Mutation-derived tumor antigens (MTA)—alternatively known as “neoantigens”—are a class of tumor antigens generated by tumor cell-intrinsic somatic DNA alterations. MTA are thought to be the predominant target of spontaneous and treatment-induced anti-tumor immunity. Direct targeting of MTA with therapeutic vaccination or other approaches is thus an active area of clinical research. However, pre-clinical studies of MTA-specific immunity have been limited in part due to a lack of reproducible targets. Furthermore, properties distinguishing effective MTA-specific immune responses, from less effective responses targeting other types of non-mutated tumor antigens have not been identified. To address this deficit, we previously reported the development of a system wherein MTA-specific CD8+ T-cell-mediated immunity could be systematically characterized. Briefly, we performed exome (WES) and RNASeq on the B16F1 and B16F10 melanoma cell lines, as well as matched normal tissue. We identified somatic single-nucleotide substitutions, insertion and deletion (INDEL), as well as larger INDEL and gene translocation events. The sequence identity and expression of somatic variants was validated by RNASeq. Standard in silico protocols were used to predict MHC-I binding affinity, which we subsequently validated via cell-free fluorescent peptide:MHC-I stability assay. Using a nanoparticle-based vaccination protocol, we identified and characterized multiple MHC-I restricted MTA in the B16F10 model system. We report an initial characterization of the vaccine-induced MTA-specific CD8+ T-cell response and demonstrate activity of vaccine monotherapy following subcutaneous tumor challenge. We extend prior observations were expanded upon in the following manner: First, the sensitivity and specificity of MTA-specific CD8+ T-cells was determined by in vitro stimulation with target MTA peptide and matched wildtype-sequence peptide followed by intracellular flow cytometry. Vaccine-induced CD8+ T-cells exhibit selectivity to MTA peptide targets ranging perfect specificity to moderate selectivity. A cell-based MHC-I binding assay was used to measure the relative affinity of MTA and matched wildtype peptides. The selectivity of vaccine-induced CD8+ T-cells to MTA peptides was observed to depend both on peptide:MHC-I as well as TCR:pMHC interactions. With respect to sensitivity of CD8+ T-cells towards target antigen, CD8+ T-cells targeting MTA were found to be more sensitive than those targeting nonmutated tumor antigens. We assessed direct recognition of target antigen endogenously presented by autologous tumor cells and describe tumor cell-intrinsic, as antigen-specific features affecting lymphocyte recognition and effector function. Second, we longitudinally profiled the surface receptor phenotype, and transcription factor usage of MTA-specific CD8+ T-cells by single-cell mass cytometry (CyTOF). We describe the phenotype of peripheral, as well as tumor-infiltrating MTA-specific CD8+ T-cells. Using a genetically-encoded fluorescent reporter we identify MTA-specific CD8+ T-cells actively engaged with antigen in situ and describe the molecular and cellular characterization of these tumor-reactive MTA-specific CD8+ T-cells. In summary, we report the identification and detailed characterization of multiple previously-undescribed MHC-I restricted MTA relevant to the widely-utilized B16F10 model. We characterize the phenotype and function of MTA-specific CD8+ T-cells. Finally, we describe phenotypic and functional properties of MTA-specific CD8+ T-cells associated with therapeutic activity in vivo.

Citation Format: John P. Finnigan, Andrew S. Ishizuka, Geoffrey M. Lynn, Alex Rubinsteyn, Robert A Seder, Nina Bhardwaj. Molecular and cellular properties of neoantigen-specific CD8+ T-cells on interaction with B16F10 melanoma in situ [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B108.