Abstract TiTAN™ is an open-label, multi-center Phase1/2a trial evaluating safety, tolerability, T-cell persistence and proliferation, and clinical activity in patients with some solid tumors. Adoptive T-cell therapies (ACT) have resulted in durable clinical responses in some patients, but many are resistant. Resistance may be due to multiple factors including antigen heterogeneity or loss, immune editing, exhausted immune responses or naturally occurring immune suppressive T-cell responses. Tumors can also express Inhibigens™, antigenic targets of suppressive T cells, which may be inadvertently expanded in the non-specific ACT manufacturing process. In animal models, Inhibigen-specific responses drive tumor hyperprogression. GEN-011, a neoantigen-targeted, autologous peripheral T cell (NPT) therapy, contains tumor-specific T cells with broad neoantigen specificity. Patients undergo sequencing of tumor from fixed tissue and selection of neoantigens by ATLAS™, an ex vivo assay that directly identifies immunogenic neoantigens for use in manufacturing NPTs, and also Inhibigens for exclusion. The patient’s peripheral T cells and monocyte-derived dendritic cells are incorporated into the proprietary PLANET™ manufacturing process where they are specifically stimulated with up to 30 ATLAS-confirmed neoantigens in a scalable, closed system. The TiTAN clinical trial is testing a low dose regimen of GEN-011 without lymphodepletion and a single dose of GEN-011 with lymphodepletion and IL-2. To date, 19 patients with assorted solid tumors have been screened with ATLAS, prioritizing an average of 12 neoantigens (range 0-43) and excluding 14 Inhibigens (range 1-55) per patient. Of the 10 patients entering PLANET, 100% have successfully yielded a released drug product. To date, 5 patients with NSCLC or SCCHN have been dosed with escalating cell numbers and lymphodepletion/IL-2 regimens without DLT. Early data show effector and central memory T-cell proliferation by day 5 post infusion, which peak between days 8 and 15. Neoantigen-specific T cells remain detectable in the peripheral blood for at least 36 days. Early best response from 4 evaluable patients are one PD and one mixed response in the low dose cohort, and in the more intense regimen a SD with reduction in tumor with resolution of pain and neuropathy extending for 2 months, and the fourth had stable disease. Maximum grade 2 CRS and one grade 2 ICANS peaked around day 8 in parallel to cell expansion and no patients required tocilizumab or corticosteroids. Upcoming patients will receive more intensive lymphodepletion and then higher dose IL-2. Taken together, these early data support the biological activity of GEN-011. Using a personalized immune assay to identify neoantigens, and to exclude Inhibigens, to generate tumor specific T cells may offer a more accessible and promising ACT for treating solid tumors. Citation Format: Maura Gillison, Jiaxin Niu, Daniel Olson, Mark Stein, David Aggen, Utkarsh Acharya, Benjamin Creelan, Richard Hernandez, Jessica Price, Kevin J. Mancini, Louisa Dowal, James Foti, Vijetha Vemulapalli, Mara Shainheit, Masoud Golshadi, Raymond D. Stapleton, Jessica B. Flechtner, Thomas A. Davis. TiTAN: a phase 1 study of GEN-011, a neoantigen-targeted peripheral blood-derived T cell therapy with broad neoantigen targeting [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 CT153.

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.

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.

Abstract Candidate adoptive T cell therapies (ACT), such as tumor infiltrating lymphocyte (TIL) treatments, have resulted in unprecedented and durable efficacy in clinical trials. Despite this, the manufacturing requires viable tumor resection, and TIL expansion conditions have the potential to promote T cell exhaustion. Moreover, a large proportion of patients do not respond to treatment, possibly due to exhaustion or to bystander T cells that are not tumor-specific present in the product candidates. In addition, we have shown that naturally occurring pro-tumor T cell responses to tumor-specific antigens we term Inhibigens™ are generated in nearly every subject with cancer; these T cells may be inadvertently expanded in the non-specific TIL manufacturing process. In animal models, Inhibigen-specific responses drive tumor hyperprogression. To avoid these pro-tumor T cells and improve upon ACT limitations, we are developing GEN-011, a neoantigen-targeted, peripheral T cell (NPT) therapy. GEN-011 is designed to contain primarily tumor-specific T cells with broad specificity and limited exhaustion, starting from easily accessible peripheral blood. Putatively beneficial neoantigen targets and deleterious pro-tumor Inhibigen targets are identified through measurement of cytokines in the assay supernatants of an in vitro ATLAS™ screen, in which each mutation identified in a patient's tumor is screened with the patient's own peripheral CD4+ and CD8+ T cells in a recall (overnight) assay, without algorithm prioritization. Next, the patient's peripheral T cells and monocyte-derived dendritic cells are incorporated into the PLANET™ manufacturing process where they are specifically stimulated with up to 30 ATLAS-verified neoantigens, avoiding Inhibigens, in a scalable, closed system. Development and engineering runs performed at scale show that the NPTs are up to 96% tumor-specific, with responses maintained for up to 89% of the intended neoantigen targets. They are non-exhausted effector and central memory T cells that express both proliferative and tissue homing markers. In addition to being highly polyfunctional, secreting multiple combinations of IFNγ, Granzyme B, TNFα, and MIP1α in response to specific neoantigens, they are also cytolytic in vitro and express memory-progenitor stem-like cell markers. The TITAN™ clinical trial evaluating GEN-011 NPTs is ongoing (NCT04596033). TiTAN is an open-label, multi-center Phase1/2a trial evaluating safety, tolerability, T cell persistence and proliferation, and clinical efficacy. The TiTAN clinical trial is testing two dosing regimens, a repeated lower dose regimen of GEN-011 without lymphodepletion and a single high dose administration of GEN-011 NPTs after lymphodepletion. Both groups will receive interleukin-2 after GEN-011 NPT dosing. By enriching healthy, broadly-specific neoantigen-targeted T cells and avoiding Inhibigens, the GEN-011 NPTs may represent an accessible and promising ACT for treating solid tumors. Citation Format: James Perry, Pranay D. Khare, Mercay Reuter, Daniel B. DeOliveira, Manish Jain, Colleen Winstead, Hubert Lam, Thomas Davis, Ray Stapleton, Jessica B. Flechtner. GEN-011: A neoantigen-targeted peripheral blood-derived T cell therapy that has broad neoantigen specificity and high T cell purity while avoiding pro-tumor T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P005.

Background: Neoantigen (neoAg)-specific T cells can be found in the peripheral blood of patients with solid tumors, and the infiltration of fresh, peripherally derived T cells into tumors has been associated with successful outcomes after checkpoint blockade therapy. We previously described the development of our PLANET™ manufacturing process to create NPTs, based on the empirical identification of neoAgs using the ex vivo ATLAS™ bioassay, for adoptive transfer into patients with cancer. We have initiated the TiTAN™ clinical trial (NCT04596033) to evaluate this candidate immunotherapy, GEN-011, in subjects with checkpoint refractory solid tumors. Here we report the successful manufacturing of NPTs to support the ongoing trial. Methods: Apheresis, FFPE tumor and saliva samples were procured from subjects who consented to participate in the trial. Monocytes and T cells were isolated and cryopreserved. Patient-specific neoAgs against which their T cells were responsive were identified with ATLAS, and up to 30 neoAgs were prioritized for manufacture; pro-tumor Inhibigens™ were excluded. Cryopreserved peripheral blood monocytes and T cells were thawed, monocytes derived into dendritic cells, and then neoAg-specific T cells expanded in the fully closed PLANET manufacturing process. The NPTs were formulated and cryopreserved for administration to patients after release testing. Results: Subjects with five different solid tumor types contributed to these data: CSC, NSCLC, SCLC, SCCHN, and melanoma. A median of 2.1bn monocytes and 6bn T cells were cryopreserved from apheresis products (N=17). The median TMB was 1.8mut/Mb (range 0.01-36.6) with 473 somatic mutations (range 32-8893); the number of non-synonymous mutations ranged from 9 to 767. ATLAS screens identified a mean of 13±4 neoAgs and 11±3 Inhibigens, resulting in an average of 13 (range 2-30) unique neoAgs in each PLANET manufacturing process. Upon conclusion of manufacturing, the mean yield per patient was 1.6±0.3bn NPTs across runs (N=9) completed by the time of data cutoff. Characterization tests revealed the NPTs were consistently 80-90% CD8+ and 10-20% CD4+ T cells, of which 97% (range 64.9-99.8%) were effector memory and 1% (range 0.1-32.3%) central memory. The median purity at release was 98% with 82% viability. By functional assessments, the NPTs retained specificity for 91% (range 82-100%) of their intended neoAg targets (N=4). Administratable doses were successfully manufactured for 100% of patients to date. Conclusions: NPTs can routinely be manufactured in a GMP setting to treat patients with solid tumors. By expanding fresh, non-exhausted NeoAg-specific T cells with known tumor specificity from the periphery, GEN-011 has the potential to provide clinical benefits of TIL with greater accessibility and minimal irrelevant T cells. The TiTAN trial is ongoing. Citation Format: Harshal Zope, Rounak Nande, Manish Jain, Charley Hubbard, Louisa Dowal, James Foti, James Loizeaux, Crystal Cabral, Daniel B. DeOliveira, Guohan Yang, Mercay Reuter, Jessica Baker Flechtner, Raymond Stapleton. The PLANET manufacturing process reproducibly generates high-quality neoantigen-targeted peripheral T cells (NPTs) for adoptive T cell therapy in the TiTAN clinical trial [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 2745.

BackgroundSelecting neoantigens that generate robust anti-tumor T cell responses remains a challenge for cancer immunotherapy design. The ATLASTM platform, a functional recall assay using patient autologous cells, identifies both stimulatory and inhibitory (Inhibigen) neoantigens via up- or downregulation of T cell cytokine secretion.1 We propose that stimulatory neoantigens are ideal targets for cancer vaccines and T cell therapies. In contrast, data suggest that Inhibigens be excluded, due to their association with accelerated tumor growth and dampened immunity in a murine melanoma model.2 While detrimental to cancer immunotherapy, the Inhibigen-associated downregulation of cytokine production may be beneficial in the context of autoimmunity.MethodsATLAS screens were performed as previously described.1 2 Peptide vaccines containing tumor-specific neoantigens ± Inhibigens were evaluated in prophylactic and therapeutic B16F10 melanoma tumor models for immunogenicity and efficacy. RNAseq analysis was performed on T cells sorted from draining lymph nodes of vaccinated tumor-bearing mice. For experimental autoimmune encephalomyelitis (EAE) studies, mice were administered a vaccine containing MOG peptide ± the melanoma MMP9FS Inhibigen. Immune responses and phenotypic analyses for both models were measured by flow cytometry, ELISPOT, and immunohistochemistry.ResultsIn the melanoma model, inclusion of the Inhibigen MMP9FS accelerated tumor growth in a non-dose dependent manner and abrogated immune responses. RNAseq of T cells from tumor-bearing mice vaccinated with MMP9FS showed a higher level of differentially expressed genes (adjusted P value of <0.05) in TCR-signaling regulation and suppressor GO pathways (>5 distinct pathways/gene) as compared to stimulatory controls, indicating Inhibigen-specific effects on T cells. In the EAE model of autoimmunity, animals treated with MOG peptide + MMP9FS exhibited dampened anti-MOG immune responses, delayed disease onset, reduced disease incidence and scoring (average 1 vs. 3) and decreased spinal cord immune infiltration as compared to control vaccination. These data indicate that Inhibigen administration has the potential to ameliorate autoimmune sequelae, independent of cognate antigen expression.ConclusionsFunctional identification and exclusion of Inhibigens from cancer immunotherapies may be critical to protective immunity since their inclusion can result in quelling of otherwise beneficial immune responses. Conversely, Inhibigen-specific responses can dampen destructive autoimmune sequelae. Mechanistic studies show altered T cell signaling pathways in the context of therapeutic Inhibigen vaccination. These data suggest that Inhibigen-specific responses, while detrimental for the treatment of cancer, may have a therapeutic benefit in other disease contexts.ReferencesNogueira C, Kaufmann JK, Lam H, Flechtner JB. Improving cancer immunotherapies through empirical neoantigen selection. Trends in Cancer 2018;4(2):97–100.Lam H, et al. An empirical antigen selection method identifies neoantigens that either elicit broad anti-tumor response or drive tumor growth. Cancer Discovery 2021;11(3):696–713.Ethics ApprovalAll animal studies were undertaken in conformity with the Cambridge, MA Ordinance 1086 of the city's Municipal Code and in accordance with the policies and protocols approved by Genocea's Institutional Animal Care and Use Committee (IACUC).

Abstract The current study explores the mechanism of Inhibigens: a new class of immune-dampening cancer neoantigens. Inhibigens are identified through Genocea's ATLASTM platform, an empirical and unbiased bioassay that utilizes autologous patient cells to characterize tumor mutations that elicit anti-tumor CD4+ and CD8+ immune responses. Unexpectedly, ATLAS also identifies inhibitory neoantigens (Inhibigens) that lead to downregulation of T cell inflammatory cytokine secretion. Previous studies demonstrated that when a single ATLAS-identified Inhibigen was administered to B16F10 tumor-bearing mice, it completely abrogated immunogenicity and efficacy of a protective vaccine. Thus, it is critical that detrimental Inhibigens be excluded from targeted immunotherapies and further studied to understand their global importance in cancer. Here we report progress in uncovering mechanisms of Inhibigen function. In ATLAS screening assays, patient-derived antigen-presenting cells (APCs) are fed with E. coli bacteria expressing individual mutations from the patient's mutanome with or without pore-forming listeriolysin O (cLLO), and subsequently exposed to patient-derived CD8+ or CD4+ T cells, respectively; T cell cytokine secretion determines antigen-specific responses. Unlike in silico approaches, ATLAS accounts for the complexity of antigen processing, the diversity of MHC class I and II alleles across races and ethnicities, and the diversity of T cell repertoires across individuals. ATLAS screening of B16F10 mouse melanoma previously identified stimulatory neoantigens and Inhibigens that were either protective or deleterious to tumor progression when included in vaccines, respectively. Current studies explore the tumor microenvironment (TME) and systemic immunity in Inhibigen-administered mice ± checkpoint inhibitors (CPI). Inhibigens accelerated tumor growth compared to protective vaccines, abrogated vaccine-driven infiltration of T cells and myeloid cells into the TME, and impaired the synergistic effects of vaccination and CPI therapy with corresponding deficiencies in the TME. In vitro studies thus far exclude cell killing and MHC competition as Inhibigen mechanisms of action. Ongoing studies evaluating Inhibigen modulation of APC and T cell function in vitro and in vivo will be presented. Our data enforce the necessity of identifying Inhibigens empirically and excluding them from cancer vaccines and immunotherapies. Genocea's GEN-009 and GEN-011 phase 1/2 clinical trials utilize ATLAS to identify optimal neoantigens and omit Inhibigens from cancer vaccines and T cell therapies. Ongoing exploration of Inhibigen phenotypes and mechanisms will illuminate new paradigms of cancer immunology and potentially pave the way for novel cancer immunotherapies. Citation Format: Hanna Starobinets, Victoria L. Devault, Stephanie Rinaldi, Julie Arnold, Osaruese Odeh, Cindy Nguyen, Jessica B. Flechtner, Hubert Lam. InhibigensTM subvert otherwise-efficacious cancer vaccines and immunotherapies in conjunction with alterations in the tumor microenvironment [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 1762.

Background GEN-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). Methods Fourteen 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. Results GEN-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. Conclusions Vaccination 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. References Lam 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 Approval ETHICS STATEMENT This study was approved by Western Institutional Review Board, approval number 1-1078861-1

Adoptive T cell therapy using ex vivo expanded autologous tumor-infiltrating lymphocytes (TIL) has resulted in notable tumor regression across multiple solid tumor indications. Despite their success, TIL harvest requires invasive surgery and sterile isolation. In addition, extracted cells from an immunosuppressive tumor microenvironment (TME) often have impaired functions, low neoantigen-specificity and require high dose cytokine for expansion. All possible reasons for non-responders or post-treatment relapse. Generating T cell therapies specifically expanded against neoantigen targets and derived from peripheral blood lymphocytes may overcome these limitations. The ATLAS bioassay identifies patient-specific neoantigen targets of T cells, and Inhibigens™, tumor antigens that are detrimental to protective immune responses. Using PLANET™, a robust and scalable closed manufacturing process, T cells specific for up to 30 ATLAS-identified neoantigens (excluding Inhibigens) are expanded, providing considerable breadth of tumor recognition and the potential to limit tumor escape. Use of peripheral T cells leverages robust cells not suppressed by the TME and eliminates the need for invasive tumor resection. Our data show that NPTs are specific for up to 89% of targeted neoantigens, consist of non-exhausted effector and central memory cells, and express proliferative and tissue homing markers. NPTs are highly polyfunctional, secreting multiple combinations of IFNγ, Granzyme B, TNFα, and MIP1α in response to specific neoantigens. Evaluation of markers for memory-progenitor stem-like features in NPTs are being explored and will be discussed. The TITAN™ clinical trial evaluating GEN-011 NPTs is ongoing (NCT04596033).

Background Effective 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. Methods Tumor 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. Results On 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. Conclusions Monotherapy 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. Acknowledgements We would like to acknowledge and thank the patients and their families for participating in this study. References Lam 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

The ATLAS™ platform screens a patient’s tumor mutanome with autologous T cells and antigen presenting cells to identify targets for cancer immunotherapy. Tumor mutations that lead to increased T cell responses are deemed stimulatory (neoantigens), while those rendering decreased responses are defined as Inhibigens. In B16F10 mouse melanoma, therapeutic neoantigen vaccination resulted in durable protection, while Inhibigen co-administration resulted in a remarkable reversal of protection and abrogation of anti-tumor responses. The Inhibigen-related pro-tumor effects were not reduced by αPD-1 therapy, and administration of αCTLA-4 resulted in modest benefit, suggesting that Inhibigen-specific responses can overpower current standard-of-care immunotherapies. The tumor microenvironment of Inhibigen-vaccinated mice was immune cold, with dramatic decreases in infiltrating T cells and myeloid cells relative to controls. Inhibigen effects were not associated with classical inhibitory mechanisms such as an overabundance of CD4+CD25+Foxp3+Tregs, MHC competition or downregulation. In addition, T cell proliferation appeared unaffected by Inhibigen administration. Time course experiments revealed that Inhibigen-specific phenotypes (e.g. defective T cell IFNγ production) occur as early as 4 days post vaccination, suggesting deficiencies related to T cell priming. RNAseq analyses showed distinct changes in T cell transcriptional patterns between neoantigen and Inhibigen-experienced T cells. Taken together, these data reveal a potential non-classical inhibitory mechanism by which responses to naturally occurring cancer mutations may promote tumor growth and reverse beneficial anti-tumor immune responses.

2613 Background: GEN-009 is an adjuvanted personalized cancer vaccine containing up to 20 neoantigens selected by ATLAS, an ex vivo bioassay screening autologous T cells for immune responses against both neoantigens as well as Inhibigens. Inhibigen-specific T cells suppress immunity and have been shown to accelerate tumor progression in mice and are avoided in GEN-009. In cohort A, all patients immunized in the adjuvant setting with GEN-009 monotherapy developed immune responses. Nearly all (99%) of selected peptides were immunogenic: ex vivo CD4+ and CD8+ fluorospot responses specific for 51% and 41% of immunized peptides, respectively. Seven of 8 patients continue without progression with a median follow up of 18 months. Methods: GEN-009 is being evaluated in patients (pts) 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 a PD-1 CPI. Patients who progressed prior to vaccination received alternative salvage therapy followed by GEN-009 in combination. Peripheral T cell responses were measured by fluorospot assays in ex vivo and in vitro stimulation. Results: 15 pts received GEN-009 in combination with a PD-1 inhibitor; 1 patient received GEN-009 monotherapy. Median number of neoantigens per vaccine was 14 (5-18). GEN-009-related adverse events were limited to vaccine injection site reactions and mild myalgias or fatigue. Longitudinal evaluation of ex vivo T cell responses revealed that sequential vaccination with GEN-009 had an overall additive effect on the robustness of IFNγ secretion and responses were persistent for at least 6 months in some patients. Epitope spread was detected in CPI sensitive patients, but not in CPI refractory patients receiving salvage therapy. Three patients who responded to PD-1 inhibition followed by disease stabilization then demonstrated further reduction after GEN-009 vaccination that could represent vaccine effect. Eight of 9 CPI responsive patients are progression-free from 3 to 10 months after first vaccine dose. Four of 7 CPI refractory patients have experienced unexpected prolonged stable disease after vaccination of up to 8 months after vaccination. 2 of 2 patients with available samples lost all evidence of circulating tumor DNA including non-targeted neoantigens. Conclusions: Vaccination with GEN-009 in combination with anti-PD-1 CPI in patients with advanced solid tumors shows little additive toxicity. Preliminary data demonstrate induction of broad 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. Clinical trial information: NCT03633110.

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

3107 Background: Tumor-specific neoantigens provide personalized targets for immunotherapy. Vaccines against epitopes predicted by in silico approaches very rarely induce CD4+ and CD8+ ex vivo T cell responses regardless of formulation. ATLAS selects neoantigens for vaccine inclusion using ex vivo screening of all patient-specific mutations to identify pre-existing CD4+ or CD8+ T cell responses and to exclude Inhibigens, which are inhibitory peptides that suppress immunity and accelerate tumor progression. The Inhibigen burden correlates with patient outcomes in observational studies and rapid tumor progression in mouse models. Methods: GEN-009-101 is a phase 1/2a study testing safety, immunogenicity and clinical activity in immune responsive tumors. After next-generation tumor sequencing and ATLAS testing of autologous leukocytes, up to 20 stimulatory synthetic long peptides adjuvanted with poly-ICLC comprise each personalized vaccine. Eight vaccinated patients have been followed for sustained immunological responses and clinical outcomes. Results: The 40 doses given across patients have induced only mild local discomfort and no DLT. Vaccination has generated immune responses against 99% of administered peptides, with both CD8+ and CD4+ responses in ex vivo fluorospot assays. To date, no patients have developed recurrent disease. Broad immunity develops as early as Day 29 and is sustained for over 12 months. Immune response against individual peptides is correlated with peptide concentration (OR = 1.26, p≤0.0001) but not with other classifiers such as GRAVY index (Grand Average of Hydropathy), tumor type, injection site or sex. The Inhibigen burden prior to treatment again correlates with disease progression. Conclusions: GEN-009 identifies tumor specific immune targets from the individual patient’s tumor mutagens. Initial clinical data show that ATLAS antigen selection may be critical to the induction of broad, rapid and sustained immunity against tumor specific neoantigens. Clinical vaccination with PD-1 blockade is in process. Clinical trial information: NCT03633110 . [Table: see text]

Background GEN-009 is an adjuvanted personalized cancer vaccine containing up to 20 neoantigens selected by ATLAS™, an ex vivo bioassay screening autologous T cells to identify both neoantigens as well as Inhibigens™ empirically and without in silico predictions. Inhibigen-specific T cells suppress immunity and have been shown to accelerate tumor progression in mice. Inhibigens are avoided in GEN-009. Previous data from patients treated with GEN-009 monotherapy showed 99% of selected peptides generated immune responses including ex vivo CD4+ and CD8+ fluorospot responses specific for 51% and 41% of immunized peptides respectively. Methods GEN-009 is being evaluated in patients (pts) with advanced cancer who received standard-of-care (SOC) PD-1 inhibitor as monotherapy or in combination therapy during vaccine manufacturing; they subsequently received 5 vaccine doses over 24 weeks in combination with the PD-1 inhibitor. Patients who progressed prior to vaccination could receive alternate therapy followed by GEN-009 combined with an appropriate salvage regimen. Peripheral T cell responses were evaluated pre-and post-vaccination by dual-analyte fluorospot assays measured both directly ex vivo and after in vitro stimulation. Results As of August 18, 2020, 15 pts received GEN-009 in combination with a PD-1 inhibitor. Their median TMB was 1.37Mut/mb (range 0.31–6.55), with a median of 24 (6–99) neoantigens and 16 (1–86) Inhibigens. The number of neoantigens in each manufactured vaccine ranged from 4–18 (median 13). GEN-009-related adverse events were limited to Grade 1 injection site reactions. Ex vivo T cell responses peaked after the third vaccination for IFNγ and some patients showed evidence of epitope spread. The initial 5 patients are evaluable for antitumor activity with at least 3 months follow up after first vaccination. Three patients experienced early tumor responses followed by stabilization on PD-1 inhibitor SOC and demonstrated a further reduction in tumor volume after GEN-009 vaccination (figure 1). One patient experienced a complete response prior to vaccination and the 5th patient had progression on SOC, but had a Partial Response to salvage and remains stable after vaccination. Conclusions Vaccination with GEN-009 in combination with PD-1 CPI is feasible for patients with advanced solid tumors with little additive toxicity. Preliminary data demonstrate induction of robust, neoantigen-specific immune responses and a potential expansion of stimulatory targets with epitope spreading in the presence of PD-1 inhibitor. Possible additive antitumor activity in combination with PD-1 inhibitors is suggested by tumor shrinkage following GEN-009 dosing. More mature response and immunogenicity data on 10 additional patients is anticipated for November. Trial Registration ClinicalTrials. gov NCT03633110 Ethics Approval The study was approved by Western Institutional Review Board, approval number 1-1078861-1.

Background ATLASTM is a cell-based bioassay that utilizes a cancer patient‘s own monocyte-derived dendritic cells and CD4+ and CD8+ T cells to screen their mutanome and identify neoantigens that elicit robust anti-tumor T cell responses, as well as, deleterious InhibigensTM.1 GEN-009, a personalized vaccine comprised of 4–20 ATLAS-identified neoantigens combined with Hiltonol®, harnesses the power of neoantigen-specific T cells to treat individuals with solid tumors. The safety and efficacy of GEN-009 is being assessed in a phase 1/2a clinical trial (NCT03633110). Methods A cohort of 15 adults with solid tumors were enrolled in the study. During the screening period, patients received standard of care PD-1-based immunotherapies appropriate for their tumor type. Subsequently, patients were immunized with GEN-009 with additional doses administered at 3, 6, 12, and 24 weeks. Peripheral blood mononuclear cells (PBMCs) were collected at baseline, pre-vaccination (D1), as well as 29, 50, 92, and 176 days post first dose. Vaccine-induced immunogenicity and persistence were assessed by quantifying neoantigen-specific T cell responses in ex vivo and in vitro stimulation dual-analyte fluorospot assays. Polyfunctionality of neoantigen-specific T cells was evaluated by intracellular cytokine staining. Additionally, potential correlations between the ATLAS-identified profile and vaccine-induced immunogenicity were assessed. Results GEN-009 augmented T cell responses in 100% of evaluated patients, attributable to vaccine and not checkpoint blockade. Furthermore, neoantigen-induced secretion of IFNγ and/or TNFα by PBMCs, CD4+, and CD8+ T cells was observed in all patients. Responses were primarily from polyfunctional TEM cells and detectable in both CD4+ and CD8+ T cell subsets. Some patients had evidence of epitope spreading. Unique response patterns were observed for each patient with no apparent relationship between tumor types and time to emergence, magnitude or persistence of response. Ex vivo vaccine-induced immune responses were observed as early as 1 month, and in some cases, persisted for 176 days. Clinical efficacy possibly attributable to GEN-009 was observed in several patients, but no correlation has yet been identified with neoantigen number or magnitude of immune response. Conclusions ATLAS empirically identifies stimulatory neoantigens using the patient‘s own immune cells. GEN-009, which is comprised of personalized, ATLAS-identified neoantigens, elicits early, long-lasting and polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses in individuals with advanced cancer. Several patients achieved clinical responses that were possibly attributable to vaccine; efforts are underway to explore T cell correlates of protection. These data support that GEN-009, in combination with checkpoint blockade, represents a unique approach to treat solid tumors. Acknowledgements We are grateful to the patients and their families who consented to participate in the GEN-009-101 clinical trial. Trial Registration NCT03633110 Ethics Approval This study was approved by Western Institutional Review Board, approval number 1-1078861-1. All subjects contributing samples provided signed individual informed consent. Reference DeVault V, Starobinets H, Adhikari S, Singh S, Rinaldi S, Classon B, Flechtner J, Lam H. Inhibigens, personal neoantigens that drive suppressive T cell responses, abrogate protection of therapeutic anti-tumor vaccines. J. Immunol 2020; 204(1 Supplement):91.15.

Identification of neoantigens that can elicit strong anti-tumor responses has become critical to vaccine design for cancer immunotherapy. Conventional methods for in silico neoantigen identification have yielded poor predictive value, highlighting the need for methods that identify bona fide neoantigen targets. The Genocea ATLAS™ platform uses autologous antigen presenting cells and T cells to identify pre-existing CD4+ and/or CD8+ T cell responses to patient-specific mutations and therefore selects confirmed neoantigens that can be used for vaccines and cell therapies. Screened neoantigens are characterized as stimulatory or inhibitory based on up- or downregulation of inflammatory cytokine secretion compared to baseline controls. In the mouse B16F10 melanoma model, therapeutic immunization with stimulatory neoantigen peptides arrested tumor growth whereas in contrast, inhibitory antigen immunization (henceforth referred to as inhibigens) resulted in accelerated tumor progression. The presence of an inhibigen in an otherwise protective vaccine formulation completely abolished protection. Global IFNγ responses to neoantigens were abrogated in these mice as measured by ELISpot suggesting that inhibigen pro-tumor responses can be immunodominant. Analysis of tumor-infiltrating lymphocytes (TILs) from mice immunized with ATLAS-identified neoantigen ± inhibigen peptide vaccines revealed significant alterations in the tumor microenvironment. Inclusion of inhibigens resulted in low T cell infiltration into tumors and increased expression of TIL inhibitory surface markers (e.g. PD-1, LAG-3). Immunological mechanisms of the inhibitory phenomenon are currently being explored. These data promote rational methods for neoantigen identification and highlight the potential advantages of excluding deleterious inhibigens from cancer vaccines and immunotherapies. GEN-009, a personalized cancer vaccine filtered for inclusion of only ATLAS-identified neoantigens (excluding inhibigens) is currently being evaluated in a Phase 1/2a clinical trial (NCT03633110). Citation Format: Victoria L. DeVault, Hanna Starobinets, Sanmit Adhikari, Simran Singh, Stephanie Rinaldi, Brendan Classon, Jessica B. Flechtner, Hubert Lam. Inclusion of inhibitory neoantigens can abolish efficacy of otherwise protective therapeutic anti-tumor vaccines [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 6680.

Background Personalized cancer immunotherapies can generate potent antitumor responses yet finding the right targets remains challenging. The ATLASTM platform employs ex vivo functional screening of tumor mutations using autologous cells to identify patient-specific neoantigens. Stimulatory neoantigens are identified by upregulation of inflammatory cytokine secretion and can be employed in vaccines or cell therapies. Conversely, ATLAS also identifies inhibitory neoantigens (termed Inhibigens) that lead to cytokine downregulation, and in murine models accelerate tumor growth and abrogate the efficacy of otherwise-protective vaccines. Here we further explore Inhibigen mechanism of action in humans and mice including whether checkpoint inhibition (CPI) can ameliorate Inhibigen-accelerated tumor growth. Methods Human and mouse ATLAS screens were performed as previously described.1 ATLAS-identified stimulatory or Inhibigen peptide vaccines were evaluated in a therapeutic B16F10 melanoma tumor model ± CPI. Immune responses were measured using ELISPOT, flow cytometry, and immunohistochemistry (IHC). Results In the GEN-009 personalized neoantigen vaccine trial (NCT03633110), Inhibigens were observed in 92% of patients (N=39). Of total mutations screened, 16% (1.8 - 47.5%) were classified as Inhibigens, which were found more often in the CD4 + (mean 10.3%; 0.5 - 42%) versus CD8 + T cell subset (mean 6.1%; 1.2–23%). No relationship between Inhibigen-specific responses and tumor type or mutational burden were observed. To study the functional effects of Inhibigen vaccination in vivo, a B16F10 mouse melanoma model was employed. Inclusion of Inhibigens in an otherwise protective vaccine abrogated efficacy and correlated with decreased T cell responses to vaccine antigens as well as a global depression of T cell cytokine secretion. Early experiments suggest that these decreases are not due to MHC competition. In addition, administration of a therapeutic vaccine containing an Inhibigen led to reduced tumor infiltration of CD8 + T cells and myeloid populations. A corresponding increase of classical Tregs in the tumor or periphery was not observed. Surprisingly, preliminary data show combination therapy with anti-CTLA4 partially ameliorated Inhibigen-accelerated tumor growth but anti-PD1 provided no additional benefit. Conclusions The nearly ubiquitous presence of Inhibigens in human cancer patients and the demonstrated pro-tumor effects in mice suggest that ATLAS-identified Inhibigens must be considered and omitted in the design of cancer immunotherapies. Furthermore, in mice, CPI co-administration has a modest (anti-CTLA4) or no (anti-PD1) effect on Inhibigen-accelerated tumor growth suggesting that Inhibigen profiling could guide CPI selection or predict clinical outcome. These data confirm the benefits of the ATLAS platform for neoantigen and Inhibigen identification. Ethics Approval All animal studies were undertaken in conformity with the Cambridge, MA City Ordinance 1086 of the city’s Municipal Code and in accordance with the policies and protocols approved by Genocea’s Institutional Animal Care and Use Committee (IACUC). Reference Nogueira C, Kaufmann JK, Lam H, Flechtner JB. Improving cancer immunotherapies through empirical neoantigen selection. Trends in Cancer 2018 Feb;4(2):97–100

Therapeutic cancer vaccine efficacy is reliant on tumor-specific neoantigens. Empirical methods to identify relevant neoantigens may overcome poor predictive values of current in silico approaches. The ATLAS™ platform is a personalized bioassay that uses high-throughput screening of autologous APCs and T cells against a patient’s mutanome to identify neoantigens that are characterized as stimulatory or inhibitory (inhibigens) based on changes in T cell cytokine secretion. In a B16F10 murine model, therapeutic vaccination with ATLAS-identified neoantigens resulted in arrested tumor growth with durable immune responses that protected mice from re-challenge. Mice that were vaccinated with an inhibigen and neoantigen combination had significantly abrogated tumor protection relative to controls. IFNγ ELISpot analysis revealed that inhibigen vaccination suppressed all antigen-specific immune responses, suggesting that inhibigens can be immunodominant. Inhibigen vaccinated mice had multiple tumor microenvironment changes including reduced T cell infiltration, altered CD8+/CD4+ T cell ratios and increased inhibitory marker expression relative to controls. These results indicate that the presence of inhibigens in an otherwise protective vaccine can alter the tumor microenvironment and abolish T cell-mediated protection. Epitope mapping of neoantigens and inhibigens and tetramer design for differential gene signature analysis of inhibigen-specific T cells is being performed. These data suggest that identification and exclusion of inhibigens from cancer vaccines is critical to prevent unintended pro-tumor responses.