Development of antigen multimers for CAR T cell detection and functional profiling

项目总结 CAR T细胞疗法为复发/难治性B细胞恶性肿瘤患者提供了新的希望，并已成为 抗癌战争中的另一个武器。尽管汽车疗法取得了成功，也令人兴奋，但敏感和 在研究中，准确的CAR T细胞检测和图谱需要通用的车标试剂， 工业，以及在临床上。为了提高CAR T细胞治疗的疗效，科学家、临床医生和药剂师 行业需要准确和多功能的汽车染色试剂来进行下游分析，进行 临床前研究，患者生物标本概况，并开发新的汽车。为了克服这一技术差距，我们 开发了抗原多聚体-高亲和力CAR检测试剂。类似于传统的MHC多聚体，我们的 初步数据表明，抗原多聚体敏感和特异地检测CD19导向的CAR T 商品输液产品、外周血中的细胞以及弥漫性大B细胞淋巴瘤的肿瘤活检组织中的细胞 病人。此外，我们发现CD19-多聚体可以很容易地用于从患者身上分离CAR T细胞。 不同处理阶段的标本进行下游流式细胞术和单细胞多组学分析。在我们的 应用程序，基于我们令人兴奋的初步观察，我们建议测试总体假设 抗原多聚体将使CAR T细胞检测和分离成为可能，并促进下游的单细胞分析 从病人的生物标本中提取CAR T细胞。我们的抗原多聚体可以用来产生新的知识 关于CAR T细胞的功能和体内疗效。 在特定的目标1，我们建议开发抗原多聚体用于高灵敏度的CAR T细胞检测和 与世隔绝。除了通过生成4种不同的汽车检测来展示我们技术的灵活性之外 ，我们还将开发同时分离和重新刺激CAR T细胞的方法 从患者样本中提取，以便进行下游表型分析和功能分析。 在特定目标2中，我们建议开发抗原多聚体使能的单个CAR T细胞多色流 细胞学和多组学。我们将首先使用CD19-多聚体来确定CAR的表达，细胞数量， 多色流式细胞术检测CAR T细胞的分化、功能表型和持久性。接下来，我们将 使用CD19多聚体从应答者和无应答者中分离CAR T细胞，用于后续的单次... 细胞多组学分析。利用两名具有互补专业知识的调查人员的经验 免疫工程(Huang)和翻译/临床CAR T细胞治疗研究(Kline)，以及 UChicago细胞治疗生物库，其中包含数百名淋巴瘤和骨髓瘤患者的治疗样本 通过CD19或BCMA导向的CAR T细胞治疗，拟议的实验将建立抗原多聚体为 一类具有广泛研究和临床应用的新型CAR染色试剂。

PROJECT SUMMARY CAR T cell therapy offers new hope to patients with relapsed/refractory B cell malignancies and has become another weapon in the war on cancer. Despite the success and excitement around CAR therapies, sensitive and universal CAR-labeling reagents are required for accurate CAR T cell detection and profiling in research, industry, and in the clinic. To improve CAR T cell therapy efficacy, scientists, clinicians, and the pharmaceutical industry need accurate and multifunctional CAR-staining reagents to perform downstream assays, conduct preclinical studies, profile patient biospecimens, and develop new CARs. To overcome this technology gap, we developed antigen-multimers – high-avidity CAR detection reagents. Akin to conventional MHC multimers, our preliminary data demonstrate that antigen-multimers sensitively and specifically detect CD19-directed CAR T cells in commercial infusion products, in peripheral blood, and in tumor biopsies of diffuse large B cell lymphoma patients. Furthermore, we show that CD19-multimers can be readily employed to isolate CAR T cells from patient samples at different treatment stages for downstream flow cytometry and single-cell multi-omics analyses. In our application, based on our exciting preliminary observations, we propose to test the overarching hypothesis that antigen-multimers will enable CAR T cell detection and isolation and facilitate downstream single-cell analyses of CAR T cells from patient biospecimens. Our antigen-multimers can be used to generate new knowledge regarding CAR T cell function and in vivo efficacy. In Specific Aim 1, we propose to develop antigen-multimers for high-sensitivity CAR T cell detection and isolation. In addition to demonstrating the flexibility of our technology by generating 4 different CAR-detecting antigen-multimers, we will also develop approaches for simultaneously isolating and restimulating CAR T cells from patient samples in order to enable downstream phenotyping and functional analyses. In Specific Aim 2, we propose to develop antigen-multimer-enabled single CAR T cell multicolor flow cytometry and multi-omics. We will first use CD19-multimers to determine CAR expression, cell number, differentiation, functional phenotype, and persistence of CAR T cells by multicolor flow cytometry. We will next use CD19-multimers to isolate CAR T cells from responder and non-responder patients for subsequent single- cell multi-omics analyses. Leveraging the experience of two investigators with complementary expertise in immunoengineering (Huang) and translational/clinical CAR T cell therapy research (Kline), as well as the UChicago Cell Therapy Biobank which contains hundreds of lymphoma and myeloma patient samples treated with CD19- or BCMA-directed CAR T cell therapy, the proposed experiments will establish antigen-multimers as a new class of CAR-staining reagents with broad research and clinical applications.