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: 9034735
• 负责人: David William Scott
• 金额: $ 23.14万
• 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 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9034735
• 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; CD8B1 gene; Cell Proliferation; Cells; Coagulation Process; Collaborations; Complement; Complex; Cytotoxic T-Lymphocytes; Disease; Effector Cell; Engineering; Epitopes; Factor VIII; Frequencies; Genes; Goals; Hemophilia A; Hemorrhage; Histocompatibility; Human; Hybridomas; Immune response; In Vitro; Incubated; 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; 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; recombinant antihemophilic factor VIII; 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(申请人提供)：我们实验室的重点一直是开发诱导耐受的新方法，以便它可以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细胞克隆中分离出来的T细胞受体，从正常捐赠者的PBMC中创建抗原特异性T细胞群(T效应器和调节性T细胞/Tregs)。转导的Tregs同时抑制效应T细胞的增殖和多种细胞因子的产生。重要的是，这些经过改造的Tregs还在体外阻断了对FVIII的抗体反应(Kim等人。血125：1107,2015)。在Christoph Königs博士和Anja Naumann的合作下，我们还将一种单链(ScFv)抗FVIII抗体植入扩增的PBMC中，并表明转导的Tregs特异性地抑制T效应细胞向FVIII的增殖。在这项建议中，我们希望通过利用逆转录病毒转导带有ScFv抗FVIII或FVIII结构域的细胞毒性T细胞来扩展这一方法，以创建能够杀死抗原结合B细胞(包括小鼠和人)的CAR CD8T细胞，从而进一步抑制抑制反应。因此，我们将结合两种方法来创造特定的CD8细胞毒细胞，以直接针对B细胞和携带FVIII特异性受体的浆细胞。单链抗体 识别免疫优势的FVIII结构域将被插入到逆转录病毒载体中，类似于CAR策略，具有跨膜和信号序列，当识别特定B细胞捕获的FVIII时，将允许触发CD8T细胞的细胞毒性。一个 第二组CAR CD8 T细胞将被转导具有免疫优势的FVIII A2和C2结构域。我们的假设是，识别这些区域的B细胞在与转导的CD8 T细胞结合时，将成为自杀的目标。我们将通过将FVIII转导的人T细胞与血友病患者的B细胞和FVIII特异性杂交瘤细胞培养在体外直接测试细胞毒性，并通过将转导的小鼠T细胞过继转移到免疫接受者体内来测试细胞毒性。因此，我们的具体目标是：(1)建立逆转录病毒载体，在人和小鼠CD8 T细胞中表达抗FVIII或FVIII结构域的单链抗体；(2)检测FVIII特异性的工程化CD8T细胞对FVIII特异性B细胞及其抑制物形成的影响。这些研究将提供CD8CAR T细胞可以被设计成有效地消除对FVIII的抑制反应的原理证据。