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

项目摘要 CAR T细胞疗法为复发性/难治性B细胞恶性肿瘤患者提供了新的希望， 是对抗癌症的另一种武器尽管CAR疗法取得了成功和令人兴奋，但敏感和 研究中需要通用CAR标记试剂用于准确的CAR T细胞检测和分析， 工业，在诊所。为了提高CAR T细胞治疗的疗效，科学家，临床医生和制药公司 工业上需要精确和多功能的CAR染色试剂来进行下游测定， 临床前研究、分析患者生物样本并开发新的汽车。为了克服这一技术差距， 开发了抗原-多聚体-高亲合力CAR检测试剂。类似于传统的MHC多聚体，我们的 初步数据表明，抗原多聚体敏感且特异地检测CD 19指导的CAR T， 商业输注产品、外周血和弥漫性大B细胞淋巴瘤的肿瘤活检中的细胞 患者此外，我们表明CD 19多聚体可以容易地用于从患者中分离CAR T细胞， 在不同的治疗阶段的样本进行下游流式细胞术和单细胞多组学分析。在我们 应用程序，基于我们令人兴奋的初步观察，我们建议测试的总体假设， 抗原多聚体将能够检测和分离CAR T细胞，并促进下游单细胞分析 的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细胞功能和体内功效。 在具体目标1中，我们提出开发用于高灵敏度CAR T细胞检测的抗原多聚体， 隔离除了通过生成4种不同的CAR检测来展示我们技术的灵活性之外， 抗原多聚体，我们还将开发同时分离和再刺激CAR T细胞的方法 以使下游表型和功能分析成为可能。 在具体目标2中，我们提出开发抗原多聚体使能的单个CAR T细胞微流 流式细胞术和多组学。我们将首先使用CD 19多聚体来确定CAR表达，细胞数量， 通过流式细胞术检测CAR T细胞的分化、功能表型和持久性。我们接下来将 使用CD 19多聚体从应答者和非应答者患者中分离CAR T细胞，用于随后的单- 细胞多组学分析。利用两名具有互补专长的调查员的经验， 免疫工程（Huang）和翻译/临床CAR T细胞治疗研究（Kline），以及 芝加哥大学细胞治疗生物库，其中包含数百个淋巴瘤和骨髓瘤患者样本治疗 使用CD 19或BCMA导向的CAR T细胞疗法，所提出的实验将建立抗原多聚体， 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.