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Supplementary Table from An Empirical Antigen Selection Method Identifies Neoantigens That Either Elicit Broad Antitumor T-cell Responses or Drive Tumor Growth

Neoantigens are critical targets of antitumor T-cell responses. The ATLAS bioassay was developed to identify neoantigens empirically by expressing each unique patient-specific tumor mutation individually in Escherichia coli, pulsing autologous dendritic cells in an ordered array, and testing the patient's T cells for recognition in an overnight assay. Profiling of T cells from patients with lung cancer revealed both stimulatory and inhibitory responses to individual neoantigens. In the murine B16F10 melanoma model, therapeutic immunization with ATLAS-identified stimulatory neoantigens protected animals, whereas immunization with peptides associated with inhibitory ATLAS responses resulted in accelerated tumor growth and abolished efficacy of an otherwise protective vaccine. A planned interim analysis of a clinical study testing a poly-ICLC adjuvanted personalized vaccine containing ATLAS-identified stimulatory neoantigens showed that it is well tolerated. In an adjuvant setting, immunized patients generated both CD4+ and CD8+ T-cell responses, with immune responses to 99% of the vaccinated peptide antigens.Significance:Predicting neoantigens in silico has progressed, but empirical testing shows that T-cell responses are more nuanced than straightforward MHC antigen recognition. The ATLAS bioassay screens tumor mutations to uncover preexisting, patient-relevant neoantigen T-cell responses and reveals a new class of putatively deleterious responses that could affect cancer immunotherapy design.This article is highlighted in the In This Issue feature, p. 521

Supplementary Figure from An Empirical Antigen Selection Method Identifies Neoantigens That Either Elicit Broad Antitumor T-cell Responses or Drive Tumor Growth

The emergence of SARS-CoV-2 variants are a persistent threat to the efficacy of currently developed prophylactic vaccines and therapeutic antibodies. These variants accumulate mutations in the spike protein which encodes the epitopes necessary for neutralizing antibody binding. Moreover, emerging evidence suggest that robust antibody responses are insufficient to prevent severe disease and long-lasting viral immunity requires T cells. Thus, understanding how the T cell antigen landscape evolves in the context of these emerging variants remains crucial. T cells responses are durable and recognize a wider breadth of epitopes reducing the possibility of immune escape through mutation. Here, we deploy the ATLAS™ assay which identifies CD4+ and CD8+ T cell antigens by utilizing the endogenous HLA class-I and class-II peptide processing pathways. Profiling of T cells from exposed and unexposed donors revealed rich and complex patterns which highlighted the breadth of antigenic potential encoded in SARS-CoV-2. ATLAS revealed several common or frequent antigenic regions as well as an abundance of responses in the unexposed cohort potentially the result of pre-exposure to related coronaviruses. ORF10 was a common CD4+ response in the unexposed cohort while spike was identified as a common and frequent target in both cohorts. Moreover, the spike response profiles allowed us to accurately predict the impact of Omicron spike mutations. This analysis could thus be applied to study the impact of future emerging VOCs.

Neoantigens are critical targets of antitumor T-cell responses. The ATLAS bioassay was developed to identify neoantigens empirically by expressing each unique patient-specific tumor mutation individually in Escherichia coli, pulsing autologous dendritic cells in an ordered array, and testing the patient's T cells for recognition in an overnight assay. Profiling of T cells from patients with lung cancer revealed both stimulatory and inhibitory responses to individual neoantigens. In the murine B16F10 melanoma model, therapeutic immunization with ATLAS-identified stimulatory neoantigens protected animals, whereas immunization with peptides associated with inhibitory ATLAS responses resulted in accelerated tumor growth and abolished efficacy of an otherwise protective vaccine. A planned interim analysis of a clinical study testing a poly-ICLC adjuvanted personalized vaccine containing ATLAS-identified stimulatory neoantigens showed that it is well tolerated. In an adjuvant setting, immunized patients generated both CD4+ and CD8+ T-cell responses, with immune responses to 99% of the vaccinated peptide antigens. Significance: Predicting neoantigens in silico has progressed, but empirical testing shows that T-cell responses are more nuanced than straightforward MHC antigen recognition. The ATLAS bioassay screens tumor mutations to uncover preexisting, patient-relevant neoantigen T-cell responses and reveals a new class of putatively deleterious responses that could affect cancer immunotherapy design. This article is highlighted in the In This Issue feature, p. 521

BackgroundGEN-009 adjuvanted personalized cancer vaccine contains up to 20 neoantigens selected by ATLAS™, an ex vivo bioassay screening autologous T-cells for immune responses against both neoantigens and Inhibigens™. Inhibigen-specific T-cells suppress immunity, have been shown to accelerate tumor progression in mice, and are excluded from GEN-009. In cohort A, all patients immunized in the adjuvant setting with GEN-009 monotherapy developed immune responses. Ninety-nine percent of selected peptides were immunogenic: ex vivo CD4+ and CD8+ fluorospot responses specific for 51% and 41% of immunized peptides, respectively.1 Six of 8 patients continue without progression with a median follow up >2 years.MethodsGEN-009 was administered to patients with advanced cancer who received standard-of-care (SOC) PD-1 inhibitor as monotherapy or in combination therapy during vaccine manufacturing. Five vaccine doses were administered over 24 weeks in combination with single agent anti-PD-1. Patients who progressed prior to vaccination received salvage therapy followed by GEN-009 in combination. Peripheral T-cell responses were measured by ex vivo and in vitro stimulated fluorospot assays. Circulating tumor (ct) DNA levels were evaluated in a subset of patients pre- and post-GEN-009 administration.Results15 patients received GEN-009 in combination with PD-1 inhibitor; 1 patient received GEN-009 monotherapy. Median number of neoantigens per vaccine was 14 (range 5–18). GEN-009-related adverse events were limited to vaccine injection site reactions, mild myalgias or fatigue. Sequential vaccination with GEN-009 had an additive effect on the magnitude of ex vivo T-cell responses, that persisted in some patients for 12+ months post first vaccine dose. An association between proportion of peptides eliciting significant cytokine responses and RECIST response is apparent. Epitope spread was detected in CD8+ T-cells from CPI-sensitive patients, but not refractory patients. Four patients who responded to PD-1 inhibition followed by disease stabilization then demonstrated further tumor reduction after GEN-009 vaccination. Seven of 9 CPI responsive patients are progression-free 7 to 18 months after first vaccine dose. Three of 7 CPI-refractory patients have experienced unexpected prolonged stable disease, with 2 PR and 1 SD after vaccination lasting up to 10 months. Plasma ctDNA kinetics mirrored RECIST responses in each tested patient; in some responders, all evidence of ctDNA disappeared, including non-targeted antigens.ConclusionsVaccination with GEN-009 alone or in combination with anti-PD-1 was well tolerated. Preliminary data demonstrate induction of robust, durable neoantigen-specific immune responses and epitope spreading in the presence of PD-1 CPI. Broad immunity against tumor specific targets and encouraging patient outcomes support further study.Trial Registration clinicaltrials.gov identifier: NCT03633110ReferencesLam H, et al. An empirical antigen selection method identifies neoantigens that either elicit broad anti-tumor response or drive tumor growth. Cancer Discovery 2021 March;11(3):696–713.Ethics ApprovalThis study was approved by Western Institutional Review Board, approval number 1-1078861-1

Genocea’s ATLAS platform is an empirical bioassay that uses patient autologous immune cells to identify both true neoantigens and Inhibigens࣪ for inclusion in or exclusion from neoantigen-targeted vaccines and cell therapies, respectively. In ATLAS, patient-derived antigen-presenting cells (APCs) are pulsed with E. coli expressing individual mutations identified from the patient mutanome ± listeriolysin O, enabling interrogation of both CD8+ and CD4+ T cell recognition. True neoantigens induce T cell activation and cytokine release, while Inhibigens lead to a downregulation of T cell responses and thus can promote tumor growth. Previous ATLAS screening of CD8+ T cells from mice carrying B16F10 mouse melanoma tumors identified both neoantigens and Inhibigens. Upon therapeutic vaccination, adjuvanted neoantigens generated immunogenicity and anti-tumor efficacy1. In contrast, therapeutic vaccination with multiple ATLAS-identified Inhibigens, alone or in combination with an otherwise-protective vaccine, led to accelerated tumor growth, impaired T cell responses, and abrogated tumor immune infiltration. Our current study further explores the mechanism of Inhibigen-specific responses through adoptive transfer of vaccine-experienced T cells into tumor-bearing recipient mice, as well as through analysis of T cell gene expression. Additionally, in order to determine whether Inhibigen identification and treatment translates into pro-tumor effects universally across tumor models, we performed ATLAS screening on CD4+ and CD8+ T cells isolated from mice bearing orthotopic KPC pancreatic cancer. Out of 73 total non-synonymous mutations, we successfully identified 14 CD4+ and 15 CD8+ true neoantigens, and 16 CD4+ and 18 CD8+ Inhibigens. This is the first known comprehensive characterization of endogenous antigens in this model. Therapeutic administration of neoantigens as adjuvanted peptide vaccines in KPC tumor-bearing mice led to smaller tumor sizes and reduced ascites volumes, whereas Inhibigen vaccination accelerated tumor growth. Mouse studies are ongoing and additional data will be presented. Taken together, our data from human cancer patients and two mouse cancer models support the importance of appropriate neoantigen selection and Inhibigen identification and exclusion from cancer therapies. Genocea’s GEN-011 neoantigen-targeted peripheral T cell (NPT) therapy candidate, designed using ATLAS-identified neoantigens and omitting Inhibigens, is being evaluated in an ongoing clinical trial (NCT04596033). Continued exploration of mechanisms of action of Inhibigen-specific responses may reveal new paradigms of cancer immune evasion. 1H Lam et al, Cancer Discov 2021;11:1-18 Citation Format: Hanna S. Starobinets, Victoria L. DeVault, Zoe C. Schmiechen, Ebony A. Miller, Eduardo Cruz, Meagan R. Rollins, Adam L. Burrack, Stephanie J. Rinaldi, Julie Arnold, Emily Tjon, Kyle Gonzalez, Dimitry Lineker, Hubert Lam, Ingunn M. Stromnes, Jessica B. Flechtner. ATLAS-identified Inhibigen-specific responses accelerate tumor growth in mouse melanoma and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2088.

BackgroundGEN-009, a personalized vaccine candidate comprised of ATLAS™-prioritized neoantigens combined with Hiltonol®, is currently being evaluated in a Phase 1/2a clinical trial (NCT03633110). ATLAS™ is a cell-based recall assay that, without predictions, screens each patient‘s mutanome to identify neoantigens for vaccine inclusion and deleterious Inhibigens™ for exclusion. In the Part A monotherapy cohort, vaccine-specific immune responses were generated in all subjects, against 99% of administered peptides.1 Here we characterize immune responses and their association with reduction in tumors in Part B of the study, in which patients were treated with GEN-009 combined with anti-PD-1-based checkpoint inhibitors (CPI).MethodsFourteen adults with solid tumors were enrolled in the study. During the screening and manufacturing period, patients received standard of care anti-PD-1 CPI. Subsequently, patients were immunized with GEN-009 in combination with anti-PD-1. CPI refractory patients received salvage therapy prior to GEN-009. Peripheral blood mononuclear cells were collected at baseline, pre-vaccination (D1), as well as multiple days post first dose. The magnitude and durability of vaccine-induced immune responses were assessed by quantifying neoantigen-specific responses in fluorospot assays. Proliferation of neoantigen-specific T cells and T cell phenotypes were evaluated by flow cytometry. Circulating tumor DNA (ctDNA) levels were monitored pre- and post-GEN-009 dosing to assess its potential as a predictive biomarker.ResultsGEN-009 immunization induced neoantigen-specific T cell responses in all evaluable patients, with ex vivo responses emerging as early as 1 month and persisting up to 366 days in some subjects. Comparing RECIST responders (PR, CR) to non-responders (SD, PD), the median breadth of statistically positive responses to vaccine antigens at day 50 was greater in non-responders ex vivo (29 vs. 75%, respectively), however, by IVS assay the proportions inverted (83% vs. 38%). Longitudinal evaluation of neoantigen-specific responses revealed an association between the magnitude and kinetics of cytokine secretion and increased activated and proliferating Ki-67+ T cells and TEM cells in both T cell subsets. Quantification of ctDNA in a subset of patients supported the RECIST readouts in association with the enhanced neoantigen-specific T cell responses.ConclusionsVaccination with GEN-009 combined with anti-PD-1-based therapy induced early, durable, and neoantigen-specific CD4+ and CD8+ T cell responses with pronounced Ki-67+ and TEM cell populations. Overall, a greater breadth of response to vaccine neoantigens was associated with improved clinical benefit, which was further supported by ctDNA levels. These data support that GEN-009, in combination with checkpoint blockade, represents a unique approach to treat solid tumors.ReferencesLam H, et al. An empirical antigen selection method identifies neoantigens that either elicit broad anti-tumor response or drive tumor growth. Cancer Discovery 2021 March; 11(3):696–713.Ethics ApprovalETHICS STATEMENTThis study was approved by Western Institutional Review Board, approval number 1-1078861-1

BackgroundEffective immune checkpoint blockade (ICB) treatment is dependent on T-cell recognition of patient-specific mutations (neoantigens). Empirical identification of neoantigens ex vivo has revealed shortcomings of in silico predictions.1 To better understand the impact of ICB treatment on T cell responses and differences between in silico and in vitro methods, neoantigen-specific T cell responses were evaluated in patients with non-small cell lung cancer undergoing first-line therapy with pembrolizumab ± chemotherapy.MethodsTumor and whole blood samples were collected from 14 patients prior to and after immunotherapy; seven each in monotherapy and combination therapy cohorts. The ex vivo ATLAS™ platform was used to profile neoantigen-specific T-cell responses. Patient-specific tumor mutations identified by next-generation sequencing (NGS) were expressed individually as ATLAS clones, processed patient-specific autologous antigen presenting cells, and presented to their T cells in vitro. ATLAS-verified antigens were compared with epitope predictions made using algorithms.ResultsOn average, 150 (range 37–339) non-synonymous mutations were identified. Pre-treatment, ATLAS identified T cell responses to a median of 15% (9–25%) of mutations, with nearly equal proportions of neoantigens (8%, 5–15%) and Inhibigens™, targets of suppressive T cell responses (8%, 3–13%). The combination therapy cohort had more confirmed neoantigens (46, 20–103) than the monotherapy cohort (7, 6–79). After treatment, the median ratio of CD4:CD8 T cells doubled in the monotherapy but not combination cohort (1.2 to 2.4 v. 1.6 to 1.3). Upon non-specific stimulation, T cells from patients on combination therapy expanded poorly relative to monotherapy (24 v. 65-fold, p = 0.014); no significant differences were observed pre-treatment (22 v. 18-fold, p = 0.1578). Post-treatment, the median number of CD8 neoantigens increased in the combination therapy cohort (11 to 15) but in monotherapy were mostly unchanged (6 to 7). Across timepoints, 36% of ATLAS-identified responses overlapped. In silico analysis resulted in 1,895 predicted epitopes among 961 total mutations; among those, 30% were confirmed with ATLAS, although nearly half were Inhibigens, which could not be predicted. Moreover, 50% of confirmed neoantigens were missed by in silico prediction.ConclusionsMonotherapy and combination therapy had differential effects on CD4:CD8 T cell ratios and their non-specific expansion. A greater proportion of neoantigens was identified than previously reported in studies employing in silico predictions prior to empirical verification.2 Overlap between confirmed antigens and in silico prediction was observed, but in silico prediction continued to have a large false negative rate and could not characterize Inhibigens.AcknowledgementsWe would like to acknowledge and thank the patients and their families for participating in this study.ReferencesLam H, McNeil LK, Starobinets H, DeVault VL, Cohen RB, Twardowski P, Johnson ML, Gillison ML, Stein MN, Vaishampayan UN, DeCillis AP, Foti JJ, Vemulapalli V, Tjon E, Ferber K, DeOliveira DB, Broom W, Agnihotri P, Jaffee EM, Wong KK, Drake CG, Carroll PM, Davis TA, Flechtner JB. An empirical antigen selection method identifies neoantigens that either elicit broad antitumor T-cell responses or drive tumor growth. Cancer Discov 2021;11(3):696–713. doi: 10.1158/2159- 8290.CD-20-0377. Epub 2021 January 27. PMID: 33504579. Rosenberg SA. Immersion in the search for effective cancer immunotherapies. Mol Med 27,63(2021). https://doi.org/10.1186/s10020-021-00321-3

We have previously reported the molecular signature of murine pathogenic TH17 cells that induce experimental autoimmune encephalomyelitis (EAE) in animals. Here we show that human peripheral blood IFN-γ+IL-17+ (TH1/17) and IFN-γ−IL-17+ (TH17) CD4+ T cells display distinct transcriptional profiles in high-throughput transcription analyses. Compared to TH17 cells, TH1/17 cells have gene signatures with marked similarity to mouse pathogenic TH17 cells. Assessing 15 representative signature genes in patients with multiple sclerosis, we find that TH1/17 cells have elevated expression of CXCR3 and reduced expression of IFNG, CCL3, CLL4, GZMB, and IL10 compared to healthy controls. Moreover, higher expression of IL10 in TH17 cells is found in clinically stable vs. active patients. Our results define the molecular signature of human pro-inflammatory TH17 cells, which can be used to both identify pathogenic TH17 cells and to measure the effect of treatment on TH17 cells in human autoimmune diseases., CD4+ T cells secreting interleukin-17 (TH17) have diverse functions in modulating autoimmune diseases. Here the authors show via transcriptome analyses that a subset of human TH 17 co-expressing interferon-γ (TH1/17) has a molecular signature similar to “pathogenic” mouse TH 17 but distinct from “non-pathogenic” mouse TH 17.