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.

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.