Reprogramming T cell function with multiplexed genome engineering to develop next-generation immunotherapy

Project Summary: Adoptive cellular immunotherapy utilizing T cells engineered with chimeric antigen receptor (CAR) has shown durable clinical responses in hematologic malignancies. However, relapse remains a challenge, urging for better CAR designs. CAR, analogous to the T cell receptor (TCR), is specifically engineered to redirect T cell specificity

towards tumor antigens. Optimal regulation on TCR dynamics is crucial for proper TCR function. Altered dynamics of TCR leads to disfunction or even immune disorders. While the importance of control TCR dynamics has been well established, there is a notable lack of comprehensive studies focusing specifically on CAR

dynamics, including endocytosis, recycling/degradation, and its impact on CAR-T function. My previous work demonstrated that modulating molecular dynamics of CAR greatly improves CAR-T function. By utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail (CCT), I

reprogrammed CAR dynamics and substantially enhanced CAR-T efficacy in vivo. To control CAR dynamics more precisely and elucidate its impact on CAR-T function, further modulation on CCT fusion and investigation on cell-intrinsic regulators to control CAR dynamics are needed. In Aim 1 of this K99/R00 application, I propose

to identify the optimal position and functional motif of CCT fusion. Completion of this aim will not only develop a novel CAR engineering approach but also provide a deeper understanding of how altering CAR dynamics contributes to improved CAR-T function. In Aim 2, I plan to uncover cell-intrinsic regulator of CAR dynamics

using CRISPR-based gene perturbation. This aim will serve to fill the gap in our understanding of the improved anti-tumor efficacy of CAR-CCT but also uncover novel regulators on CTLA-4 dynamics. In Aim 3, I propose to develop self-regulated CAR with CCT fusion in response to antigen stimulation. This novel engineer approach

will enable precise control over the timing and duration of CAR signaling, which is crucial for maintaining CAR- T efficacy with limited toxicity. All aims center on engineering CAR dynamics with CCT fusion to develop novel immunotherapies. The innovative insights and versatile engineering approach proposed in this study will also

contribute significantly to addressing the existing knowledge gap in CAR engineering. This grant will facilitate my career development during both the postdoctoral training phase and transition phase as an independent faculty and group leader.