Natural Form of Noncytolytic Flexible Human Fc as a Long-Acting Carrier of Agonistic Ligand, Erythropoietin

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.

人源免疫球蛋白G1 Fc段（Human IgG1 Fc）已被广泛用作生物偶联载体，但在应用于激动性蛋白时存在诸多缺陷，包括抗体及补体介导的细胞毒性，以及融合分子生物活性降低等问题。本研究构建了一种由免疫球蛋白D（IgD）与免疫球蛋白G4（IgG4）组成的非免疫原性、非溶细胞性且具有高柔性的嵌合Fc段（hybrid Fc，简称hyFc），并通过促红细胞生成素（erythropoietin, EPO）偶联物EPO-hyFc对其功能进行了验证。尽管IgD Fc与IgG4 Fc的氨基酸同源性仅为20.5%，但EPO-hyFc仍保留了经典的“Y型”结构；向食蟹猴体内反复静脉给予EPO-hyFc，未引发针对该偶联物的特异性抗体应答。此外，与EPO-IgG1 Fc相比，EPO-hyFc无法结合Fcγ受体I（FcγR I）与补体C1q（C1q）。同时，相较于EPO-IgG1 Fc，EPO-hyFc在大鼠体内外均表现出更优异的生物活性，推测这一现象与IgD段的高柔性密切相关。再者，高唾液酸含量的EPO-hyFc变体EPO-hyFc(H)的平均血清半衰期在大鼠体内约为高糖基化EPO，即达依泊汀α（darbepoetin alfa）的2倍。更为关键的是，在正常大鼠与顺铂诱导的贫血大鼠模型中，皮下注射EPO-hyFc(H)不仅可使血清血红蛋白水平的升高幅度显著高于达依泊汀α，还展现出延迟的血清峰浓度达峰时间，以及翻倍的最终曲线下面积（AUClast）。综上，相较于免疫球蛋白G1 Fc段，hyFc作为激动性蛋白/多肽的偶联载体更具应用潜力，因其可在延长融合分子半衰期的同时，不会引发宿主效应性功能，亦不会阻碍融合分子的生物活性。此外，头对头对比实验证实，相较于高糖基化策略，hyFc融合策略可更有效地提升EPO的体内生物活性。