Modulating extracellular TCR-CD3 interactions to identify new melanoma immunotherapy targets

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ABSTRACT Melanoma is one of the deadliest and aggressive form of skin cancer. Main treatment options in metastatic melanoma involve T cell immunotherapy using checkpoint blockade (anti-PD-1, anti-CTLA-4 and others) or adoptive T cell therapy (ACT) with modified patient T cells. Although, durable response is only observed in a fraction of patients. Further progress can be made by studying and targeting different T cell signaling pathways. One such pathway is the T cell receptor (TCR) signaling pathway. T cell recognition of antigen by the TCR and the resulting proximal signaling through the surrounding CD3 subunits are key steps in the initiation of tumor-killing. Identification of the specific extracellular contacts between the TCR and CD3 subunits gives precise guidance for immunotherapeutic strategies that modulate T-cell immunity by targeting signaling through the TCR-CD3 complex. Previous studies that targeted the antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity. Based on our recent TCR-CD3 structure, we mutated specific CD3 interacting TCR-residues that resulted in different T cell cytokine responses. Our hypothesis is that by modulating TCR-CD3 interactions in specific ways, immune-mediated cytotoxicity to tumor antigens can be increased without losing specificity for the cancer antigen. To test our hypothesis, in Aim 1, an in vitro retroviral TCR display method and a novel CD3-tetramer assay will be used to mutate specific TCR-residues and identify TCRs that interact with CD3 with increased affinity, resulting in T cells that mediate more effective functionality. In Aim 2, a structure-based TCR design will be used to identify TCR mutants with enhanced CD3 binding ability in silico. In both instances, identified mutations will be introduced into gp100-specific TCRs and their in vitro/in vivo tumor killing efficacy will be analyzed to validate the anti-tumor potential of new TCRs with the goal of developing new wave of effective T cell therapies against melanoma.