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).

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

Background In recent years, the FDA has approved engineered autologous T cell therapies with remarkable efficacy against hematological cancers. In addition, non-engineered tumor infiltrating lymphocyte (TIL) therapies have shown unprecedented benefit against solid tumors in early clinical trials. Despite their success, TIL products have limitations including the need for specialized surgery to obtain sterile tumor for T cells, low neoantigen breadth, and the potential for T cells that may be pro-tumor, exhausted, or not tumor-specific. These limitations may hinder efficacy and accessibility for certain patients. We have developed an autologous, peripheral blood-derived non-engineered T cell therapy, GEN-011, that embraces the advantages of TIL while improving on their limitations by targeting true tumor-specific neoantigens identified by the ATLASTM bioassay and avoiding potentially pro-tumor InhibigensTM.1 Methods Peripheral blood mononuclear cells and a tumor biopsy are collected from each subject; tumor DNA is sequenced by WES. The ATLAS bioassay is used to individually screen each tumor mutation with the patient‘s own T cells to identify neoantigen targets of pre-existing CD4+ and/or CD8+ T cell responses. The robust clinical scale manufacturing process, PLANETTM, expands the patient‘s peripheral blood T cells on ATLAS-identified stimulatory neoantigens. Results The PLANET process produces GEN-011 drug products (DP) containing billions of antigen-specific, cytolytic T cells. Development and engineering runs using peripheral blood T cells from cancer patients and healthy donors resulted in DPs containing >97% T cells, >90% of which were central and effector memory phenotypes. A median 534-fold increase in antigen-specific T cells was observed in GEN-011 DPs over their starting frequency in peripheral blood with up to 67% of cells upregulating activation markers upon antigen recognition. Additionally, DP T cells secrete up to 50,000 pg/mL of IFN-gamma in response to antigen stimulation. In cancer patient samples, DPs respond to up to 89% of intended neoantigen targets compared to <10% reported recently for TIL products.2 3 Conclusions GEN-011 is an autologous, neoantigen-specific T cell product, with key advantages over TIL therapy. First, GEN-011 has an unparalleled breadth of neoantigen coverage, targeting up to 30 relevant neoantigens with non-exhausted CD4+ and CD8+ memory T cells to overcome non-tumor specific ‘passenger’ T cells. Second, GEN-011 avoids pro-tumor Inhibigens that may be detrimental to clinical responses. Third, GEN-011 does not require extra surgery or viable tumor for manufacturing. In conclusion, GEN-011 is a first-in-class transformational T cell therapy candidate with characteristics that should improve accessibility and efficacy for patients with solid tumors. Ethics Approval Informed consent was obtained from all individuals providing samples for this study. References 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. Fraser H, Pike R, Thirkell S, Arshad A, Jide-Banwo S, Bartley H, Rologi E, Pruchniak M, Patel S, Mootien J, Robertson J, Craig A, Salm M, Newton K, Goodsell L, Chan F, Wilson G, Frenk S, Ali I, Peggs K, Lowdell MW, Del Rosio L, Hayes A, Turajlic S, Islam F, Lawrence D, Jamal-Hanjani M, Forster MD, Samuel E. The development of a personalized autologous clonal neoantigen T cell therapy for the treatment of solid cancer using the VELOS™ manufacturing platform generates highly potent and reactive CD8+ and CD4+ T cells for clinical use [abstract]. Virtual Annual Meeting II of the American Association for Cancer Research; 2020 Jun 22–24. Creelan B, Wang C, Teer J, Toloza E, Mullinax J, Yao J, Koomen J, Kim S, Chiappori A, Saller J, Tanvetyanon T, Landin AM, Fang B, Yu X, Saltos A, Thompson Z, Noyes D, Conejo-Garcia J, Chen T-D, Haura E, Antonia S. Durable complete responses to adoptive cell transfer using tumor infiltrating lymphocytes (TIL) in non-small cell lung cancer (NSCLC): a phase I trial [abstract]. Virtual Annual Meeting II of the American Association for Cancer Research; 2020 Jun 22–24.