表达反式嵌合性抗原受体的T细胞抗肿瘤作用的实验研究

HER-2/neu amplification and overexpression is identified in many cancer types and correlated with poor prognosis and shorter overall survival of tumor patients. Thus, HER2/neu has become an accepted tumor marker and attractive target for tumor immunotherapy. Despite the remarkable impact of anti-HER2 monoclonal antibody Herceptin on HER2-positive cancer, acquired resistance to Herceptin develops in most patients, and a large subset never respond. Thus, the novel treatment for HER2-expressing cancer is urgently needed. Adoptive cell therapy with genetically modified T cells expressing chimeric antigen receptors (CARs) appears to be a promising and powerful approach. CARs are composed of a single-chain variable-fragment (scFv) domain regions fused typically to the cytoplasmic signaling domain of the T cell receptor (TCR) CD3ζ chain. Once CARs are expressed at the surface of a modified T cells, upon binding of the scFv to its antigen an activating signal is transmitted into the T cells which in turn triggers its effector functions against the target cell in a non-MHC-restricted way.However, in a previous report, on-target off-tumour toxicity was found when a patient received the injection of T cells engineered by the chimeric antigen receptor consisted of (subnanomolar affinity) HER2-specific trastuzumab scFv. In this study, we described an efficient and reliable way to construct a HER2-specific trans-CAR using a humanized anti-HER2 A21 scFv and a anti-Fra scFv. We will evaluate the anti-HER2 trans-CAR expression on human T cells and the phenotype of trans-CAR T cells by flow cytometry. Moreover, we will evaluate the anti-tumor activity of HER2 trans-CAR T cells. Further, we investigate and compare the anti-tumor activity of HER2 trans-CAR T cells injected intravenously and intra-tumorly in murine model including subcutaneous, metastatic intraperitoneal models of human breast SKBR3 and ovarian SKOV3 cancer. Thus, this study will support that adoptive transfer of HER2-specific trans-CAR T cells may be a safe immunotherapy for HER2-positive malignancies in clinical setting.

嵌合性抗原受体（CAR）为基础的T细胞抗肿瘤治疗将抗体与抗原特异性结合功能与T细胞的杀伤功能结合于一体，特异性强，是目前肿瘤治疗的研究热点。原癌基因人上皮生长因子2（HER-2/neu）是CAR T细胞抗肿瘤治疗的理想靶点,但前期靶向HER2的CAR T细胞的安全性受到挑战。反式嵌合性抗原受体是探索提高CAR T细胞安全性的新方式。本课题拟利用人源化的抗HER2 的A21scFv构建A21 CAR T细胞；并在前期工作的基础上构建反式嵌合性抗原受体A21 CAR/FRα CART细胞，分析顺式及反式嵌合性抗原受体在人外周血T细胞表面的表达及T细胞分化表型，评价表达顺式及反式CAR T细胞体外抗瘤作用；利用HER2及FRα过表达的乳腺癌细胞SKBR3及卵巢癌细胞SKOV3分别建立小鼠皮下移植瘤及腹腔内癌性腹水模型，并设阴性对照，评价靶向HER2的顺式及反式CAR T细胞的抗肿瘤效果及安全性。

胃癌在中国发病率高且预后不良，现缺乏有效的治疗手段。我们尝试将以chA21为基础的CAR T细胞应用到HER2高表达胃癌的治疗中。我们研究发现，chA21 scFv-4-1BB-CD3ζCAR T细胞在体外特异性肿瘤抗原的刺激下能分泌Th1型细胞因子并有效杀伤HER2高表达的人胃癌细胞。而且CAR T细胞产生细胞因子的量与杀伤靶细胞的能力均与肿瘤细胞表面HER2受体表达水平呈正相关。在我们构建的人胃癌小鼠皮下移植瘤模型及人胃癌小鼠腹膜种植瘤模型中，chA21 scFv-4-1BB-CD3ζCAR T细胞能有效抑制HER2高表达肿瘤的生长并延长荷瘤小鼠的生存期，同时对HER2低表达的肿瘤无脱靶作用。另外，我们通过流式分析及免疫组化确定了CAR T细胞在静脉输注后能在小鼠体内循环中定位到特异性肿瘤位点浸润、聚集并发挥抗肿瘤作用。我们的研究结果为进一步的探究chA21 scFv-4-1BB-CD3ζCAR T细胞应用于HER2高表达胃癌以及其他HER2高表达实体瘤的临床治疗提供了前期有利的证据。