Production of biologically active equine interleukin 12 through expressionof p35, p40 and single chain IL-12 in mammalian and baculovirus expressionsystems

Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediate d immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verif y biologically activity of the products expressed from equine interleukin-1 2 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p 35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 a nd p40 subunits or single chain IL-12 enhanced IFN-gamma production in cell s derived from equine lymph nodes. Preincubation of IFN-gamma inducing prep arations with anti-p40 monoclonal antibody resulted in a significant decrea se in IFN-gamma induction capacity. Medium recovered from p35 and p40-expre ssing baculovirus infected cultures enhanced target cell IFN-gamma producti on and proliferation. Experimental studies in mice and other animals have r evealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectio us disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation o f its potential application in equine species.