Janda5, S. Van Gassen2, 3, 4, J. Philippé2, 5, 6, Y. Saeys2, 3, 4, T. Kerre1, 2, 6, 7 1Ghent University, Department of Internal Medicine & Pediatrics, Ghent, Belgium, 2Cancer Research Institute Ghent (CRIG), Ghent, Belgium, 3VIB Center for Inflammation Research, Data Mining and Modeling for Biomedicine, Ghent, Belgium, 4Ghent University, Department of Applied Mathematics, Computer Science & Statistics, Ghent, Belgium, 5Ghent University Hospital, Department of Clinical Biology, Ghent, Belgium, 6Ghent University, Department of Diagnostic Sciences, Ghent, Belgium, 7Ghent University Hospital, Department of Hematology, Ghent, Belgium
Text Relapsed acute myeloid leukemia (AML) has a poor prognosis. To lower relapse incidence, it is imperative to identify high-risk patients at diagnosis and reliably detect measurable residual disease (MRD) after therapy. The current 2017 ELN risk stratification, based on molecular and genetic tests, classifies patients into favorable, intermediate and poor risk groups. We want to improve risk stratification by including flow cytometric analysis, as it has already shown its importance in classification and MRD detection of AML. We developed a computational pipeline for analyzing flow data. By linking flow data to clinical outcome data, we aim to identify both new prognostic markers and therapeutic targets for AML.
Flow data (fcs) at diagnosis (EuroFlow panel) and clinical data at diagnosis and during follow-up were collected from a total of 198 patients diagnosed with AML at Ghent University Hospital (Belgium) between 2015-2019. Fcs files were first pre-processed computationally: margin events, doublets and aberrant timeframes during acquisition were removed using the PeacoQC algorithm and debris was automatically removed based on FlowSOM clustering. Resulting clean fcs files were used to build a FlowSOM model. Patients were divided into groups based on ELN risk category or clinical outcome and statistical analysis was used to compare these groups.
Preliminary results confirm previously published observations. Presence of an increased frequency of CD34+CD38- leukemic stem cells (LSC) at diagnosis marked patients with a worse prognosis. Also, a significantly higher percentage of LSC was found in poor and intermediate risk versus favorable risk patients (based on ELN risk stratification). Additionally, intermediate/poor risk patients with a higher frequency of CD34+CD7+ AML cells at diagnosis, more often had residual disease after induction therapy. We are currently analyzing other cell populations which are more/less abundantly present at diagnosis in patients with worse outcomes.
Computational analysis is a promising method for objective analysis of flow cytometry data. Moreover, combining flow data with clinical data could result in the detection of specific cell populations (e.g. LSC) present at diagnosis and responsible for MRD or relapse in AML, therefore linked to a worse prognosis. This would help optimize risk stratification and treatment strategy for patients with AML at diagnosis. Furthermore, markers expressed on these high-risk cell populations could be identified as novel therapeutic targets for immunotherapy of AML. We are currently also developing a 20-color panel for analysis of AML samples, including a variety of LSC markers, to further aid in identifying novel targets for AML immunotherapy.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-42
Targeting replication stress response upregulates PD-L1 expression and potentiates the effect of immune checkpoint blockade by modulating the immune microenvironment in small cell lung cancer T. Sen Memorial Sloan Kettering Cancer Center, Medicine, New York City, United States
Text Background: Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, are urgently needed. SCLC has a relatively immunosuppressed phenotype with relatively low levels of infiltrating T-cells. Only a minority of SCLC patients responds to immune checkpoint blockade (ICB). Therefore, there is a critical need to develop strategies to enhance the efficacy of immunotherapy in SCLC. We previously discovered that replication stress response (RSR) proteins are overexpressed in SCLC; targeting RSR has activity in preclinical models of SCLC. We hypothesize that tumor associated neoantigen expression is suppressed in SCLC by several mechanisms, including RSR machinery and that targeting RSR can enhance immunogenicity, antitumor immunity and response to ICB. Results: In SCLC models, inhibition of RSR proteins (CHK1, PARP, ATR) by genetic knockdown and pharmacological inhibition increased protein levels of immune checkpoint ligand, PD-L1. We next tested whether co-targeting RSRi+PD-L1 enhances the anti-tumor effect in immune-competent SCLC model. B6129F1 mice were injected with murine SCLC cells harboring conditional deletion of Trp53, Rb1, p130. When the tumor volume reached 120mm^3, mice were treated with either IgG (control), RSRi, anti-PD-L1 (300ug, 1/7) or combination of RSRi and anti-PD-L1 antibody. Single agent treatment with anti-PD-L1 antibody did not cause tumor regression in these models. However, we observed compete tumor regression with combining RSRi and anti-PD-L1. Flow cytometry-based profiling of top T-cell characterization and cytokine markers demonstrated that co-targeting RSRi+PD-L1 significantly increases the level of CD8+ cytotoxic-T cell tumor infiltration, decreases exhausted T cell population and enhances innate immune signaling in SCLC models. We demonstrated that RSRi+PD-L1 blockade and inactivates GSK3β and stabilizes PD-L1 thus providing mechanistic insight into PD-L1 regulation in SCLC. Discussion: Unlike other cancer types, immunotherapy has objective responses in only a minority of SCLC patients. Our study demonstrates, for the first time, the potential for combining the two promising modalities, immune checkpoint targeting and RSR inhibition in SCLC. The immune profiling and correlative biomarker data further provide valuable mechanistic insight and indicate the subset of the patient population who are most likely to respond to these treatments. This study suggests intriguing possibilities for therapeutic synergy and scientific rationale for the evaluation of PD-L1 / PD-1 blockade with RSRi in clinical trials for SCLC, a disease with dismal 5-year survival rate of < 5%, which has remained unchanged for >30 years.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-43
Citrullinated epitopes identified on tumour MHC class II by peptide elution stimulate both regulatory and Th1 responses and require careful selection for optimal anti-tumour responses P. Symonds1, A. Marcu2, 3, K. Cook1, R. Metheringham1, L. Durrant1, 4, V. Brentville1 1Scancell Ltd, Nottingham, United Kingdom, 2University of Tubingen, Department of Immunology, Tubingen, Germany, 3University of Tubingen, Cluster of Excellence iFIT (EXC 2180) "Image-Guided and Functionally Instructed Tumor Therapies", Tubingen, Germany, 4University of Nottingham, Division of Cancer & Stem Cells, Nottingham, United Kingdom
Text Somatic mutations or post-translational modifications of proteins result in changes that enable immune recognition. One such post-translational modification is citrullination, the conversion of arginine residues to citrulline by peptidylarginine deiminase (PAD) enzymes. Citrullinated peptides can be presented on MHC class II (MHCII) via autophagy which is upregulated in times of cellular stress. Tumour cells are subject to a number of cellular stresses and upregulate autophagy thus making them targets for immune attack. Citrullinated peptides presented on MHCII molecules were eluted from B16 melanoma model expressing HLA-DP4 and analysed by mass spectrometry in order to profile the presented repertoire. Seven of the identified citrullinated peptides were used in combination to vaccinate HLA-DP4 transgenic mice. The combined vaccine induced only weak anti-tumour therapy. Immune responses revealed that citrullinated matrix metalloproteinase-21 peptide (cit MMP21) induced an IFNγ response and cytochrome p450 peptide (citCp450) induced an IL-10 response. The remaining peptides failed to produce either cytokine. The dominant response to cit MMP21 peptide was not due to stronger MHC-II binding as all seven peptides showed similar binding to HLA-DP4. Individual peptide vaccination showed similar responses to the immunisation with the combination with citMMP21 inducing IFNγ responses and citCp450 stimulating only IL- 10. Surprisingly citrullinated Glutamate receptor ionotropic (citGRI) peptide also stimulated an IFNγ response as a single immunogen. Of particular interest was that both citMMP21 and citGRI peptides individually, stimulated strong anti-tumour responses that were significantly better than the combined vaccine. To determine if the citCp450 peptide, inducing the IL-10 response, was dominantly suppressing the anti-tumour response of the combined vaccine it was excluded from the combination vaccine but this failed to recover a strong anti-tumour response. As all the peptides in the combined vaccine have similar binding affinities to MHC-II, they may compete for binding and present a lower dose of each individual peptide on antigen presenting cells. Indeed, the avidity of the T cell response to citGRI was significantly lower than the citMMP21 response and lower doses of citGRI peptide failed to elicit an immune response. These results suggest that characterisation of responses from epitopes identified by peptide elution is necessary to optimise candidate selection for tumour therapy.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-44
Inhibition of the autophagy related protein Vps34 reprograms cold into hot inflamed tumors and improves anti-PD-1/PD-L1 immunotherapy M. Z. Noman1, S. Parpal2, Y. Yu2, M. Andersson2, A. De Milito2, B. Janji1 1Luxembourg Institute of Health, Department of Oncology - Tumor Immunotherapy and Microenvironment group, Luxembourg City, Luxembourg, 2Sprint Bioscience, Stockholm, Sweden
Text One of the major challenges limiting the efficacy of anti-PD-1/PD-L1 therapy in non responding patients is the failure of T cells to penetrate the tumor microenvironment. These patients’ tumors are referred to as immune cold tumors. There is an unmet clinical need to develop therapies enhancing the infiltration of cytotoxic immune cells into tumors. We evaluated the impact of inhibiting the autophagy related lipid kinase Vps34 on the immune landscape and the therapeutic benefit of anti-PD-L1/PD-1 in melanoma and colorectal cancer (CRC). Both genetic or pharmacological inhibition of Vps34 kinase activity using SB02024 or SAR405 (Vps34i) decreased the tumor growth and significantly improved mice survival in multiple tumor models by inducing a massive infiltration of NK, CD8+ and CD4+ T effector cells, notably in melanoma and CRC tumors. Such infiltration resulted in the establishment of a T cell inflamed tumor microenvironment, characterized by the upregulation of pro-inflammatory chemokines and cytokines, CCL5, CXCL10 and IFN gamma. Levels of CCL5 and CXCL10 were increased in the blood of melanoma and CRC bearing mice treated with SB02024. Mechanistically, Vps34i treatment induced STAT1 and IRF7 which are involved in the up-regulation of CCL5 and CXCL10. Combining Vps34i improved the therapeutic benefit of anti-PD-L1/anti- PD1 in melanoma and CRC and prolonged mice survival. Our study revealed for the first time that targeting Vps34 can turn immune cold tumors into immune hot inflamed tumors, thus enhancing the efficacy of anti- PD-L1/anti-PD-1 blockade.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-45
Accurate detection of tumor-specific fusion genes with EasyFuse reveals strongly immunogenic personal neo-antigens D. Weber1, J. Ibn-Salem1, P. Sorn1, M. Suchan1, C. Holtsträter1, U. Lahrmann2, I. Vogler2, K. Schmoldt2, F. Lang1, B. Schrörs1, M. Löwer1, U. Sahin1, 2, 3 1Translational Oncology, Mainz, Germany, 2BioNTech, Mainz, Germany, 3Johannes Gutenberg University Mainz, Mainz, Germany
Text Cancers are driven by genetic alternation such as small variants (point mutations, small insertions and deletions) and large structural variants of which the vast majority is unique to individual patients. While small variants are already considered in personalized therapy approaches, individual structural variants giving rise to fusion genes, a potential rich source for highly immunogenic neo-antigens, are currently not considered. Their exploitation as targets for personalized cancer immunotherapy is limited by a lack of validated computational tools allowing transcriptome-wide identification of unique fusion genes in an accurate and sensitive manner.
Here, we present EasyFuse, a computational pipeline, to detect individual and cancer-specific fusion genes in next-generation-sequencing transcriptome data obtained from human cancer samples. EasyFuse utilizes several prediction strategies in parallel to provide best sensitivity. We experimentally validated >1500 individual fusion candidates in cell lines and relevant FF and FFPE patient samples by qRT-PCR to establish the performance of EasyFuse. Using this data, we trained a machine learning classifier to improve specificity to of the predictions: On independent test data, we observed a performance of 95% positive predicted value at a sensitivity of 50%. Using this unprecedented ground-truth dataset, EasyFuse clearly outperforms other fusion detection tools in terms of specificity and sensitivity. We further investigated 30 selected fusion gene candidates for pre-existing immunogenicity in autologous blood lymphocytes from cancer patients. Here, we detected pre-established CD4+ and CD8+ T cell responses for 10 of 21 (48%), and for 1 of 30 (3%) of identified fusion genes, respectively.
In conclusion, we demonstrate accurate detection of cancer-specific fusion genes from transcriptome sequencing data. Furthermore, the high frequency of T cell responses detected in cancer patients supports the relevance of individual fusion genes as neo-antigens for personalized immunotherapies, especially for tumors with low point mutation burdens.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-46
The impact of glucocorticoid producer HSD11B1 on the antitumor immune response in renal cancer H. Poinot1, 2, E. Dupuychaffray1, 2, M. Alvarez1, 2, J. Tachet1, 2, G. Disner1, 2, E. Olesti1, 2, O. Ezzar1, 2, V. Gonzalez Ruiz1, 2, S. Rudaz1, 2, C. Bourquin1, 2, 3, A. Pommier1, 2 1University of Geneva, School of Pharmaceutical Sciences, Geneva, Switzerland, 2University of Geneva, Institute of Pharmaceutical Sciences of Western Switzerland, Geneva, Switzerland, 3University of Geneva, Department of Anaesthetics, Pharmacology, Intensive Care and Emergencies, Faculty of Medicine, Geneva, Switzerland
Text Glucocorticosteroids modulate many biological functions, but how their metabolism affects the antitumor immune response is not fully understood. By correlating gene expression data and clinical outcomes from the TCGA, we identified a set of genes involved in the metabolism of glucocorticoids and associated with poor survival in patients with renal cancer. One of these genes, 11beta-hydroxysteroid dehydrogenase type 1 (hsd11b1), regenerates inactive glucocorticoids into active glucocorticoids that are well known to be immunosuppressive. Furthermore, we showed that the substrate for HSD11B1 was highly produced in mouse kidney. We hypothesized that intratumoral HSD11B1 could generate active glucocorticoids in renal cancer, which then impair antitumor immunity. In order to examine how HSD11B1 activity affects the antitumor immune response, we have designed a murine coculture assay composed of dendritic cells, T cells and renal cancer cells. We showed that HSD11B1 activity inhibits T-cell activation and killing of cancer cells and that this inhibition is reversed by pharmacological HSD11B1 inhibitors. Moreover, in a human antigen recall assay where PBMC were stimulated with antigen, the antigen-specific T cell activation decreased with HSD11B1 activity and was restored with the addition of HSD11B1 inhibitors. Importantly, combination of HSD11B1 inhibitors with an anti-PD-1 immune checkpoint inhibitor improved the response to the checkpoint inhibitor in these mouse and human assays. We are currently investigating the impact of HSD11B1 inhibitor and anti-PD1 combination on the antitumor immune response in mice. In summary, our data suggest that intratumoral HSD11B1 may impair the antitumor immune response and that inhibiting HSD11B1 may improve the efficacy of immunotherapy in patients with renal cancer.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-47
ANV419 is a new generation CD122-biased IL-2/anti-IL-2 antibody fusion protein with potent stimulatory function on immune effector cells in vitro and in vivo P. Murer1, B. Brannetti2, J.-M. Rondeau2, S. Popp2, C. Regnier2, L. Petersen1, N. Egli1, A. Katopodis1, C. Huber1 1ANAVEON AG, Basel, Switzerland, 2Novartis Institutes for Biomedical Research, Basel, Switzerland
Text Novel interleukin-2 (IL-2)-based therapeutic modalities with preferential signaling through the IL-2 β/γ receptor (IL-2Rβ/γ) are entering clinical trials and have the potential to substantially increase the therapeutic index of recombinant IL-2 (aldesleukin) for cancer therapy. We developed ANV419, a uniquely engineered antibody-cytokine fusion protein, in which IL-2 is grafted in the L-CDR1 of a humanized anti-IL-2 antibody, able to block the receptor alpha (IL-2Rα, CD25) binding site.
Resolution of the ANV419 crystal structure proves that the IL-2Rα binding site of IL-2 is blocked by the antibody, while the IL-2Rβ/γ sites remain accessible. Hence, ANV419 displays high affinity binding for IL2-Rβ and for the heterodimeric IL-2Rβ/γ, but no affinity for IL-2Rα.
The novel IL-2-antibody fusion protein selectively induces STAT5 phosphorylation of CD8+ effector T cells and NK cells after incubation with murine splenocytes or human and non-human primate peripheral blood monocyte cultures. Mice receiving a single intravenous administration of ANV419 exhibited a dose- dependent expansion of CD8+ T cells and NK cells, but not of CD4+CD25+ regulatory T cells. This was observed by a significant increase of the proliferation marker Ki67 in these effector cells, as well as higher absolute cell numbers in the spleens and peripheral blood of animals.
In checkpoint sensitive (H22) as well as in checkpoint resistant (Renca and B16F10) syngeneic murine tumor models ANV419 monotherapy led to significant tumor growth retardation compared to vehicle. The combination treatment with anti-HER2 antibody trastuzumab led to an increased anti-tumor activity of the monoclonal antibody in the gastric cancer N87 xenograft model.
In non-human primates, ANV419 was well tolerated and did not induce vascular leak syndrome, which is observed in patients receiving high-dose aldesleukin treatments. The unique structure of the ANV419 antibody-cytokine construct defines its selectivity towards IL-2Rβ/γ expressing cells. Aligned to its potent proliferative effect on CD8 and NK cells, ANV419 induces strong anti- tumor responses in various mouse models of cancer. This pre-clinical data and the marked safety window of ANV419 in non-human primates support its translational development as immunotherapeutic agent in oncology.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-48
An RNA-encoded IL-2 variant with an extended half-life mediates synergistic anti-tumor activity when combined with immune checkpoint blockade M. Vormehr1, L. M. Kranz1, A. Muik1, D. Eisel1, S. Fellermeier-Kopf1, J. Diekmann1, S. Witzel2, Ö. Türeci1, U. Sahin1 1BioNTech SE, Mainz, Germany, 2TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz gGmbH, Mainz, Germany
Text The potential of cytokine therapies in oncology remains largely untapped. Recombinant interleukin-2 (IL-2) has been used for the treatment of late stage malignant melanoma and renal cell cancer since the 1990s. Most patients with complete responses after IL-2 treatment remain regression-free for more than 25 years after initial treatment, but overall response rates are low. IL-2 is known to support the differentiation, proliferation, survival and effector functions of T cells. However, efficient treatment with recombinant cytokines is complicated by its short half-life which is in the range of minutes and thus requires high level and frequent dosing, which in turn potentiates side effects. A particular challenge of IL-2 for cancer treatment is the preferential stimulation of regulatory T (Treg) cells in addition to activated tumor specific effector T cells already at low doses. These immunosuppressive cells constitutively express the trimeric high-affinity IL-2 receptor (IL-2R) comprising α, β, and γ subunits, whereas naïve T cells express the medium-affinity IL-2Rβγ. Likewise, T cells that have previously been activated with cognate antigen revert to expressing only IL-2Rβγ following a transient period of IL2-Rα upregulation shortly after antigen encounter. We designed a novel class of lipid nanoparticle-formulated, nucleoside-modified RNA therapeutics called RiboCytokines®. For preferential expansion of effector T cells over Treg cells, we engineered a RiboCytokine (BNT151) which encodes a mutant variant of human IL-2 with enhanced affinity for IL-2Rβ and reduced binding to IL-2Rα. Continuous translation of RiboCytokine in the liver as well as the fusion to human serum albumin resulted in a substantially extended serum half-life and an increased bioavailability in the tumor and tumor draining lymph node in mice. Intravenous injection of BNT151 induced a potent increase in CD8+ effector T cells and natural killer cells while Treg cell expansion was not detected. Notably, strong, synergistic therapeutic activity was observed when BNT151 treatment was combined with anti-PD-1/PD-L1 checkpoint blockade. Tumor control of the combination treatment was accompanied by increased numbers of neoantigen-specific T cells and a T cell phenotype previously associated with successful cancer immunotherapy. An open-label, Phase I/II, first-in-human trial in patients with advanced solid tumors was recently initiated to further explore the clinical safety and efficacy of BNT151 as monotherapy or in combination with other anti-cancer agents (NCT04455620).
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-49
An RNA-encoded IL-2 variant with an extended half-life enhances the anti-tumor activity of RNA-based cancer vaccines M. Vormehr1, L. M. Kranz1, A. Muik1, D. Eisel1, S. Fellermeier-Kopf1, J. Diekmann1, S. Witzel2, Ö. Türeci1, U. Sahin1 1BioNTech SE, Mainz 55131, Germany, 2TRON gGmbH, Mainz 55131, Germany
Text Interleukin-2 (IL-2) is known to support the differentiation, proliferation, survival and effector functions of T cells. Recombinant IL-2 has been used in the treatment of late-stage malignant melanoma and renal cell carcinoma since the 1990s. Most patients with complete responses after IL-2 treatment remain regression- free for more than 25 years after initial treatment, but overall response rates are low. A key hurdle to the success of recombinant IL-2-based cancer immunotherapies is its short plasma half-life, which necessitates frequent administration of high cytokine doses, leading to severe side effects such as vascular leak syndrome. Moreover, treatment with IL-2 preferentially triggers the expansion of regulatory T cells (Tregs) in addition to activated tumor-specific effector T cells already at low doses. These immunosuppressive cells constitutively express the trimeric high-affinity IL-2 receptor (IL-2R) comprising α, β, and γ subunits, whereas naïve T cells express the medium-affinity IL-2Rβγ. Likewise, T cells that have previously been activated with cognate antigen revert to expressing only IL-2Rβγ following a transient period of IL2-Rα upregulation shortly after antigen encounter. To leverage the full therapeutic potential of cytokines, we designed a novel class of lipid nanoparticle-formulated, nucleoside-modified ribonucleic acid (RNA) therapeutics with a substantially extended serum half-life called RiboCytokines®. The RiboCytokine BNT151 encodes an IL-2 variant as a fusion to human serum albumin resulting in an increased bioavailability in tumors and tumor draining lymph nodes of mice. For preferential expansion of effector T cells over Treg cells, the IL-2 variant was designed to have an enhanced affinity for IL-2Rβ and reduced binding to IL-2Rα. Upon intravenous injection, BNT151 mediated strong expansion of murine CD8+ effector T cells and natural killer cells while Treg frequencies remained unchanged. In syngeneic tumor-bearing mice, BNT151 as a single-agent and in combination with RNA-based cancer vaccination elicited a potent anti-tumor response. The effects of the combination were more pronounced when compared to either treatment alone; tumor control in mice was accompanied by an increased number of neo-antigen specific T cells and an increased CD8+ T cell to Treg ratio. These results demonstrate the ability of BNT151 to address the limitations of recombinant IL-2 therapy, making it a promising therapeutic agent and combination partner for cancer treatment. An open-label, Phase I/II, first-in- human trial in patients with advanced solid tumors was recently initiated to further explore the clinical safety and efficacy of BNT151 (NCT04455620).
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-50
Alternative pre-mRNA splicing as a source of tumor neoantigens A. Knudsen1, 2, J. Becker3, 4, L. Gallego-Paez1, C. Esdar5, A. Kulozik3, 4, 6, J. Mauer1 1BioMed X Institute, Heidelberg, Germany, 2Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany, 3Molecular Medicine Partnership Unit, Heidelberg University, Heidelberg, Germany, 4European Molecular Biology Laboratory, Heidelberg, Germany, 5Merck KGaA, Translational Innovation Platform Oncology, Darmstadt, Germany, 6Heidelberg University Children's Hospital, Department of Pediatric Hematology and Oncology, Heidelberg, Germany
Text Robust identification of neoantigens is crucial for the efficacy and safety of immunotherapy. Current approaches have provided limited numbers of immunogenic and cancer-specific targets, thus preventing the broad application of immunotherapy. Here, the focus on somatic mutation-derived neoantigens often overlooks possible neoepitopes originated from mRNA processing events. A potential new source for neoantigens is alternative pre-mRNA splicing, a process which is widely dysregulated in several cancer subtypes. However, there is limited insight regarding the potential of alternative splicing to generate peptides that are also presented on the cell surface. Thus, we aimed to investigate how dysregulation and common mutations found in cancer contribute to the neoepitope repertoire in tumor cells.
To explore alternative splicing-derived neoantigens, we choose a direct approach to determine the immunopeptidome of wild-type RPE-1 cells and RPE-1 cell lines harboring common cancer mutations. To facilitate presentation of alternative splicing-derived neoepitopes, we treated these cell lines with the splicing inhibitor GEX1A. We then performed direct HLA-I immunopurification to recover HLA-I-bound peptides of these cells, followed by peptide identification through mass spectrometry. To be able to identify non-canonical peptides from mass spectra, we generated sample-specific custom reference databases consisting of splice junction-spanning, exonic, and intronic peptide sequences derived from matched RNA-seq data. This strategy allowed us to identify more than 5,000 HLA-I-presented peptides per cell line. We then analyzed the proportion of peptides originating from aberrant alternative splice events. By performing differential splicing analysis between the various conditions, we obtained thousands of differentially regulated exon and junction events. Particularly in cells treated with the splicing inhibitor GEX1A, alternative splicing analysis revealed numerous novel splice events. To examine whether these dysregulated events were translated into novel peptides, we subsequently mapped our candidate peptides to the identified splice events. With this strategy, we were able to identify several alternative splicing-derived peptides that can now be further explored as potential neoantigens.
Taken together, we showed that small molecule-induced aberrant splicing has the potential to promote presentation of neoantigens derived from alternative splice junctions, alternative exons, and retained introns. Our findings have potential implications for immunotherapy of cancer types with low tumor mutational burden, where exploration of the splicing-derived neopeptidome could reveal novel therapeutic targets.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-51
RNA-encoded IL-2 and IL-7 with extended half-lives synergize to modulate T cell responses and enhance anti-tumor efficacy L. Kranz1, M. Vormehr1, D. Eisel1, J. Dieckmann1, A. Muik1, S. Fellermeier-Kopf1, S. Witzel2, M. Diken1, 2, S. Kreiter1, 2, Ö. Türeci1, U. Sahin1 1BioNTech SE, Mainz, Germany, 2TRON gGmbH – Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany
Text Interleukin-2 (IL-2) is known to support effector T cell differentiation, proliferation and survival. A particular challenge of IL-2 for cancer treatment is the preferential stimulation of regulatory T cells (Tregs) constitutively expressing the high-affinity IL-2 receptor alpha chain (CD25). Tregs can undermine the anti-tumor activity of antigen-specific effector T cells that only transiently express CD25. IL-7 is a homeostatic cytokine that plays an important role in lymphopoiesis, survival and memory formation, and can increase the expression of CD25 on antigen-specific effector T cells. Prompted by their complementary effects, here we investigated the potential synergy between IL-2 and IL-7 on the T cell level and on anti-tumor activity.
We designed a novel class of lipid nanoparticle (LNP)-formulated, nucleoside-modified ribonucleic acid (RNA) therapeutics which encode cytokine-serum albumin fusion proteins for intravenous delivery, called RiboCytokines®. Continuous translation of RiboCytokine in the liver as well as the fusion to human serum albumin resulted in a substantially extended serum half-life and an increased bioavailability in tumors and tumor draining lymph nodes in mice. The ability of human IL-7 (BNT152) and IL-2 (BNT153) to enhance the anti-tumor activity of a liposome-formulated RNA vaccine was determined for both RiboCytokines as single agents and in combination. We demonstrated that both BNT152 and BNT153 increase the number and frequency of RNA vaccine-induced antigen-specific CD8+ T cells. BNT152 expanded CD8+ T cells with specificities for targets other than the vaccine-encoded antigen, and this effect was more robust than BNT153. As expected, BNT153 expanded the number and frequency of Tregs. In contrast, BNT152 reduced the fraction of Tregs among CD4+ T cells. The combination of BNT152 with BNT153 proved to be powerful in overcoming their limitations as single agents as it boosted the number of antigen-specific CD8+ T cells beyond the levels of BNT153 alone, and further improved the ratio of antigen-specific CD8+ T cells over Tregs. These findings translated into superior anti-tumor activity of the combination in multiple subcutaneous mouse tumor models.
Based on the complementary mode of action of BNT152 and BNT153 and the promising preclinical data, clinical investigation will be pursued in a planned open-label, Phase I first-in-human trial (NCT04710043).
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-52
Targeted Repolarization of Tumor-Associated Macrophages via Imidazoquinoline-Linked Nanobodies M. Scherger1, E. Bolli2, S. M. Arnouk2, J. A. Van Ginderachter2, L. Nuhn1 1Max-Planck-Institute for Polymer Research, Mainz, Germany, 2Vrije Universiteit Brussel, Lab of Cellular and Molecular Immunology, Brussels, Belgium
Text Tumor-associated macrophages (TAMs) promote the immune suppressive microenvironment inside tumors and are, therefore, considered as alternative promising target for next generation cancer immunotherapies. To repolarize their phenotype into a more pro-inflammatory and tumoricidal state, we established immunochemical tools to couple the small molecule Toll-like receptor 7/8 agonist imidazoquinoline IMDQ site-specifically and quantitatively to single chain antibody fragments, so-called nanobodies, targeting the macrophage mannose receptor (MMR or CD206) on TAMs. Intravenous injection of these well-defined bioconjugates resulted in a tumor- and cell-specific delivery of IMDQ into MMRhigh TAMs, causing a significant decline in tumor growth. This was accompanied by a repolarization of TAMs towards a pro- inflammatory phenotype and an increase in anti-tumor T cell responses. Combination treatments with PD-1 checkpoint inhibition further synergized these antitumor responses. Consequently, the therapeutic benefit of precise nanobody-immunodrug conjugates may pave the road towards effective macrophage re-educating cancer immunotherapies. (Adv. Sci. 2021, 2004574)
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-53
RNA-encoded extended half-life IL-2 enhances vaccine-specific T-cell responses and boosts anti-tumor activity in combination with RNA vaccination D. Reidenbach1, L. Kranz2, M. Vormehr2, S. Witzel1, D. Eisel2, M. Diken1, 2, S. Kreiter1, 2, Ö. Türeci2, U. Sahin2 1TRON gGmbH, Mainz, Germany, 2BioNTech SE, Mainz, Germany
Text Interleukin-2 (IL-2) is a key cytokine and pleiotropic mediator of the innate and adaptive immune system, and supports T cell proliferation, differentiation and survival. Therapeutic efficacy of IL-2 is limited by its short half-life and peak dose-mediated toxicities. To overcome these limitations, we used lipid/polymer-formulated nucleoside-modified RNA encoding a murine IL-2-albumin (Alb) fusion protein (mAlb-mIL2) for intravenous injection. Here, we characterize the pharmacokinetics and biodistribution of the translated fusion protein, and investigate the potential of mAlb-mIL2 to promote vaccine-specific T-cell responses and anti-tumor activity in combination with a T cell inducing liposome-formulated RNA vaccine in mice. Administration of mAlb-mIL2 clearly extended the serum half-life of the translated protein in comparison to administration of RNA encoding mIL-2 only, or recombinant mIL-2. Additionally, formulated RNA encoding a reporter-mAlb fusion protein resulted in increased bioavailability of RNA-translated reporter protein in tumors and tumor-draining lymph nodes. In combination with antigen-encoding RNA vaccination, treatment with mAlb-mIL2 significantly improved the induction of antigen-specific CD8+ T cells against foreign and self- antigens in mouse models compared to RNA vaccination alone. Notably, mAlb-mIL2 administered once weekly was as effective as the standard repetitive regimen with recombinant IL-2. mAlb-mIL2 preferentially expanded antigen-specific CD8+ T cells, which demonstrated enhanced and prolonged expression of high affinity IL-2 receptor α (CD25) in response to mAlb-mIL2 treatment. A substantially higher fraction of splenic CD8+ T cells produced the effector cytokines granzyme B and interferon-γ, and the induced CD8+ T cells displayed a stronger cytotoxic potential towards tumor target cells ex vivo after treatment of mice with mAlb- mIL2 and an RNA vaccine, compared to the RNA vaccine alone. In the subcutaneous CT26 colon carcinoma model, mAlb-mIL2 clearly increased the proportion of complete responses and survival in combination with an RNA vaccine and anti-PD-L1 antibody, which correlated with a higher number of tumor antigen-specific CD8+ T cells. Here, we demonstrate the potency of mAlb-mIL2 to boost antigen-specific T cell expansion, functionality and anti-tumor activity in combination with RNA vaccination in mice. RNA-encoded, extended half-life cytokines are anticipated to enhance the tolerability of cytokine therapy, broaden its therapeutic window and potentiate its anti-tumor efficacy.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-54
Novel algorithm discovers up to 35% more epitopes translated from non-coding regions in cold tumours C. Bogaert, L. Van Oudenhove, B. Fant MYNEO NV, GENT, Belgium
Text Currently, most neoantigen pipelines often focus on the detection of neoantigens derived from mutations in the coding regions of the genome. However, in some cancer indications, the number of mutations detectable in tumours can be very low (low tumour mutational burden). This limits the number of actionable neoantigens and results in so-called “cold” tumours. In these cases, non-canonical neoantigens resulting from alterations in non- coding regions of the human genome could represent a high potential alternative for treatment.
Indeed, recent research has revealed that previously presumed non-coding regions of the human genome, such as long non-coding RNAs (lncRNAs), can contain translatable small open reading frames (smORFs) generating micropeptides. Some of these micropeptides have already been shown to be involved in cancer development, but these small peptides could also represent a high potential source of non-canonical neoantigens for personalised therapy.
Here, we present smORFin, a machine learning algorithm specifically trained to identify smORFs in transcripts and to assess their coding potential. While most tools are focused on longer sequences, smORFin is specifically developed to target small ORFs (<303 nucleotides). Furthermore, smORFin also accounts for smORFs with alternative initiation codons, thereby improving its sensitivity for the detection of novel unannotated smORFs. As a result, the smORFin model reaches a precision of 0.98 and an accuracy of 0.95 on its testing dataset. Using this new prediction tool, a library of human smORFs was assembled, the so-called smORFeome. This library of smORFs, and their associated proteins, was evaluated as a reference for spectrum to peptide matching in mass spectrometry data (MS) analysis. Indeed, the evaluation of seven MS samples revealed and validated the presence of smORFeome-related micropeptides and HLA-I-associated epitopes originating from smORFs.
The impact of mutations in allegedly non-coding regions of tumour genomes and its influence on the neoantigen repertoire, was evaluated through integration of smORFin in a neoantigen identification pipeline targeting lncRNA- derived mutated epitopes; lncRNeos. It was observed that these epitopes only represent a minor fraction of the total neoantigen load. Strikingly, when only focusing on tumours with a low neoantigen load, lncRNeos represented up to 27% of the total neoantigen load. This indicates that for tumours with a low TMB, and therefore with a low neoantigen load, lncRNeos allows to significantly expand the neoantigen repertoire. Biological in vivo/in vitro validation remains necessary to assess the existence, presentation, and actionability of lncRNeos.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-55
Novel machine-learning tools improve cost-effective development of personalised immunotherapies: lowering false positive rates in the search for actionable (personalised and largely shared) immunogenic neoantigens C. Bogaert, L. Pfitzer, N. Adell Mill, B. Fant MYNEO NV, GENT, Belgium
Text Personalised immunotherapy approaches rely on the ability of tumour-derived neoantigens to elicit a T-cell immune reaction able to recognise and kill the tumour cells expressing them. Clinical attempts to leverage the power of neoantigens have however yielded mixed results. This can mostly be attributed to the difficulty of finding truly immunogenic peptides from the set of novel peptides generated by mutations in a given cancer patient. In silico approaches can help alleviate this heavy cost by reducing the neoantigen search space, prioritising epitopes based on various parameters such as epitope expression or MHC binding likelihood. Here we present a suite of tools aimed at further assisting clinicians in selecting the most actionable peptides from a set of potential candidates. We developed neoMS, a neural network algorithm able to predict epitope presentation at the cell surface with unparalleled performance. The model achieves up to 0.61 precision at recall 0.4 on its test set, vastly outperforming the current industry standards. In addition, due to his sequence-based comparison method, neoMS exhibits extrapolation capabilities, achieving non-zero predictive power when evaluated on ground truth ligandome data derived from an HLA allele completely absent from the training set. In some cancer indications we showed, moreover, that the neoMS-predicted rate of neoantigen presentation can be used in combination with tumour mutational burden as a high-specificity predictor of response to immune checkpoint inhibitor treatment. Furthermore, the neoIM algorithm is able to discriminate, in an HLA-agnostic fashion, which of the presented peptides will elicit a T-cell immune reaction. This first-in-class algorithm is a random forest classifier specifically trained to classify short peptides of length 9-11 amino acids as immunogenic or non- immunogenic. neoIM vastly outperforms the currently available methods and can predict peptide immunogenicity with high accuracy (AUC=0.88). Interestingly, neoIM confirmed ELISPOT data obtained by Dillon et al. (2017) showing a response in 4 out of 11 breast cancer patients to a vaccine consisting of 9 MHC class-I restricted breast cancer-associated peptides. The 2 antigens that resulted in a CD8+ T-cell specific response were predicted by neoIM as the highest scoring showing its potential in finding the truly immunogenic neoantigens. Taken together, these tools decrease false positive rates significantly as they enable improved identification of immunogenic peptides and the predictions correlate with intensity of immune response and clinical benefits. As such, these tools represent a cost-efficient preliminary step in the search for actionable, immunogenic neoantigens.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-56
The serine/threonine kinase CAMK1D confers immune resistance of PD-L1 refractory tumor cells against tumor-reactive cytotoxic T cells V. Volpin1, 2, T. Michels1, 2, 3, A. Sorrentino1, 2, A. Menevse1, G. Knoll4, M. Ditz1, V. Milenkovic5, C.-Y. Chen1, A. Rathinasamy1, K. Griewank6, M. Boutros7, S. Haferkamp8, M. Berneburg8, C. Wetzel5, A. Seckinger9, D. Hose10, H. Goldschmidt11, M. Ehrenschwender4, M. Witzens-Harig12, A. Szoor13, G. Vereb13, N. Khandelwal3, P. Beckhove1, 2, 14 1Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany, 2German Cancer Research Center (DKFZ), Translational Immunology, Heidelberg, Germany, 3iOmx Therapeutics AG, Martinsried/Munich, Germany, 4Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany, 5Department of Psychiatry and Psychotherapy, Molecular Neurosciences, University of Regensburg, Regensburg, Germany, 6West German Cancer Center, University Duisburg-Essen and the German Cancer Consortium, Department of Dermatology, University Hospital Essen, Essen, Germany, 7German Cancer Research Center (DKFZ), Division Signalling and Functional Genomics, Heidelberg, Germany, 8Department of Dermatology, University Hospital Regensburg, Regensburg, Germany, 9Labor für Myelomforschung, Medizinische Klinik V, Universitätsklinikum Heidelberg, Heidelberg, Germany, 10Department of Hematology and Immunology, Myeloma Center Brussels, Jette, Belgium, 11Department of Internal Medicine V and National Center of Tumor Diseases (NCT), University Hospital Heidelberg,, Heidelberg, Germany, 12Department of Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Heidelberg, Germany, 13Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 14Department of Hematology, Oncology, Internal Medicine 3, University Hospital Regensburg, Regensburg, Germany
Text Background: Endogenous T cell responses against tumor antigens occur frequently in a broad variety of cancer types. However, often this does not rescue patients from tumor progression. As functional tumor reactive T cells are found in many patients refractory to anti- PD-L1/PD-1 therapy, additional immune regulatory interactions may impose dominant levels of protection against immune destruction. Indeed, immune resistance could also be achieved by tumor intrinsic mechanisms of resistance against the cytotoxic T cell attack. Therefore, the identification of novel immune-checkpoints and defining the subsequent molecular mechanisms of inhibition is essential for further improvement in cancer immunotherapy. Materials and methods: We conducted a genetic high-throughput (HT) RNAi screen to identify genes that mediate resistance against cytotoxic T lymphocytes in PD-L1 refractory human tumors. We transfected stable luciferase expressing multiple myeloma cells with a siRNA library targeting 2887 genes and co-cultured the knocked down tumor cells with HLA-A2-matched patient-derived marrow- infiltrating lymphocytes (MILs). T cell-mediated killing of tumor cells was assessed by measuring the remaining luciferase activity. Candidate genes were validated in a secondary screen that allowed to distinguish between genes altering tumor resistance towards MIL-mediated killing and those impairing MILs activity. Luminex analyses were performed to investigate the underlying molecular pathways. Results: The HT-screen revealed 128 genes whose knockdown in tumor cells substantially increased T cell-mediated tumor cell death. Among them, the serine/threonine protein kinase CAMK1D was co-expressed with PD-L1 and played a key role in protection against T cell-induced tumor cell killing not only in multiple myeloma but also in other PD-1/PD-L1-therapy refractory human cancer types. We found that CAMK1D supports the intrinsic tumor resistance against Fas mediated apoptosis. Upon CAMK1D activation through FasL induced calcium influx, CAMK1D bound and phosphorylated the effector caspases 3, 6 and 7, thereby inhibiting their activation and activity resulting in markedly reduced tumor cell apoptosis after cytotoxic T cell attack. Consistently, CAMK1D mediated resistance of murine colorectal cancer cells in vivo. The pharmacological inhibition of CAMK1D on the other hand, restored the sensitivity towards FasL treatment in human multiple myeloma and uveal melanoma cells in vitro. Conclusions: We report a mechanism of rapid inhibition of the terminal apoptotic cascade by a kinase expressed in PD-L1 refractory tumors that can be tuned through T cell activity and may contribute to tumor immune resistance upon immune-checkpoint blockade.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-57
Unraveling molecular networks of resistance against immune checkpoint inhibition J. Sax1, V. Volpin1, A. N. Menevse1, P. Beckhove1, 2 1Regensburg Center for Interventional Immunology (RCI), University Hospital, Regensburg, Germany, 2University Hospital, Department of Hematology, Oncology, Internal Medicine 3, Regensburg, Germany
Text Understanding the function of immune resistance genes in cancer is essential for the development of immunotherapies like immune checkpoint blockade therapy (ICBT). Unfortunately, the majority of patients don’t respond to such therapies resulting in relapse and progressed disease. We hypothesize that tumor cells use so far unknown complementary or redundant pathways to evade immune response. Identifying patterns of gene co- expression will facilitate the understanding of molecular circuits that cancer cells might use. Furthermore, it provides important information about reasons why treatment is failing in some patient cohorts or might even predict disease outcome.
Our group established a genome wide high-throughput screen to identify genes that regulate tumor cell rejection by cytotoxic T cells. So far, in these screens >500 genes in six different tumor entities were identified that regulated tumor cell killing. This set of genes was subjected to a weighted gene co-expression network analysis (WGCNA). As data input, we used RNA seq data from The Cancer Genome Altas (TCGA), the Genotype-Tissue expression project (GTEx) and bulk and single cell RNA seq data from publications. WGCNA enables network analysis to detect modules of co-expressed genes. After identification of gene clusters, the level of gene expression was compared in different patients to investigate individual differences. Furthermore, information of clinical traits was integrated in the analysis, if available.
The co-expression network analysis revealed clusters of co-expression of putative immune regulatory genes in different tumor entities and healthy tissues. The identified clusters were in part restricted to specific tumor types and differed between healthy and malignant tissues. However, some clusters were also used across different tumor entities. We found largely homogeneous usage of co-expression clusters in single tumor cells of the same tumor but strong inter-individual differences in the usage of specific co-clustered genes among different patients with the same tumor type. Preliminary data suggested that some genes can also be related to survival after immunotherapy. Interestingly, known immune checkpoint genes like PD-L1 were not among those genes with a clear correlation.
The results demonstrate a high homogeneity of co-expression of various putative immune regulatory genes within the same tumor and a strong heterogeneity in cluster formation in tumor and healthy tissues and in cluster usage by single patients. These findings point to avenues towards a better understanding of individual differences in tumor resistance and patient responsiveness or unresponsiveness to current ICBT and for their underlying molecular mechanisms.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-58
A humanized In Vivo System for the Preclinical Evaluation of Potential Cytokine Release Syndrome and Efficacy of Bispecific Antibodies D. Cai1, J. Jiao1, A. Flora2, K. Draheim1, M. Cheng1, J. Keck1 1The Jackson Laboratory, Sacramento, United States, 2The Jackson Laboratory, Bar Harbor, United States
Text With their ability to recruit specific immune cell populations on tumor cells, bispecific antibodies (BiAbs) have become the fastest-growing immunotherapy reagents class. The preclinical development of these molecules poses some unique challenges linked to their high specificity towards human targets and, therefore, to the lack of relevant in vivo models to test their efficacy and toxicity. A significant concern is that the accelerated recruitment of effector cells on the tumor site mediated by BiAbs could lead to an exaggerated immune response in patients and, eventually, to serious adverse events linked to cytokine release syndrome (CRS). To decrease the risk of triggering CRS events in patients, drug developers engineer the epitope-binding sites of BiAbs to reduce their affinity towards the tumor cells or the immune effector cell. This approach, however, might result in a loss of efficacy. Therefore, it is necessary to perform a thorough assessment of the CRS potential of BiAbs in relationship to their efficacy to select the most promising therapeutic leads. In this poster, we discuss the development of a new in vivo model for the rapid evaluation of the toxicity and efficacy of bispecific antibodies. This system is based on the reconstitution of immunodeficient mice with human PBMCs and, when required, human tumor cells. By expressing both the tumor and the effector immune cell human targets, this system allows for the direct evaluation of therapeutic BiAb candidates' efficacy and immunotoxicity without the need to use surrogate molecules. Our humanized model shows dose-dependent cytokine release and tumor-killing activity as in the clinical setting. Importantly, this humanized system recapitulates crucial clinical observations, such as donor-to-donor variability and increased cytokine release when BiAbs are combined with checkpoint inhibitors. To conclude, we describe the development and validation of a novel humanized in vivo model to assess quickly and efficiently the efficacy and immunotoxicity of bispecific antibodies alone or in combination with other therapeutic molecules.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-59
Extending the Mass Spectrometry-Detectable Landscape of the Immunopeptidome M. Bernhardt1, Y. Cruz Garcia2, A. Rech3, F. Erhard4, B. Schilling3, A. Schlosser1 1Universität Würzburg, Rudolf Virchow Center, Würzburg, Germany, 2Julius-Maximilians-University Würzburg, Department of Biochemistry and Molecular Biology, Würzburg, Germany, 3University Hospital Würzburg, Department of Dermatology, Venereology, and Allergology, Würzburg, Germany, 4Julius- Maximilians-University Würzburg, Institute for Virology and Immunobiology, Würzburg, Germany
Text We established an optimized protocol for isolating MHC peptides using Restricted Access Material (RAM) that improves peptide recovery and extends the landscape of mass spectrometry-detectable peptides by hydrophobic peptides. So far, either ultrafiltration or C18 solid phase extraction (C18-SPE) have been applied for separating MHC peptides from MHC proteins. Although recent studies report that C18-SPE is superior compared to ultrafiltration in terms of peptide recovery, there are still two major drawbacks of this approach. First, large capacity C18-SPE columns have to be applied in order to allow binding of both, MHC peptides and MHC proteins after affinity-purification of peptide-MHC complexes (pMHC). The use of large capacity columns generally results in decreased peptide recovery. In addition, crosslinking of antibodies to sepharose beads is mostly inevitable to prevent overloading C18-SPE columns. Second, elution of MHC peptides is strictly limited to ~30% acetonitrile (ACN), because higher acetonitrile concentration would elute MHC proteins. To overcome these limitations, we have evaluated the use of RAM-SPE for separating MHC peptides from MHC proteins. RAM is a porous reversed phase chromatographic material with a molecular weight cut-off of ~10kDa that is coated with methylcellulose, resulting in a hydrophilic outer surface and a hydrophobic inner surface. While peptides can access the pores, proteins have no access to the hydrophobic inner surface and will pass the column without binding This allows the use of higher concentration of ACN for eluting MHC peptides. Since proteins do not concurrently bind to the RAM column, less chromatographic material is required and antibody cross-linking can be omitted. We performed comparative experiments for class I and class II MHC peptides from human PBMCs and murine melanoma cells. After affinity purification of pMHC, separation of MHC peptides and MHC proteins was performed either with C18-SPE or RAM-SPE. Isolated MHC peptides were analyzed by nanoLC-MS/MS on an Orbitrap Fusion and identified by combining de novo sequencing and database matching using the software tool Peptide-PRISM. In total, we identified about 30-100% more MHC peptides and observed increased intensities of about a factor of two for all MHC peptides using RAM-SPE compared to C18-SPE. Remarkably, only with RAM-SPE we identified a unique fraction of highly hydrophobic MHC peptides. In summary, our data indicates, that RAM-SPE extends the detectable immunopeptidome landscape especially by hydrophobic and low abundance MHC peptides. Thus, in particular immunopeptidome analyses with limited input material and of cells with hydrophobic MHC allomorphs will benefit from applying RAM-SPE.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-60
Role of different particle carriers for intraperitoneal drug delivery on disease control in a murine ovarian cancer model. R. Wouters1, 2, S. Westrøm2, A. Vankerckhoven1, G. Thirion1, T. B. Bønsdorff2, I. Vergote3, 4, A. Coosemans1 1Laboratory of Tumor Immunology and Immunotherapy, Department of Oncology, Leuven Cancer Institute, KU Leuven, Leuven, Belgium, 2Oncoinvent AS, Oslo, Norway, 3Department of Obstetrics and Gynecology, University Hospitals Leuven, Leuven Cancer Institute, Leuven, Belgium, 4Department of Oncology, Gynecological Oncology, KU Leuven, Leuven, Belgium
Text Novel treatment strategies are needed to provide a better prognosis for ovarian cancer. For this purpose, the current study was designed to evaluate the effects of different types of particle drug carriers on tumor response and on the immune system in the tumor microenvironment (TME) after intraperitoneal (IP) administration in a murine tumor model. ID8-fLuc ovarian cancer mice were injected with IP drug carriers after IP tumor inoculation. Different types of particle drug carriers designed to keep the carriers with the drugs in the peritoneal cavity were evaluated and included calcium carbonate (CaCO3), hydroxyapatite, polystyrene and poly(lactic-co-glycolic acid) (PLGA) microparticles as single treatments (day 1) or in combination (day 13) with carboplatin and pegylated liposomal doxorubicin chemotherapy (day 14). Readouts were survival, time to ascites development, flow cytometry analysis for IP immune cell alterations and Luminex multiplex analysis for cytokine analysis of IP fluid.
Our results showed that 5 mg of different formulations of CaCO3 microparticles significantly shortened survival compared to vehicle control. Moreover, different degrees of hyperprogression were observed between the different formulations of CaCO3. A repeated dose of 10 mg of CaCO3 aggravated the hyperprogression, compared to a single dose of 5 mg, indicating a dose dependent effect for this type of drug carrier. The hyperprogression caused by CaCO3 microparticles was driven by an innate immunosuppression, reflected by an increase in M2 macrophages, myeloid-derived suppressor cells (MDSC) and the immune checkpoint T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3). Furthermore, similar effects were seen after IP injection of hydroxyapatite, polystyrene and PLGA particles. Interestingly, with a lower dose of 3 mg, the hyperprogression for all drug carriers only caused an acceleration in ascites development without affecting survival. When combining 5 mg CaCO3 or PLGA with chemotherapy, the chemotherapy was not able to sufficiently counteract the progression caused by the drug carriers. This research points towards the importance of developing the right drug type of carrier before using it in a therapeutic setting, since drug carriers can detrimentally influence tumor progression and immune status of the TME. Reducing the amount or optimizing the product formulation can reduce its negative effects. The hyperprogression that is observed after IP injection of particle drug carriers in the ovarian cancer mouse model seems to be driven by an upregulation of innate immunosuppression. However, it remains to be determined if the hyperprogression in this model will be of relevance in other models or in humans.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-61
Utilizing NeoFox for a comparative analysis of published neoantigen features F. Lang1, B. Schrörs1, M. Löwer1, P. Riesgo Ferreiro1, U. Sahin2, 3 1TRON-Translationale Onkologie an der Universitätsmedizin der Johannes Gutenberg-Universität Mainz gemeinnützige GmbH, Mainz, Germany, 2BioNTech SE, Mainz, Germany, 3University Medical Center of the Johannes Gutenberg University, Mainz, Germany
Text Mutations can generate neoantigens if their breakdown products (neoepitopes) are presented on tumor cells and recognized by T cells. It has been shown that neoantigens are predictive for benefit of checkpoint inhibition-based therapies and contribute to their anti-tumoral effect. Individualized neoantigen vaccination target selected neoantigens of cancer patients and recently achieved promising results. These approaches heavily depend on successful vaccine design. To this end, individual mutation profiles are detected with Next-Generation Sequencing (NGS)-based sequencing of DNA from tumor and matched normal tissue and the mutations are selected based on their potential to elicit a T cell response. Several immunogenicity prediction algorithms have been published. We integrated and re-implemented the corresponding algorithms into a toolbox called NeoFox (NEOantigen Feature toolbOX). NeoFox enables the annotation of neoantigen candidates with 16 neoantigen features. Furthermore, we performed a standardized and comparative evaluation of these features. We collected immunogenicity data sets and raw sequencing and clinical outcome data from checkpoint blockade studies. The raw data was applied to our standardized analysis platform to detect individual mutations and neoantigen candidates in a best practice manner. Our dataset compilation enables us to compare the performance of neoantigen features and analyze them in the context of the type of T-cell response. Our new toolbox NeoFox and comparative analysis may serve as a starting point for future developments in the neoantigen prediction community.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-62
Production and testing of a novel bispecific nanobody construct targeting NK cells and EGFR expressing malignancies E. C. Toffoli1, A. Sheikhi1, 2, L. Roeland1, L. King1, J. Tuynman1, J. Spanholtz3, H. Verheul4, H. van der Vliet1, 5 1Amsterdam UMC, Amsterdam, Netherlands, 2Dezful University of Medical Sciences, Department of Immunology, Dezful, Iran, Islamic Republic of, 3Glycostem, Oss, Netherlands, 4Radboud university medical center, Department of Medical Oncology, Nijmegen, Netherlands, 5Lava Therapeutics, Utrecht, Netherlands
Text The ability to kill tumor cells with an acceptable toxicity profile, makes Natural Killer (NK) cells promising assets for cancer therapy. However, strategies to enhance the preferential accumulation and activation of NK cells in the tumor microenvironment would likely increase the efficacy of NK cell-based therapies. In this study, we show a novel bispecific nanobody-based construct (biVHH) targeting both CD16A (low-affinity Fc receptor: FcRγIIIA) on NK cells and EGFR on tumors of epithelial origins. Higher levels of NK cell activity and subsequent tumor cell lysis were found in vitro in the presence of the biVHH and were dependent on the expression of both CD16A and EGFR while they were independent of the KRAS mutational status of the tumor. Increased NK cell activity was found in NK cells derived from colorectal cancer (CRC) patients when co-cultured with the biVHH and EGFR expressing tumor cells. Finally, higher levels of cytotoxicity were found against patient-derived metastatic CRC cells in the presence of the biVHH and autologous peripheral blood mononuclear cells or allogeneic NK cells. Based on our results, the bispecific CD16A and EGFR targeting VHH construct could be a useful tool in combination with various NK cell-based therapies.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-63
Bovine Meat and Milk Factors (BMMF) in Cancer- and Chronic Diseases: BMMF as Zoonotic Infectious Driving Agents for Cancer and Chronic Diseases of the CNS T. Bund, E.-M. de Villiers, H. zur Hausen German Cancer Research Center (DKFZ), Episomal-persistent DNA in Cancer- and Chronic Diseases, Heidelberg, Germany
Text The early-in-life consumption of products from Eurasian cattle has been associated with colon and breast cancer formation, but also with neuronal diseases like Multiple Sclerosis (zur Hausen et al. 2014-2017). Bovine Meat and Milk Factors (BMMF) are a group of >100 plasmid-like DNA molecules isolated from bovine sera and milk products and from Multiple Sclerosis brain (de Villiers et al. 2019). BMMF have been proposed as zoonotic infectious agents and etiological risk factors for cancer formation (zur Hausen et al. 2018). We observed BMMF DNA replication together with transcription/expression of a BMMF-encoded, conserved replication (Rep) protein in human cell lines (Eilebrecht et al., 2018). Tissue staining with anti-Rep antibodies showed Rep expression in the tumor-free mucosa, but not in tumor tissue regions of colorectal cancer (CRC) patients after immunohistochemistry (Bund et al. 2021). The Rep protein localized in CD68+ macrophages in the interstitial lamina propria between the Crypts of Lieberkühn. An increased number of Rep+ and CD68+ macrophages was shown for CRC patients when compared to healthy donors. BMMF DNA was isolated exclusively from the tissue parts with Rep expression (de Villiers et al. 2019, Bund et al. 2021). Due to the association of Rep with macrophages and oxygen/nitrogen radicals, we proposed that BMMF contribute to chronic inflammation (CI) in the pre-cancerous tissue resulting in increased radical formation. Replicating cells in the neighboring colonic crypts are exposed to the radicals over a long period. This will lead to elevated levels of random DNA mutation in the crypt cells, which later develop into progenitor cells for adenoma (polyps) and cancer (zur Hausen et al. 2018). Current studies focus on extended case vs. control analysis and functional characterization of BMMF based on in vitro and in vivo models. Rep-immunoscoring is utilized to analyze a correlation of BMMF with clinical parameters including patient survival in CRC and additional cancer types. We use in silico BMMF screening of cancer and non-cancer cohorts for BMMF profiling and identification of biomarkers. We also analyze a possible role of Rep multimerization in the context of cancer and CNS diseases of the brain. Several types of cancer show decreased incidences after usage of non-steroidal anti-inflammatory drugs (like e.g. aspirin) including colon, liver, breast, lung, prostate, and pancreas cancer. This points towards a crucial role of CI in carcinogenesis. BMMF might fuel CI and act as indirect carcinogens inducing cancer after latency of several decades. Based on BMMF in vitro and in vivo models, new avenues for diagnosis, prevention, and therapy are now available for experimental validation.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-64
Investigating the role of RasGRP1 and TGF-β signaling in regulatory and conventional T cell survival C. Cunha1, M. Takami1, 2, 3, M. Iwashima1, 3 1Loyola University Chicago, Microbiology and Immunology, Chicago, United States, 2Chiba University, Japan, Department of Medical Immunology, Chiba, Japan, 3Loyola University Chicago, Van Kampen Cardio Pulmonary Research Laboratory, Chicago, United States
Text Activation induced cell death (AICD) occurs after expansion of antigen-stimulated T cells to reduce the number of these T cells and maintain immunological homeostasis. In the tumor microenvironment, AICD of tumor-specific T cells has been shown to prevent successful anti-tumor immunity. Another barrier to tumor- specific T cell responses is suppression by regulatory T cells (Tregs). The mechanisms controlling AICD and the balance between Tregs and conventional T cells remain largely unknown. We previously reported a form of AICD, which is dependent on p53 (p53-induced CD28-dependent T-cell apoptosis, PICA). Under conditions that induce PICA in vitro, Tregs expand robustly while conventional T cells undergo apoptosis. Resistance of Tregs to PICA is dependent on TGF-β signaling. We hypothesized that there are TCR- associated signaling differences between Tregs and conventional T cells that allow for Tregs to resist PICA. We found that the expression of RasGRP1 (Ras guanyl-releasing protein) is required for PICA, as conventional T cells isolated from RasGRP1-deficient mice become resistant to PICA. After continuous stimulation, Tregs express a substantially lower amount of RasGRP1 compared to conventional T cells. This reduced expression of RasGRP1 is dependent on TGF-β, as RasGRP1 expression in Tregs increases when TGF-β signaling is inhibited. Together, these data show that RasGRP1 expression is repressed in Tregs by TGF-β signaling and suggests that reduced RasGRP1 expression is critical for Tregs to resist apoptosis caused by continuous stimulation. Tregs may utilize TGF-β to maintain low RasGRP1 expression, which could enable survival and prevent anti-tumor immunity. For conventional T cells, low RasGRP1 expression may be critical for survival in the tumor microenvironment. We are currently investigating the role of RasGRP1 in tumor-specific T cell AICD using CAR (chimeric antigen receptor) T cells. We predict that low RasGRP1 expression could prevent AICD in tumor-specific CAR T cells, which could enable enhanced tumor-killing and survival of CAR T cells.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-65
Ipilimumab, maraviroc and nivolumab in advanced metastatic colorectal and pancreatic cancer. Preliminary results from the phase 1 LUMINESCENCE trial. A. Williams1, J. Schreiber2, S. Grauling-Halama1, 3, L. Apostolidis1, U. Prüfer1, A. Ahmed1, 3, G. M. Haag1, C. Springfeld1, D. Jäger1, N. Halama1 1National Center for Tumor Diseases (NCT), University Hospital Heidelberg, Medical Oncology, Heidelberg, Germany, 2University Hospital Tübingen, Radiooncology, Tübingen, Germany, 3German Cancer Research Center (DKFZ), Heidelberg, Germany
Text Introduction Current treatment options for advanced colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are limited. Immune checkpoint inhibitor treatment alone has not been effective in microsatellite-stable disease. We conducted a phase 1 trial to assess the safety and efficacy of ipilimumab and nivolumab in combination with the CCR-5 inhibitor maraviroc in patients with previously treated microsatellite-stable CRC or PDAC with liver metastases.
Procedures In this monocentric phase 1 trial 25 patients with CRC after all standard treatment options and 25 patients with PDAC after at least platinum-containing treatment were enrolled between 11 February 2019 and 11 December 2020. Known liver metastases were an inclusion criterion. Patients received ipilimumab 1 mg/kg every 6 weeks, nivolumab 240 mg every 2 weeks and maraviroc 300 mg twice a day until disease progression or the development of intolerable toxicity for a maximum of 24 months. Paired biopsies from liver metastases were optional. Prespecified endpoints of the study included safety, objective (tumor) response, tissue response, progression-free survival (PFS) and overall survival (OS). Exploratory endpoints were biomarker analyses within liver metastases, cytokine analyses and microbial composition within stool samples.
Summary of data With a data cut-off on 11/01/2021 a total of 50 patients (age range 38-79) who had received at least 3 prior lines of treatment for advanced colorectal cancer and at least first line treatment for advanced pancreatic cancer have been enrolled. Our preliminary data revealed confirmed objective responses in 2 patients per RECIST 1.1 criteria, including a complete response in a CRC patient and a partial response in a PDAC patient. 44 patients have discontinued treatment due to progressive disease, 3 patients due to toxicity. 3 patients are still on treatment, including the CRC patient with an ongoing complete response. PFS, OS and exploratory data are pending. The majority of adverse events have been grades 1-2 according to CTCAE version 4.0 criteria. There have been 10 grade 3 or higher treatment-related adverse events of which 9 were considered to be immune-related and 1 possibly related to maraviroc. Immune-related events comprised 4 cases of hepatitis, 2 cases of pneumonitis, and 1 case each of adrenalitis, colitis and pericarditis. Two treatment- related deaths have occurred, one from suspected immune-related constrictive pericarditis, one following complications of immune- related hepatitis.
Conclusions Preliminary analyses demonstrate antitumor activity for combined ipilimumab, nivolumab and maraviroc without excessive toxicity in pretreated patients with microsatellite-stable CRC and PDAC.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-66
Identification of shared tumor epitopes from endogenous retroviruses inducing high avidity cytotoxic T cells for cancer immunotherapy P. Bonaventura1, 2, V. Alcazer1, 3, V. Mutez4, L. Tonon5, J. Martin6, N. Chuvin4, R. Boulos4, Y. Estornes4, J. Valledeau- Guilemond1, A. Viari5, C. Caux1, 2, S. Depil1, 2, 4, 7 1Cancer Research Center of Lyon, UMR INSERM U1052 CNRS 5286, Lyon, France, 2Léon Berard Center, Lyon, France, 3Lyon Sud hospital, Civil hospital of lyon, Clinic Hematology department, Lyon, France, 4ErVaccine Technologies, Lyon, France, 5Synergie Lyon Cancer, bioinformatic platform "Gilles Thomas", Lyon, France, 6CNRS, institut de biologie et chimie des proteines UMR5086, Lyon, France, 7Université de Lyon, Claude Bernard Lyon 1, Lyon, France
Text Human endogenous retroviruses (HERVs) are aberrantly expressed by tumor cells and may represent a source of T cell epitopes.
Using TCGA pancancer RNAseq data (n=8,893 samples), we developed a bioinformatics-based method to select cancer-specific HERVs associated with a cytotoxic T cell response (“cyt-HERVs”) and identify shared T cell epitope candidates. A T cell priming assay against 6 selected HLA-A2 epitope candidates was performed using peripheral blood mononuclear cells (PBMCs) from HLA-A2+ healthy donors. Peptide-specific dextramers were used to sort and expand specific CD8+ T cell clones and determine their TCR sequences and avidity. Cytotoxicity was assessed against HERV- expressing tumor cell lines and patient-derived organoids using Incucyte and Nanolive technologies.
In a pancancer analysis, we identified 192 cyt-HERVs and 57 HML-2/HERV-K HLA-A*0201 epitope candidates from 27 distinct open reading frames. Six shared HLA-A2 strong binders 9-mer peptides, present on multiple HERVs located on different chromosomes, and with translational evidence found in mass spectrometry public datasets, were selected and synthetized. In vitro HLA binding assay confirmed peptide-HLA affinity. Priming assays showed the presence of specific CD8+ T cells leading to polyfunctional IFN-γ+ TNF-α+ T cell responses with upregulation of the degranulation marker CD107A upon co-culture with peptide-pulsed T2 cells. The functionality of the sorted T cell clones was confirmed using an Elispot assay (GrzB+ IFN-γ+). Their sequenced TCRs were predicted to stably interact with their respective MHC- peptide complexes in a 3D model. This was confirmed by measurement of the functional avidity, which was in the same order as CMV-specific T cell clones. HERV-specific CD8+ T cells induced specific cell death of MDA-MB-231 (HLA-A2+ breast cancer cell line), associated with IFN-g production, in a HLA-A2 restricted manner. Finally, pre-existing HERV- specific CD8+ T cells were identified using dextramers among tumor infiltrating lymphocytes (TILs) from cancer patients. HERV-specific T cells co-cultured with patient derived organoids showed signs of activation with lysis of the organoid.
Our bioinformatics-based approach allowed us to identify shared HERV-derived CD8+ T cell epitopes specifically expressed by tumor cells and inducing high avidity T cell clones able to kill tumor cells in a class I-restricted manner. The detection of TILs recognizing HERV peptides suggests natural presentation of these epitopes in the tumors. These HERV-derived epitopes may thus represent relevant targets for the development of new cancer vaccines or T cell- based therapies, especially in tumors with low mutational burden.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-67
A rationally designed IL-2 variant with an improved receptor binding profile combines enhanced stimulation of effector T cells and natural killer cells with reduced stimulation of regulatory T cells A. Muik1, S. Fellermeier-Kopf1, M. Vormehr1, L. M. Kranz1, B. G. Lui1, S. Witzel2, Ö. Türeci1, U. Sahin1 1BioNTech SE, Mainz, Germany, 2TRON gGmbH, Mainz, Germany
Text Interleukin-2 (IL-2) is a key mediator of cellular immunity, supporting differentiation, proliferation, survival and effector functions of T cells. Although recombinant IL-2 has been approved for treatment of renal cell cancer and late stage melanoma for nearly 30 years, it’s therapeutic potential has not been fully realized. Besides rapid clearance of recombinant IL-2 from circulation, which necessitates high and frequent dosing, one major drawback of IL-2 as anti-cancer treatment is its preferential stimulation of immune-suppressive regulatory T
(Treg) cells, which can attenuate the effects of activated tumor-specific T cells. Constitutive expression of the high-affinity heterotrimeric IL-2 receptor (IL-2Rαβγ) confers high sensitivity towards recombinant IL-2 to Treg cells. In contrast, naïve T cells express the intermediate-affinity IL-2Rβγ and only transiently up-regulate the IL-2Rα subunit after activation by cognate antigen. To improve the potential of IL-2 as a cancer therapeutic, we rationally designed a next-generation IL-2 variant. We introduced mutations to stabilize IL-2 in an optimized receptor-binding conformation, resulting in an almost 20-fold increased binding affinity to IL2-Rβ. Furthermore, we also exchanged charged amino acids at the IL-2:IL2-Rα binding interface with amino acids of opposite charge, resulting in 8-fold decreased binding affinity to human IL2-Rα in surface plasmon resonance spectroscopy. The reduced capacity to bind IL-2Rα and enhanced binding to IL-2Rβ were confirmed by ELISA. We integrated the engineered IL-2 variant (IL2var) into our Ribocytokine® platform of lipid-nanoparticle-formulated, nucleoside-modified RNA therapeutics and fused it to serum albumin (Alb) for increased serum half-life and bioavailability in tumor and tumor-draining lymph nodes. Using an in vitro assay of STAT5 phosphorylation, we demonstrated that Alb- IL2var stimulated IL-2Rβγ positive primary human effector T cells and natural killer cells with >10-fold higher potency than its wild-type counterpart. In contrast, Alb-IL2var displayed markedly reduced stimulation of IL-
2Rαβγ positive Treg cells. Owing to its optimized receptor binding characteristics, IL2var has the potential to support antitumor immunity by enhancing effector T-cell responses while limiting the immune-suppressive function of Treg cells. An open-label, Phase I/II, first-in-human trial in patients with advanced solid tumors was recently initiated to further explore the clinical safety and efficacy of IL2var RiboCytokine® (also known as BNT151) as monotherapy or in combination with other anti-cancer agents (NCT04455620).
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-68
Preferential recognition of neoantigens over non-canonical peptides in cancer patients M. Lozano-Rabella1, A. Garcia-Garijo1, J. Palomero1, F. Erhard2, J. Martín-Liberal3, 4, I. Matos3, J. J.Gartner5, S. A. Rosenberg5, M. Ghosh6, F. Canals7, I. Braña3, E. Muñoz8, E. Garralda3, A. Schlosser9, A. Gros1 1Vall d'Hebrón Institute of Oncology, Tumor Immunology and Immunotherapy, Barcelona, Spain, 2Julius- Maximilians-University Würzburg, Institute for Virology and Immunobiology, Würzburg, Germany, 3Vall d'Hebrón Institute of Oncology, Early Drug Development Unit (UITM), Barcelona, Spain, 4Institut Català d'Oncologia (ICO), Immunotherapy Program Sarcoma, Melanoma and GU Malignancies Unit, Barcelona, Spain, 5National Cancer Institute (NCI), National Institutes of Health, Surgery Branch, Bethesda, Maryland, United States, 6University of Tübingen, Interfaculty Institute for Cell Biology, Department of Immunology, Tübingen, Germany, 7Vall d'Hebrón Institute of Oncology, Proteomics Laboratory, Barcelona, Spain, 8Vall d'Hebron Hospital, Department of Medical Oncology, Melanoma and other skin cancers Unit, Barcelona, Spain, 9Julius-Maximilians-University Würzburg, Rudolf Virchow Center for Experimental Biomedicine, Würzburg, Germany
Text Despite recent advances in exome and RNA sequencing to identify tumor-rejection antigens including neoantigens, the existing techniques fail to consistently identify the landscape of epitopes targeted by tumor-reactive cells. A growing number of studies suggest that HLA-I peptides derived from cryptic open reading frames or derived from allegedly non- coding regions can contribute to tumor immunogenicity. These tumor antigens have been overlooked in immunological screening assays, and their importance in tumor rejection is unclear. Here we used proteogenomics to identify personalized candidate canonical and non-canonical tumor-rejection antigens and to evaluate their contribution to cancer immune surveillance in patients. We performed whole exome sequencing to identify non-synonymous somatic mutations (NSM) and immunopeptidomics to identify the HLA-I peptides presented in 9 patient-derived tumor cell lines (TCL). Peptide PRISM proteogenomics pipeline was used to identify both canonical and non-canonical (nonC) peptides, including those derived from NSM in coding regions. All 33 canonical mutated peptides identified as well as 61 peptides derived from antigens known to be overexpressed in tumor cells were selected for T cell screening. In addition, of 840 nonC peptides initially detected with 1% FDR, 38% were filtered out to select for nonC peptides enriched in the tumor (TE) by excluding normal ORFs known to be translated based on and immunopeptidomics dataset from several healthy tissues. NonC-TE peptides were identified in all TCL studied, ranging from 0.5% to 5.4% of the total presented peptides. As described previoulsy, ORF from nonC peptides were shorter compared to canonical, and 5’UTR were the main source. Of note, the tumor type did not have an impact on the frequency of presented nonC peptides but rather the presence of HLA-A*03:01 and HLA-A*11:01 was the major determinant.The selected candidate peptides were synthesized, pulsed onto autologous APCs and co-cultured with tumor-reactive ex vivo expanded TIL to asses immune recognition. Out of the 33 canonical putative neoantigens detected, at least 11 were able to elicit an immune response. On the contrary, neither of the 290 nonC-TE candidate peptides tested thus far, including one mutated cryptic peptide were immunogenic. Our results show that although HLA-I nonC peptides were frequently presented in all tumor cell lines studied, neoantigens derived from mutations in canonical coding regions were preferentially recognized by TILs. The identification of mutated nonC antigens using whole genome sequencing to identify mutations in non-coding regions warrants further examination. Still, the specificity of many tumor-reactive TILs remains unknown.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-69
Immune receptor inhibition through enforced phosphatase recruitment R. Fernandes1, 2, L. Su2, Y. Nishiga3, J. Ren2, A. M. Bhuiyan4, N. Chen2, C. J. Kuo2, L. K. Pikton2, S. Ohtsuki2, R. G. Majzner2, S. P. Rietberg2, C. L. Mackall2, Q. Yin2, L. R. Ali5, X. Yang2, C. S. Savvides2, J. Sage3, M. Dougan4, 5, K. C. Garcia2 1University of Oxford, Oxford, United Kingdom, 2Stanford University School of Medicine, Stanford, United States, 3Stanford University, Stanford, United States, 4Massachusetts General Hospital, Boston, United States, 5Harvard Medical School, Boston, United States
Text Phosphorylation of intracellular Tyrosine residues present in inhibitory and switch motifs of the PD-1 receptor results in the recruitment of cytosolic phosphatases that dampen activatory signals initiated by the T cell receptor (TCR) and CD28. Anti-PD-1 antibody blockade - checkpoint blockade - prevents binding to PD- L1/L2 and revolutionized immunotherapy by eliciting potent antitumor T cell responses. However, despite significant progress, most patients fail to benefit from checkpoint blockade. We found that tonic or ligand- independent signalling by PD-1 reduces T cell function, even in the absence of ligand-binding. Notably, ligand-independent signalling fails to respond to anti-PD-1 antibody blockade. To overcome ligand- dependent and independent signalling, we engineered a bi-specific molecule to recruit CD45, an abundant and promiscuous receptor tyrosine phosphatase, to within close proximity of PD-1 using bispecific diabodies. With this approach, the phosphatase domain of CD45 acts intracellularly, in cis, on the phospho-Tyr residues of the PD-1 signalling motif, thus inhibiting sustained signalling. We have shown that Receptor Inhibition by Phosphatase Recruitment (RIPR) potentiates T cell activity beyond that seen with PD-1/PD-L1 antagonist antibodies, both in the presence and absence of PD-1 ligand-binding. RIPR-PD1 also showed increased therapeutic efficacy over anti-PD1 in mouse tumour models. Lastly, we have shown that the RIPR strategy extends to other immune-receptor targets containing activating or inhibitory phospho-Tyr motifs; for example, inhibition of the macrophage SIRPα ‘don’t eat me’ signal with a RIPR-SIRPα molecule potentiates antibody- dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-70
BXQ-350, a first-in-human nanovesicle formulation of the lysosomal protein Saposin C, reprograms the tumor microenvironment and synergizes with Immune Checkpoint Inhibitors G. Tapolsky, R. Takigiku Bexion Pharmaceuticals, Inc., Covington, United States
Text Immune checkpoint inhibitors have revolutionized cancer treatment; however, there is still a clinical need to improve response rates and clinical outcomes. Strategies to mitigate the limits of immune checkpoint inhibitors focus on targets that may further enhance the activity of immuno-effector cells (Tcells, M1 macrophages, dendritic cells) or inhibit the function of immune-suppressive cells (myeloid derived suppressor cells, regulatory T cells, Tumor associated macrophages) of the tumor microenvironment. BXQ-350 is an innovative nanovesicle formulation of the lysosomal regulatory protein Saposin C and dioleoylphosphatidylserine (DOPS), a synthetic phospholipid similar to cellular membrane components. BXQ-350 has demonstrated a strong safety and tolerability profile in a Phase 1 all-comers solid tumor trial, with evidence of durable single-agent activity. BXQ-350 is thought to target tumor cell membranes and tumor lysosomes. Targeting is believed to occur with phosphatidylserine expressed on tumor cells. Phosphatidylserine (PS) has been recognized as a global immunosuppressive signal which prevents local and systemic immune activation, inhibiting the development of an antitumoral immune response. After targeting, BXQ-350 alters sphingolipid metabolism, likely within the tumor lysosomes, and elevates intratumoral ceramide concentrations. Ceramides play a pivotal role in cell signaling and strongly affect the development of an immune response in the tumor microenvironment. Preclinical and clinical results will be presented and show that BXQ-350 reprograms the immune tumor microenvironment towards an anti-tumoral state, reducing the frequency of MDSCs and the levels of TGF- beta, and IL-10, IL-8, IL-6 and increasing the levels of IFN-gamma, TNF-alpha and IL-2. BXQ-350 also repolarizes tumor-associated macrophages (TAMs) from a predominant M2 to a predominant M1 phenotype, and promotes maturation of dendritic cells (DCs) and induces CD4+ and CD8+ Tcells expansion and enhances the cytotoxicity of T-cell immunity. As a result, the combination of BXQ-350 with an anti-PD-1 immune checkpoint inhibitor results in a synergistic effect.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-71
Identifying tumor-specific, recurrent, cryptic HLA-I peptides as potential new targets for cancer immunotherapy F. Erhard1, A. Rech2, A. Marcu3, L. Bichmann3, L. Kuchenbecker4, O. Kohlbacher4, A. Nelde3, J. S. Walz3, H.-G. Rammensee3, M. Neidert5, B. Schilling2, A. Schlosser6 1Institute for Virology and Immunobiology, Julius-Maximilians-University Würzburg, Würzburg, Germany, 2University Hospital Würzburg, Department of Dermatology, Venereology, and Allergology, Würzburg, Germany, 3Interfaculty Institute for Cell Biology, University of Tübingen, Department of Immunology, Tübingen, Germany, 4University of Tübingen, Dept. of Computer Science, Tübingen, Germany, 5Kantonsspital St.Gallen KSSG, Department of Neurosurgery, Sankt Gallen, Switzerland, 6University of Würzburg, Rudolf Virchow Center, Würzburg, Germany
Text We have analyzed a large number of immunopeptidome data sets from tumor as well as benign tissues in order to identify tumor-specific, recurrent, cryptic HLA-I peptides (TURCs) as potential new targets for cancer immunotherapy. We applied our recently introduced data analysis workflow that combines de novo peptide sequencing with sequence matching in ultra-large databases (Peptide-PRISM), such as the 6-frame translated human genome, and a rigorous FDR filtering strategy. This workflow enables fast, reliable and sensitive identification of HLA-I peptides directly from mass spectrometric (MS) raw data. In particular, Peptide-PRISM enables the identification of cryptic HLA peptides that derive from allegedly non-coding cryptic open reading frames (ORFs) without the need for using patient-specific RNA-seq data. We analyzed 2500 MS raw data files from 300 different patients/cell lines and identified more than 350,000 unique HLA-I peptides (1% false discovery rate (FDR), only NetMHCpan 4.0-predicted binder), presented on more than 140 different HLA allomorphs. Tumor data comprises samples from melanoma, meningioma, acute myeloid leukemia, colorectal cancer, ovarian cancer, glioblastoma and a number of other entities. Benign control data includes the complete data set of the HLA ligand atlas project (hla-ligand-atlas.org) comprising samples from 29 tissues from 21 subjects (14 autopsy subjects and seven donors), a large variety of samples from healthy blood donors, and some matched benign tissue samples. Among these HLA peptides are more than 16,000 cryptic peptides with more than 50% of these being presented on HLA-A03, A11, A31, A68 or B07. We mapped all identified peptides on the human genome and the corresponding open reading frames (ORFs) and classified those ORFs as tumor-specific from which HLA peptides are detected exclusively in tumor samples. Whereas only a few percent of the protein-coding ORFs are tumor- specific (mostly well-known cancer-testis or differentiation antigens), about 2/3 of all identified cryptic HLA peptides derive from tumor-specific ORFs. We observed that many tumor-specific, cryptic peptides are recurrently identified in different patients. Whereas some of these TURCs were exclusively identified in a certain tumor entity (e.g. melanoma), other TURCs were identified among different tumor entities. A significant part of the TURCs derived from tumor-associated lncRNAs (according to MiTranscriptome, GEPIA). In summary, our data indicates that a significant proportion of cryptic HLA peptides is recurrently and exclusively identified on tumor cells. These cryptic peptides might represent a new class of non-private neoantigens exploitable for cancer immunotherapy.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-72
Simultaneous costimulatory T-cell engagement and checkpoint inhibition by PRS-344/S095012, a PD-L1 / 4-1BB bispecific compound for tumor localized activation of the immune system J. Peper-Gabriel1, A. Morales-Kastresana1, L. Pattarini2, M. Pavlidou1, C. Barthels1, E.-M. Hansbauer1, R. S. Bel Aiba1, B. Bossenmaier1, A. Scholer-Dahirel2, T. Jaquin1, C. Gallou2, V. Blanc2, C. Rothe1, S. Olwill1 1Pieris Pharmaceuticals, Hallbergmoos, Germany, 2Institut de Recherches Servier, Croissy sur Seine, France
Text Preclinical and clinical data suggest that 4-1BB (CD137), a costimulatory immunoreceptor mainly expressed by cytotoxic cells, represents a promising therapeutic target in cancer. A combination of checkpoint blockade with further T-cell activation mediated by 4-1BB co-stimulation may increase response rates and durability of response. However, the efficacy of systemic 4-1BB stimulation is limited by on target peripheral toxicity. PRS-344/S095012 has been designed to provide the potential of a combinatorial therapy in one molecule and favor the localized stimulation of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity. Anticalin proteins are 18 kDa protein therapeutics derived from human lipocalins. We utilized phage display technologies to generate an Anticalin protein that binds to 4-1BB with high affinity and specificity. PRS- 344/S095012 was generated by recombinant fusion of two 4-1BB-specific Anticalin proteins to a PD-L1- targeting monoclonal antibody with a modified IgG4 backbone that avoids Fc gamma receptor interactions and restricts 4-1BB agonism to PD-L1 positive tissues. We demonstrate that the bispecific fusion protein PRS-344/S095012 is capable of binding 4-1BB and PD-L1 simultaneously while retaining its ability to block PD-1/PD-L1 receptor-ligand interaction with similar potency to the parental PD-L1 antibody. In relevant in vitro assays using primary human immune cells, PRS- 344/S095012 enhances T cell effector functions only in the presence of PD-L1 positive cells, in line with the desired mechanism of action. We demonstrate that PRS-344/S095012 activity is superior to PD-L1 antibodies, and to the combination of clinically relevant 4-1BB and PD-L1 antibodies. In a human 4-1BB knock in mouse model, subcutaneously implanted with a human PD-L1 expressing tumor, PRS- 344/S095012 showed clear superiority to anti-PD-L1 alone and a robust antitumor response that leads to the complete regression of implanted tumors and extension of survival. We report potent costimulatory T-cell engagement of the immunoreceptor 4-1BB in a PD-L1-dependent manner, utilizing the PD-L1/4-1BB bispecific PRS-344/S095012. This approach has the potential to provide a localized costimulation of the immune system with high efficacy and reduced peripheral toxicity. Furthermore, dual mechanism of action combining PD-L1-dependent 4-1BB agonist activity with simultaneous PD-1/PD-L1 pathway blockade provides an additional therapeutic benefit in preclinical models. Taken together our data outlines proof of concept of PRS-344/S095012 and supports further development.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-73
Novel combination immunotherapy for boosting and priming immune responses in pancreatic cancer: strong anti-tumour effects with interleukin-15 and CD40 agonist treatment J. Van Audenaerde1, 2, E. Marcq1, B. von Scheidt2, A. Davey2, A. Oliver2, J. De Waele1, D. Quatannens1, J. Van Loenhout1, P. Pauwels1, 3, G. Roeyen1, 4, F. Lardon1, C. Slaney2, 5, M. Peeters1, 6, M. Kershaw2, 5, P. Darcy2, 5, E. Smits1, 7 1University of Antwerp, Center for Oncological Research, Wilrijk, Belgium, 2Peter MacCallum Cancer Centre, Cancer Immunotherapy and Immune Innovation Laboratory, Melbourne, Australia, 3Antwerp University Hospital, Department of Pathology, Edegem, Belgium, 4Antwerp University Hospital, Department of Hepatobiliary, Endocrine and Transplantation Surgery, Edegem, Belgium, 5University of Melbourne, Sir Peter MacCallum Department of Oncology, Melbourne, Australia, 6Antwerp University Hospital, Department of Oncology and Multidisciplinary Oncological Centre Antwerp, Edegem, Belgium, 7Antwerp University Hospital, Center for Cell Therapy and Regenerative Medicine, Edegem, Belgium
Text Background: with the poorest 5-year survival of all cancers, improving treatment for pancreatic cancer is one of the biggest challenges in cancer research. In this era of combination immunotherapies, we sought to explore the potential of combining both priming and activation of the immune system. To achieve this, we combined a CD40 agonist with interleukin-15 and tested its potential in pancreatic cancer.
Methods: two different mouse models of pancreatic cancer were used to assess the potential of this combination regimen. Therefore, effects on tumour growth kinetics and survival were charted. Differential effects on immune signatures was investigated using RNA sequencing. Functional immune subset involvement was tested using different immune depletion experiments and multicolour flow cytometry in different relevant immune sites. Immune memory was checked using re-challenge experiments.
Results: we demonstrated profound reduction in tumour growth and increased survival of mice with the majority of mice being cured when both agents were combined, including an unprecedented dose reduction of CD40 agonist without losing any efficacy. RNA sequencing analysis showed involvement of natural killer cell and T cell mediated anti-tumour responses and the importance of antigen-presenting cell pathways. This combination resulted in enhanced infiltration of tumours by both cytotoxic T cells and natural killer cells, as well as a striking increase in the ratio of CD8+ T cells over T regulatory cells. We also observed a significant increase in numbers of dendritic cells in tumour draining lymph nodes, particularly CD103+ dendritic cells with cross-presentation potential. A critical role for CD8+ T cells and involvement of natural killer cells in the anti-tumour effect was highlighted. Importantly, strong immune memory was established, with an increase in memory CD8+ T cells only when both interleukin-15 and the CD40 agonist were combined.
Conclusion: we demonstrated profound synergistic anti-tumour effects upon combination of CD40 agonist and interleukin-15 treatment in mouse models of pancreatic cancer. This preclinical data supports initiation of a first-in-human clinical trial with this combination immunotherapy strategy in pancreatic cancer.
eTalk Session 1
Therapeutic Vaccination & New Targets and New Leads
S1-74
Siglec 5 - a novel checkpoint receptor in T cells A. Vuchkovska, M. Iwashima Loyola University Chicago, Microbiology and Immunology, Maywood, United States
Text Checkpoint receptors have become a major target for designing immunotherapies. Across 14 different malignancies, the FDA has approved 6 different drugs targeting 3 different inhibitory checkpoint receptors. Even though signal 1 and signal 2 are the major requirements for T cell activation, it is the engagement of different checkpoint receptors that dictate the functional outcome of the particular T cell. Here we describe a new checkpoint receptor, Siglec 5, expressed on activated T cells only. To date, Siglec 5 is defined as innate inhibitory receptor specific for sialic acids, a type of self-associated molecular patters responsible for dampening the immune system activation against self. We found that Siglec 5 is a unique late activation marker of T cells, upregulated at 48hrs, with peak expression at 72hrs post TCR stimulation. Because the intracellular domain of Siglec 5 has an ITIM and SWITCH domains, both well described for their inhibitory functions, we hypothesized that Siglec 5 serves as an inhibitory checkpoint receptor that suppresses T cells activation. Activation of Siglec 5 in T cells results in potent inhibition of T cell activation measured by NFAT and AP-1 activity, and subsequent cytokine production and activation marker expression. Interestingly, melanoma cell lines, among other cancer cell lines, express high levels of the ligands for Siglec 5. Having in mind that the cancer glyco-code is often exploited as an immune escape mechanism, this raises the question whether Siglec 5 plays a role in cancer mediated immune cell suppression. Using melanoma specific T cells, we set up an in vitrosystem to measure the T cell specific response against melanoma cancer cell lines when the Siglec 5 signaling axis is interrupted. We found that blocking the availability of Siglec 5 ligands, using a Siglec 5-Fc chimeric protein, enhances the functionality of the T cells. Currently we are working on establishing an in vivo mouse model to test the hypothesis that Siglec 5 is a mechanism of cancer immune evasion.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-75
Interleukin-2 signals converge in a lymphoid–dendritic cell pathway that promotes anticancer immunity M. E. Raeber1, R. A. Rosalia1, D. Schmid1, U. Karakus1, O. Boyman1, 2 1University Hospital Zurich, Department of Immunology, Zurich, Switzerland, 2University of Zurich, Faculty of Medicine, Zurich, Switzerland
Text Dendritic cells (DC) are a subgroup of professional antigen-presenting cells considered indispensable in orchestrating T cell responses to intracellular pathogens and tumors. Although tumor-infiltrating DCs have correlated with effective anticancer immunity and improved responsiveness to anti-programmed cell death protein 1 (PD-1) checkpoint immunotherapy, the upstream drivers of DC expansion and intratumoral accumulation remain uncertain. We report that administering interleukin-2 (IL-2) in both humans (i.e., in a clinical trial) and mice caused the pronounced expansion of type-1 and type-2 DCs, including migratory and cross-presenting DC subsets. However, neither their precursors nor mature DCs expressed functional IL-2 receptors. In mechanistic studies, IL-2 signals stimulated innate lymphoid cells, natural killer cells, and T cells to synthesize FMS-like tyrosine kinase 3 ligand (FLT3L), colony-stimulating factor 2 (CSF2), and tumor necrosis factor (TNF). These cytokines redundantly caused DC expansion and activation, which resulted in improved antigen processing and T cell activation. In transplantable and inducible mouse melanoma models, treatment with IL-2 but not anti-PD-1 increased intratumoral DC infiltration and correlated with favorable antitumor responses. Moreover, melanoma patients with high intratumoral IL-2 gene signatures correlated with DC infiltration and prolonged survival. Although immune checkpoint inhibitors have transformed modern oncology by supporting durable responses in selected metastatic cancers, only patients with highly immune cell-infiltrated, or so-called “hot” tumors, respond to immune checkpoint blockers. Our study revealed the complementary difference and unappreciated advantage of IL-2 immunotherapy compared to treatment with anti-PD-1 immune checkpoint inhibitors. IL-2 immunotherapy expanded tumor-infiltrating antigen-presenting DCs and favored the conversion of poorly immunogenic into 'hot' tumors. Such insights could help to overcome primary and secondary tumor resistance to immune checkpoint inhibitors and provide a strong rationale for using combinatorial strategies.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-76
Regulation of effektor cell function by cytosolic transport T. Baumann1, A. Dunkel2, C. Schmid3, S. Schmitt4, M. Hiltensperger5, K. Lohr1, V. Laketa6, S. Donakonda1, U. Ahting7, B. Lorenz-Depiereux8, J. E. Heil9, J. Schredelseker10, L. Simeoni11, C. Fechner12, N. Körber13, T. Bauer13, N. Hüser14, D. Hartmann14, M. Laschinger14, K. Eyerich15, S. Eyerich16, M. Anton1, M. Streeter17, B. Schraven11, D. Spiegel18, F. Assaad19, T. Misgeld12, H. Zischka20, P. Murray21, A. Heine22, M. Heikenwälder23, T. Korn5, C. Dawid3, T. Hofmann24, P. Knolle1, B. Höchst25, 26 1Institute of Molecular Immunology & Experimental Oncology, Klinikum rechts der Isar, School of Medicine, Technical University of Munich (TUM), München, Germany, 2Leibniz-Institute of Food Systems Biology at the TUM, München, Germany, 3Chair of Food Chemistry and Molecular Sensory Science, Technische Universität München, München, Germany, 4Institute for Toxicology and Environmental Hygiene, Technische Universität München, München, Germany, 5Department of Experimental Neuroimmunology, Klinikum rechts der Isar, School of Medicine, Technical University of Munich (TUM), München, Germany, 6Department of Infectious Diseases, Heidelberg, Germany, 7Institute of Human Genetics, Stoffwechsel-Zentrum, Klinikum rechts der Isar, School of Medicine, Technical University of Munich (TUM), München, Germany, 8Institute of Human Genetics, Helmholtz Zentrum München, München, Germany, 9Carl Zeiss Microscopy GmbH, Göttingen, Germany, 10Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, Ludwig-Maximilians-Universität München, München, Germany, 11Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg, Germany, 12Institute of Neuronal Cell Biology, Munich Cluster for Systems Neurology and German Center for Neurodegenerative Diseases, München, Germany, 13Institute of Virology, Helmholtz Zentrum München, München, Germany, 14Department of Surgery, Klinikum rechts der Isar, School of Medicine, Technical University of Munich (TUM), München, Germany, 15Department of Dermatology and Allergy, Technische Universität München, München, Germany, 16ZAUM-Center of Allergy and Environment, TUM and Helmholtz Center Munich, München, Germany, 17Department of Chemistry, Yale University, New Haven, United States, 18Department of Pharmacology, Yale University, New Haven, United States, 19Botany, Plant Science Department, Centre for Life Science, TUM, München, Germany, 20Institute for Toxicology and Environmental Hygiene, Klinikum rechts der Isar, School of Medicine, Technical University of Munich (TUM), München, Germany, 21Max-Planck Institute for Biochemistry, Martinsried, Germany, 22Medical Clinic III for Oncology, Hematology and Rheumatology, University Hospital Bonn, Bonn, Germany, 23Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany, 24Chair of Food Chemistry and Molecular Sensory Science, School of Life Sciences, TUM, München, Germany, 25Klinikum rechts der Isar, Technische Universität München, Institute of molecular immunology and experimental oncology, München, Germany, 26Institute of Molecular Immunology, School of Life Sciences, TUM, München, Germany
Text Myeloid derived suppressor cells (MDSCs) migrate /develop in chronical inflamed and cancer tissue where they control effector immune cell responses. Identification of these cells is difficult due to the lack of markers, and the suppressive mechanism has not been fully uncovered. Here we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl-radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase (SSAO), to cause the metabolic phenotype of MDSCs and MDSC- mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl-activity overcame MDSC-mediated T cell-suppression and together with checkpoint inhibition improved efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as marker metabolite for MDSCs that mediates T cell paralysis and can serve as target to improve cancer immune therapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-77
Prediction of neo-epitope immunogenicity reveals TCR recognition determinants and provides insight into immunoediting J. Schmidt1, A. Smith2, M. Magnin1, J. Racle3, J. Devlin2, S. Bobisse1, J. Cesbron1, S. Carmona3, F. Huber1, G. Ciriello3, D. Speiser3, M. Bassani-Sternberg1, G. Coukos1, B. Baker2, A. Harari1, D. Gfeller3 1Lausanne University Hospital, Lausanne, Switzerland, 2Notre Dame University, Notre Dame, United States, 3University of Lausanne, Lausanne, Switzerland
Text CD8+ T cell recognition of peptide epitopes plays a central role in immune responses against pathogens and tumors. However, the rules that govern which peptides are truly recognized by existing T cell receptors (TCRs) remain poorly understood, precluding accurate predictions of neo-epitopes for cancer immunotherapy. Here, we capitalize on recent (neo-)epitope data to train a predictor of immunogenic epitopes (PRIME), which captures molecular properties of both antigen presentation and TCR recognition. PRIME not only improves prioritization of neo-epitopes but also correlates with T cell potency and unravels biophysical determinants of TCR recognition that we experimentally validate. Analysis of cancer genomics data reveals that recurrent mutations tend to be less frequent in patients where they are predicted to be immunogenic, providing further evidence for immunoediting in human cancer. PRIME (http://prime.gfellerlab.org/) will facilitate identification of pathogen epitopes in infectious diseases and neo- epitopes in cancer immunotherapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-78
Dictating phenotype, function, and fate of human T cells with co-stimulatory antibodies presented by filamentous immune cell mimics M. Schluck, L. Eggermont, B. Pilzecker, J. Weiden, C. Popelier, M. Verdoes, C. Figdor, R. Hammink RadboudUMC, Tumour Immunology Lab, Nijmegen, Netherlands
Text Potent T cell activation is dependent on co-stimulatory signals in addition to T cell receptor (TCR) signaling. While most studies focus on the co-stimulatory receptor CD28, little is known about the role of the other co- stimulatory receptors in T cell signaling. A deeper understanding of how co-stimulatory receptor signaling cooperates with TCR signaling could improve our ability to control T cell function and benefit the design of T- cell based immunotherapies. Artificial antigen presenting cells (aAPCs), which mimic DCs in their ability to activate T-cells, enable tight control over the signals given to T-cells. In this study, we used polyisocyano peptide based immunofilaments as nanosized aAPCs to study how the engagement of six distinct co-stimulatory molecules (αCD2, αCD27, αCD28, αCD134, αCD137, and αCD150) affects T-cell phenotype, function, and fate. Our immunofilaments highlighted important roles for CD28 and CD2 signaling in both CD4+ and CD8+ T-cell priming, proliferation, cytokine production, and multifunctionality. Particularly, immunofilaments presenting αCD2, and to a lesser extent immunofilaments presenting αCD28, induced high perforin expression which correlated with the immunofilament signal. Additionally, these αCD2 or αCD28 presenting immunofilaments induced a significant population of CD4+ T cells expressing the T helper cytokine IL-17. Taken together, we provide insights into the role of combined TCR and co-stimulation on T-cell phenotype, function, and fate using polyisocyano peptide based immunofilaments. As such, our findings on the roles of co-stimulatory molecule function can be used for the rational design of future cancer immunotherapies.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-79
TIGIT and PD1 co-blockade restores ex vivo functions of human tumor-infiltrating CD8+ T cells in hepatocellular carcinoma Z. Ge1, G. Zhou1, L. Campos Carrascosa1, E. Gausvik1, P. Boor1, L. Noordam1, M. Doukas1, Q. Pan1, W. Polak1, T. Terkivatan1, R. Takkenberg2, J. Verheij2, J. Erdmann2, J. IJzermans1, M. Peppelenbosch1, J. Kraan1, D. Sprengers1, J. Kwekkeboom1 1Erasmus Medical Center, Rotterdam, Netherlands, 2Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands
Text Introduction: TIGIT is a co-inhibitory receptor and its suitability as a target for cancer immunotherapy in HCC is unknown. PD1 blockade is clinically effective in about 20% of advanced HCC patients. Here we aim to determine whether co-blockade of TIGIT/PD1 has added value to restore functionality of HCC tumor-infiltrating T cells (TILs). Materials and methods: Mononuclear leukocytes were isolated from resected tumors, paired tumor-free liver tissues (TFL) and peripheral blood of HCC patients, and used for flowcytometric phenotyping and functional assays. CD3/CD28 T-cell stimulation, antigen-specific T-cell stimulation and cytotoxicity assays were used to study the ex vivo effects of TIGIT/PD1 single or dual blockade on tumor-derived T-cell functions. Results: Terminally exhausted PD1highCD8+ TILs which co-expressed co-inhibitory receptors TIM3 and LAG3, were found in tumors of 68% of HCC-patients (n=44), which were called High PD1-expressers. CD8+ TILs of Low PD1 expressers showed only intermediate PD1 expression. PD1highCD8+ TILs demonstrated higher TOX expression and lower TCF-1, granzyme B and perforin expression than PD1intCD8+ TILs. Moreover, PD1highCD8+ subset showed decreased capacity to produce IFN-g and TNF-a in response to PMA/Ionomycin restimulation than PD1intCD8+ TILs. On the average, TIGIT was expressed on 70% of CD8+ TILs of High PD1 expressers and 40% CD8+ TILs of Low PD1 low expressers, whereas expression of its co-stimulatory counterpart CD226 was reduced on CD8+ TILs of High PD1 expressers compared to Low PD1 expressers. In High PD1 expressers, CD8+PD1hi TILs showed the highest TIGIT expression and the lowest CD226 expression. Expression of TIGIT-ligand CD155 was expressed on tumor-infiltrating monocytes/macrophages and dendritic cells, and its expression was upregulated on cancer cells compared to hepatocytes in TFL. Single PD1 antibody blockade enhanced ex vivo proliferation and cytokine production of CD3/CD28-stimulated CD8+ TILs from most High PD1 expressers, but not from the majority of Low PD1 expressers. On the contrary, co-blockade of TIGIT and PD1 improved ex vivo responses of CD8+ TILs from Low PD1 expressers but not from High PD1 expressers. Co-blockade also enhanced ex vivo CD8+ TILs responses against HCC tumor antigens MAGE-C2 and glypican-3, and CD8+ TIL cytotoxicity against an HCC cell line. The effect of TIGIT blockade on CD8+ TILs proliferation and IFN-g production could be abrogated by CD226 blockade. Conclusion: Ex vivo, co-blockade of TIGIT and PD1 can improve functionality of CD8+ TILs from HCC-patients that do not respond to single PD1 blockade. Therefore co-blockade of TIGIT/PD1 could be a promising immune therapeutic strategy for this subset of HCC patients.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-80
CD137 (4-1BB) costimulation of CD8 T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation. I. Otano1, A. Azpilikueta2, J. Glez-Vaz2, P. Ellmark3, S. Fritzell3, G. Hernandez-Hoyos4, M. Hase-Nelson4, M. Alvarez2, M. d. C. Ochoa2, E. Bolaños2, D. Cuculescu5, P. Jauregui2, S. Sanchez5, I. Etxeberria2, M. E. Rodriguez-Ruiz5, M. F. Sanmamed2, A. Teijeira2, P. Berraondo2, I. Melero2 1Hospital 12 de Octubre, Madrid, Spain, 2CIMA-Universidad de Navarra, Pamplona, Spain, 3Alligator Bioscience, Lund, Sweden, 4Aptevo Therapeutics, Seattle, United States, 5Clinica Universitaria de Navarra, Pamplona, Spain
Text CD137 (4-1BB, TNFSFR9) costimulation provides antigen-primed T cells with increased survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being harnessed for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signalling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cells is far superior to that provided in- trans in terms of T-cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mice and humans. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-kB signalling and provides a more robust induction of cell-cycle and DNA damage repair gene expression programs. Superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which provide costimulation either in cis or in trans.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-81
Induction of apoptosis and Natural Killer cells activation by Pereskia bleo towards cervical cancer cells N. Ismail1, A. Acosta2, S. F. Mohd-Salleh1 1Universiti Sains Malaysia, Department of Pharmacology, Kota Bharu, Malaysia, 2Universiti Sains Malaysia, School of Health Sciences, Kota Bharu, Malaysia
Text Cancer is one of the leading causes of death in the majority of world populations. Medicinal plants serve as an alternative source in the development of cancer therapy with a promising effects on elimination of cancer cells and less harm to the patients. Anti-cancer agents with cytotoxic actions and capability in boosting immune response would be an ideal approach in combating the disease. Traditionally, an edible plant, Pereskia bleo (P. bleo) is a popular remedy for various ailments and has been claimed by the locals for its anti-cancer properties. Therefore, it is important to emphasize the mechanisms of actions of this plants for its apoptosis induction and anticancer-immune response as this information is still insufficient. In this study, the leaves of P. bleo were extracted using different methods and solvent polarities. GC-MS analysis was employed to identify the phytochemicals presented in each of the extracts. The extracts were tested for its cytotoxic effects on cancer cell lines (HeLa, MDA-MB-231, SW480) and NIH/3T3 mouse normal fibroblast using MTT assay. The most potent cytotoxic extract and its corresponding cancer cells were investigated for their cell death induction through cell cycle arrest, apoptotic cells detection (Annexin V/PI) and measurement of apoptotic proteins (Bax, Bcl-2, caspase 3 and p53) using flow cytometry. In addition, they were tested for the ability to modulate immune response by activating NK cells to enhance the target cells killing through the detection of perforin, granzyme B, IFN-γ and IL-2 secretion via flow cytometry. Results showed the presence of compounds such as terpenoids, sterols, alkaloids, flavonoids, phenols, fatty acids, sugar and vitamin E in the leaves of P. bleo. Ethyl acetate extract of P. bleo leaves (PBEA) exhibited the potent cytotoxic effects
(IC50 value = 14.37±8.40 µg/ml) against cervical cancer cells (HeLa). Treatment with PBEA for 72h has resulted in inhibition of HeLa cell progression at G0/G1 phase and detection of apoptosis evidenced by occurrence of apoptosis events, the presence of fragmented nuclei, condensation of chromatin, suppression of Bcl-2, upregulation of Bax, caspase-3 and p53. PBEA also was found to stimulate NK cells from cervical cancer patients for its cytotoxic activity towards HeLa cells as observed by increase of apoptotic cells and detection of granzyme B, INF-γ and IL-2 secretion. Thus our findings showed that PBEA demonstrated cytotoxic activity by inducing apoptosis and cell cycle arrest in the cervical cancer cells (HeLa) and might possesses stimulatory effects on NK cells derived from cancer patients.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-82
Targeting intra-tumoral crosstalk between adaptive and innate immunity enhances cancer immune control N. Kirchhammer1, M. Trefny1, M. Natoli1, D. Brücher2, S. Smith2, F. Werner3, V. Koch4, D. Schreiner4, E. Bartoszek4, M. Buchi4, D. Breu5, K. Hartmann2, P. Zaytseva5, D. Thommen6, H. Läubli4, M. Stanczak7, A. Kashyap8, A. Plückthun2, A. Zippelius4 1University Hospital Basel, Department of Biomedicine, Basel, Switzerland, 2University Zurich, Zurich, Switzerland, 3General Hospital Vienna, Vienna, Austria, 4University Hospital Basel, Basel, Switzerland, 5University Hospital Zurich, Zurich, Switzerland, 6The Netherlands Cancer Institute, Amsterdam, Netherlands, 7Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany, 8Boehringer Ingelheim Pharmaceuticals, Ridgefield, United States
Text Targeting T cells for cancer immunotherapy occasionally generates robust tumor control. Harnessing additional orchestrators of the immune response against tumors may enhance and broaden the clinical benefit. Here, we demonstrate that therapeutic targeting of the Interferon γ (IFNγ)- Interleukin 12 (IL-12) pathways efficiently amplifies the anti-tumoral T cell - Natural Killer (NK) cell- Dendritic cell (DC) crosstalk for successful tumor rejection. Utilizing an engineered adenoviral platform for paracrine delivery in the tumor microenvironment, we show that IL-12 enhances a functional DC-CD8 interaction to generate profound anti- tumor immunity. This effect was dependent on the abundance of intra-tumoral NK cells, particularly their capacity to produce the DC chemoattractant CCL5. Treatment failure in response to IL-12 and other IFNγ- inducing therapies such as immune checkpoint blockade could be overcome by the attraction of cDC1s induced by intra-tumoral CCL5 delivery. Our findings reveal new therapeutic avenues that enhance T cell- NK cell-DC crosstalk and thereby enforce tumor-eliminating positive feedback mechanisms to promote anti- tumor immunity and overcome resistance.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-83
P38 MAPK signaling in M1 macrophages results in selective elimination of M2 macrophages by MEK inhibition D. Baumann1, 2, J. Drebant1, T. Hägele1, B. Luisa1, C. Serger1, P. Dudys3, G. Erdmann3, R. Offringa1, 2 1German Cancer Research Center, Molecular Oncology of Gastrointestinal Tumors, Heidelberg, Germany, 2Heidelberg University Hospital, Department of Surgery, Heidelberg, Germany, 3NMI TT Pharmaservices, Berlin, Germany
Text M2 macrophages promote tumor progression and therapy resistance, whereas pro-immunogenic M1 macrophages can contribute to the efficacy of cytostatic and immunotherapeutic strategies. The abundance of the M2 macrophages in the immune infiltrate of many cancer types has prompted the search for strategies to target and eliminate this subset. From our prior experiments in syngeneic mouse tumor models, we learned that pharmacological inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) did not merely result in tumor cell death, but also in modulation of the tumor immune infiltrate. This included a prominent decrease in the numbers of macrophages as well as an increase of the M1/M2 macrophage ratio. Investigation of the mechanism underlying this finding in primary murine macrophage cultures revealed that M2 macrophages are significantly more sensitive to MEK inhibition-induced cell death then their M1 counterparts. Further analyses showed that the p38 MAPK pathway, which is activated in M1 macrophages only, renders these cells resistant to death by MEK inhibition. In conclusion, the dependency of M2 macrophages on the MEK/ERK pathway empowers MEK inhibitors to selectively eliminate this subset from the tumor microenvironment.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-84
Therapeutic blockade of myeloid-derived suppressor cells in melanoma by IL-6/STAT3 pathway inhibition R. Weber1, A. Kurzay1, 2, J. Utikal1, V. Umansky1 1DKFZ Heidelberg and University Medical Center Mannheim, Skin Cancer Unit, Mannheim, Germany, 2University of Heidelberg, Faculty of Biosciences, Heidelberg, Germany
Text Myeloid-derived suppressor cells (MDSC) are major players in the immunosuppressive melanoma microenvironment. Under chronic inflammatory conditions, these cells are generated by a constant production of inflammatory cytokines, chemokines and growth factors, including IL-6. Here we studied the effects of IL-6 on the immunosuppressive capacity of MDSC and the potential of the IL-6/STAT3 pathway as immunotherapeutic target. In murine MDSC, IL-6 upregulated the expression and activity of arginase-1, by a STAT3 dependent mechanism, as well as the production of reactive oxygen species. Furthermore, MDSC generated in the presence of IL-6 showed increased inhibition of CD8+ T cell proliferation compared to MDSC generated without IL-6. Interestingly, a correlation between IL-6 levels and tumor-infiltrating MDSC was demonstrated in the RET transgenic melanoma mouse model. Surprisingly, IL-6 overexpressing tumors grew significantly slower in mice accompanied by CD8+ T cell activation and the absence of MDSC accumulation. Moreover, melanoma-bearing mice treated with IL-6 blocking antibodies showed significantly accelerated tumor development. Analyzing the STAT3 signaling, we found that the STAT3 inhibitor Napabucasin abrogated suppressive capacity of MDSC on CD8+ T cells in vitro. Moreover, a stimulation of T cell proliferation was observed. Napabucasin induced MDSC apoptosis and potentially upregulated the antigen-presenting machinery, as well as downregulated inducible nitric oxide synthase (iNOS) expression. However, it did not impair CD8+ T cell survival or their proliferation after polyclonal activation. Therefore, our findings indicated that IL-6 induced a strong immunosuppressive activity of MDSC via STAT3, highlighting this pathway as a promising target for melanoma immunotherapy. However, IL-6 blocking therapy did not prove to be effective in RET transgenic melanoma-bearing mice but rather aggravated tumor progression. Since the STAT3 inhibitor Napabucasin demonstrated a significant inhibition of MDSC immunosuppressive capacity in vitro without negative effects on T cells, we will further study the anti-tumor effects of Napabucasin in tumor-bearing mice and potentially in cancer patients.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-85
The impact of obesity on the response to cancer immunotherapy E. Dupuychaffray1, 2, M. Alvarez1, 2, B. Taskoparan1, 2, H. Poinot1, 2, C. Bourquin1, 2, 3, A. Pommier1, 2 1University of Geneva, School of Pharmaceutical Sciences, Geneva, Switzerland, 2University of Geneva, Institute of Pharmaceutical Sciences of Western Switzerland, Geneva, Switzerland, 3University of Geneva, Department of Anaesthetics, Pharmacology, Intensive Care and Emergencies, Faculty of Medicine, University of Geneva, Switzerland
Text Obesity is associated with many disorders and in particular with a higher incidence of cancer. However, recent clinical studies suggest that obese cancer patients, especially men, have a better outcome when treated with immune checkpoint inhibitors (ICI) compared to non-obese patients. Our aim is to understand the relationship between obesity, gender and the antitumor immune response, in particular in the context of ICI. We hypothesized that steroid hormones such as estrogens and androgens may be a determining factor of the response to ICI, since the level of steroid hormones is known to be gender- and BMI-dependent. The impact of both obesity and sex on the antitumor efficacy of ICI was investigated in male and female mice fed with a high-fat diet to induce obesity or a control diet. Obese and non-obese mice were subcutaneously injected with B16-F10 tumor cells and treated with anti-PD-1 after development of palpable tumors. Treatment efficacy was assessed by tumor growth and FACS immunophenotyping. As previously described, obese mice developed larger tumors than non-obese mice. Among females, both obese and non-obese mice responded to ICI treatment. However, in males, only obese mice responded to ICI whereas no effect of the treatment was observed in non-obese males. These experimental results support observations in melanoma patients suggesting that the response to ICI treatment is modulated by obesity. To further investigate how obesity affects the antitumor immune response, the impact of the androgen/estrogen ratio on antigen presentation, T-cell activation and T-cell cytotoxicity was assessed in a coculture assay of murine dendritic cells, antigen-specific CD8+ T cells and tumor cells exposed to the estrogen estradiol (E2) and the androgen dihydrotestosterone (DHT). Our results show that E2 enhances the immune response whereas DHT impedes it. We confirmed these effects of steroid hormones on human monocyte-derived dendritic cells generated from healthy donors in presence of E2 or DHT and whose activation profile was evaluated by FACS phenotyping and IL-6 production. Further experiments will be performed to characterize the effect of steroid hormones on the immune compartment and on the tumor microenvironment in the context of ICI. With this, we hope to identify biomarkers to improve the stratification of the ICI-responsive population and to find new therapeutic targets to enhance ICI efficacy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-86
Combined immune checkpoint blockade: in vivo validation of in vitro results E. Marcq1, S. Rovers1, J. R. M. Van Audenaerde1, J. De Waele1, C. Merlin1, P. Pauwels1, 2, S. Fisher3, 4, J. P. Van Meerbeeck1, 5, E. L. J. Smits1 1Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON) - University of Antwerp, Wilrijk, Belgium, 2Department of Pathology - Antwerp University Hospital, Antwerp, Belgium, 3National Centre for Asbestos Related Diseases (NCARD) - The University of Western Australia,, Perth, Australia, 4School of Biomedical Sciences - The University of Western Australia, Perth, Australia, 5Department of Pulmonology - Antwerp University Hospital, Antwerp, Belgium
Text Introduction Malignant pleural mesothelioma (MPM) is an aggressive cancer that is causally associated with asbestos exposure. Due to its aggressive nature and despite the effectiveness of conventional anti-cancer treatment, the prognosis of MPM patients remains dismal. Data from us and others on the presence of the immune checkpoint-related molecules PD-1, PD-L1, TIM-3 and LAG-3 in MPM lay the basis to evaluate their suitability as immunotherapeutic targets.
Material & methods Human cell lines representative for the epithelioid and sarcomatoid subtypes of MPM were placed in allogeneic co-cultures with healthy donor peripheral blood mononuclear cells. The co-cultures were treated with anti PD-1 or anti PD-L1 in combination with anti TIM-3 or anti LAG-3. Supernatant was collected and enzyme-linked immunosorbent assays and multiplex electrochemo- luminescence were used to assess the secretion of 7 cytokines; IFNg, IL-2/5/6/10, IL-1b and TNF-a, as well as the enzyme granzyme B. Statistical analysis was done to investigate the differences between the treatment conditions. Our in vitro results were validated in vivo using the AB1-HA BALB c/J mesothelioma mouse model. Tumor cells were injected subcutaneously and immune checkpoint blocking antibodies were injected intraperitoneally.
Results & discussion Treatment with immune checkpoint blockers as monotherapy or in combination resulted in a significant increase in the secretion of granzyme B and the cytokines IFNg, IL-2, IL-5 and IL-10. Although the increased secretion was not always statistically significant for all 3 MPM cell lines of the two subtypes, the same trends were observed. Interestingly, highest concentrations of granzyme B and these 4 cytokines were noticed for anti PD-1 and anti PD-L1 monotherapy and their combination with anti TIM- 3. Statistical analysis showed no significant difference between PD-1 or PD-L1 monotherapy and their combination with anti TIM-3 or anti LAG-3. Our in vivo data are in line with our in vitro findings, showing a significant survival benefit for mice treated with anti PD-L1.
Conclusion Statistical analysis showed that TIM-3 or LAG-3 blockade had no added value to PD-1 or PD-L1 monotherapy in vitro. These findings were confirmed in vivo. PD-L1 blockade resulted in a significant survival benefit in the AB1-HA mesothelioma mouse model while there were no significant effects for TIM-3 or LAG-3 blockade. These data support the idea to look beyond combined immune checkpoint blockade and to investigate the combination of anti PD-L1 with other treatment strategies.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-87
Immunomodulation by cisplatin chemotherapy in an immunocompetent intraductal model for triple-negative breast cancer J. Steenbrugge1, 2, N. Vander Elst1, K. Demeyere1, J. De Vliegher1, A. Van der Aa3, E. Ciamporcero3, T. Perera3, E. Meyer1, 2 1Ghent University, Faculty of Veterinary Medicine, Department of Pharmacology, Toxicology & Biochemistry, Merelbeke, Belgium, 2Cancer Research Institute Ghent (CRIG), Ghent, Belgium, 3OCTIMET Oncology NV, Beerse, Belgium
Text Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype showing limited immunotherapeutic efficacy due to a highly immunosuppressed tumor microenvironment. Some chemotherapeutics such as the platinum-based agent cisplatin have been reported to alleviate this immunosuppression. More recently, cisplatin was suggested to even synergize with the inhibition of immune checkpoint proteins programmed death (PD)-1 and PD-ligand (L)1. Our goal was to investigate this immunomodulation by cisplatin at the local tumor and systemic level using an immunocompetent intraductal model that recapitulates the complete TNBC disease process. Lactating female BALB/c mice were inoculated with triple-negative, syngeneic and bioluminescent 4T1 mammary tumor cells through the mammary teat canal of the third mammary gland pair. At 3 weeks after the inoculations, tumor cells broke through the ductal epithelial barrier and invaded the mammary fat pad, providing a clinical indication for chemotherapy. The mice therefore received cisplatin intraperitoneally every 5 days for 3 weeks, after which primary tumors, axillary lymph nodes, lungs, spleen and blood were isolated and immune cell subtypes as well as PD-(L)1 expression were analyzed through flow cytometry. The immune-related biomarkers chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) were analyzed locally and systemically to verify disease progression. Cisplatin treatment significantly reduced tumor and metastatic growth, corroborated by decreased CHI3L1 and LCN2 levels in primary tumors, spleen and serum. Immunosuppressive CD11b+ myeloid cells that accumulated in body fluids and tissues were not uniformly eliminated by cisplatin. CD11b+Ly6C-Ly6G+ granulocytic myeloid- derived suppressor cells, the most abundant myeloid subpopulation, were significantly reduced in spleen and axillary lymph nodes, ensuring decreased immunosuppression in these compartments. Focusing on adaptive immune cell subtypes, axillary lymph nodes showed a significant decrease in CD3ε+CD4+CD8α- helper T cells and even complete absence of CD3ε+CD4-CD8α+ cytotoxic T cells following cisplatin treatment, representing a drawback for immunotherapeutic efficacy. In contrast, helper T cells, cytotoxic T cells and CD19+ B cells were enhanced in the spleen, creating an anti-tumorigenic niche. The expression of PD-(L)1 was also significantly increased in the cisplatin-exposed spleen. In conclusion, our study provides a unique detailed characterization of the modulation of TNBC progression by cisplatin treatment and associated immune cell and immune checkpoint content across multiple tissues, identifying the spleen as most positively affected and potential driver for synergy with immunotherapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-88
Selective IL-2 Immunotherapy to Fuel the Anti-Tumor Immune Response D. Sahin, O. Boyman University of Zurich and University Hospital Zurich, Department of Immunology, Zurich, Switzerland
Text Interleukin-2 (IL-2) was the first approved immunotherapy to show efficacy in advanced cancer. However, use of IL-2 immunotherapy was hampered by the short in vivo half-life of IL-2, dose-dependent toxicity and stimulation of immunosuppressive regulatory T cells. Complexing IL-2 to specific IL-2 receptor β (CD122)- directed antibodies is a promising strategy for cancer immunotherapy, dissecting the beneficial from the unwanted effects. To make this approach amenable for clinical development we have generated NARA1, an anti-human IL-2 (hIL-2) antibody biasing IL-2 away from IL-2 receptor α (CD25)-expressing regulatory T and endothelial cells and toward CD122+ effector immune cells. However, such hIL-2/NARA1 complexes can dissociate in vivo, undermining selective IL-2 receptor stimulation. We have now developed a novel strategy to permanently graft natural hIL-2 to its antigen-binding groove on NARA1, thereby generating NARA1leukin. NARA1leukin was completely shielded from binding to CD25 and thus showed a better selectivity for CD122+ effector immune cells than hIL-2/NARA1 complexes in vitro and in vivo. This translated into potent anti-tumor responses in several pre-clinical cancer models with superior effects particularly on metastatic lesions. Collectively, NARA1leukin combines and further exceeds the advantages of IL-2/NARA1 complexes in a single and stable molecule, which will also facilitate the generation of bispecific antibodies and combination immunotherapy strategies.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-89
Stiffening cancer cells enhances T-cell mechanical force and anti-tumor efficacy K. Lei1, A. Kurum1, L. Tang1, 2 1EPFL, Institute of Materials Science and Engineering, Lausanne, Switzerland, 2EPFL, Institute of Bioengineering, Lausanne, Switzerland
Text Cancer cell softening accompanies malignancy and promotes tumor progression. However, the impact of cancer cell softening on T-cell mediated cytotoxicity and thus the therapeutic outcome of immunotherapy remains elusive. Here, we identified that cancer cells enrich cholesterol in plasma membrane for cell softening leading to their resistance to T-cell mediated cytotoxicity. To overcome such resistance, we depleted cholesterol from plasma membrane to stiffen cancer cells. Stiffening cancer cells significantly enhanced T-cell mediated cytotoxicity against cancer cells both in vitro and in multiple preclinical murine tumor models, including a highly aggressive B16F10 melanoma model. In mechanism studies, we found that stiffening cancer cells had a negligible effect on canonical biochemical pathways underlying T-cell mediated cytotoxicity. Instead, primary cytotoxic T cells exerted higher cellular force on stiffer substrates mimicking stiffened cancer cells. Besides, inhibiting cellular force generation in primary cytotoxic T cells using cytoskeleton inhibitors abrogated the enhanced T-cell mediated cytotoxicity against stiffened cancer cells. These results indicated that stiffening cancer cells augmented T-cell mediated cytotoxicity via enhancing T- cell mechanical forces. In summary, we discover that cancer cell softness is a novel immune checkpoint of mechanical basis inhibiting T-cell mediated cytotoxicity, and this mechanical immune checkpoint can be therapeutically targeted to enhance the efficacy of T-cell-based immunotherapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-90
A Manganese Phosphate Nanocluster Activates the cGAS-STING Pathway forEnhanced Cancer Immunotherapy M. Gao, Y. Xie, K. Lei, L. Tang École polytechnique fédérale de Lausanne (EPFL), Lausanne, Switzerland
Text Targeting the stimulator of interferon genes (STING) pathway with cyclic dinucleotides (CDNs), the natural STING agonists, is a promising immunotherapeutic strategy to boost anticancer immunity. However, the clinical application of natural CDNs as therapeutics is greatly hindered by their intrinsic properties including negative charges, small molecular weight, and high susceptibility to enzymatic degradation. Manganese(II) ions recently have been discovered to directly activate the cyclic GMP-AMP synthase (cGAS) and augment cyclic GMP-AMP (cGAMP)-STING binding affinity. Here, we developed a PEGylated manganese(II) phosphate (MnP-PEG) nanocluster that exhibited high biocompatibility and potent stimulating capacity of the cGAS-STING pathway. MnP-PEG nanoclusters activated the immature bone marrow-derived dendritic cells leading to the production of a 57.3-fold and 13.3-fold higher amount of interferon β (IFN-β) and interleukin-6 (IL-6) than cGAMP, respectively. The superior STING activation capacity was likely due to the highly efficient cellular internalization of MnP-PEG nanoclusters by dendritic cells (DCs) and acid-triggered release of Mn2+ ions in the endolysosomes. Intratumoral administration of MnP-PEG nanoclusters led to markedly enhanced tumor infiltration and maturation of DCs and macrophages, as well as increased activation and cytotoxicity of T cells and natural killer cells. MnP-PEG nanocluster as a monotherapy or in combination with a checkpoint blockade antibody resulted in significant tumor regression in the subcutaneous B16F10 murine melanoma model without overt toxicities. Compared to natural CDNs, the slightly negatively charged surface as well as the suitable nanosize of MnP-PEG nanoclusters render them potentially improved pharmacokinetics and retention in the tumor. MnP-PEG nanoclusters are resistant to enzymatic degradation and show good stability in buffers mimicking physiological conditions. The synthesis of MnP-PEG nanoclusters is a facile, scalable, and solution-based method, which might be compatible with large scale manufacturing processes. Hence, MnP-PEG could be a potential activator for the cGAS-STING pathway for cancer immunotherapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-91
Metabolic reprogramming of terminally exhausted CD8+ T-cells by interleukin-10 enhances anti-tumor immunity Y.-Q. Xie, Y. Guo, L. Tang École polytechnique fédérale de Lausanne, Institute of Bioengineering, Lausanne, Switzerland
Text Cancer immunotherapy represented by immune checkpoint blockades has achieved remarkable clinical success. However, an outstanding challenge remains that a great majority of patients fail to respond to this therapy. This is in part due to the fact that tumor-infiltrating lymphocytes (TILs) become exhausted and eventually incapable to control tumor progression. Among exhausted CD8+ TILs, a subpopulation termed terminally exhausted CD8+ T-cells, which hold a certain level of anti-tumor cytotoxicity but largely reduced proliferation capacity, contribute directly to tumor growth control. However, this subpopulation does not respond to immune checkpoint blockades or most existing immunotherapies and is difficult to be reactivated. Here, we show that a half-life extended interleukin (IL)-10/Fc fusion protein directly expands the terminally exhausted CD8+ TILs and sustains their effector functions in vivo. In a syngeneic B16F10 mouse melanoma model, IL-10/Fc treatment markedly and selectively expanded the PD-1+TIM-3+ double positive but not the PD-1+ single positive subset, with substantially increased frequencies as well as 10.5- and 3.3-fold greater cell counts (combination treatment vs. ACT alone) of adoptively transferred PMEL and endogenous CD8+ T- cells, respectively, leading to the eradication of established solid tumors and durable cures in a majority of treated mice when combined with adoptive T-cell transfer (ACT) immunotherapy. The combination therapy consistently induced complete tumor regression and durable cures in 90% of treated mice without overt toxicities in this poorly immunogenic and highly aggressive mouse melanoma model. Our results provide preclinical evidence that IL-10/Fc is a safe and highly effective therapy that acts on a specific subset of CD8+ TILs distinct from those responding to immune checkpoint blockades. Thus, IL-10/Fc could complement and synergize with existing cancer immunotherapies for enhanced efficacy and response rates. Importantly and for the first time, we find that IL-10/Fc enhances T-cell proliferation and functionality by promoting oxidative phosphorylation through the mitochondrial pyruvate carrier (MPC), as the effect of IL-10/Fc on enhancing proliferation of the PD-1+TIM-3+ CD8+ T-cells was abrogated by the treatment of MPC inhibitor UK5099 or when MPC was knocked-out. Our research suggests that metabolic reprogramming is sufficient to revitalize terminally exhausted CD8+ TILs. This finding provides new insight into the crucial role of metabolic profiles in T-cell exhaustion and reinvigoration and lays the foundation for further identification of metabolic switches for T-cell activity.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-92
Targeting GITR enhances ex vivo human tumour-infiltrating T cell functionality in mismatch repair proficient primary colorectal carcinoma and liver metastases Y. Rakké1, L. Campos Carrascosa2, A. van Beek2, V. de Ruiter2, M. Doukas3, S. ter Borg4, P. Doornebosch5, M. Vermaas5, E. van der Harst6, P. Coene6, D. Grünhagen1, C. Verhoef1, J. IJzermans1, D. Sprengers2, J. Kwekkeboom2 1Erasmus MC Cancer Institute, Surgery, Rotterdam, Netherlands, 2Erasmus MC Cancer Institute, Gastroenterology and Hepatology, Rotterdam, Netherlands, 3Erasmus MC Cancer Institute, Pathology, Rotterdam, Netherlands, 4Pathan BV, Rotterdam, Netherlands, 5IJsselland Hospital, Surgery, Capelle aan den IJssel, Netherlands, 6Maasstad Hospital, Surgery, Rotterdam, Netherlands
Text BACKGROUND. Immune checkpoint blockade (ICB; e.g. anti-PD-1/-CTLA-4) has been proven to be clinically effective in mismatch repair deficient (dMMR) colorectal carcinoma (CRC). Yet, the majority of patients caries mismatch repair proficient (pMMR) CRC, especially those with liver metastasis, and does not respond to ICB. Here, we studied the effect of immune checkpoint stimulation via GITR targeting on ex vivo functionality of human tumour-infiltrating lymphocytes (TIL) isolated from pMMR primary CRC and liver metastases (CRLM). METHODS. Human TIL were isolated from freshly resected pMMR tumours of patients with primary CRC (stage 1- 3) or liver metastases. GITR expression on TIL was determined using flow cytometry and compared to leukocytes isolated from blood (PBMC) and tumour-free surrounding tissues (tumour-free colon/liver, resp. TFC and TFL). Ex vivo functional assays were used to assess TIL expansion, activation and cytokine secretion upon CD3/CD28 bead activation and co-stimulation using an antibody-crosslinked recombinant trimeric GITR ligand (GITRL). RESULTS. GITR was overexpressed on TIL when compared to other stimulatory immune checkpoints (4-1BB, OX40). Its expression was enhanced on CD4+ and CD8+ TIL compared to PBMC and TFC or TFL compartments in both primary CRC and CRLM. Among CD4+ TIL, GITR expression gradually increased on CD45RA+/- FoxP3- helper T (Th), CD45RA- FoxP3int activated helper T (aTh), and CD45RA- FoxP3hi activated regulatory T cells (aTreg), respectively. Within CD8+ TIL, GITR expression was predominantly present on TOX+ PD1Hi and putative tumour-reactive CD103+ CD39+ TIL. Enhanced HLA-DR and Ki67 expression among GITR+ vs. GITR- PD1hi CD8+ TIL confirmed in situ activation. Yet, impaired effector cytokine production upon ex vivo PMA/ionomycin stimulation was observed in CD4+ and CD8+ GITR-expressing TIL, indicating functional exhaustion of the target population. However, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers and proinflammatory cytokine secretion in a dose-dependent manner. Treg depletion did not fully abrogate the stimulatory effect of GITR ligation on CD4 and CD8 T cell expansion, demonstrating partial direct effect on effector TIL. Importantly, GITR-ligation also enhanced expansion of purified CD8+CD39+ TIL. Dual treatment with GITR ligand and nivolumab (anti-PD-1) further enhanced CD8+ TIL responses compared to GITR ligand monotherapy, whereas nivolumab alone did not show any effect. CONCLUSIONS. Agonistic targeting of GITR enhances ex vivo human TIL functionality in pMMR CRC and might therefore be a promising approach for novel mono- or combinatorial immunotherapies in primary pMRR CRC and CRLM.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-93
Preclinical mechanism of action and anti-tumor activity of DuoBody-PD-L1×4-1BB (GEN1046) A. Muik1, K. Schödel1, I. Altintas2, F. Gieseke1, A. Sette2, J. Blum3, E. Stanganello4, F. Vascotto4, S. Burm2, A. Toker1, K. Sasser3, M. Diken1, 4, S. Kreiter1, 4, M. Fereshteh3, Ö. Türeci1, E. Breij2, T. Ahmadi3, U. Sahin*1, M. Jure-Kunkel*3 1BioNTech SE, Mainz, Germany, 2Genmab B.V., Utrecht, Netherlands, 3Genmab U.S., Inc., Princeton NJ, United States, 4TRON, Mainz, Germany *Contributed equally
Text Checkpoint inhibitors targeting the PD-1/PD-L1 axis (CPI) have changed the treatment paradigm and prognosis for patients with advanced solid tumors; however, many patients experience limited benefit due to treatment resistance. 4-1BB co-stimulation can activate cytotoxic T- and NK-cell-mediated anti-tumor immunity and was shown to synergize with CPI in preclinical models. DuoBody-PD-L1×4-1BB is a first-in- class, Fc-inert bispecific next-generation immunotherapy designed to target both pathways. Here we present preclinical evidence for the mechanism of action (MoA) of DuoBody-PD-L1×4-1BB and provide proof-of- concept for antitumor activity using double knock-in mice that express the extracellular domain of human PD- L1 and human 4-1BB (hPD-L1/h4-1BB dKI mice). MoA studies were conducted using immunofluorescence and live cell imaging, in vitro assays of primary human T-cell activation and effector functions and expansion of tumor-infiltrating lymphocytes (TIL) from patient-derived non-small cell lung cancer (NSCLC) specimens. DuoBody-PD-L1×4-1BB prolonged interactions between PD-L1+ dendritic cells (DC) and 4-1BB+ T cells in co-culture compared to monospecific antibody controls, and clustered with LFA-1, a marker of the immunological synapse. DuoBody-PD-L1×4-1BB enhanced activation of CD8+ T cells that were stimulated by cognate antigen, allogeneic DC, or anti-CD3 antibodies in vitro, leading to increased proliferation and pro- inflammatory cytokine production. DuoBody-PD-L1×4-1BB further induced upregulation of granzyme B and the degranulation marker CD107a in CD8+ T cells, resulting in augmented antigen-specific T-cell-mediated cytotoxicity of MDA-MB-231 tumor cells in vitro. Enhanced proliferation and effector functions were dependent on crosslinking with PD-L1+ cells and superior to CPI or the combination of PD-L1- and 4-1BB- specific antibodies. In patient-derived NSCLC tissue, DuoBody-PD-L1×4-1BB enhanced expansion of CD8+ TIL compared to CPI, which also showed increased activation upon restimulation with autologous tumor. In hPD-L1/h4-1BB dKI mice, DuoBody-PD-L1×4-1BB induced complete regression of subcutaneous MC38 tumors expressing human PD-L1 (5 mg/kg, 2QWx3) in vivo, and conferred protection against secondary tumor challenge. In conclusion, DuoBody-PD-L1×4-1BB crosslinks 4-1BB+ T cells with PD-L1+ cells and enhances T-cell activation, expansion, and effector functions through conditional 4-1BB agonist activity combined with constitutive PD-1/PD-L1 blockade. Moreover, DuoBody-PD-L1×4-1BB exhibits anti-tumor activity and induces memory immune responses in vivo. DuoBody-PD-L1×4-1BB is currently being evaluated in patients with advanced solid tumors in a first-in-human clinical trial (NCT03917381).
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-94
Oncolytic Virotherapy with recombinant VSV-NDV synergizes with anti-CTLA-4 immune checkpoint blockade in a RIG-I dependent manner J. Marek, T. Krabbe, L. Hanesch, T. Nedelko, S. Heidegger, J. Altomonte Klinikum rechts der Isar der TU München, Klinik und Poliklinik für Innere Medizin II, München, Germany
Text Immune checkpoint inhibitors are an exciting new aspect of immune oncology and have demonstrated substantial improvements in survival for a variety of malignancies. The monoclonal antibody, anti-CTLA-4, was the first immune checkpoint inhibitor approved by the Food and Drug Administration in 2011 for the treatment of malignant melanoma. However, its efficacy is generally limited to those tumors with a highly inflamed microenvironment. Oncolytic viruses represent an attractive mechanism to synergize with immune checkpoint blockade (ICB), as the innate immune response to viral infection can modulate the tumor microenvironment, transforming the immunologically “cold” tumor into an inflamed (“hot”) lesion that is sensitized to ICB therapy. RIG-I is a sensor of viral RNA that has been shown to be crucial in the anti-tumor immune response to anti-CTLA-4 therapy. In this study, we hypothesized that treatment with the oncolytic hybrid virus, VSV-NDV, could synergize with anti-CTLA-4 therapy through activation of RIG-I and by induction of complementary therapeutic mechanisms, such as viral-mediated oncolysis and induction of immunogenic cell death. We first characterized recombinant VSV-NDV in vitro for its ability to replicate and cause cytotoxicity in wildtype and RIG-I knockout B16.OVA melanoma cells. Not only could VSV-NDV rapidly kill B16.OVA melanoma cells in a RIG-I independent manner, but the dying cells were highly efficient in activating dendritic cells (DCs), indicating that the cells died via an immunogenic cell death. We next implanted B16.OVA WT or RIG-I-/- cells subcutaneously in contralateral flanks of female C57BL/6J mice. Interestingly, monotherapy with VSV-NDV by direct intratumoral injection into the tumor on the right side delayed tumor growth, both locally and in the contralateral tumor, leading to prolonged survival. This effect was shown to be further improved by combination with intraperitoneally injected anti-CTLA-4. These data are consistent with the observation of enhanced activation and proliferation of tumor-specific T cells from peripheral blood in combination treated mice. Strikingly, the improved therapeutic effects of the combination treatment could only be observed in the wildtype tumors, while RIG-I knockout tumors showed little benefit from anti-CTLA-4 therapy. In contrast, the therapeutic effects of VSV-NDV appeared to be independent of RIG-I status. VSV-NDV, therefore, represents a promising oncolytic virus platform for development as an immunotherapeutic agent, which has the potential to synergize with anti-CTLA-4 therapy via multiple mechanisms, including activation of RIG-I, which is essential for the responsiveness of tumors to ICB using anti-CTLA-4.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-95
Single-cell transcriptomics reveals changes in the immune landscape following IL-2 variant and anti-PD-1 treatment in a mouse tumor model M. Streuber1, E. Diken1, L. Kolb1, M. Vormehr2, L. M. Kranz2, D. Eisel2, M. Diken1, 2, Ö. Türeci2, U. Sahin2 1TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz gGmbH, Mainz, Germany, 2BioNTech SE, Mainz, Germany
Text Inhibition of programmed death receptor 1 (PD-1) is an approved treatment across various cancer indications; however, only a small fraction of patients develop durable anti-tumor responses. Interleukin-2 (IL-2) is an approved immunotherapy treatment, but its efficacy and utility in a clinical setting is limited by its short in vivo half-life, severe toxicity and propensity to amplify immune suppressive T regulatory (Treg) cells in addition to effector natural killer (NK) and T cells. Thus, novel approaches to overcome the limitations of IL-2 therapy are required. We demonstrated therapeutic efficacy in mouse models of BNT151, a novel lipid nanoparticle-formulated, nucleoside- modified ribonucleic acid (RNA) encoding a mutant variant of human IL-2 fused to human serum albumin to extend its serum half-life and allow preferential expansion of effector T cells over Treg cells.
We performed single-cell RNA sequencing (scRNA-seq) to explore the efficacy and mode of action of BNT151 alone or in combination with anti-PD-1 (aPD-1) in the MC38 mouse tumor model. Single-cell data analysis of >9,000 CD45+ tumor infiltrating lymphocytes (TILs) revealed 13 distinct TIL subpopulations, namely macrophages (two subsets), neutrophils, monocytes, mature dendritic cells, NK cells, NKT cells, γδ T cells, CD8 and CD4 double negative (DN) T cells, CD4+ T cells and CD8+ T cells (three subsets). Overall, tumors of mice treated with BNT151 alone or in combination with aPD-1 had an increased number of CD8+ T cells, CD4+ T cells, γδ T cells, DN T cells, NK cells and macrophages compared to Control. NK cells showed a heightened expression of genes related to cytotoxicity (e.g., Gzmf, Gzme, Gzmd), inhibitory immune checkpoint receptors (e.g., Ctla4) or interferon (IFN) response (e.g., Ifitm2). Proliferation of macrophages and all T cell populations was mediated by BNT151 and enhanced upon addition of aPD-1. The expression of markers related to TIL exhaustion (e.g., Tox, Lag3, PD-1) was strongly reduced with combination treatment. More CD8+ T cells expressed Ifng upon BNT151 treatment, which was further enhanced in combination with aPD-1. Importantly, BNT151 increased the expression of the transcription factor Tcf7, particularly on DN T cells and a subset of CD8+ T cells, which has been associated with improved cancer immunotherapy outcomes.
In conclusion, scRNA-seq revealed that BNT151 increased the number of TILs of a less exhausted phenotype and induced the expression of genetic markers commonly associated with therapeutic success. Both effects were further enhanced by the combination with aPD-1.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-96
Combination immunotherapy overcomes resistance in MHC class I-deficient cancer J. D. Beck1, M. Vormehr2, M. Diken1, 2, M. Suchan1, M. Streuber1, E. Diken1, L. Kolb1, F. Vascotto1, T. Beißert1, Ö. Türeci2, S. Kreiter1, 2, U. Sahin2 1TRON gGmbH – Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany, 2BioNTech SE, Mainz, Germany
Text Cancer immune evasion through loss of major histocompatibility complex (MHC) class I expression has been frequently associated with resistance to immunotherapy and is a major barrier to designing effective anti- cancer therapies. Here, we show that MHC class I deficiency leads to profound immune desertification of the tumor microenvironment and resistance to immune checkpoint blockade (ICB) and chemotherapy in syngeneic mouse tumor models. Combination treatment with a monoclonal antibody (mAb) against a tumor cell surface marker and interleukin-2 (IL-2; mAlb/IL-2) induces strong immune cell infiltration and an accentuated interferon (IFN) signature in the tumor microenvironment. mAb/IL-2 promotes tumor rejection and significantly prolongs survival of mice with MHC class I-deficient tumors, as well as preventing emergence of acquired resistance to ICB and reversing chemotherapy resistance. Additionally, we show that therapeutic responses depend on cellular crosstalk between CD8+ T cells and macrophages. The tumor- infiltrating CD8+ T cells are a main source of IFNγ, which is associated with marked M1 polarization of the macrophage compartment. Our findings suggest that mAb/IL2 therapy may represent a versatile approach to counteract immune escape during the course of conventional cancer treatment and immunotherapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-97
Neoantigen CD8 T cell recognition is broadened and activated in relation to disease control in the peripheral blood of patients with metastatic urothelial carcinoma following PD-L1 blockade J. S. Holm1, S. A. Funt2, 3, A. Borch1, K. K. Munk1, A.-M. Bjerregaard1, J. L. Reading4, C. Maher2, 3, 5, A. Regazzi2, 3, P. Wong2, 3, 5, H. Al-Ahmadie6, G. Iyer2, 3, T. Tamhane1, A. K. Bentzen1, N. O. Herschend1, S. De Wolf2, A. Snyder2, 3, T. Merghoub2, 3, 5, J. D. Wolchok2, 3, 5, 7, M. Nielsen1, J. E. Rosenberg2, 3, D. F. Bajorin2, 3, S. R. Hadrup1 1Technical University of Denmark, Department of Health Technology, Kgs. Lyngby, Denmark, 2Memorial Sloan Kettering Cancer Center, Department of Medicine, New York, NY, United States, 3Weill Cornell Medical College, New York, NY, United States, 4University College London Cancer Institute, Cancer Immunology Unit, Research Department of Hematology and Cancer Research UK, London, United Kingdom, 5Parker Institute for Cancer Immunotherapy, San Francisco, CA, United States, 6Memorial Sloan Kettering Cancer Center, Department of Pathology, New York, NY, United States, 7Memorial Sloan Kettering Cancer Center, Human Oncology and Pathogenesis Program, New York, NY, United States
Text Proliferation of CD8 T cells can be detected in the blood of cancer patients following a single dose of immune checkpoint blockade (ICB) and tends to be more robust in patients with clinical response. Furthermore, CD8+ T cell reactivity towards tumor mutation-derived neoantigens is widely believed to facilitate the antitumor immunity induced by ICB. However, the capacity to mount a neoantigen T cell response and the kinetics in relation to ICB remain poorly understood.
Here, we longitudinally interrogated peripheral CD8+ T cell recognition of patient-specific predicted neopeptide libraries using DNA barcode-labelled pMHC multimers in a cohort of 24 patients with metastatic urothelial carcinoma (mUC) treated with PD-L1-blockade. Additionally, phenotypic characteristics of both bulk CD8, neoantigen-recognizing (NARTs) and virus-epitope specific T cells were investigated. Patient PBMC samples were screened pre- and post-treatment (n = 85 PBMC samples) and kinetics of CD8 NARTs were assessed for association with disease control (patients with best RECIST 1.1 of Stable Disease, Partial or Complete Response).
Neoepitope peptide libraries of between 200-587 peptides were generated per patient (mean = 260 peptides per patient), with HLA-coverage of 31 out of a combined 56 possible patient HLA types (55%, mean = four HLAs per patient). Patients with disease control experienced a significant increase in the number of NART responses from pre- to 3 weeks post- treatment compared to progressive disease patients. NARTs derived from patients with disease control displayed a PD1+ Ki67+ effector phenotype 3 weeks post-treatment, underlining the understanding of rapid immune invigoration post-treatment. Meanwhile, NARTs expressed increased CD39 levels compared to bystander bulk- and virus-antigen reactive CD8+ T cells, now also evident for tumor-specific CD8 T cells in the blood.
The completed study provides novel insights into the mechanisms and kinetics of NART activity following ICB and provides evidence that early-stage NART recognition and activation are associated with response to ICB in patients with mUC.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-98
Modulation of iNKT cells in pediatric neuroblastoma N. Lehmann1, 2, C. Paret1, 2, N. Ziegler1, 2, K. El Malki1, 2, L. Seidmann3, A. Russo1, 2, A. Wingerter1, 2, R. Sandhoff4, J. Faber1, 2 1University Medical Center of the Johannes Gutenberg-University Mainz, Pediatric Hematology and Oncology, Mainz, Germany, 2University Medical Center of the Johannes Gutenberg-University Mainz, University Cancer Center (UCT), Mainz, Germany, 3University Medical Center of the Johannes Gutenberg- University Mainz, Institute of Pathology, Mainz, Germany, 4German Cancer Research Center, Lipid Pathobiochemistry, Heidelberg, Germany
Text Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. The monoclonal antibody dinutuximab has demonstrated the potential for immunotherapy to improve overall NB outcomes, however the 5-year overall survival of high-risk patients is only 50%. Therefore, new therapeutic options are a matter of utmost urgency. Pre-clinical data indicates that invariant natural killer T cells (iNKT)-based immunotherapies have promise in the treatment of solid tumors. iNKT cells represent a very small but highly variable population of 0.1% to 1% of all T cells in the blood. iNKT cells recognize lipid antigens, which are presented via CD1d. Moreover, iNKT cells act as a mediator between the adaptive and innate immune system by recruiting other cells of the immune system through the secretion of various cytokines. In addition, these cells play an important role in the immune surveillance of tumors by exercising their anti-tumor function through the direct recognition of CD1d-expressing tumor cells or via other CD1d-expressing cells within the tumor microenvironment. Here, we analyzed the iNKT cell infiltration and CD1d expression in pediatric neuroblastoma. Tumor-infiltrating lymphocytes were isolated using Percoll density centrifugation from fresh tumor specimens and iNKT cells were detected by flow cytometry. iNKT cells were identified in seven out of eight neuroblastoma samples. In particular, patients within the first 12 months of life showed a high level of iNKT cell infiltration. CD1d expression analyses of 12 neuroblastoma samples were performed using qRT-PCR. CD1d expression could not be detected in any of the 12 tumor samples. Our data indicate that neuroblastoma are often infiltrated by iNKTs. The frequency of iNKTs has been associated with improved outcomes in several malignancies. However, neuroblastoma cells themselves are CD1d negative and cannot be recognized and eliminated directly by iNKT cells. Combination with HDAC inhibitors such as Vorinostat could be used to induce CD1d expression as shown in other tumor entities. Furthermore, combination with immune checkpoint inhibitors could increase the cytotoxic efficiency of iNKT.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-99
Lactobacillus casei modulates intestinal microbiota and anti-tumor immune responses in a syngeneic colon adenocarcinoma model in mice G. Aindelis, A. Tiptiri-Kourpeti, K. Spyridopoulou, P. Ypsilantis, K. Chlichlia Democritus University of Thrace, Alexandroupolis, Greece
Text Probiotic lactic acid bacteria are known to exert strain-specific beneficial effects against a plethora of gastrointestinal disorders, including colon carcinoma. Here we aimed to investigate the impact of Lactobacillus casei consumption on the intestinal microbiota of mice during a syngeneic tumor model and any possible association between the composition of the gut micro flora and anti-tumor activity. BALB/c mice received viable probiotic bacteria per os daily for ten days and then subcutaneous CT26 tumors were established. Faeces were collected at the end of the probiotic administration and microbial content was determined with Next Generation Sequencing (NGS) analysis. Developing tumors, small intestine content and Peyer’s patches were isolated at various time points. L. casei administration resulted in a significant tumor volume reduction, accompanied by accumulation of CD8+ cytotoxic T lymphocytes in the tumor. Secretion of IgA was found to be elevated in the small intestine of probiotic fed mice and cytokine production in Peyer’s patches was modulated, with detection of increased IFN-γ. In addition, the composition of the microbiota was altered as evident by the separation of samples when evaluating beta diversity with Bray- Curtis and weighted UniFrac distance. However, alpha diversity of animals was not significantly impacted by the consumption of L. casei for the indicated time period. Sequences belonging in the family Muribaculaceae were found to be increased in mice that had received the probiotic microorganism, an observation that was also confirmed using the linear discriminant analysis (LDA) effect size (LEfSe) method. Next we tried to identify two groups of taxa whose relative abundance was associated with the tumor volume, implementing a forward selection method. The balance between families Bacteroidaceae and Muribaculaceae was identified as more closely associated with tumor volume such as that mice with larger tumors exhibited higher balance scores. Our findings suggest that L. casei administration modulated the intestinal microbiota of BALB/c mice and enhanced the anti-tumor immune response against subcutaneous colon carcinoma. Further studies are underway to better understand to what extend the intestinal microbiota and anticancer immunity are associated in this syngeneic tumor model.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-100
Fully automated and closed manufacturing of gene-engineered T cells including a simplified way to final formulate and fill drug products for fresh and/or frozen use in adoptive T cell therapy M. Maluski1, A. Engelhorn1, B. Weidemann1, C. Radek1, M. Flügge1, J. Moer1, L. Kuschnierz1, D. Sell1, J. Raasch1, J. Lüdemann1, B. Kauling1, A. Schultz1, E. Rendler2, P. Rakoczy2, S. Schallenberg1, M. Essl2, N. Mockel-Tenbrinck1, A. Kaiser1 1Miltenyi Biotec B.V. & Co.KG, R&D Immunotherapy, Bergisch Gladbach, Germany, 2Miltenyi Biotec B.V. & Co.KG, Clinical Products Cell Factory, Bergisch Gladbach, Germany
Text Adoptive immunotherapy using gene-modified T cells redirected against cancer has proven clinical efficacy and tremendous potential in several medical fields. However, such personalized medicine faces a number of challenges in the complexity associated with the current clinical manufacturing methods. Conventionally, the preparation of autologous gene-modified T cells is labour intensive and comprises many open handling steps such as the final formulation and filling of drug products either for fresh application or cryopreservation. Furthermore the complexity and synchronisation of allocated quality control testing during the manufacturing process, and even more importantly during the preparation of the drug product, has a substantial impact on the successful release.
We have developed a T Cell Transduction application including an automated formulation and filling process (TCTf) for gene-modified T cells to enhance the standard T Cell Transduction (TCT) process on the CliniMACS Prodigy. The CliniMACS Formulation Unit as accessory and a new single-use closed tubing set (TS521) with several attached product bags allow the filling of ready-to-use fresh drug products as well as cryopreservation while preparing respective Quality control samples. A refined application software including automated in-process sampling and final formulation of the drug product dependent on patient-specific characteristics enables a simplification of the process with less user interactions.
Here, we demonstrate that the new TCTf application has an equivalent culture performance as the standard TCT process. Furthermore, the automated final formulation step generates CAR T cell products with indiscriminate characteristics that meet demands for fresh formulated drug products such as cell fitness, functionality, as well as stability.
Taken together, besides being capable of manufacturing a gene-modified cell product, the novel fully- automated TCTf application on the CliniMACS Prodigy is able to final formulate and fill drug products in a simplified manner. This improvement will further simplify CAR T cell preparations and increase robustness for the release of cell products for patient treatments.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-101
Smac mimetics (SM) can manipulate cell death resistance of rhabdomyosarcoma (RMS) cells sensitizing them towards natural killer (NK) cell mediated killing L. M. Reindl1, 2, V. Särchen3, J. Heck1, 2, M. Vogler3, T. Bukur4, M. Suchan4, V. Bukur4, T. Klingebiel1, S. Fulda3, 5, 6, E. Ullrich1, 6, 7 1University Hospital Frankfurt, Goethe University, Department for Children and Adolescent Medicine, Frankfurt, Germany, 2University Hospital Frankfurt, Goethe University, Department for Children and Adolescent Medicine, Experimental Immunology, Frankfurt, Germany, 3Institute for Experimental Cancer Research in Pediatrics, Goethe University, Frankfurt, Germany, 4TRON – Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany, 5German Cancer Consortium (DKTK), Heidelberg, Germany, 6German Cancer Research Center (DKFZ), Heidelberg, Germany, 7University Hospital Frankfurt, Goethe University, Department for Children and Adolescents Medicine, Frankfurt, Germany
Text One mechanism how RMS cells evade the onset of cell death is by dysregulating inhibitor of apoptosis proteins (IAPs). Strategies to restore the apoptotic machinery comprise of the treatment with a small molecule, termed Smac mimetic (SM), mimicking the endogenous IAP antagonist Second mitochondria- derived activator of caspases (Smac). The restoration of cell death mechanisms sensitize RMS cells to an exploitable NK cell mediated killing. Identification of molecular mechanisms, responsible for the sensitizing effect, were investigated using an exploratory transcriptome analysis by RNA sequencing. Validation of the sequencing data were underwent using the Fluidigm platform, in-house RT-qPCR, Western blot and flow cytometry. Cell death was microscopically measured using a PI/Hoechst staining. Further, a three-dimensional RMS spheroid was used to model an in vivo situation. Spheroids were assessed by measuring the GFP fluorescence with an ImageXpress Micro XLS microscope. Treatment with SM leads to a rapid degradation of cIAPs and activation of NF-kB signaling pathways. Several target genes of the NF-kB signaling pathway were identified by RNAseq, e.g. BIRC2, BIRC3, NFKB2, NFKBIA, TRAF1, leading to a putative transcriptionally bimodal feedback loop of the canonical and non-canonical NF-kB signaling pathways. Further, SM induces a sensitization of the RMS cells to a TRAIL mediated killing mechanism. Apart from NF-kB signaling, SM upregulate the TRAIL-R2 on the cell surface of RMS cells. When co-cultivated with NK cells, SM pre-treated RMS spheroids show a higher sensitivity towards NK cell mediated killing and faster killing kinetics, compared to untreated spheroids. RNAseq analysis of NK cells, post co-culture with RMS cells, revealed upregulation of several biological process, e.g. cell differentiation, chemotaxis and response to cytokines.In conclusion, we can provide evidence for a complex sensitizing effect of SM on RMS cells, manipulating cell death inducing pathways. Furthermore it drives a sensitizing mechanism towards NK cell mediated killing.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-102
Novel insights from multi-parametric flow cytometry: Tumor-specific antigen levels modulate polyfunctionality of chimeric antigen receptor T cells B. Xie1, J. Euesden2, S. Lekomtsev1, B.-P. Kloke1, S. Brett1, M. Davies1 1GSK R&D, Oncology Cell Therapy-Research Unit, Stevenage, United Kingdom, 2GSK R&D, Research Statistics, Stevenage, United Kingdom
Text The polyfunctionality of chimeric antigen receptor (CAR) T cells is defined as the ability of T cells to simultaneously produce a range of functional cytokines, and has been suggested to correlate with favourable clinical outcomes. This study used flow cytometry and computational methods to examine the polyfunctionality of CAR T cells in response to cancer cell lines expressing different antigen levels. Multi-parametric flow cytometry was used to measure the number and expression intensity of intracellular cytokines, including interleukin-2 (IL2), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα), on CD3+, CD4+ and CD8+ cells. CAR T cells were co-cultured with cells expressing high or low levels of antigen at an E:T ratio of 1:1 for 24hr. For basal level comparison, CAR T cells and untransduced T (UT) cells were cultured alone. A computational algorithm (FlowSOM) was used qualitatively to gain a picture of polyfunctional profiles of T cell subsets in all conditions. At the single-cell level, logistic regression with mixed effects was used to model the difference in polyfunctional profile between UT/CAR T alone and co-culture conditions. In each model, the cytokine positivity of each cell was regressed on all conditions, with random effects of the donor and sample. FlowSOM analysis showed that both CD8+ and CD4+ CAR T cells had a broader polyfunctional profile, when CAR T cells were co-cultured with cells expressing high levels of antigen. At the single-cell level, CAR T cells showed a significantly higher degree of polyfunctionality in presence of target antigen, compared to T cells cultured alone. Specifically, co-culturing CAR T cells with high expressing target cells resulted in 355 times more CAR T cells simultaneously expressing 2+ cytokines than CAR T cells cultured alone (P<0.001); and 210 times more CAR T cells with 3+ cytokines than CAR T cells cultured alone (P<0.001). Compared to UT cells, CAR T cells also showed substantially more polyfunctionality following co-culture with both low and high antigen expressing cells. In conclusion, a complete polyfunctional profile for the CAR T product was assessed. Results can be visualised qualitatively at the T cell subset level using FlowSOM and evaluated thoroughly at the single-cell level using logistic regression. The majority of CAR T cells produced a single cytokine with a higher fraction of polyfunctional cells in co- culture conditions, especially when antigen level was higher. This indicates that polyfunctionality of these CAR T cells is governed by the tumour-specific antigen level. These methods can be widely applied to analyze polyfunctionality of engineered T cells towards specific antigen induction following immunotherapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-103
StraightFrom® MicroBeads: Fast T cell isolation for CAR T cell manufacturing without density gradient centrifugation S. Lennartz, D. Lock, C. Kolbe, G. Winkels, S. Rösch, M. Assenmacher, A. Kaiser Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
Text In recent years, CAR T cell-mediated immunotherapy has shown clinical efficacy in the fight against cancer. Increased research efforts to address several challenges associated with CAR T cell therapy for solid tumor treatment for instance, such as poor efficacy and unwanted side effects, are required to progress current approaches. Fast, efficient and flexible CAR T workflows can offer the opportunity to progress such research. At Miltenyi Biotec we have established an optimized CAR T cell research workflow, combining cell isolation, culture and analysis tailored for CAR T cell generation that can be transferred also into clinical setting. Using our StraightFrom® MicroBeads, the fundamental step of isolating high quality T cells as starting material to generate functional CAR T cells, can be performed directly from whole blood, buffy coat and blood products without density gradient centrifugation. Thereby the experimental procedure is accelerated and allows for automation on platforms such as the MultiMACS™ Cell24 Separator Plus and the MultiMACS™ X, whilst maintaining the quality, functionality and downstream compatibility of the isolated T cells, resulting in an ideal starting population for the generation of fully functional CAR T cells. In conclusion, our innovative tools can reduce experimental time and increase the reproducibility and quality of data obtained in your CAR T cell research efforts in immuno-oncology and immunotherapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-104
Early survival prediction framework in CD19-specific CAR-T cell immunotherapy using a quantitative systems pharmacology model A. Mueller-Schoell1, 2, N. Puebla-Osorio3, R. Michelet1, M. Green3, A. Künkele4, 5, W. Huisinga6, P. Strati3, B. Chasen7, S. S. Neelapu3, C. Yee8, 9, C. Kloft1 1Freie Universitaet Berlin, Institute of Pharmacy, Dept. of Clinical Pharmacy and Biochemistry, Berlin, Germany, 2Graduate Research Training Program PharMetrX, Berlin/Potsdam, Germany, 3The University of Texas MD Anderson Cancer Center, Division of Cancer Medicine, Department of Lymphoma and Myeloma, Houston, United States, 4Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt - Universität zu Berlin, and Berlin Institute of Health, Department of Pediatric Oncology and Hematology, Berlin, Germany, 5Berlin Institute of Health (BIH), Berlin, Germany, 6University of Potsdam, Institute of Mathematics, Potsdam, Germany, 7The University of Texas MD Anderson Cancer Center, Division of Diagnostic Imaging, Department of Nuclear Medicine, Houston, United States, 8The University of Texas MD Anderson Cancer Center, Department of Melanoma Medical Oncology, Houston, United States, 9The University of Texas MD Anderson Cancer Center, Department of Immunology, Houston, United States
Text Chimeric antigen receptor (CAR)-T cell therapy significantly improved the treatment of relapsed/refractory Non-Hodgkin lymphoma (NHL). Still, 36%-60% of patients ultimately relapse, warranting early response prediction. Factors associated with favourable outcomes have been proposed in correlative analyses. Still, their impact on distinct CAR-T cell kinetic phases and relative contribution to the high unexplained interindividual variability in CAR-T cell expansion remains unclear. We previously presented a literature-based mathematical model of CD19-specific CAR-T cell immunotherapy. While this model was well in line with literature knowledge, it lacked clinical evaluation. Thus, we used clinical data of 133 CAR-T cell phenotype, 1943 cytokine, and 48 metabolic tumour volume measurements to review and refine this model. The data originated from 19 ‘real-world’ NHL patients treated with axicabtagene ciloleucel with follow-up of ≤24 months. To identify patient-/therapy-related factors explaining the high interindividual variability in CAR-T cell expansion, we amalgamated insights from immunology, physiology, adoptive cell therapy, and mathematical nonlinear mixed-effects modelling to jointly characterise the clinical kinetics and dynamics of CAR-T cell phenotypes (naïve, central memory, effector memory and effector CAR-T cells) and CD19+ metabolic tumour volume. We analysed clinical endpoints in different patient subpopulations, and developed a clinical composite score framework for early survival prediction, based on but not requiring the use of the model. The updated quantitative systems pharmacology model only required minor refinement of the literature-based model and precisely predicted the clinical CAR-T cell and CD19+ metabolic tumour volume concentrations. Moreover, a previous autologous stem cell transplantation (ASCT) and the ratio of CD4+/CD8+ CAR-T cells explained 2/3 of the interindividual variability in CAR-T cell expansion. Leveraging the model, we revealed a subpopulation with a poor prognosis with no previous ASCT and significantly lower model-predicted CAR-T cell expansion capacities. Model-predicted maximum CAR-T cell expansion capacities were highly correlated with clinical composite scores of ‘Maximum naïve CAR-T cell concentrations-to-baseline tumour burden’ ratio (CCSTN) (r=0.98) and
CCSTN>0.00136 (cells/µL)/mL were identified as the best predictor for survival. This systems-driven CAR-T cell model characterised, for the first time, to the best of our knowledge, clinical concentration-time profiles of four CAR-T cell phenotypes and CD19+ metabolic tumour volume and identified patient characteristics explaining 67% of the interindividual variability in CAR-T cell expansion.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-105
BNT221, an autologous neoantigen specific T cell product for adoptive cell therapy of metastatic melanoma D. Lenkala1, B. McCarthy1, J. Kohler1, M. Nelson1, J. McGee2, D. Kallin2, J. Sridar3, P. Turcott3, D. Harjanto4, C. Nijenhuis5, J. van den Berg5, R. Gaynor6, M. van Buuren1 1BioNTech US Inc, T cell Immunology, Cambridge, United States, 2BioNTech US Inc, Peptide Chemistry, Cambridge, United States, 3BioNTech US Inc, Biochemistry, Cambridge, United States, 4BioNTech US Inc, Bioinformatics, Cambridge, United States, 5Netherlands Cancer Institute, Biotherapeutics Unit, Amsterdam, Netherlands, 6BioNTech US Inc, Research and Development, Cambridge, United States
Text Background Neoantigens are tumor-specific antigens derived from somatic mutations that have been shown to be important in the anti-tumor immune response. These antigens are not subject to central immune tolerance and are therefore potentially more immunogenic than tumor-associated antigens. Here, we present the results of multiple successful process engineering runs using leukapheresis from metastatic melanoma patients. Neoantigen specific T cell products (BNT221/NEO-PTC-01) were generated using our ex vivo induction process, NEO-STIM™. These T cell products contain specific CD8+ and CD4+ T cell responses targeting multiple neoantigens from each individual patient’s tumor.
Methods Patient-specific neoantigens were predicted using our RECON® bioinformatics platform, and the predicted high- quality neoantigens were used in our ex vivo induction protocol, NEO-STIM, in multiple successful process engineering runs. NEO-STIM is used to prime, activate, and expand memory and de novo T cell responses from both the CD8+ and CD4+ compartments. In-depth analysis was performed to characterize the specificity and functionality (cytokine production and cytolytic capacity) of the induced T cell responses through high throughput flow cytometric analysis.
Results Here we present the successful induction of CD8+ and CD4+ T cell responses in peripheral blood mononuclear cells from melanoma patients using NEO-STIM in multiple successful process engineering runs. We then extensively characterized these T cell responses and demonstrate that these responses are functional, specific and have cytolytic capacity. Moreover, the induced T cells can recognize autologous tumor.
Conclusions NEO-STIM is a novel platform that is capable of generating ex vivo T cell responses to high-quality neoantigen- targets. A first in human study with this adoptive cell therapy, BNT221, in patients with metastatic melanoma is now underway.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-106
Talimogene laherparepvec (T-VEC)-induced tumor oncolysate as a source of tumor antigens and maturation factors for blood-isolated myeloid dendritic cells J. Tijtgat1, 2, J. De Munck3, I. Dufait4, 5, J. K. Schwarze1, 2, K. Breckpot6, B. Neyns1, 2, J. L. Aerts3, S. Tuyaerts1, 2 1Universitair Ziekenhuis Brussel (UZ Brussel), Department of Medical Oncology, Brussels, Belgium, 2Vrije Universiteit Brussel (VUB), Laboratory of Medical and Molecular Oncology (LMMO), Brussels, Belgium, 3Vrije Universiteit Brussel (VUB), Laboratory for Neuro-Aging and Viro-Immunotherapy, Brussels, Belgium, 4Vrije Universiteit Brussel (VUB), Laboratory of Translational Radiation Oncology, supportive care and Physics (TROP), Brussels, Belgium, 5Universitair Ziekenhuis Brussel (UZ Brussel), Department of Radiotherapy, Brussels, Belgium, 6Vrije Universiteit Brussel (VUB), Laboratory for Molecular and Cellular Therapy, Brussels, Belgium
Text Dendritic cell (DC) tumor vaccines have been pursued for a long time. Recently blood-isolated “natural myeloid DC” were found to be more potent immunostimulants compared to monocyte-derived DC. Intratumoral administration of unmanipulated myeloid DC (myDC) in combination with the oncolytic virus Talimogene laherparepvec (T-VEC) is currently being investigated in patients with advanced melanoma (NCT03747744). In this project, we aim to mimic these cellular interactions in vitro and explore the effect of a T-VEC-induced tumor lysate on the phenotype and function of myDC. CD1c (BDCA-1)+ myDC or the combination of CD1c (BDCA-1)+/CD141 (BDCA-3)+ myDC are isolated from peripheral blood mononuclear cells (from leukapheresis) using the CliniMACS Prodigy®. Melanoma cell lines are cultured in the presence of T-VEC at a different multiplicity of infection (MOI) to induce cell death. Inhibition of cell growth is visualized using IncuCyte® live cell imaging. Uptake of pHrodo™-labelled dead tumor cells is assessed by IncuCyte® live cell imaging and flow cytometry. The phenotype of myDC is measured by flow cytometry and cytokine secretion by multiplex analysis using the MesoScale Discovery system. BDCA-1/3+ myDC that have taken up dying tumor cells will be co- cultured with autologous T cells electroporated with a T cell receptor recognizing a given tumor antigen in a specific HLA type to assess antigen processing and presentation. Melanoma cell lines 624-mel and 938-mel are susceptible to T-VEC-induced oncolysis in a dose-dependent manner. BDCA-1+ and BDCA-1/3+ myDC are not susceptible to T-VEC infection, as no secretion of GM-CSF could be demonstrated, even when infected at a MOI of 10. Melanoma cells undergo immunogenic cell death, as evidenced by partial maturation of myDC upon co-culture with the supernatant of the dying cells. Currently we are assessing the cytokine secretion pattern of these myDCs cultured in the presence of supernatant of dying tumor cells. Next, we plan to investigate the phagocytosis of dying tumor cells by BDCA-1/3+ myDC, wherein we will evaluate if both myDC subtypes take up the dying tumor cells and which receptor(s) are implicated. Finally, we will assess whether the myDCs are capable of processing and cross-presenting phagocytosed tumor antigens to T cells. In conclusion, T-VEC has no cytolytic effect on BDCA-1/3+ myDC and supernatant of T-VEC-lysed melanoma cells induces partial maturation of BDCA-1/3+ myDC. In ongoing experiments, we are investigating which myDC subtypes phagocytose T-VEC oncolysed tumor cells and which receptors are implicated in this process. Furthermore, we will evaluate whether myDCs are capable of processing and presenting phagocytosed tumor material to T cells.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-107
Combining T-cell–specific activation and in vivo gene delivery through CD3-targeted lentiviral vectors A. M. Frank1, 2, A. H. Braun3, L. Scheib3, S. Agarwal3, I. C. Schneider4, F. Fusil5, S. Perian5, U. Sahin6, F. B. Thalheimer3, E. Verhoeyen5, 7, C. J. Buchholz1, 2, 3 1Paul-Ehrlich-Institut, Medical Biotechnology, Langen, Germany, 2Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany, 3Paul-Ehrlich-Institut, Molecular Biotechnology and Gene Therapy, Langen, Germany, 4Irene Schneider Consulting, Hanau, Germany, 5International Center for Infectiology, Research Team Enveloped Viruses, Vectors and Innate Responses, INSERM, Unité 1111, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5308, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, University of Lyon, Lyon, France, 6Translational Oncology at the University Hospital Mainz, Mainz, Germany, 7Université Côte d'Azur, INSERM, Centre Méditerranéen de Médecine Moléculaire, Nice, France
Text Genetic modification of T lymphocytes is a key issue in research and therapy. Conventional lentiviral vectors (LVs) are neither selective for T cells nor do they modify resting or minimally stimulated cells, which is crucial for applications, such as efficient in vivo modification of T lymphocytes. Here, we introduce novel CD3- targeted LVs (CD3-LVs) capable of genetically modifying human T lymphocytes without prior activation. For CD3 attachment, agonistic CD3-specific single-chain variable fragments were chosen. Activation, proliferation, and expansion mediated by CD3-LVs were less rapid compared with conventional antibody- mediated activation owing to lack of T-cell receptor costimulation. CD3-LVs delivered genes not only selectively into T cells but also under non-activating conditions, clearly outperforming the benchmark vector vesicular stomatitis virus-LV glycoproteins under these conditions. Remarkably, CD3-LVs were properly active in gene delivery even when added to whole human blood in absence of any further stimuli. Upon administration of CD3-LV into NSG mice transplanted with human peripheral blood mononuclear cells, efficient and exclusive transduction of CD3+ T cells in all analyzed organs was achieved. Finally, the most promising CD3-LV successfully delivered a CD19-specific chimeric antigen receptor (CAR) into T lymphocytes in vivo in humanized NSG mice. Generation of CAR T cells was accompanied by elimination of human CD19+ cells from blood. Taken together, the data strongly support implementation of T-cell–activating properties within T-cell–targeted vector particles. These particles may be ideally suited for T-cell–specific in vivo gene delivery.
References: A.M. Frank et al., Blood Advances 2020, doi: 10.1182/bloodadvances.2020002229
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-108
Electron beam irradiation-based inactivation of CAR-NK-92 cells for AML therapy L. Walcher1, A.-K. Kistenmacher1, R. Kitte1, S. Ulbert2, U. Köhl3, 4, A. Dünkel1, S. Fricke1 1Fraunhofer Institute for Cell Therapy and Immunology, Department for GMP Process Development, Leipzig, Germany, 2Fraunhofer Institute for Cell Therapy and Immunology, Department for Vaccines and Infection models, Leipzig, Germany, 3Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany, 4Leipzig University, Institute for Clinical Immunology, Leipzig, Germany
Text NK-92 cells are promising effector cells for chimeric antigen receptor (CAR)-based therapies due to their fast and reproducible off the shelf availability. In clinical trials, gamma-irradiation (10 Gy) is applied as a safety precaution to eliminate the proliferative capacity of the cell line and prevent secondary tumor formation. In this project, we investigate CD123-directed CAR-modified NK-92 cells as a treatment option against acute myeloid leukemia (AML). We focus on the efficient inactivation of these cells by irradiation and propose low energy electron irradiation (LEEI) as an alternative to state-of-the-art gamma-irradiation. We show that the proliferation of CAR-NK-92 cells was fully inhibited by LEEI, and that the in vitro cytotoxic activity was maintained for up to three days post-irradiation. In vivo, CAR-NK-92 cell doses of up to 2 x 107 cells per application in a total of five applications were well tolerated and showed no toxic effects in NSG mice. We therefore proceeded to test in vivo efficacy in a KG-1luc cell-based AML mouse model and observed an extended median survival, which was statistically significant for gamma-irradiated CD123- directed CAR-NK-92 cells. In conclusion, LEEI is a safe and efficient alternative to gamma irradiation for the inactivation of CAR-NK-92- cellular therapeutic products. As a future perspective, LEEI holds great potential since it enables a fast, accurate, and reproducible irradiation, and can be easily integrated as an in-line tool in GMP-processes due to low shielding requirements.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-109
Assessing off-target toxicity of cellular immunotherapies V. Murcia Pienkowski, G. Mazzocco, A. Sanecka-Duin, I. Niemiec, P. Król, O. Myronov, P. Skoczylas, S. Stachura, D. Wojciechowski, P. Stępniak, J. Kaczmarczyk, A. Blum Ardigen, Karkow, Poland
Text a) Introductory sentence indicating the purposes of the study Despite their great clinical potential, cell-based cancer immunotherapies are characterized by a high risk of generating off-target immunotoxicity against healthy tissues, which can have serious health consequences for patients undergoing such therapies. Unfortunately, identifying the epitopes responsible for inducing these off-target toxicities using experimental methods is costly, time-consuming, and limited to a restricted subspace of sequences, since probing the entire space experimentally is practically unfeasible. Here, we present ArdImmune Tox - a computational tool capable of evaluating potential off-target toxicities by leveraging recent advances in computational immunology and Artificial Intelligence (AI). b) Brief description of pertinent experimental procedures Given a target peptide and its associated HLA type, a large collection of putative off-target epitopes (OTEs) having up to a predefined number of amino acid differences with respect to the original peptide, is generated computationally. Putative OTEs are then filtered according to their predicted probability of HLA presentation. Then, the probability that each amino acid interacts with the TCR is evaluated for each OTE and the TCR-facing residues are compared with the corresponding ones in the target peptide, based on selected physico-chemical descriptors. Next, the genomic position of each OTE is annotated and used to extract frequent variants from a dataset encompassing approximately 76,000 genomes. Performing this step, several alternative OTE variants are obtained. The expression patterns (both related to mRNA and protein) of OTEs are reported, as highly expressed off-targets are more likely to induce toxicity. c) Summary of the data (equivalent to results) We evaluated the predictive performance of ArdImmune Tox on three OTEs obtained from the literature, all having experimentally confirmed off-target toxicity. In all cases, our method correctly identified the OTEs ranking them among the two highest scoring potential off-target peptides. We compared our results with those produced by other methods in which the OTEs were positioned much lower in the rank list or were missing altogether. d) Statement of the conclusions
Our results suggest that ArdImmune Tox represents an effective computational approach for the identification of peptide- based off-target toxicity and cross-reactivity of T lymphocytes, with potential applications in the development of immunotherapies and in the study of molecular mimicry. We believe ArdImmune Tox may help improve the safety profile of epitope-based cancer immunotherapi
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-110
Tumor specific antigens derived from non-mutated non-coding regions of the genome can be targeted by T-cell receptor- transgenic T cells. A. Schleicher1, A. Kuhlenkamp1, Q. Zhao2, K. Vincent2, C. Perreault2, S. Milosevic1, T. Franceschetti1, D. Sommermeyer1 1Medigene Immunotherapies GmbH, Planegg-Martinsried, Germany, 2Université de Montréal, Montreal, Canada
Text T cells genetically modified with a TCR can mediate the elimination of cancer cells in patients. A main challenge for a successful therapy is the identification of immunogenic antigens that are specific for the cancer cells. Significant efforts are being made to identify novel tumor specific antigens (TSAs), investigating not only the known proteome, but also non-coding regions of the genome. We identified 44 TSAs derived from the translation in canonical and non-canonical reading frames of non-mutated non-coding genomic regions like introns, intergenic regions and 5’- and 3’-untranslated regions, by comparing the MHC-presented peptidome and RNA expression in tumor and healthy tissues. These TSAs are shared among patients and solid tumor types, thus representing ideal targets if they are recognized by T cells. The immunogenicity of 44 TSAs in the context of five common HLA types was analyzed. Blood samples were not available to assess T cell responses of patients to these diverse pHLA complexes, therefore we used our established high throughput functional screening platform employing cells of partially HLA-matched healthy donors. Dendritic cells were transfected with minigene constructs harboring the TSA sequences and co-cultured with autologous CD8+ T cells. Reactive T cells were enriched using the activation marker CD137, sorted as single cells and expanded. TSA-reactive clones were identified by measuring cytokine release upon co-culture with TSA- positive and -negative cell lines. Using this procedure, ten immunogenic TSAs derived from a variety of non-coding regions were identified; including at least one immunogenic TSA for each of the five analyzed HLAs. TCR chain sequences of TSA-reactive clones were identified by NGS, cloned in a retroviral vector and transduced into CD8+ T cells. The TSA-specificity of more than 20 tested TCRs was confirmed after expression in T cells through recognition of TSA-peptide-loaded cells. In addition, internally processed and presented TSAs expressed from minigene constructs were recognized by most of the TCR-transgenic T cells. TCRs showing positive results during these first analyses were screened for the recognition of tumor cell lines from selected cancer indications. TCR-transgenic T cells released cytokines in response to ovarian cancer cells confirming TSA expression in these cells. In conclusion, our high throughput screening approach successfully identified ten immunogenic TSAs and several TCRs specific for these non-mutated TSAs representing a key tool in the development of TCR-based cancer immunotherapies targeting this novel class of TSAs. The high relevance of the identified TCRs was confirmed by the specific recognition of tumor cells.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-111
T cells expressing a highly potent PRAME-specific T cell receptor equipped with a PD1-41BB switch receptor show a favorable preclinical safety profile, strong anti-tumor reactivity and superior fitness N. Sailer, I. Fetzer, M. Salvermoser, D. Brechtefeld, M. Bürdek, K. Mutze, M. Braun, S. Wilde, D. Sommermeyer Medigene Immunotherapies GmbH, Planegg-Martinsried, Germany
Text The success of immunotherapies with T cell receptor (TCR)-modified T cells (TCR-Ts) depends on both the choice of a target antigen well expressed in tumors with limited or no expression in normal tissues and the selection of a TCR with high antigen specificity and sensitivity. An additional challenge in the treatment of solid tumors is the immunosuppressive tumor microenvironment (TME) that negatively influences efficacy, fitness and persistence of TCR-Ts. In addition to inhibitory cytokines and deprivation of essential metabolic factors, T cells face the inhibitory checkpoint PD-1/PD-L1 axis that reduces T cell infiltration and causes their exhaustion. Thereby, new strategies are needed to equip TCR-Ts with traits to overcome the inhibitory TME. The cancer/testis antigen PRAME is well expressed in several solid cancers and normal tissue expression is mainly limited to testis, providing a relevant antigen for immunotherapies with TCR-Ts. TCRs with high specificity and sensitivity for HLA-A2-restricted PRAME-derived peptides were isolated from non-tolerized T cell repertoires using high-throughput TCR generation and characterization processes, yielding a lead TCR selected from among 30 unique specific TCRs by multi-parameter screening for specificity and activity. To enhance clinical efficacy, a chimeric receptor consisting of the extracellular domain of PD-1 and the intracellular co-stimulatory domain of 4-1BB was developed to turn an inhibitory PD-1-PD-L1 interaction into additional T cell co-stimulation. Co-expression of the PD1-41BB switch receptor with the PRAME-TCR on TCR-Ts led to enhanced proliferation, cytokine release and cytotoxicity in response to HLA-A2/PRAME/PD- L1-positive tumor cell lines compared to TCR-Ts with the TCR alone. Superiority of TCR-Ts co-expressing PD1-41BB was obvious with repeated exposure to tumor cells, showing increased fitness upon chronic antigen stimulation. A high specificity profile was demonstrated with lack of recognition of mismatched peptides, no cross-recognition of common allogeneic HLAs, nor any off-target toxicity for a panel of normal cells. Even when PD-L1 was overexpressed on target cells, TCR-Ts with PD1-41BB maintained the favorable preclinical safety profile of TCR-Ts with the PRAME-TCR alone. In summary, we combined a TCR of high natural anti-tumor reactivity for an HLA-A2-restricted PRAME- epitope with a chimeric PD1-41BB switch receptor that enhances T cell function and counteracts checkpoint- mediated dysfunction. This is a promising strategy to develop more effective TCR-T drug products for the treatment of solid cancers.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-112
Automated ex vivo isolation, transduction and expansion of CD137 positive tumor-reactive T cells on the CliniMACS Prodigy® B. Heemskerk, C. Völzke, L. Ehrhardt, E. Criado-Moronati, P. Maul, C. Wenzel, A. Kaiser, I. Hardy, A. Dzionek Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
Text Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) have shown remarkable results in patients with metastatic melanoma but only a small fraction within the TIL population reacts against the tumor. Therefore, enrichment of tumor-specific T cells and subsequent ex vivo expansion may improve the efficiency of ACT therapies. In addition, tumor-reactive T cells (TRTs) circulating in the blood have been found in low frequencies, which represents a challenge for their isolation, but also an advantage over TIL therapy since blood is a more accessible source than tumor excisions. Another drawback of ACT is the rapid expansion protocol (REP) that constitutes a highly laborious process. We developed an automated large scale ex vivo T cell isolation and expansion procedure in order to simplify the manufacturing of TRTs for ACT. To allow for genetic modifications of TILs/TRTs, the process includes an option for transduction of the isolated cells. The CliniMACS Prodigy is a controlled system that allows magnetic cell separation and cell culture under GMP conditions in a closed system. We have optimized an automated process that allows the cultivation of single-cell suspensions of tumor digest or peptide-stimulated leukapheresis, followed by isolation of T cells expressing the activation marker CD137 using clinical grade reagents. Subsequently, the tubing set is cleaned and the positive fraction can be expanded and potentially transduced in the CentriCult Unit after addition of MACS GMP CD3 pure, excess of irradiated feeders, and high doses of MACS GMP Recombinant Human IL-2. We used tumor digests as well as healthy donor leukapheresis and obtained high purities and optimal recovery of antigen-reactive CD137-positive T cells following stimulation (using viral or shared antigen MACS GMP PepTivators in the case of healthy donor leukapheresis). The isolated fractions (as low as 2x104 up to 2x106 T cells) could be expanded using the conditions mentioned above in an automated fashion in TexMACS GMP medium with 3% AB serum and IL-2; optimal shaking and feeding strategies were defined using an activity matrix that allows for high flexibility regarding culture parameters. Expansions ranged from ~1,000 to 17,000 fold, which mainly depended on the starting cell number. In summary, these data provide proof of concept for the isolation and expansion of antigen-reactive T cells from tumor digest or peripheral blood in a closed, automated manner using the GMP-compliant CliniMACS Prodigy platform, allowing for the desired efficiency, simplicity and automated production of cellular products for ACT therapies against cancer.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-113
Ex vivo expanded NK cells show potent anti-tumour activity against melanoma A. van Vliet, A.-M. Georgoudaki, D. Vodegel, D. Steenmans, J. Spanholtz Glycostem, research, Oss, Netherlands
Text Current therapies for metastatic melanoma fail to cure patients since they develop resistance to the treatments. In addition, immune infiltrates are often found in melanoma, but inactive due to the immunosuppressive environment. Taken this together, it makes melanoma a good target candidate for adoptive cell therapies. We investigated whether melanoma is a good target for our stem cell ex vivo generated Natural Killer (NK) cell product GTA002 and explored the killing mechanisms against melanoma. CD34+ hematopoietic stem cells were isolated from umbilical cord blood, expanded and differentiated into active NK cells in vitro. Cytotoxic capacity of the NK cells from 9 different donors were determined against a panel of 9 different human melanoma cell lines during a 5-hour and a 20-hour co-culture assay with an E:T ratio of 1:1. In parallel, degranulation together with intracellular levels of perforin and Granzyme B, as well as production pro-inflammatory cytokines IFNγ and TNFα were measured in the 5-hour co-culture using flow cytometry. NK cell cytotoxic responses were observed towards all 9 melanoma cell lines in a 5-hour co-culture, with varying killing susceptibility ranging from 7.8% to 40.2% killing, depending on the melanoma cell line. The 20-hour co-culture assay showed only a small increase in killing, suggesting that the NK cells exert quick cytotoxic response in the first hours. Intracellular levels of perforin and granzyme B in NK cells were decreased already after 5 hours of co-culture with the melanoma cell lines compared to no-target controls, indicating release of lytic granules quickly after target engagement. Increased levels of IFNγ and TNFα were observed in NK cells after co-culture to varying degrees depending on the melanoma cell line. Interestingly, the relative cytotoxic performance of an individual NK cell donor was consistent throughout the melanoma target cell panel. However, we still observed differences in killing between melanoma cell lines, suggesting that this could be attributed to different expression profiles of NK cell receptor ligands on the target cells. Therefore, detailed phenotypic profiling for both NK cell activating and inhibitory receptors as well as for ligands expressed on the melanomas is currently on-going. Our data show that GTA002 NK cells exert efficient anti-tumour activity against melanoma cell lines in vitro. In order to better understand the differences in susceptibility to NK cell mediated killing between the different melanoma cell lines, we will further investigate the involvement of different killing mechanisms taking into account the contribution of the individual ligand expression profile of melanomas to NK cell activation.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-114
Novel TIL stimulator cocktail improves TIL product and shortens expansion time. C. Friese, C. Heeke, N. Kirketerp-Møller, S. Færch, M. Juul Nielsen, A. Hey, U. Cordes Cbio A/S, Søborg, Denmark
Text Adoptive cell transfer (ACT) with autologous tumor-infiltrating lymphocytes (TILs) has proven to be one of the most successful immune therapy modalities with overall response rates of around 50% in patients with malignant metastatic melanoma including complete responses in up to 20% of the patients. Current protocols combine a first expansion of TILs from tumor fragments or tumor digest with high-dose IL- 2, followed by further expansion with a rapid-expansion-protocol (REP) using allogeneic feeder cells, αCD3 and IL-2. Following this protocol, TIL production takes 4-7 weeks, and many patients deteriorate before they can receive therapy. With success rates of TIL expansion ranging from 70-90%, a TIL product cannot be generated for every patient. Furthermore, clinical response to TIL therapy is lower in other solid tumors such as Ovarian Cancer, likely due to a lower number of expanded TILs and lower frequencies of tumor reactive CD8+ T cells. Therefore, there is a clinical need for improvement of current TIL expansion protocols to make this therapy available to more cancer patients. In this project, the addition of various TIL stimulators and the use of novel culture vessels have been tested in T cell expansion from tumor fragments of patients with various solid tumors. TIL expansion time, viability, cell number, composition and phenotype were compared to TILs expanded following the standard protocol. Using this novel culture protocol, success rates of expansion across all tumor types increased from 70% to >95%. Additionally, significantly higher frequency and total numbers of viable CD8+ T cells per fragment were obtained compared to standard expansion with IL-2 alone. The majority of these CD8+ T cells exhibit an effector memory phenotype with similar levels of activation markers but elevated levels of exhaustion markers. This could be caused by the ability of the TIL stimulators to reinvigorate exhausted T-cells from the tumor fragments in the first expansion step leading to an activated T cell product with potentially higher anti- tumor activity. Overall, the initial expansion time could be reduced from 2-5 weeks to 10-14 days and the rapid expansion time from 2 weeks to 8-12 days. Thus, the total culture time was shortened from 4-7 weeks to 18-26 days. With this study, we show that by adding a cocktail of different TIL stimulators, we can shorten expansion time while simultaneously improving the characteristics of the TIL product.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-115
Baboon envelope pseudotyped lentiviral vectors: a highly efficient tool to generate CAR-NK cells for clinical use M. Sohmen1, S. Müller1, J. Kostyra1, A. Mekes1, M. Nitsche1, C. Zhang1, R. Pfeifer1, R. Bari2, W. Leung3, N. Möker1 1Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany, 2Lentigen Technology Inc., Gaithersburg, United States, 3Miltenyi Biomedicine Inc., Gaithersburg, United States
Text Clinical evidence has shown that natural killer (NK) cells hold the promise for successful adoptive immunotherapy for cancer. Regulated by their germ-line encoded activating and inhibitory receptors, NK cells can recognize and eliminate tumor cells rapidly without prior sensitization. To further enhance their anti- tumor efficacy, NK cells can also be modified by chimeric antigen receptors (CARs). One of the major obstacles to use NK cells in immunotherapy is the lack of an efficient gene transfer method for primary NK cells. Viral gene delivery to primary NK cells has always proven challenging and less efficient than other cells of the hematopoietic system. We developed a platform for the clinical grade manufacturing of CAR NK cells using baboon envelope glycoprotein pseudotyped lentiviral vectors for highly efficient transduction. This was transferred to an automated process for CAR NK cell generation under good manufacturing practice (GMP)-compliant conditions in a closed system by using CliniMACS Prodigy device. The process covers the complete procedure, including separation, activation, gene modification and expansion/cultivation. High purity of NK cells (mean 93%) could be achieved with CD3-positive cell depletion followed by CD56- positive cell enrichment. Purified NK cells were activated, transduced and cultivated with a feeder cell-free protocol using NK MACS GMP Medium. After 14 days of culture a mean transduction efficiency of 49.8% CAR+ cells was achieved, with a stable CAR expression over the whole cultivation period. The CAR NK cells generated in this system show highly specific and efficient cytotoxic activity. In summary, we enabled efficient transduction of NK cells in a closed GMP compatible system within a novel fully automated process including NK purification and cultivation. The high level of automation enables standardized, consistent and operator independent genetic engineering of NK cells for therapeutic applications.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-116
Improved Method for Culturing TILs From Fine Needle Aspirate Biopsies N. Kirketerp-Møller, C. Friese, C. Heeke, U. Cordes Cbio A/S, Copenhagen, Denmark
Text Adoptive cell therapy (ACT) using ex-vivo expanded autologous tumor-infiltrating lymphocytes (TILs) has been shown to mediate complete regression of some human cancers such as metastatic melanoma. The standard method for TIL expansion involves two steps, where IL-2 is added to tumor fragments in a first step followed by a second rapid expansion protocol (REP) using allogeneic feeder cells, anti-CD3 and IL-2. However, widespread use of the TIL technology across cancer types has been limited due to the lower success rate of the standard expansion protocol and lower clinical response rates in other cancers than metastatic melanoma. In addition, the standard protocol requires 1-3 cm3 of tumor tissue as starting material, cut into at least 50 fragments to increase the likelihood of successful ex-vivo TIL expansion. This has excluded patients with in-operable or hard-to-operate tumors from the TIL technology. In this study, we have developed a novel method to expand TILs from tumor fragments by adding a cocktail of TIL stimulators to the first expansion step. Here we demonstrate that we can generate successful TIL products from significantly smaller tumor fragments down to the size of a fine needle aspirate (FNA) biopsy. Tumor tissue from five different cancer types including metastatic melanoma, head and neck-, ovarian-, cervical-, and colorectal cancer were used. By using the next generation of culture vessels (G-Rex) and a new cocktail of T-cell stimulators, we managed to successfully expand 77% (n=18) of the TILs from 5 small fragments with a total starting volume of only 0.5-5 mm3 tissue. In comparison, using the standard TIL protocol, only 3% (n=29) of the cultures were successful.The capability to grow TIL cultures from FNAs opens for several new ways to use the TIL therapy in treating patients. First of all, FNA biopsy is a much less invasive intervention than surgery. In addition, sampling tumor tissues from multiple lesions could potentially facilitate the expansion of a more heterogenous TIL product. Last but not least, patients that progress with a new lesion after TIL therapy could be retreated with a new batch of TILs grown from that lesion potentially providing the patient with another chance of combatting the disease. In conclusion, we have improved the method of culturing and activating TILs and were able to successfully grow TILs from as little as 1% of the tissue compared to the standard TIL protocol in a range of different cancer types. The reduction in starting material means that cultures can be started from a FNA biopsy, which enables the TIL technology to expand into hard-to-operate or inoperable tumors whilst generally reducing the invasiveness of the extraction procedure.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-117
Cytokine-producing Tumor-infiltrating lymphocyte products can be effectively generated from pre-treated, late stage non- small-cell lung cancer (NSCLC) lesions R. de Groot1, 2, S. Castenmiller1, 2, A. Guislain1, 2, K. Monkhorst3, K. Hartemink3, E. Smit3, J. Haanen3, M. Wolkers1, 2 1Sanquin Research, Hematopoiesis, Amsterdam, Netherlands, 2Oncode Institute, Utrecht, Netherlands, 3Netherlands Cancer Institute/Antoni van Leeuwenhoek hospital, Amsterdam, Netherlands
Text Non-small cell lung cancer (NSCLC) is the second most occurring type of cancer. Because of the high mortality rate with the current treatment regimens, novel therapeutic approaches are warranted. The high mutational rate of the tumors, and the high level of T cell infiltrates should render NSCLC patients eligible for autologous T cell therapy. We recently showed that tumor infiltrating lymphocytes (TILs) from treatment-naive, stage I/II NSCLC tumors can be effectively expanded and reprogrammed into a tumor-reactive T cell product for adoptive T cell therapy. Whether this promising finding translates into late stage NSCLC patients, and whether pre-treatment regimens and/r the location of metastatic lesions influences the generation and quality of tumor-reactive TIL products is not known, yet a pre-requisite for bringing this treatment regimen to the clinic. We here analysed TILs from metastastic lesions isolated from different locations, and from patients with different pre-treatment regiments. The immune infiltrates from metastatic NSCLC lesions (n=25) and in particular the CD3 compartment did not substantially differ from early stage primary NSCLC lesions (n=25). Furthermore, all metastatic lesions contained PD1hiand CD39+ T cells, which is indicative for the presence of tumor-reactive T cells. However, the percentage of regulatory T cells was substantially higher in metastatic lesions. Nevertheless, with the clinically approved TIL expansion protocol, we reached a similar expansion rate as described for early stage NSCLC tumors and metastatic melanoma lesions, demonstrating thatobtaining the required cell numbers to generate TIL products for the clinic is feasible. Importantly, the TIL products were not only functional but the majority also displayed anti-tumoral activity, as determined by cytokine production upon exposure to tumor digest. In summary, our study shows that irrespective of the pretreatment, metastatic NSCLC lesions can provide the source to generate tumor-reactive TIL products for the clinic. We are therefore currently preparing a clinical trial to test the feasibility of autologous T cell therapy as treatment for metastatic NSCLC patients.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-118
Inducible Medigene T cell receptor (iM-TCR): Controlled cytotoxicity of tumor-specific TCR-modified T cells with improved avidity through control of TCR surface expression A. Turqueti Neves1, A. Acs1, J. Ogonek1, B. Loesch1, D. J. Schendel2, S. Milosevic1 1Medigene Immunotherapies GmbH, a subsidiary of Medigene AG, Technology and Innovation, Planegg- Martinsried, Germany, 2Medigene Immunotherapies GmbH, a subsidiary of Medigene AG, Planegg- Martinsried, Germany
Text Several approaches employing adoptive cell transfer (ACT) of patient lymphocytes have been tested with clinical success. These include ex vivo genetic modification of patient lymphocytes with chimeric antigen receptors (CAR-T therapy), ex vivo expansion of patient tumor infiltrating lymphocytes (TIL therapy) and genetic transfer of tumor antigen-specific T cell receptors in patient lymphocytes (TCR-T therapy). Although TCR-T immunotherapy can eliminate tumor cells, the TCRs used for genetic modification of patient lymphocytes can, on occasion, also unexpectedly recognize healthy tissues and be responsible for life- threatening symptoms. This has been seen in several instances with TCRs specific for the MAGE-A3 antigen. Inducible Medigene TCRs (iM-TCRs) are modified so that full control of TCR surface expression can be achieved and thereby cytotoxicity against tumor cells can be allowed while potential unwanted toxicity against normal cells can be controlled if needed. By introducing specific mutations in the TCR constant alpha and beta regions we achieved an impairment of their pairing capacity while keeping the interaction with all CD3 subunits intact. We engineered both constant chains with truncated estrogen receptor domains at the C-terminus, which allow for fast dimerization and membrane localization of the TCR after induction with Tamoxifen or Endoxifen. Our results indicate that the engineered cells rapidly express the TCR after induction with the dimerizing drug. Already low doses of Endoxifen within clinical range can induce enough TCR membrane expression to drive specific recognition of pMHC and T cell response comparable to or even superior to the wild type version of the TCR. Importantly, upon removal of the drug the TCR is rapidly downregulated, which comes with prompt incapacity of the T cell to recognize antigen and secrete cytokines. The genetic transfer of tumor antigen-specific TCRs in the form of iM-TCRs enables the risk of off-tumor reactivity of tumor-specific TCR-modified T cells to be reduced through tight control of TCR surface expression. Furthermore, engineering TCR-Ts with the use of iM-TCRs adds an intrinsic control mechanism to ACT that is independent of expression of additional genes so that every modified T cell that is transferred can be turned on or off.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-119
Preliminary report on a Purified CD19 targeted Chimeric Antigen Receptor T-cell therapy for Aggressive B-cell Malignancies N. Hossain1, P. Stiff1, G. Scurti2, T. Moore3, A. Dalheim3, S. Quinn3, R. Ochoa4, K. Pilman4, S. Tsai1, P. Hagen1, S. Smith1, W. Adams2, 5, M. Nishimura3 1Loyola University Chicago, Medicine - Division of Hematology/Oncology, Maywood, United States, 2Loyola University Chicago, Department of Medical Education, Maywood, United States, 3Loyola University Chicago, Department of Surgery, Maywood, United States, 4Loyola University Chicago, Cancer Center Clinical Trials Office, Maywood, United States, 5Loyola University Chicago, Department of Public Health Sciences, Maywood, United States
Text Growing experience with CD19 chimeric antigen receptor (CAR) therapy has highlighted challenges, including toxicities and non-durable responses. One theory is that side effects are related to infusion of impure CAR-T cells, typically range from 10-60% purity. The delivery of substantial numbers of untransduced T cells may increase toxicity due to inclusion of potential activated autoreactive T cells and reduce longevity due to competition of CAR T cells with untransduced T cells for the T cell niche. To explore the hypothesis that a purified CAR-T cell infusion might be associated with fewer toxicities, we developed a novel approach to produce a highly purified chimeric antigen receptor product by leveraging an incorporated modified CD34 cassette fused to our CD19 CAR. We validated our production process that allows for purification by cell selection technologies of transduced T-cells using our truncated non-signaling CD34t tag, generating products that are generally >90% CAR-positive T-cells. To date, we have treated four patients with a fixed dose of fresh, non-cryopreserved, enriched CD19 CAR T-cells (1x106/kg of calculated adjusted body weight for the first three patients and 1.5x106/kg of calculated adjusted body weight for the fourth patient) for their relapsed/refractory large B-cell lymphomas after lymphodepletion with standard Fludarabine/Cyclophosphamide. The initial transduction efficacy ranged from 7-19.9% and the final product contained 87.3-96.5% transduced T-cells. For all patients, the infused cells showed encouraging expansion kinetics and persisted at assessment at four weeks after infusion based on tracking using the CD34t tag. All of the evaluable patients had significant tumor regression as assessed by PET/CT approximately 4 weeks after infusion, with a 67% overall response rate (ORR) with 33% complete remission (CR) and 33% partial remission (PR) rate. Importantly, 0% of the patients had clinically significant adverse events, including cytokine release syndrome (CRS) or neurotoxicity (NT) as of last follow up, March, 2021.. To our knowledge, this represents the first report of the successful administration of a highly purified CD19 CAR T-cell product to patients with B-cell malignancies. Our preliminary results support the hypothesis that treatment with a fresh gene-modified T cell product purified using our CD34t tag, results in encouraging clinical responses and minimal toxicity, including cytokine release syndrome, neurotoxicity and prolonged cytopenias. Furthermore, we have demonstrated the ability to produce a standardized, highly purified product even for patients whose T-cells initially have low transduction efficiency.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-120
Optimizing CD70 specific nanobody based CARs for the treatment of solid and liquid malignancies S. De Munter1, 2, A. Van Parys3, G. Goetgeluk1, J. Ingels1, S. Bonte2, 4, M. Pille1, L. Billiet1, H. Jansen1, T. Kerre1, 2, 4, 5, J. Tavernier3, B. Vandekerckhove1, 2 1Ghent University, Department of Diagnostic Sciences, Ghent, Belgium, 2Cancer Research Institute Ghent (CRIG), Ghent, Belgium, 3Orionis, Ghent, Belgium, 4Ghent University, Department of Internal Medicine and Pediatric, Ghent, Belgium, 5Ghent University Hospital, Department of Hematology, Ghent, Belgium
Text Recent clinical trials have proven that adoptive chimeric antigen receptor (CAR)-T cell therapy is a very potent and often curative option in the treatment of advanced B cell malignancies. This strategy uses synthetic chimeric receptors introduced in T cells consisting of an antibody-based extracellular part to recognize specific antigens expressed on the surface of tumor cells and an intracellular part containing (co)stimulatory signals derived from CD3ζ, CD28 and/or 4_1BB. While CAR T cells can be highly functional in hematologic malignancies, efficacy in solid tumors has yet to be demonstrated. The limited therapeutic effect could be the result of various barriers present in the tumor micro-environment. CD70 has been identified as tumor associated antigen for hematological leukemias such as acute myeloid leukemia (AML) as well as different solid malignancies such as clear cell renal cell carcinoma, head and neck squamous cell carcinoma and ovarian carcinoma.
We generated four CD70 binding CARs, in which the antigen binding domain is formed by a CD70 specific nanobody or VHH. The nanoCARs differ in co-stimulatory domains: 4_1BB, CD28, CD28 or 4_1BB plus IL2RB plus a STAT3 binding site (4_1BB:β:ζ* and CD28:β:ζ*, respectively). They also differ in extracellular spacer: CARs containing 4_1BB use the CD8α hinge while CARs containing intracellular CD28 use the CD28 hinge. All four nanoCARs were highly expressed on the cell membrane of retrovirally transduced T cells. We observed differences in cytokine production upon stimulation with a CD70+ cell line which could be translated in vivo. In NSG mice that were subcutaneously inoculated with the CD70+ cell line SKOV3, 4_1BB:β:ζ* CAR-T cells were unable to eliminate the tumor mass but could control tumor growth. The other CARs eliminated the tumor mass. However, when the CAR-T cell dose was lowered, only 4_1BB and CD28 CAR-T could eradicate the tumor in 5 out 5 mice. When the spleens of the mice were analyzed 70 days post CAR T cell injection, we observed significantly higher CAR-T cell numbers in mice treated with 4_1BB CAR-T cells compared to those treated with CD28 CAR-T cells, suggesting a co-stimulation-dependent maintenance of the in vivo CAR T cell population. At the moment, we are generating PDX models of CD70+ malignancies derived from AML, pancreatic and renal carcinomas. This way we will be able to determine the best working nanoCAR and eventually start up a clinical trial.
In conclusion, we did not detect a beneficial effect of an additional costimulatory domain that phosphorylates STAT3 and STAT5 in our model for solid tumors.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-121
Identification of tumor-specific T cell receptors with high protective capacity outside the tumor K. Schober1, A. Purcarea2, S. Jarosch2, D. Busch2 1University Hospital of Erlangen and Friedrich-Alexander-University (FAU) Erlangen-Nürnberg, Institute of Clinical Microbiology, Immunology and Hygiene, Erlangen, Germany, 2Technical University of Munich, Institute of Medical Microbiology, Immunology and Hygiene, Munich, Germany
Text During tumor disease, the composition of the T cell receptor (TCR) repertoire of tumor-specific CD8+ T cells changes in space and over time. Although TCR affinity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells, experimental evidence for this is still scarce. Using a library with TCRs of distinct affinities in a pre-clinical tumor model, we investigated both affinity-dependent tumor protection through monoclonal TCR-engineered T cells and tracked the evolution of defined polyclonal tumor-specific TCR repertoires within and outside the tumor. Further, we analyzed the transcriptional profile and TCR repertoire of human neo-antigen-reactive T cells from the tumor and peripheral blood of a melanoma patient. In both the murine and human setting, we could identify converging biomarkers of T cell receptors with high protective capacity outside the tumor. Deciphering the TCR affinity- dependent trajectory of tumor-specific T cells has major consequences for the design and monitoring of immunotherapies.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-122
Synthetic biology-based therapeutics to target or re-educate glioma-associated macrophages J. de Sostoa1, 2, 3, E. Marinari1, 2, 3, V. Widmer1, 2, 3, V. Dutoit1, 2, 3, D. Migliorini1, 2, 3, 4 1University of Geneva, Brain Tumor and Immune Cell Engineering Laboratory, Geneva, Switzerland, 2University of Geneva, Translational Research Center in Oncohaematology, Geneva, Switzerland, 3Swiss Cancer Center Léman, Lausanne, Switzerland, 4Geneva University Hospital, Department of Oncology, Geneva, Switzerland
Text Glioblastoma (GBM) is an aggressive disease with a life expectancy of less than 24 months with standard of care treatment. It is a highly heterogeneous tumor, whose immunosuppressive tumor microenvironment (TME) is a major determinant of resistance to the therapeutic drugs used in the clinic. Transcriptomic data at single-cell resolution and flow cytometry data reveal that tumor-associated macrophages (TAMs) represent a major component of the tumor bulk and are endowed with an anti-inflammatory pro-tumoral profile. Thus, to target or re-educate glioma-associated macrophages towards a pro-inflammatory phenotype is a potential approach to restore an efficient antitumor immunity. Oncolytic viruses (OVs) represent a promising class of anticancer therapeutics that promote antitumor activity by both selective cancer cell killing and induction of durable immune responses. Here, we propose that TAMs targeted by the oncolytic adenovirus ICO15K can lead to altered macrophage polarization and abolish the TAM-induced immunosuppressive microenvironment. Moreover, preconditioning of the TME with ICO15K can potentially improve effectiveness of subsequent CAR T-cell therapy, leading to overall enhanced tumor efficacy and antitumor immunity. Our preliminary results show that ICO15K is able to infect, replicate and exert a significant cytotoxicity effect in a panel of glioma patient-derived cell lines. In addition, M1 and M2-like macrophages populations, which have been successfully obtained and characterized in vitro, are currently being tested for ICO15K-induced transition from a pro-tumoral M2 towards an M1-like pro-inflammatory phenotype. In parallel, in an effort to directly target TAMs, we aim at generating anti-TAMs CAR T cells. To this aim, we analyzed gene expression data of sorted immune cell populations found in the TME of tissue specimens from non-tumor brain tissue, IDH-mutated, and wild type gliomas to identify potential surface markers that are more expressed in TAMs compared to macrophages in healthy tissue and to other immune cell type populations. We then cross-referenced our hits on a single-cell RNAseq dataset. This exploratory analysis resulted in the identification of five potential macrophage targets for the design of anti-TAM CAR T cells. Altogether, here we present preliminary data of ICO15K-mediated TME modulation strategy and identification of potential specific targets for glioma-associated macrophages. Both strategies could definitely constitute promising therapeutic options for improved GBM patient outcome.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-123
Deciphering cellular communications at single-cell resolution for developing efficient and targeted immunotherapies for solid tumors R. Weingarten1, F. Schulze Bockeloh1, M. Rezaei1, K. Kirchhoff1, H. Kleine-Brüggeney1, B. Altvater2, S. Kailayangiri2, S. Bühren1 1evorion biotechnologies GmbH, Muenster, Germany, 2University Children’s Hospital Muenster, Department of Pediatric Hematology and Oncology,, Muenster, Germany
Text In the tumor microenvironment (TME), cancer cells directly interact with stroma and infiltrating immune cells. These interactions play a decisive role in tumor outgrowth and the control of tumor progression. A deep understanding of the crosstalk (juxtacrine or paracrine) between cancer cells and immune cells within the heterogeneous tumor microenvironment is crucial for developing efficient cancer- immunotherapies and improving cancer diagnostic procedures.
To investigate these cellular communication, we developed a unique microfluidic chip-based technology that enables live-cell interaction analysis in physiologically relevant 3D microenvironment at single-cell resolution. Our novel functional phenomics technology has an unmatched advantage that allows researchers for the first time to perform temporal secretion analysis and link the secretion profile directly to the underlying dynamic cellular interaction. The whole approach is highly parallelized, providing a comprehensive analysis of thousands of cellular interactions and underlying secretion profiles at the same time.
By applying our technology, we have performed a multiparametric analysis of cellular interactions between endothelial cells and breast cancer cells showing time-resolved material transfer between these different cell types. In addition, we analyzed the interaction between CAR-T cells and cancer cell populations at single-cell resolution. This study showed heterogeneities in interactions of CAR-T cells and cancer cells in 3D microenvironment by identifying serial killer CAR-T cells with the underlying cytokine secretion profiles. A deep characterization of the cellular communication between cancer cells and immune cells within the heterogeneous TME by our technology will revolutionize the development of immunotherapies
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-124
Antibiotic Therapy Induced Gut Microbiome Injury Correlates with Decreased Survival After Treatment with CD19. CAR T-Cells V. Blumenberg*1, 2, 3, M.-L. Schubert*4, E. Zamir*5, S. Schmidt5, R. Rohrbach5, D. Bozic4, H. Poeck6, C. Schmidt1, V. Bücklein1, 2, C. Müller-Tidow4, 7, P. Dreger4, M. von Bergwelt1, 3, M. Schmitt4, M. Subklewe1, 2, 3, C. Stein-Thöringer5, 7 1University Hospital LMU Munich, Department for Internal Medicine III, Hematology Oncology, Munich, Germany, 2Gene Center of the LMU Munich, Laboratory for Translational Cancer Immunology, Munich, Germany, 3German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany, 4University Clinic Heidelberg, Department of Internal Medicine V, Heidelberg, Germany, 5German Cancer Research Center (DKFZ), Research Division Microbiome and Cancer, Heidelberg, Germany, 6University Hospital Regensburg, Department of Internal Medicine III, Hematology and Oncology, Regensburg, Germany, 7NCT Heidelberg, Department of Medical Oncology, Heidelberg, Germany
Text The CD19 specific chimeric antigen receptor (CAR) T-cell products Axicabtagene-Ciloleucel (Axi-cel) and Tisagenlecleucel (Tisa-cel) are approved for the treatment of refractory/relapsed (r/r) B-cell precursor ALL (BCP-ALL) and Diffuse Large B-cell lymphoma (DLBCL). Despite high response rates, long term remission is only achieved in a subgroup of patients. Given the importance of the gut microbiome in T-cell based immunotherapies, we hypothesize that the gut microbiome also affects the outcomes of CAR T-cell therapy. Notably, exposure to broad-spectrum antibiotics has been associated to a decreased gut microbial diversity with a loss of beneficial commensals and/or a bloom of facultative pathobionts, which in turn may impair treatment responses in CAR-T cell patients. Patients with r/r BCP-ALL and DLBCL were treated with Axi-cel or Tisa-cel or in-house manufactured CD19-targeted CD28-4-1BB-CD3ζ CAR T-cells. 252 fecal biospecimens from 53 patients were collected and 16S rRNA- and shotgun metagenomic sequencing was performed. Results and clinical metadata were integrated into a patient-centered “hospitalome” including concomitant anti-infective therapy and outcome. Patients receiving anti-infectives up to two weeks prior to CAR T-cell transfusion displayed a significantly lower progression free survival (PFS) compared to patients, who had been treated with antibiotic and / or -mycotic treatment after day 0 (univariate p=0.01, multivariate p<0.001). Using 16S rRNA gene sequencing, we observed that treatment with anti-infectives either before or after CAR-T cell infusion induced a loss of microbial diversity. Associating diversity with clinical outcomes, non-responding patients (NR) showed a lower microbiome diversity at baseline compared to responders (R, p=0.016). Furthermore, lower diversity correlated with lower overall survival (p=0.004) and PFS (p=0.01). Assessment of beta diversity (i.e. variation between samples) demonstrated changes of the taxonomic composition with a mono-domination pattern by Enterococcus species (i.e., rel. abundance ≥ 30% per sample), again facilitated by antibiotic treatment. A network analysis of metagenomic sequencing data revealed beneficial microbial species such as ruminococci or bifidobacteria with a higher abundance in R compared to NR. Broad-spectrum antibiotics drives diversity loss of the gut microbiome in CAR T-cell patients. Decreased alpha diversity is associated with the bloom of facultative pathobionts after CAR T-cell transfusion, particularly Enterococcus species. This analysis of the gut microbial taxa indicates an impact of the species composition on the outcome of CAR T-cell therapy and might enable early microbiome-based patient assessment.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-125
Toggling of NKG2A expression drives adaptive reprogramming and functional specialization of iPSC-derived CD19 CAR NK cells M. Kanaya1, C. Philippon1, A. Cieslar-Pobuda1, F. Cichocki2, M. Sætersmoen1, S. Mahmood3, Q. Hammer4, M. Vincenti1, M. T. Wiiger1, S. Z. Krokeide1, H. J. Hoel1, E. H. Ask1, M. Kosugi-Kanaya1, L. Kveberg1, S. Gaidarova3, R. Bjordahl3, H.- y. Chu3, B. Groff3, M. Denholtz3, J. S. Miller2, T. Lee3, D. S. Kaufman5, J. P. Goodridge3, B. Valamehr3, K.-J. Malmberg1, 4 1Oslo University Hospital, Institute for Cancer Research, Department of Cancer Immunology, Oslo, Norway, 2University of Minnesota, Department of Medicine, Division of Hematology, Oncology and Transplantation, Minneapolis, United States, 3Fate Therapeutics Inc., San Diego, United States, 4Karolinska Institute, Center for Infectious Medicine, Department of Medicine, Huddinge, Stockholm, Sweden, 5University of California San Diego, Division of Regenerative Medicine, Department of Medicine, La Jolla, United States
Text Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against hematological malignancies. A unique benefit of iPSC-derived immune effector cells is the possibility to perform multiple precision editing steps at the single cell level to achieve a homogenous effector cell population tailored to target a desired cancer type and equipped with selected functional properties. These functional edits are superimposed on the innate reactivity of NK cells to stress ligands and MHC downregulation (missing self). The ability of NK cells to sense missing self is based on a functional calibration to self MHC during a process termed NK cell education, the latter being critically dependent on signaling through canonical inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). Whereas the differentiation of NK cells into mature effector cells from iPSCs has been well characterized, the role of natural variation in inhibitory receptor expression and NK cell education remains poorly defined in iNK cells.
We monitored functional responses by a range of genetically engineered iPSC-derived NK cell lines, including iNK-CAR19 cells, against K562, K562.HLA-E and CD19+ Nalm-6 cells. Subset stratification showed that NKG2A+ iNK cells had superior functionality compared to NKG2A- iNK cells across all iNK cell lines tested. CRISPR knock out of b2microglobulin (b2m) in iNK cells revealed that the functional potency of NKG2A+ iNK cells was independent of NKG2A-HLA-E interactions in cis or trans during differentiation. NKG2A+ iNK cells expressed higher levels of Eomes, granzyme B, KIR and DNAM-1, suggesting a more differentiated phenotype that could explain the enhanced functional potency of this iNK cell subset. CRISPR-mediated ablation of NKG2A led to a reduced global functional responsiveness but was associated with increased levels of the activating receptor NKG2C. Interestingly, NKG2C+ iNK cells showed functional specialization with increased IFN-g production in response to receptor ligation, similar to conventional adaptive NK cells.
Our results shed light on the regulatory gene circuits and cellular programs that determine functional potential in iPSC-derived NK cells products. Specifically, our results point to a crucial role for NKG2A-driven acquisition of a mature effector cell phenotype and highlight the need to consider subset diversity and receptor interactions in the design of iNK cell therapy strategies.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-126
Adoptive T cell therapy is complemented by oncolytic virotherapy with fusogenic VSV-NDV in combination treatment of murine melanoma T. Krabbe1, J. Marek1, T. Groll2, K. Steiger2, J. Altomonte1 1Kinikum rechts der Isar, 2. Medizinsiche Klinik und Poliklinik, Gastrlabor IV, München, Germany, 2Technische Universität München, Institut für Pathologie, 81675, Germany
Text With immunotherapeutic approaches gaining ground in the clinic, combination strategies are pursued to target multiple mechanisms of the complex anti-tumor immune response, with the aim of developing a curative treatment regimen. In particular, immune checkpoint inhibitors show great potential and have demonstrated impressive response rates in specific subsets of patients with an existing anti-tumor immune response. Alternative immunotherapeutic approaches, that are able to actively induce an anti-tumor immune response, such as adoptive cell transfer (ACT) and oncolytic virotherapy (OV) have evolved into promising immunotherapeutic agents and shown clinical success in the treatment of leukemia and melanoma, respectively. We investigated the combination effect of the hybrid oncolytic VSV-NDV platform with adoptively transferred antigen-specific T cells. VSV-NDV is a versatile hybrid virus platform based on the vesicular stomatitis virus (VSV) backbone, in which the VSV glycoprotein is replaced by the surface proteins from Newcastle disease virus (NDV). The induction of cell fusion-mediated immunogenic cell death by VSV- NVD provides an enhanced immunotherapeutic platform. Based on the immune-competent B16 melanoma mouse model, we evaluated the immunotherapeutic potential of oncolytic VSV-NDV virotherapy, adoptive T cell transfer and their combination effects in vitro and in vivo. We demonstrate that combination of VSV-NDV treatment has beneficial effects on the suppressive microenvironment through upregulation of MHC-I and maintaining low expression levels of PD-L1 on tumor cells. Combination therapy was able to improve recruitment of T cells to virus-infected tumor cells in vitro. In vivo, we observed substantial delays in tumor growth and evidence of absocpal effects, as well as prolongation of overall and median survival in the B16 model in response to combination therapy administered in a clinically relevant dosing scheme, where mono- therapies show no treatment effect. Our results indicate, that treatment with oncolytic VSV-NDV, combined with adoptive T cell therapy, induces multi-mechanistic and synergistic tumor responses, which supports the further development of this promising translational approach.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-127
CRISPR/Cas9-mediated TβRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro K. Alishah1, 2, R.-H. Voss3, M. Diken2, M. Birtel4, S. Asad1, J. Hadjati5, U. Sahin2, 6, 7 1University of Tehran, Biotechnology, Tehran, Iran, Islamic Republic of, 2TRON–Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH,, Mainz, Germany, 3University Medical Center of the Johannes Gutenberg University Mainz, Research Center for Immunotherapy (FZI), Mainz, Germany, 4Biopharmaceutical New Technologies (BioNTech) Corporation, BioNTech Cell & Gene Therapies GmbH, Mainz, Germany, 5Tehran University of Medical Science, Immunology, Tehran, Iran, Islamic Republic of, 6University medical center (UMC) of the Johannes Gutenberg University, Research Center for Immunotherapy (FZI), Mainz, Germany, 7Biopharmaceutical New Technologies (BioNTech) Corporation, BioNTech Cell & Gene Therapies GmbH, Mainz, Germany
Text Only recently, approved in 2017, it became obvious that CAR T cell therapy could be one of the most promising cancer therapies in lymphoma cases. The innovative idea of combining efficient, sensitive and HLA-independent antigen recognition properties of antibodies and killing mechanism of T-cells made this system extraordinary in detecting and eradicating tumor cells. However, unfortunately, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Lack of suitable tumor specific antigens, poor tumor infiltration of CAR T cells, and the immunosuppressive tumor microenvironment (TME) hinder successful CAR T therapy in solid tumors. Among the immunosuppressive components of TME, cytokines with an inhibitory impact on the immune system such as TGFβ and IL-10 are of great importance: TGFβ is a pleiotropic cytokine, which induces or promotes metastasis and neoangiogenesis and potently suppresses the immune system, secreted by a couple of TME resident cells, and also by tumor cells. It has been shown that overexpressing the dominant negative TGFβ receptor along with PSMA specific CARs in T cells, increase proliferation of these lymphocytes and enhance cytokine secretion. We hypothesized that knocking out the TGFβ receptor gene, could further improve CAR T cell functions in vitro and in vivo. For this purpose, we used the novel genomic editing machinery, CRISPR/Cas9, to knockout the TGFβ receptor II gene. Here in this study, we electroporated Cas9 coding mRNA and O-methyl protected synthetic gRNAs to T cells and we were able to monitor efficient gene knock out by T7 endonuclease assay and sequencing. In the next step we overexpressed ovarian cancer-specific CARs which target the tumor antigen mesothelin or Claudin 6 using in vitro transcribed mRNA. We assessed poly-functionality of these TβRII KO CAR T cells in terms of proliferation, cytokine secretion and cytotoxicity. Our experiments demonstrated that these TβRII KO CAR T cells fully retained their capabilities in killing tumor antigen positive target cells. More intriguingly, these TβRII KO CAR T cells could resist the anti-proliferative effect of exogenous TGFβ in vitro and thus, outperformed wildtype CAR T cells. Noteworthy, no antigen or growth factor-independent proliferation of these TβRII KO CAR T-cells has been recorded. Moreover, we generated evidences that TβRII KO CAR T-cells could appreciably resist the suppressive effect of iTregs on proliferation, while WT CAR T cells failed. In conclusion, genomic knock out of the TGFβRII may become a promising approach in immunotherapy of solid tumors, as it may override one of the key negative regulatory signaling pathways in T-cells.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-128
MHC class II-restricted transgenic T cell receptor therapy targeting mutant capicua transcriptional repressor in experimental gliomas M. Kilian1, 2, 3, M. Friedrich1, 2, K. Sanghvi1, 2, 3, E. Green1, S. Pusch4, 5, A. von Deimling4, 5, W. Wick6, 7, M. Platten1, 2, 8, L. Bunse1, 2 1German Cancer Research Center (DKFZ), CCU Neuroimmunology and Brain Tumor Immunology, Heidelberg, Germany, 2Medical Faculty Mannheim, MCTN, University of Heidelberg, Department of Neurology, Mannheim, Germany, 3Heidelberg University, Faculty of Biosciences, Heidelberg, Germany, 4German Cancer Research Center (DKFZ), DKTK Clinical Cooperation Unit Neuropathology, Heidelberg, Germany, 5Heidelberg University Hospital, University of Heidelberg, Department of Neuropathology, Heidelberg, Germany, 6Heidelberg University Hospital, University of Heidelberg, Neurology Clinic and National Center for Tumor Diseases, Heidelberg, Germany, 7German Cancer Research Center (DKFZ), DKTK Clinical Cooperation Unit Neuro-Oncology, DKTK Clinical Cooperation Unit Neuro-Oncology, Germany, 8Helmholtz Institute for Translational Oncology (HI-TRON), Mainz, Germany
Text Glioma subtypes are recognized by characteristic mutations and show a high degree of resistance to standard therapeutic modalities such as radiotherapy and alkylating chemotherapy. Some of these characteristic mutations have shown to generate immunogenic neoepitopes suitable for targeted immunotherapy. 70% of oligodendrogliomas carry capicua transcriptional repressor (CIC) inactivating mutations. In a screen for potential immunogenic glioma neoepitopes we identified recurrent CIC hotspot mutations at position 215 (CICR215W/Q) expressed in a subset of oligodendrogliomas as an immunogenic major histocompatibility complex (MHC) class II-restricted neoepitope. Peptide-based vaccination of MHC- humanized mice resulted in the generation of robust mutation-specific T cell responses against CICR215W/Q, restricted to MHC class II. Droplet-based single cell T cell receptor (TCR) sequencing from CICR215W-specific T cell lines enabled retrieval of MHC class II-restricted CICR215W-reactive TCRs. By retroviral transduction of T cells, we established a flow cytometry-based testing platform of retrieved TCRs and were able to show the top reactive TCR against CICR215W to be shared between individual mice. Using a newly developed and mutationally defined glioma model in MHC-humanized mice induced by CRISPR- based knockout of tumor suppressor genes, we show that intraventricularly transferred CICR215W-specific TCR-transgenic T cells infiltrate the tumor and exert anti-tumor responses against CICR215W-expressing syngeneic gliomas. The integration of immunocompetent MHC-humanized orthotopic glioma models in the discovery of shared immunogenic glioma neoepitopes facilitates the identification and preclinical testing of HLA-restricted neoepitope-specific TCRs for locoregional TCR-transgenic T cell adoptive therapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-129
High Yield human iPSC-derived monocytes and macrophages are functionally comparable to primary cells D. Cui1, A. Franz1, S. A. Fillon1, L. Jannetti1, T. Isambert2, K. Fundel-Clemens3, H. J. Huber3, C. Viollet3, A. Ghanem1, A. Niwa4, B. Weigle1, S. Pflanz1, 5 1Boehringer Ingelheim, Cancer Immunology and Immune Modulation, Biberach an der Riss, Germany, 2Boehringer Ingelheim, Medicinal Chemistry, Biberach an der Riss, Germany, 3Boehringer Ingelheim, Global Computational Biology and Digital Sciences, Biberach an der Riss, Germany, 4Kyoto University, Center for iPS Cell Research and Application, Kyoto, Japan, 5Boehringer Ingelheim, Venture Fund, Ridgefield, United States
Text Macrophages are pivotal effectors of host immunity and regulators of tissue homeostasis. Understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human iPSC-derived monocytes and macrophages, as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of macrophage biology and implication in human diseases. In this study, we present a fully optimized differentiation protocol of hiPSC-derived monocytes and GM-CSF or M-CSF macrophages. We present characterization of iPSC-derived myeloid lineage cells at phenotypic, functional and transcriptomic levels, in comparison with corresponding subsets of peripheral blood-derived cells. We also highlight the application of hiPSC-derived monocytes and macrophages as a gene-editing platform for functional validation in research, drug screening, and the study also provides a reference for cell therapies.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-130
A T cell receptor targeting a shared neoantigen mediates efficient rejection of patient-derived acute myeloid leukemia in vivo E. Giannakopoulou1, 2, M. Lehander3, S. Virding Culleton3, W. Yang1, 2, Y. Li1, 2, A. Hillen3, M. Bill4, M. Griffioen5, T. Karpanen1, T. J. Gjerdingen1, 2, S. E. W Jacobsen3, 6, 7, 8, P. S Woll3, J. Olweus1, 2 1Oslo University Hospital Radiumhospitalet, Department of Cancer Immunology, Oslo, Norway, 2University of Oslo, K.G. Jebsen Center for Cancer Immunotherapy, Institute of Clinical Medicine, Oslo, Norway, 3Karolinska Institute, Department of Medicine Huddinge, Center for Hematology and Regenerative Medicine, Stockholm, Sweden, 4Aarhus University Hospital, Department of Hematology, Aarhus, Denmark, 5Leiden University Medical Center, Department of Hematology, Leiden, Netherlands, 6Karolinska Institute, Department of Cell and Molecular Biology, Stockholm, Sweden, 7Karolinska University Hospital, Stockholm, Sweden, 8University of Oxford, MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Oxford, United Kingdom
Text Acute myeloid leukemia (AML) is the most frequent leukemia in adults, and adoptive therapy with T cells genetically modified to express immune receptors targeting shared neoantigens could be an attractive treatment option. We identified a TCR reactive to the FLT3 D835Y mutation, a recurrent mutation in AML patients screened for in routine diagnostics, restricted by the frequently expressed HLA-A*02:01(HLA-A2) allele (FLT3mut TCR), by use of our previously described technology to stimulate healthy donor T cells. T cells redirected with the FLT3mut TCR recognized target cells pulsed with mutated but not wild type peptide at concentrations as low as 10-100pM, indicating high functional avidity. No reactivity was observed against a panel of 25 HLA-A2 positive cell lines of different tissue origins and expressing a variety of HLA types, unless the cells were pulsed with the mutant peptide or electroporated with relevant mRNA. FLT3mut TCR T cells successfully recognized and eliminated leukemia cells harboring the relevant mutation in samples from 7 patients, while sparing normal lymphoid cells. No reactivity was observed in response to HLA-A2 patient leukemia cells carrying different amino acids in the same position (WT or D835E) or with mutations in other positions (FLT3/ITD). We next tested in vivo efficacy of the FLT3mut TCR T-cells in treatment of leukemia in mouse models. Mice engrafted with BV173 leukemia cells with induced expression of the D835Y mutation did not show evidence of leukemia at end of experiment (d60) whereas mock TCR treated mice had to be sacrificed by d21 due to high leukemia burden. To investigate the in vivo impact on FLT3 D835Y primary AML cells, mice stably engrafted with FLT3 D835Y primary AML cells from two patients were treated with control or FLT3mut TCR T-cells. For both patients, mutated AML cells could not be detected in the bone marrow of FLT3mut TCR T-cell-treated mice by ddPCR, and endogenous mouse hematopoiesis suppressed by the AML cells was restored. In contrast, the leukemia burden in control mice was high. As not all leukemia-propagating cells might harbor the D835Y mutation, we finally investigated whether the leukemia cells with engrafting potential are killed by the FLT3mut TCR T-cells. Following 48h in vitro TCR-T cell co-culture with patient leukemia cells, cells were injected into NSG mice. No engraftment was observed in mice injected with FLT3mut TCR-treated cells at 26 weeks, while all mock-TCR T cell-treated mice showed leukemic engraftment. In conclusion, we have identified a TCR specific for a peptide encoded by the recurrent FLT3 D835Y mutation presented on HLA-A2 that mediates efficient and specific in vivo rejection of AML harboring this mutation.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-131
Identification and characterisation of neoantigen-specific TCRs from autologous peripheral blood using single cell transcriptome and TCR repertoire sequencing J. Untch1, E. Bräunlein1, F. Füchsl1, S. Jarosch2, N. de Andrade Krätzig3, 4, O. Baranov3, 4, D. H. Busch2, R. Rad3, 4, A. M. Krackhardt1, 5 1Technische Universität München, School of Medicine, Klinik und Poliklinik für Innere Medizin III, Klinikum rechts der Isar, Ismaningerstr. 22, Munich 81675, Germany, 2Technische Universität München, Institute for Medical Microbiology, Immunology and Hygiene, Trogerstr. 30, Munich 81675, Germany, 3Technische Universität München, Institute of Molecular Oncology and Functional Genomics, TUM School of Medicine, Ismaningerstr. 22, Munich 81675, Germany, 4Technische Universität München, Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Einsteinstr. 25, Munich 81675, Germany, 5German Cancer Consortium (DKTK), partner-site Munich; and German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg 69120, Germany
Text Immunotherapy has become a hallmark of anti-tumour therapy, especially fuelled by the success of immune checkpoint inhibitors based on the natural immunogenicity of malignant cells. Targeting neoantigens with specific T cells promises a highly specific and safe approach that can lead to durable regression in various types of cancer. However, since not all patients benefit from standard immunotherapies, it will be important to better understand determinants affecting the recognition of tumour cells. Within a previous project, primary material from a melanoma patient was used to identify mutated peptide ligands by mass spectrometry and in silico prediction. Using tumour infiltrating lymphocytes and peripheral blood we thereby discovered seven novel neoantigen specific T-cell receptors (TCRs) targeting three neoantigens (KIF2CP13L, SYTL4S363F, NCAPG2P333L) and subsequently characterised their anti-tumour properties. Functional analysis demonstrated that all TCRs showed distinct differences in their binding avidities to the mutated peptide-human leukocyte antigen (HLA) complex whilst lacking any reactivity against the wildtype counterpart. However, TCRs with lower functional avidity showed comparable tumour rejection efficacy in vivo, dominant spatial and temporal distribution as well as lower activation patterns upon primary in-vitro stimulation. To gain more insight into the functional relationship between neoantigen and TCR we performed single cell transcriptome and TCR repertoire/VDJ sequencing of CD8+ T cells from this patient. T cells were stimulated with two mutated peptide ligands (KIF2CP13L and SYTL4S363F). We developed a method to quickly enrich PBMCs for peptide reactive TCRs using an autologous dendritic cell assay combined with CD137+-based selection followed by rapid expansion. Enriched cells were restimulated after eleven days with autologous antigen-presenting cells and respective mutated peptides. The 10X Genomics Chromium Single Cell workflow was applied to gain transcriptome and VDJ data from both an unenriched, unstimulated and an enriched, restimulated condition for comparison. Using the new protocol, we were able to detect an enrichment of all previously identified neoantigen reactive TCR clonotypes and comparison showed differences of distinct trancriptomic features associated to their antigen specificity. Furthermore we successfully detected two previously unknown TCRs with specificity to KIF2CP13L based on analysis of clonotype enrichment.In conclusion, using this approach we were able to gain deeper insights into transcriptomic signatures of antigen-dependent T-cell activation. Moreover, this protocol could serve as a sensitive tool for the detection of neoantigen specific TCRs.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-132
CD47 and phosphatidylserine contribute to the interaction between antigen presenting cells and the allogeneic cell-based relapse vaccine DCP-001 H. Zuo1, S. K. Singh1, M.-J. van Lierop1, J. Kaspers1, R. Bos1, A. Kamermans2, H. E. de Vries2, T. D. de Gruijl3, A. M. Kruisbeek1, E. H. Manting1 1DCprime bv, Leiden, Netherlands, 2Amsterdam UMC, Department of Molecular Cell Biology and Immunology, Amsterdam, Netherlands, 3Amsterdam UMC, Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam, Netherlands
Text DCP-001 is a cancer relapse vaccine derived from the DCOne® human leukemic cell line. During manufacturing, DCOne® cells are shifted towards a mature dendritic cell (mDC) phenotype, combining an endogenous tumor antigen repertoire (e.g. WT-1, RHAMM and PRAME) with a mDC costimulatory profile and providing the basis for the highly immunogenic vaccine DCP-001. In a phase I clinical study in acute myeloid leukemia (AML), DCP-001 demonstrated to be safe and to induce multifunctional antitumor immune responses. It has also been reported that DCP-001 induces antitumor immunity against multiple myeloma cells in peripheral blood mononuclear cells (PBMC) from patients and that DCP-001 antigenic material is transferred to host antigen presenting cells (APC), possibly via extracellular vesicles. However, the direct interaction between DCP-001 and host APC has not yet been investigated. To further elucidate the mode of action of DCP-001, we studied the interactions of DCP-001 with human PBMC and immature monocyte- derived DCs (iMoDC) in in vitro co-culture studies. We found that DCP-001 stimulates the secretion of various proinflammatory cytokines (IL-1β, GM-CSF, IFN-γ, IL-2, TNF-α, and IL-6) and chemokines (IL-8 and RANTES) in PBMC. In addition, we demonstrate that DCP-001 is efficiently taken up by iMoDC via direct cell-cell interactions and that this phagocytic process is influenced by “eat-me” and “don’t eat me” signalling pathways. Blocking of the “eat-me” signals calreticulin and phosphatidylserine inhibited the uptake of DCP- 001, whereas blockade of the “don’t eat me” signal CD47 enhanced DCP-001 uptake. Our data suggest a vital role for host antigen presenting cells in the triggering of immune responses upon DCP-001 vaccination. In addition, these data provide a rationale for potential combination therapies based on DCP-001 and inhibitors of the CD47 pathway.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-133
Targeting telomerase with an HLA class II-restricted TCR for cancer immunotherapy. A. Winge-Main1, 2, P. Dillard2, H. Köksal2, S. M. Maggadottir2, S. Pollard2, M. Menard3, M. R. Myhre2, G. Mælandsmoe4, V. A. Flørenes5, G. Gaudernack6, G. Kvalheim2, S. Wälchli2, E. M. Inderberg2 1Oslo University Hospital, Department of Oncology, Oslo, Norway, 2Oslo University Hospital, Department of Cellular Therapy, Oslo, Norway, 3Oslo University Hospital, Department of Radiation Biology, Oslo, Norway, 4Oslo University Hospital, Department of Tumour Biology, Oslo University Hospital, Norway, 5Oslo University Hospital, Department of Pathology, Oslo, Norway, 6Oslo University Hospital, Department of Cancer Immunology, Oslo, Norway
Text T-cell receptor (TCR) -engineered T-cell therapy is a promising cancer treatment approach. Telomerase (hTERT) is overexpressed in the majority of tumors and a potential target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR redirected primary CD4+ and CD8+ T cells demonstrated in vitro efficacy producing inflammatory cytokines and killing hTERT+ melanoma cells in both 2D and 3D settings, as well as ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. We are currently testing Radium-4 in a patient derived melanoma xenograft model. Against NSCLC we have advanced further and are about to perform an mRNA-based clinical trial of hTERT TCR using Radium-4. Since hTERT is a universal cancer antigen and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumours.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-134
A Novel CD22-CAR against B-cell malignancies A. Faane1, N. P. Casey1, B. Caulier1, C. H. Klee1, A. Graczyk-Jarzynka2, K. Fidyt2, M. Krawczyk2, P. Dillard1, M. Winiarska2, E. B. Smeland3, G. Kvalheim1, E. M. Inderberg1, S. Wälchli1 1Translational Research Unit, Section for Cellular Therapy, Oslo University Hospital, Oslo, Norway, 2Department of Immunology, Medical University of Warsaw, Warsaw, Poland, 3Section for Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway
Text Chimeric antigen receptor (CAR) modified T cell therapy has led to great success in treating patients with B cell malignancies. CD19, which is highly expressed in B-cell lymphoma, has successfully been targeted with CAR T-cells and revolutionized the treatment of acute lymphoblastic leukemia (ALL). Despite this success, a subset of patients receiving CD19-targeted immunotherapy relapse due to loss of the targeted antigen or mutation at the antibody recognition site. Thus, there is not only an obvious requirement to target alternative B-cell antigens, but also to diversify the spectrum of epitopes within the same antigen. CD22 is now well accepted as a substitute target for CD19 relapse; one antibody, anti-CD22 clone m971, has so far been widely validated and used in clinic. It has been claimed that the efficiency of this clone was due to its epitope position being located within a membrane-proximal domain. Comparative studies, with a CAR derived from an antibody targeting the very distal domain of CD22, demonstrated the superiority of m971. Here we have compared m971-CAR with a CAR derived from IS7, an antibody targeting an epitope located in a medium to distal position. We first showed that IS7-CAR was active and specific against CD22 positive targets, including primary patient samples. We then performed a set of experiments and observed that while IS7- CAR killed less rapidly than m971-CAR in vitro, it was as efficient in controlling lymphoma xenograft models in vivo. Thus, IS7-CAR is a potential alternative candidate for treatment of refractory B-cell malignancies.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-135
Flow cytometric assays for CAR T cell manufacturing and patient monitoring, involving specific CAR detection reagents, stabilized pre-mixed cocktails, and automated data acquisition and analysis M. Mues, M. Winkels, K. Lange, L. Stiem, E. Janz, S. Biedermann, M. Niemöller, D. Missing, N. Borgelt, T. Holzer, C. Dose, C. Siewert, A. Richter Miltenyi Biotec, Bergisch Gladbach, Germany
Text Unprecedented efficacy in targeting cancer has been demonstrated with CAR T cell therapy. However, the CAR T cell manufacturing process is still highly complex and has extensive demands on personnel and infrastructure. Using a device for automated cell processing, the CliniMACS Prodigy, helps to overcome these hurdles, allowing generation of CAR T cells in a single automated and closed system. However, also the necessary QC procedures and any accompanying CAR T cell phenotyping efforts require robust and reliable assays. Therefore, to streamline the assessment of CAR T cells during manufacturing and patient monitoring, we aimed to establish a set of flow cytometric assays prepared from recombinant antibodies along with CAR detection reagents. These assays were complemented by stabilized pre-mixed cocktails and additional tools for automated data acquisition and analysis, to achieve the highest level of standardization, reproducibility, and convenience.
Our flow cytometric assays were developed for 1) in-process control, QC release testing, and CAR T cell phenotying during cell manufacturing, and 2) for determination of CAR T cell persistance and phenotyping during patient immunomonitoring. Currently already used in clinical trials, these assays allow to determine the general immune cell composition, the CAR transduction efficiency, and functional CAR T cell phenotyping like differentiation, activation, or exhaustion status. To further improve assay standardization, to simplify and speed up the IPC/QC process, and to reduce risks from manual handling, we developed stabilized pre-mixed antibody cocktails for these flow assays, featured in a single-tube format. In addition, so-called Express Modes were programmed that allow an automated sample acquisition and analysis on MACSQuant Analyzers. These Express Modes feature predefined experiment settings, automate the sample measurement, and apply a fully automated gating strategy with computational gate adjustment. In conclusion, elaborate flow assays specifically designed for CAR T cells, run with specific CAR detection reagents and high-quality recombinant antibodies in a stabilized pre-mixed format, along with fully automated flow analysis, provide a robust assessment for cell manufacturing and patient immunomonitoring. This will help with establishing complex individualized therapies and will enable us to understand in greater detail the phenotypic changes occuring throughout the life time of a CAR T cell.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-136
Targeting KRAS mutations with MHC class II-restricted TCRs for the treatment of solid tumors N. Casey1, P. Dillard1, S. Pollman1, P. Vernhoff1, G. Gaudernack2, G. Kvalheim1, S. Wälchli1, E. M. Inderberg1 1Oslo University Hospital-The Norwegian Radium Hospital, Translational Research Unit, Department of Cellular Therapy, Oslo, Norway, 2Institute for Cancer Research, Oslo University Hospital, Department of Cancer Immunology, Oslo, Norway
Text T-cell receptor (TCR) redirected T-cell therapy is considered as the next generation of care for the treatment of numerous solid tumors. KRAS mutations are driver neoantigens that are expressed in over 25% of all cancers, and thus are regarded as ideal targets for Adoptive Cell Therapy (ACT). We have isolated four KRAS-specific TCRs from a long-term surviving pancreatic cancer patient who had been vaccinated with a mix of mutated KRAS peptides. The sequence of these TCRs could be identified and expressed in primary cells. We demonstrated stable expression of all TCRs, as well as target-specific functionality, when expressing T cells were co-incubated with target cells presenting KRAS peptides. In addition, these TCRs were all partially co-receptor independent since they were functional in both CD4 and CD8 T cells, thus indicating high affinity. Interestingly, we observed that certain TCRs were able to recognize several KRAS mutations in complex with their cognate MHC, suggesting that in these cases, the point mutations were less important for the human leukocyte antigen (HLA) binding and TCR recognition. Finally, we demonstrated that these peptides were indeed processed and presented, since HLA-matched antigen-presenting cells exogenously loaded with KRAS proteins were recognized by TCR-transduced T cells. Taken together, our data demonstrate that KRAS mutations are immunogenic for CD4 T cells and are interesting targets for TCR- based cancer immunotherapy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-137
Mutation-specific T cell receptors from tumor-bearing hosts display optimal functionality L. Rosenberger1, L. Hansmann2, 3, 4, K. Borutta1, V. Anastasopolou1, 3, 4, S. Wolf5, 6, X. Feng7, G. Cao7, J. Huang7, E. Strønen8, 9, T. Kato10, P. Yin Yew11, Y. Nakamura12, J. Olweus8, 9, G. Willimsky1, 3, 4, T. Blankenstein1, 13, H. Schreiber5, 6, 14, M. Leisegang1, 3, 4, 6 1Charité-Universitätsmedizin, Institute of Immunology, Berlin, Germany, 2Charité-Universitätsmedizin, Department of Hematology, Berlin, Germany, 3German Cancer Consortium (DKTK), Berlin, Germany, 4German Cancer Research Center (DKFZ), Heidelberg, Germany, 5The University of Chicago, Department of Pathology, Chicago, United States, 6The University of Chicago, David and Etta Jonas Center for Cellular Therapy, Chicago, United States, 7The University of Chicago, The Pritzker School of Molecular Engineering, Chicago, United States, 8Oslo University Hospital Radiumhospitalet, Institute for Cancer Research, Oslo, Norway, 9University of Oslo, K.G. Jebsen Center for Cancer Immunotherapy, Institute of Clinical Medicine, Oslo, Norway, 10Osaka University Graduate School of Medicine, Department of Urology, Osaka, Japan, 11The University of Chicago, Center for Personalized Therapeutics, Chicago, United States, 12Japanese Foundation for Cancer Research, Cancer Precision Medicine Center, Tokyo, Japan, 13Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany, 14The University of Chicago, Committee on Cancer Biology and Committee on Immunology, Chicago, United States
Text Mechanisms of central and peripheral tolerance prune the repertoire of T cells directed against self-antigens. The impact of prolonged exposure to neoantigens on specific T cells in cancer patients, however, remains unclear. We developed a mouse cancer model that allows for tetracycline-controlled gene expression of the H-2Kb-presented neoantigen mp68 in established, progressively growing tumors. Expression of mp68 was induced 3 weeks after transplantation of tumor cells to avoid T cell priming due to the inflammatory environment after subcutaneous injection. CD8+ mp68-specific T cells were detectable in the population of tumor-infiltrating lymphocytes (TILs, approx. 1%) when tumors were analyzed 3, 5, and 12 weeks after inducing antigen expression in tumor cells. Sequences of T cell receptors (TCRs) obtained by analysis of single mp68-specific TILs were diverse and functionally comparable to each other and to TCRs isolated from tumor-free mice that generated mp68-specific T cells after immunization. Thus, chronic exposure to neoantigen that lasted for up to 3 months had no curtailing effect on the functionality of TCRs obtained from TILs of tumor-bearing mice. These results were confirmed by experiments with human TCRs recognizing the HLA-A2-presented neoantigen CDK4-R24L. To identify even small differences in functionality, we examined TCRs derived from patients, healthy third-party donors, or humanized mice in a syngeneic mouse cancer model of adoptive T cell therapy. The patient-derived TCRs were able to destroy established tumors with similar efficiency as TCRs derived from a tumor-free environment. The results provide important evidence that neoantigen-specific TCRs when isolated from cancer patients can be used for T cell therapy without compromising efficacy.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-138
Adoptive T cell therapy during chemotherapy with or without Interferon-a in patients with recurrent ovarian cancer. E. Verdegaal1, M. Visser1, L. van der Minne1, L. de Bruin1, I. Roozen2, P. Meij3, J. Kroep2, S. van der Burg1 1Leiden University Medical Center, Medical Oncology and Oncode Institute, Leiden, Netherlands, 2Leiden University Medical Center, Medical Oncology, Leiden, Netherlands, 3Leiden University Medical Center, Center for Cellular and Gene Therapy, Leiden, Netherlands
Text Epithelial ovarian cancer (EOC) has a poor prognosis, with a 5-year survival rate of 35%. The clear correlation between T cell infiltration and disease progression in EOC suggests that EOC may be sensitive to adoptive cell therapy with autologous Tumor Infiltrating Lymphocytes (TIL), provided that myeloid derived suppressor cell- and M2 macrophage--mediated immune suppression is reduced. Carboplatin-paclitaxel (CP) chemotherapy can directly kill tumor cells but can also alleviate such immunosuppression, potentially creating a window of opportunity for T cell-based immunotherapy. Furthermore, there is evidence that interferon-α (IFNα), which we use as a mild preconditioning regimen, may additionally support persistence of infused TIL. Based on this, we initiated a phase I/II trial to study the feasibility and safety of TIL administration during CP-chemotherapy with or without IFNα in patients with recurrent platinum-sensitive EOC. TIL were expanded ex vivo and administered two weeks after the second, third and fourth CP-chemotherapy course. The first cohort of six patients were treated with TIL and CP-chemotherapy and IFNα was added in the second cohort for twelve weeks in total, starting one week before the first TIL infusion. TIL could be successfully cultured from tumor samples or biopsies for all included patients. Currently, fifteen patients are on treatment. While treatment with TIL during chemotherapy was safe the addition of IFNα added to chemotherapy-induced toxicity. The data thus far, confirm that CP-chemotherapy alleviates immunosuppression, reflected by reduced plasma IL-6 levels and circulating myeloid cell numbers. The optimal reduction in the myeloid/lymphocyte ratio is obtained at 1-2 weeks after the second CP- chemotherapy. As additional objective, signs of clinical activity are recorded according to RECIST1.1. Clinical activity could not be evaluated in two patients who experienced rapid disease progression precluding completion of treatment with TIL infusions. For the eleven evaluable patients, ten complete or partial responses (91%) and one stable disease were recorded. Interestingly, a progression free survival that exceeded the previous platinum-free interval was observed in at least two patients. These preliminary data suggest additional clinical benefit of the infusion of TIL during CP-chemotherapy. In depth studies on immune modulation by chemotherapy and by TIL plus IFNα, and correlations between phenotypic characteristics of infused TIL and clinical outcome is ongoing. In conclusion, our preliminary data suggest that properly timed TIL infusions during CP-chemotherapy may result in clinical benefit for EOC patients.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-139
Aberrant expression of splicing factors as mechanism of resistance to CART-19 immunotherapy N. Ziegler1, C. Paret1, F. Alt1, S. Attig2, K. El Malki1, N. Lehmann1, L. Roth1, J. Faber1 1Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Zentrum für Kinder- und Jugendmedizin, Mainz, Germany, 2Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Translationale Onkologie & Immunologie, Mainz, Germany
Text Acute lymphoblastic leukaemia (ALL) is the commonest childhood cancer and B-cell ALL (B-ALL) accounts for 80-85% of childhood ALL cases. The prognosis of B-ALL in developed countries has dramatically improved during the last decades with overall cure rates of about 90%. However, the prognosis of children with relapsed or therapy refractory disease remains poor. Chimeric antigen receptor-armed T cells (CART) targeting the B cell antigen CD19 have resulted in an unprecedented complete and durable clinical response in children with chemotherapy-resistant disease, however, unfortunately relapses occur in 10% to 20% of responders. Epitope loss under therapy pressure has been suggested as one mechanism of tumor cells to escape the recognition from CART-19 and may derive from splice variants of CD19 lacking the exon 2 and therefore the epitope targeted by CART-19. Abnormal expression of splicing factors interferes with normal splicing of pre-mRNA, resulting in cancer. Accordingly, deregulation in splicing factors in B-ALL may contribute to resistance to CART-19 therapy. To support this hypothesis, leukemic blasts and normal B cells of pediatric patients were isolated by flow cytometry and expression analysis of different splicing factors was performed by qRT-PCR. The Serine/arginine-rich splicing factor 3 (SRFS3) was one factor being downregulated in leukemic blasts. SRSF3 is generally considered as a proto-oncogene and is overexpressed in various types of cancer. However, specific loss-of-function in some tumor types has also been described. Nevertheless, previous data suggest that splicing of CD19 transcripts leading to the loss of exon 2 is mediated by SRSF3. Our data support the hypothesis that deregulation of SRSF3 in leukemic blasts of some patients can contribute to alterations in CD19 splicing. Understanding the mechanisms of abnormal mRNA processing via SRSF3 and other factors could provide new insights that might support the development of effective therapeutic strategies against CAR-T-resistant B-ALL.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-140
BNT211: A phase I/IIa trial to evaluate safety and efficacy of CLDN6 CAR-T and CARVac-based in vivo expansion to improve treatment of patients with CLDN6-positive advanced solid tumors. C. Koenecke1, A. Mackensen2, J. Haanen3, A. Desuki4, E. Wagner-Drouet4, D. Heudobler5, W. Alsdorf6, P. Borchmann7, E. Wiegert8, C. Schulz9, B. Rengstl9, L. Preußner9, U. Sahin9, Ö. Türeci9 1Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany, 2University Hospital Erlangen, Department of Internal Medicine 5, Hematology/Oncology, Erlangen, Germany, 3Netherlands Cancer Institute, Division of Medical Oncology, Amsterdam, Netherlands, 4University Medical Center Mainz, 3rd Medical Department, Hematology, Oncology and Pneumology, Mainz, Germany, 5University Hospital Regensburg, Department of Internal Medicine III, Haematology and Oncology, Regensburg, Germany, 6University Medical Center Eppendorf, Department of Oncology, Hematology and Bone Marrow Transplantation with Division of Pneumology, Hamburg, Germany, 7University Hospital of Cologne, Department I of Internal Medicine, Cologne, Germany, 8Bexon Clinical Consulting, Upper Montclair, United States, 9BioNTech SE, BioNTech Cell & Gene Therapies, Mainz, Germany
Text BioNTech’s first chimeric antigen receptor (CAR)-T cell product candidate (BNT211) targets the tumor specific antigen Claudin-6 (CLDN6) and was developed in combination with a CAR-T cell Amplifying RNA Vaccine (CARVac). Preclinical studies demonstrated that CARVac treatment leads to in vivo expansion of adoptively transferred CAR-T cells, resulting in improved persistence and superior functionality. In this first-in-human, open-label, multi-center, Phase I/IIa trial, the safety and efficacy of increasing dose levels of CLDN6 CAR-T cells with or without CARVac after lymphodepleting chemotherapy will be assessed. Secondary endpoints are objective response rate, disease control rate and duration of response. A comprehensive biomarker program including assessment of cytokines, tumor markers and CAR-T cell frequency complements the clinical data package. Patients with CLDN6-positive relapsed or refractory solid tumors without further standard treatment options are eligible for recruitment. Dosing of first cohort with starting flat dose of CLDN6 CAR-T cells has already been completed. There were no manufacturing failures. The underlying diseases of the first three patients were ovarian carcinoma, desmoplastic round small cell tumor and yolk sac carcinoma. No acute toxicities occurred after CAR-T cell administration, and clinical manifestation of cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) have not been observed. In total, no dose-limiting toxicities (DLTs) have been reported, neither prolonged cytopenia nor acquired infections. There have been no AEs ≥ grade 3 related to the drug product. Transient and moderate elevation of IL-6 and CRP serum levels was detected. Analysis of CLDN6 CAR-T cell frequency in peripheral blood revealed robust engraftment in all patients. While CAR-T cell frequency of patient 1 declined after reaching a peak at day 17, CAR-T cells of patient 2 expanded until day 24 remaining in a stable plateau. Data from additional time points and further results for patient 3 are pending. The first and second tumor assessment based on RECIST 6 and 12 weeks post CLDN6 CAR-T administration indicates stable disease for patient 1. In case of patient 2, stable disease with evidence of tumor shrinkage (-19.7%, target lesions) is observed. Clinical follow-up for patient 3 is pending. In conclusion, dosing of the first cohort with CLDN6 CAR-T cell monotherapy was completed with no clinical manifestations of CRS, ICANS or other DLTs. Robust engraftment of CLDN6 CAR-T cells and first signs of efficacy could already be observed in initial dose cohort. Enrollment into higher dose levels and cohorts for combination with CARVac is currently ongoing.
eTalk Session 2
Improving Immunity & Cellular Therapy
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-141
In-depth characterization of tumor-infiltrating CD8+ T cells for clinical application E. Criado-Moronati1, 2, A. Gosselink1, A. Singh1, L. N. Küster1, P. Zjablovskaja1, R. Offringa3, A. Dzionek1, B. Heemskerk1 1Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany, 2University of Heidelberg, Faculty of Bioscience, Heidelberg, Germany, 3German Cancer Research Center, Department of Molecular Oncology of Gastrointestinal Tumors, Heidelberg, Germany
Text Tumor-infiltrating T lymphocytes (TILs) have been successfully used in adoptive cell therapies for the treatment of various cancer types, although only a small fraction of TILs mediate antitumor responses. Therefore, the enrichment and expansion of tumor-reactive T cells may improve the efficacy of TIL treatments. The identification of tumor-reactive T cell phenotypes constitutes an appealing approach to develop isolation strategies of ex vivo TILs based on surface markers independently of the target antigen. For this purpose, we investigated the phenotypes of CD8+ TILs in different regions of several tumor tissues by using the highly multiplexed immunofluorescent MACSima™ Imaging Platform. In addition, flow cytometry and single-cell RNA sequencing analysis of sorted CD8+ T cells from tumor digests were performed to complement the characterization of CD8+ TILs. The MACSima data highlighted the complexity of the tumor microenvironment and the presence of CD8+ T cell subsets with a tissue-resident memory phenotype. We observed a tendency to find activated/exhausted CD8+ T cells, characterized by the expression of PD-1, TIM-3, CD137 or CD39, in close proximity to tumor cells. In one ovarian patient, T cells were mainly found in tertiary lymphoid structures in the periphery of the tumor, displaying an activated state. Flow cytometry data revealed similar phenotypes and highlighted the presence of a CD39+ CD8+ T cell subset which highly co- expressed activation markers and it may thus contain tumor-reactive T cells. To test this hypothesis, we isolated and in vitro expanded CD39+ CD8+ T cells of one lung and two ovarian cancer samples. We could detect tumor-reactive T cells in an ovarian cancer patient, which were present in the expanded CD39+ but not in the negative counterpart. Moreover, differences in the TCR repertoires and phenotypes among the CD39 T cell subsets were also observed at single-cell RNA level. In conclusion, we used the MACSima™ Imaging System to decipher relevant phenotypes of CD8+ TILs, such as CD39, in different tumor regions. The further development of enrichment strategies of tumor-reactive T cell subsets will ultimately shed light on the feasibility of this approach for clinical use.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-142
Enhancing functionality of NK cells towards colorectal cancer cell lines N. Lamers-Kok, Y. van Waardenburg, A. van Vliet, D. Vodegel, L. Kučerová, J. Spanholtz, A. Duru Glycostem, Research, Oss, Netherlands
Text The World Health Organisation reported 935,000 deaths over the year 2020 caused by colorectal and rectal cancer. There is a high need to develop novel curative treatments for patients especially with metastatic disease non-responsive to current treatments. Allogeneic natural killer (NK) cells have shown to be safe and effective in targeting tumors and they do not cause graft versus host disease. However, the treatment of solid tumors is challenging due to their immunosuppressive microenvironment. Introduction of chimeric antigen receptors (CAR) to NK cells, stands out as one of the most promising approaches to overcome this resistance. In this study, we investigated the potency of our umbilical cord blood derived NK cell (UCB-NK) product (GTA002) against various colorectal cancer cell lines. Tumor antigen positive cell lines were subsequently targeted by CAR NK cells (GTA102) to investigate the efficiency of antigen-specific targeting of resistant tumor cells.
Potency of GTA-002 against the colorectal cancer cell lines SW480, HT29, HCT116, LOVO, LS174T, SW48 and COLO205 was tested in a luminescence cell viability assay; in the IncuCyte live cell imaging platform (2D, n=7 and 3D, n=6) and in the Cytoflex LX flowcytometer. CARs were developed and delivered in UCB derived CD34+ cells by a 3rd generation lentiviral system. CAR mediated anti-tumor responses and cellular avidity was measured by flowcytometry and the Z-movi, respectively.
Luminescence data showed refractoriness of the tumor cell lines HT29 and HCT116 to GTA-002, which was confirmed by the kinetic analysis (2D) and flowcytometry data. Potency of GTA-002 was observed against tightly formed spheroid CRC cultures in a kinetic image-based analysis. Additionally, we developed an NK cell line (KHYG1) based reporter system and demonstrated significant CAR mediated enhanced targeting of the antigen+ resistant tumor cell lines when compared to unmodified cells. Initial transduction experiments in CD34+ cells, showed stable expression of eGFP and CAR during the first weeks of expansion and differentiation into CD56+ NK-cells and no impairment of innate NK cell potency.
In conclusion, CRC cell lines exhibited differential sensitivity/responsiveness profile in the 2D and 3D settings. It is essential to evaluate the potency of cell-based immunotherapy products in both 2D and 3D settings to target solid tumors efficiently. Most importantly, our results demonstrate that introduction of CAR to NK cells significantly improves targeting of UCB-NK cell resistant antigen+ CRC cells and stands out as a potential cell therapy product for the treatment of colorectal cancer patients.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-143
TCR-engaging antigen-presenting scaffolds for targeted expansion of functionally improved T-cells for adoptive cell therapy S. Tvingsholm1, V. M. Rafa1, M. S. Frej2, M. Ormhøj1, U. K. Hansen2, M. Kadivar1, T. Tamhane1, A. K. Bentzen1, J. W. Kjeldsen3, A. Kverneland3, O. Met3, M. Donia3, I.-M. Svane3, S. N. Jakobsen4, S. R. Hadrup1 1Danmarks Tekniske Universitet (DTU), Health Technology, Kgs. Lyngby, Denmark, 2pokeAcell Aps, Copenhagen, Denmark, 3Copenhagen University Hospital Herlev, Center for Cancer Immunotherapy, Herlev, Denmark, 4Tetramer Shop Aps, Copenhagen, Denmark
Text Precise targeting of tumors can be achieved through specific T-cells recognizing tumor antigens displayed on the surface of tumor cells in the context of major histocompatibility complexes (MHC). Once the landscape of tumor antigens is defined, an additional challenge is to mount a functional T-cell response against them. Current T-cell expansion techniques provide no efficient strategy for antigen-specific stimulation and result in terminally differentiated and exhausted T-cells. Here we present a new technology for ex vivo peptide-MHC (pMHC)-directed expansion of T-cells using artificial antigen-presenting scaffolds (Ag-scaffolds). The Ag-scaffold is designed to support a favorable functional profile of the expanded T-cells, by providing both antigen-specific stimulation and growth and co-stimulatory signals via pMHCs, cytokines and co-stimulatory molecules co-attached to a dextran-polysaccharide backbone. Unlike conventional adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs), Ag-scaffolds can selectively expand tumor-specific T-cells directly from peripheral blood and therefore bypasses the need for tumor material. Here we demonstrate the value of the Ag-scaffold-based expansion, both by the expansion of virus- and tumor antigen-specific CD8+ T-cells from peripheral blood. The resulting T-cell product exhibits a favorable profile characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion and a multifunctional cytokine profile upon antigen-challenge. Comparing various combinations of cytokines and co- stimulatory molecules, allowed us to identify an optimal Ag-scaffold with pMHC and cytokines IL-2 and IL-21. Using Ag-scaffolds, multiple different antigen-specific T-cell populations can be expanded simultaneously in a single culture. With the desire to design a shared Ag-scaffold for patient with metastatic melanoma, we identified the 30 most abundant shared HLA-A0201-restricted melanoma antigens in a cohort of 87 patients and combined these in a Top-30 Ag-scaffold product. Using this product, we were able to expand tumor-specific T-cells from 60- 80% of melanoma patients in three cohorts. In some patients, we expanded up to 8 of the 30 shared antigens and 25% specific cells. This study presents a new technology for targeted expansion of functionally enhanced antigen-specific CD8+ T- cells as a promising alternative to existing ACT strategies. Since T-cell specificity, phenotype and functionality are known to be important parameters affecting long-lasting clinical responses to ACT, it is plausible that an Ag- scaffold-expanded T-cell product may improve treatment outcome.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-144
Efficient scale-up and pre-clinical evaluation of NKG2C+ adaptive NK cell expansions for therapy against high-risk AML/MDS A. Haroun-Izquierdo1, M. Vincenti2, H. van Ooijen3, 4, M. Wiiger2, S. Li2, B. Zhang5, F. Cichocki5, M. Felices5, H. J. Hoel2, H. Netskar2, A. Björklund1, 6, J. Goodridge7, L. Kveberg2, B. Valamehr7, B. Onfelt3, 4, 8, J. S. Miller5, K.-J. Malmberg1, 2, E. Sohlberg1 1Karolinska Institutet, Center for Infectious Medicine, Stockholm, Sweden, 2Institute for Cancer Research, Department of Cancer Immunology, Oslo, Norway, 3Science for Life Laboratory, Department of Applied Physics, Stockholm, Sweden, 4KTH Royal Institute of Technology, Stockholm, Sweden, 5University of Minnesota, Masonic Cancer Center, Minneapolis, United States, 6Karolinska Institutet, Department of Hematology, Stockholm, Sweden, 7Fate Therapeutics Inc., La Jolla, United States, 8Karolinska Institutet, Department of Microbiology, Tumour and Cell biology, Stockholm, Sweden
Text Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against haematological malignancies, including acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). NK cell recognition of allogeneic tumours is tightly regulated by inhibitory killer cell immunoglobulin-like receptors (KIR) that bind to groups of HLA class I alleles. High frequencies of alloreactive NK cells in the donor correlate with reduced relapse rates in adoptive NK cell therapy against AML. However, KIR expression on NK cells is highly diverse due to genetic variation and stochastic expression in individual cells. Hence, the number of allogeneic NK cells isolated and expanded from random donors is variable and sometimes negligible, limiting the potential of missing self- recognition. In this study, we describe an 11-day protocol to enrich adaptive NKG2C+ NK cells with a homogenous expression of one single self-HLA specific KIR (self-KIR). By screening >200 healthy donors, we established the prerequisites for robust expansion of adaptive NK cells from peripheral blood of CMV+ donors and found that donors with >20% pre-existing adaptive NK cells (termed superdonors) showed efficient expansion of adaptive NK cells. After GMP-transfer we have so far implemented this protocol on apheresis products from six identified superdonors to achieve robust expansion of alloreactive adaptive NK cells, with an average frequency of >85% NKG2C+ self-KIR+ cells in the end product. The current donor-bank is sufficient to allow dose-escalation and expansion Phase I/II studies in up to 185 patients. Flow cytometry-based assays combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) revealed retention of an adaptive state with high contents of effector molecules and a high degranulation capacity of the expanded cells, which correlated with efficient killing of mismatched tumour cell lines and primary AML blasts. Importantly, we observed negligible killing of normal bystander cells. Finally, the expanded adaptive NK cells showed potent re-directed killing against the CD33+ HL-60 cell line and primary AML blasts when combining with Tri-specific Killer cell Engagers (TriKEs) directed towards CD33. These pre-clinical data demonstrate the feasibility of off-the-shelf therapy with a non- engineered and yet highly specific NK cell population, representing the first route to clinical testing of missing self- recognition.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-145
Preclinical characterization of a CD20-specific TCR: focusing on safety in vivo and in vitro Z. Foldvari1, 2, K. Borutta3, W. Yang1, 2, C. Knetter Hoel1, 2, T. J. Gjerdingen1, 2, F. Lund-Johansen2, 4, R. Klopfleisch5, A. Kolstad2, 6, M. Leisegang*3, 7, 8, 9, J. Olweus*1, 2 1Oslo University Hospital Radiumhospitalet, Institute for Cancer Research, Department of Cancer Immunology, Oslo, Norway, 2University of Oslo, K. G. Jebsen Center for Cancer Immunotherapy, Institute for Clinical Medicine, Oslo, Norway, 3Charité - Universitätsmedizin Berlin, Institute of Immunology, Berlin, Germany, 4University of Oslo and Oslo University Hospital, Department of Immunology, Oslo, Norway, 5Freie Universität Berlin, Institute of Veterinary Pathology, Berlin, Germany, 6Oslo Univeristy Hospital Radiumhospitalet, Department of Oncology, Oslo, Norway, 7The University of Chicago, David and Etta Jonas Center for Cellular Therapy, Chicago, United States, 8German Cancer Consortium (DKTK), partner site Berlin, Berlin, Germany, 9German Cancer Research Center (DKFZ), Heidelberg, Germany *Contributed equally
Text TCR-engineered T cells hold promise for cancer treatment, but can pose a risk of on- and off-target toxicity. Here, we present a pipeline for a pre-clinical in vitro and in vivo efficacy and safety testing of TCR candidates. Two TCRs (TCR1 and TCR2) reactive to a peptide of the B-cell specific CD20, presented on HLA-A*02:01 (HLA-A2) were identified earlier. They efficiently kill HLA-A2+ CD20+ cell lines and primary cancer cells while spare HLA-A2+ CD20- cells of various tissue origins. To study the in vivo efficacy of TCR1 and -2 and to a certain degree safety, we used a syngeneic, HLA-A2-transgenic cancer mouse model (HHDxRag1-/-). T cell therapy of HHDxRag1-/- mice bearing tumors with stable high levels of the CD20/HHD target resulted in tumor rejection in all treated mice (TCR1 n=17, TCR2 n=19). Relapse was observed only in one TCR2 treated mouse. In a more stringent setup targeting tumors expressing low HLA-A2 levels, 50% of the TCR1- treated mice still rejected the tumors (n=4/8) while all TCR2 treated mice relapsed (n=11). The HHDxRag1-/- model allows to also analyze TCR safety as normal tissues of mice present a multitude of HLA-A2-presented murine peptides. As murine and human proteomes overlap, this provides valuable safety data. Pathological study of vital organs collected from mice 10 weeks after successful therapy did not reveal off-target toxicity. We next used an off-target reactivity screening strategy for TCR-T cells. First, we mapped residues critical for TCR recognition by a peptide library where every amino acid (AA) position in the cognate peptide was substituted with every possible AA, one at a time. Next, the human proteome was screened for natural peptides containing combinations of allowed AA exchanges that were tested for T-cell response. Finally, we determined if remaining peptides were naturally processed and presented on HLA-A2. Cell lines expressing RNA constructs encoding 30-mer AA sequences with the candidate cross-reactive peptide or cell lines naturally expressing candidate cross-reactive proteins were used. After mapping the TCR1 recognition profile, 19 potentially cross-reactive peptides were found. By applying the algorithm above, we reduced the list to three. Cell lines expressing high levels these proteins failed to activate TCR1. Safety testing of the clinically approved NY-ESO-1 reactive 1G4 TCR, included as a control, similarly failed to reveal clinically relevant off-target effects. We show that TCR1 is effective in killing tumor cells in vivo without off-target toxicity. The pre-clinical screening strategy established here is useful to characterize safety and efficacy of potential therapeutic TCRs, fundamental to clinical translation of new immunotherapies.
eTalk Session 2
Improving Immunity & Cellular Therapy
S2-146
First Anti-Tumor Activity Associated with Robust Biological Activity Observed in Early Phases of Dose Escalation Across Three TCR-T Trials A.-M. Tsimberidou1, A. Mohamed2, A. Mayer-Mokler3, A. Satelli2, C. Reinhardt4, D. Araujo1, D. Maurer3, G. J. Blumenschein1, H. Singh4, K. Guenther3, M. Kalra2, M. Chatterjee5, M. Wermke6, N. Hilf3, R. Mendrzyk3, S. Walter2, S. Eck2, T. A. Holderried7, T. Weinschenk3, V. Morris1, C. Britten4 1MD Anderson Cancer Center, Houston, United States, 2Immatics Biotechnologies, Houston, United States, 3Immatics Biotechnologies, Tübingen, Germany, 4Immatics Biotechnologies, Munich, Germany, 5Universitätsklinikum Würzburg, Würzburg, Germany, 6Universität Dresden, Dresden, Germany, 7University Hospital Bonn, Department of Hematology, Oncology, Immunoncology and Rheumatology, Internal Medicine III, Bonn, Germany
Text Despite recent treatment successes in advanced cancers with high mutational load and hematological cancers by immune checkpoint inhibitors and CAR-T therapy, a high unmet medical need still exists for most patients with solid tumors. ACTengine® IMA201, IMA202 and IMA203 product candidates utilize genetically engineered T cells modified to express novel T cell receptors (TCRs) against HLA-A*02:01-presented peptides derived from the cancer testis antigens MAGEA4/8, MAGEA1 or PRAME for infusion of recurrent or refractory solid cancer patients after multiple lines of prior therapies. HLA-A*02:01- and target-positive patients, with a variety of different cancers, undergo leukapheresis and autologous cellular products are manufactured for each patient. Safety, biological activity and anti-tumoral efficacy are closely monitored throughout and following lymphodepletion with Fludarabine and Cyclophosphamide, T cell infusion and low dose IL-2 treatment. Each of the three first-in-human trials is currently recruiting patients at the dose escalation cohort (Phase 1a), which will be followed by the dose expansion cohort at the highest tolerated dose (Phase 1b). As of 16 February 2021, 14 heavily pre-treated, patients with recurrent/ refractory solid cancers have been treated with one ACTengine® product candidate of the IMA200 series at dose level 1 or 2. Doses ranged from 80 to 650 million transduced CD8 T cells, which is below the anticipated target dose level. Most frequent adverse events were transient, cytopenias expected from the lymphodepletion regimen and transient Grade 1-2 cytokine release syndrome (CRS) following T cell infusion. Disease control was observed in 90% (9/10) of treated patients with at least one available tumor response assessment. Tumor mass decreased in 8/10 patients, including one unconfirmed partial response (RECIST1.1) as of data cut-off. Robust T cell engraftment, persistence until end of observation period (up to 9 months) and tumor infiltration was observed in all analyzed patients. Two patients with strongest reduction in tumor load showed remarkable peak levels of target-specific T cells (11% and 32% of CD8 T cells) after infusion, despite the low doses of 350 million and 189 million transduced CD8 T cells infused. Taken together, treatment-emergent adverse events for ACTengine® product candidates were transient and manageable and in line with published data for autologous cell therapy in solid cancers. First anti-tumor activity in heavily pre-treated patients after infusion of low cell doses was unexpected, and together with robust biological activity warrants exploration of IMA201, IMA202 and IMA203 product candidates at target dose.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-147
Soluble immune checkpoints and T-cell subsets in blood as biomarkers for resistance to immunotherapy in melanoma patients D. Machiraju1, M. Wiecken1, 2, 3, N. Lang1, I. Hülsmeyer1, J. Roth1, R. Eurich4, N. Halama4, A. Enk1, J. C. Hassel1 1University Hospital Heidelberg, Department of Dermatology and National Center for Tumor Diseases (NCT), Heidelberg, Germany, 2University of Heidelberg, Faculty of Biosciences, Heidelberg, Germany, 3University Medical Center and Medical Faculty Mannheim, University of Heidelberg, Department of Dermatology, Venereology and Allergology, Mannheim, Germany, 4University Hospital Heidelberg, Medical Oncology, National Center for Tumor Diseases (NCT), Heidelberg, Germany
Text Different mechanisms lead to immune checkpoint inhibitor (ICI) resistance. Identifying clinically useful biomarkers might improve drug selection and patients’ therapy. We analyzed the soluble immune checkpoints sPD1, sPDL1, sLAG3, and sTIM3 using ELISA and their expression on circulating T cells using FACS in pre-and on-treatment blood samples of ICI treated melanoma patients. In addition, pre-treatment melanoma metastases were stained for TIM3 and LAG3 expression by immunohistochemistry. Results were correlated with treatment response and progression-free survival (PFS). We found that, resistance to anti- PD1 treatment (n=48) was associated with high pre-treatment serum levels of sLAG3 (DCR: p=0.009; PFS: p=0.018; ROC cut-off > 148 pg/ml) but not sPD1, sPDL1 or sTIM3. In contrast, resistance to ipilimumab plus nivolumab (n=42) was associated with high levels of sPD1 (DCR: p=0.019, PFS: p=0.046; ROC cut-off > 167 pg/ml) but not sPDL1, sLAG3 or sTIM3. Both treatment regimens shared a profound increase of sPD1 serum levels with treatment (p<0.0001). FACS analysis revealed reduced frequencies of CD3+CD8+PD1+ T cells (p=0.028) in anti-PD1-resistant patients, whereas increased frequencies of CD3+CD4+LAG3+ T cells characterized patients resistant to ipilimumab plus nivolumab (p=0.033). Unlike anti-PD1 monotherapy, combination blockade significantly increased proliferating T cells (CD3+CD8+Ki67+ T cells; p<0.0001) and eosinophils (p=0.03). In melanoma metastases, an increased infiltration with TIM3+ or LAG3+ T cells in the tumor microenvironment correlated with a shorter PFS under anti-PD1 treatment (TIM3: p=0.019, LAG3: p=0.07). In summary, different soluble immune checkpoints characterized checkpoint inhibitor-resistant melanoma. Measuring these serum markers is easy and has the potential to be used in clinical routine.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-148
Uncoupling of T-cell bispecific antibody-induced cytokine release and cytotoxicity revealed by differential activities of mTOR, JAK1/2 and Src inhibitors. G. Leclercq1, H. Haegel1, A. Schneider1, A.-M. Giusti1, V. Pulko1, C. Ferlini1, A. Toso2, T. Zimmermann2, L. Green2, N. Steinhoff1, J. Sam1, P. Umaña1, M. Bacac1, C. Klein1 1Roche Innovation Center Zurich, Roche Pharma Research and Early Development (pRED), Schlieren, Switzerland, 2Roche Innovation Center Basel, Roche Pharma Research and Early Development (pRED), Basel, Switzerland
Text T cell bispecific antibodies (TCBs) are potent T cell engagers, harboring a 2+1 format with one binder to the CD3ε chain and two binders to specific tumor antigens. Crosslinking of CD3 with tumor antigens triggers T cell activation and proliferation, cytokine release and tumor cell killing. TCB treatment is sometimes associated with safety liabilities due to on-target on-tumor or on-target off-tumor cytotoxicity and cytokine release. Off-tumor activity of the TCB may occur if the targeted tumor antigens are expressed on healthy cells, which may potentially result in tissue damages and compromise the patient’s safety. Patients treated with TCBs may also experience a Cytokine Release Syndrome (CRS), characterized by fever, hypotension and respiratory deficiency and associated with the release of pro-inflammatory cytokines such as IL-6, TNF- α, IFN-γ, and IL-1β. Tyrosine kinases such as Src, mTOR and JAK1/2 are involved in downstream signaling pathways after engagement of the T cell receptor. Using an in vitro model of target cell killing by human peripheral blood mononuclear cells stimulated with TCBs, we assessed the effects of mTOR, JAK and Src kinase inhibitors on T cell activation, tumor cell killing and cytokine release. In line with previous reports for CAR-T cells, dasatinib (a src inhibitor) was found to fully switch off TCB-induced T cell functionality. In contrast, temsirolimus, sirolimus, everolimus (mTOR inhibitors) and ruxolitinib, baricitinib, tofacitinib, fedratinib (JAK1/2 inhibitors) were found to more potently prevent TCB-induced cytokine release without blocking TCB-mediated target cell killing. These results provide evidence that the mechanisms of TCB- dependent cytokine release and tumor cell killing can be uncoupled. The FDA-approved mTOR and JAK1/2 inhibitors could represent potential alternatives to the blockade of IL-6/IL-6R and TNF-α or the use of corticosteroids for the mitigation of CRS. The Src inhibitor dasatinib could rather stand as a potential antidote for on-target off-tumor activity or high-grade CRS. The data are being validated in vivo.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-149
Fast automated microfluidic-based multiplexed immunofluorescence for tumor microenvironment analysis A. Kehren1, M. G. Procopio1, B. Pelz1, Z. Siddiqui1, K. Roman2, S. Adnane1, S. Brajkovic1, C. Hoyt2, D. G. Dupouy1, A. Soltermann3 1Lunaphore Technologies SA, Tolochenaz, Switzerland, 2Akoya Biosciences, Phenoptics, Marlborough, United States, 3ADMED Pathology, Neuchâtel, Switzerland
Text Background and objectives Immuno-oncology and targeted molecular therapies have acquired a central role in the treatment of multiple cancers. Consequently, high-throughput biomarker analysis and tumor immune profiling have seen an increased demand. Multiplexed immuno-assays are a powerful tool to address these needs, but still time- and resource-consuming. Our goal is to develop a fast and automated high-plex fluorescent immunostaining procedure, using a microfluidic-based device, that can be easily implemented as routine assay.
Method Protocol optimization has been performed on FFPE sections of human tonsil. Slides were manually deparaffinized before being entirely processed (antigen-retrieval, staining, elution and counterstaining) by Lunaphore’s autostainer, LabSatTM. The OPAL® tyramide signal amplification (TSA) system was used as detection method. Signal analysis was done on Mantra® workstation. The 6-plex panel was composed of FoxP3, PD-L1, PD-1, CD68, CD8 and pan-CK, plus DAPI counterstaining. Protocols were subsequently transferred on NSCLC representative specimens and finally assessed on a TMA cohort.
Results Our platform allowed to reduce drastically the incubation times due to active transport of reagents across the tissue. Thereby, the automated 6-plex assay could be performed in less than 4h30min, within the timeframe of a single IHC standard assay. Protocol optimization resulted in high signal-to-background ratio for each marker and removal of previous step antibodies over 99%. LabSatTM also guaranteed remarkable signal uniformity, even over large tissue sections with less than 10% signal gradient over 1 cm. On NSCLC samples, the detected pattern and expression level for all six biomarkers were comparable to the standard chromogenic stainings performed with standard automated tissue stainer.
Conclusion LabSatTM autostainer enables multistaining runs in a timely manner, opening the perspective of rapid simultaneous detection of multiple markers in their morphological context on a routine-based approach. This versatile analysis tool can offer a better and more quantitative understanding of tumor heterogeneity and microenvironmental interactions, allowing advances in targeted therapy for lung cancer as well as broader spectrum of malignancies.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-150
Effect of whole body hyperthermia on NK cell function in breast cancer patients J. Krüger, S. Van Gool, J. Makalowski, V. Schirrmacher, W. Stücker, S. Van Gool Immun-Onkologisches Zentrum Köln, Köln, Germany
Text Cancer patients regularly have low natural killer (NK) cell function. Whole body hyperthermia (WBH) is a type of modulatory immunotherapy aimed to increase NK cell function. We aimed to study this effect in patients with breast cancer and treated with individualized multimodal immunotherapy (IMI). WBH was administered when the NK cell function was below the reference range. Breast cancer patients were sampled for retrospective analysis until the end of August 2020. The evolution of NK cell function was compared between patients without (WBH-) or with (WBH+) WBH (39°C for 45-60 minutes) as part of IMI. Results of NK cell function before and after at least two WBH, and available over a time window of < 5 months, were selected for the WBH+ group. The results of the NK cell function in WBH- patients were derived from analyses before and after 2 DC vaccines or before and during IMI also within a time window of < 5 months. NK cell function was categorized as “less than normal” (L), “normal” (N), or “higher than normal” (H) for the given reference range. 38 and 40 patients could be selected in the WBH- and WBH+ group. The duration of disease and number of events prior to IMI was significantly higher in the WBH- group. The age and the hormone receptor profiles between both groups were similar. The distribution of the first NK cell function in the WBH- group showed 14 L, 4 N and 20 H, in contrast to 35 L, 2 N and 3 H in the WBH+ group. In the WBH+ group, the NK cell function increased to H in 17 patients and to N in another 3 patients after two WBH treatments, while 16 patients remained with low NK cell function. The increase was most visible in hormone receptor+ Her2Neu- patients. In 10/28 WBH+ patients with more than 2 WBH treatments the NK cell function remained high at last measurement. In another 10/28 patients, the increase of NK cell function was only transient, and these patients returned to L. In contrast, only 6 patients of the WBH- group kept high NK cell function during IMI, while NK cell function normalized in 4 H patients. However, the NK cell function became low in 13 N+H patients and remained low in 10 L patients. Overall 26/38 WBH- patients ended with low NK cell function over the time window of study, and 10/15 patients were low at last measurement. WBH has a positive effect to restore the NK-cell function of breast cancer patients, which was maintained during further disease course in 36% of patients. IMI without WBH could not sustain NK cell function. WBH can be recommended as modulatory immunotherapy.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-151
Evaluating the proficiency of laboratories in detecting T-cell responses using ELISpot assay R. Yding Tingleff, C. Essendrup Dam, L. Brix Immudex, Virum, Denmark
Text The ability to monitor antigen-specific T-cell responses and compare results generated by laboratories worldwide is a powerful tool to ensure alignment and drive improvements in immunotherapeutic research and development. Supported by CIC and CIMT, Immudex are conducting Proficiency Panels yearly allowing laboratories worldwide to assess their performance within monitoring of antigen-specific T-cell responses using direct human IFN-γ ELISpot assay.
In the T-cell ELISpot Proficiency Panel 2020, 30 laboratories from 12 different countries participated. Pretested PBMC samples, peptide pools and instructions were sent to the participating laboratories. Each participant conducted IFN-γ ELISpot assay to determine the number of IFN-γ secreting antigen-specific cells per 200.000 viable cells after stimulation with standardized peptide pools comprising CMV and mixed virus epitopes.
To assess the proficiency of the participants, the relative accuracy (how close each participants measurement is to the median of all participant measurements) was compared. For the PBMC sample with the highest frequency of antigen-specific cells (median of 308 spots), 56% of the participants obtained results within the average range (1.5 times lower or higher than the median). For the other two PBMC samples with lower frequency of antigen-specific cells (median of 111 and 13 spots), the results were less aligned. Here, 30% and 26%, respectively, of the participants were within the average range. Furthermore, a PBMC sample with no specific response was included in testing as a negative sample. 93% of the participants were within the average range of the panel median when analyzing this negative sample, which demonstrates a general alignment and low risk of false positive measurements.
The 2020 T-cell ELISpot Proficiency Panel shows that T-cell ELISpot assays are more harmonized across different laboratories when looking at high-frequent T-cell responses than low-frequent responses. Proficiency Panels are a useful tool to evaluate the proficiency of immune monitoring assays across different laboratories to ensure comparable results in e.g. multicenter trials.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-152
A flow-cytometry-based method for assessment of functional antigen-specific CD4+ and CD8+ T cells by staining activated integrins A. Schöllhorn1, J. Schuhmacher1, L. Besedovsky2, R. Fendel3, T. Lange4, H.-G. Rammensee1, 5, 6, J. Born2, 7, 8, S. Dimitrov2, C. Gouttefangeas1, 5, 6 1University of Tübingen, Institute for Cell Biology, Department of Immunology, Tübingen, Germany, 2University of Tübingen, Institute of Medical Psychology and Behavioral Neurobiology, Tübingen, Germany, 3University of Tübingen, Institute of Tropical Medicine, Tübingen, Germany, 4University of Lübeck, Department of Rheumatology and Clinical Immunology, Lübeck, Germany, 5German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) partner site Tübingen, Tübingen, Germany, 6University of Tübingen, Cluster of Excellence iFIT (EXC2180) "Image-Guided and Functionally Instructed Tumor Therapies", Tübingen, Germany, 7German Center for Diabetes Research (DZD), Tübingen, Germany, 8Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tübingen (IDM), Tübingen, Germany
Text Many T-cell based immunotherapy approaches, e.g. anti-cancer vaccines, are designed to activate simultaneously CD8+ and CD4+ T cells. CD4+ T cells support CD8+ T cell activation, expansion, and memory formation, but can also kill tumors in a direct or indirect way. Therefore, robust and sensitive identification and monitoring of functional antigen-specific CD8+ and CD4+ T cells is essential for basic research, but also during immunotherapy. Commonly used methods for functional assessment of antigen-specific T cells rely on de novo expression of activation markers like cytokines after several hours of stimulation. Inactive ß2-integrins, like LFA-1, are highly expressed on the cell surface of antigen-experienced T cells, and get activated upon TCR-mediated signaling by a process called “inside out” signaling. Activation induces conformational change and clustering of LFA-1, leading to an increase in affinity and valency which allows interaction with its ligand ICAM-1. We previously described a flow cytometry-based method that allows identification of functional antigen-specific CD8+ T cells within minutes of stimulation using fluorescent multimeric ICAM-1 (mICAM-1). Here, we present a modified experimental setup of this method that can be applied for assessment of both antigen- specific CD8+ and CD4+ T cells. Instead of ICAM-1 multimers, we now use a monoclonal antibody (clone m24) specific for the open high affinity conformation of ß2-integrins. We show that antigen-specific CD4 T cells require longer stimulation times than CD8+ T cells for maximal activation of LFA-1 (hours vs. minutes for CD4+ and CD8+ T cells, respectively). Activated LFA-1 on both CD4+ and CD8+ T cells identifies the fraction of cells producing cytokines after stimulation. m24 antibody staining is combinable with intracellular and surface marker stainings, and co-staining with m24 and CD154 antibodies allows identification of extremely low frequencies of antigen-specific cells ex vivo. We show that m24 Ab stains cells with different differentiation status. The protocol is robust with a low intra- and inter-assay variability, and is compatible with a 96 well plate format, thereby increasing the throughput. In conclusion, detection of activated LFA-1 is a simple method for sensitive and simultaneous identification of antigen- specific CD4+ and CD8+ T cells and is suitable for monitoring low frequency antigen-specific T cell responses e.g. during immunotherapy.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-153
Evaluating the performance of immune monitoring laboratories using MHC multimer assay R. Yding Tingleff, C. Essendrup Dam, C. Halgreen, L. Brix Immudex, Virum, Denmark
Text Disease-specific T-cell immune monitoring is becoming increasingly important in immunotherapeutic research and development. MHC multimer assays have been shown to be reliable and accurate tools for monitoring antigen-specific T-cell immunity thanks to the harmonization efforts by the CIC and CIMT. Supported by CIC and CIMT, Immudex are conducting Proficiency Panels allowing laboratories worldwide to assess their performance in enumerating antigen-specific T-cell responses using MHC multimer assay and flow cytometry. The ability to compare results generated by different laboratories is a powerful tool to ensure alignment and drive improvements within immune monitoring.
In the MHC Multimer Proficiency Panel 2020, 20 laboratories from 8 countries participated. Each participant received pretested PBMC samples with low, medium or high frequencies of T cells specific for predefined FLU- or EBV- epitopes. All laboratories analyzed samples using our instructions and their own MHC multimer assay protocol and reported their results back as number of MHC multimer-specific CD8+ T cells out of total number of CD8+ T cells analyzed.
To assess the performance of the participants, we compared how close each participant measurements was to the average value reported by all participants. Overall, 80% of all reported measurements were in the average range (defined as 1.5x lower or higher than the median) or near the average range (defined as 1.6 - 2.0x higher and 0.50 - 0.65x lower than the median). In general, the participating laboratories showed similar duplicate results both for the positive and negative MHC multimer specificities.
Overall, the results of this MHC multimer Proficiency Panel show that the MHC multimer assay is well harmonized across different laboratories. The MHC multimer assay is equally harmonized when looking at high-frequent T-cell responses and low-frequent T-cell responses. It is a useful tool for evaluating treatment response in immunotherapeutic research and development.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-154
Automated analysis of multiplexed flow cytometry data with open source tools M. Helm, K. Lange, M. Mues, J. Kollet Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
Text The integration of flow cytometry data in automated multi-omics analysis pipelines for immunomonitoring in clinical trials is not straight forward. One major hurdle is, flow data is typically analyzed using standalone software, which requires manual data upload, analysis, and exports of results, whereas open source tools provide solutions for integrated analysis and downstream processing. Here we tested R and programs therein to generate an automated workflow to analyze flow cytometry data. Our example data sets were generated with bead-based kits, the MACSPlex cytokine kit human or mouse. We developed a pipeline which enables batch analysis and seamless integration in multimodal data analysis. We also implemented a shiny app for facilitated interactive analysis or as a central web service. For the application, a fully automated analysis is initiated at the click of a button. First, single bead events are selected by singlet gating. Then, individual bead populations are identified. Finally, standard curves are fitted and used to calculate the concentrations of each cytokine for all samples. The singlet gate and bead populations with their corresponding cytokines are displayed in separate plots for interactive exploration. In addition, the standard curves for calculation of the cytokine concentrations are shown. The calculated cytokine concentrations are summarized for each sample as a table. The results, consisting of measured concentrations, corresponding intensities and bead counts can be exported as an Excel file. In addition, all plots can be downloaded. Alternatively, the program could be initiated automatically in the background, and the results could be integrated directly as R objects in multi-omics analysis pipelines. Using the open source solution we were able to quickly implement a different detection method – a functionality, typically not provided by standard software. For example, we observed robust detection of bead populations using clustering instead of standard methods. This enabled reliable classification for measurements obtained with different instruments despite variations in fluorescence intensities. In summary, the automation of the analysis of bead-based flow data, from retrieval to downstream integration of the results in other pipelines, was feasible with R and programs therein. Additional advantages were the flexible inclusion of alternative analysis methods and the option to use an interactive web application. The development of automated analysis pipelines with open source tools provide promising perspectives for the rapid interpretation of multi-omics immunomonitoring data in concomitant research of clinical trials.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-155
Phenotypic and functional investigation of γδ T-cells in melanoma under PD-1 checkpoint blockade N. Beucke1, J. Bochem1, T. Amaral1, B. Weide1, G. Pawelec2, 3, F. Meier4, P. Terheyden5, M. Löffler2, 6, 7, T. Eigentler1, K. Wistuba-Hamprecht1, 8 1University Medical Center, Department of Dermatology, Tübingen, Germany, 2Interfaculty Institute for Cell Biology, Department of Immunology, Tübingen, Germany, 3Health Sciences North Research Institute, Sudbury, Canada, 4Skin Cancer Center, Medical Faculty and University Hospital Carl Gustav Carus, Department of Dermatology, Dresden, Germany, 5University of Lübeck, Department of Dermatology, Lübeck, Germany, 6University Hospital Tübingen, Department of General, Visceral and Transplant Surgery, Tübingen, Germany, 7University Hospital Tübingen, Department of Clinical Pharmacology, Tübingen, Germany, 8Clinical Bioinformatics, Department of Internal Medicine I, Tübingen, Germany
Text The global incidence of melanoma, the deadliest form of skin cancer, has risen rapidly since the mid-1950s. Although immune checkpoint therapy using the antagonistic PD-1 antibodies nivolumab and pembrolizumab significantly improved the survival of melanoma patients, prognosis for patients with distant metastasis remains poor. About 20% of γδ T cells express PD-1, making this numerically minor population of unconventional T cells a potential target of checkpoint blockade. Interest in γδ T cells is growing rapidly due to their diverse functions and involvement in tumor immunity, immunological disorders and infectious diseases. The γδ T cell population comprises about 1-10% of T cells in the peripheral blood and encompasses heterogeneous subsets with various different functions ranging from cytotoxic activity and cytokine secretion to antigen presentation and induction of immunoglobulin class switching. Unlike αβ T cells, γδ T cells are not MHC-restricted, and their ligands and mode of antigen-recognition are not yet well- characterized. A detailed understanding of these mechanisms and the role of γδ T cells in immunity is required to assess their prognostic, predictive and therapeutic potential in human cancer. We have previously shown that high frequencies of Vδ1 γδ T cells in peripheral blood are associated with poor survival in metastatic melanoma patients. Our current investigations confirm this correlation and show that this is associated with a differentiation profile dominated by late-differentiated Vδ1 T cells and higher frequencies of CD57+ cells, a putative marker of replicative senescence. Under immune checkpoint blockade we observed a decline in the frequency of CD57+ Vδ1 γδ T cells, suggesting an expansion of CD57- Vδ1 γδ T cells. Functional studies revealed a reduced proliferative capacity of Vδ1 as well as Vδ2 γδ T cells compared to healthy donors. Taken together, our data indicate a phenotypic and functional alteration of γδ T cells in late-stage melanoma patients and a possible contribution of γδ T cells to melanoma immunosurveillance.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-156
Deep phenotyping of antigen specific T-cells at a single-cell resolution using MHC dCODE Dextramer® (RiO) reagents designed for the BD®Rhapsody™ Single-Cell Analysis system K. D. Jacobsen1, C. Halgreen2, L. Brix1 1Immudex aps, Virum, Denmark, 2Immudex aps, R&D, Virum, Denmark
Text Background: Multiomics approaches are revolutionizing the field of immunology, allowing deep characterization, and understanding of the functions of individual cells and their heterogeneity. The power of multiomics assays allow simultaneously measurement of multiple parameters of single immune cells, including gene expression and surface marker identification in a single readout. Here, we show deep phenotyping of antigen-specific T cells at single cell level by integrating Immudex MHC dCODE Dextramer® (RiO) reagents with the BD Rhapsody™ Single-Cell Analysis workflow. Methods A panel of 8 MHC I dCODE Dextramer® (RiO) reagents each displaying different viral epitopes (+ 5 negative control dCODE® reagents), and 16 BD® AbSeq™ antibodies along with the targeted BD® Rhapsody™ Immune Response panel (395 genes total) was used to profile a human PBMC sample. HPBMCs were labeled using above reagents, as well as CD8/BV421 antibody and viability stain for FACS sorting. The sorted cells were introduced into the BD Rhapsody™ Single-Cell Analysis system, and 3 DNA libraries were generated: 1) dCODE® library encoding information on TCR recognition, 2) AbSeq™ library encoding surface marker expression info, and 3) mRNA library providing info on Immune response gene expression. The sequence data was analyzed using FlowJo SeqGeq™ analysis software. Due to the low frequency of antigen specific T cells in contrast to high expressed surface marker and mRNA expression, a specific dCODE library amplification step was introduced, separating the dCODE® library form the AbSeq™ and mRNA expression libraries, allowing control over the sequencing depths for each library independently. Results 7 antigen-specific T-cell populations at high resolution were detected in the HPBMC sample. Each positively verified by conventional flow cytometry. Despite the disparity in the number of cells analyzed on the two platforms, the results showed that the frequencies of positive cells found by the Rhapsody™ workflow, were in concordance with those found by conventional Flow cytometry. Information on surface marker and gene expression allowed detailed characterization of the antigen-specific T cells. Conclusions: The results show detection of antigen-specific T cell at high resolution, aided by the dCODE specific library amplification step. Data allows for deep phenotyping of the antigen specific T cells by the dCODE® RiO reagents together with BD® AbSeq™ antibodies and the BD® Immune response panel expression profiles.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-157
Peptide pools for target antigen identification, immune monitoring and cellular therapy M. Eckey1, P. Holenya1, T. Teck1, J. Zerweck1, T. Knaute1, M. Schulz1, M. Drosch1, K. Schnatbaum1, U. Reimer1, H. Wenschuh1, F. Kern1, 2 1JPT Peptide Technologies GmbH, Berlin, Germany, 2Brighton and Sussex Medical School, Brighton, United Kingdom
Text There is an ever increasing need for peptide tools in experimental and clinical immunology, particularly for personalized immunotherapies. The most important applications include target protein identification, epitope mapping/optimization, and T-cell response monitoring. Clinical grade peptides are essential for (neo-)epitope based vaccines and immunotherapies. Even for experienced users, a multitude of possible specifications make it difficult to choose the best peptide format for a specific application. Peptide pools are produced in different specifications. Purities range from crude (for discovery) to highly purified (for clinical applications). Crude peptide pools for antigen target discovery are produced by high- throughput peptide synthesis allowing the assembly of tens of thousands of peptides covering whole antigen families or even entire pathogens. Peptide pools for immune monitoring, by contrast, are manufactured using traditional peptide synthesis methods with added measures that avoid contaminations that either impede T- cell stimulation or cause it by themselves. Clinical grade peptides representing tumor-associated (neo- )antigens or pathogen-derived antigens are produced to meet the highest specifications in terms of purity and absence of contaminations. While no peptide format is universally applicable, several tailored formats were developed that proved to be useful in key applications requiring T-cell stimulation. For example, unpurified but well-characterized peptide pools and individual peptides are used to measure T-cell responses against known target proteins, to discover new target proteins, and to identify stimulating peptides from a large number of candidate peptides. Only purified peptides and fully characterized pools, by contrast, provide the robust assay data needed for cell-based immune monitoring. For clinical applications peptide and pool manufacturing are performed under line clearance in a low bioburden environment with full analytical coverage. In addition, full process traceability and documentation as well as stability testing are performed to fulfill regulatory requirements. When deciding, which peptide pool format to use for a specific purpose, the following aspects need to be considered: peptide length, number and amount of peptide(s), purity, avoidance/elimination of contaminations, stability, QC/QA measures, and last but not least cost. A good decision can help to speed up development projects as exemplified with vaccine development projects for SARS-CoV-2.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-158
Engraftment of neoantigen-reactive CD8+ T cells affects the clinical outcome in patients receiving adoptive transfer of tumor-infiltrating lymphocytes N. Pagh Kristensen1, C. Heeke1, S. A. Tvingsholm1, A. Borch1, A. Draghi2, M. D. Crowther2, I. Carri3, K. K. Munk1, J. Sejerø Holm1, A.-M. Bjerregaard1, A. Kai Bentzen1, A. M. Marquard1, Z. Szallasi4, N. McGranahan5, R. Andersen2, M. Nielsen1, 3, G. B. Jönsson6, M. Donia2, I. M. Svane2, S. Reker Hadrup1 1Technical University of Denmark, Department of Health Technology, Kgs. Lyngby, Denmark, 2National Center for Cancer Immune Therapy (CCIT-DK), Copenhagen University Hospital, Herlev, Denmark, 3Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Buenos Aires, Argentina, 4Danish Cancer Soceity Research Center, Copenhagen, Denmark, 5University College London Cancer Institute, London, United Kingdom, 6Lund University Cancer Center, Lund, Sweden
Text Neoantigen-driven recognition and T cell-mediated killing are suggested to contribute to tumor clearance following adoptive cell therapy (ACT) with Tumor-Infiltrating Lymphocytes (TILs). Consistently, tumor mutational burden and mutation-derived neoantigen load have been described as predictors of successful TIL-ACT. However, the impact on patient survival of diversity and frequency of expanded neoepitope-specific CD8 T cells has not yet been fully determined. Using barcoded pMHC multimers, we provide a comprehensive mapping of the presence of CD8+ T cells recognizing neoepitopes in TIL infusion products and blood samples from 26 metastatic melanoma patients who received ACT. We identify 106 neoepitopes within TIL infusion products corresponding to 1.8% of all predicted neoepitopes. We observe neoepitope- specific recognition to be virtually devoid in TIL infusion products given to patients with progressive disease outcome. Moreover, we find that the estimated frequency of neoepitope-specific CD8+ T cells in TIL infusion products correlates with increased survival. We observe that 62.5% of the neoepitope-specific T cell populations in the TIL infusion product is also detected within peripheral blood post-treatment, and that such engrafted neoepitope-specific CD8+ T cells are unique to responders of TIL-ACT. Finally, we find that lymphocyte activity within the tumor microenvironment correlates with the frequency of neoepitope-specific CD8+ T cells in the infusion product, suggesting this as an important parameter for the generation of a clinically successful TIL infusion product. These data propose that successful TIL-ACT is associated with an expansion of neoepitope-specific CD8+ T cells and that strategies to boost such neoepitope-specific T cell populations in the infusion products could increase the effectiveness of TIL-ACT.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-159
Simultaneous identification of antigen-specificity and T cell receptor sequence in single cells A. K. Bentzen, H. R. Povlsen, A. M. Marquard, M. Kadivar, U. K. Hansen, J. S. Holm, M. Nielsen, S. R. Hadrup Technical University of Denmark, Department of Health Technology, Kongens Lyngby, Denmark
Text T cells are essential for immune protection and play a critical role in the immune response to cancer, where they directly kill malignant cells or orchestra the response of other immune cells. A T cell response is initiated once a T-cell receptor (TCR) engages specifically with a peptide antigen (p) displayed in the MHC. Accurate specificity and broad coverage of pMHC antigens is obtained through somatic recombination of the genetic loci, V(D)J, that encodes the α and β chains of the heterodimeric TCR. The process creates an extensively variable and dynamic repertoire, with an estimated 107 distinct αβ TCRs in an individual. This variability combined with the vast number of potential pMHC targets makes it challenging to accurately assign a given TCR sequence with its antigen specificity. We are currently applying DNA barcode-labeled MHC multimers in a single cell platform, to allow identification of the T cell receptor sequence along with knowledge of the DNA barcode encoding the specific TCR-pMHC interaction. This provides a direct link of the αβ TCR sequence with the corresponding antigen specificity. We generate an array of MHC multimers, each labeled with a unique DNA barcode that constitutes a tag for the given pMHC specificity. Single T cells are isolated after incubation with such DNA barcode-labeled MHC multimers and the sequencing read-out integrates the TCR sequence and the antigen- specificity. We have applied this strategy to stain for >100 specificities in parallel and are currently implementing it at larger scale. We will use this platform for high-throughput capture of disease relevant TCRs from neo-and shared antigens in tumor infiltrating lymphocytes of patients receiving adoptive cell therapy. This will provide TCR sequences for potential therapeutic use, and provide novel insight to TCR diversity for a range of tumor antigens. By providing a large data set of TCR sequences matched with cognate pMHC specificity, this will moreover open the possibility for modeling of TCR interaction with pMHC. Thus, it will provide a first step towards resolving the relationship between the TCR sequence and the pMHC binding motif, which is a major gap in our understanding of T-cell reactivity.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-160
Monitoring the immune activation in recurrent glioblastoma patients treated with Nivolumab and Bevacizumab S. K. Skadborg1, S. Maarup2, A. Draghi3, A. Borch1, B. Hasselbalch2, H. S. Poulsen2, U. Lassen2, S. R. Hadrup1 1Technical University of Denmark, Health Technology, Kgs. Lyngby, Denmark, 2Rigshospitalet, Department of Oncology, Copenhagen, Denmark, 3Herlev Hospital, National Center for Cancer Immune Therapy, Herlev, Denmark
Text Glioblastoma (GBM) is a high grade glioma, and thus patient with this cancer type has a very low survival rate of 3% 5-year survival. As standard treatment in the primary setting GBM patients receive Stupp’s regime (radiotherapy and concomitant and adjuvant Temozolomide) after possibly neurosurgical resection. However, when patients relapse, treatment in the recurrent setting shows very limited effect.
We are monitoring the immune system of patients participating in a phase II clinical trial, where patients with recurrent GBM receive Nivolumab and Bevacizumab, an anti-PD1 – and anti-VEGF drug respectively. The clinical trial consists of two arms; arm A includes patients who are able to have the tumor removed through surgery, and arm B includes patients who are only able to receive medical treatment. Arm A has received Nivolumab 7 days prior to surgery.
Blood samples has been collected from patients through the course of treatment, wherefrom PBMCs has been purified. Additionally, single cells suspension was produced from the resected tumors. We are analyzing the collected patient samples using multi-color flow cytometry. With four different flow cytometry panels covering markers to distinguish a variety of cell types, including T cells, NK cells, B cells and myeloid cells, as well as detecting activing and inhibitory molecules, we can monitor the immune cells in the periphery and in the tumor.
Preliminary data has shown an infiltration of the tumor with T cells expressing CD103 as a tissue resident marker. Additionally, a general higher expression of inhibitory molecules was observed on such tumor infiltrating T cells. Additional flow cytometry data will be obtained prior to the conference and the clinical outcome of patients will be available for analyses in association with the immune characteristics.
With this project and clinical trial we will gain further knowledge of the “general” immune system’s role in clearance of tumors in the brain, and the capacity for infiltration to brain tumor lesions. This will guide us to the use, benefit and further development of immunotherapy in brain cancer.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-161
Longitudinal analysis of anti-tumor T-cell responses in vaccinated glioblastoma patients with improved survival N. Mensali1, I. Snapkov2, B. Mikkelsen Saberniak1, H. Vidarsdotter Juul1, S. Mjelstad Olafsrud1, D. Josefsen1, I. Langemoen3, 4, G. Kvalheim1, V. Greiff2, E. Vik-Mo3, 4, S. Wälchli1, E. M. Inderberg1 1Oslo University Hospital-The Norwegian Radium Hospital, Cellular Therapy, Oslo, Norway, 2Oslo University Hospital-Rikshospitalet, Department of Immunology, Oslo, Norway, 3Oslo University Hospital-Rikshospitalet, Department of Neurosurgery, Oslo, Norway, 4University of Oslo, Faculty of Medicine, Oslo, Norway
Text Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults with a median survival of 12–15 months. Several immunotherapeutic strategies are under investigation in GBM, but have not yet shown sufficient clinical efficacy. We previously performed a clinical trial investigating dendritic cell (DC)-based vaccine targeting cancer stem cells (CSC) in GBM (NCT00846456). mRNA from autologous CSCs in addition to mRNA encoding universal tumor antigens (telomerase and survivin) were transfected into autologous DCs and then injected intradermally. We detected specific T cell responses in the majority of the patients and three were long term survivors. To link survival with immune response, we investigated the T-cell repertoire at different locations. We detected both survivin- and telomerase-specific T-cell proliferation post-vaccination. The antigen specificity was confirmed in functional assays and we analyzed T-cell composition by TCR high-throughput sequencing (TCR-seq). In one patient, we further analysed cerebrospinal fluid (CSF) and biopsies taken >5 years after start of vaccination. T cells from CSF and tumor were predominantly CD4+ and displayed an effector memory phenotype with few dominant clones. TCR-seq analysis revealed substantial overlap between the TCR repertoire in blood samples and leukapheresis product pre-and post-vaccination. Tumor infiltrating lymphocytes (TILs) demonstrated high clonality and overlap with post-vaccination samples whereas T cells from CSF showed minimal overlap. Interestingly, TILs from a later biopsy upon tumor progression showed increased TCR diversity and phenotypic heterogeneity indicating reduced tumor specificity. Survivin-specific CD4+ T-cell clones were isolated from peripheral blood and CMV-specific CD4+ T cells from CSF. TCRs isolated from these clones and expressed in donor T cells were functional in CD4+ and, surprisingly, also in CD8+ T cells, producing TNF-α and IFN-γ upon recognition of cognate peptide. Interestingly, CD8+T cell clones isolated from TILs demonstrated reactivity against HLA-matched CSC. Work is still ongoing to identify the target antigen of these CD8+ TILs. Our data suggest that effector T cells were induced by vaccination and could be detected both in the periphery and tumor. Following these encouraging data, a randomized study (NCT03548571) comparing DC vaccine and standard treatment was initiated. Additional CSF samples and biopsies are obtained post-vaccination and are currently analysed to confirm specific clonal expansion of T cells. This will provide insight into how a successful anti-tumor response is constituted in GBM and can lead to the design of more efficient immunotherapy approaches.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-162
Characteristics of neoepitope-reactive CD8+ T cells as a predictive biomarker for clinical outcome to checkpoint inhibition therapy across various cancer types K. H. Moss, U. K. Hansen, S. R. Hadrup Technical University of Denmark, Health Technology, Kongens Lyngby, Denmark
Text Recent preclinical and clinical studies have highlighted the role of immune checkpoint blockade molecules in tumor-driven immune suppression. Immune checkpoint inhibitors have been approved as first-line or second- line therapies for an ever-expanding list of malignancies. However, these forms of therapies are not effective for the majority of patients. There is a growing interest in characterizing the role of T cells and identifying relevant antigens that play a part in the response to immune checkpoint inhibition, as T cells recognizing mutation-derived neoepitopes are hypothesized to be key mediators of tumor elimination. Tumor mutational burden has been thought to be a potential predictive biomarker for assessing the inclusion of patients in such immunotherapeutic trials. However, the presence, properties and dynamics of neoepitope-reactive T cells in the context of immune checkpoint inhibition are still minimally understood, especially in a pan-cancer setting. To explore this, tumor biopsies and peripheral blood were obtained pre- and post-treatment from 20 patients with solid metastatic tumors in a Phase I clinical trial. From whole-exome sequencing and RNAseq data, the total tumor mutational burden and number of non-synonymous mutations were obtained. Patient-specific libraries of neopeptides were predicted and screened with DNA barcode-labeled MHC multimers for T cell reactivity. An antibody phenotype panel was used in conjunction with MHC multimers to enable exploration into key characteristics of the neoepitope-reactive T cells. We observe a tendency for a higher mutational burden in the non-progressive disease group at both time points and a significantly higher number of predicted neopeptides. We detect neoepitope-reactive T cell populations across all patients. In the peripheral blood and tumor-infiltrating lymphocytes, the estimated frequency of neoepitope-reactive T cells increases after treatment initiation. Through dimensionality reduction techniques, we identify phenotypic differences in the neoepitope-reactive CD8 T cells between the non-progressive and progressive disease groups. Our preliminary data indicate the possibility of monitoring the breadth and magnitude of T cell immune responses during immune checkpoint inhibition whilst evaluating phenotypic properties of the neoepitope-reactive T cells, in order to guide patient selection and to facilitate the design of tailored precision immunotherapeutic approaches.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-163
A signature of peripheral blood T cells recognizing tumor-associated antigens in advanced melanoma patients under PD-1 blockade correlates with clinical outcome A. Gaißler1, C. Heeke2, S. Babaei3, T. S. Meldgaard2, S. A. Tvingsholm2, J. Bochem1, J. Spreuer1, T. Amaral1, R. Klein4, C. Garbe1, F. Meier5, G. Pawelec6, M. Claassen3, B. Weide1, T. Eigentler1, S. R. Hadrup2, K. Wistuba-Hamprech1, 3, 6 1University Medical Center Tübingen, Department of Dermatology, Tübingen, Germany, 2DTU health tech, Department of Health Technology, Copenhagen, Denmark, 3University Medical Center Tübingen, Department of Internal Medicine I, Tübingen, Germany, 4Eberhard Karls University Tübingen, Department of Immunology, Tübingen, Germany, 5Technical University Dresden, Dermatology, Dresden, Germany, 6Eberhard Karls University Tübingen, Department of Immunology, Tübingen, Germany
Text Metastatic melanoma is the deadliest form of skin cancer, hard to treat until the advent of checkpoint blockade (CB). Standard-of-care treatment now commonly includes the employment of antibodies blocking the binding of programmed death protein-1 (PD-1) on the T cell surface with its ligands. However, many patients progress or do not respond after treatment, and most do not experience remission. Due to their high mutational burden, melanomas present many neoepitopes as potential T cell targets, some of which are unique to the patient. However, to study neoepitope-specific T cells, tumor DNA needs to be sequenced, neoepitopes predicted and T cell reactivity tested for each individual patient. An alternative approach is to use a preselected set of peptide- loaded MHC multimers to test for the presence of T cell receptors specific for shared melanoma-associated epitopes (MAEs), which can be used for most patients and thus facilitate routine transfer to the clinic. The aim of the present study was to screen for peripheral T cells recognizing a preselected set of 167 MAE using a DNA-barcoded multimer-based, high throughput approach. We screened ex vivo cryopreserved peripheral blood mononuclear cells from 36 HLA-A*0201-positive stage IV melanoma patients before and during CB at a median of 42 days after initiation. In this cohort we detected a total of 121 MAE-specific T cell clones and determined individual T cell profiles per patient. We found that patients with an increase in the overall number of MAE-specific T cell clones under CB experienced a prolonged overall survival (OS) compared to those with constant or declining numbers thereof; a phenomenon that might be explained through CB-induced epitope spreading. Next, we applied a machine learning-based approach to search for shared MAE that are associated with the patients’ OS. Seven MAE-specific T cell clones were identified and used to develop a surrogate model. The latter indicated that a loss under CB of at least one of the seven MAE-specific T cell clones was associated with significantly worse OS. This led us to the conclusion that these MAE might play a special role in cancer rejection across different individuals. Validation of the results and functional investigations of these MAE-specific T cell responses is currently ongoing. We hypothesize that combining CB with vaccination against these particular MAEs might be advantageous for the majority of such patients in future.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-164
Mapping of HPV-specific CD8+ T cells and immune signatures in cervical intraepithelial neoplasia and cancer M. C. Viuff1, M. Kadivar1, D. Blirup1, J. Bonde2, B. Kirschner3, K. M. Jochumsen4, S. K. Kjær5, 6, S. R. Hadrup1 1Technical University of Denmark, Department of Health Technology, Kgs. Lyngby, Denmark, 2Copenhagen University Hospital, Department of Pathology, Hvidovre, Denmark, 3Copenhagen University Hospital, Department of Gynaecology and Obstetrics, Hvidovre, Denmark, 4Odense University Hospital, Department of Gynaecology and Obstetrics, Odense, Denmark, 5Danish Cancer Society Research Center, Copenhagen, Denmark, 6Rigshospitalet, Copenhagen University Hospital, Department of Gynecology, Copenhagen, Denmark
Text The development of cervical cancer is caused by persistent HPV infection and the oncogenic activity of the viral proteins E6 and E7. The disease progresses gradually from cervical intraepithelial neoplasia (CIN) to invasive cancer. Although most individuals clear an HPV infection, others are incapable of raising a sufficient immune response. In this study, we investigated the immunophenotypes characterizing HPV-related late-stage CIN3 and cervical cancer compared to healthy individuals; and recognition of specific HPV peptides by CD8+ T cells using a large library of peptides predicted for binding towards 14 different HLA types (n=1161). We analyzed the immunophenotypes in cervix samples and PBMCs collected from healthy donors (n=10) and from patients diagnosed with either CIN3 (n=10) or cervical cancer (n=15) prior to receiving therapy. Moreover, we compared cervix samples obtained from biopsies and the less invasive liquid-based cytology (LBC) method. Using multicolor flow cytometry, we identified a large proportion of tumor infiltrating CD8+ T cells exhibiting signs of late activation/exhaustion in the cervical cancer patients that could be associated to the cancer stage. In biopsies and LBC samples, these phenotypic characteristics were mainly limited to the cancer patients, whereas no considerable difference in immunophenotypes was observed between healthy individuals and CIN3 patients, suggesting that the anti-tumor immune response was not yet established in these HPV positive individuals. Furthermore, the expression of exhaustion markers was found to be comparable between biopsies and LBC samples, but not with PBMCs. Additionally, using DNA barcode-labelled pMHC multimers, we performed high-throughput screening of HPV peptides recognized by circulating CD8+ T cells in the same cohort (n=28). The MHC-I restricted HPV peptide library was generated from the viral proteins E2, E6 and E7 from both type 16 and 18 HPV. PBMCs from healthy donors and patients were screened for recognition of HPV peptides predicted to bind to their respective HLA types. CD8+ T cell recognition of E2-derived peptides was most predominant, and recognition of similar peptide sequences was seen across multiple individuals, suggesting these are immunogenic epitopes of potential clinical relevance. Taken together, these results show that cervical cancer patients elicit an ongoing but impaired immune response towards the tumor, and that they have raised a CD8+ T cell response towards HPV, which might be relevant for future immunotherapy.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-165
Langerhans islets induce anti-tumor immunity at the expense of glycemic control and predict chemotherapy response in pancreatic cancer A. Ahmed1, 2, P. Charoentong1, 2, R. Klotz3, S. Köhler1, M. Suarez-Carmona2, N. A. Valous1, 2, D. Ferber1, M. Heikenwälder2, N. Giese3, T. Hackert3, F. Klupp3, M. Schneider3, T. Schmidt3, C. Springfeld1, Y. Tanriver4, C. S. Falk5, L. Zitvogel6, I. Zörnig1, 2, D. Jäger1, 2, N. Halama1, 2 1National Center for Tumor Diseases, Heidelberg, Germany, 2German Cancer Research Center, Heidelberg, Germany, 3University Hospital Heidelberg, Heidelberg, Germany, 4University of Freiburg, Freiburg, Germany, 5University of Hannover, Hannover, Germany, 6Université Paris XI, Paris, France
Text We intended to investigate immunological and metabolic features of pancreatic ductal adenocarcinoma (PDA) patients to identify enhancers of anti-tumor immunity, decipher their interaction with glycemic control, and consequently improve clinical management strategies.
Patient-derived PDA tissues (n= 51) were used to analyze expression profiles of cytokines and immune cell infiltration patterns. Corresponding clinical information was used to perform survival analyses. Mechanistic studies were conducted in a human PDA explant model and by using human Langerhans islet cells. Multiplex protein assays and immunohistochemistry were performed to assess immune and metabolic phenotypes. Intracellular signalling alterations were analysed by multiplex phosphoprotein quantification. PDA cases (n= 2264) were screened for information about neoadjuvant chemotherapy, presence of type 2 diabetes mellitus (T2DM) and extractable glycated haemoglobin (HbA1c) levels.
We found that intratumoral C-C motif chemokine ligand 27 (CCL27) expression and tumour infiltration by
+ CD8 T cells are significantly associated with improved survival. CCL27 promotes a TH1 phenotype in the microenvironment resulting in an enhanced effector T cell infiltration. CCL27 is expressed by Langerhans islet cells and its expression is associated with T2DM. On a cellular level, regulated via STAT3, tumoural stress leads to increased CCL27 and impaired insulin secretion. T2DM and rise of HbA1c levels are significantly associated (p<0.0001) with response to neoadjuvant chemotherapy with fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX).
Langerhans islets promote anti-tumour immunity via CCL27, which is paralleled by a loss of glycemic control. These findings led to the identification of metabolic parameters (T2DM, HbA1c levels) as response markers for neoadjuvant chemotherapy with FOLFIRINOX.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-166
Blockade of PD-L1 installs an immunosuppressive TNF-α-linked response in lung tumor-infiltrating monocytes K. De Ridder1, H. Locy1, M. Ibarra Zuazo2, R. Maximillian Awad1, S. Verhulst1, Y. De Vlaeminck1, Q. Lecocq1, E. Reijmen1, W. De Mey1, L. De Beck1, T. Ertveldt1, D. Escors2, 3, M. Keyaerts4, K. Breckpot1, C. Goyvaerts1 1Vrije Universiteit Brussel, Laboratory for Molecular and Cellular Therapy, Jette, Belgium, 2Navarrabiomed- UPNA-IdiSNA, Immunomodulation Group, Pamplona, Spain, 3University College London, Rayne Institute, Division of Infection and Immunity, London, United Kingdom, 4Vrije Universiteit Brussel, In vivo Cellular and Molecular Imaging laboratory, Jette, Belgium
Text Introduction Clinical blockade of Programmed Death-1 (PD-1) or its ligand PD-L1 revolutionized the survival forecast for advanced non-small cell lung cancer (NSCLC). Yet, up to 75% of patients are refractory, signifying room for improvement. Recent observations hint towards a pivotal role for PD-(L)1+ lung tumor- infiltrating myeloid cells. Methods We evaluated the abundance, phenotype and function of 11 different myeloid subsets within an orthotopic squamous Lewis lung carcinoma (LLC) model over the course of anti-PD-L1 monoclonal antibody (mAb) treatment. Significant findings were correlated with human data from the lung squamous cell cancer (LUSC) Cancer Genome Atlas (TCGA) cohort. Results We confirm that LLC-bearing mice could serve as a model for anti-PD-L1 mAb treatment refractory LUSC since no clear therapeutic benefit was shown. The fractions of MHC-IIlow macrophages, Ly6Chi and Ly6Clow monocytes did significantly decrease in lung tumor tissue upon PD-L1 blockade. Furthermore, the serological concentration of TNF-α elevated significantly while gene set enrichment analysis of lung-sorted monocytes implied responsiveness to this TNF-α elevation. Combined TNF-α and anti-PD-L1 mAb treatment of monocytes resulted in an immunosuppressive phenotype, characterized by reduced CD80 and TIGIT expression next to an increase in the alternative checkpoint molecules VISTA, SIRPα, TIM-3 and LAG-3. Finally, we were able to validate this correlation between TNF-α expression and the latter checkpoint molecules within the human LUSC TCGA cohort. Conclusions These findings provide a rationale for combined blockade of TNF-α and PD-L1 to ameliorate the clinical response rates of squamous NSCLC patients to current PD-L1-targeted immunotherapy.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-167
Utility of the T-cell fraction metric in cancer prognosis using the immunoSEQ Assay S. Kaewert, P. Fields Adaptive Biotechnologies, Seattle, United States
Text Recent advances in immunosequencing have allowed for accurate quantitation of the T-cell immune repertoire, both in circulating cells and tumor infiltrating T cells. Immunosequencing has been used widely in the context of oncology, infectious disease and autoimmunity, among others, to both assess changes over time and as a read-out of immune competence. One metric that can be ascertained from immunosequencing is T-cell fraction (TCF), which is defined as the total number of T cells out of the total number of nucleated cells. T-cell fraction can be an informative metric for use in cancer studies, both on its own and combined with other measures, such as repertoire clonality or diversity. The current standard for measuring TCF is immunohistochemistry, which depends on staining and imaging and is restricted to single regions. While data generated by the immunoSEQ Assay lacks the spatial specificity of immunohistochemistry, it can capture a broader sampling. Also, since it is based on the presence of recombined T-cell receptors, it does not depend on staining efficiency. Here we demonstrate the utility of immunoSEQ T-cell fraction to provide insights into tumor-infiltrating T cells and its prognostic value in melanoma, colorectal cancer and squamous cell carcinoma. In all three cancer types, higher T-cell fraction is associated with better outcome in response to standard-of-care therapy. Together these studies demonstrate the broad applications of assessing T-cell fraction via the immunoSEQ Assay. Combining T-cell fraction with other immune repertoire metrics such as clonality or clonal expansion may allow for further insights in monitoring immune repertoire dynamics in response to oncology therapies and other treatments.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-168
Tumor-infiltrating plasmacytoid dendritic cells are associated with survival in human colon cancer I. Plesca1, M. Kießler1, U. Sommer2, R. Wehner1, 3, 4, F. Wilczkowski1, L. Müller1, A. Tunger1, 3, X. Lai1, A. Rentsch5, K. Peuker6, 7, S. Zeissig6, 7, A. M. Seifert3, 8, L. Seifert3, 8, J. Weitz3, 4, 8, M. Bachmann3, 4, 5, 7, 9, M. Bornhäuser3, 4, 5, 6, 7, D. Aust2, 3, 4, G. Baretton2, 3, 4, M. Schmitz1, 3, 4, 7 1Institute of Immunology, Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany, 2Institute of Pathology, University Hospital Carl Gustav Carus, Dresden, Germany, 3National Center for Tumor Diseases (NCT), Partner Site Dresden, Dresden, Germany, 4German Cancer Consortium (DKTK), Partner Site Dresden, and German Cancer Research Center (DKFZ), Heidelberg, Germany, 5University Cancer Center, University Hospital Carl Gustav Carus, Dresden, Germany, 6Department of Medicine I, University Hospital Carl Gustav Carus, Dresden, Germany, 7Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany, 8Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Dresden, Germany, 9Institute of Radiopharmaceutical Cancer Research, Helmholtz Center Dresden-Rossendorf (HZDR), Dresden, Germany
Text Plasmacytoid dendritic cells (pDCs) play a key role in the induction and maintenance of anti-tumor immunity. Conversely, they can act as tolerogenic DCs by inhibiting tumor-directed immune responses. Therefore, pDCs may profoundly influence tumor progression. To gain novel insights into the role of pDCs in colon cancer, we investigated the frequency and clinical relevance of pDCs in primary tumor tissues from colon cancer patients. Immunohistochemical stainings were performed to explore the frequency of colon cancer-infiltrating BDCA-2+ pDCs. Statistical analyses were conducted to determine an association between the pDC density and clinicopathological characteristics of the patients. Furthermore, we utilized multiplex immunofluorescence stainings to evaluate the localization and phenotype of pDCs in stroma and tertiary lymphoid structures (TLS) of colon cancer tissues. An increased density of infiltrating pDCs was associated with lower UICC stages. Furthermore, a higher pDC frequency was significantly correlated with increased progression-free and overall survival of colon cancer patients. Moreover, a lower number of colon cancer-infiltrating pDCs was significantly and independently linked to worse prognosis. In addition, we found that a proportion of pDCs shows a nuclear expression of the transcription factor interferon regulatory factor 7 (IRF7), which is characteristic for an activated phenotype. In various tumor stroma regions, IRF7+ pDCs were located in the neighborhood of granzyme B-expressing CD8+ T cells. Moreover, pDCs were identified as a novel component of the T cell zone of colon cancer-associated TLS, which are major regulators of adaptive anti-tumor immunity. A proportion of TLS-associated pDCs displayed a nuclear IRF7 expression and was preferentially distributed in the proximity of CD4+ T cells. These results indicate that higher densities of tumor-infiltrating pDCs are associated with prolonged survival of colon cancer patients. Moreover, colon cancer-infiltrating pDCs may represent a novel prognostic factor. The co-localization of activated pDCs and T cells in tumor stroma and within TLS may contribute to the correlation between higher pDC densities and better prognosis. In addition, our findings may have implications for the design of novel immunotherapeutic strategies that are based on targeting colon cancer-infiltrating pDCs.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-169
Characterization of pre-existing and post-infectious immune responses to SARS-CoV-2 in cancer patients T. Bilich1, 2, 3, M. Roerden2, 3, 4, Y. Maringer1, 2, 3, A. Nelde1, 2, 3, J. S. Heitmann1, M. L. Dubbelaar1, 2, 3, 5, A. Peter6, S. Hörber6, J. Bauer1, 2, 3, J. Rieth1, 2, M. Wacker1, 2, 3, S. Walz7, E. Erne7, F. Berner8, L. Flatz8, 9, S. Held10, P. Brossart10, M. Märklin1, 3, H.-G. Rammensee2, 3, 11, H. R. Salih1, 3, J. S. Walz1, 2, 3, 12 1Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Department of Internal Medicine, University Hospital Tübingen, Tübingen, Germany, 2Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen, Germany, 3Cluster of Excellence iFIT (EXC2180) “Image- Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany, 4Department of Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tübingen, Tübingen, Germany, 5Quantitative Biology Center (QBiC), University of Tübingen, Tübingen, Germany, 6Institute for Clinical Chemistry and Pathobiochemistry, Department for Diagnostic Laboratory Medicine, University Hospital Tübingen, Tübingen, Germany, 7Department of Urology, University Hospital Tübingen, Tübingen, Germany, 8Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen, Switzerland, 9Department of Dermatology, University Hospital Tübingen, Tübingen, Germany, 10Department for Hematology and Oncology, University Hospital Bonn, Bonn, Germany, 11German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), partner site Tübingen, Tübingen, Germany, 12Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and Robert Bosch Center for Tumor Diseases (RBCT), Stuttgart, Germany
Text Cancer patients are at increased risk for critical illness upon COVID-19. We here assessed antibody and T cell responses in unexposed and SARS-CoV-2 infected cancer patients to characterize SARS-CoV-2 immunity and to identify immunological parameters contributing to COVID-19. Immune responses were assessed in unexposed (n = 199) and SARS-CoV-2-infected cancer patients (n = 17), comprising different hematological malignancies (HM) and solid tumors. SARS-CoV-2 convalescents without malignant disease (n = 193) and unexposed healthy volunteers (HV, n = 94) served as reference groups. Whereas pre-existing SARS-CoV-2 cross-reactive CD4+ T cell responses were detectable in a high proportion of HV (78%) and similarly also in solid tumor patients (77%), preexisting T cell responses were substantially lower in unexposed HM patients (34%) as assessed by in IFN-y ELISPOT assays using previously defined cross-reactive SARS-CoV-2 T cell epitopes (Nelde et al., Nature Immunol, 2021). This was in line with an increased proportion of CD4+ T cells expressing PD-1, LAG-3, and TIM-3 in HM compared to solid tumor patients and HV, pointing to T cell exhaustion as underlying cause for reduced T cell reactivity. Within SARS-CoV-2 convalescents, no difference in antibody positivity was observed between HM and solid tumors as well as cancer patients in general and HV. With regard to T cell responses, no significant difference in recognition frequency to SARS-CoV-2-specific HLA class I and HLA-DR epitopes was observed, whereas the frequency of T cell responses to the HLA-DR cross-reactive epitopes was significantly reduced in cancer patients compared to HV. Alike in unexposed cancer patients, this could be attributed to a markedly reduced frequency of cross-reactive CD4+ T cell responses in patients with HM (38%) compared to both, solid tumor patients (78%) and HV (87%). Analyses of SARS-CoV-2 T cell responses to single HLA-DR peptides after in vitro expansion revealed reduced expandability of SARS-CoV-2 T cells for 73% of SARS-CoV-2-derived peptides in COVID-19 cancer patients compared to HV. Moreover, the diversity of SARS-CoV-2 CD4+ T cell responses, i.e. the recognition of multiple different T cell epitopes, was significantly reduced in COVID-19 HM patients (20%) compared to both, solid cancer patients (35%) and HV (50%). Alike in non-cancer convalescents, reduced T cell diversity associated with a more severe course of COVID-19 in cancer patients. Together, our results identify impaired SARS-CoV-2 T cell immunity as determinant for dismal outcome of COVID-19 in cancer patients in particular in patients suffering from HM, which guides development of therapeutic measures and vaccines for this vulnerable patient population.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-170
Pan-cancer expression of Cancer Germline Antigens is related to mechanisms of T cell evasion D. Kortleve, R. Coelho, D. Hammerl, R. Debets ErasmusMC, Medical Oncology, laboratory of Tumor Immunology, Rotterdam, Netherlands
Text Cancer Germline Antigens (CGAs) are recognized for their expression during gametogenesis in immune- privileged germline tissues, while the expression of CGAs is epigenetically silenced in somatic tissues. Notably, CGAs are re-expressed in tumors and directly promote oncogenesis, for which reason certain CGAs have been explored as targets for immune therapy. We questioned whether CGA expression is linked to immune evasion in a pan-cancer manner, and whether CGAs differentially affect mechanisms of T cell evasion. To this end, we have interrogated gene transcripts of tumors of the skin, head-and-neck, bladder, prostate, breast, lung and pancreas, and demonstrated that a subset of CGAs significantly correlates with the presence or absence of T cells. Zooming in on potential underlying mechanisms, we identified 7 CGAs, namely ACRBP, IL13RA2, TMEM108, GPATCH2, IMP3, TSGA10, ZNF165, the expression of which showed a, for some previously unrecognized, relationship with either aberrant influx, antigen-recognition and/or function of T cells in multiple tumor types. Here, we present the selection of these 7 CGAs, and provide an overview of reports on functional determinants that link this set of CGAs to T cell evasion, as well as the relevance of these CGAs to future immune therapeutic interventions.
Key words: Cancer Germline Antigens (CGA); Tumor Infiltrating Lymphocytes (TILs); Pan-cancer; CD8 T cell evasion; Influx; Function; Antigen recognition
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-171
The role of TLR4 signaling in myeloid cell-mediated immune suppression in malignant melanoma F. G. Özbay Kurt1, 2, 3, R. Weber1, 2, J. Utikal1, 2, V. Umansky1, 2, 3 1German Cancer Research Center (DKFZ), Skin Cancer Unit, Heidelberg, Germany, 2University Medical Center Mannheim, Department of Dermatology, Venereology and Allergology, Mannheim, Germany, 3University of Heidelberg, Faculty of Biosciences, Heidelberg, Germany
Text Immunotherapeutic strategies in malignant melanoma have not yet reached their full potential mainly due to the resistance developed by a considerable subset of patients. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid cells, have been shown to accumulate in the melanoma microenvironment. With the ability to inhibit anti-tumor T cell responses, MDSCs are likely to be one of the key drivers of the local immunosuppression, promoting tumor progression and tumor cell resistance to therapeutic strategies. TLR4 signaling has been shown to be involved in the upregulation of PD-L1 expression and induction of immunosuppressive properties of human CD14+ cells. Nevertheless, the role of TLR4 signaling in the acquisition of MDSC immunosuppressive properties remains to be better elucidated. Hence, this study aims to evaluate the role of TLR4 signaling on MDSC-mediated immune suppression in malignant melanoma. MDSCs were purified from peripheral blood of late-stage melanoma patients and bone marrow of melanoma- bearing mice. For in vitro generation of MDSCs with IL-6, GM-CSF, and TLR4 agonist LPS, peripheral blood monocytes of healthy volunteers and bone marrow cells from wild type mice were used. The immunosuppressive capacity of MDSCs was assessed in the functional assays with T cells. In addition, TLR4 inhibitor (Resatorvid) was tested in both human and murine T cell suppression assays. The blockade of TLR4 signaling led to substantial attenuation of the suppression of T cell proliferation mediated by in vitro generated MDSCs. This effect was independent of the presence of TLR4 ligand LPS. On the other hand, Resatorvid did not alter the immunosuppression in murine T cell suppression assays established with MDSCs either generated in vitro or derived from tumor-bearing mice. In the absence of GM- CSF and IL-6, the treatment with TLR4 ligand alone did not convert normal monocytes into immunosuppressive MDSCs. However, monocytes treated with TLR4 ligand possessed a lower capacity to stimulate T cell proliferation in the presence of anti-CD3 monoclonal antibodies as compared to untreated monocytes. We will further investigate a possible correlation between the amount of TLR4 ligands present in the serum of patients or tumor-bearing mice and MDSC immunosuppressive capacity. Furthermore, the effect of IL-6 and GM-CSF in our in vitro system on the production of TLR4 ligands and TLR4 signaling will be studied.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-172
A standardized analysis of tertiary lymphoid structures in human melanoma: disease progression- and tumor site-associated changes with germinal center alteration F. Werner1, C. Wagner1, M. Simon1, K. Glatz2, K. D. Mertz3, 4, H. Läubli5, J. Griss6, S. N. Wagner1 1Laboratory of Molecular Dermato-Oncology and Tumor Immunology, Department of Dermatology, Medical University of Vienna, Vienna, Austria, 2Institute of Medical Genetics and Pathology, University Hospital Basel, University Basel, Basel, Switzerland, 3Institute of Pathology, Cantonal Hospital Baselland, Liestal, Switzerland, 4University Hospital Basel, Basel, Switzerland, 5Laboratory for Cancer Immunotherapy, Department of Biomedicine and Medical Oncology, Department of Internal Medicine, University Hospital Basel, Basel, Switzerland, 6Department of Dermatology, Medical University of Vienna, Vienna, Austria
Text There is increasing evidence that tertiary lymphoid structures (TLS) not only control local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. As in secondary lymphoid organs, lymphoid structures in cancer can undergo different maturation stages with variable cellular composition and differentiation. In human melanoma, however, a comprehensive analysis of TLS density, phenotypes and spatial distribution in different disease stages is lacking. Here we used seven color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution was determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely form germinal centers and mostly locate intratumorally within 1 mm distance to the invasive tumor front. The presence of these TLS was not associated with established clinicopathological prognostic factors. In contrast, melanoma metastases have a significantly increased density of secondary follicular TLS. They appear preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varies over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lack the presence of BCL6+ lymphatic cells as well as canonical germinal center polarity with formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotype, density and spatial distribution. The frequent lack of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. In view of the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach. This provides a high degree of reproducibility and should contribute to the development of a future generally accepted evaluation standard for TLS analysis in human cancer tissues.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-173
Human ovarian cancer ascites impairs anti-tumor effector functions of NK cells A. Hrvat, M. Schmidt, N. Mallmann-Gottschalk, S. Brandau University Hospital Essen, Exp and Translational Research, Dept. of Otorhinolaryngology, Essen, Germany
Text Ovarian cancer is often detected in an advanced stage when the already developed metastasis and ascites cause significant tumor burden, immunosuppression, and therapy failure. In this project, we wanted to mechanistically analyze the effects of the ascites on NK cell function. To this end, we obtained human ascites samples from patients with ovarian cancer and investigated their effect on tumor and NK cell interaction. Biochemical and biophysical characterization of ascites fluid revealed substantial differences between ascites and human control serum or cell culture medium. Cytokine analysis showed high levels of immunosuppressive cytokines in ascites. NK effector functions, such as IFN-γ secretion, degranulation, conjugation to target cells as well as tumor lysis were inhibited by ascites. Subsequent studies elucidated several candidate molecules that potentially mediated these suppressive effects. Due to the anti-adhesive properties of the ovarian cancer ascites, long-chained mucin molecules were suspected to cause steric interference during cellular interactions. Although some of the candidates like IgGs and albumin are normal constituents of the human serum, they were still found to induce an immunosuppressive effect. Furthermore, human serum samples were shown to be inhibitory in the coculture assay, albeit to a smaller degree. These results would imply the existence of an innate or more physical immunosuppressive and immunoregulatory mechanism, present in various body compartment fluids. This mechanism could present a difficult obstacle in moving the cancer immunotherapy from in vitro to in vivo and could explain why immunotherapy often performs with low efficacy and demands much higher doses of therapeutic molecules compared to in vitro conditions. Based on our findings, we propose a model in which ascites components inhibit the conjugate formation of NK cells to tumor targets. This leads to antibody-dependent cellular cytotoxicity (ADCC) failure and impaired natural cytotoxicity.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-174
T and B cell abundance are strongly reduced in the immune microenvironment of post-transplant malignancies. R. R. Datta1, 2, S. Schran2, O.-D. Persa3, C. Aguilar1, M. Thelen2, J. Lehmann2, M. A. Garcia-Marquez2, K. Wennhold2, E. Preugszat2, A. Quaas4, C. Bruns1, C. Mauch3, H. Löser4, D. Stippel1, H. A. Schlößer1, 2 1University Hospital Cologne, Department of General, Visceral and Cancer Surgery, Cologne, Germany, 2University Hospital Cologne, Center for Molecular Medicine Cologne, CMMC, Cologne, Germany, 3University Hospital Cologne, Department of Dermatology, Cologne, Germany, 4University Hospital Cologne, Department of Pathology, Cologne, Germany
Text Immunosuppressive medication is mandatory in the majority of solid organ transplant recipients to reduce the risk of allograft rejection. An increased risk to develop cancer is a negative side effect of long-term immunosuppression. However, the impact of immunosuppression on the tumor immune microenvironment (TME) is poorly understood. We aimed to elucidate differences in the TME of post-transplant malignancies and non- immunosuppressed cancer patients. 117 primary tumor samples (formalin fixed and paraffin embedded, FFPE) from 80 organ recipients (kidney, heart, lung and liver) were included. Immunohistochemistry of FFPE whole section slides was used for digital image analysis to assess abundance and localization of T cells (CD3, CD8) and B cells (CD20) in the TME of 14 different cancer types. These data were used to calculate the Immune-score and to quantify tertiary lymphoid structures in the TME. Impaired HLA-I expression and expression of PD-L1 as important immune escape mechanisms were also included. Results were compared to a matched cohort of cancer samples from non-immunosuppressed control patients. In line with an increased risk of cancer in solid organ transplant recipients we found a remarkably reduced immune infiltrate in the center (CT) as well as the invasive margins (IM). T cell abundance was significantly decreased in IM and CT of skin and non-skin tumors when compared to non-immunosuppressed controls. These differences were more pronounced in the IM than in the CT and larger when assessing the abundance of CD8+ T cells. The Immune-score integrating results from CT and IM was also decreased in transplant recipients. Similar to the results observed for T cells, B cell abundance and density of tertiary lymphoid structures were lower in cancer samples of transplant recipients. Decreased expression of HLA-I was more common in transplant recipients whereas the fraction of samples with PD-L1 expression seemed to be higher in controls. Our study supports the hypothesis of reduced anti-tumor immune response as important mechanism underlying increased risk of cancer in solid-organ recipients. This could be a result of impaired immune recognition as well as impaired attraction of lymphocytes into the TME. Optimized immunosuppressive protocols may reduce cancer incidence and cancer therapies need to consider the distinct immune microenvironment of post-transplant malignancies.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-175
Subtyping for B cell-and antibody secreting cell-subsets in human melanoma reveals age-, metastasis- and tumor stage- associated changes M. Chen1, F. Werner1, C. Wagner1, M. Simon1, E. Richtig2, K. D. Mertz3, 4, J. Griss5, S. N. Wagner1 1Laboratory of Molecular Dermato-Oncology and Tumor Immunology, Department of Dermatology, Medical University of Vienna, Vienna, Austria, 2Department of Dermatology, Medical University of Graz, Graz, Austria, 3Institute of Pathology, Cantonal Hospital Baselland, Liestal, Switzerland, 4University Hospital Basel, Basel, Switzerland, 5Department of Dermatology, Medical University of Vienna, Vienna, Austria
Text The role of tumor-associated B cells in human cancer is only starting to emerge. B cells typically undergo a series of developmental changes in phenotype and function, however, data on the composition of the B cell population in human melanoma are largely absent including changes during tumor progression and their potential clinical significance. In this study, we compared the number and distribution of six major B cell and antibody secreting cell subpopulations in whole tumor sections of 154 human cutaneous melanoma samples (53 primary tumors without subsequent metastasis, 44 primary tumors with metastasis, 57 metastatic samples) obtained by seven color multiplex immunohistochemistry and automated tissue imaging and analysis. In primary melanomas, we observed the highest numbers for plasmablast-like, memory-like and activated B cell subtypes. These B cell subpopulations showed a patchy distribution in the paratumoral and sometimes intratumoral stroma. Plasma cell-like cells were hardly detected, germinal center- and transitional/regulatory- like B cells not at all. Of the major clinicopathological prognostic factors for primary melanomas, metastasis was associated with decreased memory-like B cell numbers and a higher age and Breslow depth associated with high plasmablast-like cell numbers. When we compared the composition of the B cell population in primary melanomas and metastatic samples, we found a significantly higher proportion of plasma cell-like cells at distant metastatic sites and a higher proportion of memory-like B cells at locoregional than distant metastatic sites. These data provide a first comprehensive and comparative morphological analysis of major B cell and antibody secreting cell subpopulations in human melanoma and describe age-, metastasis- and tumor stage- associated changes, an important premise for B cell-related biomarker and therapy studies.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-176
Immune microenvironment profiling of breast cancer brain metastases using multiplex immunofluorescence A. Tosi1, G. Griguolo1, 2, V. Guarnieri1, 2, M. V. Dieci1, 2, V. Rossi3, S. Fineberg4, A. Ventura2, L. Bauchet5, J. Jacob6, V. Rigau7, W. Jacot8, A. Darlix8, P. F. Conte1, 2, A. Rosato1, 2 1University of Padua, Department of Surgery, Oncology and Gastroenterology, Padova, Italy, 2Veneto Institute of Oncology, Padova, Italy, 3University of Padua, Padova, Italy, 4Albert Einstein Collage of Medicine/Montefiore Medical Center, Bronx, United States, 5Neurosurgery CHU Montpellier, Montpellier, France, 6Department of Pathology - Beth Israel Deaconess Medical Center, Boston, United States, 7Pathological Department CHU Montpellier, Montpellier, France, 8Department of Medical Oncology, Institute du Cancer de Montpellier, Montpellier, France
Text Despite potential clinical implications, the complexity of immune microenvironment in breast cancer (BC) brain metastases (BM) is still poorly understood. Multiplex immunofluorescence (mIF) allows simultaneous visualization and quantification of several markers while maintaining spatial information. This novel technique can be used to comprehensively describe BCBM immune microenvironment, potentially providing useful information to guide novel therapeutic approaches. BM samples were collected for 60 BC patients undergoing neurosurgery (2003-2018) at three institutions. BCBM immune contexture was characterized using two custom mIF panels, including cell subtyping (CD4, CD8, FoxP3, CD68), activation (Granzyme B), localization (cytokeratin for tumor recognition) markers and immune checkpoint and co- inhibitory molecules (PD1, PD-L1, TIM3, LAG3, CD163). Associations between immune cell densities, spatial proximity metrics, BC subtype and overall survival (OS) from BM diagnosis were studied. Sixty BCBM samples were analyzed: 32% HR+/HER2-, 38% HER2+, 30% HR-/HER2-. At a median follow-up of 43 months, the only clinical variable associated with OS was BC subtype (shortest for HR-/HER2- and longest for HER2+, log-rank p=0.02). In the tumor area, HR+/HER2- tumors showed higher density of CD68+ cells compared to other subtypes, while a higher percentage of CD8+Granzyme B+ T lymphocytes was observed in HR-/HER2-. High CD163+ M2-polarized macrophage density was significantly associated with worse OS, even after correction by BC subtype. In the HR-/HER2- BCBM subgroup, high intra-tumoral densities of CD4+, CD8+ and CD8+Granzyme B+ T lymphocytes, high percentage of tumor cells within a 25 µm radius from CD8+ cells and low percentage of CD3+ cells within a 20 µm radius from CD163+ macrophages, were associated with longer OS. In the subgroup of patients with HR+/HER2- BCBM, high TIM3+ cell density in the stroma, low percentage of CD3+PD-1+ T cells and a longer distance between intra-tumoral CD163+ macrophages and CD3+ T lymphocytes were associated with increased OS. Furthermore, low density of CD3+PD-1+LAG3+ T cells and low percentage of intra-tumoral PD-1+ cells within a 20 µm radius from PD-L1+ cells were associated with better OS in HER2+ BCBM subgroup. In conclusion, in BCBM the immune infiltrate differs according to BC subtype. M2 macrophage polarization is consistently associated with worse OS across all BC subtypes, and activated T cells infiltration and checkpoint expression are correlated with patient outcome. These results suggest that a more tolerogenic immune microenvironment is associated with worse OS, and might represent a potential target for optimization of immunotherapy for BCBM patients.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-177
Gangliosides shift as prerequisite and mechanism of resistance by anti-GD2 therapy C. Paret1, A. Wingerter1, R. Sandhoff2, L. Seidmann3, N. Lehmann1, K. El Malki1, A. Russo1, H. Otto1, J. Faber1 1University Medical Center of the Johannes Gutenberg-University Mainz, Department of Pediatric Hematology/Oncology, Mainz, Germany, 2German Cancer Center (DKFZ), Lipid pathobiochemistry, Heidelberg, Germany, 3University Medical Center of the Johannes Gutenberg-University Mainz, Institute of Pathology, Mainz, Germany
Text Gangliosides are sialic acid-containing glycosphingolipids that are particularly abundant in the nervous system. The synthesis of gangliosides is a stepwise process regulated by the cross-talk of different enzymes and the pattern of gangliosides undergoes dramatic changes during brain development. In embryonic brains, the predominant gangliosides are simple GM3 and GD3 and the intermediate product GD2. As the brain develops, the expression of these gangliosides is down-regulated and complex gangliosides such as GM1, GD1a, GD1b and GT1b are up-regulated. During tumorigenesis, simple gangliosides are re-expressed on neuroectodermal-derived cancers and sarcoma, where they are considered as oncofetal markers. Due to this expression pattern, GD2 has been long considered a relevant target for cancer immunotherapy. The anti-GD2 antibodies naxitamab and dinutuximab are already released for the treatment of neuroblastoma and CAR-T cells are in clinical testing also in other tumor entities. However, tumors may become resistant under the therapy. Here we hypothesized that the perturbation of the gangliosides synthesis is required for achieving clinically relevant amounts of GD2 on tumor cells, but it can also account for resistance to anti-GD2 therapy. To assess this hypothesis, we analysed the expression of gangliosides by mass spectrometry and flow cytometry in longitudinal samples of an Ewing’s Sarcoma patient treated off-label with dinutuximab. While GD2 was not expressed at diagnosis, its expression increased stepwise under standard treatment with chemotherapy, supporting an off-label application of dinutuximab. Under dinutuximab treatment, a metastasis developed in the skull. The metastasis was partially GD2 negative due to the stop of the synthesis of gangliosides at the level of GM3. In conclusion, a shift in the gangliosides profile toward GD2 under chemotherapy allowed the treatment of the patient with dinutuximab. However, a further shift allowed resistance to the same therapy. GM3 is also a cancer specific antigen, therefore co-targeting GM3 and GD2 may represent a powerful and safe option to avoid the development of resistances. It remains to be elucidated if this mechanism is specific for Ewing’s Sarcoma or applies also to other tumor entities.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-178
MicroRNA-mediated generation of myeloid-derived suppressor cells in malignant melanoma S. Lasser1, 2, L. Fritz1, 2, J. Utikal1, 2, P. Altevogt1, 2, V. Umansky1, 2 1University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Department of Dermatology, Venereology and Allergology, Mannheim, Germany, 2German Cancer Research Center (DKFZ), Skin Cancer Unit, Heidelberg, Germany
Text Avoiding immune destruction through the generation of myeloid-derived suppressor cells (MDSC) is a crucial process in the successful treatment of malignant melanoma. An important mechanism of MDSC generation was found to be linked to extracellular vesicles (EV), constitutively produced by melanoma cells. Melanoma- derived EV contain various microRNAs (miRs) that can influence the expression of target genes or have a signaling function. Here we studied how miRs found in melanoma-derived EV can stimulate the acquisition of MDSC function by normal myeloid cells. The exposure of murine immature myeloid cells (IMC) to miR-125a and miR-125b led to an upregulation of PD-L1, indicating the acquisition of an immunosuppressive phenotype. Since certain miRs can bind to endosomal Toll-like receptors (TLR), thereby inducing a prometastatic inflammatory response, TLR7- deficient IMC were tested for their capability to respond to miR treatment. Interestingly, upregulation of PD- L1 by miR-125a and miR-125b was also observed in TLR7-deficient IMC. Analyzing further the involvement of TLR signaling, IMC lacking either MyD88 or both MyD88 and TRIF were treated with miR-125a or miR- 125b. However, PD-L1 upregulation on IMC appeared to be independent of MyD88 and TRIF. Since the miRs seem to be a strong trigger for immunosuppressive functions of the myeloid cells, we will further investigate the underlying mechanism that could provide novel approaches to target the generation of MDSC.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-179
How miRNAs affect the expression of immune checkpoint molecules T. Kordaß1, 2, T.-Y. Chao1, W. Osen1, S. Eichmüller1 1DKFZ, GMP & T-Zelltherapie, Heidelberg, Germany, 2University Heidelberg, Faculty of Biosciences, Heidelberg, Germany
Text Expression of immune checkpoint molecules by cancer cells can counter-act tumor reactive immune responses, for example by reducing the susceptibility of tumor cells to cytotoxic T cell (CTL) mediated cytolysis, thereby promoting tumor immune evasion. miRNAs are small non-coding RNAs involved in post- transcriptional regulation. Thus, binding of miRNAs to the 3`-UTR of target mRNAs can block translation or lead to degradation of the targeted mRNA. Cancer cells often exhibit aberrant miRNA expression profiles, thus tumor derived miRNAs can be utilized as biomarkers for early tumour detection. Within this study we conducted a FACS-based human miRNA library screen and determined miRNAs affecting surface expression of the immune checkpoint molecules NT5E (CD73), ENTPD1 (CD39) and PD-L1 (CD274) on the human tumor cell lines SK-Mel-28 (melanoma) and MDA-MB-231 (breast cancer). We could identify a set of potential tumour-suppressor miRNAs that decreased expression of the immune checkpoints molecules in these cell lines, as well as a group of potential oncomirs that induced increased immune checkpoint molecule expression. The results of the high-throughput screen for NT5E were verified in a validation process including up to 12 distinct cancer cell lines. Thus, four candidate miRNAs (candidate #1 to #4) were determined as potent inhibitors of NT5E expression. Luciferase-based reporter assays proved that these NT5E inhibitory miRNAs act through direct binding to the NT5E 3’-UTR. Using functional malachite-green assays the net effect of reduced NT5E expression on adenosine production caused by candidates #1 and #2 was assessed. As adenosine is known to inhibit effector function of cytotoxic T cells, miRNA mediated alterations in NT5E expression might affect the susceptibility of cancer cells to T cell mediated cytolysis. Moreover, we determined another three candidate miRNAs that could enhance NT5E expression most effectively. Notably, the observed increase of NT5E was very consistent across a panel of melanoma and breast cancer cell lines. Different from the NT5E inhibiting miRNAs above, we suspect indirect mechanisms for the miRNA-mediated enhancement of NT5E expression. In summary, microarray expression profiling was performed on miRNA transfected tumor cells to unravel the mechanisms responsible for the up-regulated NT5E expression observed.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-180
Tumor-cell-intrinsic HMGB1 impairs the antitumor immune response B. Taskoparan1, 2, V. Puddinu1, 2, L. Spagnuolo1, 2, 3, C. Bourquin1, 2, 4 1University of Geneva, School of Pharmaceutical Sciences, Geneva, Switzerland, 2University of Geneva, Institute of Pharmaceutical Sciences of Western Switzerland, Geneva, Switzerland, 3The Netherlands Cancer Institute, Division of Tumor Biology and Immunology, Amsterdam, Netherlands, 4University of Geneva, Faculty of Medicine, Department of Anaesthetics, Pharmacology, Intensive Care and Emergencies, Geneva, Switzerland
Text The High Mobility Group Box 1 (HMGB1) protein is a highly conserved nuclear protein with multiple intracellular and extracellular functions, including transcriptional regulation, as well as the promotion of inflammation and cell migration. In this study, we aimed to determine the impact of cancer-cell-intrinsic HMGB1 on anticancer immunity. Wild type or HMGB1low CT26 mouse cancer cells were injected subcutaneously to induce tumors in mice. We observed that the levels of HMGB1 profoundly influenced tumor development: Downregulation of intratumoral HMGB1 blocked the growth of tumors. Importantly, HMGB1low tumors showed increased infiltration of CD4+ T cells and CD11b+ F4/80+ myeloid cells, as well as an increase in the numbers of tumor-specific cytotoxic T cells. Spleens of mice bearing HMGB1low tumors showed higher numbers of total CD4+ and CD8+ T cells than those of mice bearing wild-type tumors, which suggests a systemic modulation of the immune landscape. When induced in immune-deficient NSG mice, HMGB1low tumors grew at the same rate as WT tumors, demonstrating that growth inhibition of these tumors in immunocompetent mice was immune-mediated. RNA sequencing analysis of the bulk tumors, as well as the cell lines and isolated tumor cells from the tumor microenvironment further supported the fact that the reduction in tumor growth was immune-mediated. While HMGB1low bulk tumors displayed enrichment of differentially expressed genes in pathways such as antigen presentation, TcR, and cytokine signaling, HMGB1low cell lines and isolated tumor cells showed enrichment in pathways such as focal adhesion, integrin signaling, and ECM-receptor interaction. Further investigations are ongoing to decipher the underlying mechanism. Overall, we propose that tumor-cell-intrinsic HMGB1 plays a critical role in limiting anticancer immune responses and that HMGB1 can become a novel target for improving the outcome of cancer immunotherapy.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-181
Epithelial to Mesenchymal Transition Regulates Surface PD-L1 via CMTM6 and CMTM7 Induction in Breast Cancer M. Xiao, M. Hasmim, B. Janji, M. Z. Noman Luxembourg Institute of Health, Department of Oncology - Tumor Immunotherapy and Microenvironment group, Luxembourg City, Luxembourg
Text CMTM6 is a critical regulator of cell surface expression of PD-L1 in tumor cells, but little is known about the transcriptional regulation of CMTM6. Here we report that the expression of CMTM6 positively correlates with the epithelial to mesenchymal transition (EMT) score in breast cancer cell lines and with the major EMT marker Vimentin in triple negative breast cancers (TNBC). We showed that CMTM6 is concomitantly overexpressed with PD-L1 in breast mesenchymal compared with the epithelial cells. Driving a mesenchymal phenotype in SNAI1-inducible MCF-7 cells (MCF-7Mes cells) increased both PD-L1 and CMTM6. CMTM6 silencing in MCF-7Mes cells partially reduced cell surface expression of PD-L1, indicating that a proportion of the PD-L1 on the surface of MCF-7Mes cells depends on CMTM6. We also found a positive correlation between CMTM3 and CMTM7 expression with EMT score in breast cancer cells, and with Vimentin in TNBC patients. Dual knockdown of CMTM6 and CMTM7 significantly decreased PD-L1 surface expression in MCF- 7Mes cells, indicating that both CMTM6 and CMTM7 regulate the expression of PD-L1. This study highlights the importance of CMTM6 and CMTM7 in EMT-induced PD-L1 and suggests that EMT, CMTM6 or CMTM7 modulators can be combined with anti-PD-L1 in patients with highly aggressive breast cancer.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-182
Combining immunohistochemistry with gene expression analysis to gain insight in tumor-infiltrating lymphocytes in breast cancer H. Locy1, S. Verhulst2, W. Cools3, W. Waelput4, S. Brock4, L. Cras4, A. Schiettecatte5, J. Jonckheere5, L. A. van Grunsven2, M. Vanhoeij6, K. Thielemans1, K. Breckpot1 1Vrije Universiteit Brussel, Laboratory for Molecular and Cellular Therapy, Brussel, Belgium, 2Vrije Universiteit Brussel, Liver Cell Biology Research Group, Brussel, Belgium, 3Vrije Universiteit Brussel, Biostatistics & Medical Informatics Research group, Brussel, Belgium, 4Universitair Ziekenhuis Brussel, Department of Anatomo-Pathology, Brussel, Belgium, 5Universitair Ziekenhuis Brussel, Department of Radiology, Brussel, Belgium, 6Universitair Ziekenhuis Brussel, Department of Surgery, Brussel, Belgium
Text Activating the immune system is a promising treatment strategy in breast cancer, as shown for HER2 positive and triple negative breast cancer. To date, the correlation between various subpopulations of tumor- infiltrating lymphocytes (TILs) and response to therapy is still under debate. To understand therapy response, it is critical to study the composition of TILs before and after therapy. In this study, we evaluated TIL scoring based on immunohistochemistry and nanoString nCounter® gene expression analysis. Formalin- fixed paraffin-embedded and fresh frozen core needle biopsies of 12 breast cancer patients were included in this study. TILs were scored on tissue sections stained with standard hematoxylin-eosin or antibodies specific for CD45, CD3, CD4 or CD8. Scoring was performed twice by three pathologists with a wash-out period of three days. Increasing intra- and inter-rater variability was observed with higher TIL numbers for all methods with the highest reproducibility observed on tissue sections stained for CD3 and CD8 positive TILs. These TIL scores correlated well with the TIL scores obtained through nanoString nCounter® gene expression analysis. Additionally, nanoString nCounter® gene expression analysis revealed 62 genes that are inversely related to both TIL scoring methods. This study argues for the use of gene expression analysis to corroborate immunohistochemistry data as well as to provide additional information that could provide insight into the TILs’ contribution to controlling breast cancer progression.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-183
Paracrine signalling with stromal fibroblasts drives recovery of cancer cells after chemotherapy treatment A. Maia dos Santos Leite1, Z. Gu1, A. Koch2, M. Berdiel-Acer1, R. Will1, M. Schlesner1, S. Wiemann1 1German Cancer Research Center (DKFZ), Heidelberg, Germany, 2Department of Women’s Health, University of Tübingen, Tübingen, Germany
Text Introduction The main cause of death for cancer patients is the development of metastasis. These arise mainly due to irresponsiveness of cancer cells to therapy, which fails to eliminate all cancer cells present in the patient. To overcome this problem, it is essential to understand which mechanisms are involved in the lack of treatment response. We investigate how the tumour microenvironment (TME) affects the response of cancer cells to chemotherapy (CTX) and how it can be modulated to improve the outcome of patients.
Materials and Methods Co-culture of CTX-treated breast cancer cell lines with primary fibroblasts isolated from breast cancer patients was performed to investigate if fibroblasts affect the response of tumour cells to commonly used CTX agents. Recovery of cells was assessed using colony formation assays and cell cycle profiling by EdU and the FUCCI system. To further explore the complex crosstalk between cancer cells and fibroblasts in the context of CTX, gene expression analysis of both cell types was done using next generation sequencing. Validation and evaluation of the biological impact of the identified pathways was done using RT-qPCR, western-blot and perturbation experiments. Lastly, publicly available datasets for breast cancer were used to investigate the clinical relevance of our findings.
Results and Discussion We show that cancer cells utilize paracrine signalling with stromal fibroblasts to drive their recovery after treatment withdrawal. Cell cycle analysis and RNA-sequencing revealed an increase in cell cycle re-entry of cancer cells in co-culture with fibroblasts. In addition, we have successfully shown that cancer cells treated with CTX upregulate IFNβ1 which modulates fibroblasts into a pro-tumorigenic state. Silencing of IFNβ1 in cancer cells abolished their fibroblast-dependent recovery potential. Analysis of human breast carcinomas supported the proposed role of IFNβ1 since its expression is inversely correlated with recurrence free survival (RFS). Moreover, expression of the gene signature identified in fibroblasts in co-culture with CTX-treated cancer cells was equally associated with higher recurrence rates and a worse outcome in breast cancer patients.
Conclusion CTX-induced secretory profile of cancer cells orchestrates the reprogramming of fibroblasts into a pro-tumorigenic state, which drives the expansion of cancer cells. Our study unravels a novel paracrine communication between cancer cells and stromal fibroblasts that ultimately results in the escape of malignant cells to treatment, highlighting the importance of the TME in drug response. Targeting of this axis could potentially improve the outcome of breast cancer patients to CTX treatment.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-184
IL-17-producing CD8+ T cells associate with poor survival in PDAC and promote carcinogenesis via induction of inflammatory cancer associated fibroblasts F. Picard1, V. Lutz1, 2, A. Brichkina3, H.-R. Chung4, A. Hupfer3, P. Mayer5, H. Raifer1, C. Bauer2, M. Gaida6, M. Lauth3, M. Huber1 1Philipps-University Marburg, Medical Microbiology and Hospital Hygiene, Marburg, Germany, 2Philipps- University Marburg, Dept. of Gastroenterology, Marburg, Germany, 3Philipps-University Marburg, Institute for Molecular Biology and Tumor Research (IMT), Marburg, Germany, 4Philipps-University Marburg, Institute for Medical Bioinformatics and Biostatistics, Marburg, Germany, 5University of Heidelberg, Clinics for Diagnostic and Interventional Radiology, Heidelberg, Germany, 6Johannes-Gutenberg University, Institute for Pathology, Mainz, Germany
Text Pancreatic ductal adenocarcinoma is characterized by highly desmoplastic stroma, suppression of antitumoral immune response and resistance against chemotherapy. Recent publications show a role of pro- inflammatory cytokine IL-17 not only in context of autoimmune diseases and systemic inflammation, but also in the development of pancreatitis and a shift from pancreatic intraepithelial neoplasia (PANins) to the onset of PDACs. Intratumoral CD8+ T-cells are of special interest, either as canonical antitumoral cytotoxic T- lymphocytes (CTLs) or as IL-17 producing Tc17 cells. Modulation of these immune cells in combination with classical antitumor therapies might open up new avenues of treatment. Our data reveal increased intratumoral Tc17 abundance associates with strongly decreased survival and advanced tumor stage in PDAC. Consistently with this finding in patients, also in the mouse model Tc17 cells enhanced pancreatic tumor growth. Mechanistically, Tc17 promoted tumorigenesis in an indirect manner via induction of inflammatory cancer associated fibroblasts (iCAF). IL-17A and F produced by Tc17 cells directed iCAF phenotype, which in turn enhanced tumor cell growth in vitro and in the mouse model. Our RNA-seq analysis revealed that Tc17-driven iCAF changed the transcriptional program of pancreatic tumor cells towards expression of genes associated with positive regulation of cell proliferation, cell-cell adhesion, cholesterol biosynthesis and negative regulation of extrinsic apoptosis. These genes were also present human PDAC.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-185
Tumor microenvironment characterization of Human papillomavirus-positive and -negative oropharyngeal squamous cell carcinomas B. Parisatto1, A. Tosi1, P. Boscolo-Rizzo2, 3, A. Rosato4, 5 1University of Padua, Department of Surgery, Oncology and Gastroenterology, Padova, Italy, 2University of Padua, Department of Neuroscience, Padua, Italy, 3Treviso Regional Hospital, Treviso, Italy, 4University of Padua, Department of Surgery, Oncology and Gastroenterology, Padua, Italy, 5Istituto Oncologico Veneto, Padua, Italy
Text The Human papilloma virus (HPV) infection is the cause of a subset of oropharyngeal squamous cell carcinoma (OPSCC). We characterized density and spatial distribution of immune cells in HPV+ and HPV- OPSCC microenvironment, in primary tumors and in the corresponding neck metastases. In particular, ten HPV+ and nine HPV- OPSCC samples were analysed with a 9-color multiplex immunofluorescence (mIF) panel including markers for cell subtyping (CD68, CD8, CD103), functionality (FoxP3, CD163), immune checkpoint molecule (PD-1, PD-L1) and localization (pan-cytokeratin for tumor recognition). Cell densities, percentages and cell distances were evaluated for each sample. Ten (53%) patients harboured a transforming HPV infection defined by HPV DNA and p16INK4a double positivity. No significant difference in immune cell densities was observed between primary tumors and the corresponding lymph node metastases, either in HPV+ or HPV- patients. HPV+ primary tumors and metastases showed a higher infiltration of CD8+ T cell, CD8+CD103+, CD8+PD-1+ cells, and CD8+CD103+PD-1+ T compared to HPV- samples. A shorter mean distance between tumor cells and CD8+, CD8+CD103+ and CD8+PD-1+ T cells and an increased percentage of tumor cells within a 15 µm radius from CD8+ T lymphocytes were observed in HPV+ primary tumors and metastases. Moreover, CD8+ T lymphocytes in HPV+ specimens were closer to CD163+ macrophages and to FoxP3+ cells. In both HPV+ primary tumors and metastases, the density of PD-L1+ cells was higher, with most abundant PD-L1 expression on cancer cells, but also on infiltrating macrophages. PD-L1+ tumor cells and CD8+PD-1+ T cells resulted closer in HPV+ samples. Independently from HPV status, a better disease-free survival (DFS) was associated with higher densities of CD8+CD103+PD-1+ (log rank p = 0.048), FoxP3+ cells (log rank p = 0.031) and a lower density of CD163+PD-L1+ (log rank p = 0.050) in primary tumors. Patients with a major density of CD8+PD-1+ lymphocytes in metastasis showed a better DFS (log rank p = 0.048). These results suggest that mIF technique allowed the characterization of close interactions between cells expressing immunotherapy-targeted molecules within OPSCC microenvironment. Indeed, we observed that HPV+ OPSCC samples were more infiltrated, characterized by potentially active host immune response, but blocked by checkpoint molecules interactions. In these setting, the PD-L1 blockade immunotherapy might be a useful treatment strategy to allow the tumor-specific T cells to overcome the shield formed by PD-L1+ tumor cells and to exert cytotoxic activity. Our study supports the rationale for the administration of PD-1/PD-L1 targeted therapy as first-line treatment in HPV+ OPSCC patients.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-186
Cellular and molecular profiling of the human tumor microenvironment in the IMMUcan consortium H. Hong1, R. Liechti2, D. Schulz3, 4, S. Tissot5, M. Morfouace6 1Merck Healthcare KGaA, Darmstadt, Germany, 2Swiss Institute for Bioinformatics, Lausanne, Switzerland, 3University of Zurich, Zurich, Switzerland, 4Eidgenössische Technische Hochschule, Zurich, Switzerland, 5Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, 6European Organisation for Research and Treatment of Cancer, Brussels, Belgium
Text Unraveling the complexities of the human tumor microenvironment (TME) requires extensive profiling efforts, not limited to single technological modalities as well as adequate sample sizes to account for the substantial heterogeneity. The EU-funded, public-private IMMUcan consortium is contributing to this task by combining broad molecular profiles of the tumor with extensive, spatially resolved cellular information of the TME in up to 3,000 patients. In detail, all tumor samples obtained by the consortium undergo the same analytical pipeline consisting of whole exome sequencing, total RNA sequencing, multiplex immunofluorescence (IF) and imaging mass cytometry (IMC) analyses. Notably, whereas the IF platform facilitates the quantification of immune cells defined by up to 7 markers on whole sections, IMC provides high-dimensional, single cell read- outs with up to 40 simultaneously captured cellular markers. To fully leverage the power of these two imaging techniques, we developed and implemented an IF data-guided method to reproducibly select regions-of-interest for IMC analyses. The combination of all these technologies enable us to integrate the mutational landscape of the tumor with matching gene expression data and a detailed survey of the infiltrating immune cell populations at quantitative and qualitative level. Most of the patients are recruited prospectively in IMMUcan and all were diagnosed with one of these five core indications: non-small cell lung cancer (NSCLC), breast cancer, head and neck squamous cell carcinoma (HNSCC), colorectal cancer (CRC) and renal cell carcinoma (RCC). In contrast to previous large-scale tumor-profiling efforts, the selection of patients in each of these indications focuses on unmet medical need, including late-stage, metastatic disease as well as tumors unresponsive to treatment with immune checkpoint inhibitors in a HNSCC sub-cohort. To potentially offer more immediate benefit to clinicians and patients, molecular reports based on DNA and RNA sequencing data are generated for all prospectively enrolled patients within 6 to 8 weeks. Molecular tumor boards are held to discuss potential actionable molecular alterations and therapeutic options. Once completed, the IMMUcan dataset will represent a rich resource facilitating explorative biomarker analyses as well as discovery research. At the time of abstract submission, 274 [HH1] patients have been included in IMMUcan and preliminary findings from the study will be presented. Funding: IMI2 JU grant agreement 821558, supported by EU’s Horizon 2020 and EFPIA.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-187
Unravelling neoantigen-specific T cell responses in low mutation burden colorectal cancers. J. van den Bulk1, M. Ijsselsteijn1, M. van der Ploeg1, D. Ruano1, R. van der Breggen1, K. Peeters1, T. Duhen2, N. de Miranda1 1Leiden University Medical Centre, Leiden, Netherlands, 2Earle A. Chiles Institute - Providence Cancer Institute, Portland, OR, United States
Text Innovative treatment options are required for patients diagnosed with advanced colorectal cancer. In tumours with low mutation burden, such as mismatch repair proficient (MMR-p) colorectal cancers, the scarcity of mutated antigens (neoantigens) is most likely responsible for their insensitivity to T cell checkpoint blockade therapies. Nevertheless, we and others have identified the presence of neoantigen-specific T cells infiltrating MMR-p colorectal cancers. This observation raises the question as to why these cells are not able to mediate therapeutic responses to checkpoint blockade but also whether they can be exploited by specific therapeutic approaches. We investigated the phenotypes of tumour-infiltrating immune cells in colorectal cancers using single-cell and imaging CyTOF analysis and demonstrated the presence of T cells expressing CD103 and CD39, markers associated with tumour-reactive T cells, in both MMR-proficient and -deficient tumours. In order to determine an association between neoantigen-specificity and the expression of CD39 and CD103 on T cells, we sorted tumour-infiltrating CD8+ T lymphocytes of ten MMR-p colorectal cancers based on the single or combined expression of CD39 and CD103. Each T cell subset was expanded independently and was interrogated for its potential to evoke an immune response upon co-culture with peptides corresponding to neoantigens predicted by exome and transcriptomic sequencing of the tumours. As hypothesized, we were able to validate neoantigen-directed immune reactivity only in the CD39+CD103+ CD8 T cell subset for MMR-p colorectal cancer patients. These observations highlight the possibility of undertaking immunotherapeutic strategies in MMR-p colorectal cancer patients, for instance, through the enrichment of neoantigen-specific T cells for the development of therapeutic T cell products.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-188
Dual CRISPR screen reveals novel regulators of tumor resistance and sensitivity to cytotoxic T cell mediated killing A.-K. Mattes1, E. Martin2, L. Boryn3, P. Baskaran4, H. Huber4, M. Schuler2, L. K. Swee1 1Boehringer Ingelheim Pharma GmbH & Co. KG, Cancer Immunology + Immune Modulation, Biberach, Germany, 2Boehringer Ingelheim Pharma GmbH & Co. KG, Drug Discovery Sciences, Biberach, Germany, 3Ardigen S.A., Krakow, Poland, 4Boehringer Ingelheim Pharma GmbH & Co. KG, Global Computational Biology and Digital Sciences, Biberach, Germany
Text Cytotoxic CD8+ T cells (CTLs) are the key player of adaptive anti-tumor immunity based on their ability to specifically recognize and destroy tumor cells. Many cancer immunotherapies rely on unleashing CTL function, however, tumors can evade killing through strategies which are not yet fully elucidated. Since resistance to cancer immunotherapy occurs in many indications, our goal was to provide deeper insight into tumor evasion mechanisms. In order to study this in an antigen dependent manner, we established a human co-culture system including tumor and immune cells. Based on this system, we systematically investigated tumor intrinsic regulators of resistance and sensitivity, respectively, by conducting a dual CRISPR screen approach for the first time. By harnessing CRISPR activation (CRISPRa) and CRISPR KO technology simultaneously, we investigated gene gain-of-function as well as loss-of-function across genes with annotated function. Depletion of calcium-associated and transport related pathways in the CRISPRa screen as well as depletion of pathways related to housekeeping function in the CRISPR KO screen suggested that gene activation and editing took place with reasonable efficiency. Applying the dual CRISPR screen approach covering around 10.000 genes in a human colon carcinoma cell line, we identified new tumor intrinsic regulators which could be potential targets for new therapeutic approaches. CRISPRa screen resulted in 186 and CRISPR KO in 704 gene hits (FDR <5 %) with 60 of them overlapping. These data confirmed previously identified genes involved in interferon and tumour necrosis factor response (e.g. IFNGR1, STAT1, JAK1/2, PTPN2, SOCS1, TNFRSF1A), autophagy (e.g. ATG3/10/12/13/101), and apoptosis (e.g. CASP8, CFLAR). Our results also highlighted genes playing a role in regulating cell cycle (e.g. CCND1) and PI3K/Akt/mTOR pathway (e.g. PIK3CA, AKT1, MAPKAP1, mTOR) that contribute to phenotypic changes associated with evasion of CTL killing. Novel gene hits found in the dual CRISPR screen are currently under further investigation.
Collectively, the data of our dual CRISPR screen approach provide new insight into regulation of tumor evasion pathways to CTL mediated killing. Based on this, further investigations can be initiated to identify potentially important new immune regulators which may open new avenues for therapeutic opportunities.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-189
The role of TIM3, VISTA and PD1 on macrophages in the sensing of dying cancer cells to guide cancer chemo- immunotherapy I. Vanmeerbeek1, J. P. A. Sprooten1, D. M. Borras1, L. Boon2, M. Mazzone3, 4, S. Tejpar5, A. D. Garg1 1Cell Stress & Immunity (CSI) Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium, 2Bioceros, Utrecht, Netherlands, 3Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium, 4Laboratory of Tumor Inflammation and Angiogenesis, VIB center for Cancer Biology, Leuven, Belgium, 5Molecular Digestive Oncology unit, Department of Oncology, University Hospital Leuven, Leuven, Belgium
Text Immune checkpoint (IC)-blockers (ICBs) have revolutionized clinical cancer immunotherapy, however only a subset of the patients responds to ICBs-monotherapy targeting CTLA4/PD(L)1. Combinatorial application of ICBs with cytotoxic therapies (e.g., chemotherapy) can extend these remarkable responses to non-responders. Currently this strategy is not entirely accounting for the potential ‘skewing’ effects of dying cancer cells, since these can strongly facilitate/engage ICs on macrophages, the most abundant tumoral immune-cells. An integrated single-cell (sc)RNAseq interrogation across human glioma, head & neck cancer, lung cancer, melanoma and colorectal cancer (CRC) tumours showed that, of all the typical CD8+T cell exhaustion-linked (surface) ICs (PD1, CTLA4, LAG3, TIGIT, TIM3, VISTA), TIM3/VISTA were substantially enriched in myeloid cells, especially macrophages. Deep analysis of TIM3+VISTA+macrophages scRNAseq data in CRC patients showed that, this macrophage-subset is highly phagocytic and propagates wound healing-like suppression. Cell ontology enrichment analyses across >300 pan-disease scRNAseq-studies with CRC TIM3+VISTA+macrophages found these to be frequently enriched in human cancers, especially lung cancer. In line with this, analysis of longitudinal human tumor transcriptomics data showed that the expression of ICs was ‘skewed’ after cytotoxic therapy in 3 different cancer-types, with a specific up-regulation of TIM3/VISTA, that also gained high correlation with phagocytic macrophage signatures (post-cytotoxic therapy). We translated these results in vitro with J774 reporter macrophages, bone-marrow derived (BMD) macrophages and tumor associated macrophages (TAMs). An increase of TIM3/VISTA/PD1 levels on both J774/ BMD-macrophages after co-incubation with apoptotic (cisplatin or paclitaxel induced) cancer cells (LLC lung cancer or MC38 CRC cells) was seen. Same phenomenon was seen on the level of cancer cells, where ligands for TIM3/VISTA/PD1 were upregulated post-chemotherapy. When we looked at the effect of blocking these ICs on macrophages, a significant decrease in phagocytosis of dying cancer cells after blockade with either TIM3/VISTA was seen. An opposite effect was seen for PD1, which after blockade increased phagocytosis. Furthermore, a specific increase in NF-kB activity in J774 macrophages in co-culture with (dying) cancer cells was seen after TIM3/VISTA blockade, while PD1 had a general macrophage sensitizing impact. Therefore, we believe that blocking TIM3 or VISTA, should be prioritised in combination with chemotherapy, and guided by corresponding biomarkers, to maximally ablate the myeloid compartment within tumours.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-190
Common clonal origin of conventional T cells and induced regulatory T cells in breast cancer patients M. Xydia1, R. Rahbari2, E. Ruggiero3, I. Macaulay2, S. Mastitskaya4, N. Halama4, M. Schmidt3, C. von Kalle3, F. Schütz5, T. Voet2, P. Beckhove1 1RCI Regensburg Center for Interventional Immunology, Regensburg, Germany, 2Wellcome Sanger Institute, Hinxton, United Kingdom, 3NCT and DKFZ, Heidelberg, Germany, 4NCT, Heidelberg, Germany, 5University Hospital of Heidelberg, Heidelberg, Germany
Text Breast cancer patients harbor Tumor Antigen (TA)-reactive effector T cells (Teff), a subset of conventional T cells (Tconv), which recognize and kill tumor cells showing great therapeutic potential. However, regulatory CD4+ T cells (Treg) inhibit tumor eradication by TA-reactive Teff and release pro-tumorigenic factors, supporting cancer progression. Importantly, Treg numbers increase in cancer patients and correlate with poor survival, comprising a major obstacle of cancer immunity. In tumor-bearing mice Treg enrichment stems from thymic Treg expansion but also from Tconv conversion into induced Treg (iTreg) under tumor-suppressive stimulation conditions. Nevertheless, the mechanisms of Treg accumulation in cancer patients remain largely unclear, despite their importance for the design of efficient cancer immunotherapies. Here we analyzed Treg clonal origin in breast cancer patients and healthy individuals, as negative control. Therefore, we performed T-Cell Receptor (TCR) and deep Single-Cell (SC)-transcriptome sequencing of Tconv and Treg from blood and tumors after SC-FACS sorting or Immunofluorescence and SC-Laser Microdissection. In breast cancer patients Treg bear a few highly-expanded clones in contrast to polyclonal Treg in healthy individuals, suggesting a tumor-specific immune-suppressive response against a few TAs in patients. Despite the massive expansion of a few TA-reactive Teff, in patients’ blood both TA-reactive Teff and total Tconv show only minor clonal overlap with Treg, arguing against the existence of iTreg in the circulation. While Treg from blood and tumor are clonally distinct, circulatory Tconv clones, including TA-reactive Teff, are detected in both compartments, suggesting their migration from the blood into the tumor. Within the tumor CD4+ T cells accumulate in five distinct transcriptome clusters comprising early activated Tconv, cytotoxic Th1 Teff, uncommitted Teff, suppressive Treg and pro-tumorigenic Treg. SC- transcriptome developmental trajectory analysis predicts that in the tumor early activated Tconv differentiate not only into tumor-antagonistic Th1 Teff, as expected, but also into suppressive and pro-tumorigenic Treg, enhancing cancer development. Interestingly, this coincides with the expression on early activated Tconv of genes known to be required for the conversion of Tconv into iTreg. Most importantly, in the tumor Tconv, activated Tconv and Treg share dominant highly-expanded clones contributing up to 65% of the intratumoral Treg population. Thus, our data demonstrate that tumor-infiltrating Treg in breast cancer patients are largely shaped by the conversion of antigen- experienced Tconv into suppressive pro-tumorigenic iTreg upon intratumoral activation.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-191
Multi-omics dissection of CD8+T-cell states in checkpoint therapy S. Naulaerts1, D. Borras2, A. Antoranz Martinez3, J. Messiaen3, Y. Van Herck4, L. Gelens5, T. Venken6, I. Vanmeerbeek2, S. More7, J. Sprooten2, O. Bechter4, G. Bergers8, A. Liston9, S. De Vleeschouwer10, B. J Van Den Eynde1, D. Lambrechts6, J. Borst11, F. Bosisio12, S. Tejpar13, F. De Smet3, A. D Garg2 1Ludwig Institute for Cancer Research, Brussels, Belgium, 2KU Leuven, Cell Stress and Immunity, Leuven, Belgium, 3KU Leuven, Laboratory for Precision Cancer Medicine, Translational Cell and Tissue Research, Department of Imaging & Pathology, Leuven, Belgium, 4UZ Leuven, Laboratory of Experimental Oncology, Department of Oncology, Leuven, Belgium, 5KU Leuven, Laboratory of Dynamics in Biological Systems, Department of Cellular & Molecular Medicine, Leuven, Belgium, 6KU Leuven, Laboratory of Translational Genetics, Department of Human Genetics, Leuven, Belgium, 7KU Leuven, Laboratory of Cell Death Research & Therapy, Department of Cellular & Molecular Medicine, Leuven, Belgium, 8KU Leuven, Laboratory of Tumor Microenvironment and Therapeutic Resistance, Department of Oncology, VIB-Center for Cancer Biology, Leuven, Belgium, 9VIB, VIB Center for Brain and Disease Research, Leuven, Belgium, 10University Hospitals Leuven, Department of Neurosurgery, Leuven, Belgium, 11Leiden University Medical Center, Department of Immunology and Oncode Institute, Leiden, Netherlands, 12KU Leuven, Translational Cell & Tissue Research, Department of Imaging & Pathology, Leuven, Belgium, 13KU Leuven, Laboratory for Molecular Digestive Oncology, Department of Oncology, Leuven, Belgium
Text Exhausted CD8+T-cell clinical characteristics being immunotherapy-responsive have been well characterized in checkpoint therapy context for multiple cancer types. However, there still exist immunotherapy-non-responsive, dysfunctional, CD8+T-cells that are enigmatic in clinical context. To better understand this sub-category of dysfunctional CD8+T-cells we here present a large-scale multi-omics integration analysis across several cohorts of cancer patients that identified the CD8+T cell dysfunctional and exhausted niches across 6 different human cancers. We identified tumor and organ-specific immune modulational niches of CD8+Tcell states. Using this as a continuum of CD8+T cell states we centered our additional research on melanoma and + + + glioblastoma as cancer representatives of exhausted and dysfunctional CD8 T-cell states, respectively (CD8 Tex/CD8 Tsdf). Using transcriptomic (RNA-seq/scRNA-seq) and epigenomic (ATAC-seq) multi-omics data analysis of patient samples from these cancer types we identified effector/cytolytic dysfunctions that, combined with dysregulated effector/memory trajectories, + + highlighted cell-cycle alterations and pro-death stress defects in CD8 Tsdf. CD8 Tex did not show the same cell-cycle and + cytolytic alterations and presented an active immune-checkpoint expression profile. In contrast, our results show that CD8 Tsdf presented a depleted antigen-priming profile. These results were further corroborated using tumor-spatial transcriptome (LCMD) + and spatial single-cell protein colocalization (MILAN) on tumor biopsy slides where we show a striking convergence of CD8 Tex + + towards vascularized regions and a divergent CD8 Tsdf along with dysfunctional CD4 T cell-cell interactions between melanoma + and glioblastoma. Furthermore, we confirmed these tendencies at the single-cell expression landscape where CD8 Tsdf cells showed increased dysfunctionality of IFNG and IL2 in intra tumoral CD4+T to CD8+T cell interactions as well as immunosuppressive TGFb and wound healing. Overall, our findings are in line with immune-oncology clinical trials where + checkpoint therapy such as anti-PD1 were not able to reactivate the CD8 Tsdf. Contrary to that, we showed that anti-PD1 therapy produced increased effector dysfunctionality and TGFb and wound healing signals. In conclusion, we provided a comprehensive integrational study of CD8+T cell states identified across multi-omics assays that are driven by tumor and organ- specific niches and can further determine the potential of checkpoint therapy responses.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-192
Immunohistochemical expression of PD-L2 in TILs correlates with improved survival outcomes and immune cell infiltration in Triple-Negative Breast Cancer (TNBC) C. H. C. Ong1, J. W. Lu1, 2, D. Y. Lee1, 3, B. Lee4, J. P. S. Yeong1, 4, J. X. Lim1, A. A. Thike1, 2, J. C. T. Lim1, 5, P. H. Tan1, 2, J. Iqbal1, 2 1Singapore General Hospital, Department of Anatomical Pathology, Singapore, Singapore, 2Duke-NUS Medical School, Singapore, Singapore, 3Nanyang Technological University, School of Biological Sciences, Singapore, Singapore, 4Agency of Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), Singapore, Singapore, 5Agency of Science, Technology and Research (A*STAR), Institute of Molecular Cell Biology (IMCB), Singapore, Singapore
Text Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype among breast tumours. To date, TNBC patients suffer from worse prognosis along with limited therapeutic options, lacking reliable molecular markers. Patients with high expression of programmed death ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) have been found to have better prognosis and increased responsiveness to anti-PD-1/PD-L1 immunotherapy. However, the role programmed death ligand 2 (PD-L2), the other known ligand of PD-1, plays in PD-1/PD-L1 checkpoint pathway has not been well studied. Therefore, this study aims to investigate i) the relationship between PD-L2 expression in tumour-infiltrating lymphocytes (TILs) and tumour cells (TCs), ii) whether PD-L2 can serve as a predictor of patient survival outcomes, and iii) the association of PD-L2 expression with relevant immune cell types in the tumour microenvironment.
The study comprises 296 TNBC cases diagnosed at the Department of Anatomical Pathology, Singapore General Hospital, from 2003 to 2013. Tissue microarrays (TMA) were constructed, and immunohistochemistry (IHC) was performed using antibody to PD-L2, coupled with Nanostring measurement. Semi-quantitative H-scores denoted low (H score <1) and high (H score ≥1) in both tumour and immune infiltrating cell expression of PD-L2 were correlated with patient survival outcomes and relevant immune cell types. Disease-free survival (DFS) and overall-survival (OS) were estimated using Kaplan-Meier analysis and compared between groups with the log-rank test.
Univariate analysis reported that TNBC patients with PD-L2+ TILs expression to have improved DFS and OS (HR = 0.51, P = 0.036 and HR = 0.43, P = 0.038, respectively). Significant association of programmed death ligands (PD-L1 and PD-L2) expression in both TILs and TCs was reported as well (P = 0.022 and P = 0.013, respectively). PD-L2+ TILs was correlated with CD3+ T cells (P = 0.008) and CD20+ B cells (P = 0.001) infiltration in the stromal compartments and intratumoural CD38+ plasma cells (P = 0.049) infiltration. Furthermore, patients bearing PD-L2+ TNBCs reported a 30% difference increase in mRNA expression level between above and below-median expression of 7 immunoglobulin gene profiles (IGHG1, IGHM, IGHV4OR15- 8, IGKC, IGKV1-37, IGKV1OR2-108, IGKV1OR2-118) and CXCL9.
Our results demonstrate that both programmed death ligands (PD-L1 and PD-L2) are expressed not only in the immune cells, but also in tumour cells. Similar to PD-L1, PD-L2 positive TILs were found to indicate a better prognosis compared to patients with PD-L2 negative tumours, suggesting PD-L2 targeting as a potential viable breast cancer therapeutic approach.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-193
Hypoxia-regulated Carbonic Anhydrase IX (CAIX) protein is an independent prognostic indicator in Triple-Negative Breast Cancer (TNBC) C. H. C. Ong1, D. Y. Lee1, 2, B. Lee3, J. P. S. Yeong1, 3, H. Y. Lau1, J. X. Lim1, A. A. Thike1, 4, J. C. T. Lim1, 5, P. H. Tan1, 4, J. Iqbal1, 4 1Singapore General Hospital, Department of Anatomical Pathology, Singapore, Singapore, 2Nanyang Technological University, School of Biological Sciences, Singapore, Singapore, 3Agency of Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), Singapore, Singapore, 4Duke- NUS Medical School, Singapore, Singapore, 5Agency of Science, Technology and Research (A*STAR), Institute of Molecular Cell Biology (IMCB), Singapore, Singapore
Text Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype among breast tumours. To date, clinical outcomes in TNBC remain poor with limited therapeutic options, lacking reliable molecular markers. The effect of extracellular microenvironment (hypoxia and pH) has been regarded as a key hallmark in cancer progression. This study aims to investigate key hypoxia-inducible markers such as i) Carbonic anhydrase IX (CAIX) and ii) Hypoxia inducible factor 1 (HIF-1) and their effect on hypoxia-related mechanisms in TNBC, while predicting clinicopathological parameters and survival outcomes.
The study comprises 323 TNBC cases diagnosed at the Department of Anatomical Pathology, Singapore General Hospital, from 2003 to 2013. Tissue microarrays were constructed, and immunohistochemistry was performed using an antibody to CAIX, coupled with Nanostring measurement. Semi-quantitative H-scores denoted low (<1) and high (≥1) CAIX membranous expression were correlated with clinicopathological parameters using X2 or Fisher’s exact tests. Disease-free survival (DFS) and overall-survival (OS) were estimated using Kaplan-Meier analysis and compared between groups with the log-rank test.
Significant associations were found between CAIX+ tumour cells and clinicopathological features such as larger tumour size (P < 0.001) and higher histological grade (P < 0.001). Multivariate analysis reported that patients with CAIX+ TNBC to have significantly worse DFS and OS after adjustment to age, grade, tumour size and lymph node positivity (HR = 2.99, 95% CI 1.784 to 5.022, P < 0.0001 and HR = 2.56, 95% CI 1.409 to 4.652, P = 0.002, respectively). Multivariate analysis further confirmed protein co-expression of CAIX and HIF-1α in TNBC patients to have worse DFS and OS after adjustment to age, grade, tumour size and lymph node stage (HR = 4.46, 95% CI 2.26 to 8.81, P < 0.0001 and HR = 3.3, 95% CI 1.57 to 6.94, P = 0.002, respectively). Furthermore, gene ontology enrichment analysis revealed 6 significantly enriched cellular functions (secretion, cellular component disassembly, regulation of protein complex assembly, glycolytic process, cellular macromolecular complex assembly, positive regulation of cellular component biogenesis) associated with genes differentially expressed (CAIX, SETX, WAS, HK2, DDIT-4, TUBa4A, ARL1) between CAIX high-low groups in TNBC.
Our results demonstrate that CAIX can be a reliable surrogate hypoxia marker and prognostic indicator for TNBC. The identification of CAIX-linked 7 gene-signatures and their relationship with 6 enriched cellular functions further support the implication and influence of hypoxia-mediated CAIX expression in tumour microenvironment and breast cancer development.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-194
Tertiary lymphoid structures in PDAC resemble lymphoid follicles in secondary lymphoid organs by organization, function and gene expression J. Lehmann1, M. Thelen1, S. Schran1, K. Wennhold1, M. Garcia-Marquez1, P. Lohneis2, S. Kruger3, S. Böck3, S. Ormanns3, M. Rudelius3, F. Popp4, M. von Bergwelt-Baildon1, 5, 6, 7, C. Bruns8, A. Quaas9, H. A. Schlößer1, 8 1Center for Molecular Medicine Cologne, Cologne, Germany, 2University Hospital Cologne, Pathology, Cologne, Germany, 3Ludwig-Maximilians-University of Munich, Munich, Germany, 4University Hospital Cologne, Cologne, Germany, 5Ludwig-Maximilians-University of Munich, Department of Medicine III, Munich, Germany, 6German Cancer Consortium (DKTK), Heidelberg, Germany, 7Ludwig-Maximilians-University of Munich, Gene Center, Munich, Germany, 8University Hospital Cologne, Department of General, Visceral and Cancer Surgery, Cologne, Germany, 9University Hospital Cologne, Institute of Pathology, Cologne, Germany
Text Tertiary lymphoid structures (TLS) are gaining attention in the recent years as secondary lymphoid organs (SLO) like structures, which are involved in induction and enhancement of anti-tumor immune responses. These TLS are induced or enriched in the tumor microenvironment and are assumed to represent hotspots for T cell priming, B cell activation and differentiation, which cause cellular and humoral anti-tumor immune responses. We performed immunohistochemistry staining of 120 primary pancreatic ductal adenocarcinoma (PDAC) patients for CD20, CD3, CD8, FOXP3, HLA-ABC, and follicular dendritic cells (FDCs) to analyze spatial distribution of tumor-infiltrating lymphocytes. To investigate structural components of TLS in comparison to lymphoid follicles in SLOs 5-color immunofluorescence staining was implemented. Tissue was punched out of membrane slides by laser microdissection and Nanostring-base RNA expression analysis was conducted to compare gene expression in TLS, PDAC, SLOs and normal pancreatic tissue. TLS were found in 85 % of all patients, but frequency of TLS differed between patients. While TLS were mainly localized in a 2000 µm invasive tumor margin 50 % of all patients showed at least a few intratumoral TLS. TLS distribution will be correlated with clinical parameters, Immunoscore and immune escape mechanisms. FOXP3+ regulatory T cells (Tregs) infiltration of TLS differed between patients with most patients having low infiltration, while in some patients Tregs made up to 18 % of the cells in TLS, suggesting inhibition of antitumor immune responses in these TLS. In 90 % of analyzed TLS a network of FDCs was found similar in structure of SLO B cell follicle. SLO like organization and functions of TLS was revealed by 5-color immunofluorescence. Nanostring analysis confirmed similarities of TLS and SLOs by largely overlapping expression patterns. Taken together our results indicate the close similarities of TLS and SLOs in organization, function and gene expression patterns.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-195
B cells with CD86 expression forming antigen-presenting clusters that induce specific T cell responses in Cancer K. Wennhold1, J. Lehmann1, M. Thelen1, S. Schran1, E. Preugszat1, M. Garcia-Marquez1, A. Lechner2, 3, A. Shimabukuro-Vornhagen4, M. S. Ercanoglu5, F. Klein5, F. Thangarajah6, S. Eidt7, H. Löser8, C. Bruns9, A. Quaas8, M. von Bergwelt-Baildon1, 3, 10, 11, H. A. Schlößer1, 9 1Center for Molecular Medicine Cologne, Cologne, Germany, 2Ludwig-Maximilians-University of Munich, Department of Otorhinolaryngology, Head and Neck Surgery, Grosshadern Medical Center, Munich, Germany, 3Ludwig-Maximilians-University of Munich, Gene Center, Munich, Germany, 4University Hospital Cologne, Department I for Internal Medicine, Cologne, Germany, 5University Hospital Cologne, Institute of Virology, Laboratory of Experimental Immunology, Cologne, Germany, 6University Hospital Cologne, Department of Gynecology, Cologne, Germany, 7St. Elisabeth Hospital, Institute of Pathology, Cologne, Germany, 8University Hospital Cologne, Institute of Pathology, Cologne, Germany, 9University Hospital Cologne, Department of General, Visceral and Cancer Surgery, Cologne, Germany, 10Ludwig-Maximilians- University of Munich, Department of Medicine III, Munich, Germany, 11German Cancer Consortium (DKTK), Heidelberg, Germany
Text In the recent years B cells are getting more into focus regarding immunotherapies due to their anti-tumor immunity. Beside the secretion of antibodies, the effector functions of B cells also include the presentation of antigens to T cells. A CD86+ CD21- B cell subset was recently described in humans as antigen-presenting B cells (BAPCs) with strong immunostimulatory properties. To analyze the abundance and spatial distribution of BAPCs in blood (PBMC) and tumor samples of 238 cancer patients multicolor flow cytometry and immunohistochemistry was performed. Antigen-specific T cell responses upon incubation with cancer testis antigens and sorted BAPCs was determined by Fluorospot assay for selected patients. In nine out of ten analyzed cancer types BAPCs were increased in the tumor microenvironment with site- specific variations. While in renal cell carcinoma BAPCs were not increased, we found a systemic increase with elevated fractions in TILs and PBMC of colorectal cancer and gastro-esophageal adenocarcinoma patients. In these cancer types BAPCs were localizing in lymphoid follicle like tertiary lymphoid structures (TLS). In tumors with increased numbers of TLS, BAPC were also enriched. Increased percentage of tumor antigen-specific B cells were found in isolated BAPCs from tumor draining lymph nodes of cancer patients, which induced a strong response of autologous T cells in vitro. In summary our findings highlight the relevance of BAPCs as professional antigen-presenting cells in tumor immunity and provides explanations for the recently observed correlation of response to immune checkpoint inhibition and B cell abundance.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-196
CXCL5-mediated accumulation of SiglecFhigh neutrophils in lung tumor tissues impairs CD8 T cell responses and limits the efficacy of PD-L1 checkpoint blockade. F. Simoncello1, N. Caronni2, T. Battini1, F. Benvenuti1 1International centre for genetic engineering and biotechnology, Trieste (TS), Italy, 2San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy, Milano (MI), Italy
Text Neutrophils (neu) have a key role in defence against infection and in regulation of innate and adaptive immunity. In cancer, tumor-associated neutrophils (TANs) can exert dual function depending on the tumor type, the cellular microenvironment and the constellation of immune modulating factor. In the KrasG12D/+;Trp53-/- mouse lung adenocarcinoma (KP) two subsets of neutrophils were identified, which can be separated based on the expression of the sialic-acid-binding protein SiglecF. SiglecFhigh neutrophils preferentially accumulate in tumors and are associated with a pro-tumorigenic behaviour, whereas SiglecFlow counterpart populates healthy lungs and was not associated to tumor growth. However, the mechanism of neu-SiglecFhigh recruitment and their impact on T cell response is not fully elucidated. Here, we used a transplantable KP line to investigate the role of the C-X-C motif chemokine 5 (CXCL5) in recruitment and reprogramming of the microenvironment of lung tumors. By genome editing we abrogated the expression of CXCL5 in KP cells (KP-CXCL5ko) and we characterized neutrophils frequencies and T cell activation. We observed a drastic decrease of neu-SiglecFhigh in KP-CXCL5ko tumors proving that the chemokine is a key player for their accumulation. Phenotypic and functional analysis of the T cell infiltrate revealed a significant expansion of IFN-γ producing tumor specific T cells. Worth notes that growth of KP-CXCL5ko tumors was delayed as compared to CXCL5wt tumors, showing that neu-SiglecFhigh inhibit tumor containment. Preliminary immunofluorescence analysis of lung tissues shows tight CD8 T cell-neutrophils interactions, suggesting a contact-mediated mechanism of inhibition. Yet, neu-SiglecFhigh, due to a high expression of PD-L1, may contribute to immune-suppression by supplying T cell inhibitory ligands. In order to evaluate if lack of neu- SiglecFhigh in tumors increases sensitivity to checkpoint blockade, we administered antibodies to PD-L1 during challenge. Indeed, treatment reduced growth of KP-CXCL5ko tumors, while having a limited impact on the growth of KP-CXCL5wt tumors. We infer the neu-SiglecFhigh hamper the full activity of checkpoint blockade and that targeting the CXCL5-axis could be a viable improvement to existing immunotherapy.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-197
Self-replicating RNA vectors expressing galectin-3 inhibitors for the treatment of pediatric osteasarcoma. G. Herrador Cañete1, 2, M. Zalacain2, 3, 4, D. de la Nava2, 3, 4, I. Ausejo2, 3, 4, M. M. Alonso2, 3, 4, C. Smerdou1, 2 1Cima Universidad de Navarra, Gene Therapy, Pamplona, Spain, 2Health Research Institute of Navarra (IDISNA), Pamplona, Spain, 3Clínica Universidad de Navarra, Department of Pediatrics, Pamplona, Spain, 4Cima Universidad de Navarra, Program in Solid Tumors, Pamplona, Spain
Text Osteosarcoma is an aggressive bone tumor, primarily arising in the pediatric age. Despite years of intensive research, outcome for metastatic and non-responder patients is very poor and has not improved in the last 30 years. These tumors harbor a highly immunosuppressive environment, making the existing immunotherapies ineffective. Inhibition of galectin-3 (Gal3), a protein involved in immunosuppression, adhesion of tumor cells and metastases, has demonstrated to reduce tumor progression in different tumor models, including osteosarcoma. On the other hand, virotherapy based on recombinant Semliki Forest Virus (SFV), a self-replicating RNA virus, has shown therapeutic effect in orthotopic osteosarcoma mouse models. With this premise, we constructed recombinant SFV vectors expressing inhibitors of Gal3 based on truncated forms of this protein. These constructs included the Gal3 carboxy-terminal domain (SFV-Gal3-C) and its amino-terminal domain by itself (SFV-Gal3-N) or fused to the Gal3 inhibitor peptide C12 (SFV-Gal3-N-C12). An additional construct expressed only the C12 peptide (SFV-C12). We confirmed the activity of our recombinant constructs in vitro by an assay based on blocking the migration of tumor cells. We then analyzed Gal3 expression in different murine tumor cell lines and selected osteosarcoma K7M2 cells, which showed high expression of Gal3, for in vivo studies. Orthotopic osteosarcoma tumors, induced in mice by intratibial injection of K7M2 cells, were treated with SFV vectors expressing Gal3 inhibitors or SFV expressing luciferase (SFV-Luc) and PBS as controls. SFV-Gal3-N-C12 vector showed the highest antitumor activity, being able to significantly reduce tumor growth in comparison with control mice that received PBS. In fact, this vector was able to prolong animal survival, leading to 40% of complete regressions. Among the other vectors, SFV-Gal3-N and SFV-C12 were also able to transiently decrease tumor growth, although they had no impact on animal survival. Moreover, the number of spontaneous lung metastasis were reduced in mice treated with SFV vectors expressing Gal3 inhibitors. Preliminary mechanistic studies showed an increase of CD3 cells infiltration in tumors treated with SFV-Gal3-N-C12 and SFV-Gal3-N vectors. Despite the antitumor effect observed with SFV-Gal3-N-C12, no protection against tumor rechallenge was observed in cured mice, indicating that a strong memory immune response had not been induced, and suggesting that this type of therapy might benefit from other immunostimulatory approaches. In summary, we believe that the SFV-Gal3-N- C12 vector could constitute a potential new therapeutic strategy for patients affected by osteosarcoma expressing high levels of Gal3.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-198
FcγRIIB-mediated inhibition of antibody-mediated target cell depletion is independent of ITIM signalling A. Simpson1, A. Roghanian1, R. Oldham1, C. Chan1, C. Penfold1, J. Kim1, T. Inzhelevskaya1, I. Mockridge1, K. Cox1, Y. Bogdanov1, S. James1, A. Tutt1, D. Rycroft2, P. Morley2, I. Teige3, B. Frendeus3, S. Beers1, M. Cragg1 1University of Southampton, Antibody and Vaccine Group, Cancer Sciences, Southampton, United Kingdom, 2GSK, Biopharm Discovery, Stevenage, United Kingdom, 3BioInvent International, Lund, Sweden
Text The anti-CD20 monoclonal antibody (mAb) rituximab has significantly improved patient outcomes in non-Hodgkin’s lymphoma and a growing body of autoimmune disorders. It elicits its therapeutic effects by engaging activatory Fc gamma receptors (FcγR), resulting in depletion of CD20+ B cells. These activatory FcγRs are negatively influenced by the single inhibitory FcγR, FcγRIIB. FcγRIIB has been shown to reduce the efficacy of mAbs through impairment of activatory FcγR function on immune effector cells. Another unique FcγRIIB-mediated mechanism of inhibition is driven by the internalisation of the FcγRIIB-rituximab-CD20 complexes from the B cell surface.
FcγRIIB contains an intracellular immunoreceptor-tyrosine-based inhibition motif (ITIM), which delivers inhibitory signalling. Whether the ITIM is required for all FcγRIIB-mediated inhibitory activities is not yet clear. To address this issue, we developed and characterised a novel transgenic mouse model (NoTIM), in which the inhibitory mouse (m)FcγRII is replaced with a non-signalling ITIM mutant human (h)FcγRIIB. Cells from mice expressing the NoTIM were no longer able to elicit phosphorylation of the FcγRIIB ITIM or prevent anti-IgM-mediated calcium flux, confirming the expected phenotype.
To understand how FcγRIIB inhibitory signalling impacted mAb-mediated depletion of target cells, NoTIM mice were treated with an anti-mouse CD20 mAb. The extent of depletion was compared to mice either lacking mFcγRII or where mFcγRII was replaced with a signalling competent hFcγRIIB (hFcγRIIB Tg). B cell depletion was reduced in both the NoTIM and hFcγRIIB Tg mice compared to those lacking mFcγRII. Subsequent experiments revealed this was not due to accelerated mAb internalisation from the B cell surface in the NoTIM mice, nor enhanced clearance from the serum. Using a series of adoptive transfer models we determined that the NoTIM hFcγRIIB was mediating inhibition through its expression on myeloid cells. Flow cytometric analysis indicated inhibition was achieved by binding the depleting mAb at the myeloid cell surface, preventing engagement with activatory FcγRs.
These findings were then assessed with respect to other target cells as well as malignant B cells in the Eu-TCL1 syngeneic mouse tumour model using the same anti-CD20 mAb. It was found that depletion of both Treg and malignant B cells were also negatively impacted by the expression of FcγRIIB at the myeloid surface rather than by its ability to signal. These data indicate that signalling through the ITIM is not critical for the ability of hFcγRIIB to prevent mAb- mediated depletion of target cells, questioning current paradigms for its mechanism of action.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-199
Comparison of murine adenocarcinoma MC38 cell lines from two different sources reveals substantial differences B. Schrörs1, B. J. Hos2, I. G. Yildiz1, M. Löwer1, F. Lang1, C. Holtsträter1, J. Becker1, M. Vormehr3, U. Sahin3, 4, F. A. Ossendorp2, M. Diken1, 3 1TRON - Translational Oncology at the University Medical Center of the Johannes Gutenberg-University Mainz non-profit GmbH, Mainz, Germany, 2Leiden University Medical Center, Department of Immunohematology and Blood Transfusion, Leiden, Netherlands, 3BioNTech SE, Mainz, Germany, 4University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany
Text Murine MC38 is an adenocarcinoma cell line with high mutational burden and a commonly used model for colorectal carcinoma. The cell line is of particular interest for pre-clinical investigations with respect to cancer immunotherapy as it is sensitive to immune checkpoint immunotherapy and endogenous CD8+ T cell responses against neoantigens were already reported. Here, we re-sequenced exomes and transcriptomes of MC38 cells from two different sources, namely Kerafast (originating from NCI/NIH) and Leiden University Medical Center, and found substantial differences that were far beyond of what is expected for cell lines which differ by passage time only. A distinct structural composition in the two genomes and differences in the ploidy of two cell lines was determined (Kerafast: diploid, Leiden: pentaploid). Further, in the Leiden cell line, about 1.3-fold more SNVs and small insertions and indels in exons were observed than in Kerafast cells. Both cell lines harboured numerous private non- synonymous variants (Kerafast: 1141, Leiden: 2210) and only 35.3% of the non-synonymous variants were shared. In addition, only five fusion genes (10.0%) were common to both cell lines. The prevalent base substitution in both cell lines was C>A/G>T. However, distinct mutational signatures could be found. Transcript expression values of both cell lines correlated well, but specific differences might be relevant for individual studies depending on the genes of interest as we found different pathways enriched in the genes that were differentially upregulated in the Leiden or Kerafast cells, respectively. This holds true also for experiments targeting reported neoantigens as some of them (e.g. Rpl18, Adpgk) were present only in Leiden cells and thus, only Leiden cells were recognized by neoantigen-specific T cells. In summary, our comparison shows that the MC38 cell line consists of at least two sub-cell lines. Our analyses can be used as a reference to select neoantigens for immunotherapeutic interventions.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-200
Actin cytoskeleton-mediated polarization of inhibitory ligands promotes tumor immune evasion H. Wurzer1, 2, L. Filali1, C. Hoffmann1, M. Krecke1, 2, F. Kleine-Borgmann3, J. Mastio1, A. M. Biolato1, 2, E. Ockfen1, 2, 3, M. Mittelbronn1, 3, 4, 5, C. Thomas1 1Luxembourg Institute of Health, Oncology, Luxembourg, Luxembourg, 2University of Luxembourg, Esch-sur- Alzette, Luxembourg, 3Laboratoire national de santé (LNS), Dudelange, Luxembourg, 4Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg, 5Luxembourg Centre of Neuropathology (LCNP), Dudelange, Luxembourg
Text Cancer immune evasion is a major obstacle to effective anticancer immunotherapies. Significant progress has been made in investigating the numerous strategies that arise during tumor immunoediting that lead to evasion of recognition and destruction by the immune system. However, approaches on antagonizing these immune escape strategies are often overcome in a short time. Downregulation of major histocompatibility complex class I (MHC-I) and upregulation of immune checkpoints, such as PD-L1, are among the best investigated immune evasion strategies. Here, we describe a new layer of cancer immune escape from natural killer (NK) cells that is characterized by extensive actin cytoskeleton remodeling at the cancer cell side of the immunological synapse (IS). While reorganization of actin filaments (F-actin) in cytotoxic lymphocytes is a crucial prerequisite for the formation of IS and directed release of cytotoxic granules, these observations were so far limited to the immune cell side. Our findings indicate that rapid accumulation cancer cell actin filaments at the immunological synapse promotes resistance against natural killer (NK) cell, but also CD8+ T lymphocyte attack. We termed this observation “actin response”. Cancer cells that are competent to respond to cell-cell contact with a cytotoxic immune cell with an actin response survive cytotoxicity assays and detach from the immune cell without undergoing cell death. We found that the actin response drives the polarization of inhibitory ligands, such as classical and non-classical MHC-I or PD-L1, to the synaptic region. Clustering of inhibitory ligands towards the immunological synapse prevents immune cell activation and decreases degranulation efficiency. Remarkably, immune cell activation can be restored by interference with the cancer cell’s actin cytoskeleton dynamics which prevents inhibitory ligand polarization. We report similar findings in various types of solid and hematological cancer, both in cell lines and patient-derived tumor cells, as well as different mouse cancer models, supporting the hypothesis that the actin response is a fundamental and highly conserved process underlying tumor immune evasion. Furthermore, the frequency of the actin-based immune evasion mechanism also impacts the tumor growth in in vivo mouse models and the immune cell infiltration and activation status. Altogether, our data provide a new mechanism of immune escape of tumor cells, and identification and selective targeting of linker proteins between immune checkpoint ligands and the actin cytoskeleton could further improve immune checkpoint inhibitor therapy.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-201
GARP as a potential uniform marker for glioblastoma stem-like cells N. Zimmer1, E. Trzeciak1, B. Sprang2, 3, E. Kim2, 3, A. Tuettenberg1 1University Medical Center Mainz, Department of Dermatology, Mainz, Germany, 2University Medical Center Mainz, Department of Neurosurgery, Mainz, Germany, 3University Medical Center Mainz, Translational Neurooncology Research Group, Mainz, Germany
Text Glioblastomas are known for their aggressive behavior and tumor cell heterogeneity. They are prone to infiltrating surrounding tissue and therefore have a low chance of complete removal by surgical intervention. In addition to surgery, standard therapy consists of radiotherapy and temozolomide. In spite of these therapeutic options, patient survival is only extended for 14.6 months (median). Due to their heterogeneity, glioblastomas are particularly hard to target as they can quickly adapt to new environmental conditions and therapies. It is thought, that this high adaptability and resistance can be attributed to glioblastoma stem-like cells (GSCs). GSCs are capable of self-renewal and differentiation, making them the main driver for therapy resistance and tumor recurrence. This heterogeneous nature also results in difficulties distinguishing GSCs from glioblastoma tumor cells. Several markers like CD133, CD44, and CD15, were associated with GSCs in the past but failed to universally identify GSCs. Glycoprotein A repetitions predominant (GARP) is a protein, known to be expressed on the surface of activated regulatory T cells and platelets. Recently, we demonstrated that GARP has strong regulatory and anti- inflammatory properties in vitro and in vivo. GARP led to the induction of peripheral Treg and the inhibition of tumor antigen-specific CD8+ T cells. We were also able to show that GARP is expressed by different tumor entities, including glioblastoma. In this study, we investigated GARP as a potential stable marker for GSCs. We tested patient-derived GSC cell lines from (1) different regions of the same tumor, (2) from a patient before and after radio- and chemotherapy, and (3) from tumors, which display differences in differentiation capacity. These patient-derived GSC cell lines were analyzed for GARP and CD133 expression via flow cytometry, confocal microscopy, Imagestream, and by immunoprecipitation western blot. We could observe that GARP is expressed by all tested cell lines. This expression was more stable than the expression of CD133, indicating that GARP expression is less affected by external influences. Notably, GARP expression was observed to be elevated in patient-derived GSC lines in comparison to a control human glioblastoma cell line, T98G. Additionally, we were able to confirm our previous results, which showed that GARP is also expressed in the nuclei glioblastoma cells. This suggests, for the first time, that GARP is post-translationally modified and differentially localized to the nuclei of tumor cells. Altogether, our data suggests that GARP could be a novel stable and uniformly expressed marker for GSCs, which enables easier identification of stem-like cells in glioblastoma.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-202
The role for Interleukin-18 signaling for intratumoral T cell dysfunction in a murine pancreatic cancer model V. Lutz1, F. Picard2, V. Hellmund1, M. Klein3, H.-R. Chung4, T. Bopp3, T. Gress1, M. Huber2, C. Bauer1 1Philipps-University Marburg, University Hospital Marburg, Department of Gastroenterology, Endocrinology, Metabolism and Infectiology, Marburg, Germany, 2Philipps-University Marburg, Institute for Medical Microbiology and Hygiene, Marburg, Germany, 3Johannes Gutenberg-University Mainz, Institute for Immunology and Department of Dermatology, University Medical Center, Mainz, Germany, 4Philipps- University Marburg, Institute of Medical Bioinformatics and Biostatistics, Marburg, Germany
Text Introduction: Adaptive immune response in pancreatic cancer is characterized by immune escape mechanisms that render intratumoral CD8+ cytotoxic T cells (CTLs) dysfunctional. This phenomenon, also known as exhaustion, is characterized by impairment of cytokine production as well as upregulation of co- inhibitory receptors such as PD1 and TIM3, resulting in loss of CTL effector function. Proinflammatory cytokines, like IL-18, might play an important role in the induction of this dysfunctional state. Here, we investigate the role of NLRP3 mediated IL-18 signaling on cytotoxic T cell responses in a murine model of pancreatic cancer.
Methods: Antigen specific CTLs were generated from transgenic OT-I mice or from IL-18R-deficient OT-I mice. CTLs were adoptively transferred into mice bearing pancreatic carcinoma cells expressing the model antigen OVA (PancOVA). Effector function of these transferred cells was evaluated by flow cytometry analysis of coinhibitory receptors (PD1, TIM3), transcription factors as wells as restimulation capacity ex vivo. RNA-sequencing of adoptively transferred CTLs was performed.
Results: Our data revealed an immunosuppressive effect of IL-18 receptor signaling on intratumoral CTLs. Analysis of transferred IL-18 receptor deficient CTLs ex vivo showed reduced expression of coinhibitory receptors and improved restimulation capability compared to transferred WT CTLs. RNAseq data indicated that IL-18-induced T cell exhaustion is mediated by the IL-2/STAT5-pathway.
Conclusion: T cellular signaling of NLRP3-dependent IL-18 induces T cell exhaustion. Our results indicate that efficacy of checkpoint inhibitor therapy for PDAC patients might be improved by use of concomitant anti- IL-18 treatment strategies.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-203
Benchmarking Syngeneic Tumor Models to Explore Immuno-Pathogenesis by Depletion of a Series of Immune Lineages A. Xiaoyu An, Y. Jin, B. Mao, D. Xuesong Ouyang, L. Bourre, H. Q. Li Crown Bioscience Inc, San Diego, United States
Text Background Syngeneic in vivo models are commonly used for proof of concept and mechanistic exploration of immuno-oncology (I/O) therapies, e.g. immune-checkpoint inhibitors (ICIs). Each model responds differently to ICIs, which is believed to be dependent on the unique intrinsic tumor immunity of each model, as well as unique mechanisms of action for each ICI, in individual tumors. However, knowledge on these mechanisms remains limited due to large experiment variation and a lack of comprehensive benchmarking of these models, preventing optimal model utilization.
Material and methods This study investigated a panel of syngeneic models, including MC38, Hepa1-6, EMT6 and CT26.WT, to benchmark their intrinsic tumor immunity by depleting key immune subsets and to monitor the associated pharmacodynamics of tumor- infiltrating lymphocytes (TILs) by flow cytometry, and the impact on anti-PD-1 therapeutic efficacy.
Results Efficient depletion was achieved for CD8+, CD4+, and NK cells, while a single dose of anti-CD25 potentially only led to a transient Treg depletion, which was no longer detectable at the efficacy endpoint. Macrophage depletion was also inefficient which was likely due to quick re-population; however, liposome treatment did slow tumor growth in all four models. CD8+ T cell depletion promoted tumor growth in MC38 and EMT6, but had little impact on the growth kinetics of Hepa 1-6. CD4+ T cell depletion remarkably accelerated tumor growth of Hepa 1-6, and in contrast, it induced tumor growth inhibition in MC38 and EMT6. Although transient, Treg depletion by anti-CD25 produced antitumor activities in all models except Hepa 1-6. However, treating Foxp3-DTR mice with diphtheria toxine (DT) induced a strong tumor inhibition effect, which indicated that anti-CD25 may also hit activated CD4+ and CD8+ cells, thus compromising the effect generated by removing Treg in WT mice. Macrophage depletion consistently inhibited tumor growth, while NK cell depletion had minor effects on the tumor growth of all four models. The immunophenotyping data of TILs by flow cytometry largely correlated with the efficacy data for each treatment.
Conclusions Benchmarking the baseline and dynamic immunophenotypes of syngeneic models will help to select optimal models to evaluate I/O pharmacology and also potentially translate into understanding patient disease mechanisms and strategies for I/O therapies in the clinic.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-204
Tumoral MHC class II expression in gliomas drives T cell exhaustion Y.-C. Chih1, 2, K. Sahm1, 3, A. Sadik4, T. Bunse1, 3, N. Trautwein5, S. Pusch6, 7, S. Stevanovic5, C. Opitz4, A. Deimling6, 7, W. Wick8, 9, F. Sahm6, 7, L. Bunse1, 3, M. Platten1, 3 1German Cancer Research Center (DKFZ), Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, Heidelberg, Germany, 2Heidelberg University, Faculty of Biosciences, Heidelberg, Germany, 3Medical Faculty Mannheim, Department of Neurology, Mannheim, Germany, 4German Cancer Research Center (DKFZ), Brain Cancer Metabolism, Heidelberg, Germany, 5University of Tübingen, Department of Immunology, Tübingen, Germany, 6University Hospital Heidelberg, Department of Neuropathology, Heidelberg, Germany, 7German Cancer Research Center (DKFZ), Clinical Cooperation Unit Neuropathology, Heidelberg, Germany, 8University Hospital Heidelberg, Department of Neurology, Heidelberg, Germany, 9German Cancer Research Center (DKFZ), Clinical Cooperation Unit Neurooncology, Heidelberg, Germany
Text Neoepitopes are presented on MHCII molecules. In glioma, for instance, the recurrent mutation IDH1R132H was shown to bear an MHCII-restricted epitope in preclinical and clinical vaccine studies. The general relevance of MHCII expression in glioma for antitumor immunity, however, remains unknown. Here we evaluate stromal and tumoral MHCII expression, functionality, and its association with survival in gliomas. Immunohistochemistry and immunofluorescence of human glioma tissues were used to identify tumoral and stromal MHCII expression and to enumerate T cell infiltrates. To gain insights into tumoral MHCII, bulk transcriptomic data from TCGA and single-cell transcriptomic data from publicly available datasets were analyzed. Ligandome analyses of an MHCII+ glioma cell line and human glioma tissues were used to determine the functionality of tumoral MHCII. Functional in vitro co-culture assays with an HLA-DR-matched tetanus toxoid (TT) epitope-overexpressing glioma cell line and in vitro-expanded TT-reactive T cells from healthy donors were used to examine direct target recognition by T helper cells. CRISPR-Cas9-mediated knockout of MHCII in preclinical hypermutant glioblastoma cell line GL261 was employed to further validate the consequences of tumoral MHCII expression and to probe potential clinical intervention with existing therapies. Interestingly, MHCII is expressed in the majority of gliomas and associated with increased infiltration of T cells. In 10% of the analyzed glioma tissues and a subset of single cells, tumoral MHCII expression is detected. Clinical and transcriptomic data reveal that tumoral MHCII is associated with poor prognosis, cytokine responses, immune inhibition and T cell differentiation. Ligandome analyses evidence presentation of peptides by MHCII molecules on glioma cells. In in vitro co-culture assays, TT-reactive T helper cells specifically produce IFN-γ when co-cultured with MHCII+ glioma cells upon co-stimulation. In agreement with the clinical data, preclinical murine models demonstrate that tumoral MHCII expression leads to reduced survival. Co-culture assay shows that tumoral MHCII results in PD-1 upregulation on T helper cells antigen-specifically. Concordantly, immune checkpoint blockade (ICB) therapy slows the disease progression of mice carrying MHCII+ tumors. Overall, MHCII is expressed in gliomas by a subset of tumor cells. Although tumoral MHCII is functional, it is associated with poor survival in both clinical and preclinical data. T cell exhaustion induced by tumoral MHCII expression can, in part, be overcome by ICB in vivo. Further experiments are required to decipher tumor cell intrinsic and microenvironmental consequences of tumoral MHCII expression.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-205
Discovery of neoantigen-specific TCRs and evaluation of their anti-tumoral functionality I. G. Yildiz1, K. Reinhard2, M. Vormehr3, K. Wunsch2, Y. Ouchan2, T. Omokoko2, S. Bardissi2, S. Kreiter1, M. Diken1, U. Sahin1, 4, 5 1TRON - Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany, 2BioNTech, Cell and Gene Therapies, Mainz, Germany, 3BioNTech, RNA Pharmaceuticals, Mainz, Germany, 4BioNTech SE, Mainz, Germany, 5University Medical Center of Johannes Gutenberg University Mainz, Mainz, Germany
Text Neoantigens, products of non-synonymous mutations arising during tumorigenesis, have gained significant importance for cancer immunotherapy primarily due to their unique expression in tumor cells, which enables neoantigen-reactive T cells bypass central thymic tolerance unlike self-antigen-reactive T cells. Both our preliminary work and literature revealed neoantigen-specific T cells might possess anti-tumoral activities especially for tumors with high mutational burden. It was also shown that majority of neoantigens were recognized by CD4 T cells. However, high anti-tumoral potency of neoantigen-specific T cells, especially the function of neoantigen-specific MHCII restricted CD4 T cells is not well understood. To decipher the mechanisms of neoantigen-specific T cell responses and interplay between neoantigen-specific CD4 and CD8 T cells, we rely on ex vivo reprograming of bulk T cells with TCR (T cell receptor) sequences recognizing the neoantigen of interest. These TCR sequences were identified using an easily applicable and high throughput single-cell TCR sequencing (scTCRseq) platform, which we integrated together with a fully automated data analysis pipeline. Using this platform, a library of different neoantigen-specific TCRs, both MCHI and MHCII restricted, from different tumor models was generated. Particularly, we rely on MC38, mouse colon 38, tumor model for which both MHCI and MHCII restricted TCRs were identified and evaluated for their functionality based on IFNg secretion upon antigen specific stimulation. Functionally top ranked TCRs were then selected and used to reprogram T cells for further investigation of anti-tumoral activities both in vitro and in vivo.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-206
Synaptic filopodium-like protrusions protect tumor cells against cytotoxic lymphocyte-mediated killing L. Filali1, C. Hoffmann1, F. Kleine Borgmann2, H. Wurzer1, M. Mittelbronn2, C. Thomas1 1Luxembourg Institute of Health, Oncology department, Luxembourg, Luxembourg, 2Luxembourg Center of Neuropathology, Oncology department, Luxembourg, Luxembourg
Text Cytotoxic lymphocytes (CLs), in particular cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, are key effector anti-tumor immune cells. Although their cytolytic functions is regulated by different types of receptors, both CTLs and NK cells physically interact with prospective target cells through a highly specialized cell-to-cell junction termed the immunological synapse (IS). The IS is a cell-to-cell interface necessary for recognition of the target cell, activation of the lymphocyte, and killing of the target cell. For several years now, lymphocytes have been the focus of immunotherapy research aiming to boost their activity, but the efficiency and durability of these therapies are still limited. We previously reported that resistant cancer cells respond to NK cell attack by fast remodeling of their actin cytoskeleton and massively accumulation of actin filaments at the IS, a process we termed “actin response”. We showed that the actin response potently protects tumor cells against NK cell-mediated killing and that its ablation is sufficient to convert cancer cells into a highly susceptible phenotype and inhibit tumor growth in immunocompetent mice. Using correlative light electron microscopy, we found that the actin response is associated with long actin- rich protrusions that project and accumulate into the synaptic cleft. These protrusions are filled with long actin filaments organized in a main bundle and closely resemble the filopodial extensions found at the leading edge of migrating cells. Our investigations indicate that such synaptic filopodium-like protrusions (SFPs) correlate with morphologically abnormal synapses with fewer cell-to-cell adhesion points and aberrantly large synaptic clefts. In addition, we found that SFPs are heavily decorated with inhibitory ligands, such as PD-L1, that polarize to the IS in tumor cells with an actin response. Our pioneering work provides evidence that conjugates tumor cells do not passively wait for their death but actively engage in fighting by rapidly remodeling their own actin cytoskeleton and assembling membrane protrusions that likely alter IS formation and activity. By redirecting attention to the tumor cell side of the IS our observations pave the way for the development of new therapeutic strategies to restore a potent anti- tumor immune response and improve the efficacy of existing immunotherapies.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-207
Enhanced T cell infiltration in the normal colonic mucosa of Lynch syndrome tumor patients and tumor-free LS carriers L. Bohaumilitzky1, 2, K. Kluck3, R. Hüneburg4, 5, J. Nattermann4, M. Kirchner3, L. Wagner1, 2, F. Echterdiek6, N. Müller1, 2, H. Bläker7, T. Seppälä8, 9, J.-P. Mecklin10, 11, M. von Knebel Doeberitz11, 2, A. Stenzinger3, J. Budczies3, M. Kloor1, 2, A. Ahadova1, 2 1University Hospital Heidelberg, Institute of Pathology, Department of Applied Tumor Biology, Heidelberg, Germany, 2German Cancer Research Center (DKFZ), Clinical Cooperation Unit Applied Tumor Biology, Heidelberg, Germany, 3University Hospital Heidelberg, Institute of Pathology, Heidelberg, Germany, 4University Hospital of Bonn, Department of Internal Medicine I, Bonn, Germany, 5University Hospital of Bonn, Center for Hereditary Tumor Syndromes, Bonn, Germany, 6Klinikum Stuttgart, Department of Nephrology and Autoimmune Diseases, Stuttgart, Germany, 7University Hospital Leipzig, Institute of Pathology, Leipzig, Germany, 8Helsinki University Hospital and Helsinki University, Department of Gastrointestinal Surgery, Helsinki, Finland, 9Johns Hopkins University, Department of Surgical Oncology, Baltimore, MD, United States, 10University of Jyväskylä and Jyväskylä Central Hospital, Department of Sport and Health Sciences, Jyväskylä, Finland, 11Central Finland Central Hospital, Department of Education and Research, Jyväskylä, Finland
Text Due to the high load of immunogenic frameshift neoantigens, tumors arising in the context of Lynch syndrome (LS), the most common inherited cancer syndrome, are characterized by a pronounced immune infiltration. Immune interactions with frameshift neoantigens were described to shape the mutational pattern of arising tumors. Moreover, systemic frameshift neoantigen-specific immune reactions were detected even in tumor-free LS carriers, supporting the hypothesis of immune surveillance. Whereas the immune milieu of LS colorectal cancer (CRC) has been extensively studied before, the knowledge about immune infiltration of normal colonic mucosa is largely lacking. We asked whether evidence for immune activation could be found in normal, tumor-distant mucosa of individuals with LS. CD3 and FOXP3-positive T cells were quantified in a total of 227 normal colonic mucosa samples from LS tumor-free carriers, LS CRC patients, sporadic MSI CRC and MSS CRC patients. In addition, LS CRCs (n=24) were analyzed for CD3 and FOXP3-positive T cell densities. Gene expression analysis with Nanostring nCounter PanCancer Human IO360 Panel was performed in a subset of samples from each group. We observed significantly elevated CD3-positive (p<0.0001) and FOXP3-positive (p<0.001) T cell densities in the normal colonic mucosa of LS carriers and LS CRC patients compared to MSS CRC patients. Among LS specimens, gene expression data suggested overrepresentation of Tregs and neutrophils in CRC specimens compared to normal mucosa of LS individuals. In contrast, CD45-positive, exhausted CD8-positive, NK, Mast and B cell populations were overrepresented in the normal mucosa of LS carriers compared to CRC specimens. Interestingly, analysis of gene expression profiles indicated that normal mucosa from tumor-free LS carriers and normal mucosa from LS CRC patients form two distinct clusters exemplified by overrepresentation of CD45, exhausted CD8, NK and mast cells. Our observations suggest that LS is characterized by alterations of the mucosal immune environment even in the absence of manifest cancers. The mechanisms underlying these observations are unclear, however they may be related to neoantigen-triggering mutational activity in LS colonic mucosa. Moreover, the mucosal immune environment may modify CRC risk in LS.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-208
Tumor-stroma cross-talk in an organotypic human melanoma-in-skin model promotes immune suppression by hampering dendritic cell differentiation E. Michielon1, M. López González2, J. L. A. Burm1, T. Waaijman1, E. S. Jordanova3, S. Gibbs1, 4, T. D. de Gruijl2 1Amsterdam UMC, Vrije Universiteit, Department of Molecular Cell Biology and Immunology, Amsterdam, Netherlands, 2Amsterdam UMC, Vrije Universiteit, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, Department of Medical Oncology, Amsterdam, Netherlands, 3Center for Gynecologic Oncology Amsterdam (CGOA), Amsterdam UMC, Vrije Universiteit, Amsterdam, Netherlands, 4Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit, Department of Oral Cell Biology, Amsterdam, Netherlands
Text Melanoma immune escape is facilitated by the presence in the tumor microenvironment (TME) of immune suppressive factors, which hinder dendritic cell (DC) differentiation and promote the enrichment of tolerogenic M2 macrophages and myeloid-derived suppressor cells. This effectively hampers T cell priming and effector T cell activity, and so interferes with the efficacy of immunotherapies, contributing to their failure in more than half of the treated patients. Counteracting this suppression is thus critical to activate anti-tumor immune responses and improve therapeutic strategies. The preclinical human models currently employed to study tumor-mediated myeloid suppression often fail to reflect the complexity of the TME. Here, we established an in vitro three-dimensional (3D) reconstructed organotypic human melanoma-in-skin (Mel-RhS) model by co-seeding epidermal cells and the BRAF- and PTEN-mutated SK-MEL-28 melanoma cell line onto a fibroblast-populated dermal equivalent, allowing the monitoring of tumor growth and progression up to six weeks. Tumor nests developed over time and spread towards the dermis, resembling the early stages of in vivo melanoma invasion. Significantly higher levels of the immune suppressive cytokines IL-10, M-CSF, and TGF-β were secreted by the Mel-RhS, in comparison to its healthy counterpart. Indeed, Mel-RhS-derived culture supernatants interfered with monocyte-to-DC differentiation, leading to the formation of M2-like macrophages, characterized by a lack of CD1a and concerted upregulation of CD14, CD163, BDCA3, CD16, PD-L1, and PD-L2. The use of neutralizing antibodies against IL-10, M-CSF, and TGF-β prevented this suppression to varying degrees with differential effects on surface marker expression and development of monocyte-derived immune subsets. In particular, blockade of IL-10 significantly increased CD1a expression and reduced all M2-associated markers, while upregulating CD80 levels, as shown by t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis. Interestingly, despite not detecting IL-10 in two-dimensional (2D) SK-MEL-28 monolayers, RNA in situ hybridization revealed de novo IL-10 expression in the melanoma cells, as well as in the surrounding keratinocytes and fibroblasts. We conclude that the physiologically relevant 3D configuration of the Mel-RhS encompasses melanoma-associated features that are otherwise lost in 2D cultures, involving cellular cross-talk between tumor and stroma. This allows for the uncovering of meaningful TME-induced immune evasion mechanisms and immune suppressive cytokine-related effects. Further immune reconstitution in this melanoma model may provide a novel in vitro tool for preclinical testing of immune modulatory agents.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-209
Actin accumulation at the immunological synapse protects CLL cells from natural killer cell-mediated lysis H. Wurzer1, L. Filali1, C. Hoffmann1, M. Krecke1, A. M. Biolato1, J. Mastio1, S. Dewilde2, J. H. François2, A. Largeot1, J. Paggetti1, E. Moussay1, G. Berchem1, 3, C. Thomas1 1Luxembourg Institute of Health, Department of Oncology, Luxembourg, Luxembourg, 2Centre Hospitalier du Luxembourg, Luxembourg, Luxembourg, 3Centre Hospitalier du Luxembourg, Department of Hemato- Oncology, Luxembourg, Luxembourg
Text Chronic lymphocytic leukemia (CLL) has been described as one of the cancer types with the highest incidence in adults from the United States and Europe. The disease progression is slow, however, even in the early stages, the adaptive immune system is already heavily impacted by the constant pressure of CLL cell exposure. Natural Killer (NK) cells are cytotoxic effector lymphocytes belonging to the innate immune system displaying a critical anti-tumor effect against the malignancy. Nevertheless, they fail to control the disease progression over long term. During their encounter with CLL cells, NK cells have to form a specific contact area known as the immunological synapse (IS). This cell-cell interface and its effector functions rely mainly on the dynamics of the actin cytoskeleton on the NK side and lead to a controlled release of cytotoxic molecules such as perforin and granzyme B. Here, we introduce a novel intrinsic immune evasion mechanism of CLL cells from cytotoxic NK cells. We report for the first time cytoskeletal changes on the CLL side during target-effector interaction and show that rapid actin accumulation at the IS is closely associated with resistance against NK cell-mediated cytotoxicity. This synaptic actin filament accumulation or "actin response", can be abrogated in CLL cells by targeting pharmacologically the key actin regulator CDC42, leading to a restauration of CLL cell susceptibility to NK cell cytotoxicity. After confirming the actin response in various CLL cell lines, we validated this evasion mechanism in primary patient-derived CLL cells. Together our result provide compelling evidence that the actin cytoskeleton plays a critical role in CLL resistance against NK cell cytotoxicity and suggest that the actin response can be used as a therapeutic target to inhibit CLL immune evasion.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-210
Diversification of circulating and tumor-infiltrating pDCs towards the P3 (CD80+ PDL1-)-pDC subset negatively impacts clinical outcome in melanoma patients E. Sosa Cuevas1, 2, N. Bendriss-Vermare3, S. Mouret4, F. De Fraipont5, J. Charles1, 4, J. Valladeau-Guilemond3, L. Chaperot1, 2, C. Aspord1, 2 1INSERMU1209/UGA, Immunology and immunotherapy in chronic diseases, Grenoble, France, 2EFS- Auvergne-Rhone-Alpes, R&D, Grenoble, France, 3Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de Recherche en Cancérologie de Lyon, Lyon, France, 4CHU Grenoble Alpes, Dermatology Department, Pôle Pluridisciplinaire de Médecine,, Grenoble, France, 5Medical Unit of Molecular genetic (hereditary diseases and oncology), Grenoble University Hospital, Grenoble, France
Text pDCs play a critical yet enigmatic role in antitumor immunity through their pleiotropic immunomodulatory functions. Despite proof of pDC diversity in several physiological or pathological contexts, pDCs have been studied as a whole population so far in cancer. The assessment of diverse pDC subsets is needed to fully grasp their involvement in cancer immunity, especially in melanoma where pDC subsets are largely unknown and remain to be uncovered. In this study, we explored the features of diverse circulating and tumor- infiltrating pDC subsets in melanoma patients using multi-parametric flow cytometry, and assessed their clinical relevance. Based on CD80, PDL1, CD2, LAG3, and Axl markers, we provide an integrated overview of the frequency, basal activation status, and functional features of pDC subsets in melanoma patients together with their clinical impact. Strikingly, we demonstrate that P3-pDCs (CD80+PDL1-) accumulate within tumor of melanoma patients and negatively impact clinical outcomes. The basal activation status, diversification towards P1-/P2-/P3-pDCs, and functionality of several pDC subsets upon TLR7-/TLR9- triggering were perturbed in melanoma patients, and differentially impacted clinical outcome. Our study shed light on the phenotypic and functional heterogeneity of pDCs in the blood and tumor of melanoma patients, and their key role in dictating clinical outcomes. Such novelty brightens our understanding of pDC complexity, and prompts the further deciphering of pDCs’ features to better apprehend and exploit these potent immune players. It enlightens the importance of considering pDC diversity when developing pDC- based therapeutic strategies to ensure optimal clinical success.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-211
Dysfunctional BTN3A together with deregulated immune checkpoints and type I/II IFN dictate defective interplay between pDCs and γδT cells in melanoma patients which impacts clinical outcomes P. Girard1, E. Sosa Cuevas1, 2, B. Ponsard1, S. Mouret3, F. De Fraipont4, J. Charles2, 3, O. Manches1, 2, L. Chaperot1, 2, C. Aspord1, 2 1EFS-Auvergne-Rhone-Alpes, R&D, Grenoble, France, 2INSERMU1209/UGA, Immunology and immunotherapy in chronic diseases, Grenoble, France, 3CHU Grenoble Alpes, Dermatology Department, Pôle Pluridisciplinaire de Médecine,, Grenoble, France, 4Medical Unit of Molecular genetic (hereditary diseases and oncology), Grenoble University Hospital, Grenoble, France
Text Objectives: pDCs and γδT cells emerge as potent immune players participating in the pathophysiology of cancers, yet still remaining enigmatic while harboring a promising potential for clinical translations. Despite strategic and closed missions, crosstalk between pDCs and γδT cells have not been deciphered yet in cancers, especially in melanoma where the long-term control of the tumor still remains a challenge. Methods: This prompted us to explore the interplay between pDCs and γδT cells in the context of melanoma, investigating the reciprocal features of pDCs or γδT cells, the underlying molecular mechanisms and its impact on clinical outcomes. Results: TLRL-activated pDCs from the blood and tumor-infiltrate of melanoma patients displayed an impaired ability to activate, modulate immune-checkpoints, and trigger the functionality of γδT cells. Conversely, γδT cells from the blood or tumor-infiltrate of melanoma patients activated by PAg were defective in triggering pDCs’ activation and modulation of immune-checkpoints, and failed to elicit the functionality of pDCs. Reversion of the dysfunctional cross-talks could be achieved by specific cytokine administration and immune-checkpoint targeting. Strikingly, we revealed an increased expression of BTN3A on circulating and tumor-infiltrating pDCs and γδT cells from melanoma patients, but stressed out its potential functional impairment. Conclusion: Our study uncovered that melanoma hijacked pDCs/γδT cells’ bidirectional interplay to escape from immune control, and pointed out BTN3A dysfunction. Such understanding will help harness and synergize the power of these potent immune cells to design new therapeutic approaches exploiting their antitumor potential while counteracting their skewing by tumors to improve patient outcomes.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-212
Immune escape mechanisms developed in lung cancer following target therapy resistance: Role of the RNA-binding protein HuR A. Nigro1, I. Salvato2, L. Ricciardi1, M. J. Lamberti3, G. Caramori2, V. Casolaro1, C. Stellato1, J. Dal Col1 1University of Salerno, Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, Baronissi, Italy, 2University of Messina, Department of Biomedical Sciences, Dentistry, Morphological and Functional Imaging, Messina, Italy, 3Instituto de biotecnologia ambiental y salud (INBIAS), Cordoba, Argentina
Text Tyrosine kinase inhibitors (TKIs) including gefitinib are first-line treatment for advanced non-small cell lung cancers (NSCLC) with sensitive mutations in epidermal growth factor receptor (EGFR). However, acquired resistance invariably develops and is associated with an immune suppressive phenotype involving expression of immuno-related proteins, such as programmed death ligand-1 (PD-L1) and CD47, and secretion of proinflammatory and tumor resistance- promoting cytokines. The RNA-binding protein HuR targets these factors in cancer types including NSCLC by binding to 3’-untranslated region (UTR) and modulate mRNA stability/translation. Our aim was to define HuR role in immune escape following TKI-resistance in NSCLC. We investigated in vitro the expression of HuR and its regulatory role on immune checkpoints and cytokine expression, as well as migration of NSCLC cell lines in the absence/presence of gefitinib-induced resistance (PC9 and HCC827 cells with EGFR-sensitizing mutations; H1975 with EGFR-resistant secondary mutation and PC9GR and HCC827GR with in vitro acquired gefitinib-resistance). PC9 HuR-KO cell line was generated by CRISPR/Cas9 technology. Nuclear/cytoplasmic HuR levels were evaluated by Western blot and HuR binding to 3’- UTR of CD47 and PD-L1 mRNA by biotin pull-down assay. Expression of CD47 and PD-L1 was assessed by flow cytometry, IL-6 and IL-8 levels by specific ELISA, cell migration by wound healing assay. Gefitinib treatment reduced HuR expression in PC9 cells but not in PC9GR. While remaining mostly nuclear in PC9 cells, HuR increased in cytoplasmic fraction in PC9GR and H1975 cells. IL-6 and IL-8 release were significantly increased in PC9GR and HCC827GR cell lines while their secretion was significantly reduced in TKI-untreated and treated PC9 HuR- KO cells. Additionally, PC9 HuR-KO cells did not acquire TKI-resistance in vitro and wound closure was significantly delayed in these cells compared to PC9 parental cells. Surface CD47 expression was increased in PC9GR and HCC827GR; PD-L1 was significantly upregulated in PC9GR, while it was downregulated in HCC827GR compared to parental cells. Biotin pull-down identified HuR association with CD47 and PD-L1 mRNAs. Our results showed that HuR may regulate key factors involved in immune escape following TKI-induced resistance in NSCLC. Dissecting the contribution of HuR in this context would support the development of pharmacological inhibitors of specific interactions between HuR and its target mRNAs reverting the immune suppressive phenotype promoted by TKI-resistance.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-213
Capturing tumor-promoting macrophage transition in a 3D model of the early melanoma microenvironment G. Pizzurro, C. Liu, K. Bridges, A. Alexander, M. Bosenberg, M. Mak, K. Miller-Jensen Yale University, New Haven, United States
Text Anti-tumor immune responses are shaped by the tumor microenvironment (TME), which often evolves to be immunosuppressive, promoting disease progression and metastasis. Melanomas display high numbers of tumor-associated myeloid cells (TAMs), usually correlating with a worse prognosis. However, the functional plasticity of this immune cell population confers them the potential to restore their anti-tumor activity through targeted therapies. In order to target TAMs, there is a need to understand the early events in cell-cell interactions that shape their tumor-promoting profile in order to determine critical communication networks in the TME. Standard in vitro bone-marrow-derived macrophage (BMDM) polarization states do not accurately reproduce the diverse functional profiles observed in TAMs. In the present study, we built and optimized 3D in vitro culture systems in a collagen-I matrix using the Yale University Mouse Melanoma (YUMM) model to recreate the early TME and study macrophage response at the single-cell level. In these cocultures, we studied how the interaction with fibroblasts and tumor cells modulates macrophage immune activity throughout early timepoints. We monitored BMDM behavior and interactions through time-lapse imaging and characterized their activation through expression of functional markers and multiplex cytokine/chemokine secretion. We found that stromal cells induce a rapid functional activation and ‘scouting’ mode on BMDMs, driven by their initial distance to tumor cells and their location within the collagen gel over time. Fibroblasts displayed an active role in supporting macrophage survival and proliferation, and in the collagen matrix remodeling. Their presence in the 3D in vitro TME accelerated some aspects of the functional evolution of BMDMs. Most importantly, over the course of 7 days, BMDMs cultured in this 3D in vitro TME acquired a phenotypic and functional profile that resembles melanoma immunosuppressive TAMs of later timepoints. Overall, the direct cell-cell interactions of BMDMs with melanoma and stromal cells in a 3D collagen-I environment shape their transition towards a TAM-like state. Using a 3D in vitro TME system, we captured the initial macrophage-stromal crosstalk that builds the immunosuppressive melanoma TME. This system will allow us to study further TAM changes and responses when the TME is exposed to perturbations, such as immune effector cells, or their functional cytokines, and therapies targeting the myeloid compartment or the stroma.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-214
CCL21-loaded 3D synthetic hydrogels as artificial lymph nodes for T cell culture E. Pérez del Río1, 2, F. Santos1, 2, M. Castellote2, 3, J. Veciana1, 2, I. Ratera1, 2, J. Guasch2, 3 1Institute of Materials Science of Barcelona (ICMAB-CSIC), Bellaterra (Barcelona), Spain, 2CIBER-BBN, Bellaterra (Barcelona), Spain, 3Institute of Materials Science of Barcelona (ICMAB-CSIC), Max Planck Partner Group "Dynamic Biomimetics for Cancer Immunotherapy", Bellaterra (Barcelona), Spain
Text A family of three-dimensional (3D) poly(ethylene) glycol (PEG) hydrogels covalently combined with heparin (Hep) were engineered to resemble the extracellular matrix of the lymph nodes. In these hydrogels, PEG provides the needed structural and mechanical properties, whereas heparin is used as an anchor for positively charged cytokines, such as the cytokine CCL21, which can affect cell homing and proliferation. After optimizing the concentration of the reactants, 3%wt PEG-Hep hydrogels were found to be the more adequate ones with pore sizes around 50 um, as measured by environmental scanning electron microscopy. The shear modulus of the hydrogels was measured to be 1.1±0.1 KPa, which is in agreement with in vivo values, as also observed for the structural properties. Finally, the 3D PEG-Hep hydrogels were loaded with the cytokine CCL21, which remained immobilized in the hydrogels, as demonstrated by different techniques including fluorescence spectroscopy. Finally, the 3D PEG-Hep hydrogels were used to culture primary human CD4+ T cells, resulting in an increase in cell viability and proliferation compared to the state-of-the-art expansion systems consisting of artificial antigen presenting cells, such as Dynabeads, in suspension. Moreover, we were able to tune the cellular phenotypes obtained by modifying the physicochemical properties of the hydrogels. Thus, we present a new tool which could be used to 1) culture T cells in a way that better resembles the in vivo situation than the standard expansion systems to be used in ACT or 2) mimic metastatic lymph nodes, if the physicochemical properties of the hydrogels are properly tuned.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-215
Actin remodeling drives vesicle-mediated tumor immune escape A. M. Biolato1, F. Kleine Borgmann1, 2, L. Filali1, H. Wurzer1, C. Hoffman1, M. Mittelbronn1, 2, C. Thomas1 1Luxembourg Institute of Health (LIH), Oncology, Luxembourg, Luxembourg, 2Luxembourg Centre of Neuropathology, Luxembourg, Luxembourg
Text Natural killer (NK) cells and CD8+ T lymphocytes (CTLs) are cytotoxic lymphocytes with potent activity in eliminating tumor cells. They both directly kill transformed cells and have accordingly a great potential for cancer immunotherapy. However, the resistance mechanisms mounted by cancer cells and immunosuppressive tumor microenvironments remain major hurdles. Although their activation is regulated by different molecular interactions with tumor cells, NK cells and CTLs kill their targets using common mechanisms. One potent killing modality involves the formation of a specific contact interface with the target cell, known as the immunological synapse, and the directed secretion of lytic granules containing cytotoxic mediators, such as Perforin and Granzyme B (GrB). We recently uncovered a pivotal role of the actin cytoskeleton in mediating tumor cell resistance to NK cell-mediated lysis. Our findings show that, upon NK cell attack, a massive accumulation of actin near the synaptic region rapidly takes place in resistant target cells. This so-called "actin response" protects NK cell-conjugated target cells from apoptosis by decreasing intracellular levels of GrB. Our unpublished data support that the actin response also provides resistance against tumor-specific CTLs, and considerably facilitates tumor growth in immunocompetent mice. Currently, we are characterizing the mechanism by which the actin response prevents GrB accumulation in target tumor cells, and thus their subsequent cell lysis. Our imaging flow cytometry data demonstrate an enrichment of endosomal and lysosomal vesicles towards the synaptic region in tumor cells with an actin response and suggest that drives local increase of inhibitory signals and, possibly, enhanced degradation of cytotoxic molecules. Moreover, correlative light electron microscopy (CLEM)-based investigations revealed the presence of many small vesicles, resembling exosomes, in the synaptic region of cancer cells with an actin response. Their role in tumor immune evasion is under investigation.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-216
CD3-slow release DARPin® (CD3-SRD) – a smart solution for reducing cytokine release syndrome while maintaining optimal anti-tumor efficacy J. K. Ahlskog, A. Bosshart*, D. Schiegg, A. Eggenschwiler, S. Fontaine, D. Villemagne, M. Franchini, M. Matzner, W. Ali, M. Hänggi, Y. Kaufmann, I. Schlegel, C. Zitt, T. Hospodarsch, A. Neculcea, T. Lekishvili, H. Poulet, M. Paladino, C. Herzog, A. Schlegel, T. Looser, B. Schlereth Molecular Partners AG, Schlieren-Zurich, Switzerland *Contributed equally
Text T-cell engagers (TCEs) are a very potent class of drugs and may exhibit acute toxicities, such as endothelial activation, neurotoxicity and cytokine release syndrome (CRS), manifesting especially after the administration of the first dose. These toxicities narrow the therapeutic window of TCEs, often preventing them from reaching an efficient dose. Mitigation strategies include pre-medication with immune-suppressive steroids, treatment with IL-6 receptor antagonists, step-dosing or continuous intravenous infusion. Despite these strategies, severe side effects including death in extreme cases are still observed in the clinic, and as a consequence require suboptimal dosing schedules.
Here we describe a novel conceptual design to prevent overstimulation of T-cells and ultimately CRS. We have generated a non-covalent, anti-idiotypic DARPin® binder (hereafter termed blocker), that masks the CD3 effector moiety of a DARPin®- based TCE, thus preventing the TCE from interacting with CD3. Following administration, the blocker disengages the half-life extended, bioactive TCE with predictable first-order kinetics, slowly unmasking it in the circulation. This slow release mechanism avoids high maximal concentration (Cmax) peaks of the active TCE at treatment start, provides continuously increasing bioactivity, and mitigates the potential to trigger excessive immune activation.
A panel of blockers with various affinities for the CD3 effector moiety ranging from single-digit to triple-digit picomolar (pM) was tested for the ability to block and progressively release the CD3 engager molecule in in vitro assays. We demonstrate here that these blockers were able to initially inhibit the T-cell mediated tumor cell killing induced by the DARPin®-based TCE. The activity of the TCE and induction of tumor cell killing was progressively recovered, at a rate determined solely by the off-rate of the blocker molecule.
We also show that cytokine release was strongly reduced in an ex vivo preclinical study in a human whole-blood test system when using a slow activation, transiently blocked TCE compared to an active, non-blocked one. Finally, transiently blocked TCE showed reduced cytokine release in comparison to the non-blocked TCE while maintaining anti-tumor efficacy in a set of in vivo preclinical studies in human PBMC-engrafted, tumor-bearing NOG mice.
In summary, the slow release concept for DARPin®-based T-cell engagers resulted in reduced cytokine levels, while showing equivalent anti-tumor efficacy. This approach holds great promise beyond T-cell engagers and could theoretically be applied to any protein drug where acute side effects originate from the fast onset of drug effect.
J. Ahlskog and A. Bosshart contributed equally
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-217
Agonistic CD40 antibody therapy induces tertiary lymphoid structures but impairs the response to immune checkpoint blockade in glioma L. van Hooren1, A. Vaccaro*1, M. Ramachandran1, K. Vazaios1, S. Libard1, 2, T. van de Walle1, M. Georganaki1, H. Huang1, I. Pietilä1, J. Lau3, M. H. Ulvmar1, M. C.I. Karlsson4, M. Zetterling5, S. M. Mangsbo6, A. S. Jakola7, 8, T. Olsson Bontell9, 10, A. Smits5, 8, M. Essand1, A. Dimberg1 1Uppsala University, Immunology, genetics and pathology, Uppsala, Sweden, 2Uppsala University Hospital, Uppsala, Sweden, 3Uppsala University, Medical Cell Biology, Uppsala, Sweden, 4Karolinska Institutet, Microbiology, Tumor and Cell Biology, Stockholm, Sweden, 5Uppsala University, Neuroscience, Neurology, Uppsala, Sweden, 6Uppsala University, Pharmaceutical Biosciences, Science for Life Laboratory, Uppsala, Sweden, 7Sahlgrenska University Hospital, Neurosurgery, Gothenburg, Sweden, 8University of Gothenburg, Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy, Gothenburg, Sweden, 9Sahlgrenska University Hospital, Clinical Pathology and Cytology, Gothenburg, Sweden, 10University of Gothenburg, Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, Gothenburg, Sweden *Contributed equally
Text Gliomas are brain tumors characterized by immunosuppression. Immunostimulatory agonistic CD40 antibodies (αCD40) are in clinical development for solid tumors but are yet to be evaluated for glioma. Here, we demonstrate that systemic delivery of αCD40 in preclinical glioma models induced the formation of tertiary lymphoid structures (TLS) characterized by a follicle-like organization, consisting of a B cell core, T cells, antigen presenting cells and follicular dendritic cells. TLS were also observed in meningeal regions in treatment-naïve glioma patients, where their presence correlated with increased intratumoral T cell infiltration. However, αCD40 treatment also led to impaired T cell responses, which were defined by reduced proliferation and activation of CD8+ and CD4+ T cells. This phenotype could not be rescued by co- administrating checkpoint inhibitors (CPIs). Instead, combinatorial treatment with αCD40 resulted in impaired responses to CPI therapy. This was associated with a systemic induction of suppressive CD11b+ B cells post αCD40 treatment. In summary, our study demonstrates that systemic αCD40 therapy results in impaired T cell responses and reduces the efficacy of CPIs in preclinical glioma models. Our work also reveals for the first time that TLS are present in association with brain tumors in patients and that immunotherapies can modulate these structures in murine glioma models.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-218
Comparison of the immune landscape and tumor mutational burden of primary and metastatic lesions of melanoma patients treated with immune checkpoint blockade M. A. J. Gorris1, L. L. van der Woude1, 2, K. F. Bol3, L. I. Kroeze2, K. Verrijp2, A. L. Amir2, J. C. Textor1, I. J. M. de Vries*1, C. G. Figdor*1 1Radboudumc, Tumor Immunology, Nijmegen, Netherlands, 2Radboudumc, Pathology, Nijmegen, Netherlands, 3Radboudumc, Medical Oncology, Nijmegen, Netherlands
Text Approximately 20% of metastatic melanoma patients have a long- term response when treated with ipilimumab. However, the majority of melanoma patients do not benefit, leading to disease progression and exposure to non- effective, expensive treatment with potential severe side effects. Therefore, there is a strong need for biomarkers that can accurately predict whether a patient may benefit from therapy or not. High tumor mutational burden (TMB), and programmed cell death 1 ligand 1 (PD-L1) expression as well as the number of tumor infiltrating lymphocytes (TILs) have been associated with response to immunotherapy in melanoma patients. When studying the tumor microenvironment in melanoma, often tumor samples from metastatic sites are used. However, these samples can be very heterogenous as they are found at different anatomical sites such as skin, lymph node and other distant organs. Other studies have focused on biomarker development in primary tumors. Currently, it is not known how similar or dissimilar the tumor microenvironment (TME) is between primary tumors and their respective metastases. Here we study the primary and metastatic lesions of melanoma patients to get insight in TME characteristics during the course of disease. TMB was assessed through targeted sequencing of DNA from primary and metastatic melanoma using the TSO500 panel. Tumor tissues were subjected to 7-color multiplex immunohistochemistry for the detection of TIL subsets (B cells, cytotoxic-, helper- and regulatory T cells) and analyzed using a machine-learning algorithm. We observed a high correlation between TMB derived from primary and metastatic melanoma lesions. By contrast, TIL density and composition between primary and metastatic melanoma lesions were very dissimilar. However, when analyzing two different metastatic melanoma lesions within a single patient, TIL density and composition correlated well, even when studying different anatomical locations. High TMB, either derived from primary or metastatic melanoma tissue, directly correlated with response to ipilimumab, whereas lymphocyte density or composition did not. We conclude that during the course of disease, TMB, as opposed to TIL, is similar between primary and metastatic melanoma lesions. Furthermore, since the strong link between high TMB and response to immunotherapy, independent of the timepoint and location of acquisition, TMB remains the most reliable biomarker for use in clinical practice.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-219
Immune Cell Type Deconvolution by RNA Sequencing Data P. Pandey1, J. Mohr1, C. Hotz1, N. Salomon2, F. Vascotto2, M. Vormehr1, M. Lang1, U. Sahin1, 2, 3 1BioNTech, Mainz, Germany, 2TRON, Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz gGmbH, Mainz, Germany, 3Research Center for Immunotherapy (FZI), University Medical Center at the Johannes Gutenberg University Mainz, Mainz, Germany
Text Tumor infiltrating lymphocytes (TILs) are an integral constituent of the tumor microenvironment and play essential roles in cancer development and progression. TILs are heterogeneous mixture of immune cells that include innate and adaptive immune populations, and cell types linked with active (e.g. cytotoxic T Cells) and suppressive (e.g. regulatory T Cells) immune functions. Their abundance varies with tumor type and stage, and they have been associated with prognosis and response to therapy. Characterizing TILs can reveal the mechanisms underlying the anti-cancer immune response and help to assess the immunogenic effects of therapies. Methods such as immunohistochemistry or flow cytometry are laborious and challenging to standardize. Therefore, in silico tools such as regularization methods have been developed to profile these immune subsets from bulk-RNA sequencing (bulk-RNAseq) data, using a set of immune specific marker gene expression signatures. We have predicted TILs from bulk-RNAseq of murine tumors in three different experimental settings. We used support vector regression and elastic net along with mouse specific immune gene signatures. Predicted immune cell type enrichments explained tumor suppression observed in the treatment groups compared to controls. Our results were concordant with biological expectations that matched with laboratory outcomes. Further, we have tested both of these methods on the phase I study (NCT02035956) of melanoma patients. These patients could be clustered into immune subtypes and compared with the clinical outcomes reported in the study.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-220
Exploring the immune infiltrate of rhabdoid tumors: from basic knowledge to the design of effective therapies. V. Manriquez1, L. Niborski1, 2, M. Vandenbogaert2, A. Leruste2, Z. Han2, Y. Gerber1, W. Richer1, J. Tosello1, R. Ramos1, J. Denizeau1, C. Sedlik1, P. Benaroch1, J. Helft1, J. Waterfall2, F. Bourdeaut2, E. Piaggio1 1Institut Curie, INSERM unit 932, Paris, France, 2Institut Curie, INSERM unit 830, Paris, France
Text Rhabdoid tumors (RT) are aggressive solid tumors of infancy characterized by the loss of only one gene, SMARCB1, which is a component of a chromatin-remodeling complex. We have recently shown that SMARCB1 loss-of-function induces the expression of endogenous retroviruses, activating the dsRNA- sensing pathway in the tumor, and inducing RT immunogenicity despite their inherent genetic similarity to self; positioning RT as an interesting model system for studying epigenetically driven tumors. By FACS and scRNAseq analysis of the tumor-infiltrating immune cells, we show that both human and mouse RTs: i) are highly infiltrated by clonally expanded CD8+ T and myeloid cells; ii) bear activated CD8+ T cell subpopulations expressing druggable inhibitory checkpoints, such as PD-1, Tim-3 and LAG-3; and iii) that blockade of the PD-1/PDL-1 pathway induced the regression of established RTs in mice. Deeper characterization of the myeloid infiltrate indicated that tumor-associated macrophages (TAMs) were the most represented subpopulation. Depletion of macrophages (using CD64-hDTR mice) delayed tumor growth, highlighting the negative impact of this population on tumor control. Based on the observed high expression of TLR3 in both human and mice myeloid cells in our RT samples, we treated RT-bearing mice by intratumoral administration of poly(I:C), which induced a significant delay of tumor growth. In-depth analysis using FACS, scRNAseq and immunofluorescence showed that poly(I:C) treatment: i) increased tumor infiltration by anti-tumoral neutrophils and inflammatory monocytes; ii) decreased the number of protumoral macrophages and skewed the phenotype of remaining TAMs into a pro-inflammatory one; iii) induced the accumulation of dendritic cells (cDC1) in tumor-draining lymph nodes ; and iv) induced the expansion of precursor exhausted CD8+ T cells (TPEX), which have been described as the main population responding to anti-PD-1. As expected, combination treatment of poly(I:C) with anti-PD1 induced durable total tumor rejection and full memory against tumor rechallenge. Altogether, our study suggests that combined targeting of myeloid and T cells represent a promising immunotherapy strategy for RT patients.
eTalk Session 3
Immunomonitoring & Tumor Biology and Interaction with the Immune System
S3-221
High-dimensional imaging mass cytometry reveals the spatial orchestration of CD8+ T cells and dendritic cells in oropharyngeal squamous cell carcinoma patients with tumor-specific immunity Z. Abdulrahman1, S. J. Santegoets2, M. E. Ijsselsteijn3, A. Somarakis4, M. J. Welters2, N. F. de Miranda3, S. H. van der Burg2 1Leiden University Medical Center, Departments of Medical Oncology and Pathology, Oncode Institute, Leiden, Netherlands, 2Leiden University Medical Center, Department of Medical Oncology, Oncode Institute, Leiden, Netherlands, 3Leiden University Medical Center, Department of Pathology, Leiden, Netherlands, 4Leiden University Medical Center, Department of Radiology, Leiden, Netherlands
Text Background: Oropharyngeal squamous cell carcinoma (OPSCC) is the most prevalent type of head and neck cancer. It can develop either through exposure to carcinogens (HPV- OPSCC) or following human papillomavirus infection (HPV+ OPSCC), the incidence of the latter is increasing. Previously we showed that the presence of tumor-infiltrating tumor-specific T cells (immune response positivity, IR+, ~60%) accurately correlated with survival. To gain comprehensive insight in the immune composition of OPSCCs with and without an anti-tumor immune response, a high-dimensional exploratory study of their immune microenvironment was performed.
Methods: Formalin-fixed paraffin-embedded tissue of OPSCC patients (n=20) collected from a prospective observational study were analyzed by 33-plex imaging mass cytometry (IMC, Hyperion). The images obtained by IMC were analyzed with a newly developed comprehensive bioinformatical pipeline, employing semi-supervised deep learning for signal normalization, cell segmentation and tissue segmentation, and HSNE based cell clustering. This enabled high-dimensional phenotyping of all cells in the TME as well as in-depth analysis of their spatial interactions.
Results: Almost half a million individual cells were characterized, and 51 unique cell clusters were identified, comprising 14 tumor, 15 lymphoid, 15 myeloid, and 7 other stromal cell clusters. The HPV+IR+ tumors displayed the highest immune cell content and were richly infiltrated by CD4+ and CD8+ T cells, M1 and M2 macrophages as well as dendritic cells. In contrast, the OPSCC of HPV16+IR- and HPV- patients contained relatively more tumor cells, fibroblasts and blood vessels, indicative for a more tumor promoting environment. Interestingly, a clear distinct spatial interactome composition was found between HPV16+IR+ and HPV16+IR- OPSCC, which retained after permutation based correction for differences in cell frequencies. While CD8+ T cells had strong direct spatial interactions with two clusters of dendritic cells and with tumor cells in HPV16+IR+ OPSCC, the CD8+ T cells more frequently spatially interacted with classical CD68+CD163+CD14- tumor associated M2 macrophages in HPV16+IR- OPSCC. Analysis of the 360° spatial compositions of the differential interactions sustained the notion that direct lymphocyte-tumor cell and lymphocyte-myeloid cell interactions occurred dominantly in HPV16+IR+ tumors, and were absent or scarcely present in HPV16+IR- tumors.
Conclusion: The presence of higher numbers of orchestrated intra-tumoral micro immune aggregates comprising CD8+ T cells and dendritic cells distinguishes OPSCC patients with tumor specific immunity from those without, and is associated with superior patient survival.