Novel CHO-cells derived IFN alpha analogs with enhanced in vivo biological activity

NATALIA CEAGLIO; MARCOS OGGERO; MARINA ETCHEVERRIGARAY; RICARDO KRATJE

Coolum, Sunshine Coast. Queensland. Australia

Conferencia; Cell Culture Engineering XI; 2008

Engineering Conferences International (ECI)

Human alpha interferons (hIFN-alpha) comprise a family of closely related proteins that exhibit antiviral, growth inhibitory and immune modulator functions. The recombinant cytokine has proved efficacy for the treatment of a diversity of human viral diseases and cancers. However, due to its rapid clearance, patients have to be treated by daily or thrice weekly injections, which in turn bring about significant side effects. Besides, as therapeutic levels are difficult to sustain, the effectiveness of hIFN-alpha in the therapy of solid tumors is severely compromised. To address this problem, a long-acting IFN-alpha mutein was designed by introduction of N-linked glycosylation consensus sequences via site-directed mutagenesis. Fourteen mutants were prepared focusing on amino acid substitutions that were believed not to be critical for the protein´s structure and function. Each IFN analog was expressed in CHO cells and, taking into account the retained specific biological activity and the higher carbohydrate content, five N-glycosylation positions were selected to be sequentially introduced into the cytokine. Analogs with increased size and charge were concomitantly obtained with each addition of a functional consensus sequence, resulting in highly glycosylated molecules with four and five N-glycosylation sites (4N-IFN and 5N-IFN). 4N-IFN and 5N-IFN muteins were found to have a similar 20-fold decrease in systemic clearance rate and a 25-fold increase in elimination half-life after subcutaneous inoculation in rats, compared to the non-glycosylated rhIFN-alpha. Moreover, in spite of its lower in vitro activity, the 4N-IFN showed a noteworthy enhanced in vivo potency as shown by its anti-proliferative effect on human prostate carcinoma implanted in nude mice. Carbohydrate structure analysis of the 4N-IFN variant through HPAEC-PAD and MALDI TOF MS evidenced the presence of high amounts of tetraantennary and triantennary structures, both with no, one, two and three N-acetyllactosamine repeat units, as well as small amounts of Man6GNAc2 with one phosphate and diantennary glycans. These structures are very similar to those found in many recombinant glycoproteins expressed in CHO cells, which share the feature of exhibiting a greater in vivo biological activity than their non-glycosylated counterparts. Therefore, the CHO cells-derived 4N-IFN analog turns out to be a potential drug for clinical use, combining less frequency of administration with enhanced therapeutic efficacy. Carbohydrate structure analysis of the 4N-IFN variant through HPAEC-PAD and MALDI TOF MS evidenced the presence of high amounts of tetraantennary and triantennary structures, both with no, one, two and three N-acetyllactosamine repeat units, as well as small amounts of Man6GNAc2 with one phosphate and diantennary glycans. These structures are very similar to those found in many recombinant glycoproteins expressed in CHO cells, which share the feature of exhibiting a greater in vivo biological activity than their non-glycosylated counterparts. Therefore, the CHO cells-derived 4N-IFN analog turns out to be a potential drug for clinical use, combining less frequency of administration with enhanced therapeutic efficacy.