靶向GPC3的第四代嵌合抗原受体T细胞 (分泌IL-7和CCL19) 的构建以及功能

Construction and function of Glypican-3-targeted fourth-generation chimeric antigen receptor T cells (secreting IL-7 and CCL19)

摘要:

基于嵌合抗原受体修饰的T细胞 (CAR-T) 的过继免疫疗法已被证明是治疗恶性肿瘤最有希望的策略 之一，但是目前其在实体瘤中的应用依然有限。研究表明磷脂酰肌醇蛋白聚糖3 (GPC3) 对肝细胞癌来说是一种有意义的诊断、治疗和预后生物标志物，且已有利用第二代/第三代GPC3靶向的CAR-T细胞治疗肝细胞癌的研究报道。为了进一步提高治疗效果，构建同时表达GPC3 CAR、人源IL-7和CCL19细胞因子的第四代慢病毒载体，转染293T细胞包装慢病毒并感染人T淋巴细胞制备靶向GPC3的第四代CAR-T细胞 (GPC3-BBZ-7×19)。利用细胞计数、趋化小室、荧光素酶生物发光法以及流式细胞术等比较其与第二代GPC3 CAR-T细胞 (GPC3-BBZ)在增殖、迁移、杀伤以及亚型分布方面的区别，评估GPC3-BBZ-7×19 CAR-T细胞对免疫缺陷小鼠体内GPC3阳性的肝细胞癌腹腔移植瘤生长的作用。结果表明与GPC3-BBZ CAR-T细胞相比，GPC3-BBZ-7×19 CAR-T细胞具备更强的增殖能力、趋化能力以及记忆干细胞 (Stem memory T cell，Tscm) 组成比 (P值均<0.05)，而在体外特异性杀伤GPC3阳性的肝细胞癌细胞以及细胞因子分泌水平方面无显著差异。此外，GPC3-BBZ-7×19 CAR-T细胞能够明显抑制免疫缺陷小鼠体内的GPC3阳性肝细胞癌移植瘤模型。因此靶向GPC3的第四代CAR-T细胞 (分泌IL-7和CCL19)有望比第二代GPC3靶向的CAR-T细胞更持久有效地对抗肿瘤，并产生针对肿瘤的记忆功能，为以后的临床试验提供了临床前研究基础。

Abstract:

Adoptive immunotherapy based on chimeric antigen receptor-modified T cells (CAR-T) is one of the most promising strategies to treat malignant tumors, but its application in solid tumors is still limited. Glypican-3 (GPC3) is a meaningful diagnostic, therapeutic, and prognostic biomarker for hepatocellular carcinoma (HCC). The second/third generation GPC3-targeted CAR-T cells are generated to treat HCC. In order to improve the therapeutic effect, we constructed a fourth-generation lentiviral vector to express GPC3 CAR, human interleukin-7 (IL-7) and CCL19. Then the lentiviral vector and packaging plasmids were co-transfected into HEK293T cells to generate CAR lentiviral particles. Human T lymphocyte cells were transduced with CAR lentiviral to develop the fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19). In vitro, we used cell counting, transwell assay, luciferase bioluminescence assay and flow cytometry to compare the proliferation, chemotaxis, cytotoxicity and subtype distribution between GPC3-BBZ-7×19 CAR-T cells and the second generation GPC3-targeted CAR-T cells (GPC3-BBZ). In vivo, we established GPC3-positive HCC xenograft model in immunodeficient mice, then untransduced T cells (non-CAR-T) or GPC3-BBZ-7×19 CAR-T cells were injected. Tumor growth in mice was observed by bioluminescence imaging. Results showed that compared with GPC3-BBZ CAR-T, GPC3-BBZ-7×19 CAR-T cells had stronger proliferation, chemotactic ability, and higher composition of memory stem T cells (Tscm) (P values<0.05). However, there were no significant difference in cytotoxicity and cytokine secretion between them. In addition, GPC3-BBZ-7×19 CAR-T cells could significantly eliminate GPC3-positive HCC xenografts established in immunodeficient mice. Therefore, the fourth-generation GPC3-targeted CAR-T cells (secreting IL-7 and CCL19) are expected to be more durable and effective against HCC and produce tumor-specific memory, to provide a preclinical research basis for future clinical trials.