The development of novel chimeric antigen receptor gamma‐delta T cells against multiple myeloma and single‐cell RNA sequencing analysis

Chimeric antigen receptor (CAR) gamma-delta (γδ) T cells offer a major histocompatibility complex-independent, off-the-shelf alternative to conventional CAR alpha-beta (αβ) Tcells, but their efficacy in multiple myeloma (MM) remains underexplored. In this work, we developed a series of novel CAR γδ T cells redirected to B-cell maturation antigen (BCMA) and performed functional assays compared to CAR αβ T cells. As a result, novel CAR γδ T cells demonstrated similar activation upon antigen stimulation compared with CAR αβ T cells but showed lower proliferative capacity and less durable cytotoxicity. In vivo, higher doses of CAR γδ T cells were required to achieve tumour control comparable to CAR αβ T cells. Among CAR constructs, those with cluster of differentiation 3 (CD3ε or CD3ζ) intracellular domain exhibited superior cytotoxicity and in vivo efficacy. Single-cell transcriptomic profiling revealed the low ratio of cycling CAR γδ T cells after antigen stimulation limited the persistence of CAR γδ T cells. In conclusion, our developed novel CAR γδ T cells effectively eliminated MM cells in vitro, but exhibited limited in vivo persistence compared to traditional CAR αβT cells. These findings establish a baseline for utilizing γδ T cells as an off-the-shelf platform and highlight that addressing the proliferation bottleneck through further engineering is essential for future clinical translation.