Engineered CARs Targeting FVIII-specific T and B Cells

 描述（由申请人提供）：我们实验室的重点是开发诱导耐受性的新方法，以便其可用于预防或逆转不良免疫反应，包括血友病抑制剂的形成。我们最近调整了CAR（嵌合抗原受体）方法，通过从血友病受试者的FVIII特异性T细胞克隆分离的T细胞受体的逆转录病毒转导，从正常供体的PBMC中产生抗原特异性T细胞群（T效应子和调节性T细胞/T细胞）。经转导的TcR抑制效应T细胞增殖和多种细胞因子的产生。重要的是，这些工程化的Tcl 3还在体外阻断了对FVIII的抗体应答（Kim等人，Blood 125：1107，2015）。与Christoph Königs博士和Anja Naumann合作，我们还将单链（scFv）抗FVIII抗体工程化到扩增的PBMC中，并显示转导的TcR特异性抑制T效应细胞增殖为FVIII。在该提议中，我们希望通过利用具有scFv抗FVIII或FVIII结构域的细胞毒性T细胞的逆转录病毒转导来扩展该方法，以产生能够杀死抗原结合B细胞（小鼠和人）的CAR CD 8 T细胞，从而进一步抑制抑制剂应答。因此，我们将结合联合收割机两种方法来产生特异性CD 8细胞毒性细胞，以直接靶向携带FVIII特异性受体的B细胞和浆细胞。单链抗体 识别免疫显性FVIII结构域的抗体将被插入逆转录病毒载体中，类似于CAR策略，具有跨膜和信号传导序列，其将允许在识别由特异性B细胞捕获的FVIII时触发CD 8 T细胞中的细胞毒性。一 第二组CAR CD 8 T细胞将用免疫显性FVIII A2和C2结构域转导。我们的假设是识别这些结构域的B细胞在与转导的CD 8 T细胞结合时将被靶向以实施“自杀”。我们将通过将FVIII转导的人T细胞与血友病受试者的B细胞和FVIII特异性杂交瘤一起培养来直接在体外测试细胞毒性，并通过将转导的鼠T细胞过继转移到免疫受体中来在体内测试细胞毒性。因此，我们的具体目标是：（1）开发逆转录病毒载体以在人和小鼠CD 8 T细胞中表达scFv抗FVIII或FVIII结构域，以及（2）确定工程化FVIII特异性CD 8 T细胞对FVIII特异性B细胞和抑制剂形成的影响。这些研究将提供CD 8 CAR T细胞可以被工程化以有效消除对FVIII的抑制剂应答的原理证明。

 DESCRIPTION (provided by applicant): The focus of our lab has been to develop novel approaches for the induction of tolerance so that it can be applied to the prevention or reversal of undesirable immune responses, including the formation of hemophilia inhibitors. We recently adapted the CAR (chimeric antigen receptor) approach to create antigen-specific T-cell populations (T effectors and regulatory T cells/Tregs) from PBMC of normal donors by retroviral transduction of T-cell receptors isolated from a hemophilia subject's FVIII-specific T-cell clone. The transduced Tregs suppressed both effector T-cell proliferation and production of multiple cytokines. Importantly, these engineered Tregs also blocked the antibody response to FVIII in vitro (Kim et al. Blood 125: 1107, 2015). In collaboration with Dr. Christoph Königs and Anja Naumann, we have also engineered a single chain (scFv) anti-FVIII antibody into expanded PBMC and shown that transduced Tregs specifically suppressed T effector cell proliferation to FVIII. In this proposal, we wish to extend this approach by utilizing retroviral transduction of cytotoxic T cells with scFv anti-FVIII or FVIII domains to create CAR CD8 T cells capable of killing antigen-binding B cells (both mouse and human) to further suppress the inhibitor response. Thus, we will combine two approaches to create specific CD8 cytotoxic cells to directly target B cells and plasma cells bearing FVIII- specific receptors. Single chain antibodies recognizing the immunodominant FVIII domains will be inserted into a retroviral vector, analogous to CAR strategies, with transmembrane and signaling sequences that will allow triggering of cytotoxicity in CD8 T cells upon recognition of FVIII captured by specific B cells. A second set of CAR CD8 T cells will be transduced with immunodominant FVIII A2 and C2 domains. Our hypothesis is that B cells recognizing these domains will be targeted to commit "suicide" when bound to transduced CD8 T cells. We will test cytotoxicity directly in vitro by culturing the FVIII-transduced human T cells with hemophilia subjects' B cells and with FVIII-specific hybridomas, and in vivo by adoptive transfer of transduced murine T cells into immunized recipients. Therefore, our specific aims are: (1) to develop retroviral vectors to express scFv anti-FVIII or FVIII domains in human and mouse CD8 T cells and (2) to determine the effect of engineered FVIII-specific CD8 T cells on FVIII-specific B cells and inhibitor formation. These studies will provide proof of principle that CD8 CAR T cells can be engineered to effectively eliminate inhibitor responses to FVIII.