Background: CAR-T cell therapy has shown impressive results and is now part of standard-of-care treatment of B-lineage malignancies, whereas the treatment of myeloid diseases has been limited by the lack of suitable targets. CD45 is expressed on almost all types of blood cells including myeloid leukemia cells, but not on non-hematopoietic tissue, making it a potential target for CAR-directed therapy. Because of its high expression on T and NK cells, fratricide is expected to hinder CD45CAR-mediated therapy. Due to its important roles in effector cell activation, signal transduction and cytotoxicity, CD45 knockout aimed at preventing fratricide in T and NK cells has been expected to lead to considerable functional impairment. Methods: CD45 knockout was established on T and NK cell lines using CRISPR/Cas9-RNPs and electroporation, and the successful protocol was transferred to primary T cells. A combined protocol was developed enabling CD45 knockout and retroviral transduction with a third-generation CAR targeting CD45 or CD19. The functionality of CD45koeffector cells, CD45ko/CD45CAR-T and CD45ko/CD19CAR-T cells was studied using proliferation as well as short- and long-term cytotoxicity assays. Results: As expected, the introduction of a CD45-CAR into T cells resulted in potent fratricide that can be avoided by CD45 knockout. Unexpectedly, the latter had no negative impact on T- and NK-cell proliferation in vitro. Moreover, CD45ko/CD45CAR-T cells showed potent cytotoxicity against CD45-expressing AML and lymphoma cell lines in short-term and long-term co-culture assays. A pronounced cytotoxicity of CD45ko/CD45CAR-T cells was maintained even after four weeks of culture. In a further setup, we confirmed the conserved functionality of CD45kocells using a CD19-CAR. Again, the proliferation and cytotoxicity of CD45ko/CD19CAR-T cells showed no differences from those of their CD45-positive counterparts in vitro. Conclusions: We report the efficient production of highly and durably active CD45ko/CAR-T cells. CD45 knockout did not impair the functionality of CAR-T cells in vitro, irrespective of the target antigen. If their activity can be confirmed in vivo, CD45ko/CD45CAR-T cells might, for example, be useful as part of conditioning regimens prior to stem cell transplantation.
背景：CAR-T细胞疗法已展现出显著疗效，成为B系恶性肿瘤的标准治疗方案之一，而髓系疾病的治疗因缺乏合适靶点而受限。CD45在包括髓系白血病细胞在内的几乎所有血细胞类型中均有表达，但在非造血组织中不表达，这使其成为CAR导向治疗的潜在靶点。由于CD45在T细胞和NK细胞中高表达，预期同源杀伤效应会阻碍CD45CAR介导的疗法。鉴于CD45在效应细胞活化、信号转导和细胞毒性中的重要作用，旨在预防T细胞和NK细胞同源杀伤的CD45敲除预期会导致显著功能损伤。 方法：采用CRISPR/Cas9-RNPs与电穿孔技术在T细胞和NK细胞系中建立CD45敲除模型，并将成功方案应用于原代T细胞。开发出联合方案，实现CD45敲除与靶向CD45或CD19的第三代CAR逆转录病毒转导。通过增殖实验及短期与长期细胞毒性实验，研究CD45敲除效应细胞、CD45敲除/CD45CAR-T细胞及CD45敲除/CD19CAR-T细胞的功能特性。 结果：如预期所示，在T细胞中引入CD45-CAR会导致强烈的同源杀伤效应，而CD45敲除可有效避免该现象。出乎意料的是，CD45敲除对T细胞和NK细胞的体外增殖未产生负面影响。此外，在短期与长期共培养实验中，CD45敲除/CD45CAR-T细胞对表达CD45的AML和淋巴瘤细胞系展现出强大细胞毒性。即使经过四周培养，CD45敲除/CD45CAR-T细胞仍保持显著的细胞毒性。在另一实验设计中，我们通过CD19-CAR验证了CD45敲除细胞的功能完整性。CD45敲除/CD19CAR-T细胞的增殖与细胞毒性在体外与其CD45阳性对照细胞无显著差异。 结论：本研究成功制备出具有持续高效活性的CD45敲除/CAR-T细胞。CD45敲除未损害CAR-T细胞的体外功能，且该现象与靶抗原无关。若其活性能在体内得到证实，CD45敲除/CD45CAR-T细胞或可作为干细胞移植前预处理方案的重要组成部分。
肿瘤(癌症)患者之家