Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030, USA.
Mol Ther. 2011 Dec;19(12):2133-43. doi: 10.1038/mt.2011.131. Epub 2011 Jul 19.
Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy.
EB 病毒（EBV）特异性细胞毒性 T 淋巴细胞（CTL）可以被修饰为异源肿瘤定向效应细胞，由于在血清阳性个体持续性感染期间表达的病毒抗原的慢性刺激，其在体内的存活时间比没有病毒特异性的肿瘤定向 T 细胞更长。我们评估了非病毒的 PiggyBac（PB）转座子系统作为修饰 EBV-CTL 以表达功能性人表皮生长因子受体 2 特异性嵌合抗原受体（HER2-CAR）的平台，从而将病毒特异性、基因修饰的 CTL 靶向 HER2 阳性癌细胞。外周血单核细胞（PBMC）用转座子进行核转染，该转座子编码 HER2-CAR 和用于选择的截断 CD19 分子，然后对 EBV-CTL 进行特异性激活和扩增。在 CD19 选择后，约 40%的 T 细胞表达 HER2-CAR，同时保留免疫表型、多克隆性和功能。HER2-CAR 修饰的 EBV-CTL（HER2-CTL）在体外杀伤 HER2 阳性脑肿瘤细胞系，在抗原特异性刺激后 HER2-CAR 表达短暂且可逆增加，并在 120 天以上稳定表达 HER2-CAR。PB 修饰的 HER2-CTL 的过继转移导致小鼠异种移植模型中的肿瘤消退。我们的结果表明，PB 可用于将病毒特异性 CTL 重定向至肿瘤靶标，这应该会延长体内肿瘤特异性 T 细胞的存活时间，从而产生更有效的免疫治疗。
