N-glycoengineering of hIFN-alpha2b by fusion of ANITVNITV peptide: impact on physicochemical properties, in vitro bioactivity and pharmacokinetics

GUGLIOTTA, AGUSTINA; CEAGLIO, NATALIA; KRATJE, RICARDO; OGGERO, MARCOS

Río de Janeiro

Simposio; VIII Simposio Latinoamericano de Tecnología de Cultivos Celulares (SLATCC 2018); 2018

Sociedad Latinoamericana de Tecnología de Cultivos Celulares (SLATCC)

IFN-α is a multifunctional cytokine since it presents antiviral, antiproliferative and inmunomodulatory activities. Therefore, it has been used as a therapeutic agent for the treatment of viral and tumor diseases. However, due to its rapid plasma clearance, rhIFN-α has to be administered in high and repeated doses in order to reach the desired effect, resulting in the appearance of some undesired symptoms. In order to overcome this kind of problems that arise not only for IFN but for other therapeutic proteins, different engineering strategies have been developed and successfully applied. One of the commonly used strategies involves the chemical and genetic fusion to a peptide, polypeptide or a synthetic polymer. In this sense, the 9-mer peptide ANITVNITV, which contains 2 potential N-glycosylation sites, has been described as a useful tool for improving FSH pharmacokinetics (Perlmann et al., 2003). Other strategy involves the mutation of the protein sequence in order to introduce potential N-glycosylation sites. rhIFN-α2b has been previously modified by our group by replacing aminoacids Pro4, Arg23, Lys70 and Asp77 by Asn. This hyperglycosylated variant -called IFN4N- showed a 25-fold increased half-life and a 20-fold reduced clearance compared to the non-glycosylated IFN-α2b (Ceaglio et al., 2008). In this work, two hIFN-α2b variants were designed and constructed by fusing ANITVNITV peptide to the N-terminus of IFNwt and IFN4N. Thus, IFNwtNter and IFN4NNter were produced in CHO-K1 cells and purified by immunoaffinity chromatography in order to study the effect of this modification on the physicochemical, biological and pharmacokinetic properties of the molecule.The physicochemical characterization of IFNwtNter and IFN4NNter was performed by SDS-PAGE and isoelectric focusing (IEF) followed by Coomasie blue staining. The comparison between modified and unmodified proteins revealed that ANITVNITV-derived glycans contributed both to the apparent molecular weight increment and to the isoelectric point reduction of both analogs. In accordance with this, the sialic acid content was higher for the peptide fusion proteins compared to the corresponding unmodified ones. As it was expected, the pharmacokinetic analysis of IFNwtNter and IFN4NNter revealed a notable improvement of parameters such as Tmax, AUC and CLapp. The in vitro biological activity analysis revealed a reduction of the antiviral specific biological activity (SBA) of IFNwtNter and IFN4NNter compared to IFNwt and IFN4N, respectively. Despite the fact that a decreased in vitro antiproliferative SBA was expected for both proteins, it was only observed for IFNwtNter. Particularly, the peptide addition to IFN4N caused a 5-fold increment of its in vitro antiproliferative SBA. To conclude, the fusion of the ANITVNITV peptide constitutes a useful strategy to improve the physicochemical and pharmacokinetic properties of both IFN variants. Moreover, this modification increased the growth-inhibitory function of IFN4N, resulting in a promising agent for the treatment of tumor diseases. ReferencesCeaglio, N., Etcheverrigaray, M., Kratje, R. y Oggero, M. (2008) Novel long-lasting interferon alpha derivatives designed by glycoengineering. Biochimie. 90:437-449.Perlman, S., van den Hazel, B., Christiansen, J., Gram-Nielsen, S., Jeppesen, C.B., Andersen, K.V., Halkier, T., Okkels, S., Schambye, H.T. (2003) Glycosylation of an N-terminal extension prolongs the half-life and increases the in vivo activity of follicle stimulating hormone. J. Clin. Endocrinol. Metab. 88(7):3227-3235.