CAREER: Seeing in the Dark--Engineering Cytotoxic T Cells to Detect and Respond to Intracellular Cancer Markers

肿瘤靶向T细胞是一种白细胞，可以从癌症患者身上采集，经过基因改造以识别肿瘤细胞，然后重新注射到相同的患者体内，已经显示出治疗晚期癌症的显着潜力。然而，T细胞治疗的一个主要挑战是肿瘤外毒性，即T细胞攻击在其表面显示与肿瘤细胞相同标记的健康组织。肿瘤外毒性可导致严重的副作用，包括在几项临床试验中观察到的患者死亡。这项研究计划旨在通过使T细胞在做出杀死决定之前询问靶细胞的内部来降低肿瘤外毒性。这将通过一种新的工程蛋白来实现，这种蛋白由T细胞产生并传递到靶细胞中，但当且仅当它们遇到靶细胞内的肿瘤特异性蛋白时才会产生毒性。该技术的成功开发将通过提高肿瘤靶向的特异性和扩大可靶向的癌症类型来提高过继性t细胞治疗癌症的安全性和有效性。拟议的研究旨在开发一类新的感知和反应蛋白质装置。称为细胞质抗原检测器和消灭触发器（cadet）？使T细胞能够在释放毒性之前在目标细胞内搜索疾病标记物。cadet将通过将细胞毒性蛋白颗粒酶B与响应特定细胞内癌蛋白的调控结构域（如sumo特异性蛋白酶1 （SENP1）和人类端粒酶逆转录酶（hTERT））相连接来构建。通过合理的设计构建了一个响应senp1的CADET，其中一个抑制结构域阻断了CADET？直到它被SENP1切割掉。这种结构可以推广到其他具有明确切割靶点的肿瘤相关蛋白酶。将开发一种高通量筛选过程，以优化对非蛋白水解配体（如hTERT）有反应的CADET分子。这一基于荧光的筛选过程将在毕赤酵母细胞中进行，而表现最好的CADET序列将在原代人T细胞中进行表征，以确定其生产和递送效率以及htert激活的毒性。最佳的CADET分子将在T细胞中与嵌合抗原受体共表达，并在小鼠中评估其选择性杀死表达癌蛋白的肿瘤细胞同时保留非肿瘤靶细胞的能力。与圣莫尼卡男孩女孩俱乐部（SMBGC）合作，该提案还将为12至14岁的儿童制定一个外展计划，以介绍拟议的研究并讨论未来工程职业的潜在途径。这项提议的技术结合了合成生物学和t细胞工程来解决一个重要的生物医学挑战，它将作为一个令人兴奋的工程职业提供的可能性的演示。

PI: Chen, Yvonne Y. Proposal Number: 1553767Tumor-targeting T cells a type of white blood cells that can be harvested from cancer patients, genetically modified to recognize tumor cells, and then re-injected into the same patients have shown remarkable curative potential against advanced cancers. However, a major challenge in T-cell therapy is off-tumor toxicity, in which T cells attack healthy tissues that display the same markers as tumor cells on their surfaces. Off-tumor toxicity can result in severe side effects, including patient deaths that have been observed in several clinical trials. This research proposal aims to reduce off-tumor toxicity by enabling T cells to interrogate the interior of a target cell before making a killing decision. This will be achieved by a new class of engineered proteins that are produced and delivered by T cells into target cells, but become toxic if and only if they encounter a tumor-specific protein inside the target cell. Successful development of the proposed technology will increase the safety and efficacy of adoptive T-cell therapy for cancer by improving the specificity of tumor targeting and by expanding the types of cancers that can be targeted. The proposed study aims develop a new class of sense-and-respond protein devices?termed Cytoplasmic Antigen Detectors and Extermination Triggers (CADETs)?that enable T cells to search inside the target cell for disease markers prior to unleashing toxicity. CADETs will be constructed by accessorizing the cytotoxic protein granzyme B with regulatory domains that respond to specific intracellular oncoproteins, such as SUMO-specific protease 1 (SENP1) and human telomerase reverse transcriptase (hTERT). A SENP1-responsive CADET has been constructed by rational design, in which an inhibitory domain blocks CADET?s enzymatic activity until it is cleaved off by SENP1. This architecture can be generalized to other tumor-associated proteases with well-defined cleavage targets. A high-throughput screening process will be developed to enable optimization of CADET molecules that respond to non-proteolytic ligands, such as hTERT. This fluorescence-based screening process will be performed in Pichia pastoris yeast cells, and top-performing CADET sequences will be characterized in primary human T cells for production and delivery efficiency as well as hTERT-activated toxicity. The optimal CADET molecule will be co-expressed with chimeric antigen receptors in T cells and evaluated for the ability to selectively kill oncoprotein-expressing tumor cells while sparing off-tumor target cells in mice. In partnership with the Boys and Girls Club of Santa Monica (SMBGC), this proposal will also develop an outreach program for children between the ages of 12 and 14 to introduce the proposed research and discuss potential paths to future careers in engineering. The proposed technology combines synthetic biology and T-cell engineering to address an important biomedical challenge, and it will serve as an exciting demonstration of the possibilities that an engineering career could offer.