Hyperglycosylated IFNalpha2b muteins with improved pharmacokinetic properties and higher in vivo antitumor activity

AGUSTINA GUGLIOTTA; NATALIA CEAGLIO; MARINA ETCHEVERRIGARAY; RICARDO KRATJE; MARCOS OGGERO

Buenos Aires

Otro; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedades de Biociencias

IFN-alpha2b is a pleiotropic cytokine that has been used for the treatment of many viral and tumor diseases. Its pharmaceutical relevance has inspired the development of many new IFN-based drugs. IFN4N constitute a hyperglycosylated IFN-alpha2b that was developed in our laboratory by glycoengineering strategies. This new molecule exhibited reduced in vitro antiviral and antiproliferative activity compared to the non-glycosylated IFN. However, IFN4N showed improved pharmacokinetic properties as well as higher in vivo antitumor activity. Since the mutation R23N has been identified as the main responsible of affecting IFN4N antiproliferative capacity, here we propone the design, production, purification and characterization of new highly glycosylated IFN-alpha2b muteins with better in vitro and in vivo biological activity. Different groups of muteins have been designed and produced in CHO-K1 cells in order to reach the goal. Group A involves IFN variants with the same amount of potential glycosylation sites but higher in vitro antiproliferative activity compared to IFN4N. Group B presents muteins with higher glycosylation degree but lower in vitro activity (R23N mutation is present). Group C combines the best mutations in new muteins that exhibited improved in vitro antiproliferative activity as well as higher glycosylation degree. Two muteins of each group were purified in order to analyze their pharmacokinetic properties in Wistar rats. The result showed that the higher the apparent molecular mass, the slower the plasmatic clearance. In vivo experiments, performed in nude mice implanted with prostate cancer derived cells, revealed that the new hyperglycosylated variants were able to reduce the growth rate of the tumors. Particularly, their weight at the end of the treatment was significantly reduced for muteins belonging to group A and C, compared to the control. Our results show the importance of pharmacokinetics as well as in vivo antitumor activity in order to improve the performance of IFN-alpha2b as a biotherapeutic.