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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：我们实验室的重点是开发诱导耐受性的新方法，以便将其应用于预防或逆转不良免疫反应，包括形成血友病抑制剂。最近，我们通过从正常供体的PBMC中通过逆转录病毒对FVIII-FVIII特异性T-Cell特异性T-Cell Clone分离出的T细胞接收器来调整CAR（嵌合抗原受体）方法，以从正常供体的PBMC中创建抗原特异性T细胞群（T效应和调节性T细胞/Treg）。转移的Treg抑制了效应子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Treg还阻止了体外对FVIII的抗体反应（Kim等人血液125：1107，2015）。与ChristophKönigs博士和Anja Naumann博士合作，我们还将单个链（SCFV）抗FVIII抗体设计为扩展的PBMC，并表明Translated Tregs特异性抑制了T效应细胞的增殖为FVIII。在该提案中，我们希望通过使用SCFV抗FVIII或FVIII结构域将细胞毒性T细胞逆转来扩展这种方法，以创建能够杀死抗原结合B细胞（小鼠和人）的CAR CD8 T细胞，以进一步抑制抑制剂。这，我们将结合两种方法来创建特定的CD8细胞毒性细胞，以直接靶向带有FVIII受体的B细胞和浆细胞。单链抗体识别免疫主导的FVIII结构域将被插入逆转录病毒载体中，类似于汽车策略，跨膜和信号序列将允许在识别特定B细胞FVIII捕获的FVIII时触发CD8 T细胞中的细胞毒性。一个第二组CD8 T细胞将使用免疫主导的FVIII A2和C2结构域转移。我们的假设是，B细胞识别这些结构域的目标是在翻译CD8 T细胞时犯下“自杀”。我们将通过用血友病受试者的B细胞和FVIII特异性杂交瘤培养FVIII转导的人T细胞，直接在体外测试细胞毒性，并通过自适应将鼠T细胞转移到免疫的受体中。因此，我们的具体目的是：（1）开发逆转录病毒载体以在人和小鼠CD8 T细胞中表达SCFV抗FVIII或FVIII结构域，以及（2）确定工程FVII特异性CD8 T细胞对FVIII特异性B细胞和抑制剂形成的影响。这些研究将提供原理证明，可以设计CD8 CAR T细胞以有效消除对FVIII的抑制剂反应。