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 γδT cells as a CAR backbone. γδT cells lack allogenecity and are safely used in haploidentical transplants. Moreover, γδT 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 γδ T cells with high purity (>98% CD3+ and >99% γδTCR+). CAR transduction efficacy of γδ T cells was equally high when compared to standard CAR-T cells (60.5 ± 13.2 and 65.3 ± 18.3%, respectively). CD19-directed γδCAR-T cells were effective against CD19+ cell lines in vitro and in vivo, showing cytokine production, direct target killing, and clearance of bone marrow leukemic cells in an NSG model. Multiple injections of γδCAR-T cells and priming of mice with zoledronate lead to enhanced tumor reduction in vivo. Unlike standard CD19 CAR-T cells, γδCAR-T cells were able to target CD19 antigen negative leukemia cells, an effect that was enhanced after priming the cells with zoledronate. In conclusion, γδCAR-T cell production is feasible and leads to highly pure and efficient effector cells. γδCAR-T cell may provide a promising platform in the allogeneic setting, and may target leukemic cells also after antigen loss.

经过改造以表达针对CD19抗原的嵌合抗原受体（CAR）的自体T细胞在当代血液肿瘤治疗中处于前沿，导致B细胞恶性肿瘤的缓解率很高。尽管有效，但主要障碍包括复杂且昂贵的个性化制造过程，而CD19靶向抗原的丢失或调节会导致CAR治疗后耐药和复发。这些限制的潜在解决方案是使用供体来源的γδT细胞作为CAR骨架。γδT细胞缺乏同种异体性，可安全用于单倍体移植。而且，已知γδT细胞介导天然抗肿瘤反应。在这里，我们描述了从外周血单核细胞开始的14天生产过程，导致高纯度γδT细胞的中位数膨胀185倍（> 98％的CD3 +和> 99％的γδTCR+）。与标准CAR-T细胞相比，γδT细胞的CAR转导效率同样很高（分别为60.5±13.2和65.3±18.3％）。CD19定向的γδCAR-T细胞对CD19 +细胞系有效体外在体内，在NSG模型中显示出细胞因子的产生，直接靶点的杀伤和骨髓白血病细胞的清除。多次注射γδCAR-T细胞并用唑来膦酸盐引发小鼠可增强肿瘤减少体内。与标准CD19 CAR-T细胞不同，γδCAR-T细胞能够靶向CD19抗原阴性白血病细胞，这种作用在用唑来膦酸盐启动细胞后得以增强。总而言之，γδCAR-T细胞的生产是可行的，并可以产生高纯度和高效的效应细胞。γδCAR-T细胞可能在同种异体环境中提供有希望的平台，并且在抗原丢失后也可以靶向白血病细胞。