Engineering chimeric antigen receptors with VLR-mediated cancer recognition in CD5 edited gamma delta T cells

在 CD5 编辑的 γ δ T 细胞中设计具有 VLR 介导的癌症识别功能的嵌合抗原受体

• 批准号: 9395532
• 负责人: Lauren Cari Fleischer
• 金额: $ 4.4万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-17 至 2020-07-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9395532
• 关键词: Acute T Cell Leukemia; Adaptive Immune System; Adverse effects; Allogenic; Antigen Receptors; Antigen Targeting; Antigens; Antineoplastic Agents; B-Cell Acute Lymphoblastic Leukemia; B-Cell Leukemia; B-Lymphocytes; Binding; CD19 gene; CD5 Antigens; CRISPR/Cas technology; Cell Line; Cell physiology; Cells; Cessation of life; Child; Chronic Lymphocytic Leukemia; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; DNA cassette; Data; Disease; Disease remission; Effectiveness; Effector Cell; Eligibility Determination; Emerging Technologies; Engineering; Engraftment; Foundations; Generations; Genetic Engineering; Goals; Guide RNA; Hematopoietic Stem Cell Transplantation; Immune; Immune Targeting; Immune response; Immunocompetent; Immunoglobulin G; Immunoglobulins; Immunologic Deficiency Syndromes; Immunotherapy; In Vitro; Injectable; Injection of therapeutic agent; Knock-out; Laboratories; Lampreys; Lentivirus Vector; Light; Lymphocyte; Malignant Neoplasms; Measurable; Measures; Mediating; Memory; Multiple Myeloma; Mus; Patients; Plasmids; Protein Engineering; Refractory Disease; Relapse; Severe Adverse Event; Signal Transduction; Specificity; Structure; Survival Rate; System; T-Cell Activation; T-Cell Leukemia; T-Lymphocyte; Technology; Testing; Therapeutic; Translating; Transplantation; Treatment Efficacy; Treatment Protocols; antigen binding; burden of illness; cancer cell; cancer therapy; cell killing; cellular transduction; chemotherapy; chimeric antigen receptor; cytotoxic; design; extracellular; gene therapy; genome editing; improved; in vitro testing; in vivo; killings; mouse model; neoplastic cell; novel; novel strategies; prevent; receptor; response; trafficking; treatment strategy; tumor; virtual; γδ T cells

PROJECT SUMMARY/ABSTRACT Current chemotherapy treatment for children with relapsed T-cell leukemia has a survival rate of less than 15%. Although children eligible for allogeneic hematopoietic stem cell transplant (HSCT) have a nearly 50% chance of survival, disease remission is necessary and is seldom achieved in relapsed T-cell leukemia. Immunotherapy using chimeric antigen receptors (CARs) is emerging as a powerful technology for targeted cancer therapeutics that can be utilized for the treatment of relapsed T-cell leukemia. CARs are predicted to provide specific targeting of tumor cells, unlike currently used anti-cancer agents, and could potentially provide children with relapsed T-cell disease the option to undergo allogeneic HSCT. Unfortunately, the clinical usefulness of CARs in numerous cancers has varied from producing curative results with virtually no side effects, to severe adverse events and even death. Clearly there is need to understand and improve upon this emerging technology. The antigen-binding domain of CARs, which generally consist of a single-chain variable fragment (scFv), is used to direct and activate genetically engineered cytotoxic immune cells. The expression of CARs, such as CD19-CARs, on T cells has demonstrated potent and successful immunotherapy for B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) and multiple myeloma in clinical trials. However, no successful CAR therapy for treating T-cell leukemia patients has been established, largely due to on-target but off-tumor effects. We propose to generate a CAR targeting the CD5 antigen on T cells and deliver this construct to innate immune γδ T cells and test the engineered cells in vitro and in vivo using a T-cell leukemia mouse model. It is proposed that γδ T cells are advantageous compared to αβ T cells because they do not form memory responses, which are predicted to be lethal when directed against T cells. Also, redirecting T cells to T-cell antigens causes CAR-modified T cells to self-activate and kill each other instead of the leukemic T cells. Therefore, we propose to knockout CD5 in γδ T cells using CRISPR/Cas9 technology. We designed and tested guide RNAs in conjunction with Cas9 using a single expression plasmid, which we showed is functional using a T-cell line. Additionally, our laboratory demonstrated the effectiveness of replacing the scFv domain of CARs with a variable lymphocyte receptor (VLR). VLRs are the main component of the lamprey adaptive immune system and are analogous to immunoglobulin, however, they have a fundamentally different structure resulting in novel binding partners inaccessible to scFvs. We have cloned an anti-CD5-VLR onto a CAR and confirmed its function on effector cells in vitro. We have also demonstrated lentiviral transduction of γδ T cells as a means of CAR delivery. Ultimately, our goal is to better understand how to engineer γδ T cells, but foremost to provide a treatment strategy to induce a state of remission for T-cell leukemia patients, allowing them to be eligible for HSCT. !

项目摘要/摘要 目前治疗儿童复发性T细胞白血病的化疗存活率低于 15%。尽管符合异基因造血干细胞移植(HSCT)条件的儿童有近50% 存活的机会，疾病的缓解是必要的，很少能在复发的T细胞白血病中实现。 使用嵌合抗原受体(Car)的免疫治疗正在成为靶向治疗的一项强大技术。 可用于治疗复发性T细胞白血病的癌症疗法。据预测，汽车将 与目前使用的抗癌药物不同，提供针对肿瘤细胞的特异性靶向，并可能提供 复发T细胞疾病的儿童可以选择接受异基因造血干细胞移植.不幸的是，临床上 汽车在多种癌症中的效用各不相同，从产生疗效到几乎没有副作用。 影响，严重的不良事件，甚至死亡。显然，有必要理解和改进这一点 新兴技术。CARS的抗原结合域，通常由单链变量组成 单链抗体(ScFv)，用于引导和激活基因工程细胞毒免疫细胞。表达方式 T细胞上的CARS，如CD19-CARS，已经证明对B细胞的免疫治疗是有效的和成功的 急性淋巴细胞白血病、慢性淋巴细胞白血病和多发性骨髓瘤的临床研究 审判。然而，目前还没有成功的CAR疗法用于治疗T细胞白血病患者，主要是 由于靶点上但肿瘤外的影响。我们建议生成一种针对T细胞上CD5抗原的CAR，并 将此构建物传递给先天免疫γδT细胞，并使用T细胞在体外和体内测试工程细胞 白血病小鼠模型。有人认为γδT细胞比αβT细胞更有优势，因为它们 不要形成记忆反应，当针对T细胞时，记忆反应被预测为致命的。另外， 将T细胞重定向到T细胞抗原会导致CAR修饰的T细胞自我激活并相互杀死，而不是 白血病T细胞。因此，我们建议使用CRISPR/CAS9技术来敲除γδT细胞中的CD5。我们 设计并测试了与CAS9结合的GUIDE RNA，使用的是单个表达载体，我们 使用T细胞系显示的是正常的。此外，我们的实验室还证明了更换 具有可变淋巴细胞受体(VLR)的CARS单链抗体结构域。VLR是VLR的主要组件 七鳃鳗适应性免疫系统和免疫球蛋白类似，然而，它们从根本上有 不同的结构导致新的结合伙伴不能与单链抗体结合。我们已经克隆了一个抗CD5-VLR 并在体外确认了其对效应细胞的功能。我们还展示了慢病毒 转导γδT细胞作为CAR传递的一种方式。最终，我们的目标是更好地了解如何 设计γδT细胞，但最重要的是提供一种诱导T细胞缓解状态的治疗策略 白血病患者，使他们有资格接受造血干细胞移植。 好了！