人源化Mesothelin嵌合抗原受体介导的T细胞抗肿瘤效果

Anti-tumor Effect of T Cells Mediated by Humanized Mesothelin Chimeric Antigen Receptor

CAR-T细胞(嵌合抗原受体T细胞)在治疗血液肿瘤领域已取得了显著的效果,但在实体瘤中的治疗应用仍存在很多阻碍。间皮素(Mesothelin)是肿瘤相关抗原,在间皮瘤、卵巢癌、胰腺癌等肿瘤细胞表面过表达,当前大多以Mesothelin为靶点的研究使用小鼠来源的scFv(单链可变识别区)进行基因编辑构建表达载体,在模拟人类疾病方面显示出诸多不足,为研究转化到临床治疗中带来障碍,因此进行人源化的研究非常必要。构建表达人源化anti-mesothelin的scFv(命名为P4)联用人41-BB共刺激结构域的P4 CAR表达载体,制备表达P4 CAR的慢病毒,感染人CD3^+T细胞,成功制备了稳定且高表达水平的P4 CAR-T细胞。PCR检测表明人宫颈癌肿瘤细胞系HeLa中表达Mesothelin。体外实验表明,P4 CAR-T细胞能有效杀伤HeLa细胞;在NSG小鼠中进行体内实验,相比对照组,注射P4 CAR-T细胞组中已形成的肿瘤生长延缓,荷瘤小鼠的生存期也得到延长。研究结果说明,P4 CAR-T细胞对于Mesothelin阳性的肿瘤具有潜在的治疗效果,为进一步改善体内治疗效果及临床治疗的增效策略研究提供了基础。

Despite that CAR-T cell (chimeric antigen receptor-modified T cell) therapy has achieved remarkable outcomes in the treatment of hematological malignancies, there are still lots of obstructions in the treatment of solid tumors. Mesothelin is a tumor associated antigen over-expressing on tumor cell surface such as mesothelioma, ovarian, pancreatic. Most of the current studies about CAR-T cells targeting Mesothelin use scFv (single-chain variable fragment) of mouse are to build expression vector by gene editing, which shows some shortcomings in simulating clinical treatment and obstacles of translation of research to clinical treatment, so it is very necessary to develop humanized study. We constructed a CAR vector with a humanized anti-mesothelin scFv called P4, packaged the lenti-virus and used them to transfect human peripheral blood CD3 + T cells. We successfully produced P4 CAR-T cells with high and steady transfection efficiency. PCR results indicated that Mesothelin was expressed in human cervical cancer tumor cell line HeLa. In vitro experiments demonstrated that our P4 CAR-T cells could effectively kill HeLa cells. In vivo experiments showed that infusion of P4 CAR-T cells slowed the growth of established tumors slightly and prolonged the survival of tumor-bearing mice in comparison to the control group. Here we reported that the P4 CAR-T cell can be used as a potential therapeutic strategy against Mesothelin positive tumor, and it provided a basis for further research on synergistic strategies to improve the therapeutic effect in vivo and clinical treatment.