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

描述(申请人提供)：过继T细胞疗法是一种很有前途的治疗策略，适用于耐受常规方法包括手术、化疗和放射治疗的癌症。特别是，通过过继转移表达肿瘤靶向嵌合抗原受体(CARS)的基因修饰的T细胞，通过将T细胞特异性重定向到惰性肿瘤，显示了临床疗效。然而，在使用CAR修饰的T细胞方面仍然存在重要的挑战，包括对正常细胞的非靶标毒性以及靶向肿瘤对突变逃逸的敏感性。这项研究的目标是通过设计更强大和更多功能的肿瘤靶向T细胞来提高过继T细胞治疗的安全性和有效性，这将通过两个特定的目标来实现。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。或门CARS可在多种肿瘤相关抗原的作用下触发T细胞介导的细胞毒作用，以降低肿瘤细胞突变逃逸(即丢失所有靶向抗原)的可能性。只有在正确的抗原组合存在的情况下才会触发细胞毒性的AND和NOT-GATE CARS将被构建成降低对正常细胞的脱靶毒性。在特定目标2中，将构建对肿瘤特定环境线索(包括缺氧和局部免疫抑制细胞因子转化生长因子β(β)浓度增加)做出反应的可诱导转录系统，以表达增强抗肿瘤免疫反应的基因产物，包括增加T细胞增殖和将天然免疫系统成分招募到肿瘤部位。这些可诱导的转录系统将与在特定目标1中开发的逻辑门CARS相结合，以产生能够执行和招募对疾病靶标的强大抗肿瘤反应的肿瘤靶向T细胞。建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组装技术来构建。新的基因结构将通过慢病毒转导稳定地整合到已建立的和原代人类T细胞中，以实现性能表征和系统优化。体外检测包括免疫印迹、细胞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)分析，以确定新受体和转录系统的表达和功能活性。在体外表现出强劲性能的构建体将在小鼠的肿瘤异种移植模型中进一步检验，以评估它们对修饰T细胞根除肿瘤的效果。这项研究旨在通过追求从头开始来解决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基因结构，从而产生具有更强大和更精确靶向的抗肿瘤活性的T细胞，用于抗癌免疫治疗。

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.