Symes J C, Siatskas C, Fowler D H, Medin J A
Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
Cancer Gene Ther. 2009 May;16(5):439-52. doi: 10.1038/cgt.2008.96. Epub 2008 Dec 19.
Adoptively transferred T cells possess anticancer activities partially mediated by T-cell FasL engagement of Fas tumor targets. However, antigen-induced T-cell activation and clonal expansion, which stimulates FasL activity, is often inefficient in tumors. As a gene therapy approach to overcome this obstacle, we have created oncoretroviral vectors to overexpress FasL or non-cleavable FasL (ncFasL) on murine T cells of a diverse T-cell receptor repertoire. Expression of c-FLIP was also engineered to prevent apoptosis of transduced cells. Retroviral transduction of murine T lymphocytes has historically been problematic, and we describe optimized T-cell transduction protocols involving CD3/CD28 co-stimulation of T cells, transduction on ice using concentrated oncoretrovirus, and culture with IL-15. Genetically modified T cells home to established prostate cancer tumors in vivo. Co-stimulated T cells expressing FasL, ncFasL and ncFasL/c-FLIP each mediated cytotoxicity in vitro against RM-1 and LNCaP prostate cancer cells. To evaluate the compatibility of this approach with current prostate cancer therapies, we exposed RM-1, LNCaP, and TRAMP-C1 cells to radiation, mitoxantrone, or docetaxel. Fas and H-2(b) expression were upregulated by these methods. We have developed a novel FasL-based immuno-gene therapy for prostate cancer that warrants further investigation given the apparent constitutive and inducible Fas pathway expression in this malignancy.
过继转移的T细胞具有抗癌活性，部分是由T细胞FasL与Fas肿瘤靶标的结合介导的。然而，刺激FasL活性的抗原诱导的T细胞活化和克隆扩增在肿瘤中通常效率低下。作为克服这一障碍的基因治疗方法，我们构建了逆转录病毒载体，以在具有不同T细胞受体库的小鼠T细胞上过表达FasL或不可裂解的FasL（ncFasL）。还对c-FLIP的表达进行了改造，以防止转导细胞的凋亡。历史上，小鼠T淋巴细胞的逆转录病毒转导一直存在问题，我们描述了优化的T细胞转导方案，包括T细胞的CD3/CD28共刺激、使用浓缩的逆转录病毒在冰上进行转导以及用IL-15培养。基因改造的T细胞在体内归巢到已建立的前列腺癌肿瘤。共刺激表达FasL、ncFasL和ncFasL/c-FLIP的T细胞在体外均介导了对RM-1和LNCaP前列腺癌细胞的细胞毒性。为了评估这种方法与当前前列腺癌治疗方法的兼容性，我们将RM-1、LNCaP和TRAMP-C1细胞暴露于辐射、米托蒽醌或多西他赛。这些方法上调了Fas和H-2(b)的表达。我们已经开发了一种新型的基于FasL的前列腺癌免疫基因治疗方法，鉴于这种恶性肿瘤中明显的组成性和诱导性Fas途径表达，值得进一步研究。
