Engineering of computational receptors and gene circuits for T-cell immunotherapy

描述（由申请人提供）：连续性T细胞疗法是一种有前途的治疗策略，用于对常规方法（包括手术、化疗和放疗）具有抗性的癌症。特别地，遗传修饰以表达肿瘤靶向嵌合抗原受体（汽车）的T细胞的过继转移已经通过将T细胞特异性重定向至惰性肿瘤而显示出临床功效。然而，在使用CAR修饰的T细胞方面仍然存在重要的挑战，包括对正常细胞的脱靶毒性和对靶向肿瘤突变逃逸的敏感性。这项研究的目标是通过工程化更强大和多功能的肿瘤靶向T细胞来提高过继性T细胞治疗的安全性和有效性，这将通过两个具体目标来实现。在具体目标1中，将开发能够对多个输入信号进行逻辑计算的下一代汽车。将开发响应于多种肿瘤相关抗原而触发T细胞介导的细胞毒性的OR门控汽车，以降低突变逃逸的概率（即，所有靶向抗原的丢失）。将构建仅在存在正确的抗原组合的情况下触发细胞毒性的AND门和NOT门汽车以降低对正常细胞的脱靶毒性。在特定目标2中，将构建对肿瘤特异性环境线索（包括缺氧和局部浓度增加的免疫抑制细胞因子转化生长因子β（TGF-β））有反应的诱导型转录系统，以表达增强抗肿瘤免疫应答的基因产物，包括增加T细胞增殖和将天然免疫系统成分募集到肿瘤部位。这些诱导型转录系统将与特定目标1中开发的逻辑门汽车相结合，以产生能够执行和招募针对患病靶标的强大抗肿瘤应答的肿瘤靶向T细胞。拟议的受体和转录系统将使用合成生物学领域开发的快速模块化DNA组装技术构建。新的遗传构建体将通过慢病毒转导稳定整合到已建立的和原代人T细胞中，以实现性能表征和系统优化。体外试验包括蛋白质印迹、表面和细胞内抗体染色， 将进行细胞因子产生谱分析和铬释放（细胞裂解）测定以确定新受体和转录系统的表达和功能活性。将在小鼠的肿瘤异种移植模型中进一步检查显示出稳健的体外性能的构建体，以评估它们对经修饰的T细胞的肿瘤根除的作用。这项研究旨在通过追求从头治疗来解决T细胞治疗癌症进展的关键障碍。 构建以前在T细胞治疗工具箱中不可用的多功能遗传构建体，从而产生具有更稳健和精确靶向的抗肿瘤活性的T细胞，用于针对癌症的免疫治疗。

DESCRIPTION (provided by applicant): Adoptive T-cell therapy is a promising treatment strategy for cancers resistant to conventional methods including surgery, chemotherapy, and radiation therapy. In particular, the adoptive transfer of T cells genetically modified to express tumor-targeting chimeric antigen receptors (CARs) has shown clinical efficacy by redirecting T-cell specificity toward indolent tumors. However, important challenges remain in the use of CAR-modified T cells, including off-target toxicity toward normal cells and susceptibility to mutational escape by targeted tumors. The goal of this research is to improve the safety and efficacy of adoptive T-cell therapy by engineering more robust and versatile tumor-targeting T cells, which will be achieved through two specific aims. In Specific Aim 1, next-generation CARs capable of logical computation of multiple input signals will be developed. OR-gate CARs that trigger T-cell-mediated cytotoxicity in response to multiple tumor-associated antigens will be developed to lower the probability of mutational escape (i.e., loss of all targeted antigens) by tumor cells. AND- and NOT-gate CARs that trigger cytotoxicity only in the presence of the correct combination of antigens will be constructed to lower off-target toxicity toward normal cells. In Specific Aim 2, inducible transcription systems responsive to tumor-specific environmental cues-including hypoxia and increased local concentrations of the immunosuppressive cytokine transforming growth factor beta (TGF-β)-will be constructed to express gene products that enhance anti-tumor immune responses, including increased T-cell proliferation and the recruitment of native immune system components to tumor sites. These inducible transcription systems will be combined with the logic-gate CARs developed in Specific Aim 1 to generate tumor-targeting T cells capable of both executing and recruiting robust anti-tumor responses to diseased targets. The proposed receptors and transcription systems will be constructed using rapid, modular DNA assembly technologies developed in the field of synthetic biology. The novel genetic constructs will be stably integrated into established and primary human T cells via lentiviral transduction to enable performance characterization and system optimization. In vitro assays including western blots, surface and intracellular antibody staining, cytokine production profiling, and chromium release (cell lysis) assays will be performed to ascertain the expression and functional activities of new receptors and transcription systems. Constructs showing robust in vitro performance will be further examined in tumor xenograft models in mice to evaluate their effects on tumor eradication by modified T cells. This research aims to address a critical barrier to progress in T-cell therapy for cancer by pursuing the de novo construction of multi-functional genetic constructs previously unavailable in the T-cell therapy toolbox, thereby generating T cells with more robust and precisely targeted anti-tumor activities for immunotherapy against cancer.