肿瘤细胞治疗研究
英文原题：Engineering single-vector logic-gated CAR T cells with transgene sizes beyond current limitations.
这些数据建立了工程化逻辑门控单载体免疫疗法的框架，并提供了生成携带超出当前尺寸限制转基因的 CAR T 细胞的优化工作流程。
背景：采用合成Notch（synNotch）受体逻辑门控回路工程化嵌合抗原受体（CAR）T细胞，可提高特异性并减轻靶向肿瘤同时损伤正常组织毒性。然而，传统synNotch系统使用两个慢病毒载体，分别编码synNotch受体和诱导型CAR，需要双重转导及细胞分选，限制临床转化。将synNotch-CAR回路整合到单一慢病毒载体可克服这一问题，但大型转基因会大幅降低慢病毒滴度和T细胞转导效率，使CAR-T制备仍具挑战。现有生产流程通过分选转导细胞弥补低转导率，进一步阻碍临床转化，因此限制了synNotch-CAR疗法的广泛开发。方法：研究构建单载体synNotch（svsNotch）系统，将传统双载体回路所有组分整合进一个慢病毒载体，以促进临床转化。针对大型svsNotch转基因导致慢病毒滴度及T细胞转导效率降低的问题，建立了优化的效应T细胞CAR-T生产流程，适用于大型慢病毒转基因。结果：优化流程使T细胞转导率最高提高14.8倍，并能生产携带超过有效包装容量上限9.2 kb的慢病毒转基因效应T细胞。作为概念验证，研究构建HER2-间皮素（MSLN）svsNotch（9.2 kb）：靶向HER2的synNotch受体调控第二代4-1BB CAR表达，使其靶向MSLN，从而选择性靶向HER2⁺MSLN⁺卵巢肿瘤。体外实验中，与传统双载体synNotch-CAR T细胞相比，该细胞特异性更佳，可选择性杀伤HER2⁺MSLN⁺而非HER2敲除、MSLN⁺肿瘤细胞。为实现体内监测，研究构建整合CBG荧光素酶的HER2-MSLN-click beetle green（CBG）svsNotch（10.1 kb）。在以组成型CAR-T细胞为对照的小鼠模型中，HER2-MSLN-CBG svsNotch在缺乏HER2时细胞毒性极低，对HER2低、MSLN高和HER2高、MSLN高肿瘤疗效更优。结论：本研究建立了工程化逻辑门控单载体免疫疗法的框架，并提供了可制备超过当前尺寸限制转基因CAR-T细胞的优化流程。
BACKGROUND: Engineering chimeric antigen receptor (CAR) T cells with logic-gated synthetic Notch (synNotch) receptor circuits can enhance specificity and mitigate on-target/off-tumor toxicity. However, the conventional synNotch system uses two lentiviral vectors encoding the synNotch receptor and inducible CAR, requiring dual transduction and cell sorting, which limits clinical translation. Integrating the synNotch-CAR circuit into a single lentiviral vector could overcome this limitation, yet manufacturing CAR T cells with large transgenes remains challenging, as increasing transgene size drastically reduces lentiviral titers and T cell transduction efficiency. Current production workflows compensate for low transduction efficiency by sorting transduced cells, further impeding clinical translation. Consequently, these constraints have limited the broader development of synNotch-CAR T cell therapies. METHODS: We engineered a single-vector synNotch (svsNotch) system that integrates all components of the conventional dual-vector circuit into one lentiviral vector to facilitate clinical translation. To overcome the low lentiviral titers and T cell transduction efficiency caused by the large svsNotch transgene, we established an optimized CAR T cell production workflow for effector T cells with large lentiviral transgenes. RESULTS: Our optimized workflow increased T cell transduction rates by up to 14.8-fold and enabled the production of effector T cells with lentiviral transgenes exceeding the effective packaging capacity limit of 9.2 kb. As a proof of concept, we engineered human epidermal growth factor receptor 2 (HER2)-mesothelin (MSLN) svsNotch (9.2 kb), in which a synNotch receptor targeting HER2 regulates the expression of a second-generation 4-1BB CAR against MSLN to enable selective targeting of double-positive HER2 + MSLN + ovarian tumors. In vitro, HER2-MSLN svsNotch T cells demonstrated superior specificity to conventional dual-vector synNotch-CAR T cells, with selective cytotoxicity against HER2 + MSLN + but not HER2 ko MSLN + tumor cells. To enable in vivo monitoring, we engineered HER2-MSLN-click beetle green (CBG) svsNotch (10.1 kb) incorporating CBG luciferase. In mouse models using constitutive CAR T cells as controls, HER2-MSLN-CBG svsNotch T cells exhibited minimal cytotoxicity in the absence of HER2 and superior efficacy against HER2 low MSLN high and HER2 high MSLN high tumors. CONCLUSION: These data establish a framework for engineering logic-gated single-vector immunotherapies and provide an optimized workflow for generating CAR T cells with transgenes that exceed current size limitations.
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