Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy

Gamma delta (γδ) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-major histocompatibility complex-associated antigens on transformed and pathogen-infected cells. In addition to their inability to mediate graft versus host disease, γδ T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of γδ T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene-edited chimeric antigen receptor (CAR)-γδ T cells for therapeutic application. Engineered CAR-γδ T cells demonstrate robust functionality in vitro and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-γδ T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-γδ T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-γδ T cell subsets to therapeutic efficacy in vivo.