Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin

• Published in: PloS one 2011-09, Vol.6 (9), p.e24574
• Main Authors: Im, Se Jin, Yang, Sang In, Yang, Se Hwan, Choi, Dong Hoon, Choi, So Young, Kim, Hea Sook, Jang, Do Soo, Jin, Kyeong Sik, Chung, Yo-Kyung, Kim, Seung-Hee, Paik, Sang Hoon, Park, Yoo Chang, Chung, Moon Koo, Kim, Yong Bum, Han, Kang-Hyun, Choi, Kwan Yong, Sung, Young Chul
• Format: Article
• Language: English
• Subjects: Acids; Amino acids; Animals; Artificial Gene Fusion - methods; Binding sites; Bioavailability; Biocompatibility; Biological activity; Biology; Carbohydrates; Cell Line; Cisplatin; Complement component C1q; Complement system; Cytotoxicity; Darbepoetin alfa; Elongation; Engineering; Erythropoietin; Erythropoietin - analogs & derivatives; Erythropoietin - genetics; Erythropoietin - metabolism; Erythropoietin - pharmacokinetics; Erythropoietin - pharmacology; Fc receptors; Half-life; Hemoglobin; Hemoglobins; Homology; Humans; Immunoglobulin D; Immunoglobulin Fc Fragments - genetics; Immunoglobulin G; Immunoglobulin Isotypes - chemistry; Immunoglobulins; Intravenous administration; Life sciences; Ligands; Male; Medicine; Models, Molecular; Molecular weight; Monkeys; Peptides; Polyethylene glycol; Protein Conformation; Proteins; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacokinetics; Recombinant Fusion Proteins - pharmacology; Toxicity; Toxicology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0024574

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.