Next generation human alpha Interferons obtained in CHO cells by O-Glycoengineering

CEAGLIO, NATALIA; CRAVERO, DIANELA; GUGLIOTTA, AGUSTINA; ETCHEVERRIGARAY, MARINA; KRATJE, RICARDO; OGGERO EBERHARDT, MARCOS

Lille

Congreso; 23rd Meeting of the European Society for Animal Cell Technology (ESACT); 2013

ESACT

Multiple biological activities of human alpha interferons (hIFN-alpha) including antiviral, antiproliferative and immunomodulating properties have motivated the development of their recombinant forms to be used for treatment of numerous viral and tumor diseases. Unfortunately, one major issue regarding the clinical use of rhIFN-alpha is its short half-life in circulation after injection of patients. For that reason, administration of high and frequent doses of the cytokine is required, leading to many adverse side effects.Our goal was to exploit the ability of O-glycans to extend the half-lives of proteins to create O-glycosylated rhIFN-alpha variants with lower in vivo clearance and preserved bioactivity. For this purpose, we fused a peptide containing four potential O-glycosylation sites derived from the carboxi-terminal sequence of the hCG beta-subunit (CTP) to the N-terminal and the C-terminal ends of rhIFN-alpha2b. CHO-K1 cells were employed as hosts to express CTP-IFN and IFN-CTP for physicochemical and biological characterization. For both molecules SDS-PAGE followed by Western blot evidenced the presence of a broad band between 28 and 37 kDa, indicating a great microheterogeneity of glycoforms with higher molecular mass with respect to wild-type IFN. This result was confirmed by isoelectric focusing/Western blot, which clearly showed a great number of isoforms with highly acidic pI due to the incorporation of the CTP and, consequently, of new carbohydrates containing terminal sialic acids. In vitro antiviral specific bioactivity of CTP-IFN and IFN-CTP ranged between 66 and 74%, respectively, compared with that of native rhIFN-alpha2b, while antiproliferative specific bioactivity remained practically invariable (92 and 112%, respectively). These preliminary results suggest that O-glycoengineering could be an attractive approach in order to increase charge and mass of IFN with the aim of improving its pharmacokinetic properties while preserving high in vitro bioactivity.