Optimizing TCR-CD3 signaling for immunotherapy of cancer

ABSTRACT The recent development of T cell-based cancer immunotherapies, including checkpoint blockade (anti-PD-1, anti-CTLA-4 and others) or adoptive T cell therapy (ACT) using modified patient T cells, has led to improved patient outcomes for a variety of cancers. However, durable responses are observed in only a fraction of patients. Further progress can be made by studying and targeting different T cell signaling pathways, such as the T cell receptor (TCR)-CD3 signaling pathway. T cell recognition of antigen by the TCR and the resulting proximal signaling through surrounding CD3 subunits are key steps in the initiation of tumor-killing. Previous studies that targeted the antigen binding site of the TCR for enhancing T cell responses to tumor antigens often lead to off- target effects and toxicity. Instead, identification of the specific extracellular interactions between the TCR and CD3 subunits could offer precise guidance for the development of immunotherapeutic strategies that modulate T cell immunity by targeting signaling through the TCR-CD3 complex without altering antigen-specificity. Our preliminary data showed that mutating residues in the constant domain of the TCR resulted in altered T cell cytokine responses. Based on our preliminary data, our hypothesis is that by modulating TCR-CD3 signaling, immune-mediated cytotoxicity to tumor antigens can be enhanced without losing specificity for the cancer antigen. To test our hypothesis, in Aim 1 we will use an in vitro retroviral TCR display method, a novel CD3- tetramer assay, and an in-silico structure-based TCR design approach to identify signal-enhancing TCR mutants that enable T cells to mediate more effective in vitro tumor killing. In Aim 2, we will identify the mechanisms that drive altered signaling evidenced in select TCR signal-enhancing and signal-hampering mutants. In Aim 3, identified mutations will be introduced into gp100-specific TCRs with different antigen affinities and their in vitro and in vivo tumor killing efficacy will be analyzed to characterize the tumor killing potential, T cell differentiation, and T cell exhaustion patterns of new signal-enhancing T cell clones with the goal of developing a new strategy for effective T cell therapies against cancer.