重定向T淋巴细胞对共表达GPC3和EGFR肝细胞肝癌的过继免疫治疗

The most common type of primary liver cancer is hepatocellular carcinoma. This type of liver cancer is the third leading cause of cancer-related deaths worldwide. Its conventional treatments include surgery, chemotherapy and radiation therapy. In spite of continuous development of treatments, the prognosis of patients with HCC still sees no major improvement. As an important member of tumor adoptive cell therapy, chimeric antigen receptor (CAR) can enable T cells to react with tumor antigens through non-MHC restricted way and redirect T lymphocytes to tumor, thus bringing hope to tumor immunotherapy..CAR T cells genetically modified method can redirect T-cells of the patient to the tumor site, by antigen-specific CAR targeting a single tumor antigen. However, during the implementation process, the researchers found that there are few truly tumor-specific antigen was confirmed during the treatment of HCC. So the normal tissues may also suffer from T cell-mediated non-specific damage. Recently, we reported that the third-generation GPC3-targeted CAR T cells could eradicate GPC3-positive HCC xenografts in preclinical studies. However, it is found that GPC3 is not only expressed in HCC, but also lowly expressed in kidney, ovarie and teste, etc. Usually this target antigen in healthy tissues has different levels of expression, so the normal tissues may also suffer from T cell-mediated non-specific damage..In the study, we constructed a double chimeric antigen receptor-modified T cells, the first generation of CAR mediated GPC3 (epidermal growth factorinal receptor, EGFR) antigen recognition. The T cell costimulatory signal is generated by chimeric costimulatory receptor (CCR) whitch recognizes another antigen EGFR. CAR, CCR simultaneously modified T lymphocytes. In this study, by combining two targets GPC3 and EGFR in the treatment of HCC, T cells can be specifically targeted to the tumor, so as to induce apoptosis of tumor cells while causing no damage to normal tissues. T cells might be a novel potential therapeutic agent for the treatment of patients with HCC.

原发性肝癌致死率在肿瘤相关疾病中高居第三位，其中肝细胞肝癌（HCC）占原发性肝癌90%以上。传统治疗随不断改进，但患者预后并无大的改善，迫切需要突破性的疗法。嵌合抗原受体（CAR）能重新定向T细胞到肿瘤细胞，为肿瘤的免疫治疗带来了曙光。.CAR 修饰的T细胞通过抗原特异性靶向单一肿瘤抗原，但鲜有肿瘤特异性的抗原，因此表达靶抗原的健康组织潜在T细胞介导损伤的风险。我们最近研究表明GPC3靶向的CAR T细胞在体内、外能有效清除GPC3+的HCC细胞。但考虑GPC3在肾脏、卵巢、睾丸等正常组织也有相当水平表达，因此潜在损伤正常组织器官的风险。.本研究中，我们尝试通过组合抗原识别的方式解决以上问题。利用第一代CAR介导GPC3 抗原识别，T细胞共刺激信号则由识别另一个抗原EGFR的嵌合共刺激受体（CCR）独立介导。CAR，CCR同时修饰T淋巴细胞。从而T细胞可以特异性靶向肿瘤，且可能不会对正常组织造成损伤。

肝细胞肝癌（HCC）占原发性肝癌90%以上。传统治疗预后差，迫切需要突破性的疗法。嵌合抗原受体（CAR）能重新定向T细胞到肿瘤细胞，为肿瘤免疫治疗带来了曙光。既往研究中我们发现第三代表达抗GPC3嵌合抗原受体的T细胞（GPC3-CAR T）对HCC具有显著治愈潜力。GPC3-CAR T在体内、外能有效清除GPC3+的HCC细胞。但肿瘤鲜有特异性的抗原，“on-target off-tumor”作用是CAR T在临床中主要问题，即对表达靶抗原的健康组织潜在T细胞介导的损伤风险。GPC3在肾脏、卵巢、睾丸等正常组织也有相当水平表达，潜在损伤正常组织器官风险。EGFR在HCC 中扩增和过表达，是HCC细胞表面重要标志之一，但人体上皮来源大部分正常组织都表达EGFR，也是非肿瘤特异性抗原。.本研究通过组合抗原识别的方式解决以上问题。利用第一代CAR介导GPC3 抗原识别（CARgpc3），T细胞共刺激信号则由识别另一个抗原EGFR的嵌合共刺激受体（CARegfr）独立介导。CARgpc3，CARegfr共修饰T淋巴细胞获得CARgpc3-egfr T细胞。使其在同时识别两种抗原时，才能被完全激活。理论上使T细胞可特异性靶向肿瘤且不会对正常组织造成损伤。.实验结果表明，只激活共刺激信号，即CARegfr T细胞对于EGRF+GPC3+肿瘤细胞并不会产生杀伤作用，而CARgpc3与CARgpc3-egfr T 对EGRF+GPC3+肿瘤细胞均能产生一定程度细胞毒性。CARgpc3-egfr T在双抗原同时阳性情况下，共刺激信号的激活，其杀伤作用优于CARgpc3 T细胞。同时研究发现与EGRF+GPC3+肿瘤细胞共培养后，CARgpc3-egfr T细胞比CARgpc3 T细胞具有更强的细胞因子分泌、增殖及抗凋亡能力。在原位移植瘤模型中，我们发现CARgpc3-egfr T细胞比CARgpc3 T细胞具有更强的肿瘤抑制作用和更长的体内存活时间，同时该组动物生存时间更长。数据表明双靶向的CARgpc3-egfr T细胞在同时识别GPC3+和EGFR+才能完全激活，维持相对有效的抗肿瘤活性。该设计既能降低潜在的on-target off-tumor效应，同时也能维持CAR T细胞的杀伤活性。