Abstract 143: Enhancing γδ CAR T cell function through alternative CD3 cytoplasmic domains.

Abstract Gamma delta (γδ) T cells are a unique subset of lymphocytes that possess innate and adaptive immune functions. Unlike conventional αβ T cells, γδ 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 γδ 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 γδ T cells that express chimeric antigen receptors (CARs), which combines their natural tumor-recognition capabilities with the targeted specificity of CARs. γδ CAR T cells offer several advantages over αβ 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 γδ 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 γδ 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, CD3δ, CD3ε, and CD3γ, 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 αβ 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-6ₗuc (luciferase expressing) xenograft model, a well-established system for evaluating next-generation cellular therapies. These experiments aim to explore whether similar benefits apply to γδ 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 γδ 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 γδ CAR T cells in cancer treatment. Citation Format: Philipp Metzger, Marina Zintchenko, Susana Minguet, Cynthia Obodozie, Holger Weber. Enhancing γδ CAR T cell function through alternative CD3 cytoplasmic domains abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (7 Suppl): Abstract nr 143.