FAS Ablation Confers Resistance to Allogeneic CAR-T Rejection By T Cells in Absence of NK Cell Sensitization

Allogeneic chimeric antigen receptor T cells (allo-CAR-T) derived from healthy donors hold potential to overcome limitations associated with autologous cancer therapies, providing immediate access to standardized, affordable batches of CAR-T with improved efficacy. Concomitant to immune reconstitution, however, allogeneic CAR-T are rejected by the host's immune system, resulting in reduced therapeutic efficacy and early relapses. To systematically identify targets overcoming allo-CAR-T rejection, we developed in vivo genome-wide CRISPR KO screens based on allogeneic T cell positive selection (screening for KO genes that promote resistance in fully mismatched immunocompetent mice). We show that Fas or B2m deletion increases the survival of allogeneic T cells in vitro and in vivo. Consequently, FAS or B2M silencing in anti-CD19 allo-CAR-T enhances their anti-leukemic activity and promotes mice survival. Nevertheless, while B2M KO allo-CAR-T become highly sensitive to NK cell-mediated rejection, FAS KO CAR-T, like control CAR-T, express normal levels of HLA-I and remain resistant to NK cell killing. We validated the results in a clinically relevant context using base-editing for multiplexing gene-KO in human CAR-T cells. CD3-FAS KO outperforms CD3-B2M KO CAR-T in the control of leukemia growth under allogeneic pressure by both T and NK cells. We conclude that CD3-FAS double-KO allogeneic CAR-T are partially protected from both T and NK cell-mediated allo-rejection, an approach with the potential to improve the efficacy of allogeneic cellular therapies in patients with B-cells neoplasms and more generally in cancer patients.

来自健康供体的同种异体嵌合抗原受体T细胞（allo - CAR - T）有潜力克服与自体癌症疗法相关的局限性，能够立即提供标准化、价格合理且疗效更佳的CAR - T批次。然而，在免疫重建的同时，同种异体CAR - T会被宿主的免疫系统排斥，导致治疗效果降低和早期复发。为了系统地确定克服同种异体CAR - T排斥的靶点，我们基于同种异体T细胞阳性选择开发了体内全基因组CRISPR基因敲除（KO）筛选（筛选在完全不匹配的免疫健全小鼠中促进抗性的KO基因）。我们发现Fas或B2m的缺失增加了同种异体T细胞在体外和体内的存活。因此，抗CD19同种异体CAR - T中FAS或B2M的沉默增强了它们的抗白血病活性并提高了小鼠的存活率。然而，虽然B2M基因敲除的同种异体CAR - T对自然杀伤（NK）细胞介导的排斥变得高度敏感，但FAS基因敲除的CAR - T与对照CAR - T一样，表达正常水平的人类白细胞抗原I（HLA - I），并对NK细胞的杀伤保持抗性。我们在临床相关背景下使用碱基编辑对人CAR - T细胞中的多基因敲除进行了结果验证。在同种异体T细胞和NK细胞的压力下，CD3 - FAS基因敲除在控制白血病生长方面优于CD3 - B2M基因敲除的CAR - T。我们得出结论，CD3 - FAS双基因敲除的同种异体CAR - T可部分免受T细胞和NK细胞介导的同种异体排斥，这种方法有可能提高同种异体细胞疗法对B细胞肿瘤患者以及更广泛的癌症患者的疗效。