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

狗作为高保真、高通量模型来评估 CAR-T 细胞功能和功能障碍

基本信息

批准号：
10314748
负责人：
Samuel Brill
金额：
$ 3.8万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-15 至 2026-09-14
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10314748
关键词：
Animal Model Animals Antibodies Antigens B lymphoid malignancy B-Cell Lymphomas Back Bar Codes Binding Biology Biopsy Breast Carcinoma CD28 gene Cancer Biology Canis familiaris Cell physiology Cells Cellular biology Cellular immunotherapy Chemicals Chimeric Proteins Clone Cells Coculture Techniques Complement Contracts Disease remission Engraftment Enzyme-Linked Immunosorbent Assay Ethics Evolution Fc Receptor Functional disorder Future Generations Genetic Health Hematologic Neoplasms Home Homing Human Immune Immune system Immunocompetent Immunodeficient Mouse In Vitro Inbreeding Incidence Individual Infusion procedures Interferon Type II Interleukin-2 Kinetics Lentivirus Vector MS4A1 gene Malignant Neoplasms Measurement Measures Methods Modeling Molecular Mus Neuroblastoma Nucleotides Patients Phenotype Procedures Production Receptor Signaling Refractory Relapse Research Sampling Scientist Solid Neoplasm T-Cell Receptor T-Lymphocyte T-Lymphocyte Subsets Testing TimeLine Training Translating Treatment Efficacy Tumor Antigens Tumor Cell Line Veterinarians Video Microscopy Xenograft Model antitumor effect base career cellular transduction chimeric antigen receptor chimeric antigen receptor T cells complement system cost design doctoral student driving force efficacy evaluation in vivo in vivo imaging system melanoma mouse model novel novel strategies osteosarcoma pre-clinical preclinical efficacy restraint single cell sequencing success trafficking transcriptomics translational model tumor tumor growth

项目摘要

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.

项目摘要该提案旨在培训双度，DVM-PHD学生的职业，从事实验室动物兽医和独立的职业科学家。该提案中概述的研究将开发一个临床前平台，以评估犬类的CAR-T治疗模型。嵌合抗原受体（CAR）-T细胞已诱导高达90％的治疗率复发/难治性B细胞恶性肿瘤。虽然小鼠对CAR-T进展有用，但CAR-T治疗固体这种近交，免疫缺陷模型阻碍了肿瘤。宠物犬是更高的忠诚转化模型由于它们的遗传学，完整的免疫系统，癌症的高入射以及类似的癌症生物学。汽车是融合蛋白完成的T细胞受体信号传导结构域，共刺激结构域和基于抗体的融合蛋白结合域。将汽车引入患者的T细胞后体内，使T细胞能够直接识别肿瘤抗原。 CAR-T细胞是一种“生活疗法”，其中治疗的效率不仅依赖于汽车的设计，还依赖于汽车的设计 CAR-T细胞如何能够进入肿瘤并引起抗肿瘤作用。该汽车帮助T细胞“识别” 肿瘤，但是这些细胞的运输，持久性和效应子功能在很大程度上依赖于内在的T细胞生物学。到充分评估体内CAR-T细胞功能，汽车的设计（AIM 1）和患者T细胞生物学（AIM 2）将是评估。 AIM 1 - 确定针对靶向肿瘤相关抗原GD2，FOLR1和CD20的最佳汽车设计。汽车构造将针对肿瘤相关的抗原GD2，FOLR1和CD20设计。这些汽车将被介绍给主要犬T细胞通过慢病毒载体。将评估CAR-T细胞的IFNγ对抗原阳性肿瘤的效率 ELISA，IL-2 ELISA和Incucyte Live细胞视频显微镜。这些结构中的每一个都将通过CAR COTUMIMUNATIO进行测试域4-1BB和CD28。具有强反应性的汽车构造将通过点头进行进一步评估伽马（NSG）小鼠异种移植模型，测量体内肿瘤生长抑制和CAR-T扩展。 AIM 2 - 确定哪个CAR-T细胞的子集优先流量，并在体内持续存在。评估 CAR-T细胞子集对体内抗肿瘤效率的各自贡献，半随机核苷酸条形码将是添加到汽车构建体中，可以在CAR-T生产，输液和后的Clonal谱系跟踪克隆谱系在小鼠中植入。使用单细胞测序，将将CAR-T输液产品的克隆多样性与克隆多样性在肿瘤内。将确定优先在肿瘤中和膨胀的CAR-T子集。这些目标共同为在犬类模型中进行CAR-T治疗的未来研究奠定了基础。 AIM 1将提供候选汽车构造将在犬模型中评估。 AIM 2将提供一种理解Car-T的方法细胞在体内肿瘤中流动并持续存在。该平台将用于筛选和完善新颖的方法高保真，高通量动物模型中的CAR-T治疗。