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. Single-cell RNA-Seq analysis of gamma-delta CAR T cells manufactured with or without feeder cells. Single-cell RNA-Seq was performed using the Chromium GEM-X Single Cell 3' kit. Libraries were constructed with Chromium Connect. Sequencing was performed using a NextSeq 2000. FASTQ files were aligned and summarized as feature-barcode matrices by cellranger v8.8.0 with the GRCh38-2024-A reference. Feature-barcode matrices were analyzed with Seurat v5.0.3 and integrated using the FindIntegrationAnchors and IntegrateData functions with 50 dimensions. UMAP was performed in the top 15 principal components, and cell cycle scoring was performed using Seurat.