Gene Therapy (1997) 4, 367–374  1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Cloning of human IL-12 p40 and p35 DNA into the Semliki Forest virus vector: expression of IL-12 in human tumor cells J Zhang 1, *, C Asselin-Paturel 2, *, F Bex 1 , J Bernard 3 , J Chehimi 4 , F Willems 1 , A Caignard 2 ,P Berglund 5 , P Liljestro ¨m 5 , A Burny 1 and S Chouaib 2 1 Universite ´ Libre de Bruxelles, Belgium; 2 CJF 94-11 INSERM, Institut Gustave-Roussy, Villejuif, France; 3 Institut Jean-Godinot, Reims, France; 4 Children’s Hospital of Philadelphia, USA; and 5 Karolinska Institute, Huddinge, Sweden IL-12 can enhance the development of effective immune express the two subunits from the same vector, the p35 responses against tumors as well as against certain infec- and the p40 cDNAs were cloned into pSFV1, each under tious agents. It is therefore a potential candidate for thera- the control of a subgenomic SFV promoter. Recombinant peutic use in cancer therapy and in the design of vaccines RNA produced by in vitro transcription of SFV-IL-12 con- against several infectious diseases. Several studies have struct, was packaged into SFV viral particles with the use demonstrated that IL-12 could efficiently induce tumor of a non-packageable helper RNA. We show that human regression in animal models. To investigate the antitumor tumor cell lines infected in vitro and in vivo with recombi- effect of direct gene transfer of human IL-12 into tumors, nant SFV-IL-12 viral particles secrete high levels of biologi- human IL-12 p35 and p40 cDNAs were cloned into the cally active heterodimeric p35/p40 IL-12, as demonstrated Semliki Forest virus (SFV) vector pSFV1. In order to using ELISA and biological assays. Keywords: human IL-12; SFV vector; gene therapy; human tumor cells administration of IL-12 has been shown to inhibit the Introduction growth of both established subcutaneous tumors and Numerous studies in animal models have indicated that hepatic metastases in mice. 12 Furthermore, local injections the expression of various cytokine genes in tumor cells of IL-12 at the periphery of the tumor have led to generally results in a dramatic alteration of tumor cell regression of established subcutaneous tumors. 12 growth and in induction of tumor-specific immunity. 1 Recently, the anti-angiogenic effect of IL-12 has also been These studies clearly established that tumor vaccines reported. 13 All these properties of IL-12 make it parti- based on the use of cytokines could be a valuable way cularly valuable in gene therapy. Interestingly, the effects of eliciting a specific antitumor response against poorly of paracrine secretion of IL-12 on tumor establishment in immunogenic tumors. vaccination models indicate that local delivery of IL-12 Interleukin-12, a heterodimeric cytokine consisting of can induce an immune response against poorly immuno- two disulfide-bonded subunits of 35 kDa and 40 kDa, 2–4 genic tumors. 14 is secreted by antigen presenting cells (APC). 5 IL-12 has The development of methods in gene transfer suitable a critical early role in the induction of T cell-mediated for tumor therapy is a major focus of research at the immunity. It binds to its receptor on T cells and natural present time. In most studies performed to date, retro- killer (NK) cells, 6,7 and it promotes the induction of prim- virus, adenovirus and adeno-associated virus vectors arily T helper 1 (Th1) response in vitro and in vivo, 8,9 lead- have been used to deliver cytokine genes in mammalian ing to cellular-mediated immunity. IL-12 appears to be a cells. 15 Vaccinations performed in the different protocols physiologic inducer of IFN production by T and NK induced efficient antitumor immunity, which signifi- cells. 10 Injecting recombinant murine IL-12 into mice cantly reduced the malignancy of parental cells inocu- increases NK activity, enhances allogeneic cytolytic T cell lated subsequently. 16–18 Recently, a new vector system responses and induces secretion of IFN. 11 Systemic based on Semliki Forest virus (SFV) was reported. 19 The SFV system is suicidal and characterized by high expression levels and broad host range. 20 This system has already been shown to be effective in recombinant vaccine studies, 21–23 suggesting that it could be successfully used for in vivo expression of cytokines. The current study was undertaken to analyze the possibility of using an SFV vector as a new method of expressing human IL-12 in Correspondence: Dr S Chouaib, CJF 94-11 INSERM, Institut Gustave- human tumor cells. Here, we first describe the construc- Roussy, PR1, 39 rue Camille-Desmoulins, 94805 Villejuif Cedex, France tion of the IL-12 vector and show its efficiency in trans- *The first two authors contributed equally to this work Received 5 November 1996; accepted 8 January 1997 ducing human tumor cell lines, both in vitro and in vivo.