Translational Research Center, Zhengzhou University People's Hospital, #7 Weiwu Road, Zhengzhou, Henan 450003, China.
J Hematol Oncol. 2013 May 9;6:33. doi: 10.1186/1756-8722-6-33.
Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain.
A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model.
Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells.
Our study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment.
嵌合抗原受体 (CAR) 修饰的 T 细胞过继转移似乎是一种很有前途的免疫治疗策略。CAR 结合了抗体的特异性和细胞毒性 T 淋巴细胞的细胞毒性，增强了 T 细胞特异性靶向抗原和有效杀伤癌细胞的能力。最近的研究努力已经将共刺激信号整合到 CAR 中，以提高抗肿瘤疗效。表皮生长因子受体变异 III（EGFRvIII）是一个有吸引力的治疗靶点，因为它在神经胶质瘤和许多其他类型的癌症中经常表达。我们目前的研究旨在研究含有诱导共刺激分子（ICOS）信号域的 CAR 修饰的 T 细胞的特异性和高效抗肿瘤作用。
生成了第二代 EGFRvIII/CAR，它包含 EGFRvIII 单链可变片段、ICOS 信号域和 CD3ζ 链。制备慢病毒 EGFRvIII/CAR，并用编码 EGFRvIII/CAR 的慢病毒感染人 CD3+T 细胞。通过流式细胞术和 Western blot 确认 CD3+T 细胞上 EGFRvIII/CAR 的表达。通过体外和体内方法（包括细胞毒性测定、细胞因子释放测定和异种移植肿瘤小鼠模型）评估 EGFRvIII/CAR+T 细胞的功能。
构建了嵌合 EGFRvIIIscFv-ICOS-CD3ζ（EGFRvIII/CAR），并制备了滴度为 106 TU/ml 的慢病毒 EGFRvIII/CAR。慢病毒 EGFRvIII/CAR 对 T 细胞的转导效率约为 70%，免疫印迹验证 EGFRvIII/CAR 蛋白的表达为约 57 kDa 的条带。四小时 51Cr 释放测定显示 EGFRvIII/CAR+T 细胞对表达 EGFRvIII 的 U87 细胞具有特异性和高效的细胞毒性。在 EGFRvIII/CAR+T 细胞与表达 EGFRvIII 的 U87 细胞的共培养上清液中检测到 IFN-γ 分泌的显著增加。EGFRvIII/CAR+T 细胞静脉内和肿瘤内注射抑制了表达 EGFRvIII 的神经胶质瘤细胞的体内生长。
我们的研究表明，EGFRvIII/CAR 修饰的 T 细胞可以以 EGFRvIII 特异性方式有效破坏神经胶质瘤细胞，并以抗原依赖性方式释放 IFN-γ。具有 ICOS 信号域的 EGFR 导向 T 细胞的特异性识别和有效杀伤活性为我们将来在癌症治疗中采用这种方法奠定了基础。
