Fakhrai H, Shawler D L, Van Beveren C, Lin H, Dorigo O, Solomon M J, Gjerset R A, Smith L, Bartholomew R M, Boggiano C A, Gold D P, Sobol R E
Sidney Kimmel Cancer Center, Clemma A. Hewitt Gene Therapy Laboratory, San Diego, California, USA.
J Immunother. 1997 Nov;20(6):437-48. doi: 10.1097/00002371-199711000-00003.
Several investigators have employed interleukin-2 (IL-2) gene transfer to enhance the immunogenicity of tumor cell vaccines. We describe in this report the construction and characterization of retroviral vectors for IL-2 gene therapy. Human IL-2 cDNA with a chimeric rat preproinsulin/IL-2 DNA leader sequence was subcloned into the pLXSN (long terminal repeat promoter) and pLNCX (cytomegalovirus [CMV] promoter) vectors to generate the plasmids pLXSN-iIL2 and pLNCX-iIL2, respectively. Human IL-2 cDNA with a chimeric human tissue factor/IL-2 DNA leader sequence was utilized to construct the vector pLXSN-tIL2. The levels of IL-2 secreted by transduced tumor cells and fibroblasts were evaluated by enzyme-linked immunosorbent assay (ELISA) of culture supernatants and compared with those of normal peripheral blood mononuclear cells (PBMC) activated in vitro with calcium ionophore and phorbol 12-myristate 13-acetate. The highest levels of IL-2 secreted by transduced tumor cells (760 units/10(6) cells/24 h), adult fibroblasts (625 units/10(6) cells/24 h), and embryonic fibroblasts (3,975 units/10(6) cells/24 h) were 150- to 1,000-fold higher than than secreted by the activated PBMC (4 units/10(6) cells/24 h). Similar levels of IL-2 were expressed by human fibroblasts transduced with pLXSN vectors employing the preproinsulin (pLXSN-iIL2) or tissue factor (pLXSN-tIL2) leader sequences (range in IL-2 units/10(6) cells/24 h pLXSN-iIL2 = 375-625 vs. pLXSN-tIL2 = 90-440). Because IL-2-transduced cells for clinical applications are generally irradiated to prevent cellular proliferation, we evaluated the effects of radiation on IL-2 production. Radiation doses between 1,500 and 10,000 cGy resulted in gradual decreases in IL-2 secretion by transduced cells. The range of the decrease in IL-2 secretion was 7-11% by day 7, 0-29% by day 14, and 25-50% by day 35. For clinical applications, stable production of the vector in high concentrations is an important consideration. The retroviral vector pLXSN-tIL2 produced the highest viral titer and was chosen for further characterization. Southern blot analysis of SacI-digested genomic DNA from the LXSN-tIL2 producer cell line and SacI-digested pLXSN-tIL2 plasmid DNA revealed the expected 3.2-kbp fragment, suggesting the absence of transgene rearrangement and the suitability of this vector as a candidate for clinical applications.
几位研究者已采用白细胞介素-2（IL-2）基因转移来增强肿瘤细胞疫苗的免疫原性。我们在本报告中描述了用于IL-2基因治疗的逆转录病毒载体的构建和特性。将带有嵌合大鼠前胰岛素原/IL-2 DNA前导序列的人IL-2 cDNA亚克隆到pLXSN（长末端重复启动子）和pLNCX（巨细胞病毒[CMV]启动子）载体中，分别产生质粒pLXSN-iIL2和pLNCX-iIL2。利用带有嵌合人组织因子/IL-2 DNA前导序列的人IL-2 cDNA构建载体pLXSN-tIL2。通过对培养上清液进行酶联免疫吸附测定（ELISA）来评估转导的肿瘤细胞和成纤维细胞分泌的IL-2水平，并与用钙离子载体和佛波醇12-肉豆蔻酸酯13-乙酸酯体外激活的正常外周血单个核细胞（PBMC）的水平进行比较。转导的肿瘤细胞（760单位/10⁶细胞/24小时）、成年成纤维细胞（625单位/10⁶细胞/24小时）和胚胎成纤维细胞（3975单位/10⁶细胞/24小时）分泌的IL-2最高水平比激活的PBMC（4单位/10⁶细胞/24小时）分泌的高150至1000倍。用采用前胰岛素原（pLXSN-iIL2）或组织因子（pLXSN-tIL2）前导序列的pLXSN载体转导的人成纤维细胞表达的IL-2水平相似（IL-2单位/10⁶细胞/24小时范围：pLXSN-iIL2 = 375 - 625对pLXSN-tIL2 = 90 - 440）。由于用于临床应用的IL-2转导细胞通常要进行照射以防止细胞增殖，我们评估了辐射对IL-2产生的影响。1500至10000 cGy的辐射剂量导致转导细胞分泌的IL-2逐渐减少。IL-2分泌减少的范围在第7天为7% - 11%，第14天为0% - 29%，第35天为25% - 50%。对于临床应用，载体在高浓度下的稳定产生是一个重要考虑因素。逆转录病毒载体pLXSN-tIL2产生的病毒滴度最高，被选用于进一步的特性分析。对来自LXSN-tIL2产生细胞系的经SacI消化的基因组DNA和经SacI消化的pLXSN-tIL2质粒DNA进行Southern印迹分析，显示出预期的3.2-kbp片段，表明不存在转基因重排，并且该载体适合作为临床应用的候选载体。
