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 (AUClast). 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.
Highlights
The application of Fc-fusion strategies to therapeutic proteins has become increasingly popular in recent years, as evidenced by the appearance of commercial products such as Orencia (CTLA4-Fc), Amevive (LFA3-Fc) and Enbrel (TNFR-Fc) [1]
The purified EPO-hybrid Fc (hyFc) protein consisted of a homodimer of approximately 130 kDa, which is slightly heavier than the EPO-IgG1 Fc protein (Fig. S1); both proteins had similar pI values, indicating comparable carbohydrate content
Using ELISA, we found that EPO-hyFc was unable to bind either protein over a concentration range of 31.2 to 2000 ng/ml for Fcc receptor I (FccR I) and 0.1 to 100 ug/ml for component 1q (C1q) (Fig. 2)
Summary
The application of Fc-fusion strategies to therapeutic proteins has become increasingly popular in recent years, as evidenced by the appearance of commercial products such as Orencia (CTLA4-Fc), Amevive (LFA3-Fc) and Enbrel (TNFR-Fc) [1]. The IgG2 amino acids flanking these substitutions are identical to those in the native IgG4 sequence; the stretches of amino acids with substitutions in IgG2 are present in IgG4 Another IgG2/IgG4 hybrid, eculizumab (Soliris), is a monoclonal antibody against the terminal complement protein C5 consisting of the hinge and CH1 regions of IgG2 and the CH2 and CH3 regions of IgG4 [11]. Without mutating or substituting sites, it is difficult to completely avoid ADCC and CDC This strategy imposes more severe steric hindrance between neighboring molecules and diminishes bioactivity due to the relatively low hinge flexibility of IgG2 (32u)
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