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

项目摘要 该提案旨在培养一个双学位，DVM-PhD学生的职业生涯作为一个实验室动物兽医和独立 科学家该提案中概述的研究将开发一个临床前平台，以评估犬的CAR-T治疗 模型嵌合抗原受体（CAR）-T细胞已诱导高达90%的治疗缓解率 复发性/难治性B细胞恶性肿瘤。虽然小鼠有助于CAR-T的进展，但CAR-T治疗对固体 这种近亲繁殖的免疫缺陷模型阻碍了肿瘤的生长。宠物狗是一个更高的保真度翻译模型 这是由于它们的远系繁殖遗传学、完整的免疫系统、高癌症发病率和相似的癌症生物学。 CAR是由T细胞受体信号传导结构域、共刺激结构域和基于抗体的结构域组成的融合蛋白。 结合域汽车被体外引入患者的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”，其中治疗的功效不仅依赖于CAR的设计，而且还依赖于CAR基因的表达。 CAR-T细胞如何能够回到肿瘤并引发抗肿瘤作用。CAR帮助T细胞“识别” 肿瘤，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内在的T细胞生物学。到 为了充分评估体内CAR-T细胞功能，CAR的设计（目标1）和患者T细胞生物学（目标2）将是 评估。 目的1 -确定靶向肿瘤相关抗原GD 2、FolR 1和CD 20的最佳CAR设计。CAR构建体 将针对肿瘤相关抗原GD 2、FolR 1和CD 20设计。这些汽车将被引入到主要的 犬T细胞通过慢病毒载体。将通过IFNγ评估CAR-T细胞对抗原阳性肿瘤的功效。 ELISA、IL-2 ELISA和Incucyte活细胞视频显微镜检查。这些构建体中的每一个将用CAR共刺激因子进行测试。 结构域4-1BB和CD 28。具有最强反应性的CAR构建体将用NOD scid 图10示出了在γ（NSG）小鼠异种移植物模型中测量肿瘤生长抑制和体内CAR-T扩增的方法。 目的2 -确定CAR-T细胞的哪个子集优先运输并在体内持续存在于肿瘤中。评价 CAR-T细胞亚群对体内抗肿瘤功效的各自贡献，半随机核苷酸条形码将是 添加到CAR构建体中，允许在CAR-T生产、输注和后处理期间跟踪克隆谱系。 小鼠中的移植。使用单细胞测序，将CAR-T输注产品的克隆多样性与 克隆多样性。将鉴定优先归巢并在肿瘤中扩增的CAR-T亚组。 总之，这些目标将为未来在犬模型中进行CAR-T治疗的研究奠定基础。目标1将提供 候选CAR构建体在犬模型中进行评估。目标2将提供一种理解CAR-T如何 细胞在体内运输到肿瘤并在肿瘤内存留。该平台将用于筛选和完善新的方法， 高保真、高通量动物模型中的CAR-T疗法。

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.