Gamma-Delta CAR-T Cells Show CAR-Directed and Independent Activity Against Leukemia

Autologous T cells engineered to express a chimeric antigen receptor (CAR) against the CD19 antigen are in the frontline of contemporary hemato-oncology therapies, leading to high remission rates in B-cell malignancies. Although effective, major obstacles involve the complex and costly individualized manufacturing process, and CD19 target antigen loss or modulation leading to resistant and relapse following CAR therapy. A potential solution for these limitations is the use of donor-derived gamma delta T cells as a CAR backbone. gamma delta T cells lack allogenecity and are safely used in haploidentical transplants. Moreover, gamma delta T cells are known to mediate natural anti-tumor responses. Here, we describe a 14-day production process initiated from peripheral-blood mononuclear cells, leading to a median 185-fold expansion of gamma delta T cells with high purity (>98% CD3+ and >99% gamma delta TCR+). CAR transduction efficacy of gamma delta T cells was equally high when compared to standard CAR-T cells (60.5 +/- 13.2 and 65.3 +/- 18.3%, respectively). CD19-directed gamma delta CAR-T cells were effective against CD19+ cell linesin vitroand in vivo, showing cytokine production, direct target killing, and clearance of bone marrow leukemic cells in an NSG model. Multiple injections of gamma delta CAR-T cells and priming of mice with zoledronate lead to enhanced tumor reductionin vivo. Unlike standard CD19 CAR-T cells, gamma delta CAR-T cells were able to target CD19 antigen negative leukemia cells, an effect that was enhanced after priming the cells with zoledronate. In conclusion, gamma delta CAR-T cell production is feasible and leads to highly pure and efficient effector cells. gamma delta CAR-T cell may provide a promising platform in the allogeneic setting, and may target leukemic cells also after antigen loss.