可控断裂的双功能连接臂介导的CAR-T细胞可“开关”型调控及应用研究

Chimeric antigen receptor engineered T cells (CAR-T) provide a potent anti-tumor response and have become a promising treatment option for various of diseases. However, due to the inability to control the interaction between CAR-T cells and tumor cell, the therapy of CAR-T are associated with significant safety challenges related to cytokine release syndrome, and how to control the activation of CAR-T cells with more efficiently and simply is the current key problem in this field. Basis on this, our project intend to design the bifunctional linker consisting of controlled release chemical group as “switch”, and use that to conjugate with CAR-T cells and target cells. Theoretically, through this methodology, the consequent activation of CAR-T (termed “turn-on”) could be dose-dependent responded to the addition of switch; and the suppression of CAR-T (termed “turn off” ) could be achieved by the use of bio-orthogonal cleavage reaction, which could disconnect between CAR-T and target cells; and by that, the aim of switch-mode control of CAR-T with more effective and flexible could be achieved. In our previous work, we have already finished the synthesis of “switch” molecule and preparation of FITC-specific CAR-T cells, and the validity of technology theory is confirmed primarily by the experiments. The further study will mainly conduct from the whole level about in-vitro and in-vivo, for the completion of this theory as a therapeutic method. In addition, the results of this project is also expect to develop as a versatile platform to comprehensively improve the controllability and safety of CAR-T therapy, and provide the new selectivity for the further clinical application.

目前，嵌合抗原受体T细胞（CAR-T）技术已经在癌症等疾病的治疗中展现了巨大的应用前景。而如何能更简便高效的对CAR-T进行激活/关闭控制，是目前该疗法在实际应用中亟待解决的关键问题。基于此，本项目拟设计双功能连接臂，并在其中间引入可控断裂的连接子，进而将其作为“开关”，用于偶联CAR-T细胞和靶细胞。理论上，通过此设计，CAR-T的激活（开）将剂量依赖于小分子开关的加入；而CAR-T的抑制（关）则可借助正交断裂反应，分离CAR-T与靶细胞，进而实现对CAR-T高效即时的“开关”式灵活调控。本项目已完成了“开关”分子的合成和特异性CAR-T制备等前期工作，并初步验证了此技术理念的可行性。项目将进一步在各阶段水平上对此技术的有效性和可控性进行验证并优化，完善其作为治疗方法的实用性。本项目的研究成果将有望作为平台技术，全面提高现有CAR-T疗法的可控性和安全性，为其进一步的临床应用提供选择性。

为提高CAR-T的可控性和安全性，研究成功设计了光控的连接臂分子FITC-O-Folate，即将FITC和作为靶头的叶酸分子通过光敏基团邻硝基苯基酯基连接在一起，光照时连接臂断裂，使CAR-T细胞与靶细胞分离，从而系统性地降低CAR-T细胞杀伤活性和多种细胞因子分泌。项目成功设计并证明了CAR-T细胞的激活（开）严格依赖于此连接臂的加入，并呈剂量依赖效应；CAR-T细胞的关闭将不依赖于连接臂分子的代谢，而可随连接臂分子的断裂进行即时且随意地调控；更进一步地，关闭后的CAR-T细胞，其杀伤效应可被补充连接臂分子再激活，实现CAR-T细胞“激活-关闭-再激活”的可循环式精确调控，从而不影响CAR-T细胞的整体作用效果。利用生物正交断裂进行可“开关”式CAR-T的调控，从概念上开拓展了现有CAR-T细胞技术的可控性，为提高CAR-T细胞安全性提供了新的研究工具。. 更进一步的，基于此通用型CAR-T，我们将连接臂分子的另一端更换为多种可识别自身反应B细胞的抗原肽，用于自身免疫病的靶向性治疗。在体外实验中，我们设计并验证了此技术的有效性和可控性，并根据自身免疫病患者的自身抗体类型，定制化杀灭分泌特异性表面抗体的不同自身反应性B细胞，实现了CAR-T对自身免疫病靶向、可控、且定制化治疗的概念验证。更进一步的，在本项目的后续研究中，基于IL-2在T细胞生长激活中的关键作用，申请人成功开发了一系列具有偏向且长效的IL-2突变体，有望与CAR-T细胞进行联合应用，提高其在自身免疫病或肿瘤治疗中的有效性，具备理想的临床转化前景。