Construction and characterization of retroviral vectors expressing biologically active human interleukin-12.
Construction and characterization of retroviral vectors expressing biologically active human interleukin-12.
复制标题
表达生物活性人白细胞介素 12 的逆转录病毒载体的构建和表征。
DOI：
10.1089/hum.1994.5.12-1493
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发表时间：
1994
影响因子：
4.2
通讯作者：
Lotze,MT
中科院分区：
文献类型：
--
作者：
Zitvogel,L;Tahara,H;Cai,Q;Storkus,WJ;Muller,G;Wolf,SF;Gately,M;Robbins,PD;Lotze,MT
白细胞介素-12（IL-12）是一种异源二聚体细胞因子，最初由其诱导溶细胞淋巴细胞成熟的能力以及其在诱导溶细胞活性中与IL-2有效协同的能力定义。最近对小鼠的研究表明，IL-12能够导致肿瘤消退并刺激治疗动物的长期抗肿瘤免疫力。为了研究IL-12直接基因转移到肿瘤中的抗肿瘤作用，我们开发了协同表达IL-12的两个亚基的逆转录病毒载体。使用基于MFG的逆转录病毒载体来产生重组逆转录病毒，其中长末端重复（LTR）驱动的多顺反子转录物编码人IL-12的两个亚基：hp 35和hp40 cDNA使用来自脑心肌炎病毒（DFG-hIL-12）的内部核糖体进入位点（IRES）连接并共表达。此外，两个IRES序列用于表达IL-12的两个亚基和来自相同多顺反子信息的新霉素抗性（neoR）选择性标记基因（TFG-hIL-12）。嗜中性DFG-hIL-12和TFG-hIL-12病毒用于感染人和鼠细胞系以及原代肿瘤培养物。在PHA母细胞增殖生物测定和ELISA中测量未选择的感染细胞的人IL-12的产生，并且范围为15至40 ng/106个细胞/24 hr。在TFG-hIL-12感染的细胞的G418选择之后，IL-12的表达水平显著更高（高达120 ng/106个细胞/24 hr）。感染细胞分泌的IL-12蛋白具有重组hIL-12的所有生物学活性：增殖活化的自然杀伤（NK）细胞和T细胞，刺激NK细胞和T细胞诱导γ-干扰素（IFN-γ），增强淋巴因子激活的杀伤（LAK）活性。这些表达人IL-12的逆转录病毒载体在评估IL-12的生物学特性以及用于癌症患者的基因治疗的临床试验中应该是有用的。
Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hlL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neoR) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/106cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/106cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-γ (IFN-γ) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer.