Project Summary Among recent breakthroughs in treating hematopoietic malignancies, chimeric antigen receptor (CAR)-T cell therapy is one of the most promising advances in cancer immunotherapy. However, CAR-T cells have shown limited success targeting solid tumors. One major hurdle is the immunosuppressive tumor microenvironment of solid tumors that promotes T-cell exhaustion. Another challenge is a poor understanding of how CAR structure impacts the signaling mechanisms driving T cell function, which has prevented the rational design of a superior CAR therapeutic with potent anti-tumor activity and resistance to exhaustion. I am a Damon Runyon Cancer Research Postdoctoral fellow at UCSF. My mentor is Dr. James Wells, an expert in chemical biology and protein engineering, and my co-mentor is Dr. Arthur Weiss, an expert in T cell biology. Tyrosine phosphorylation is the central component of the signaling pathways of CAR-T cells but the tools available to study pY modifications are very limited. During my postdoctoral training, I developed an innovative and generalizable platform called “pY-Targeting by Recombinant Antibody Pairs” or “pY-TRAP” to engineer highly specific and tight pY binding domains. This revolutionary tool is the first in vitro method for engineering specific binders against pY-modifications in three-dimensional protein structures. It provides a platform for developing strategies to engineer and to rewire pY-mediated signaling pathways in CAR-T cells. In this work, I propose to develop pY-TRAP-based strategies to address key challenges in CAR-T cell therapies. In Aim 1, I will determine how differing CAR structure modulates the CAR interactome using a novel pY-dependent proximity ligation approach. This work will reveal key differences in the CAR signalosome for various CAR designs and expose the mechanisms behind their functional divergence. In Aim 2, I will establish a screening method to determine how varying CAR structures affect the cellular response to PD-1 signaling. This work will further unveil how to rationally engineer CAR-T cells with enhanced resistance to immunosuppression. In Aim 3, I will engineer a synthetic PD-1 pathway in CAR-T cells designed to counteract the original immunosuppressive function of the pathway1. This is a fundamentally different approach relative to existing strategies to make CAR-T cells more resistant to TME-associated immunosuppressive signals. Taken together, the studies outlined in this proposal will lead to a deeper understanding of CAR-T cell biology, provide knowledge to inform future CAR designs, and expose new strategies to engineer and optimize signaling outcomes in cells to advance the development of cell therapeutics. With the support of my mentors, collaborators, consultants and the great research environment at UCSF, I will receive training in proteomics, quantitative data analysis, and T cell biology. These skills will help me reach my long-term goal of becoming the head of a ...