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

摘要 黑色素瘤是最致命和侵袭性最强的皮肤癌之一。转移性肿瘤的主要治疗选择 黑色素瘤涉及T细胞免疫治疗，使用检查点阻断(抗PD-1、抗CTLA-4等)或 采用改良的患者T细胞进行过继T细胞治疗(ACT)。尽管，持久的反应只有在 病人的一部分。通过研究和针对不同的T细胞信号转导可以取得进一步的进展 小路。其中一个途径是T细胞受体(TCR)信号通路。T细胞对抗原的识别 TCR和由此产生的通过周围CD3亚单位的近端信号是 启动肿瘤杀灭。TCR和CD3亚基之间特异性胞外接触的鉴定 为通过靶向信号调节T细胞免疫的免疫治疗策略提供准确的指导 通过TCR-CD3复合体。先前针对抗原结合部位增强T细胞的研究 对肿瘤抗原的反应往往会导致非靶点效应和毒性。根据我们最新的TCR-CD3 结构，我们突变了特定的CD3与TCR残基相互作用，从而产生了不同的T细胞细胞因子 回应。我们的假设是，通过以特定方式调节TCR-CD3相互作用，免疫介导的 在不丧失肿瘤抗原特异性的情况下，可以增加对肿瘤抗原的细胞毒性。测试我们的 假设，在目标1中，将使用体外逆转录病毒TCR展示方法和新的CD3-四聚体分析来 突变特定的TCR残基，并鉴定与CD3相互作用的TCR，亲和力增强，导致T细胞 调节更有效功能的细胞。在目标2中，将使用基于结构的TCR设计来识别 在硅胶中CD3结合能力增强的TCR突变体。在这两种情况下，已识别的突变都将是 将其导入gp100特异性TCR，并对其体外/体内肿瘤杀伤效果进行分析以验证 以开发新一波有效的T细胞疗法为目标的新型TCR的抗肿瘤潜力 黑色素瘤。

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.