CCR5-targeted allogeneic gamma-delta CD19 CAR T cells for HIV-associated B cell malignancy immunotherapy

Immune-based cell therapy offers a promising approach to cancer treatment. While autologous chimeric antigen receptor (CAR) T cells have shown success, production is time-consuming, costly, and patient-specific. Gamma-delta (γδ) T cells are promising for ‘off-the-shelf’ CAR T cell therapy. However, clinical translation of γδ CAR T cells is hampered by low frequency, resistance to genetic manipulation, and advanced differentiation after expansion, limiting therapeutic feasibility. Here, we demonstrate a method for in vitro activation and expansion of peripheral blood γδ T cells, facilitating high rates of gene editing and efficient CAR integration. Using artificial antigen-presenting cells, we produce minimally differentiated, highly functional γδ CAR T cells. By targeting an FDA-approved CD19 CAR to the CCR5 locus, we generate CCR5-deficient γδ CD19 CAR-T cells (γδ CCR5KI-CAR19), which demonstrated resistant to HIV-mediated depletion and robust antitumor responses against B cell lymphoma and leukemia. γδ CCR5KI-CAR19 T cells enable the immunotherapy of HIV-associated B cell malignancies. These studies provide preclinical evidence supporting large-scale development of potent allogeneic γδ CAR T cells for diverse immunotherapies.