Mutation-derived Tumor Research Articles

OptiPRM: A Targeted Immunopeptidomics LC-MS Workflow With Ultra-High Sensitivity for the Detection of Mutation-Derived Tumor Neoepitopes From Limited Input Material

Personalized cancer immunotherapies such as therapeutic vaccines and adoptive transfer of T cell receptor-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. Liquid chromatography mass spectrometry (LC-MS)-based immunopeptidomics is the only method to directly prove actual peptide presentation and we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for the improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft from as little as 2.5× 106cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, makes the identification of actionable neoepitopes possible from sample sizes usually available in the clinic.

Abstract 2924: In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes

Abstract Microsatellite instability is a hallmark of DNA mismatch repair (MMR)-deficient colorectal cancer (CRC), and is accompanied by genome-wide somatic hypermutation and enhanced immunogenicity. Clinical trials have demonstrated remarkable efficacy of immune checkpoint blockade (i.e. anti-PD-1) in treating MMR-deficient cancers, which is thought to be driven by an enhanced burden of mutation-derived tumor specific antigens (neoantigens). Despite these successes, tumor mutation burden is an imperfect clinical predictor of immunotherapy response, and major questions remain regarding the mechanistic role of neoantigens and other features of MMR-deficiency in the anti-tumor immune response. Notably, most genetically engineered mouse models (GEMMs) fail to recapitulate the mutation burden of their associated human cancer and lack prominent immune involvement, precluding careful study of these questions. To model microsatellite instability in colon cancer, we developed novel autochthonous GEMMs utilizing CRISPR/Cas9-based knockout of DNA MMR genes in the distal colon. Colonoscopy-guided submucosal injection was used to deliver lentivirus containing two single-guide RNAs (sgRNAs) against Apc (the initiating event in the majority of colon cancers) and MMR pathway genes (Msh2, Mlh1, Msh3, Msh6). Whole-exome sequencing of MMR-targeted colon tumors revealed a high mutation burden with signatures consistent with those found in MMR-deficient human cancer. In addition, sequencing revealed a number of recurrently mutated microsatellites, including those within putative tumor suppressor genes Asxl1, Acvr2a, and Acvr1b. To interrogate the functional relevance of these genes in MMR-deficient colon cancer, we developed a novel ex vivo organoid competition assay. Specifically, Apc null organoids expressing Cas9 were infected with lentivirus expressing the fluorophore mScarlet and sgRNAs targeting the putative tumor suppressor genes. Flow cytometry was used to longitudinally assess kinetics of the mScarlet positive fraction. Preliminary results suggest a selective advantage following deletion of Asxl1 and Acvr2a. Further assessment of tumorigenesis in vivo will be performed to confirm the role of these putative tumor suppressors. Altogether, these genetically manipulable systems can be used for rapid functional interrogation of tumor suppressor genes, facilitating downstream mechanistic studies and preclinical testing of therapies in these important subsets of CRC. Citation Format: Daniel D. Zhang, Peter M. Westcott, Olivia Smith, Nate Sacks, Haley Hauck, Mary C. Beytagh, Abbey Jin, Tyler Jacks. In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2924.

CTIM-17. PHASE I STUDY OF THE SAFETY AND IMMUNOGENICITY OF PERSONALIZED NEOANTIGEN VACCINES AND TUMOR TREATING FIELDS IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) generates mutation-derived tumor antigens, which arise from somatic variants that result in mutated protein fragments displayed to immune cells. These mutated proteins can provide targets for anti-tumor personalized neoantigen vaccine (PNV) therapy. Combining PNV with Tumor Treating Fields (TTFields), a modality that increases survival of GBM patients, is a viable option. METHODS In this phase I study (NCT03223103), PNV and TTFields are combined with the standard treatment for newly diagnosed GBM. Neoantigens are identified using the OpenVax computational pipeline, which identifies somatic variants in a patient’s DNA and ranks resulting neoantigens by their tumor RNA abundance and predicted HLA-I binding affinity. Each patient is vaccinated with up to 10 mutated peptides, receiving 14 doses of PNV administered subcutaneously with poly-ICLC as the adjuvant. RESULTS We report our preliminary results in the 8 patients with median age 59 (range 32–84) that have been vaccinated. Neoantigens were identified in each tumor sample in sufficient quantities for vaccination, with each vaccine comprising a median of 8.5 peptides (range 6–10). Patients received a median of 13 vaccines (range 8–14). Immune monitoring for one of the patients showed neoantigen-specific CD4 and CD8 T cell responses. There was radiological tumor progression in two while receiving PNV and in another, prior to PNV. The most common adverse events of the PNV were injection site reaction and flu-like symptoms. Combination with TTFields did not increase toxicity. The Kaplan-Meier estimated 12-month progression-free survival is 62.5% (s.e.= 17.1%) and the estimated 12-month survival from date of surgery is 83.3% (s.e.= 16.7%). The median survival has not been reached. CONCLUSIONS Despite a typically low mutational burden of GBM, selection of a sufficient number of neoantigens was successful for each patient’s vaccine. Treatment with PNV and TTFields is safe. The data is encouraging and updated results will be presented.

Abstract CT173: PGV-001: A phase I trial of a multipeptide personalized neoantigen vaccine in the adjuvant setting

Abstract Background: Mutation-derived tumor antigens (MTAs) arise as a result of somatic mutations, such as nucleotide substitutions and small insertions/deletions. MTAs can serve as specific targets for antitumor therapy, including neoantigen vaccines. The goal of neoantigen vaccination is to help prime T cells to recognize such tumor-specific mutations. Here we describe a phase I trial testing a personalized genomic vaccine (PGV-001) in multiple histologies in the adjuvant setting (NCT02721043). Methods: This trial included patients with histologic diagnosis of solid malignancies or multiple myeloma with a >30% risk of recurrence but no measurable disease at the time of first vaccination. For each patient, HLA typing was performed, and the patient's tumor and germline DNA and tumor RNA were sequenced. Mutated peptides containing predicted neoantigens were selected using the OpenVax computational pipeline, which prioritizes somatic mutations by expression of the mutant allele in the tumor RNA and predicted MHC class I epitope binding for the patient's HLA type. A maximum of 10 peptides was included in each patient's personalized vaccine. The vaccine was administered over the course of 6 months, given in combination with poly-ICLC as the adjuvant. The primary objectives were to determine safety and tolerability, feasibility, and immunogenicity of the PGV-001 neoantigen vaccine. Results: The neoantigen vaccine was successfully administered to 13 patients, spanning 5 different tumor types. For each patient, an average of 1730 somatic mutations were identified (range 521-5106), of which 349 were coding variants (range 68-1493), 88 were coding and expressed in the tumor RNA (range 9-233), and 71 were coding, expressed and resulted in predicted MHC class I ligands (range 8-193). The urothelial, head & neck, and lung tumors resulted in larger numbers of predicted neoantigens than in the cases of multiple myeloma or breast tumors. Despite this difference, each of the 13 patient tumor samples showed enough of a neoantigen load to enable vaccination. We have also initiated analysis of immunogenicity, and early reports demonstrate neoantigen-specific CD4 and CD8 T-cell responses. Conclusions: The PGV-001 personalized neoantigen vaccine was successfully administered to 13 patients, where each patient's neoantigen load was adequate for vaccine synthesis. While we have demonstrated the computational feasibility of identification of neoantigens for inclusion in the PGV-001 genomic vaccine, clinical outcomes will be reported separately. Citation Format: Julia Kodysh, Thomas Marron, Alex Rubinsteyn, Tim O'Donnell, John Finnigan, Ana Blazquez, Mansi Saxena, Marcia Meseck, Philip Friedlander, Nina Bhardwaj. PGV-001: A phase I trial of a multipeptide personalized neoantigen vaccine in the adjuvant setting [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT173.

Abstract CT062: A Phase I study of the safety and immunogenicity of personalized mutation-derived tumor vaccine and treatment fields in patients with newly diagnosed glioblastoma

Abstract Glioblastoma (GBM) like other cancers generates mutation-derived tumor antigens (MTA) arising from somatic mutations such as insertions, deletions, nucleotide substitutions and gene fusions. These mutations can be recognized by the immune system, providing a specific target for antitumor therapy. The use of TTFields, a modality that utilizes low intensity electric fields that disrupt cell division, increases progression-free survival and overall survival in newly diagnosed GBM. Among several mechanisms of action, TTFields potentially induces an immunogenic cell death. Therefore, we combined this modality treatment with the standard of care of adjuvant Temozolomide chemotherapy and a personalized vaccine for newly diagnosed GBM in a single-arm, open label clinical trial. Phase Ia tests the safety and immunogenicity of MTA-vaccine and phase Ib tests the safety and immunogenicity of the vaccine in conjunction with the TTFields. RNA and DNA are extracted from tumor specimens and blood, and whole-exome sequencing and mRNASeq is performed. HLA typing is obtained for each patient. MTA are identified using a computational pipeline for prediction and ranking according to both HLA binding affinity and expression. A maximum of 10 peptides is selected for the synthesis of each patient vaccine. Patients receive 14 doses of the MTA-vaccine in combination with 14 doses of poly-ICLC, the vaccine adjuvant. We have found mutation-derived tumor antigens in all patients included in phase Ia despite a low mutational load in GBM patients. The patients that took part in phase Ia have received multiple vaccinations spanning over 6 months of treatment. The vaccine has been well tolerated without serious adverse events. Quantitative and qualitative changes in epitope-specific circulating T-cell response is currently being evaluated. Citation Format: Adilia Hormigo, Alex Rubinsteyn, Julia Kodysh, Ana Blazquez, Nina Bhardwaj. A Phase I study of the safety and immunogenicity of personalized mutation-derived tumor vaccine and treatment fields in patients with newly diagnosed glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT062.

A phase I study of the safety and immunogenicity of a multi-peptide personalized genomic vaccine in the adjuvant treatment of solid tumors and hematological malignancies.

e14307 Background: Mutation-derived tumor antigens (MTAs) arise as a direct result of somatic variations that occur during carcinogenesis and can be characterized via genetic sequencing and used to identify MTAs. We developed a platform for a fully-personalized MTA-based vaccine in the adjuvant treatment of solid and hematological malignancies. Methods: This is a single-arm, open label, proof-of-concept phase I study designed to test the safety and immunogenicity of Personalized Genomic Vaccine 001 (PGV001) that targets up to 10 predicted personal tumor neoantigens based on patient’s HLA profile (ClinicalTrials.gov: NCT02721043). Results: Patients who completed vaccination with PGV001_002 (head and neck squamous cell cancer) received 10 doses of vaccine comprising 10 long peptides (LP) combined with poly-ICLC (toll-like receptor-3 agonist) intradermally. Vaccine-induced T-cell responses were determined at weeks 0 and 27 (before and after treatment, respectively), ex vivo by interferon (IFN)-g enzyme-linked immunospot assay and after expansion by intracellular cytokine staining. Overlapping 15-mer peptides (OLPs) spanning the entirety of each LP and 9-10-mer peptides corresponding to each predicted class I epitope (Min) were pooled. Ex vivo responses to these peptide pools were undetectable at week 0 but were evident at week 27 against 2 OLPs out of 10 (20%) and in 5 Min out of 10 (50%). After in vitro expansion, neoantigen-specific CD4+ and CD8+ T-cell responses were found in 5 out of 10 pooled peptides (50%). 7 out of 10 (70%) epitopes elicited polyfunctional T-cell responses (secretion of INF-g, TNF-a, and/or IL-2) from either CD4+ or CD8+ T cells. Conclusions: The PGV001 vaccine in our first patient showed both safety and immunogenicity, eliciting CD4+ and CD8+ responses to the vaccine peptides. As we enroll additional patients in this clinical trial, and perform deeper phenotyping of their tumor-reactive T cells, we will learn the determinants necessary for the successful generation of MTA-based vaccines, while informing future immunotherapeutic approaches and rational combinations. Clinical trial information: NCT02721043.

Late-differentiated effector neoantigen-specific CD8+ T cells are enriched in non-small cell lung carcinoma patients responding to atezolizumab treatment

Background: Response to checkpoint blockade immunotherapy appears to be associated with reinvigoration of CD8+ T cells specific for mutation-derived tumor neoantigens. Thus, the frequencies or characteristics of these tumor-specific cells could be useful and convenient as biomarkers of response. However, neoantigen-specific cells are difficult to reliably identify due the wide range of possible peptide-MHC epitope for any given patient, combined with the likelihood of being present only at low frequencies in blood.

Abstract A005: A phase I study of the safety and immunogenicity of a multipeptide personalized genomic vaccine in the adjuvant treatment of solid cancers

Abstract Introduction: Mutation-derived tumor antigens (MTAs) arise as a direct result of somatic variations, including nucleotide substitutions, insertions, and deletions that occur during carcinogenesis. These somatic variations can be characterized via genetic sequencing and used to identify MTAs. We developed a platform for a fully-personalized MTA-based vaccine in the adjuvant treatment of solid and hematologic malignanicies. Methods: This is a single-arm, open label, proof-of-concept phase I study designed to test the safety and immunogenicity of Personalized Genomic Vaccine 001 (PGV001) that targets up to 10 predicted personal tumor neoantigens. The single-center study will enroll 20 eligible subjects with histologic diagnosis of solid and hematologic malignancies. Subjects must have no measurable disease at time of first vaccine administration, and 5-year disease recurrence risk of > 30%. Toxicity will be defined by CTCAE v5.0. Blood samples will be collected at various time points for immune response monitoring. Each patient’s vaccine peptides are selected by identifying somatic mutations from comparison of tumor and normal exome sequencing data, phasing somatic variants with co-occurring germline variants using tumor RNA sequencing data, and ranking mutated peptide sequences ”Openvax pipeline.” The process for determining somatic variants hews closely to the Broad Institute’s “Best Practices” for cancer SNVs and indels. The phasing of somatic and germline variants is implemented in a custom bioinformatics tool called Isovar. Mutated protein sequences containing phased variants are ranked according to two criteria: expression of the mutant allele in tumor RNA and aggregated predicted affinity to the patient’s Class I MHCs. Both quantities are normalized and multiplied together to create single ranked ordering of the candidate mutant sequences. Results: PGV001_002 (head and neck squamous cell cancer), who has completed vaccination, received 10 doses of vaccine comprising 10 long peptides (25 amino acid length) combined with poly-ICLC (toll-like receptor-3 agonist) intradermally. Vaccine-induced blood T-cell responses were determined, at weeks 0 (before-treatment) and 27 (after-treatment), ex vivo by interferon (IFN)-g enzyme-linked immunospot (ELISPOT) assay and after in vitro expansion by intracellular cytokine staining (ICS). Overlapping 15-16-mer assays peptides (OLPs) spanning the entirety of each ILP and 9-10-mer peptides corresponding to each predicted class I epitope (Min) were pooled and used to monitor immunogenicity. Ex vivo responses to these peptide pools were undetectable at week 0 but were evident at week 27 against 2 OLPs out of 10 (20%) and in 5 Min out of 10 (50%). After in vitro expansion, neoantigen-specific CD4+ and CD8+ T-cell responses were found in 5 out of 10 pooled peptides (50%). 7 out of 10 (70%) epitopes elicited polyfunctional T-cell responses (secretion of INF-α, TNF-α, and/or IL-2) from either CD4+ or CD8+ T-cells. Conclusion: To identify which predicted epitopes within the peptides pools stimulated the T-cell responses, we deconvoluted all the pools by either ex vivo and in vitro expansion. Ex vivo IFN-α production was detected in 1 (15-mer) peptide out of 15 (6.7%) and in 4 (9-10-mer) peptides out of 22 (18.2%). After expansion with single peptides, of 22 (9-10-mer) peptides tested, CD8+ T-cells were reactive against 13 peptides (59%), while CD4+ responses were seen in response to 11 of 15 (15-16-mer) peptides tested. Both CD4+ and CD8+ T-cell responses were polyfunctional. The PGV001 vaccine in our first patient showed both safety and immunogenicity, eliciting both CD4+ and CD8+ responses to the vaccine peptides. As we are enrolling additional patients, the information learned from this clinical trial will instruct the next generation of MTA-based vaccines, future development of immunotherapeutic approaches and rational combinations. Citation Format: Ana B. Blazquez, Alex Rubinsteyn, Julia Kodysh, John P. Finnigan, Thomas Marron, Rachel L. Sabado, Marcia Meseck, Timothy J. O'Donnell, Jeffrey Hammerbacher, Michael Donovan, John Holt, Milind Mahajan, John Mandeli, Krysztof Misiukiewicz, Eric M. Genden, Brett A. Milles, Hooman Khorasani, Peter R. Dottino, Hanna Irie, Amy B. Tiersten, Elisa R. Port, Andrea S. Wolf, Hern J. Cho, Ashutosh Tewari, Samir S. Parekh, Sujit Nair, Matthew D. Galsky, William K. Oh, Sacha Gnjatic, Eric E. Schadt, Phillip A. Friedlander, Nina Bhardwaj. A phase I study of the safety and immunogenicity of a multipeptide personalized genomic vaccine in the adjuvant treatment of solid cancers [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A005.

Abstract B108: Molecular and cellular properties of neoantigen-specific CD8+ T-cells on interaction with B16F10 melanoma in situ

Abstract Mutation-derived tumor antigens (MTA)—alternatively known as “neoantigens”—are a class of tumor antigens generated by tumor cell-intrinsic somatic DNA alterations. MTA are thought to be the predominant target of spontaneous and treatment-induced anti-tumor immunity. Direct targeting of MTA with therapeutic vaccination or other approaches is thus an active area of clinical research. However, pre-clinical studies of MTA-specific immunity have been limited in part due to a lack of reproducible targets. Furthermore, properties distinguishing effective MTA-specific immune responses, from less effective responses targeting other types of non-mutated tumor antigens have not been identified. To address this deficit, we previously reported the development of a system wherein MTA-specific CD8+ T-cell-mediated immunity could be systematically characterized. Briefly, we performed exome (WES) and RNASeq on the B16F1 and B16F10 melanoma cell lines, as well as matched normal tissue. We identified somatic single-nucleotide substitutions, insertion and deletion (INDEL), as well as larger INDEL and gene translocation events. The sequence identity and expression of somatic variants was validated by RNASeq. Standard in silico protocols were used to predict MHC-I binding affinity, which we subsequently validated via cell-free fluorescent peptide:MHC-I stability assay. Using a nanoparticle-based vaccination protocol, we identified and characterized multiple MHC-I restricted MTA in the B16F10 model system. We report an initial characterization of the vaccine-induced MTA-specific CD8+ T-cell response and demonstrate activity of vaccine monotherapy following subcutaneous tumor challenge. We extend prior observations were expanded upon in the following manner: First, the sensitivity and specificity of MTA-specific CD8+ T-cells was determined by in vitro stimulation with target MTA peptide and matched wildtype-sequence peptide followed by intracellular flow cytometry. Vaccine-induced CD8+ T-cells exhibit selectivity to MTA peptide targets ranging perfect specificity to moderate selectivity. A cell-based MHC-I binding assay was used to measure the relative affinity of MTA and matched wildtype peptides. The selectivity of vaccine-induced CD8+ T-cells to MTA peptides was observed to depend both on peptide:MHC-I as well as TCR:pMHC interactions. With respect to sensitivity of CD8+ T-cells towards target antigen, CD8+ T-cells targeting MTA were found to be more sensitive than those targeting nonmutated tumor antigens. We assessed direct recognition of target antigen endogenously presented by autologous tumor cells and describe tumor cell-intrinsic, as antigen-specific features affecting lymphocyte recognition and effector function. Second, we longitudinally profiled the surface receptor phenotype, and transcription factor usage of MTA-specific CD8+ T-cells by single-cell mass cytometry (CyTOF). We describe the phenotype of peripheral, as well as tumor-infiltrating MTA-specific CD8+ T-cells. Using a genetically-encoded fluorescent reporter we identify MTA-specific CD8+ T-cells actively engaged with antigen in situ and describe the molecular and cellular characterization of these tumor-reactive MTA-specific CD8+ T-cells. In summary, we report the identification and detailed characterization of multiple previously-undescribed MHC-I restricted MTA relevant to the widely-utilized B16F10 model. We characterize the phenotype and function of MTA-specific CD8+ T-cells. Finally, we describe phenotypic and functional properties of MTA-specific CD8+ T-cells associated with therapeutic activity in vivo. Citation Format: John P. Finnigan, Andrew S. Ishizuka, Geoffrey M. Lynn, Alex Rubinsteyn, Robert A Seder, Nina Bhardwaj. Molecular and cellular properties of neoantigen-specific CD8+ T-cells on interaction with B16F10 melanoma in situ [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B108.

Abstract SY14-02: Personalized therapies: Neoantigen discovery and vaccination

Abstract Tumor antigens derived from somatic mutations, or indels, are referred to as mutation-derived tumor antigens (MTAs) but are also known as neoantigens,or tumor-specific antigens. Evidence suggests that MTAs play a significant role in the endogenous adaptive immune response to tumors, and in the induced response following treatment with checkpoint inhibitors such as anti-CTLA-4 or anti-PD-1. This session will cover the concept of shared neoantigens in cancer and vaccine approaches to elicit adaptive antitumor immunity. Citation Format: Nina Bhardwaj. Personalized therapies: Neoantigen discovery and vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY14-02.

Tumor Immunity and Survival as a Function of Alternative Neopeptides in Human Cancer.

The immune system exerts antitumor activity via T cell-dependent recognition of tumor-specific antigens. Although the number of tumor neopeptides-peptides derived from somatic mutations-often correlates with immune activity and survival, most classically defined high-affinity neopeptides (CDNs) are not immunogenic, and only rare CDNs have been linked to tumor rejection. Thus, the rules of tumor antigen recognition remain incompletely understood. Here, we analyzed neopeptides, immune activity, and clinical outcome from 6,324 patients across 27 tumor types. We characterized a class of "alternatively defined neopeptides" (ADNs), which are mutant peptides predicted to bind MHC (class I or II) with improved affinity relative to their nonmutated counterpart. ADNs are abundant and molecularly distinct from CDNs. The load of ADNs correlated with intratumoral T-cell responses and immune suppression, and ADNs were also strong predictors of patient survival across tumor types. These results expand the spectrum of mutation-derived tumor antigens with potential clinical relevance. Cancer Immunol Res; 6(3); 276-87. ©2018 AACR.

Cancer immunogenomic approach to neoantigen discovery in a checkpoint blockade responsive murine model of oral cavity squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCC) are an ideal immunotherapy target due to their high mutation burden and frequent infiltration with lymphocytes. Preclinical models to investigate targeted and combination therapies as well as defining biomarkers to guide treatment represent an important need in the field. Immunogenomics approaches have illuminated the role of mutation-derived tumor neoantigens as potential biomarkers of response to checkpoint blockade as well as representing therapeutic vaccines. Here, we aimed to define a platform for checkpoint and other immunotherapy studies using syngeneic HNSCC cell line models (MOC2 and MOC22), and evaluated the association between mutation burden, predicted neoantigen landscape, infiltrating T cell populations and responsiveness of tumors to anti-PD1 therapy. We defined dramatic hematopoietic cell transcriptomic alterations in the MOC22 anti-PD1 responsive model in both tumor and draining lymph nodes. Using a cancer immunogenomics pipeline and validation with ELISPOT and tetramer analysis, we identified the H-2Kb-restricted ICAM1P315L (mICAM1) as a neoantigen in MOC22. Finally, we demonstrated that mICAM1 vaccination was able to protect against MOC22 tumor development defining mICAM1 as a bona fide neoantigen. Together these data define a pre-clinical HNSCC model system that provides a foundation for future investigations into combination and novel therapeutics.

A phase I study of the safety and immunogenicity of a multipeptide personalized genomic vaccine in the adjuvant treatment of solid cancers.

TPS3114 Background: Mutation-derived tumor antigens (MTAs) arise as a direct result of somatic variations, including nucleotide substitutions, insertions, and deletions that occur during carcinogenesis. These somatic variations can be characterized via genetic sequencing and used to identify MTAs. We propose a platform for a fully-personalized MTA-based vaccine in the adjuvant treatment of solid tumors. Methods: This clinical trial is a single-arm, open label, proof-of-concept phase I study designed to test the safety and immunogenicity of the Personalized Genomic Vaccine 001 (PGV001). The single-center study will enroll 20 eligible subjects with histological diagnosis of the following tumor types: (a) head and neck squamous cell cancer, (b) non-small cell lung cancer, (c) ductal or lobular breast cancer, (d) serous carcinoma of the ovary, uterine adnexa, (e) urothelial carcinoma of renal pelvis or bladder, (f) cutaneous squamous cell cancer. Subjects must have no measurable disease at time of first vaccine administration, and 5-year disease recurrence risk of > 30%. Patients will receive 10 doses of PGV001 as well as 10 doses of poly-ICLC (toll-like receptor-3 agonist, vaccine adjuvant), administered 1 day after PGV001 vaccination. Toxicity (endpoint 1) will be defined by Common Terminology Criteria for Adverse Events v5.0. Blood samples will be collected at various time points for immune response monitoring of MTA-specific humoral and cellular immune responses. For each patient, immunogenicity (endpoint 2) will be defined as an epitope-specific T cell response, detectable in peripheral blood samples after PGV001 vaccination. The change in the frequency of vaccine-induced epitope-specific T lymphocyte populations post-vaccination relative to baseline will be determined using mixed effects linear regression modeling. Conclusions: Our clinical trial will test for the first time the safety and immunogenicity of PGV001 in patients with multiple solid cancers. The information learned from this clinical trial will instruct the next generation of MTA-based vaccines, future development of immunotherapeutic approaches and rational combinations. Clinical trial information: NCT02721043.

Dendritic Cell Strategies for Eliciting Mutation-Derived Tumor Antigen Responses in Patients.

Dendritic cells (DCs) are equipped for sensing danger signals and capturing, processing, and presenting antigens to naive or effector cells and are critical in inducing humoral and adaptive immunity. Successful vaccinations are those that activate DCs to elicit both cellular and humoral responses, as well as long-lasting memory response against the target of interest. Recently, it has become apparent that tumor cells can provide new sources of antigens through nonsynonymous mutations or frame-shift mutations, leading to potentially hundreds of mutation-derived tumor antigens (MTAs) or neoantigens. T cells recognizing MTA have been detected in cancer patients and can even lead to tumor regression. Designing MTA-specific vaccination strategies will have to take into account the adjuvant activity of DC subsets and the best formulation to elicit an effective immune response. We discuss the potential of human DCs to prime MTA-specific responses.

Patient-Specific Mutation-Derived Tumor Antigens As Targets for Cancer Immunotherapy in Multiple Myeloma

Multiple myeloma (MM) is an incurable plasma cell malignancy accounting for more than 10,000 deaths in the US each year. Novel therapeutic approaches for relapsed MM are urgently needed. Tumor-specific mutations are ideal targets for cancer immunotherapy as they can be potentially recognized as neo-antigens by mature T-cells. Targeting tumor-specific antigens harboring somatic mutations presented on major histocompatibility complex class I molecules (MHC-I) with peptides could personalize the therapeutic approach for relapsed patients. To test this possibility, we examined 6 relapsed MM tumor samples from Mount Sinai, NY to predict in silico patient-specific tumor mutations that may activate the patient's immune systems. This is the first study to utilize Whole-Exome Sequence data (WES) from relapsed MM patients to show the feasibility of using exome sequencing to identify mutation derived neo-antigens that are patient-specific.DNA and RNA from six MM patients were extracted from sorted CD138+ cells from bone marrow aspirates. At the time of sample collection all patients had relapsed following at least five lines of therapy including Autologous Stem Cell Transplantation. The exome capture for DNA sequencing was carried out using the Agilent human whole-exome SureSelect assay. RNA-seq libraries were prepared using Illumina mRNA-seq protocol. All libraries were sequenced on an Illumina HiSeq2500 to generate 100 nucleotide reads. RNA reads were aligned to human reference genome (hg19) and assembled into transcripts using Bowtie-TopHat-Cufflinks. WES data was mapped to reference genome by BWA and then processed by MuTect to detect somatic mutations. Patient-specific alleles were determined using Seq2HLA. The identified mutations lead to candidate antigenic peptides that were filtered by tumor expression level (FPKM >2) using RNA sequence data. Candidate peptides of 8-11 character long were then ranked based on peptide-MHC binding affinity prediction (IC50nM) performed in silico using NetMHCpan.We identified a total of 340 tumor-specific nonsynonymous somatic mutations expressed in the context of patient specific HLA type. 263 (77%) genes were strong binders (IC50<150nM) and 77 (23%) genes were moderate/weak binders (IC50150-500nM). The number of mutated genes that were immunogenic per patient ranged from a minimum of 6 genes to 147 genes. Further, Database for Annotation, Visualization and Integrated Discovery (DAVID) tool was used to identify potentially enriched biological processes among the 340 genes using Gene Ontology (GO) terms. Enrichment analysis of 263 genes showed that they are mainly involved in myeloid cell activation during immune response (eg.LAT2, MYO1F), cell cycle process (CDK1, CHEK2, DNM2, EP300, SETD8), cellular response to stress (RAD21, HDAC2, MAT2B), chromatin silencing (SIRT2, SMARCA4), cell apoptosis and signal transduction (KRAS, NLRP1, ING4, IGF2R). Similarly enrichment analysis of 77 genes revealed their involvement mainly in B cell activation and leukocyte differentiation (LRRC8A, CD3E, PRKDC). Examples of some of these significantly mutated genes with binding affinity and predicted peptides are shown in Table 1.In this study, we show for the first time a correlation between tumor mutations and the epitope landscape by in silico data, suggesting that somatic mutations in MM are immunogenic and could potentially confer antitumor vaccine activity. Our results support an approach in creating cancer vaccines that use tumor-specific immunogenic mutations for the development of personalized vaccines for MM patients.Table 1Immunogenic mutations in Multiple Myeloma#Patient Specific allelesPeptidesIC50Mutated GenesEffectPatient#11HLA-C*14:02CYGHTMVAF57.75LZTR1p.R284C2HLA-C*14:02LYFFGMHVQEY29.75EP300p.C1372Y3HLA-C*14:02TFNEPSSEYF114.21SMARCA4p.G1146SPatient#24HLA-B*15:01MSLHNLGTVF26.2BCRp.A1160G5HLA-A*31:01SIISDSPR149.38FcRL5p.V269I6HLA-A*31:01HYFMHLLK37.16SIRT2p.R116HPatient#37HLA-C*05:01ITDFGHSEIL25.18CHEK2p.K344EPatient#48HLA-A*11:01VVGARGVGK121.47KRASp.G12R9HLA-B*41:01LEIDQLFRI132.84CDK1p.S208LPatient#510HLA-A*31:01AVGCGFRRARR106.68MAT2Bp.P65RPatient#611HLA-A*30:01HQRVLYIEI93.61HDAC2p.D145E12HLA-A*30:01FTRCLTPLL63.19RAD21p.V397L DisclosuresChari:Array Biopharma: Consultancy, Other: Institutional Research Funding, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium/Takeda: Consultancy, Research Funding; Biotest: Other: Institutional Research Funding; Novartis: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Jagannath:BMS: Honoraria; Janssen: Honoraria; MERCK: Honoraria; Novartis Pharmaceuticals Corporation: Honoraria; Celgene: Honoraria. Dudley:NuMedii, Inc: Patents & Royalties; Janssen Pharmaceuticals: Consultancy; GlaxoSmithKline: Consultancy; Personalis: Patents & Royalties; Ayasdi, Inc: Other: Equity; Ecoeos, Inc: Other: Equity. Hammerbacher:Cloudera: Membership on an entity's Board of Directors or advisory committees; Bay Sensors: Other: Equity; Cambrian Genomics: Other: Equity; Genome Compiler Corporation: Other: Equity; Science Exchange: Other: Equity; Transcriptic: Other: Equity; Pymetrics: Other: Equity. Schadt:Pacific Biosciences: Consultancy; Berg Pharma: Other: Scientific Advisory Board; GNS Healthcare: Other: Scientific Advisory Board; Clinical Gene Networks AB: Other: Equity. Bhardwaj:Dynavax Technologies Corporation: Consultancy; Crucell: Other: Equity; Dendreon Corporation: Other: Scientific Advisory Board; Merck & Co., Inc.: Other: Scientific Advisory Board; Neostem, Inc.: Other: Equity.