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.

项目概要/摘要 免疫系统已经发展到能够识别和靶向外来或突变的细胞内蛋白质来防御 对抗癌症、病毒和感染。细胞内蛋白质主要以肽的形式存在 细胞表面的组织相容性复合物，可被 CD8+ 上的 T 细胞受体 (TCR) 识别 T细胞。 TCR 对癌症治疗很有吸引力，因为它们可以特异性靶向肽-MHC (pMHC) 在众多患者和癌症中发现的抗原，称为共享抗原。共有抗原有 已从癌症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 已从患者样本中鉴定出抗原。这些 TCR 已成功用于治疗 当在自体 T 细胞中外源表达并回输时，许多患者都出现了这种情况。同时，肿瘤反应性 TCR在治疗方面具有巨大的应用潜力，但确定肿瘤反应靶点仍然具有挑战性 TCR。这限制了可以作为治疗目标的已知共享抗原的库。在这里我们建议 一种识别 TCR 靶标的高通量方法，从而增加基于 TCR 的潜力 疗法并识别新的共享抗原。为了识别 TCR 的靶标，编码 DNA 文库 肽将用于呈现大量具有代表性的 pMHC。肽库旨在 负责大多数 TCR 接触的位置具有可变性，使文库偏向于更多 反应性的。为了以功能相关的方式确定 TCR 靶点，依靠 T 细胞的细胞毒性， 将进行共培养耗尽筛选。从屏幕上耗尽的肽被视为命中。这 命中将用于识别与 TCR 反应性相关的肽结合基序。从临床上这些主题 将确定相关的潜在目标。将使用单肽共培养杀伤来验证目标 测定和 ELISpot 用于鉴定干扰素 γ（T 细胞反应性标记物）。这项技术将 用于从肿瘤浸润淋巴细胞中识别 TCR 的临床相关靶标。