title: Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin.
abstract: 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.
authors.affiliation: Division of Molecular and Life Sciences, POSTECH, Pohang, Republic of Korea.
authors.forename: Se Jin
authors.initials: SJ
authors.lastname: Im
authors.forename: Sang In
authors.initials: SI
authors.lastname: Yang
authors.forename: Se Hwan
authors.initials: SH
authors.lastname: Yang
authors.forename: Dong Hoon
authors.initials: DH
authors.lastname: Choi
authors.forename: So Young
authors.initials: SY
authors.lastname: Choi
authors.forename: Hea Sook
authors.initials: HS
authors.lastname: Kim
authors.forename: Do Soo
authors.initials: DS
authors.lastname: Jang
authors.forename: Kyeong Sik
authors.initials: KS
authors.lastname: Jin
authors.forename: Yo-Kyung
authors.initials: YK
authors.lastname: Chung
authors.forename: Seung-Hee
authors.initials: SH
authors.lastname: Kim
authors.forename: Sang Hoon
authors.initials: SH
authors.lastname: Paik
authors.forename: Yoo Chang
authors.initials: YC
authors.lastname: Park
authors.forename: Moon Koo
authors.initials: MK
authors.lastname: Chung
authors.forename: Yong Bum
authors.initials: YB
authors.lastname: Kim
authors.forename: Kang-Hyun
authors.initials: KH
authors.lastname: Han
authors.forename: Kwan Yong
authors.initials: KY
authors.lastname: Choi
authors.forename: Young Chul
authors.initials: YC
authors.lastname: Sung
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods*; D002460:Cell Line; D000068256:Darbepoetin alfa; D004921:Erythropoietin / Q000031:analogs & derivatives / Q000235:genetics* / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000737:chemistry; D008024:Ligands; D008297:Male; D008958:Models, Molecular; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology
chemical_list: D007141:Immunoglobulin Fc Fragments; D007132:Immunoglobulin Isotypes; D008024:Ligands; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D000068256:Darbepoetin alfa