Wiznerowicz M, Fong A Z, Mackiewicz A, Hawley R G
Department of Cancer Immunology, Great Poland Cancer Center, Poznan, Poland.
Gene Ther. 1997 Oct;4(10):1061-8. doi: 10.1038/sj.gt.3300500.
The efficient genetic modification of solid tumors in situ to stimulate therapeutic immune responses against them is currently under active investigation, but is not yet possible using existing gene transfer technologies. Thus, ex vivo/in vivo vaccination strategies have been proposed in which the patient's tumor is surgically excised, single cell suspensions are prepared, the therapeutic genes are introduced and then the gene-modified cells, after being gamma-irradiated, are injected back into the patient. However, even with high-efficiency gene delivery systems, this is a labor-intensive process. Moreover, it is often difficult to obtain sufficient numbers of gene-modified primary tumor cells during short-term culturing. On the other hand, extended in vitro passaging of primary tumor explants may alter their immunophenotypic properties. One approach to overcome these limitations would be to design universal vaccines consisting of standardized gene-transduced neoplastic cell lines or mixtures of gene-transduced cell lines to be combined with autologous tumor samples if available. Melanoma, which is notable for being one of the most immunogenic human malignancies, represents a cancer where shared tumor-associated antigens have been identified. We developed and analyzed several different retroviral vectors for their ability to stably express exogenous genes at high levels in a panel of melanoma cell lines. All vectors contained a reporter gene (nlslacZ) encoding beta-galactosidase with a nuclear localization signal and the neomycin phosphotransferase (neo) gene as selectable marker. One vector, DCCMV, which carried a bicistronic nlslacZ-neo transcriptional unit under the control of the human cytomegalovirus immediate-early promoter in the U3 region of its 3' LTR, was found to perform consistently better than the other vectors. The DCCMV vector, which is an extreme example of the double-copy class of retroviral vectors, was subsequently used to generate melanoma cell lines constitutively secreting human interleukin-6 or a soluble form of the human interleukin-6 receptor for potential use in a phase II clinical vaccine trial for the treatment of melanoma patients. The DCCMV vector design may also be useful in gene therapy applications where the intent is to implant polymer-encapsulated cell lines genetically engineered to stably express high levels of bioactive proteins.
目前正在积极研究对实体瘤进行原位高效基因改造以刺激针对实体瘤的治疗性免疫反应，但利用现有基因转移技术尚无法实现。因此，已提出体外/体内疫苗接种策略，即手术切除患者肿瘤，制备单细胞悬液，导入治疗基因，然后将经γ射线照射的基因修饰细胞重新注入患者体内。然而，即使使用高效基因递送系统，这也是一个劳动密集型过程。此外，在短期培养过程中，往往难以获得足够数量的基因修饰原发性肿瘤细胞。另一方面，原发性肿瘤外植体在体外长时间传代可能会改变其免疫表型特性。克服这些局限性的一种方法是设计通用疫苗，该疫苗由标准化基因转导的肿瘤细胞系或基因转导细胞系混合物组成，如有自体肿瘤样本，可与之联合使用。黑色素瘤是最具免疫原性的人类恶性肿瘤之一，是已鉴定出共享肿瘤相关抗原的一种癌症。我们开发并分析了几种不同的逆转录病毒载体，以研究它们在一组黑色素瘤细胞系中高水平稳定表达外源基因的能力。所有载体都包含一个编码带有核定位信号的β-半乳糖苷酶的报告基因（nlslacZ）和作为选择标记的新霉素磷酸转移酶（neo）基因。一种载体DCCMV，在其3' LTR的U3区域的人巨细胞病毒立即早期启动子控制下携带一个双顺反子nlslacZ-neo转录单元，发现其表现始终优于其他载体。DCCMV载体是逆转录病毒载体双拷贝类别的一个极端例子，随后被用于生成持续分泌人白细胞介素-6或人白细胞介素-6受体可溶性形式的黑色素瘤细胞系，用于治疗黑色素瘤患者的II期临床疫苗试验。DCCMV载体设计在基因治疗应用中也可能有用，在这些应用中，目的是植入经基因工程改造以稳定表达高水平生物活性蛋白的聚合物包裹细胞系。
