基因工程T细胞改造及其在实体瘤治疗中的应用探索

过继性T细胞疗法（Adoptive T Cell Therapy，ACT）作为一种有效的免疫疗法在多种血液肿瘤的治疗中取得了显著的效果，但在实体瘤的治疗中效果不佳。主要原因在于实体瘤组织结构致密，导致T细胞浸润到肿瘤内部的数量有限，同时实体瘤中高度免疫抑制的肿瘤微环境也使T细胞难以被激活并迅速衰竭。促进T细胞向肿瘤微环境浸润是提高过继性T细胞疗法在临床治疗中的关键。

研究表明NRP1（Neuropilin-1，NRP1）广泛表达在多种恶性肿瘤细胞表面，而在T细胞表面修饰相应的NRP1配体（Neuropilin-1 Ligand，NRP1L），可以通过促进T细胞与NRP1阳性的肿瘤细胞结合并形成有利于T细胞向肿瘤内浸润的通道。课题组前期的研究已经证明了Sema3A作为一种NRP1L，能够促进Jurkat-T细胞向实体瘤的浸润。

本研究在此基础上，首先利用基因工程技术，构建了能够促进T细胞向肿瘤微环境浸润的双特异性元件：NRP1L×CD3双特异性蛋白。该蛋白一端由NRP1L的结构域组成，能够与多种恶性肿瘤表面广泛表达的NRP1结合；另外一端由anti-CD3结构域组成，能够识别并结合T 细胞表面CD3结构域。然后通过逆转录病毒构建工程化的间充质干细胞和工程化的293T细胞，通过这两类工程化细胞表达并分泌该类蛋白，验证该蛋白的表达并纯化NRP1L×CD3双特异性蛋白，之后将NRP1L×CD3双特异性蛋白与T细胞孵育，使T细胞表面能够携带NRP1L结构域，进而能够识别并结合NRP1阳性的肿瘤细胞。为了进一步验证NRP1L×CD3双特异性蛋白对于T细胞浸润能力的影响，本研究采用高表达NRP1的细胞系构建3D细胞球模拟实体瘤的3D结构，并通过荧光显微分析技术、流式细胞技术及酶联免疫分析技术对修饰后的T细胞浸润效果进行定性、定量分析。结果显示：NRP1L×CD3双特异性蛋白的修饰能够显著提升T细胞向肿瘤内部的浸润能力并对T细胞有一定的激活作用。综上，我们的研究结果表明NRP1L×CD3双特异性蛋白作为一种创新的双功能策略，可以通过对T细胞的非基因修饰，克服过继性免疫治疗的主要瓶颈，在促进T细胞向实体瘤的浸润方面有着稳定且明显的效果，为过继性T细胞疗法的应用创造条件。

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