IntroductionCombining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically “cold” tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically “cold” tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically “cold” tumor models.MethodsMice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment.ResultsAn in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the “cold” B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen.ConclusionRT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically “cold”, solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

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Antibodies can play an important role in innate and adaptive immune responses against cancer, and in preventing infectious disease. Using a high-density whole-proteome peptide array, we assessed potential protein-targets for antibodies found in sera of immune mice that were previously cured of their melanoma through a combined immunotherapy regimen with long-term memory. Using flow cytometry, immune sera showed strong antibody-binding against melanoma tumor cell lines. Sera from 6 of these cured mice were analyzed with this high-density, whole-proteome peptide array to determine specific antibody-binding sites and their linear peptide sequence. We identified thousands of peptides that were targeted by 2 or more of these 6 mice and exhibited strong antibody binding only by immune, not naive sera. Confirmatory studies were done to validate these results using 2 separate ELISA-based systems. To the best of our knowledge, this is the first study of the "immunome" of protein-based epitopes that are recognized by immune sera from mice cured of cancer via immunotherapy.

Background The in-situ vaccine using CpG oligodeoxynucleotide combined with OX40 agonist antibody (CpG + OX40) has been shown to be an effective therapy activating an anti-tumor T cell response in certain settings. The roles of tumor volume, tumor model, and the addition of checkpoint blockade in the efficacy of CpG + OX40 in-situ vaccination remains unknown. Methods Mice bearing flank tumors (B78 melanoma or A20 lymphoma) were treated with combinations of CpG, OX40, and anti-CTLA-4. Tumor growth and survival were monitored. In vivo T cell depletion, tumor cell phenotype, and tumor infiltrating lymphocyte (TIL) studies were performed. Tumor cell sensitivity to CpG and macrophages were evaluated in vitro. Results As tumor volumes increased in the B78 (one-tumor) and A20 (one-tumor or two-tumor) models, the anti-tumor efficacy of the in-situ vaccine decreased. In vitro, CpG had a direct effect on A20 proliferation and phenotype and an indirect effect on B78 proliferation via macrophage activation. As A20 tumors progressed in vivo, tumor cell phenotype changed, and T cells became more involved in the local CpG + OX40 mediated anti-tumor response. In mice with larger tumors that were poorly responsive to CpG + OX40, the addition of anti-CTLA-4 enhanced the anti-tumor efficacy in the A20 but not B78 models. Conclusions Increased tumor volume negatively impacts the anti-tumor capability of CpG + OX40 in-situ vaccine. The addition of checkpoint blockade augmented the efficacy of CpG + OX40 in the A20 but not B78 model. These results highlight the importance of considering multiple preclinical model conditions when assessing the efficacy of cancer immunotherapy regimens and their translation to clinical testing.

Introduction: Using an in situ vaccine (ISV) regimen consisting of radiation combined with immunocytokine (tumor-targeting mAb linked to IL2), we can cure mice of well established B78 melanoma tumors (B78s). Mice cured of their B78s with ISV demonstrate long-term immune memory, evidenced by rejection of engraftment of a tumor rechallenge >180 days post initial cure of tumor. Traditionally, immaune memory is thought to be mediated via CD8+ T cells, which require antigen presentation via MHC Class I (MHCI). However, B78s express little to no MHCI but do express MHCII when stimulated with IFNγ. While not commonly expressed on solid tumors, MHCII is expressed on 50-60% of melanomas in humans. Here we explored implications of MHCI and MHCII expression in both the primary and long-term memory anti-tumor responses generated with ISV. Methods: CD4+ or CD8+ T cells were depleted in mice during the primary anti-tumor response (i.e. B78 tumor bearing mice receiving the ISV regimen) or during tumor rechallenge experiments (i.e. B78-cured mice rechallenged with B78s). Tumor growth was monitored. In separate studies, tumors and tumor draining lymphnodes (TDLNs) were harvested during the primary antitumor response and analyzed via flow cytometry to assess T cell activation and immune infiltrate. Finally, TDLNs were harvested from B78-cured mice, 7 days after tumor rechallenge, to define memory T cell subsets. Results: Depletion studies revealed CD4+ T cells are required for both the antitumor response to ISV and the long-term memory response in B78-cured mice, but CD8+ T cells are not required for either of these responses. Increased CD8+ and CD4+ T cell infiltrates are observed in the tumor microenvironment during the primary anti-tumor response. In B78-cured mice, though not required for memory responses, CD8+ central memory T cells are significantly increased in the TDLNs compared to naïve or primary tumor bearing mice. The amount of CD4+ effector memory T cells are significantly increased in the TDLN of B78-cured mice compared to CD8+ effector memory T cells. Conclusion: Often not expressed on solid tumors, MHCII is expressed on some melanoma tumors, and its expression has been correlated with a positive response to immunotherapies. CD4+ cytotoxic T cells can directly engage MHCII on tumors, suggesting this interaction has an important role in the response to immunotherapy for MHCII expressing tumors. Our data suggest that CD4+ T cells drive both the primary anti-tumor and long-term immune memory responses in the B78 model when treating with this effective ISV. We are continuing our efforts to understand the characteristics of the B78 cell line that may be relevant to its response to immunotherapy and its resistance to single agent checkpoint blockade. Understanding the cellular and molecular mechanisms involved in ISV-induced immune recognition and destruction of B78s may guide future improvements of this clinically-relevant immunotherapy regimen. Citation Format: Amy K. Erbe, Arika Feils, Mackenzie Heck, Sabrina VandenHeuvel, Julianna Castillo, Alina Hampton, Lizzie Frankel, Anna Hoefges, Peter Carlson, Alex A. Pieper, Taylor Aiken, Lauren Zebertavage, David Komjathy, Dan Spiegelman, Noah Tsarovsky, Zachary S. Morris, Ravi Patel, Alexander Rakhmilevich, Paul M. Sondel. The influence of MHC class I and II on T cell responses in mouse melanoma [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 1385.