Modulating extracellular TCR-CD3 interactions to identify new melanoma immunotherapy targets

摘要 黑色素瘤是皮肤癌中最致命和最具侵略性的形式之一。转移性肿瘤的主要治疗选择 黑色素瘤涉及使用检查点阻断（抗PD-1、抗CTLA-4等）的T细胞免疫疗法，或 使用修饰的患者T细胞的过继性T细胞疗法（ACT）。虽然，持久的反应只观察到在一个 部分患者。通过研究和靶向不同的T细胞信号传导， 途径。一种这样的途径是T细胞受体（TCR）信号传导途径。T细胞对抗原的识别 TCR和通过周围CD 3亚基产生的近端信号传导是免疫应答的关键步骤。 启动肿瘤杀伤。TCR和CD 3亚基之间特异性细胞外接触的鉴定 为通过靶向信号调节T细胞免疫的免疫策略提供了精确的指导 通过TCR-CD 3复合物。以前的研究靶向抗原结合位点以增强T细胞 对肿瘤抗原的反应通常导致脱靶效应和毒性。根据我们最近的TCR-CD 3 结构，我们突变了特异性CD 3相互作用TCR残基，导致不同的T细胞细胞因子， 应答我们的假设是，通过以特定的方式调节TCR-CD 3相互作用，免疫介导的免疫应答可能会增加。 可以增加对肿瘤抗原的细胞毒性而不丧失对癌抗原的特异性。来测试我们 假设，在目标1中，体外逆转录病毒TCR展示方法和新的CD 3-四聚体测定将用于 突变特异性TCR残基，并鉴定与CD 3相互作用的TCR，其亲和力增加，导致T 细胞介导更有效的功能。在目标2中，将使用基于结构的TCR设计来识别 计算机模拟具有增强的CD 3结合能力的TCR突变体。在这两种情况下，鉴定出的突变将是 并将分析它们的体外/体内肿瘤杀伤功效以验证 新TCR的抗肿瘤潜力，目标是开发新一波有效的T细胞疗法， 黑素瘤

ABSTRACT Melanoma is one of the deadliest and aggressive form of skin cancer. Main treatment options in metastatic melanoma involve T cell immunotherapy using checkpoint blockade (anti-PD-1, anti-CTLA-4 and others) or adoptive T cell therapy (ACT) with modified patient T cells. Although, durable response is only observed in a fraction of patients. Further progress can be made by studying and targeting different T cell signaling pathways. One such pathway is the T cell receptor (TCR) signaling pathway. T cell recognition of antigen by the TCR and the resulting proximal signaling through the surrounding CD3 subunits are key steps in the initiation of tumor-killing. Identification of the specific extracellular contacts between the TCR and CD3 subunits gives precise guidance for immunotherapeutic strategies that modulate T-cell immunity by targeting signaling through the TCR-CD3 complex. Previous studies that targeted the antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity. Based on our recent TCR-CD3 structure, we mutated specific CD3 interacting TCR-residues that resulted in different T cell cytokine responses. Our hypothesis is that by modulating TCR-CD3 interactions in specific ways, immune-mediated cytotoxicity to tumor antigens can be increased without losing specificity for the cancer antigen. To test our hypothesis, in Aim 1, an in vitro retroviral TCR display method and a novel CD3-tetramer assay will be used to mutate specific TCR-residues and identify TCRs that interact with CD3 with increased affinity, resulting in T cells that mediate more effective functionality. In Aim 2, a structure-based TCR design will be used to identify TCR mutants with enhanced CD3 binding ability in silico. In both instances, identified mutations will be introduced into gp100-specific TCRs and their in vitro/in vivo tumor killing efficacy will be analyzed to validate the anti-tumor potential of new TCRs with the goal of developing new wave of effective T cell therapies against melanoma.