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. !

项目概要/摘要 目前复发性T细胞白血病儿童的化疗存活率低于 15%。尽管符合异基因造血干细胞移植（HSCT）条件的儿童有近 50% 为了提高生存机会，疾病缓解是必要的，但在复发性 T 细胞白血病中很少能实现这一目标。 使用嵌合抗原受体 (CAR) 的免疫疗法正在成为针对靶向药物的强大技术。 可用于治疗复发性 T 细胞白血病的癌症疗法。 CAR 预计将 与目前使用的抗癌药物不同，它可以特异性靶向肿瘤细胞，并且有可能提供 患有复发性 T 细胞疾病的儿童可以选择接受同种异体 HSCT。不幸的是，临床 CAR 在许多癌症中的用途各不相同，从产生几乎没有副作用的治疗结果到 的影响，导致严重的不良事件甚至死亡。显然，需要理解并改进这一点 新兴技术。 CAR的抗原结合结构域，一般由单链变量组成 片段（scFv），用于指导和激活基因工程细胞毒性免疫细胞。表达式 T 细胞上的 CAR（例如 CD19-CAR）已证明对 B 细胞具有有效且成功的免疫疗法 临床上急性淋巴细胞白血病（B-ALL）、慢性淋巴细胞白血病（CLL）和多发性骨髓瘤 试验。然而，目前尚未建立成功的 CAR 疗法来治疗 T 细胞白血病患者，主要是 由于靶向但非肿瘤效应。我们建议生成针对 T 细胞上 CD5 抗原的 CAR 将这种构建体传递给先天免疫 γδ T 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。有人提出，γδ T 细胞与 αβ T 细胞相比具有优势，因为它们 不形成记忆反应，预计当针对 T 细胞时，记忆反应将是致命的。还， 将 T 细胞重定向至 T 细胞抗原，导致 CAR 修饰的 T 细胞自我激活并互相杀死，而不是 白血病T细胞。因此，我们建议利用CRISPR/Cas9技术敲除γδT细胞中的CD5。我们 使用单一表达质粒与 Cas9 一起设计并测试了指导 RNA，我们将其 使用 T 细胞系显示出功能。此外，我们的实验室证明了替代的有效性 具有可变淋巴细胞受体（VLR）的 CAR 的 scFv 结构域。 VLR 是 七鳃鳗适应性免疫系统与免疫球蛋白类似，但是，它们具有根本性的 不同的结构导致 scFv 无法接触到新的结合伴侣。我们克隆了抗CD5-VLR 到 CAR 上并在体外证实了其对效应细胞的功能。我们还展示了慢病毒 γδ T 细胞转导作为 CAR 递送的一种手段。最终，我们的目标是更好地了解如何 改造 γδ T 细胞，但最重要的是提供诱导 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 HSCT。 ！