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

描述（由申请人提供）：过继性t细胞治疗是一种很有前途的治疗策略，用于治疗对传统方法（包括手术、化疗和放疗）有抗药性的癌症。特别是，通过基因修饰表达肿瘤靶向嵌合抗原受体（CARs）的T细胞过继转移，通过将T细胞特异性转向惰性肿瘤，显示出临床疗效。然而，car修饰T细胞的使用仍然存在重要的挑战，包括对正常细胞的脱靶毒性和靶向肿瘤突变逃逸的易感性。本研究的目标是通过设计更强大和通用的肿瘤靶向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Specific Aim 1中，将开发能够对多个输入信号进行逻辑计算的下一代car。将开发能够触发t细胞介导的细胞毒性以响应多种肿瘤相关抗原的OR-gate car，以降低肿瘤细胞突变逃逸（即所有靶向抗原的丢失）的可能性。只有在抗原正确组合的情况下才会触发细胞毒性的与门和非门car将被构建以降低对正常细胞的脱靶毒性。在Specific Aim 2中，将构建对肿瘤特异性环境线索（包括缺氧和免疫抑制细胞因子转化生长因子β (TGF-β)局部浓度增加）有反应的诱导转录系统，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募集到肿瘤部位。这些可诱导的转录系统将与Specific Aim 1中开发的逻辑门car相结合，以产生肿瘤靶向T细胞，这些T细胞能够对患病靶标执行和招募强大的抗肿瘤反应。拟议的受体和转录系统将使用合成生物学领域发展的快速模块化DNA组装技术构建。新的遗传结构将通过慢病毒转导稳定地整合到已建立的和原代的人类T细胞中，以实现性能表征和系统优化。体外检测包括western blots，表面和细胞内抗体染色，

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.