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

项目摘要/摘要 免疫系统的发展是为了识别和定位外来的或突变的细胞内蛋白来防御 对抗癌症、病毒和感染。细胞内的蛋白质主要以多肽的形式存在 CD8+表面T细胞受体(TCR)识别的细胞表面组织相容性复合体 T细胞。TCRs因能特异性靶向多肽-MHC(PMHC)而成为癌症治疗的靶点。 在许多患者和癌症中发现的抗原，称为共同抗原。共同的抗原已经 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 已从患者样本中识别出抗原。这些TCR已经成功地作为治疗在 许多患者在外源性表达自体T细胞并回输时。同时，肿瘤反应 TCRs在治疗中有很大的应用潜力，但识别肿瘤反应的靶点仍然具有挑战性 TCR。这限制了可以作为治疗靶点的已知共享抗原的保留范围。在这里，我们建议 一种高通量识别TCR目标的方法，从而提高了基于TCR的潜力 治疗和识别新的共同抗原。为了识别TCR的靶标，TCR是一个编码DNA文库 多肽将被用来呈现大量具有代表性的pMHC。多肽库的设计目的是 在负责大多数TCR联系人的位置上有变异性，偏向于库更多 被动的。根据T细胞的细胞毒性，以功能相关的方式确定TCR靶点， 将进行共培养耗竭筛查。从屏幕上耗尽的多肽被认为是热门。这个 HITS将用于识别与TCR反应性相关的肽结合基序。从临床上看这些图案 将确定相关的潜在目标。靶标将通过单肽共培养杀灭进行验证。 检测和ELISpot用于鉴定干扰素--一个T细胞反应性的标志物。这项技术将是 用于从肿瘤浸润性淋巴细胞中识别TCRs的临床相关靶点。

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.