A High Throughput Method to Determine the Target of T Cell Receptors

一种确定 T 细胞受体靶标的高通量方法

• 批准号: 10065125
• 负责人: Heather Jones
• 金额: $ 4.55万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-02 至 2023-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10065125
• 关键词: Affect; Antigen Targeting; Antigens; Autologous; Back; Binding; Biological Assay; CD8-Positive T-Lymphocytes; Cancerous; Cell surface; Cells; Coculture Techniques; Complex; Cytotoxic T-Lymphocytes; DNA Library; Data; Development; Epitopes; Genetic; Human; Immobilization; Immune system; Individual; Infection; Interferon Type II; Libraries; Link; Major Histocompatibility Complex; Malignant Neoplasms; Methods; Mutate; Mutation; Noise; Patients; Peptide Library; Peptide Synthesis; Peptide/MHC Complex; Peptides; Positioning Attribute; Proteins; Research; Sampling; Surface; System; T-Cell Receptor; T-Lymphocyte; Technology; Testing; Therapeutic; Tumor-Infiltrating Lymphocytes; Virus Diseases; Yeasts; base; cancer genome; cancer testis antigen; cancer therapy; cancer type; candidate identification; clinically relevant; cytotoxicity; design; experimental study; melanoma; novel; screening; success; targeted treatment; tool; tumor

Project Summary/Abstract The immune system has developed to identify and target foreign or mutated intracellular proteins to defend against cancers, viruses and infections. Intracellular proteins are presented as peptides in major histocompatibility complexes on the surfaces of cells, which are recognized by T cell receptors (TCR) on CD8+ T cells. TCRs are attractive for cancer therapy because they can specifically target peptide-MHC (pMHC) antigens found across numerous patients and cancers, known as shared antigens. Shared antigens have been identified from cancer testis antigens and melanoma antigens. Endogenous TCRs targeting these antigens have been identified from patient samples. These TCRs have been successful as treatment in numerous patients when exogenously expressed in autologous T cells and reinfused. While, tumor reactive TCRs have a great potential for use in therapy, it remains challenging to identify the target of tumor reactive TCRs. This limits the repertoire of known shared antigens that can be targeted for therapy. Here we propose a high throughput method to identify the targets of TCRs, thereby increasing the potential of TCR based therapies and identifying novel shared antigens. To identify the targets of TCRs, a DNA library encoding peptides will be used to present a large array of representative pMHCs. The peptide libraries are designed to have variability in positions responsible for the majority of TCR contacts, biasing the libraries to be more reactive. To determine TCR targets in a functionally relevant manner, relying on the cytotoxicity of T cells, coculture depletion screens will be performed. Peptides depleted from the screens are considered hits. The hits will be used to identify peptide binding motifs related to TCR reactivity. From these motifs clinically relevant, potential targets will be identified. The targets will be validated using single peptide coculture killing assays and ELISpot for identification of interferon gamma, a marker of T cell reactivity. This technology will be used to identify clinically relevant targets of TCRs from tumor infiltrating lymphocytes.

项目摘要/摘要 免疫系统已经开发出识别和靶向外源或突变的细胞内蛋白来防御 反对癌症，病毒和感染。细胞内蛋白作为主要的肽表示 细胞表面上的组织相容性复合物在CD8+上被T细胞受体（TCR）识别 T细胞。 TCR对癌症治疗具有吸引力，因为它们可以特异性靶向肽-MHC（PMHC） 在许多患者和癌症中发现的抗原，称为共享抗原。共享抗原具有 从癌睾丸抗原和黑色素瘤抗原中鉴定出来。针对这些的内源性TCR 已经从患者样品中鉴定出抗原。这些TCR已成功作为治疗 许多患者在自体T细胞中外源表达并重新灌注。而，肿瘤反应性 TCR具有在治疗中的巨大潜力，确定肿瘤反应性的靶标仍然具有挑战性 TCR。这限制了可用于治疗的已知共享抗原的曲目。我们在这里提出 一种识别TCR目标的高通量方法，从而增加了基于TCR的潜力 疗法和识别新颖的共享抗原。要识别TCR的目标，DNA库编码 肽将用于呈现大量代表性PMHC。肽库设计为 在负责大多数TCR接触的职位方面有差异，偏向图书馆更加 反应性。以功能相关的方式确定TCR靶标，依靠T细胞的细胞毒性， 将执行共培养耗竭筛查。从屏幕上耗尽的肽被认为是命中。这 命中将用于识别与TCR反应性有关的肽结合基序。从临床上这些图案 相关的潜在目标将被确定。目标将使用单个肽共培养杀戮来验证 测定和ELISPOT用于鉴定干扰素伽玛，这是T细胞反应性的标志。这项技术将是 用于从肿瘤浸润淋巴细胞中鉴定出TCR的临床相关靶标。