Unlocking Hidden Potential: Exploring the Complexities of Signaling in Immune Cell Activation to Optimize CAR Therapy

Chimeric antigen receptors (CARs) are synthetic, transmembrane proteins that trigger immune cell signaling following their engagement. They have been first utilized in T cells and later in natural killer (NK) cells to redirect their cytotoxicity toward a specific surface antigen. The advent of CAR technology has revolutionized the landscape of cancer immunotherapy. Despite the success of CAR T cells, they remain limited to a subset of patients, and the associated toxicities cannot be overlooked. NK cells have a high potential as a novel therapeutic, and their specificity can be redirected by engineering them with CARs. NK cells are a subset of innate lymphocytes that constitute approximately 10% of circulating lymphocytes and exhibit rapid recognition and elimination of virally infected and tumorigenic cells. NK cells offer certain benefits over T cells, as they can be readily used as an "off-the-shelf" therapy without requiring a custom manufacturing process. They exhibit a distinct cytokine profile associated with reduced risk for cytokine release syndrome and neurotoxicity. The NK92 cell line has demonstrated safety and efficacy in clinical trials, and its ease of manipulation and engineering make it a promising therapeutic agent. In this thesis document, I will first review the signaling pathways that trigger lymphocyte activation and cytotoxicity against target cells and then examine the recent advancements in CAR technology. Understanding the molecular mechanisms underlying immune cell activation is crucial for designing potent and selective next-generation CARs tailored to specific immune cell types. CARs were initially developed based on the T cell receptor's (TCR) signaling properties. Commonly used signaling domains are mainly extracted from CD3z in tandem with costimulatory domains derived from CD28 or CD137 (4-1BB). Although initially designed for T cells, these CAR constructs show activity in NK cells. However, NK cells intrinsically express various immune receptors in parallel and can process multiple signals. Signaling motifs in NK cell receptors or signaling cascades initiated from these receptors can be manipulated and integrated into CARs to fully exploit these cells' vast potential. 2B4 is a potent NK cell-activating receptor that initiates NK cell cytotoxicity upon engagement. Mechanistically, it signals through the linker for activation of T cells (LAT). LAT is known to transduce proximal signaling into distal signaling. It acts as a docking site for SH2-containing adapters (GRB2, GADS, and SLP-76) and signaling proteins (SOS1, ADAP, NCK, PLCγ, and VAV1). LAT downstream signaling pathways include calcium influx/NFAT, PI3K/AKT, PKCΞ/NFκB, RASGRP1/AP1, p38/JNK, and mTOR. During my thesis research, we developed an NK-specific CAR, where LAT was incorporated within the signaling domain of the CAR to mimic the endogenous NK activation process. Our study demonstrates that LAT can function as an activating domain within a CAR construct. Engineering NK92 cells with the LAT-based CAR enables superior tumor control compared to traditional CD28-CD3z signaling domains. Additionally, our results indicate that the LAT-based CAR can be tailored for T cells, opening new opportunities for CAR optimization and signal modulation. Our findings suggest that incorporating LAT into CAR designs holds great potential for enhancing the efficacy of CAR-based therapies in cancer treatment.