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

PI：Chen，Yvonne Y.建议编号：1553767 Tumor-靶向T细胞一种可以从癌症患者身上获取的白细胞，经过基因改造可以识别肿瘤细胞，然后重新注射到相同的患者体内，已经显示出对抗晚期癌症的显著疗效潜力。然而，T细胞治疗的一个主要挑战是非肿瘤毒性，即T细胞攻击表面显示与肿瘤细胞相同标记的健康组织。非肿瘤毒性可能会导致严重的副作用，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细胞在做出杀伤决定之前能够询问靶细胞的内部来降低肿瘤外的毒性。这将通过一类新的工程蛋白来实现，这些蛋白由T细胞产生并输送到目标细胞中，但只有当它们在目标细胞内遇到肿瘤特异性蛋白时才会变得有毒。拟议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采用T细胞疗法治疗癌症的安全性和有效性。这项拟议的研究旨在开发一种新的正义和反应蛋白质设备--被称为细胞质抗原检测器和根除触发器(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志物。学员将通过辅助细胞毒蛋白颗粒酶B与特定的细胞内癌蛋白反应的调节结构域来构建，如相扑特异性蛋白1(SENP1)和人类端粒酶逆转录酶(HTERT)。通过合理的设计，构建了一个对SENP1有反应的学员，其中一个抑制结构域阻止学员S的酶活性，直到它被SENP1切割。这一体系结构可以推广到其他具有明确切割靶点的肿瘤相关蛋白。将开发一种高通量筛选过程，以优化对非蛋白水解性配体(如hTERT)反应的CADET分子。这一基于荧光的筛选过程将在巴斯德毕赤酵母细胞中进行，并将在原代人类T细胞中鉴定最高性能的CADET序列的生产和输送效率以及hTERT激活的毒性。优化的CADET分子将与T细胞中的嵌合抗原受体共表达，并评估其选择性杀死表达癌蛋白的肿瘤细胞的能力，同时保留小鼠的肿瘤外靶细胞。这项提案还将与圣莫尼卡男孩女孩俱乐部(SMBGC)合作，为12至14岁的儿童开发一个外展计划，介绍拟议的研究并讨论未来在工程领域的潜在职业道路。这项拟议的技术结合了合成生物学和T细胞工程，以应对一个重要的生物医学挑战，它将作为一个令人兴奋的演示，展示工程职业可能提供的可能性。

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.