The expansion of neoantigen (NeoAg)-specific T cells often accompanies clinical responses to immunotherapies such as immune checkpoint blockade (ICB), adoptive cellular therapy (ACT), and personalized cancer vaccines (PCV), highlighting their importance for antitumor immunity. While the functions of NeoAg-specific CD8+ T cells have been characterized, the role of NeoAg-specific CD4+ T cells is less well understood. To study the antitumor mechanisms of NeoAg-specific CD4+ T cells, we sorted single CD4+ T cells specific for a mutated clathrin heavy chain epitope (Cltc H129Q) expressed by the murine squamous cell carcinoma VII (SCC VII) tumor. T cell receptor (TCR) sequencing analysis revealed the presence of four distinct TCR clonotypes and expression of these TCRs in primary cells was sufficient to confer preferential recognition of Cltc H129Q as compared to the corresponding wildtype Cltc epitope. Despite differences in TCR avidity, both low and high avidity CD4+ T cells were capable of proliferating to similar degrees in response to tumor antigen in vivo and enhancing primary tumor immunity in a CD8+ T cell- and CD40L-dependent manner. ACT with Cltc H129Q specific CD4+ T cells also synergized with cyclophosphamide treatment to reduce disease burden in mice with established tumors. Overall, these findings illuminate the mechanistic role of NeoAg-specific CD4+ T cells in tumor immunity, provide insights into the impact of TCR avidity on the helper functions of CD4+ T cells, and highlight the therapeutic potential of ACT with TCR-engineered CD4+ T cells.
Neoantigens (NeoAg) offer attractive therapeutic targets for directing a patient’s immune response to the immunogenic subset of mutations expressed exclusively by their cancer cells. Despite the specificity with which NeoAg enable tumor recognition, the majority of approaches for their identification rely on purely predictive methods such as calculating the ability of mutated peptides to bind to a patient’s set of HLA molecules. These methods have met with limited success in revealing natural targets present on tumor cells. We have developed a novel HLA-agnostic functional approach to NeoAg identification which combines genomic sequencing with bioinformatic analysis to nominate mutations for subsequent functional analysis using patient’s own T cells in an effort to identify natural responses generated under physiologic conditions. Using this, we identified a missense mutation (V205I) in the ribosomal protein RPS2 that is recognized by CD8+ T cells from tumor-infiltrating lymphocytes (TIL) of a metastatic HPV16+ Head and Neck Squamous Cell Carcinoma lesion. We then performed adoptive cellular therapy (ACT) using either unseparated TIL or those enriched for RPS2 V205I-specific CD8+ T cells and found the latter to be superior in controlling outgrowth of tumor of a PDX cell line generated from this lesion in NSG mice. Finally, we used single-cell transcriptomics to isolate the genes encoding the RPS2-specific TCR and show that it recognizes the mutated peptide bound to HLA-B*07:02. These results demonstrate that high-affinity NeoAg-specific T cell responses can be identified in cancer patients, that ACT of these cells can control tumor growth, and that the relevant TCR can be isolated for use in TCR engineering-based immunotherapy.
Neoantigens (NeoAg) offer attractive therapeutic targets for directing a patient’s immune response to the immunogenic subset of mutations expressed exclusively by their cancer cells. Despite the specificity with which NeoAg enable tumor recognition, the majority of approaches for their identification rely on purely predictive methods such as calculating the ability of mutated peptides to bind to a patient’s set of HLA molecules, and have met with limited success in revealing natural targets present on tumor cells. We have developed a novel HLA-agnostic functional approach to NeoAg identification which combines genomic sequencing with bioinformatic analysis to nominate mutations for subsequent functional analysis using patient’s own T cells in an effort to identify natural responses generated under physiologic conditions. Using this, we identified a missense mutation (V205>I) in the ribosomal protein RPS2 that is recognized by CD8+ T cells from tumor-infiltrating lymphocytes (TIL) of a metastatic HPV-16+ Head and Neck Squamous Cell Carcinoma (HNSCC) lesion. We then performed adoptive cellular therapy (ACT) using either unseparated TIL or those enriched for RPS2 V205>I-specific CD8+ T cells and found the latter to be superior in controlling outgrowth of tumor of a PDX cell line generated from this lesion in NSG mice. Finally, we used single-cell transcriptomics to isolate the genes encoding the RPS2-specific TCR and show that it recognizes the mutated peptide bound to HLA-B*07.02 as well as the tumor cell line expressing the RPS2 protein. These results demonstrate that high-affinity NeoAg-specific T cell responses can be identified in cancer patients, that ACT of these cells can control tumor growth, and that the relevant TCR can be isolated for use in TCR engineering-based immunotherapy. Citation Format: Martin S. Naradikian, Leslie Montero, Samantha Hall, Milad Bahmanof, Rukman Thota, Luise Sternberg, Jerome Lane, Zeynep Kosaloglu-Yalcin, Manasa Lanka, Aaron Miller, Bjoern Peters, Ezra Cohen, Stephen Schoenberger. Identification and characterization of neoantigen specific T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1439.
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