Synthetic biology toolkit for precise tuning of T cell activity

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Project Summary / Abstract. Chimeric antigen receptor (CAR) T cell-based immunotherapy has shown curative potential in patients with haematological malignancies. However, it faces significant safety issues (e.g., cytokine release syndrome and neurotoxicity) and efficacy loss arising from tonic signaling and T cell exhaustion. These undesireable features are more or less decoded in the distal end of lymphocyte activation pathway, the two-component Calcium Release-Activated Calcium (CRAC) channel composed of stromal interaction molecule (STIM) and ORAI to form a major Ca2+ entry route in T cells and control T cell activation. Upon T-cell receptor (TCR) engagement, Ca2+ depletion in the endoplasmic reticulum (ER) is sensed by stromal interaction molecule 1 (STIM1) to initiate a series of conformational changes, culminating in the activation of the pore-forming subunit of the CRAC channel, ORAI1. Ca2+ influx induces a series of processes in T cells, including the secretion of cytolytic granules and the activation of Ca2+-dependent enzymes, including calcineurin, CaMKII and Erk1/2, as well as master transcription factors, such as NF-κB and nuclear factor of activated-T cells (NFAT), that are essential for adaptive immunity. Most importantly, nuclear translocated NFAT differentially engages its binding partners to promote the activation, differentiation, anergy/exhaustion, and effector functions of various T cell subsets. Notably, tonic signaling and exhaustion observed in CAR-T cell therapy are associated with hyperactive Ca2+/NFAT signaling. Till now, no FDA-approved CRAC channel blockers are in hand to modulate the CRAC channel for therapeutic applications. There remains, therefore, a critical need to exploit novel interventional approaches by targeting the distal CRAC channels in T cells. Unlike most existing studies centered on CAR per se or the proximal signaling components in modulating CAR-T cell activation pathway, this project focuses on engineering the distal end of lymphocyte activation pathway without any modifications to the chimeric antigen receptor or proximal TCR signaling. The m-PIs propose to to develop a suite of genetically-encoded CRAC channel Blockers (CRAB) that can be precisely controlled by light or drugs (LiCRAB for Aim 1, and DiCRAB for Aim 2, respectively), thereby conferring tight control of T cell activity to fine-tune T cell efficacy and mitigate CAR-T cell tonic signaling and/or exhaustion. The successful execution of this project will explore innovative immunoengineering approaches to accelerate the design of intelligent cell-based therapies for human disease. Mechanistically, the tools can be utilized to probe the kinetic requirement of Ca2+/NFAT signaling during CAR-T cell activation. From a translational perspective, we will generate broadly-applicable genetically-encoded tools for therapeutic T cell functional tuning, which hold great promise to overcome tonic signaling / exhaustion, and curtail cytokine storm associated with existing FDA-approved CAR T-cell therapies.