Dogs as a high fidelity, high throughput model to evaluate CAR-T cell function and dysfunction

项目摘要 这项提案旨在培养一名双学位、DVM-博士生，将来成为一名独立的实验室动物兽医 科学家。这项提案中概述的研究将开发一个临床前平台来评估犬的CAR-T疗法 模特。嵌合抗原受体(CAR)-T细胞诱导的治疗缓解率高达90% 复发/难治性B细胞恶性肿瘤。虽然小鼠对CAR-T的进展起到了重要作用，但CAR-T治疗固体 这种近亲繁殖、免疫缺陷的模式阻碍了肿瘤的发展。宠物狗是一种更高保真的翻译模式 由于他们近亲繁殖的基因，完整的免疫系统，癌症的高发病率，以及相似的癌症生物学。 CAR是一种融合蛋白，由T细胞受体信号转导结构域、共刺激结构域和抗体组成 结合结构域。CARS在体外被引入患者的T细胞，使T细胞能够直接识别肿瘤抗原。 CAR-T细胞是一种“活疗法”，它的疗效不仅取决于汽车的设计，还取决于 CAR-T细胞是如何定位于肿瘤并产生抗肿瘤作用的。这辆车帮助T细胞“识别” 但这些细胞的运输、持久性和效应功能在很大程度上依赖于固有的T细胞生物学。至 充分评估CAR-T细胞在体内的功能，CAR的设计(目标1)和患者T细胞生物学(目标2)将 已评估。 目的1-确定靶向肿瘤相关抗原GD2、FolR1和CD20的最佳CAR设计。汽车构造 将为肿瘤相关抗原GD2、FolR1和CD20设计。这些汽车将被引入小学 通过慢病毒载体获得犬T细胞。干扰素γ将评估CAR-T细胞对抗原阳性肿瘤的疗效 酶联免疫吸附试验、白介素2酶联免疫吸附试验和印迹细胞活细胞视频显微镜检查。这些构造中的每一个都将用CAR共刺激进行测试 结构域4-1BB和CD28。反应性最强的CAR结构将通过NOD SCID进行进一步评估 伽马(NSG)小鼠异种移植模型，检测体内肿瘤生长抑制和CAR-T扩张。 目的2-确定CAR-T细胞的哪个亚群在体内优先通过并持续存在于肿瘤中。要评估 CAR-T细胞亚群对体内抗肿瘤疗效的各自贡献，半随机核苷酸条形码 添加到CAR结构中，允许跟踪CAR-T生产、输液和后克隆血统 在小鼠体内植入。使用单细胞测序，CAR-T输液产品的克隆多样性将与 肿瘤内克隆多样性。将确定优先在肿瘤中定位和扩展的CAR-T亚群。 总而言之，这些目标将为未来在犬模型中进行CAR-T疗法的研究奠定基础。AIM 1将提供一个 将在犬类模型中评估的候选汽车构造。目标2将提供一种方法来理解CAR-T如何 在体内，细胞运输到肿瘤并在肿瘤内持续存在。该平台将用于筛选和改进新的方法，以 在高保真、高通量的动物模型中进行CAR-T治疗。

Project Summary This proposal aims to train a dual-degree, DVM-PhD student for a career as a lab animal veterinarian and independent scientist. The research outlined in this proposal will develop a pre-clinical platform to evaluate CAR-T therapy in a canine model. Chimeric antigen receptor (CAR)-T cells have induced up to 90% remission rates for treatment relapsed/refractory B cell malignancies. While mice have been instrumental to CAR-T progress, CAR-T therapy for solid tumors have been hampered by this inbred, immunodeficient model. Pet dogs are a higher fidelity translational model due to their outbred genetics, intact immune system, high incidence of cancer, and similar cancer biology. A CAR is a fusion protein comprised of a T cell receptor signaling domain, costimulatory domain, and an antibody based binding domain. CARs are introduced to patients’ T cells ex vivo, enabling the T cells to directly recognize tumor antigen. CAR-T cells are a “living therapy” wherein the efficacy of the treatment relies not only on the design of the CAR, but also how the CAR-T cells are able to home to the tumor and elicit anti-tumor effects. The CAR helps T cells to “recognize” the tumor, but the trafficking, persistence, and effector function of these cells relies heavily on intrinsic T cell biology. To adequately assess CAR-T cell function in vivo, design of the CAR (Aim 1) and patient T cell biology (Aim 2) will be evaluated. Aim 1 – Determine optimal CAR design for targeting tumor associated antigens GD2, FolR1, and CD20. CAR constructs will be designed for the tumor associated antigens GD2, FolR1, and CD20. These CARs will be introduced to primary canine T cells via a lentiviral vector. The CAR-T cells will be evaluated for efficacy against antigen positive tumors by IFNγ ELISA, IL-2 ELISA, and Incucyte live cell videomicroscopy. Each of these constructs will be tested with CAR costimulatory domains 4-1BB and CD28. The CAR constructs with the strongest reactivity will be further evaluated with an NOD scid gamma (NSG) mouse xenograft model, measuring tumor growth inhibition and CAR-T expansion in vivo. Aim 2 – Determine which subset of CAR-T cells preferentially traffic and persist in the tumor in vivo. To evaluate the respective contribution of CAR-T cell subsets to anti-tumor efficacy in vivo, semi-random nucleotide barcodes will be added to the CAR constructs allowing for the tracking of clonal lineage during CAR-T production, infusion, and post- engraftment in mice. Using single cell sequencing, clonal diversity of the CAR-T infusion product will be compared to clonal diversity intratumorally. Subsets of CAR-T that preferentially home to and expand in the tumor will be identified. Together, these aims will set the basis for future studies of CAR-T therapy in a canine model. Aim 1 will provide a candidate CAR construct to be evaluated in a canine model. Aim 2 will provide a method for understanding how CAR-T cells traffic to and persist within a tumor in vivo. This platform will be used to screen and refine novel approaches to CAR-T therapy in a high-fidelity, high-throughput animal model.