Engineered CARs Targeting FVIII-specific T and B Cells

针对 FVIII 特异性 T 和 B 细胞的工程化 CAR

• 批准号: 9258469
• 负责人: David William Scott
• 金额: $ 19.28万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-10 至 2018-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9258469
• 关键词: Active Sites; Adoptive Transfer; Antibodies; Antibody Formation; Antibody Response; Antigens; B-Lymphocytes; Binding; Blocking Antibodies; Blood; Blood Coagulation Disorders; Blood Coagulation Factor; Blood coagulation; C2 Domain; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Proliferation; Cells; Clone Cells; Coagulation Process; Collaborations; Complement; Complex; Cytotoxic T-Lymphocytes; Disease; Effector Cell; Engineering; Epitopes; F8 gene; Factor VIII; Frequencies; Genes; Goals; Hemophilia A; Hemorrhage; Histocompatibility; Human; Hybridomas; Immune response; Immunize; In Vitro; Incubated; Intravenous; Lead; Libraries; Life; Link; Memory B-Lymphocyte; Mus; Mutation; Natural Killer Cells; Patients; Peptide Signal Sequences; Peptides; Peripheral Blood Mononuclear Cell; Phage Display; Plasma; Plasma Cells; Population; Prevention; Production; Proliferating; Recombinants; Regulatory T-Lymphocyte; Research; Retroviral Vector; Signal Transduction; Specificity; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; Transgenes; Translating; accomplished suicide; antigen binding; base; cellular transduction; chimeric antigen receptor; cytokine; cytotoxicity; cytotoxicity test; gene therapy; in vivo; inhibitor/antagonist; killings; male; novel strategies; novel therapeutic intervention; prevent; public health relevance; receptor; response; retroviral transduction; vector

 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.

 描述（由申请人提供）：我们实验室的重点是开发诱导耐受的新方法，以便将其应用于预防或逆转不良免疫反应，包括血友病抑制剂的形成。我们最近采用了 CAR（嵌合抗原受体）方法，通过逆转录病毒转导从血友病受试者的 FVIII 特异性 T 细胞克隆中分离出的 T 细胞受体，从正常供体的 PBMC 中创建抗原特异性 T 细胞群（T 效应细胞和调节性 T 细胞/Treg）。转导的 Tregs 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 Tregs 还在体外阻断了对 FVIII 的抗体反应 (Kim et al. Blood 125: 1107, 2015)。我们还与 Christoph Königs 博士和 Anja Naumann 博士合作，将单链 (scFv) 抗 FVIII 抗体工程化到扩增的 PBMC 中，并表明转导的 Tregs 特异性抑制 T 效应细胞增殖为 FVIII。在本提案中，我们希望通过利用带有 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这种方法，以创建能够杀死抗原结合 B 细胞（小鼠和人类）的 CAR CD8 T 细胞，以进一步抑制抑制剂反应。因此，我们将结合两种方法来创建特异性 CD8 细胞毒性细胞，以直接靶向带有 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 结构域的病毒将被插入逆转录病毒载体中，类似于 CAR 策略，其跨膜和信号序列将允许在识别特定 B 细胞捕获的 FVIII 后触发 CD8 T 细胞的细胞毒性。一个 第二组 CAR CD8 T 细胞将用免疫显性的 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 CD8 T 细胞结合时将有针对性地“自杀”。我们将通过将 FVIII 转导的人类 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，直接在体外测试细胞毒性，并通过将转导的鼠 T 细胞过继转移至免疫受体体内来测试细胞毒性。因此，我们的具体目标是：(1) 开发逆转录病毒载体，在人和小鼠 CD8 T 细胞中表达 scFv 抗 FVIII 或 FVIII 结构域；(2) 确定工程化的 FVIII 特异性 CD8 T 细胞对 FVIII 特异性 B 细胞和抑制剂形成的影响。这些研究将为 CD8 CAR T 细胞经过改造以有效消除 FVIII 抑制剂反应提供原理证明。