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

项目总结/摘要 免疫系统已经发展到识别和靶向外源或突变的细胞内蛋白质以防御 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，其被CD 8+上的T细胞受体（TCR）识别 T细胞。TCR对于癌症治疗是有吸引力的，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。共有抗原具有 已从癌症睾丸抗原和黑素瘤抗原中鉴定出来。内源性TCR靶向这些 已经从患者样品中鉴定出抗原。这些TCR已成功作为治疗， 当在自体T细胞中外源表达并再输注时，而肿瘤反应性 TCR在治疗中具有巨大的应用潜力，但确定肿瘤反应性受体的靶点仍然具有挑战性。 TCR。这限制了可以靶向治疗的已知共有抗原的库。在这里我们建议 一种高通量的方法来鉴定TCR的靶点，从而增加基于TCR的免疫治疗的潜力。 治疗和鉴定新的共享抗原。为了鉴定TCR的靶点， 肽将用于呈现大量代表性pMHC。肽文库被设计成 在负责大多数TCR接触的位置上具有可变性，使文库偏向于更 反应性为了以功能相关的方式确定TCR靶，依赖于T细胞的细胞毒性， 进行共培养物耗竭筛选。从筛选中耗尽的肽被认为是命中。的 命中将用于鉴定与TCR反应性相关的肽结合基序。临床上从这些图案 将确定相关的潜在目标。将使用单肽共培养物杀伤来验证靶标 用于鉴定干扰素γ（一种T细胞反应性的标志物）的测定和ELISpot。这项技术将 用于从肿瘤浸润淋巴细胞中鉴定临床相关的TCR靶点。

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.