Construction of a Double Recombinant Adenovirus Vector Expressing a Heterodimeric Cytokine: In Vitro and In Vivo Production of Biologically Active Interleukin-12

Interleukin-12 (IL-12) is a heterodimeric cytokine that plays an important role in the development of cellular immunity. Clinical applications for this lymphokine include resolution of infectious disease, cancer immunotherapy, and boosting cellular immunity in AIDS patients. When using IL-12 and other cytokines therapeutically, an approach designed to obtain localized cytokine expression would be beneficial, because this could reduce the problem of systemic toxicity. As a means of developing a suitable delivery vehicle for IL-12, we have produced double-recombinant adenovirus vectors containing the p35 subunit cDNA of murine IL-12 in early region 1 of adenovirus type 5 and the cDNA for p40 in early region 3 (AdmIL-12). Cell lines infected with AdmIL-12 produced up to 42,000 units of IL-12/10⁶ cells per 24 hr. Biological activity of the virally expressed product was demonstrated in vitro through its ability to induce proliferation of phytohemagglutinin (PHA)-stimulated lymphoblasts and to stimulate natural killer (NK) activity in naive splenocytes. Mice injected intraperitoneally with these vectors displayed serum IL-12 levels that increased proportionately with the amount of virus administered. IL-12 production in vivo caused a dose-dependent increase in splenic and lung NK cell activity. This work represents the first demonstration of a double-recombinant adenovirus vector expressing a functional heterodimeric protein. The results of these studies support the use of AdmIL-12 as an efficient delivery vehicle for IL-12, and direct studies of its ability to modulate cellular immunity in vivo are currently underway.

Replication-deficient adenovirus vectors are very useful tools for gene delivery, because these viruses are highly infectious and yield high levels of protein production. In terms of immunotherapy, adenoviral vectors can be advantageous because expression is generally localized near the site of injection, thereby reducing the likelihood of systemic toxicity. Preclinical studies have suggested that interleukin-12 (IL-12) may be effective in the treatment of cancer, AIDS, and other infectious diseases. The development of viral vectors expressing this cytokine is complicated by the fact that it is the product of two separate coding sequences. Double-recombinant adenoviruses have been constructed in which the p35 subunit cDNA of murine IL-12 was cloned in the E1 region of adenovirus type 5 and the p40 subunit cDNA in E3. These vectors expressed high levels of bioactive IL-12 both in vitro and in vivo and are currently being studied in animal models of cancer and lung infection.