Open Full Page

CCR FOCUS CCR Focus Targeting the Heterogeneity of Cancer with Individualized Neoepitope Vaccines Ozlem€ Tureci€ 1, Mathias Vormehr2, Mustafa Diken1, Sebastian Kreiter1, Christoph Huber1, and Ugur Sahin1,2,3

Abstract

Somatic mutations binding to the patient's MHC and rec- "private" mutations. Concurrence of scientificadvancesand ognized by autologous T cells (neoepitopes) are ideal cancer technological breakthroughs enables the rapid, cost-efficient, vaccine targets. They combine a favorable safety profile due to a and comprehensive mapping of the "mutanome," which is the lack of expression in healthy tissues with a high likelihood of entirety of somatic mutations in an individual tumor, and the immunogenicity, as T cells recognizing neoepitopes are not rational selection of neoepitopes. How to transform tumor shaped by central immune tolerance. Proteins mutated in mutanome data to actionable knowledge for tailoring individ- cancer (neoantigens) shared by patients have been explored ualized vaccines "on demand" has become a novel research as vaccine targets for many years. Shared ("public") mutations, field with paradigm-shifting potential. This review gives an however, are rare, as the vast majority of cancer mutations in a overview with particular focus on the clinical development of given tumor are unique for the individual patient. Recently, the such vaccines. Clin Cancer Res; 22(8); 1885–96. 2016 AACR. novel concept of truly individualized cancer vaccination See all articles in this CCR Focus section, "Opportunities and emerged, which exploits the vast source of patient-specific Challenges in Cancer Immunotherapy."

Introduction (UTR) of mRNAs, altered proteasomal cleavage sites, or protein-splicing events (7, 8). Genetic abnormalities are key drivers of cancer (1–4). A hun- Neoepitopes are exempt from central tolerance, and thus, dred years ago, Ernest Tyzzer introduced the term "somatic thepresenceofhigh-affinity T cells against them in the mutation" to describe these abnormalities. In a compellingly patient's repertoire is very likely. A considerable number of visionary review (5), he also predicted immune recognition of the neoepitopes accumulating in the course of tumor evolu- somatic mutations and their interindividual variability within tion are recognized by autologous spontaneously occurring tumors of a species, and postulated the existence of tumor-related T cells in mice and in cancer patients and may constitute the immunosuppressive mechanisms (see Box 1). Achilles' heel of tumor cells. In mice, molecular characteriza- Various types of genomic mutations exist: point mutations tion of tumor rejection antigens unraveled mutated epitopes (exchange of one nucleotide by another), insertions/deletions (9–14). Various unique immunodominant mutations were (one or more nucleotides added or removed from the original identified in human cancer patients using tumor-reactive DNA), amplifications/deletions (multiplication or loss of T-cell lines derived from tumor-infiltrating lymphocytes (TIL) copies of a chromosomal region), and translocations and or obtained by repeated stimulation of peripheral blood inversions (interchange of pieces of nonhomologous chromo- T cells with autologous tumor cell lines (refs. 15–17; reviewed somes or reversion of the orientation of a chromosomal in ref. 8). segment; ref. 6). By altering the sequence of translated gene Whereas these data clearly indicate sporadic immune rec- products, mutations may create novel immunogenic epitopes ognition of mutant neoepitopes, the overall relevance of presented on MHC molecules and recognized by T cells mutations for cancer immunotherapy remained controversial (Fig. 1). Neoepitopes may also be created by cancer-associated and ambiguous for a long time. Only recently, a series of alterations in mRNA translation, antigen processing, and pre- independent reports revealed that neoepitope-specificTcells sentation, for example, translation of untranslated regions are crucial for clinical responses mediated by adoptive transfer of autologous TILs or by immune checkpoint inhibitors [(refs. 18–23; summarized in refs. 24–27); also see reviews by Hegde and colleagues (28) and Maus and June (29) in 1TRON – Translational Oncology at the University Medical Center of Johannes Gutenberg University, Mainz, Germany. 2Research Center this CCR Focus]. In patients treated with anti–CTLA-4 or anti– for Immunotherapy (FZI), Mainz, Germany. 3Biopharmaceutical New PD-1, the mutational load in the tumor appeared to be an Technologies (BioNTech) Corporation, Mainz, Germany. important predictor of clinical benefit establishing the rele- Corresponding Author: Ugur Sahin, TRON Translational Oncology at the Uni- vance of neoepitopes for rejection of at least melanoma, non– versity Medical Center of Johannes Gutenberg University, Freiligrathstr. 12, smallcelllungcancer(NSCLC),andmismatchrepair–defi- Mainz 55131, Germany. Phone: 4906-1312-1610; Fax: 4906-13121-61100; E-mail: cient colorectal tumors, which are the "signature" cancer types [email protected] for checkpoint blockade. Concordantly, temporary expansion doi: 10.1158/1078-0432.CCR-15-1509 of mutation-specific T cells upon checkpoint blockade was 2016 American Association for Cancer Research. reported in some patients and in tumor-bearing mice (20, 30).

www.aacrjournals.org 1885

human tumor specimen was a potential MHC class I neoepi- Box 1. Relationships between somatic tope (33, 34). mutations and tumor immunity as Various attempts have been made to apply neoantigen- predicted by Tyzzer based vaccine strategies in mouse models. Matsushita and colleagues and Gubin and colleagues showed the feasibility There are marked differences in the behavior of various tumors of using genomic and bioinformatic approaches to identify on transplantation in given classes of mice. Even tumors arising in MHC class I neoepitopes as tumor rejection antigens in a homogenous races show such differences, and this may be attrib- highly immunogenic methylcholanthrene (MCA)-induced ... uted to acquisition of new characteristics, [ ] it appears logical murine sarcoma model (14, 30). In continuation of this work, ... to regard a tumor as a manifestation of somatic mutation[ ]. his laboratory demonstrated in immune-edited variants of the The tissue of the new growth has thus in certain respects become sarcoma model that the antitumor effect of checkpoint block- ... foreign to the other tissues [ ]. Malignant tumors must have ade is mediated by MHC class I neoepitope-specificTcellsand fi feeble antigenic power as well as suf cient resistance to the normal that vaccination with long synthetic peptides encoding these fl inhibiting in uences to provide continued growth in the animal neoepitopes induces the same therapeutic activity as check- fi in which they originate, otherwise reactions suf cient to destroy point inhibition. them would occur more frequently. Our group pioneered the concept of individualized neoepitope vaccines and proposed a tailored approach to exploit Ernest Edward Tyzzer, "Tumor Immunity," The Journal of the full spectrum of the individual mutations of a patient Cancer Research, 1916 (ref. 5) (14, 30, 34, 35). Vaccination studies with synthetic peptides and mRNA-encoding mutations identified by NGS in three different mouse tumor models revealed that a significant portion of nonsynonymous point mutations (21%–45%) is immunogenic. Unexpectedly,thevastmajorityofthese þ Moreover, recent studies from Laurence Zitvogel's (INSERM, neoepitopes were not recognized by CD8 but were recognized þ U805, Institut Gustave Roussy, 39 Rue Camille Desmoulins, by CD4 T cells. mRNA vaccines encoding single MHC class II F-94805 Villejuif, Paris, France) as well as from Thomas neoepitopes were capable of controlling the growth of estab- Gajewski's (Department of Pathology, University of Chicago, lished mouse B16-F10 melanoma and CT26 colon cancer Chicago, IL) laboratories convincingly demonstrated that tumors. On the basis of these observations, we developed a commensal gut microbes, such as Bacteroides, Burkholderia,or process combining computationalpredictionofrelevantMHC fi Bi dobacterium species, may positively affect the success of class II neoepitopes from NGS exome data with rapid produc- checkpoint blockade treatment in tumor-bearing mice (31, tion of synthetic poly-neoepitope mRNA vaccines. Vaccination 32). Vetizou and colleagues demonstrated that adoptive trans- with such mRNA vaccines induced an inflammatory tumor – þ fer of bacteria-restimulated T cells from anti CTLA-4 anti- microenvironment, mediated induction of a CD8 T-cell – body exposed mice is able to induce tumor growth retarda- response by antigen spread, and resulted in complete rejec- fi tion in tumor-bearing recipient mice (31). This nding indi- tion of established aggressively growing tumors. Interestingly, fi cates that microbial antigen-speci c T cells might be cross- employing the same predictive algorithm on corresponding reactive with neoantigens, thereby supporting antitumoral human cancer types demonstrated an abundance of MHC responses after checkpoint blockade. Snyder and colleagues class II epitopes indicating the applicability of this approach – proposed that anti CTLA-4 checkpoint blockade favors T-cell to human cancers. responses against neoantigens sharing common peptide sub- Duan and colleagues showed that prophylactic vaccination fi string patterns (22); however, this nding could not be against neoepitopes identified by a prediction approach select- fi con rmed in a study by Van Allen and colleagues investigat- ing neoepitopes with improved MHC class I binding inhibits ing responses in a larger cohort of cancer patients (18). growth of CMS5 or Meth A tumors in mice (36). Yadav and Further studies with isolated T-cell clones or T-cell receptor colleagues combined mass spectrometry (MS) and exome – (TCR) engineered T cells are needed to get a better under- sequencing and demonstrated prophylactic and therapeutic standing of potential common epitope patterns recognized by activity of vaccination against identified neoepitopes in the fi neoantigen cross-reactive microbial antigen-speci cTcells. MC38 tumor model (37).

Vaccines Targeting Individual Mutations Clinical Translation of Individualized Mutation-based vaccination attempts represent "off-the-shelf" approaches, as they target single or multiple frequently shared Neoepitope Vaccines neoantigens, such as mutRas, mutP53, mutVHL, mutEGFR, or Despite excitement that in conjunction with individualized mutIDH1 (Table 1), which were used to stratify patients according immunotherapy, the spectrum of potential targets is now to the presence or absence of the respective mutations in their extended by unique mutations, there is no consensus so far tumor specimen (Fig. 2). on the path to their successful clinical application. That each With the introduction of next-generation sequencing tech- and every patient's cancer would be subjected to deep sequenc- nologies (NGS), it became apparent that human cancers are ing to manufacture a vaccine of unique composition "on much more complex and bear dozens to thousands of muta- demand" differs in key aspects from conventional "off-the- tions, of which, according to the analysis with algorithms for shelf" drug approaches. Putting a mutanome-based individu- MHC-binding prediction, a considerable number in every alized treatment concept into practice requires both highly

1886 Clin Cancer Res; 22(8) April 15, 2016 Clinical Cancer Research

Normal DNA Exon Protein CGUGGA Intron G A C Altered RNA G sequence C T Gly Leu Glu His Met Lys Types of neoepitopes

Point mutation CC GGAG G G C Nonanchor residue

C C G Gly Pro Glu His Met Figure 1. Lys Anchor residue Types of genomic mutations and T-cell neoepitopes resulting from them. A, nonsynonymous point Deletion mutations in the coding sequence of a gene alter a single amino acid. By CGGGA creating an anchor residue or G C Frameshift type 1 changing the TCR-binding (original frame + new frame) properties (nonanchor residue), a C G Gly Arg Ser Arg Met neoepitope may be formed. Val Insertion of three or a multiple of 3 nucleotides in frame introduces Insertion novel amino acids into the protein Frameshift type 2 (new frame) sequence, potentially generating a G CGUGGA T-cell response. Insertion or C G A C deletions of exonic nucleotides, G C T G mutations in intronic regions that Gly Ala Gly Glu Met affect RNA splicing, or gene fusions Trp In-frame insertion (altered genes after fusion marked with an apostrophe) can cause inclusion of introns into mRNA and a shift of the open reading frame. Splice site mutation Resulting T-cell epitopes may be in

part (type 1) or fully (type 2) gtaacgag gtcggcactgttaactcg comprised of an altered amino acid Gly Ala Thr Ser Arg His sequence. In addition, T cells may Val Translated intron target neoepitopes formed by Gene fusion translated introns or fusion of distant Gene A Gene Z Gene A’ Gene Z’ exons and genes as a result of splice Exon/Gene A’ site mutations and gene fusion. B, Exon/Gene Z’ human cells express up to 6 different Ala MHC class I (2 alleles of HLA-A, -B, Gene A’ Gene Z’ Gly Ala Ser Fusion spanning Lys Thr Arg and -C) and class II molecules (2 Val alleles of HLA-DP, -DQ, and -DR), thereby presenting neoepitopes to T cells. B

β α α2 α1 1 1

β α α3 β2M 2 2

HLA-A, B, C (2x) HLA-DP, DQ, DR (2x)

www.aacrjournals.org Clin Cancer Res; 22(8) April 15, 2016 1887

Table 1. Cancer vaccine trials using single or multiple mutations as cancer vaccine targets Patients Reference/ planned Clinicaltrials. Date Sponsor Cancer entity or treated Target Vaccine format Outcome gov identifier 2015 National Center for Grade III–IV gliomas 39 IDH1R132H Peptide þ Montanide Open study NCT02454634 Tumor Diseases and topical (Heidelberg, imiquimod Germany) 2015 (75) Glioblastoma 65 EGFRvIII mutation Peptide þ KLH Improved OS — 24 months; 85% of patients analyzed have specific humoral response 2015 Duke University Recurrent grade II 24 IDH1R132H Peptide þ GM-CSF þ Ongoing study NCT02193347 (Durham, NC) glioma Montanide 2014 (76) Colorectal (38), 53 Patients' individual Peptide þ IL2 20/37 patients developed — pancreatic (11), RAS mutations a specific immune common bile duct (1), response measured lung (3) in ELISpot ond/or proliferation assay 2014 (77) KRAS-mutant lung 24 7 common RAS 4 heat-inactivated 50% of patients — cancer mutations Saccharomyces developed a T-cell cerevisiae yeast response; proof of products, each safety expressing a unique combination of 3 RAS mutations, collectively targeting 7 RAS mutations 2013 Radboud University CRC, hypermutated 25 Frameshift-derived Peptide-loaded DC Ongoing study NCT01885702 (Nijmegen, the (Lynch, MSI) neoantigens; CEA Netherlands) 2013 Dana-Farber Cancer Melanoma 20 Personal neoantigens Peptide þ Poly ICLC Ongoing study NCT01970358 Institute (Boston, MA) 2011 Celldex Therapeutics Glioblastoma 700 EGFRvIII mutation Peptide þ KLH Ongoing study NCT01480479 2011 Oryx GmbH CRC, hypermutated Frameshift-derived Synthetic peptide þ 22 patients completed; NCT01461148 (Lynch, MSI) neoantigens for Montanide induction of immune TAF1B, HT101, responses against AIM2 frameshift-derived neoantigens, 1 patient with advanced disease showed stable disease 2010 (78) Glioblastoma 18 (group); EGFRvIII mutation Peptide þ KLH Greater OS in treatment — 17 (control group compared with group) control group; 6/14 patients analyzed showed specific humoral response; 3/17 patients showed a positive DTH reaction 2009 (79) Glioblastoma 15 EGFRvIII mutation Peptide-loaded DC 10/12 vaccinated patients — showed a specific proliferative T-cell response in vitro 2008 (80) Pancreatic and CRC 12 (5 pancreas, Patients' individual 13 mer peptide Specific T-cell response — 7 colorectal) RAS mutations in 5 patients; proof of safety 2007 NCI (Bethesda, MD) Colorectal, pancreatic, Data not RAS mutations Peptide þ Detox-B Study completed; no NCT00019006 lung cancer available outcome data available 2007 (81) RAS-positive NSCLC 4 7 common RAS Peptide mix of 7 1 patient developed a — mutations peptides þ GM-CSF positive DTH reaction; proof of safety 2007 (74) Chronic myeloid 21 BCR–ABL fusion Peptides þ IFNa Improved cytogenetic NCT00466726 leukemia protein response in 13/21 patients, 6 complete molecular remissions 2006 GlobeImmune Pancreatic cancer 176 Mutated Ras Yeast-derived Study completed NCT00300950 recombinant- mutated Ras protein 2005 (82) Various cancer entities 39 Individual mutant Peptide-loaded PBMC 16 patients developed a — p53; individual specific immune mutant RAS response; proof of safety (Continued on the following page)

1888 Clin Cancer Res; 22(8) April 15, 2016 Clinical Cancer Research

Table 1. Cancer vaccine trials using single or multiple mutations as cancer vaccine targets (Cont'd ) Patients Reference/ planned Clinicaltrials. Date Sponsor Cancer entity or treated Target Vaccine format Outcome gov identifier 2002 (83) Alveolar 16 Breakpoint region Peptide-loaded PBMC 1/3 patients evaluated — rhabdomyosarcoma of the EWS/FLI-1 developed a specific breakpoint region immune response from the 2 most measured in common variants proliferation assay of this translocation and the invariant breakpoint of the PAX3/FKHR fusion 2001 (84) Melanoma 10 4 common RAS Peptide mix of 4 8 patients developed a — mutations peptides þ GM-CSF strong DTH reaction; in 2 patients, mutation- specific in vitro proliferation was observed 2001 (85) Pancreatic 48 4 common RAS 4 peptides mixed þ Specific T-cell response — adenocarcinoma mutations GM-CSF in 25/43 evaluable patients; prolonged survival of patients with specific T-cell response 2001 NCI Breast cancer, cervical Data not p53, RAS mutations Peptide, peptide-pulsed Study completed; no NCT00019084 cancer, CRC, lung available DC, in vivo–primed outcome data cancer, ovarian T cells generated available cancer, pancreatic against the p53 or cancer Ras mutation 2000 Memorial Sloan Lung adenocarcinoma Data not RAS mutations Peptide þ GM-CSF Study completed; no NCT00005630 Kettering available outcome data Cancer Center available (New York, NY) 1999 NCI Sporadic renal cell 6 VHL mutations Peptide þ IFA Terminated due to poor NCT00001703 cancer carrying accrual and peptide VHL mutations supply problems Specific T-cell response in 4/5 patients 1999 (86) Advanced solid cancer 10 (evaluable) Patients' individual Peptide þ Detox-B Specific T-cell response — RAS mutations in 3 patients 1999 Memorial Sloan Myelodysplastic Data not RAS mutations Peptide þ GM-CSF Study completed NCT00003959 Kettering syndrome available No outcome data Cancer Center available 1999 NCI Ovarian cancer 21 p53 mutations Peptide þ GM-CSF Study completed; no NCT00001827 outcome data available 1996 (87) Advanced pancreatic 5 Patients' individual Peptide-loaded APC 2 patients generated a — cancer RAS mutations specific T-cell response; proof of safety Abbreviations: APC, antigen-presenting cell; CEA, carcinoembryonic antigen; CRC, colorectal cancer; DC, dendritic cell; DTH, delayed-type hypersensitivity; GM-CSF, granulocyte macrophage colony-stimulating factor; MSI, microsatellite instability; OS, overall survival; PBMC, peripheral blood mononuclear cell; VHL, von Hippel–Lindau.

interdisciplinary research and an innovative drug development Identification, Prioritization, and Selection process. Clinical translation of this concept has just begun, and of Mutations for Design of Cancer Vaccines a small number of early clinical trials with exciting exploratory concepts are under way, data from which are expected to for Clinical Use increase the level of understanding (Table 2). After several The cancer mutanome is defined by comparing exome sequenc- decades of preclinical and clinical cancer vaccine research, the ing data obtained by NGS of individual healthy tissue with field as such has matured, and many lessons have been learned sequences from tumor-derived nucleic acids. Usually, the blood as to which aspects to consider for the next generation of cells of a patient serve as a source for healthy tissue DNA. Methods vaccines [see reviews by Whiteside and colleagues (38) and to obtain DNA from tumor tissue should preferably be compat- Bol and colleagues (39) in this CCR Focus], for example, by ible with what is available in the context of routine diagnostics. In understanding the pros and cons of potential vaccine format our experience, efficient and reproducible isolation of nucleic platforms, the use of suitable adjuvants, and synergistic com- acids in NGS-grade quality is feasible from fresh as well as frozen bination treatment protocols [also see review by Zarour (40) in or formaldehyde-fixed, paraffin-embedded biopsies. A caveat is this CCR Focus]. that, in general, a limited number of tumor lesions per patient are

www.aacrjournals.org Clin Cancer Res; 22(8) April 15, 2016 1889

PIGR PLEKHF2 NFE2 ITPRIP TACC1 MMRN1 MMP2 HPN IGSF9B FCGBP CARD11

AFAP1 LOC147464

PI3K ATP13A1

Ras EGFRvIII AFAP1 Braf IGSR RBM10 PAWR PPFIA4 PAK6 IDH1 C9orf131 ZNF485 Stratiﬁed KDM6A P53 GPR114 GPN1 Individualized RHBDF2 treatment PPFIA2 CXorf64 treatment SH2D5 CDH15 RTN4R IL18RAP ZBTB7B LOC100128674 ERCC2 NTN5 PDK1 SOS1 OVGP1 APC TNN DNAJC28

Individualized PIGR tailored drug PIGR PLEKHF2 PLEKHF2 NFE2 ITPRIP NFE2 ITPRIP TACC1 MMRN1 TACC1 MMRN1 MMP2 Not HPN MMP2 HPN Individualized IGSF9B eligible Individualized IGSF9B FCGBP tailored drug FCGBP CARD11 tailored drug CARD11 AFAP1 AFAP1 LOC147464 LOC147464 PI3K ATP13A1 Invariant PI3K ATP13A1 drug EGFRvIII EGFRvIII Ras Ras Braf AFAP1 Braf RBM10 IGSR RBM10 AFAP1 IGSR PAWR PPFIA4 PAK6 PAWR PPFIA4 PAK6 C9orf131 C9orf131 ZNF485 IDH1 IDH1 KDM6A P53 GPR114 GPN1 ZNF485 KDM6A P53 GPR114 GPN1 RHBDF2 PPFIA2 RHBDF2 PPFIA2 CXorf64 SH2D5 CDH15 RTN4R CXorf64 SH2D5 CDH15 RTN4R IL18RAP IL18RAP ERCC2 ZBTB7B LOC100128674 ZBTB7B LOC100128674 ERCC2 NTN5 NTN5 PDK1 SOS1 OVGP1 PDK1 OVGP1 APC SOS1 TNN APC DNAJC28 TNN DNAJC28

Targeting of shared mutations Targeting of multiple individual mutations

Figure 2. Stratiﬁed versus individualized mutation–based vaccine approaches. As the vast majority of cancer-associated mutations is unique to the individual patient, the common denominator of mutations shared by different patients is small. Stratiﬁed approaches are based on one or more vaccine targets shared by a considerable number of patients. Patients are screened for presence of the mutation, and only those individuals carrying the respective mutationare treated with the invariant "off-the-shelf" drug. For an individualized treatment, in contrast, neoepitopes were selected out of the patient's mutanome based on rational criteria deemed to correlate with the induction of strong immune responses. Each and every patient receives an "on-demand" manufactured vaccine.

accessible and available, and thus, the mutanome determined by mor activity in mice (30, 34–37, 42) and men (43). Even so, a these analyses may not represent the entire clonality of an indi- poly-neoepitope vaccine has the added value of covering var- vidual's disease. On another note, no consensus has been reached ious subclones, thereby mitigating the risk of clonal selection of so far about how to distinguish authentic cancer-associated muta- antigen loss variants (44–47). Moreover, as there is no way to tions from erroneous calls. Besides artifacts introduced by tissue predict relevant epitopes with absolute certainty, the polyepi- ﬁxation, sequencing, and mutation calling procedures, con- tope approach increases the likelihood of inducing antitumor tamination with healthy tissue or necrotic cells as well as tumor activity. Until now, no consensus has been reached about how heterogeneity may compromise the mutanome data. Different many mutations should be represented in a polyepitope vac- statistical approaches and algorithms were developed by us and cine and which criteria should be applied for their selection. others to determine true mutations in individual NGS data (41). The minimal requirements for selection of a mutation as T cells targeting one single mutated MHC class I or class II vaccine target are that the neoantigen is expressed in the tumor epitope have been shown to confer potent therapeutic antitu- and that it gives rise to an epitope with an altered amino acid

1890 Clin Cancer Res; 22(8) April 15, 2016 Clinical Cancer Research

Table 2. Individualized cancer vaccine trials based on the patients' personal mutanome Patients planned or Clinicaltrials.gov Date Citation/Sponsor Cancer entity treated Target Vaccine format Outcome identiﬁer 2015 University of Texas Pancreatic cancer; 40 Personal Peptide þ IFA Study not yet NCT02600949 MD Anderson CRC neoantigens recruiting Cancer Center (Houston, TX) 2015 (88) Melanoma 3 HLA A02 epitopes DC þ peptide Induction of speciﬁc NCT00683670 from individual T-cell responses nonsynonymous mutations 2015 Washington Glioblastoma, 10 Personal Peptide þ Poly ICLC Ongoing study NCT02510950 University School astrocytoma neoantigens of Medicine (St. grade IV Louis, MO) 2015 Washington Triple-negative 15 Personal Polyepitope DNA Ongoing study NCT02348320 University School breast cancer neoantigens of Medicine 2015 Washington NSCLC 12 Personal DC þ peptides þ Study not yet NCT02419170 University School neoantigens cyclophosphamide recruiting of Medicine 2015 Washington Triple-negative 15 Personal Peptide þ Poly ICLC Ongoing study NCT02427581 University School breast cancer neoantigens of Medicine 2014 BioNTech AG Triple-negative 30 Personal Polyepitope RNA Study not yet NCT02316457 (Mainz, Germany) breast cancer neoantigens and recruiting personalized shared tumor antigens 2014 BioNTech AG Melanoma 15 Personal Polyepitope RNA Ongoing study NCT02035956 neoantigens 2014 Dana-Farber Cancer Glioblastoma 20 Personal Peptideþ poly ICLC Ongoing study NCT02287428 Institute (Boston, neoantigens MA) 2014 Immatics and Glioblastoma 20 Personal Peptide þ Poly ICLC Ongoing study NCT02149225 BioNTech AG neoantigens þ GM-CSF 2014 Dana-Farber Cancer Melanoma 20 Personal Peptide þ poly ICLC Ongoing study NCT01970358 Institute (Boston, neoantigens MA) Abbreviation: CRC, colorectal cancer.

sequence presented on one of the patient's MHC molecules. patient's respective MHC allele are in fact presented by the tumor. Quantification of the mutated allele frequencies by exome MS-assisted MHC ligandome analyses may be used for this sequencing provides the clonal abundance of tumor cells carrying purpose to validate NGS-identified mutations (37). However, the respective mutation (48). due to its relatively low sensitivity as compared with T-cell The number of RNA sequencing reads of the mutated sequence recognition, failure to detect a neoepitope by MHC ligandome provides the expression level of the mutated allele, and also, the technology does not prove its lack of relevance. It is more MHC haplotypes can be determined with high accuracy by mining important for the field that validated neoepitopes with proven the patient's NGS data (49–54). Epitopes that bind to the different immunogenicity and antitumor activity are collected in public MHC alleles of an individual patient can be predicted by a variety databases and enable development of improved tools for predic- of Web-based tools (55). MHC I prediction softwares (e.g., tion of relevant neoepitopes. NetMHC or IEDB consensus) are good in enriching MHC class Preexisting T-cell responses for computationally predicted I binding epitopes with high sensitivity for a broad range of MHC neoepitopes can be assessed by testing patients' PBMCs on autol- class I alleles. Seq2MHC, an MHC haplotype prediction approach ogous antigen-presenting cells pulsed with neoepitope peptides our laboratory developed, provides information about expression or antigen-encoding RNA (58–60). Whether mutations against levels of the respective MHC alleles in the tumor sample, which which the patient has mounted an apparently inefficient response may further facilitate the selection of mutations (52). Accurate are in fact good vaccine targets needs validation by in vivo studies. prediction of MHC class II epitopes (e.g., TEPITOPE, NetMHCII- Mutant class I and class II epitopes may be combined in a pan) remains a challenge (56, 57). poly-neoepitope vaccine for synergistic effects. Whereas cyto- þ þ Many factors other than binding score determine whether a toxic CD8 T cells kill tumor cells directly, CD4 T cells have a neoepitope is eventually displayed on the tumor cells, and only a "catalytic" function orchestrating the activity of other cell minority of predicted epitopes, in fact, are processed and pre- types, such as macrophages, natural killer (NK) cells, B cells, þ sented. There is no reliable method that can be implemented into dendritic cells (DC), and CD8 T cells. Moreover, they can the clinical setting to unequivocally verify that in silico–identified modify stroma functions by intratumoral secretion of inflam- neoepitopes tested for binding on target cells expressing the matory cytokines, such as IFNg (61–63).

www.aacrjournals.org Clin Cancer Res; 22(8) April 15, 2016 1891

MHC class I MHC class II

Figure 3. TAP1 Schematic mechanism of an mRNA- based neoepitope vaccine. The mRNA vaccine–encoding parts of the neoantigen are translated in the TAP2 cytosol. Subsequently, the mutated peptides are C-terminally truncated ER by the proteasome and transported into the endoplasmic reticulum (ER), where loading of the neoepitope on MHC class I takes place. Routing Endosomal of mutated peptides into the targeting endosomal pathway allows the presentation by MHC class II Proteasome molecules.

Translation

AAAA mRNA

Once neoepitopes have been selected the patient's individual- and durable immunity against cancers by vaccination is still an ized vaccine can be manufactured. The production needs to be ongoing quest. Our group focuses on mutanome-engineered RNA "on demand," cost-effective, rapid, and compliant with Good immunotherapy (MERIT) vaccines, which are designed to display Manufacturing Practice (GMP). Currently, ongoing mutanome multiple neoepitopes separated by nonimmunogenic linkers in a vaccine trials (Tables 1 and 2) use synthetic peptides and antigen- single molecule (34). These mRNAs are taken up by DCs, are encoding DNA or RNA (Fig. 3) as formats. These vaccines types endogenously translated and, due to their intrinsic and strong þ þ can be rapidly produced in a GMP-compliant manner and are able adjuvant activity, elicit most potent CD8 as well as CD4 T-cell to deliver effective mutant MHC class I and class II neoepitopes. responses (64, 65). On the basis of the experience from ongoing early clinical studies, lead times from the start of processing of the patient's sample for determination of mutations to the release of the investigational Clinical Translation of the Concept drug product are currently about 3 to 4 months. Patients can be Clinical translation of individualized mutanome vaccines has treated with other standard or experimental compounds until some unique aspects. Regulatory challenges are associated with their personal vaccine has been produced. For both peptide the fact that each patient will receive a tailored vaccine that is and mRNA vaccine platforms, reduction of lead times to less unique. This is fundamentally different from classical molecularly than 4 weeks is expected, which is compatible with use in the vast deﬁned vaccines that can be premanufactured, released, and majority of cancer indications. stored until use for all patients in a deﬁned trial. The entire process The route of administration, bioavailability and pharma- from sample acquisition, mutation discovery, vaccine design and cokinetics, and level of uptake by DCs, as well as simultaneously production, drug administration, and clinical monitoring must be coapplied adjuvants and immune modulators, all crucially deter- run in the framework of a regulatory approved clinical trial with mine the overall potency of a given vaccine. How to achieve strong standardized processes (66).

1892 Clin Cancer Res; 22(8) April 15, 2016 Clinical Cancer Research

As the sequence composition of the individualized drug technology) must be used according to GCLP (Good Clinical product is patient specific, formal preclinical toxicity studies, Laboratory Practice) standards and documented according to which usually inform a risk-adapted safety strategy for clinical MIATA (Minimal Information about T cell Assays; ref. 69). translation, are not feasible. Instead, preclinical studies are to In the later stages of clinical development, the classical clinical be designed in accordance with regulatory guidelines to test endpoints, such as survival, will play a major role to provide the the safety of a substitute drug product generated by a repre- clinical proof of concept and to establish the risk/benefit profile as sentative process. On the basis of the mode of action of compared with the standard of care. mutation-based vaccines, the induction of T cells cross-reactive Assessment of single-agent activity and effect size of individ- with the nonmutated wild-type counterparts may be consid- ualized vaccines has just begun and will show in which settings ered a potential risk. In preclinical mouse models, we have (e.g., minimal residual disease, adjuvant setting) such vaccines occasionally detected induction of such cross-reactivity (35), can be used in their own right. Combining the individualized however, without any evidence for autoimmunity-associated mutanome vaccine approach with potent immunomodulatory toxicities. In the vast majority of cancer trials using defined therapies, such as checkpoint blockade, immunogenic cell death– mutated epitopes, no safety issues based on such cross-reac- inducing chemotherapeutics, and mutation-inducing interven- tivity were reported. However, this risk cannot be fully exclud- tions, such as radiotherapy, will open additional avenues. On ed with vaccine platforms of higher potency emerging. Poten- the other hand, the antitumor effect of checkpoint inhibitors tial on-target toxicity organs cannot be announced apriorion mediated by unleashing immune effectors may be further boosted the trial protocol level, as each patient is immunized with a by individualized vaccines, which steer the immune system with a different set of mutated epitopes, and wild-type counterparts tailored target map of the patient's tumor. of these are expressed in different sets of normal tissue. This problem can be addressed by providing investigators with patient-specificprofiles of organs with high expression levels Outlook of wild-type counterparts of chosen mutations as the basis for Ongoing clinical trials furnish clear evidence for the feasi- personalization of safety monitoring. Generally speaking, dil- bility of individualized vaccination of cancer patients with igent safety assessment of patients to capture potential auto- unique mutations. The concept holds several promises. immune-driven toxicity is advisable. Strategies for safety signal One such promise is the full exploitation of the mutational detection across trials within a development program must neoepitope repertoire of tumors, which is the richest so far take into account that treatment-related adverse event profiles identified source of otherwise narrow vaccine target discovery may vary between patients, and smart solutions for this chal- space. Mutations are the very root of cancer as supported by the lengeneedtobeworkedout. unequivocal clinical benefitofdrugsagainstsharedmutations The activity of individualized mutanome vaccines may be (e.g., imatinib, crizotinib). The relevance of mutated epitopes assessed throughout the clinical development by clinical, molec- as tumor rejection antigens is validated, for example, as clinical ular, and immunologic endpoints. Imaging of tumor lesions responses to checkpoint blockade and adoptive T-cell transfer according to the standards developed specifically for immu- have been linked to immune responses against neoepitopes. notherapies [e.g., immune-related response criteria (67)] imple- Prevalent T-cell responses against mutations are rare in cancer mented early on in clinical development assists in verifying patients and need to be boosted by vaccines. Mutanome vac- þ activity in patients with measurable disease. Potential surrogates cines may be particularly useful to induce CD4 T-cell þ for tumor cell burden, such as classical tumor serum markers (e.g., responses. Given the role of CD4 T cells as orchestrators of PSA, CA125), circulating tumor cells, and circulating nucleic immunity, such vaccines may result in immunogenicity of acids, may have a supportive value. The most immediate mea- tumors that lack spontaneous immunity, thereby rendering surements for the intended pharmacodynamic effect of the vac- tumors responsive to checkpoint blockade treatment (70, 71). cine are immunologic parameters, for example, the frequency, Second, this concept takes personalization from stratified function, and phenotype of neoepitope-specific immune cell approaches to the next level of truly individualized medicine. subsets in the peripheral blood, T-cell infiltrates at the vaccination Not any more limited by the small common denominator of site, induction of delayed-type hypersensitivity (68) reactions, neoantigen targets shared by many patients, the mutanome and infiltration of vaccine-induced, neoepitope-specific T cells vaccine approach taps the large unique antigenic target repertoire into tumor lesions. The induction of functional mutation-specific of each individual patient and provides a universally applicable T-cell responses, which are able to recognize neoepitopes in the regimen from which each and every patient can profit. The natural context, i.e., either on autologous tumor cells or on tumor- availability of multiple vaccine targets for each patient allows us derived DCs, their trafficking to the tumor as determined by to address interindividual variability and intratumor clonal het- functional immunologic readouts and TCR repertoire profiling erogeneity, which are key to the largely disappointing effects of is a straightforward strategy to establish the clinical mode-of- currently used targeted therapeutics (72). action in first-in-human trials. Longitudinal analysis of antigen- Third, generally speaking, cancer therapy is moving from a specific T cells in blood samples and in tumor biopsies before, drug-centered to a patient-centered approach with different levels during, and after vaccination will aid in understanding of the of personalization (73). This requires paradigm shifts along the immunogenicity of each individual mutation. Immunogenicity entire drug development process, including diagnostics, drug data generated from such trials will help to build knowledge production, logistics, regulatory aspects, and patient manage- around the key requirement for successful individualized vacci- ment. Fully individualized mutanome vaccination may become nation. As the assessment of treatment-emergent T-cell responses a prototype for how to open up this territory by developing is endpoint relevant, scientifically sound and qualified bioassays best-practice blueprints for the implementation of personalized (e.g., ELISpot, flow cytometric cytokine release, MHC multimer approaches.

www.aacrjournals.org Clin Cancer Res; 22(8) April 15, 2016 1893

On a related note, with health care systems becoming increas- a non-operative board member for, has ownership interest (including patents) ingly cost-conscious, one of the challenges is the pharmacoeco- in, and is a consultant/advisory board member for BioNTech. U. Sahin is CEO nomics of individualized medicine. In the clinical development of and has ownership interest (including patents) in BioNTech. No other potential conflicts of interest were disclosed. stage, individualized vaccines will be expensive, mainly due to costs for genome sequencing and manufacturing of small, patient- specific GMP drug product batches, and costs need to be reduced Authors' Contributions until commercialization. There is good reason to believe that the Conception and design: O.€ Tureci,€ C. Huber, U. Sahin concurrence of future trends, for example, dramatically decreasing Development of methodology: M. Diken genome sequencing costs, full automation, miniaturization, and Analysis and interpretation of data (e.g., statistical analysis, biostatistics, € optimization of manufacturing processes, will make individual- computational analysis): O. Tureci,€ S. Kreiter € ized mutanome approaches affordable. Writing, review, and/or revision of the manuscript: O. Tureci,€ M. Vormehr, M. Diken, S. Kreiter, C. Huber, U. Sahin Disclosure of Potential Conflicts of Interest Study supervision: S. Kreiter Other (figure preparation): M. Vormehr O.€ Tureci€ is CEO of Ganymed Pharmaceuticals. M. Vormehr is an employee of and has ownership interest (including patents) in BioNTech RNA Pharma- ceuticals. M. Diken is a consultant/advisory board member for BioNTech. Received December 2, 2015; revised February 21, 2016; accepted February 23, S. Kreiter is a consultant/advisory board member for BioNTech. C. Huber is 2016; published online April 15, 2016.

References 1. Von Hansemann D. Ueber asymmetrische Zellteilung in Epithelzellen 18. Van Allen EM, Miao D, Schilling B, Shukla SA, Blank C, Zimmer L, et al. und deren biologische Bedeutung. Virchows Arch A Pathol Anat 1890; Genomic correlates of response to CTLA-4 blockade in metastatic mela- 119:299–326. noma. Science 2015;350:207–11. 2. Boveri T. Zur Frage der Entstehung maligner Tumoren. Jena, Germany: 19. Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, et al. Gustav Fischer; 1914. Mutational landscape determines sensitivity to PD-1 blockade in non- 3. Garraway LA, Lander ES. Lessons from the cancer genome. Cell 2013; small cell lung cancer. Science 2015;348:124–8. 153:17–37. 20. Van Rooij N, Van Buuren MM, Philips D, Velds A, Toebes M, Heemskerk B, 4. Martincorena I, Campbell PJ. Somatic mutation in cancer and normal cells. et al. Tumor exome analysis reveals neoantigen-specific T-cell reactivity in Science 2015;349:1483–9. an ipilimumab-responsive melanoma. J Clin Oncol 2013;31:e439–42. 5. Tyzzer E. Tumor immunity. J Cancer Res 1916;1:125–56. 21. Lin EI, Tseng L-H, Gocke CD, Reil S, Le DT, Azad NS, et al. Mutational 6. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Kinzler KW. profiling of colorectal cancers with microsatellite instability. Oncotarget Cancer genome landscapes. Science 2013;339:1546–58. 2015;6:42334–44. 7. Hanada K-I, Yewdell JW, Yang JC. Immune recognition of a human renal 22. Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A, et al. cancer antigen through post-translational protein splicing. Nature 2004; Genetic basis for clinical response to CTLA-4 blockade in melanoma. 427:252–6. N Engl J Med 2014;371:2189–99. 8. Parmiani G, De Filippo A, Novellino L, Castelli C. Unique human tumor 23. Gros A, Robbins PPF, Yao X, Li YF, Turcotte S, Tran E, et al. PD-1 identifies antigens: immunobiology and use in clinical trials. J Immunol 2007;178: the patient-specific CD8þ tumor-reactive repertoire infiltrating human 1975–9. tumors. J Clin Invest 2014;124:2246–59. 9. De Plaen E, Lurquin C, Van Pel A, Mariame B, Szikora JP, Wolfel€ T, et al. 24. Wolchok JD, Chan TA. Cancer: antitumour immunity gets a boost. Nature Immunogenic (tum-) variants of mouse tumor P815: cloning of the gene of 2014;515:496–8. tum- antigen P91A and identification of the tum- mutation. Proc Natl Acad 25. Gubin MM, Artyomov MN, Mardis ER, Schreiber RD. Tumor neoanti- Sci U S A 1988;85:2274–8. gens: building a framework for personalized cancer immunotherapy. 10. Sibille C, Chomez P, Wildmann C, Van Pel A, De Plaen E, Maryanski JL, J Clin Invest 2015;125:1–9. et al. Structure of the gene of tum- transplantation antigen P198: a 26. Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. point mutation generates a new antigenic peptide. J Exp Med 1990; Science 2015;348:69–74. 172:35–45. 27. Fritsch EF, Hacohen N, Wu CJ. Personal neoantigen cancer vaccines: The 11. Monach PA, Meredith SC, Siegel CT, Schreiber H. A unique tumor antigen momentum builds. Oncoimmunology 2014;3:e29311. produced by a single amino acid substitution. Immunity 1995;2:45–59. 28. Hegde PS, Karanikas V, Evers S. The where, the when, and the how of 12. Dubey P, Hendrickson RC, Meredith SC, Siegel CT, Shabanowitz J, Skipper immune monitoring for cancer immunotherapies in the era of checkpoint JC, et al. The immunodominant antigen of an ultraviolet-induced regressor inhibition. Clin Cancer Res 2016;22:1865–74. tumor is generated by a somatic point mutation in the DEAD box helicase 29. Maus MV, June CH. Making better chimeric antigen receptors for adoptive p68. J Exp Med 1997;185:695–705. T-cell therapy. Clin Cancer Res 2016;22:1875–84. 13. Matsutake T, Srivastava PK. The immunoprotective MHC II epitope of a 30. Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, et al. chemically induced tumor harbors a unique mutation in a ribosomal Checkpoint blockade cancer immunotherapy targets tumour-specific protein. Proc Natl Acad Sci U S A 2001;98:3992–7. mutant antigens. Nature 2014;515:577–81. 14. Matsushita H, Vesely MD, Koboldt DC, Rickert CG, Uppaluri R, Magrini VJ, 31. Vetizou M, Pitt J, Daillere R, Lepage P, Waldschmitt N, Flament C, et al. et al. Cancer exome analysis reveals a T-cell-dependent mechanism of Anticancer immunotherapy by CTLA-4 blockade relies on the gut micro- cancer immunoediting. Nature 2012;482:400–4. biota. Science 2015;350:1079–84. 15. Wang RF, Wang X, Atwood AC, Topalian SL, Rosenberg SA. Cloning genes 32. Sivan A, Corrales L, Hubert N, Jason B, Aquino-michaels K, Earley ZM, et al. encoding MHC class II-restricted antigens: mutated CDC27 as a tumor Commensal Bifidobacterium promotes antitumor immunity and facili- antigen. Science 1999;284:1351–4. tates anti–PD-L1 efficacy. Science 2015;350:1084–9. 16. Lennerz V, Fatho M, Gentilini C, Frye RA, Lifke A, Ferel D, et al. The response 33. Segal NH, Parsons DW, Peggs KS, Velculescu V, Kinzler KW, Vogelstein B, of autologous T cells to a human melanoma is dominated by mutated et al. Epitope landscape in breast and colorectal cancer. Cancer Res neoantigens. Proc Natl Acad Sci U S A 2005;102:16013–8. 2008;68:889–92. 17. Wolfel€ T, Hauer M, Schneider J, Serrano M, Wolfel€ C, Klehmann-Hieb E, 34. Kreiter S, Vormehr M, van de Roemer N, Diken M, Lower€ M, Diekmann J, et al. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lym- et al. Mutant MHC class II epitopes drive therapeutic immune responses phocytes in a human melanoma. Science 1995;269:1281–4. to cancer. Nature 2015;520:692–6.

1894 Clin Cancer Res; 22(8) April 15, 2016 Clinical Cancer Research

35. Castle JC, Kreiter S, Diekmann J, Lower€ M, Van De Roemer N, De Graaf J, 60. Cohen CJ, Gartner JJ, Horovitz-Fried M, Shamalov K, Trebska-McGowan K, et al. Exploiting the mutanome for tumor vaccination. Cancer Res Bliskovsky VV, et al. Isolation of neoantigen-specific T cells from tumor and 2012;72:1081–91. peripheral lymphocytes. J Clin Invest 2015;125:3981–91. 36. Duan F, Duitama J, Al Seesi S, Ayres CM, Pawashe AP, Blanchard T, et al. 61. Toes RE, Ossendorp F, Offringa R, Melief CJ. CD4 T cells and their role in Genomic and bio-informatic profiling of mutational neo-epitopes reveals antitumor immune responses. J Exp Med 1999;189:753–6. new rules to predict anti-cancer immunogenicity. J Exp Med 2014;211: 62. Schoenberger SP, Toes RE, van der Voort EI, Offringa R, Melief CJ. T-cell 2231–48. help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. 37. Yadav M, Jhunjhunwala S, Phung QT, Lupardus P, Tanguay J, Bumbaca S, Nature 1998;393:480–3. et al. Predicting immunogenic tumour mutations by combining mass 63. Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can be a spectrometry and exome sequencing. Nature 2014;515:572–6. temporal bridge between a CD4þ T-helper and a T-killer cell. Nature 38. Whiteside TL, Demaria S, Rodriguez-Ruiz ME, Zarour HM, Melero I. 1998;393:474–8. Emerging opportunities and challenges in cancer immunotherapy. 64. Diken M, Kreiter S, Selmi A, Britten CM, Huber C, Tureci€ O,€ et al. Selective Clin Cancer Res 2016;22:1845–55. uptake of naked vaccine RNA by dendritic cells is driven by macropino- 39. Bol KF, Schreibelt G, Gerritsen WR, de Vries IJM, Figdor CG. Dendritic cell– cytosis and abrogated upon DC maturation. Gene Ther 2011;18:702–8. based immunotherapy: state of the art and beyond. Clin Cancer Res 65. Kreiter S, Selmi A, Diken M, Koslowski M, Britten CM, Huber C, et al. 2016;22:1897–906. Intranodal vaccination with naked antigen-encoding RNA elicits potent 40. Zarour HM. Reversing T-cell dysfunction and exhaustion in cancer. Clin prophylactic and therapeutic antitumoral immunity. Cancer Res Cancer Res 2016;22:1856–64. 2010;70:9031–40. 41. Lower€ M, Renard BY, de Graaf J, Wagner M, Paret C, Kneip C, et al. 66. Britten CM, Singh-Jasuja H, Flamion B, Hoos A, Huber C, Kallen K-J, et al. Confidence-based somatic mutation evaluation and prioritization. The regulatory landscape for actively personalized cancer immunothera- PLoS Comput Biol 2012;8:e1002714. pies. Nat Biotechnol 2013;31:880–2. 42. Schumacher T, Bunse L, Pusch S, Sahm F, Wiestler B, Quandt J, et al. A 67. Wolchok JD, Hoos A, O'Day S, Weber JS, Hamid O, LebbeC,etal. vaccine targeting mutant IDH1 induces antitumour immunity. Nature Guidelines for the evaluation of immune therapy activity in solid tumors: 2014;512:324–7. immune-related response criteria. Clin Cancer Res 2009;15:7412–20. 43. Tran E, Turcotte S, Gros A, Robbins PF, Lu Y-C, Dudley ME, et al. Cancer 68. De Vries IJM, Bernsen MR, Lesterhuis WJ, Scharenborg NM, Strijk SP, immunotherapy based on mutation-specific CD4þ T cells in a patient with Gerritsen M-JP, et al. Immunomonitoring tumor-specific T cells in epithelial cancer. Science 2014;344:641–5. delayed-type hypersensitivity skin biopsies after dendritic cell vaccination 44. Uyttenhove C, Maryanski J, Boon T. Escape of mouse mastocytoma P815 correlates with clinical outcome. J Clin Oncol 2005;23:5779–87. after nearly complete rejection is due to antigen-loss variants rather than 69. Britten CM, Janetzki S, Butterfield LH, Ferrari G, Gouttefangeas C, Huber C, immunosuppression. J Exp Med 1983;157:1040–52. et al. T cell assays and MIATA: the essential minimum for maximum 45. Khong HT, Restifo NP. Natural selection of tumor variants in the gener- impact. Immunity 2012;37:1–2. ation of "tumor escape" phenotypes. Nat Immunol 2002;3:999–1005. 70. Herbst RS, Soria J-C, Kowanetz M, Fine GD, Hamid O, Gordon MS, et al. 46. Dunn GP, Old LJ, Schreiber RD. The three Es of cancer immunoediting. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A Annu Rev Immunol 2004;22:329–60. in cancer patients. Nature 2014;515:563–7. 47. Greaves M, Maley CC. Clonal evolution in cancer. Nature 2012;481: 71. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. 306–13. PD-1 blockade induces responses by inhibiting adaptive immune resis- 48. Castle JC, Loewer M, Boegel S, Tadmor AD, Boisguerin V, de Graaf J, et al. tance. Nature 2014;515:568–71. Mutated tumor alleles are expressed according to their DNA frequency. 72. Turner NC, Reis-Filho JS. Genetic heterogeneity and cancer drug resistance. Sci Rep 2014;4:4743. Lancet Oncol 2012;13:e178–85. 49. Liu C, Yang X, Duffy B, Mohanakumar T, Mitra RD, Zody MC, et al. 73. Vonderheide RH, Nathanson KL. Immunotherapy at Large: The road to ATHLATES: accurate typing of human leukocyte antigen through exome personalized cancer vaccines. Nat Med 2013;19:1098–100. sequencing. Nucleic Acids Res 2013;41:e142. 74. Bocchia M, Ippoliti M, Defina M, Gozzetti A, Chitarrelli I, Lauria F. 50. Huang Y, Yang J, Ying D, Zhang Y, Shotelersuk V, Hirankarn N, et al. BCR-ABL derived peptide vaccines for chronic myeloid leukaemia. HLAreporter: a tool for HLA typing from next generation sequencing data. J Siena Acad Sci 2012;1:11–4. Genome Med 2015;7:25. 75. Schuster J, Lai RK, Recht LD, Reardon DA, Paleologos NA, Groves MD, 51. Warren RL, Choe G, Freeman DJ, Castellarin M, Munro S, Moore R, et al. et al. A phase II, multicenter trial of rindopepimut (CDX-110) in Derivation of HLA types from shotgun sequence datasets. Genome Med newly diagnosed glioblastoma: the ACT III study. Neuro Oncol 2015; 2012;4:95. 17:854–61. 52. Boegel S, Lower€ M, Sch€afer M, Bukur T, de Graaf J, Boisguerin V, et al. HLA 76. Rahma OE, Hamilton JM, Wojtowicz M, Dakheel O, Bernstein S, Liewehr typing from RNA-Seq sequence reads. Genome Med 2012;4:102. DJ, et al. The immunological and clinical effects of mutated ras peptide 53. Kim HJ, Pourmand N. HLA typing from RNA-seq data using hierarchical vaccine in combination with IL-2, GM-CSF, or both in patients with solid read weighting. PLoS One 2013;8:e67885. tumors. J Transl Med 2014;12:55. 54. Bai Y, Ni M, Cooper B, Wei Y, Fury W. Inference of high resolution HLA 77. Chaft JE, Litvak A, Arcila ME, Patel P, D'Angelo SP, Krug LM, et al. Phase II types using genome-wide RNA or DNA sequencing reads. BMC Genomics study of the GI-4000 KRAS vaccine after curative therapy in patients with 2014;15:325. stage I-III lung adenocarcinoma harboring a KRAS G12C, G12D, or G12V 55. Web tools | cancer immunity [Internet]. [cited 2015 Nov 30]. Available mutation. Clin Lung Cancer 2014;15:405–10. from: http://cancerimmunity.org/resources/webtools/ 78. Sampson JH, Heimberger AB, Archer GE, Aldape KD, Friedman AH, 56. Zhang L, Chen Y, Wong H-S, Zhou S, Mamitsuka H, Zhu S. TEPITOPEpan: Friedman HS, et al. Immunologic escape after prolonged progression-free extending TEPITOPE for peptide binding prediction covering over 700 survival with epidermal growth factor receptor variant III peptide vacci- HLA-DR molecules. PLoS One 2012;7:e30483. nation in patients with newly diagnosed glioblastoma. J Clin Oncol 57. Karosiene E, Rasmussen M, Blicher T, Lund O, Buus S, Nielsen M. NetMH- 2010;28:4722–9. CIIpan-3.0, a common pan-specific MHC class II prediction method 79. Sampson JH, Archer GE, Mitchell DA, Heimberger AB, Herndon JE, Lally- including all three human MHC class II isotypes, HLA-DR, HLA-DP and Goss D, et al. An epidermal growth factor receptor variant III-targeted HLA-DQ. Immunogenetics 2013;65:711–24. vaccine is safe and immunogenic in patients with glioblastoma multi- 58. Kreiter S, Konrad T, Sester M, Huber C, Tureci€ O, Sahin U. Simultaneous forme. Mol Cancer Ther 2009;8:2773–9. ex vivo quantification of antigen-specific CD4þ and CD8þ T cell responses 80. Toubaji A, Achtar M, Provenzano M, Herrin VE, Behrens R, Hamilton M, using in vitro transcribed RNA. Cancer Immunol Immunother 2007; et al. Pilot study of mutant ras peptide-based vaccine as an adjuvant 56:1577–87. treatment in pancreatic and colorectal cancers. Cancer Immunol Immun- 59. Tran E, Ahmadzadeh M, Lu Y, Gros A, Turcotte S, Robbins PF, et al. other 2008;57:1413–20. Immunogenicity of somatic mutations in human gastrointestinal cancers. 81. Meyer RG, Korn S, Micke P, Becker K, Huber C, Wolfel€ T, et al. An open- Science 2015;350:1387–90. label, prospective phase I/II study evaluating the immunogenicity and

www.aacrjournals.org Clin Cancer Res; 22(8) April 15, 2016 1895

safety of a ras peptide vaccine plus GM-CSF in patients with non-small cell 85. Gjertsen MK, Buanes T, Rosseland AR, Bakka A, Gladhaug I, Søreide O, lung cancer. Lung Cancer 2007;58:88–94. et al. Intradermal ras peptide vaccination with granulocyte-macrophage 82. Carbone DP, Ciernik IF, Kelley MJ, Smith MC, Nadaf S, Kavanaugh D, et al. colony-stimulating factor as adjuvant: Clinical and immunological Immunization with mutant p53- and K-ras-derived peptides in cancer responses in patients with pancreatic adenocarcinoma. Int J Cancer patients: immune response and clinical outcome. J Clin Oncol 2005; 2001;92:441–50. 23:5099–107. 86. Khleif SN, Abrams SI, Hamilton JM, Bergmann-Leitner E, Chen A, 83. Dagher R, Long LM, Read EJ, Leitman SF, Carter CS, Tsokos M, et al. Pilot Bastian A, et al. A phase I vaccine trial with peptides reflecting ras trial of tumor-specific peptide vaccination and continuous infusion inter- oncogene mutations of solid tumors. J Immunother 1999;22:155–65. leukin-2 in patients with recurrent Ewing sarcoma and alveolar rhabdo- 87. Gjertsen MK, Bakka A, Breivik J, Saeterdal I, Gedde-Dahl T, Stokke KT, myosarcoma: an inter-institute NIH study. Med Pediatr Oncol 2002;38: et al. Ex vivo ras peptide vaccination in patients with advanced pan- 158–64. creatic cancer: results of a phase I/II study. Int J Cancer 1996;65: 84. Hunger RE, Brand CU, Streit M, Eriksen JA, Gjertsen MK, Saeterdal I, 450–3. et al. Successful induction of immune responses against mutant ras in 88. Carreno BM, Magrini V, Becker-Hapak M, Kaabinejadian S, Hundal J, Petti melanoma patients using intradermal injection of peptides and GM- AA, et al. A dendritic cell vaccine increases the breadth and diversity of CSF as adjuvant. Exp Dermatol 2001;10:161–7. melanoma neoantigen-specific T cells. Science 2015;348:803–8.

1896 Clin Cancer Res; 22(8) April 15, 2016 Clinical Cancer Research

Özlem Türeci, Mathias Vormehr, Mustafa Diken, et al.

Clin Cancer Res 2016;22:1885-1896.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/22/8/1885

Cited articles This article cites 86 articles, 36 of which you can access for free at: http://clincancerres.aacrjournals.org/content/22/8/1885.full#ref-list-1

Citing articles This article has been cited by 14 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/22/8/1885.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/22/8/1885. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.