Enhancing gammadelta CAR T cell function through alternative CD3 cytoplasmic domains

Gamma delta (gammadelta) T cells are a unique subset of lymphocytes that possess innate and adaptive immune functions. Unlike conventional alphabeta T cells, gammadelta T cells recognize antigens in an MHC-independent manner. This allows them to detect a wide range of stress-induced ligands that are commonly expressed on tumor cells. This property makes gammadelta T cells particularly attractive for cancer immunotherapy, especially in cases where tumors evade immune surveillance by downregulating MHC molecules. Recent advances in cellular engineering have made it possible to generate gammadelta T cells that express chimeric antigen receptors (CARs), which combines their natural tumor-recognition capabilities with the targeted specificity of CARs. gammadelta CAR T cells offer several advantages over alphabeta CAR T cells, including lower graft-versus-host disease risk, suitability for allogeneic applications (“out-of-the-shelf”), and enhanced solid tumor infiltration due to tissue-homing properties. These properties provide a strong rationale for investigating gammadelta CAR T cells as a novel cancer therapy approach and underscore the importance of continued optimization to realize their full therapeutic potential. This study explores strategies to improve the intracellular signaling domain of CAR constructs with a focus on gammadelta T cells. The CD3ζ domain has traditionally been used as the primary activation motif in CAR design. However, emerging evidence suggests that it may not be optimal for all T cell subsets. Therefore, we investigated alternative CD3 subunits, specifically, CD3delta, CD3ε, and CD3gamma, as intracellular signaling domains in CAR constructs. Our in vitro data show that CARs with these alternative CD3 cytoplasmic tails are more effective than conventional CD3ζ-based CARs in terms of activation and cytotoxicity. In vivo validation of alphabeta CAR T cells incorporating alternative CD3 subunits has demonstrated enhanced antitumor efficacy. To extend these findings, in vivo experiments are currently underway using the NALM-6_luc (luciferase expressing) xenograft model, a well-established system for evaluating next-generation cellular therapies. These experiments aim to explore whether similar benefits apply to gammadelta CAR T cells. If confirmed, these results would underscore the potential of leveraging alternative CD3 signaling domains to optimize CAR T cell performance, particularly in gammadelta T cell-based therapies. Our findings provide a compelling rationale for reevaluating CAR construct design, suggesting that alternative CD3 subunits could significantly enhance the therapeutic potential of gammadelta CAR T cells in cancer treatment.