Synthetic circuits that drive infiltration of therapeutic T cells into immunologically cold tumors

项目摘要/摘要 推动治疗性T细胞向免疫排斥肿瘤渗透的工程运输电路 工程化嵌合抗原受体(CAR)T细胞尚未达到对抗实体癌的效果。 尤其具有挑战性的是免疫排斥的“冷”肿瘤，这种肿瘤不会积累大量的浸润性肿瘤。 T细胞。在这种情况下，即使治疗性T细胞在体外识别并杀死肿瘤细胞，如果它们在体内失败，它们也会失败 不能渗入肿瘤。我们建议将调节T细胞运输的合成电路作为一种 推动治疗性T细胞向免疫寒冷肿瘤侵袭的一般策略。 免疫细胞自然依赖于复杂的贩运行为。它们巡视身体以监测疾病。一次 病变组织被识别后，它们会在当地定居并局部扩张。细胞贩运计划在很大程度上 依赖于三个核心细胞功能的调节：1)趋化(调节细胞进出)，2)细胞-细胞 黏附(减少细胞外流)；3)局部增殖信号(细胞因子信号)。虽然这些机制 自然地被T细胞利用，进化的途径很容易受到许多肿瘤的抑制 机械装置。我们假设合成的调节电路直接连接肿瘤抗原信号来控制 治疗性T细胞的趋化、黏附和局部增殖信号将改善靶向渗透 免疫排除肿瘤。我们建议通过In Silico的循环来开发合成贩运电路设计 建模和体外实验。我们将测试人工贩运电路是否可以提高CAR T细胞的效率，在 体内，使用免疫排除的胰腺癌的免疫活性小鼠模型。生成的单元格 贩运电路应适用于范围广泛的实体癌症以及其他疾病。 目的1.设计和表征协同调节趋化性的合成T细胞转运电路， 肿瘤抗原识别反应中的黏附和局部增殖 1.a.使用多尺度计算模型探索可能的T细胞贩运电路的设计空间。使用 用于确定显著增加肿瘤选择性渗透的电路结构和参数的模型 1.b.使用SynNotch受体构建模块化转运电路工具箱，以控制趋化、黏附、 和增殖反应肿瘤抗原识别；构建电路组合文库。 1.c.使用测量T细胞贩运的多室组织模型在体外测试合成贩运回路 和迁徙。通过测量双侧肿瘤异种移植小鼠模型中T细胞的转运来评估体内电路。 目的2.使用工程化转运回路提高免疫排斥疗法的抗肿瘤效果 免疫活性小鼠胰腺导管腺癌模型的建立。 利用合成转运回路改善小鼠α-Mesothelin CAR-T细胞的渗透和清除 KPC胰腺导管腺癌同基因小鼠模型。使用单细胞分析评估对 肿瘤抑制细胞、间质、宿主免疫细胞浸润和CAR T细胞耗竭。

Project summary/abstract Engineering trafficking circuits that drive therapeutic T cell infiltration into immune-excluded tumors Engineered chimeric antigen receptor (CAR) T cells have yet to achieve efficacy against solid cancers. Particularly challenging are immune-excluded “cold” tumors, which fail to accumulate large numbers of infiltrating T cells. In such cases, even if therapeutic T cells recognize and kill tumor cells in vitro, they will fail in vivo if they cannot infiltrate the tumor. We propose to engineer synthetic circuits that regulate T cell trafficking as a general strategy to drive therapeutic T cell infiltration into immunologically cold tumors. Immune cells naturally rely on complex trafficking behaviors. They patrol the body to surveil for diseases. Once diseased tissue is identified, they establish local residence and focally expand. Cell trafficking programs largely rely on regulation of three core cellular functions: 1) chemotaxis (modulating cell ingress and egress), 2) cell-cell adhesion (reducing cell egress), and 3) local proliferative signaling (cytokine signaling). While these mechanisms are naturally exploited by T cells, the evolved pathways are susceptible to suppression by numerous tumoral mechanisms. We hypothesize that synthetic regulatory circuits that directly wire tumor antigen signals to control therapeutic T cell chemotaxis, adhesion, and local proliferative signaling will improve targeted infiltration of immune excluded tumors. We propose to develop synthetic trafficking circuit designs through cycles of in silico modeling and in vitro experiments. We will test if synthetic trafficking circuits can improve CAR T cell efficacy, in vivo, using an immunocompetent murine model of immune-excluded pancreatic cancer. The resulting cell trafficking circuits should be applicable to a broad range of solid cancers, as well as other diseases. AIM 1. Design and characterize synthetic T cell trafficking circuits that coordinately regulate chemotaxis, adhesion and local proliferation in response to tumor antigen recognition 1.A. Use multi-scale computational modeling to explore design space of possible T cell trafficking circuits. Use model to identify circuit architectures and parameters that robustly increase tumor-selective infiltration 1.B. Construct a toolbox of modular trafficking circuits using synNotch receptors to control chemotaxis, adhesion, and proliferation in response to tumor antigen recognition; Construct combinatorial library of circuits. 1.C. Test synthetic trafficking circuits in vitro using multicompartment tissue models that measure T cell trafficking and migration. Evaluate circuits in vivo by measuring T cell trafficking in bilateral tumor xenograft mouse models. AIM 2. Use engineered trafficking circuits to improve anti-tumor efficacy in an immune excluded immunocompetent murine model of pancreatic ductal adenocarcinoma. Leverage synthetic trafficking circuits to improve murine α-Mesothelin CAR-T cell infiltration and clearance of KPC pancreatic ductal adenocarcinoma syngeneic mouse model. Use single cell analysis to assess impact on tumoral suppressor cells, stroma, host immune cell infiltration, and CAR T cell exhaustion.