Role Of Neoantigens Research Articles

Abstract 3855: Kinetics of neoantigen-specific T cells in post-transplant relapse

Abstract Introduction: Graft-versus-leukemia effect (GVL) underpins the curative mechanism of allogeneic stem cell transplantation (alloSCT) for acute leukemias. Donor-derived T cells can recognize various antigens, including human leukocyte antigen (HLA), minor-histocompatibility antigens (miHA), over-expressed antigens, or neoantigens derived from somatic mutations of leukemia. However, the role of neoantigens has not been fully investigated in GVL. Here, we aimed to predict neoantigens and identify neoantigen-reactive T cells using samples after alloSCT. Materials: Leukemia blasts were purified from relapsed samples. Recipient saliva or T cells from pre-transplant peripheral blood (PB) were used as a recipient germline control, while donor monocyte or CD34 cells were used as donor controls. DNA/RNA isolated from these specimens were analyzed for whole exome sequencing (WES) paired with RNAseq. Somatic mutations were called using blasts and recipient germline controls by mutect and mpileup and annotation with SnpEff. Somatic mutations with variant allele frequency >20% were screened for neoantigen prediction bound to recipient HLA (IC50 < 500nM) by pVAC-seq. We also genotyped 73 SNPs associated with publicly known miHAs. Serial PB were stained for HLA-A*02:01 tetramers loaded with neoantigens or CMVpp65. T cells were stimulated with neoantigens for 10 days and tested for cytokine production in responses to target or control peptides. Results: We screened neoantigens in 8 patients relapsing after alloSCT (7 acute myeloid leukemia and 1 acute lymphoblastic leukemia) and their donors (4 matched siblings, 1 matched unrelated, 3 haploidentical). In 7 subjects, WES/RNAseq showed an average of 273 somatic mutations per patient (range 108- 609). In one subject, targeted next-generation sequencing was used to detect 2 somatic mutations. Among these mutations, seven out of eight subjects (87.5%) had potential neoantigen candidates, with a median number of 9 antigens per subject. In contrast, only 2 subjects (25%) had potential miHA. UPN8, a responder to donor lymphocyte infusion (DLI), showed dominant CMVpp65-specific T cells at the time of relapse and emergence of TP53- (neoantigen) or HA-2- (miHA) specific T cells after relapse. HA-2-specific T cells upregulated PD-1 expression at the time of graft-versus-host disease (GVHD), indicating activation of these miHA-specific T cells during GVHD. In two subjects (UPN3 and UPN8) who achieved long-term remission after DLI, CD8 T cells specific to neoantigens were detectable by IFN-γ/CD107a production in reaction to mutant peptides (SDE2.pK123X or TP53.pR248W) from post-relapse samples. Discussion: Our study showed the feasibility of in-silico neoantigens prediction and neoantigen-specific T-cell detection from a subject who developed relapse after alloSCT. Further investigation is needed to test if neoepitope burden and miHA disparity could correlate with transplant outcomes. Citation Format: Biswas Neupane, Jui-En Ray Lee, Kedwin Ventura, Kayla Parr, Kevin Quann, Sawa Ito. Kinetics of neoantigen-specific T cells in post-transplant relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3855.

Kinetics of Neoantigen Specific T Cells in Post-Transplant Relapse

Introduction: In allogeneic stem cell transplantation (alloSCT), donor-derived T cells mediate the graft-versus-leukemia effect (GVL) by recognizing antigens expressed by leukemia cells. These antigens may include either major- or minor-histocompatibility alloantigens, leukemia over-expressed antigens (leukemia-associated antigens), or neoantigens derived from leukemic somatic mutations. In solid tumors, higher somatic mutation burdens are associated with better response to checkpoint inhibitors, which underscores the importance of neoantigen-specific T-cells in cancer immunotherapy. However, mutation burdens are relatively low in acute leukemia, and the role of neoantigens in GVL remains unknown. Here, we screened potential neoantigens and identified neoantigen-reactive T cells in the patients who developed post-transplant relapse. Materials and methods: Leukemic blasts from patients in relapse were FACS-isolated from bone marrow aspirate or peripheral blood samples. Recipient T cells or saliva were isolated from pre-transplant peripheral blood as a germline control, and donor monocyte or CD34-positive cells were used as donor controls. DNA/RNA isolated from these specimens were analyzed for whole exome sequencing (WES) at 100X coverage, paired with RNAseq at 40M reads per sample. Somatic mutations were called between recipient blasts and germline controls with mutect and mpileup, followed by annotation with SnpEff. Somatic mutations with variant allele frequency >20% were subjected to pVAC-seq for prediction of neoantigen likely presented on recipient MHC (IC50 < 500nM). 73 SNPs loci associated with publicly known minor histocompatibility antigens (miHAs) were also genotyped to find the miHA disparities between donors and recipients. Serial peripheral blood mononuclear cell samples (PBMCs) were obtained before relapse if available, at the time of relapse, and after treatment for relapse. For HLA-A*02:01-restricted neoantigens, PBMCs were stained with HLA-A*02:01 tetramers loaded with neoantigens or CMVpp65 (control). Purified CD3 cells were stimulated with neoantigens for 10 days and tested for cytokine production in responses to target or control peptides. Results: Eight patients with relapsed acute leukemia after allo-SCT (7 acute myeloid leukemia and 1 acute lymphoblastic leukemia) and their transplant donors (4 matched siblings, 1 matched unrelated, 3 haploidentical) were screened for neoantigens. We analyzed samples from 7 subjects with available WES/RNAseq and detected an average of 273 somatic mutations per patient (range 108- 609) on average. Targeted next-generation sequencing was used to detect 2 somatic mutations in one subject. Among these mutations, potential neoantigen candidates were identified in seven out of eight subjects (87.5%), with a median number of 9 candidate neoantigens per subject. In contrast, only 2 subjects (25%) were found with donor:receipient miHAs at tested loci. CD8 T cells derived from UPN8, a responder to DLI, showed dominant CMVpp65-specific T cells at the time of relapse and persistent low frequencyTP53- (neoantigen) or HA-2- (miHA) specific T cells after relapse by HLA-A*02:01 tetramer staining (Figure A). HA-2-specific T cells upregulated PD-1 expression at the time of graft-versus-host disease (GVHD), indicating activation of these T cell clones during GVHD (Figure B). In two subjects (UPN3 and UPN8) who achieved long-term remission after DLI, CD8 T cells reactive to neoantigens were detectable by IFN-γ/CD107a production specific to mutant peptides ( SDE2.pK123X and TP53.pR248W respectively). Discussion: Our in-silico analysis suggested the possibility of somatic mutations in acute leukemia to serve as putative neoantigens in alloSCT. We were able to detect neoantigen-specific T cells by tetramer staining and cytokine production assays from a subject who achieved a durable remission after DLI. Although the sample size is very small, we demonstrated the feasibility of studying neoantigen-specific T cells in post-transplant specimens. Further investigation is needed to test if neoepitope burden and miHA disparity could correlate with post-relapse outcomes, especially for response to DLI.

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.

Neoantigen Controversies.

Next-generation sequencing technologies have revolutionized our ability to catalog the landscape of somatic mutations in tumor genomes. These mutations can sometimes create so-called neoantigens, which allow the immune system to detect and eliminate tumor cells. However, efforts that stimulate the immune system to eliminate tumors based on their molecular differences have had less success than has been hoped for, and there are conflicting reports about the role of neoantigens in the success of this approach. Here we review some of the conflicting evidence in the literature and highlight key aspects of the tumor-immune interface that are emerging as major determinants of whether mutation-derived neoantigens will contribute to an immunotherapy response. Accounting for these factors is expected to improve success rates of future immunotherapy approaches.

18th Association for Cancer Immunotherapy (CIMT) Annual Meeting. Virtual - May 10-12, 2021

The Association for Cancer Immunotherapy (CIMT) held its 18th annual meeting virtually due to the COVID-19 pandemic. Europe's largest specialty meeting on cancer immunology and immunotherapy research and development digitally connected over 800 researchers and clinical professionals. Along with plenary sessions, around 220 abstracts were presented online in eTalks format. The multidisciplinary program covered several topics on the cancer immunotherapy research area, with emphasis on cellular therapies (mainly CAR T and natural killer cells), the role of neoantigens and therapeutic vaccination, combination therapies, gut microbiota and its relationship with cancer immunotherapies, immunoguiding, and the role of tumor microenvironment, as well as some sessions on computational immunology and regulatory research. Furthermore, a crosstalk session was dedicated to COVID-19 and immuno-oncology. This report focuses on a selection of presentations covering treatment updates given during the conference. © 2021 Clarivate Analytics.

DENDRO: genetic heterogeneity profiling and subclone detection by single-cell RNA sequencing

Although scRNA-seq is now ubiquitously adopted in studies of intratumor heterogeneity, detection of somatic mutations and inference of clonal membership from scRNA-seq is currently unreliable. We propose DENDRO, an analysis method for scRNA-seq data that clusters single cells into genetically distinct subclones and reconstructs the phylogenetic tree relating the subclones. DENDRO utilizes transcribed point mutations and accounts for technical noise and expression stochasticity. We benchmark DENDRO and demonstrate its application on simulation data and real data from three cancer types. In particular, on a mouse melanoma model in response to immunotherapy, DENDRO delineates the role of neoantigens in treatment response.

Abstract LB-065: A high quality neoantigen fitness model cooperates with T lymphocyte infiltrates to identify glioblastoma patients with the longest survival

Abstract Malignant glioma (glioblastoma, GBM) is a lethal cancer resistant to multimodality therapeutic approaches. A high burden of tumor-specific mutant peptides (neoantigens) correlates with a more favorable prognosis and response to immunotherapies in selected solid tumors but the role of neoantigens and how they impact clinical outcome in most tumors including GBM remains unclear. Here, we exploited the similarity between tumor neoantigens and infectious disease-derived immune epitopes and applied a neoantigen fitness model for the identification of high quality neoantigens in human GBM. The neoantigen quality fitness model stratified GBM patients with more favorable clinical outcome and, together with tumor infiltration of CD8 T lymphocytes, identified a subgroup of GBM patients with the longest survival, thus exhibiting optimal performance. Conversely, neither tumor neoantigen burden from a quantity model nor the isolated enrichment of CD8 T lymphocytes could predict better survival of GBM patients. The GBM subgroup that emerged from the neoantigen quality model displays distinct genomic and transcriptomic features that should guide patient stratification for immunotherapy and maximal benefit of GBM patients. Citation Format: Jing Zhang, Francesca P. Caruso, Jason K. Sa, Sune Justesen, Do-Hyun Nam, Peter Sims, Michele Ceccarelli, Anna Lasorella, Antonio Iavarone. A high quality neoantigen fitness model cooperates with T lymphocyte infiltrates to identify glioblastoma patients with the longest survival [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 LB-065.

Neoantigen Landscape of Relapsed Acute Leukemia Following Allogeneic Stem Cell Transplantation

INTRODUCTION: The potent graft versus leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) is considered as a blueprint for cellular immunotherapy. However, failure of GVL leads to relapse of underlying leukemia, the major cause of death after allo-SCT. In solid tumors, higher tumor mutation burdens are associated with better response to check point inhibitors which implies the importance of neoantigen specific T-cell functions in cancer immunity. In contrast, the frequencies of somatic mutations in acute leukemia are generally low, therefore the role of neoantigens in GVL remains undetermined. Here, we developed a platform to screen for potential neoantigens by performing whole exome sequencing (WES) and RNA sequencing (RNAseq) in matched samples: leukemic blasts at relapse after allo-SCT, recipient T cell controls, and donor cells.METHODS: Leukemic blasts from patients in relapse were enriched by flow sorting from bone marrow aspirate or peripheral blood samples. Recipient T cells were isolated from pre-transplant peripheral blood as germline controls, and donor monocytes or CD34-positive cells were used as hematopoietic-lineage cell controls. WES was performed to 100X coverage, paired with RNAseq 40M reads per sample. Somatic mutations were detected with mutect and mpileup, followed by annotation with SnpEff. High confidence somatic mutations were subjected to pVAC-seq for neoantigen predictions.RESULTS: Six patients with relapsed acute leukemia (AML 5, ALL 1) after allo-SCT and their transplant donors (matched sibling 3, haplo-identical 3) had suitable samples available for analysis. On average, somatic mutations were identified in 297 genes (range 108- 609) by comparing leukemic blasts and germline control T cells. Among those mutations, potential candidates of neoantigen were identified in five out of six subjects. Allele frequencies of mutant genes varied. Most of neoantigens were predicted to bind HLA of both class I (median 5, range 0-15) and class II (median 6, range 0-12). One subject had only HLA class II restricted peptides as predicted neoantigens. Of interest majority of antigens were derived from molecules known to play important roles in leukemia or tumor biology which include ETV6, CCNY, IDH2, PTPN11, SF3B1, and TP53. Evolution analysis of neoantigen showed an emergence of new antigens in relapsed leukemia while a few driver gene mutations persisted after allo-SCT (Figure).CONCLUSION: Our in-silico analysis demonstrated the possibility that somatic mutation in acute leukemia could serve as putative neoantigens applicable for novel immunotherapy after allo-SCT. The binding capacity of mutant peptides to class I and II HLA implies the importance of both CD4 and CD8 contributions to anti-neoantigen immunity. Next, we will search for neoantigen specific T cells exerting an anti-leukemia effect to validate the GVL potential of these mutations in allo-SCT. DisclosuresNo relevant conflicts of interest to declare.

Abstract PR02: Identification of breast cancer neoantigens exposed by radiation therapy

Abstract Tumor-specific mutations can generate neoantigens, a class of major histocompatibility complex (MHC)-binding peptides that may be highly immunogenic because they are not expressed in healthy tissues and thus not subject to central thymic tolerance. Recent studies have highlighted the key role for neoantigens in cancer immunotherapy. We have previously shown in the 4T1 mouse model (syngeneic with BALB/c mice) of immune checkpoint blockade (ICB)-resistant metastatic breast cancer that local radiation therapy (RT) combined with CTLA-4 blockade induces the CD8+ T cell-mediated regression of irradiated tumors and limits metastatic lung colonization. Preliminary analysis of the T-cell receptor (TCR) repertoire by deep sequencing of CDR3 regions of TCRβ indicated that unique clonotypes are expanded in 4T1 tumors treated with RT and CTLA-4 blockade, suggesting that tumor rejection involves T cells reactive to a set of tumor antigens that are made available to the immune system by RT. Therefore, we hypothesize that RT, by changing the transcriptional profile of cancer cells, may expose antigenic mutations not transcribed at sufficient levels in untreated tumors, and hence promote priming of T cells to these unique mutated antigens. We performed whole-exome sequencing (WES) and RNA sequencing (RNA-seq) of 4T1 cells irradiated (8GyX3) or not (0Gy) in vitro to identify tumor-specific mutations, including nonsynonymous and frameshift mutations (insertions or deletions). Genetic variants in WES data were called using the mm10 mouse reference genome, and genomic DNA from BALB/c mouse tail was used to identify somatic mutations. We then used several algorithms (NetMHC 3.4; SMM; SYFPEITHI) to predict MHC-I (H2-Kd, -Dd, -Ld)-binding epitopes from these mutated genes, and we selected those with a predicted affinity <500 nM. In order to focus on the mutations likely to be expressed in a majority of tumor cells, we used the RNA-seq data to select for variants present at a minimum of 40% allelic frequency in both conditions (irradiated or not), but we prioritized mutated peptides that were upregulated by radiation. Using these genomic and computational analyses, we selected 17 predicted MHC-I restricted neo-epitopes, out of 309 total mutations initially identified in the 4T1 cell line. These candidate peptides were synthesized and tested in vitro for binding to H2-Ld or H2-Kd in a MHC stabilization assay using RMA-S cells expressing the appropriate MHC- I allele. We identified two mutated peptides that bind H2-Ld with high affinity. One of these neoepitopes arises from the Dhx58 gene which is significantly upregulated (fold change=3.4, p<0.001) in 4T1 cells irradiated with 8GyX3. Vaccination experiments are ongoing to test the immunogenicity of these peptides, and their ability to elicit a tumor-specific CD8+ T cell response in BALC/c mice. We demonstrate here that the use of an integrative approach combining WES, RNA-seq and in vitro binding assays allows for the identification of potential neoantigens that may be selectively unveiled by RT. Although the immunogenicity of the RT-exposed neoantigens identified here needs to be confirmed in vivo, these data provide initial proof-of-principle evidence that RT can uncover existing neoantigens to the immune system. This abstract is also being presented as Poster B85. Citation Format: Claire Lhuillier, Nils Rudqvist, Sandra Demaria. Identification of breast cancer neoantigens exposed by radiation therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR02.

Abstract 5720: Functional characterization of neoantigens determining immune checkpoint blockade response in mouse models of human melanoma

Abstract Melanoma is the deadliest form of skin cancer due to the lack of widely effective therapies for advanced disease. Recently FDA-approved immunotherapies, such as immune checkpoint blockade (ICB) by CTLA-4 and PD-1/PD-L1 antibodies, provide unprecedent durable responses but in less than 40% of late stage melanoma patients. While high mutational loads characteristic of responsive tumors has not shown predictive value of patient outcome, accumulating evidences suggest a key role for neoantigens in the response to ICB. Moreover, new T-cell transfer- and vaccine-based therapeutic strategies highlighted the relevance of an efficient identification and prioritization of highly immunogenic neoantigens to improve immunotherapies. However, mechanistic studies are not possible in humans, and the development of adequate predictive methods is still the center of intense debate in the field. Here we use two genetically engineered melanoma mouse models exhibiting distinct response to ICB. Melanomas induced by neonatal ultraviolet radiation (UV) in a HGF-transgenic mouse (HGF-tg) showed high sensitivity to anti-CTLA-4, whereas UV-induced melanomas in HGF-tg; BrafV600E; Cdkn2a+/- mouse (Braf/HGF) did not respond. We hypothesized these models will allow us to identify the neoantigen features required for ICB response including type, expression levels and allele frequency patterns. High tumor immunogenicity, assessed by in vivo vaccination assays, as well as increased T-cell infiltration upon anti-CTLA-4 treatment were correlated to greater response. Moreover, exome and RNA sequencing analyses revealed similar mutational and neoantigen load in both models, albeit with no common expressed mutations. Notably, both models expressed similar levels of antigen presentation related genes (e.g. B2m, H2-Kd, Tap1) suggesting that specific neoantigens in HGF-tg melanoma cells may contribute to their sensitivity to anti-CTLA-4. To test this, we predicted MHC-I/-II binding of HGF-tg melanoma mutated epitopes in silico and generated a “neo-epitope” library. Importantly, the expression and allele frequency of most of selected mutations were decreased in anti-CTLA-4 responder HGF melanomas. The “neo-epitope” library was transduced into Braf/HGF non-responder cells and future studies will identify the neo-epitopes lost upon ICB, representing determinants of therapeutic success. Additionally, in vivo vaccination assays using synthetic mutant peptides will be performed to validate each neoantigen candidate. We anticipate that our studies will provide insight into the role that neoantigens play in melanoma immunotherapy responses. (EPG and CPD contribute to this abstract equally). Citation Format: Eva Perez Guijarro, Chi-Ping Day, Zoe W. Ohler, Rajaa El Meskini, Howard Yang, Suman Vodnala, Cari Graff-Cherry, Sung Chin, Anyen Fon, Helen Michael, Maxwell Lee, Terry Van Dyke, Shyam Sharan, Glenn Merlino. Functional characterization of neoantigens determining immune checkpoint blockade response in mouse models of human melanoma [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 5720.

Abstract 2623: Identification of neo-antigens driving melanoma response to immune checkpoint blockers via in vivo screening

Abstract Immune checkpoint blockers (ICBs) have rendered unprecedented, durable responses in metastatic melanoma, but the heterogeneous response among patients continues to be the major obstacle for their therapeutic development. It is generally hypothesized that neoantigens derived from mutated genes are involved in tumor response to ICBs, since the latter is correlated witt mutational loads of tumors. However, direct experimental evidence showing that threshold quantity or specific properties of neoantigens drive ICB response are mostly lacking. To identify and characterize neoantigens implicated in ICB response, we have generated two UV-induced melanoma models based on the BrafV600E/Pten-knockout (Braf/PKO) and Hgf-transgenic (Hgf-tg) mouse, which displayed intrinsic resistance and high sensitivity to an anti-CTLA-4 antibody, respectively. Exome sequencing identified 216 and 291 non-synonymous mutations in the Braf/PKO and UV-Hgf melanoma cell lines, respectively. By RNA sequencing, 74 (34%) and 121 (42%) of these mutated genes were found to be expressed in each model, respectively, and there were no overlapping mutations between them. The mutations found in the “sensitive” UV-Hgf melanoma were analyzed in silico for their binding affinity to MHC-I and/or MHC-II, thus characterizing putative neoantigens. A “neo-epitope” library was generated by cloning the DNA sequences flanking non-synonymous mutations in frame with the eGFP gene in a lentiviral vector. We further showed that such eGFP-fused epitopes can be presented by the cells to induce specific T cell responses. The library will be transduced into the “resistant” Braf/PKO melanoma, which will be treated with anti-CTLA-4 in mice to identify the neoantigens required for the response. To prevent immunity against eGFP expressed by tumors, the library-transduced melanoma cells will be transplanted into the eGFP-tolerant “glowing head mice”. The results will be used to determine if one, or more, of our candidate neo-epitopes can induce a response to anti-CTLA-4. We will also analyze if the response to this ICB is an epitope-specific reaction or require multiple epitopes, which will help to identify resistance mechanisms. We anticipate that our results will provide insight into the role of neoantigens in ICB response. Moreover, our models will serve as a platform to study the specific contribution and predictive value of neoantigens for melanoma response to immunotherapy, which could help improve therapeutic strategies involving ICBs. Citation Format: Chi-Ping Day, Eva Perez-Guijarro, Rajaa El Meskini, Zoe Weaver Ohler, Maxwell Lee, Howard Yang, Suman Vodnala, Shyam Sharan, Glenn Merlino. Identification of neo-antigens driving melanoma response to immune checkpoint blockers via in vivo screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2017-2623

An open-label, phase Ib study of NEO-PV-01 + adjuvant with nivolumab in patients with melanoma, non-small cell lung carcinoma, or transitional cell carcinoma of the bladder.

TPS3116 Background: Cancer cells contain unique DNA mutations that result in altered amino acid sequences known as neoantigens. Growing evidence supports a central role for neoantigens as targets for tumor directed immune responses. Tumor mutational burden as well as neoantigen load have been associated with anti-tumor activity of checkpoint inhibitors. Vaccines targeting neoantigens offer a highly specific way to induce de novo T cell reactivity and to expand existing T cell responses against neoantigens. Here, we describe NEO-PV-01, a personalized, neoantigen vaccine designed specifically for the molecular profile of each individual’s tumor. Methods: NT-001 is a single-arm, phase IB study designed to evaluate the safety of administering NEO-PV-01 + adjuvant (Poly-ICLC) with nivolumab in patients with advanced melanoma, smoking-associated non-small cell lung carcinoma, or transitional cell carcinoma of the bladder who have received no more than one prior systemic treatment. Patients undergo a baseline tumor biopsy and HLA typing. DNA and RNA sequencing is performed on the tumors as well as peripheral blood to serve as normal DNA controls. On Day 1, patients begin treatment with nivolumab at a dose of 240 mg IV while their customized vaccine is being generated. Each vaccine is custom designed for the individual patient and contains up to 20 peptides 14-35 amino acids in length. The peptides are pooled into four groups and mixed with Poly-ICLC at the time of administration. Beginning at Week 12, patients receive five priming immunizations over a three-week period followed by booster vaccinations at Weeks 19 and 23. The primary endpoint is safety. Secondary endpoints are ORR, CBR, PFS, and assessment of response conversion between Week 12 and Week 24. Exploratory endpoints include extensive immune monitoring. Clinical trial information: NCT02897765.

Abstract 4388: The role of neoantigens in immunotherapy of cutaneous melanoma

Abstract Melanoma is increasing in incidence by up to 3% annually, and metastatic melanoma, which is resistant to most conventional therapies, has a poor prognosis with a 5-year survival rate of less than 20%. Immunotherapy is able to yield durable response in melanoma; however, the response rate is limited (20-30%). It has been shown that tumor immunogenicity is highly correlated to the response to immunotherapy. The mutational load and the characteristics of the neoantigens displayed by the tumor cells are theorized to play an important role in the immunogenicity of the tumor. We used genetically engineered preclinical mouse models to study the role of these neoantigens in melanoma response to immune therapy. Three syngeneic C57BL/6 mouse melanoma models were developed: UV induced melanoma in albino BRAF(V600E);PTEN-/- mice (1), DMBA induced melanoma in pigmented HGF-tg;CDK4(R24C) mice (2), and UV induced melanoma in pigmented HGF-tg mice. These three models were found to have varying degrees of tumor immunogenicity based on vaccination studies. The UV induced BRAF(V600E);PTEN-/- model displayed poor immunogenicity, while the DMBA induced HGF-tg;CDK4(R24C) model exhibited moderate immunogenicity and the UV induced HGF-tg model showed the highest immunogenicity. Exome sequencing results showed that high immunogenicity is associated with mutations of genes in DNA damage responses and frameshift mutations. To test the role of neoantigens, RNA from each of the melanoma models was sequenced and analyzed for mutational and expression profiles. We will be comparing the response of these three mouse models to correlate immunogenicity with response to anti-CTLA-4 therapy. We plan to analyze the pathways in which mutated genes were enriched, allowing the identification of “druggable” targets to enhance therapeutic efficacy of immune checkpoint inhibitors. CRISPR/Cas9-based screening approaches will be used to identify specific neoantigens able to influence response to immune therapy. In conclusion, our preclinical mouse models show that tumor immunogenicity may be correlated to the carcinogenic mechanisms, and RNA sequence has provided further insight into the role of mutational load and neo-epitopes on immune-based therapeutic response. (1) Provided by Marcus Bosenberg (Yale School of Medicine, New Heaven, CT), Martin McMahon (Huntsman Cancer Institute, Salt Lake City, UT). (2) Provided by Thomas Tueting (University Hospital Bonn, Bonn, Germany). Citation Format: Renee M. Thomas, Chi-Ping Day, Zoe Weaver Ohler, Rajaa El Meskini, Carri Graff-Cherry, Sung Chin, Aleksandra Michalowski, Ji Luo, Terry Van Dyke, Glenn Merlino. The role of neoantigens in immunotherapy of cutaneous melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4388.

The role of neoantigens in response to immune checkpoint blockade.

Immune checkpoint blockade has demonstrated substantial promise for the treatment of several advanced malignancies. These agents activate the immune system to attack tumor cells. For example, agents targeting CTLA4 and programmed cell death 1 (PD-1) have resulted in impressive response rates and, in some cases, durable remissions. Neoantigens are mutations that encode immunologically active proteins that can cause the immune system to recognize the affected cell as foreign. Recent data have made it clear that these mutations are, in large part, the functional targets of immune checkpoint blockade. This review summarizes the key discoveries leading up to this important conclusion and discusses possible applications of neoantigens in cancer therapy.

The Role of Neoantigens in Naturally Occurring and Therapeutically Induced Immune Responses to Cancer.

Definitive experimental evidence from mouse cancer models and strong correlative clinical data gave rise to the Cancer Immunoediting concept that explains the dual host-protective and tumor-promoting actions of immunity on developing cancers. Tumor-specific neoantigens can serve as targets of spontaneously arising adaptive immunity to cancer and thereby determine the ultimate fate of developing tumors. Tumor-specific neoantigens can also function as optimal targets of cancer immunotherapy against established tumors. These antigens are derived from nonsynonymous mutations that occur during cellular transformation and, because they are foreign to the host genome, are not subject to central tolerance. In this review, we summarize the experimental evidence indicating that cancer neoantigens are the source of both spontaneously occurring and therapeutically induced immune responses against cancer. We also review the advances in genomics, bioinformatics, and cancer immunotherapy that have facilitated identification of neoantigens and have moved personalized cancer immunotherapies into clinical trials, with the promise of providing more specific, safer, more effective, and perhaps even more generalizable treatments to cancer patients than current immunotherapies.