Novel interferon alpha glycosylated derivates with extended half-lives

NATALIA CEAGLIO; MARCOS OGGERO; MARINA ETCHEVERRIGARAY; RICARDO KRATJE

La Habana. Cuba

Simposio; BIOTECNOLOGÍA HABANA 2006: Aplicaciones Médicas de la Biotecnología; 2006

Centro de Ingeniería Genética y Biotecnología (CIGB)

Human alpha interferons (hIFN-alpha) comprise a family of closely related proteins that exhibit antiviral, growth inhibitory and immune modulator functions. Recombinant hIFN-alpha2 is used clinically to treat a diversity of human viral diseases and cancers. However, it has a short circulating half-life, which requires high doses and frequent administration to patients. With the aim of increasing the in vivo biological activity of the rhIFN-alpha2b, a glycoengineering strategy was carried out using site directed mutagenesis. Fourteen mutants were prepared by the insertion of one N-glycosylation consensus sequence into different positions of the cytokine. Mutations were focused on amino acid positions 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 introduced into the molecule. Analogs containing two to five N-linked consensus sequences were also obtained and analyzed by western blot, showing a broad profile consistent with the presence of carbohydrates heterogeneous in size. Analogs with four and five N-glycosylation sites (4N-IFN and 5N-IFN) demonstrated the higher site occupancy with a similar molecular weight ranging from 21 to 45 kDa. Pharmacokinetics studies showed a similar behavior of 4N-IFN and 5N-IFN molecules with a 20-fold increase of the area under curve after the subcutaneous inoculation compared to the non-glycosylated rhIFN-alpha2b. Besides, using the intravenous via, the half-life of the 4N-IFN was 125.8 ± 16.4 min in comparison with 14.1 ± 2.8 min of the non-glycosylated cytokine. Therefore, these IFN-alpha2 derivatives have worth as potential drugs for clinical use with enhanced in vivo activity.