Construction and Characterization of Retroviral Vectors Expressing Biologically Active Human Interleukin-12

Abstract

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-hIL-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/10(6) cells 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/10(6) cells 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-gamma (IFN-gamma) 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.

Induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12

“…14 -16 A clinical trial using a direct injection of tumors with cultured autologous fibroblasts transduced with a retroviral vector encoding both chains of human IL-12 is currently in progress for the treatment of patients with metastatic melanoma, breast, and head and neck cancer. [17][18][19] Novel gene transfer systems are now being developed to allow in vivo transduction and to overcome the time-consuming and costly need for ex vivo culture and manipulation of tumor cells or fibroblasts before the administration of the potential vectors for in vivo gene delivery. In this regard, much attention has been focused recently on the use of replication-deficient adenovirus (Ad) vectors.…”

Retroviral-mediated transfer of genes encoding interleukin-2 and interleukin-12 into fibroblasts increases host antitumor responsiveness

“…45,46 In mice, an excess of homodimeric p40 can inhibit heterodimeric IL-12. 47 In our construct, although p40 was subcloned upstream to p35 and could have been translated in excess, we did not observe this phenomenon. Using a PHA blast bioassay, the E86/ MSCV-IL-12 cell line and infected autologous fibroblasts produced moderate to high levels of bioactive mIL-12.…”

The selectable marker neo gene down-regulates gene expression from retroviral vectors containing an internal ribosome entry site

“…Indeed, IRES elements have been demonstrated to work in the context of retroviral vectors to express two or more genes. [6][7][8][9][10] Despite the wide use of neo and IRES in retroviral vector designs, factors affecting gene expression in IRES-containing retroviral vectors remain poorly understood. Here we report that the presence of the neo gene in the IRES-containing vectors significantly down-regulated expression of the second gene in the same vector, suggesting a need for a different selectable marker when a high level of gene expression is desired.…”

“…The retroviral vectors MFG was chosen because IRES has already been used to drive gene expression efficiently in this vector. 9 The amphotropic packaging line BING was transfected with respective retroviral vectors and cell-free viral supernatants were used to transduce NIH3T3 cells. Two days after transduction, culture supernatants were taken to measure the concentration of each protein by the commercially available ELISA kit.…”

“…In one type, an independent promoter, either internal or the LTR itself, is used, 5 while in the second type, the neo gene is expressed as part of a multicistronic message using an internal ribosome entry site (IRES). [6][7][8][9][10] The internal promoter-based designs were thought to suffer from the limitation of promoter occlusion where the transcription from one promoter suppresses transcription from another when the two promoters are arranged in the same orientations. [11][12][13] IRESbased designs avoid such problems by expressing multiple genes in one message from a single promoter.…”