Engineered Soluble Monomeric IgG1 Fc with Significantly Decreased Non-Specific Binding

Abstract

Due to the long serum half-life provided by the neonatal Fc receptor (FcRn) recycling, the IgG1 Fc has been pursued as the fusion partner to develop therapeutic Fc-fusion proteins, or as the antibody-derived scaffold that could be engineered with antigen-binding capabilities. In previous studies, we engineered the monomeric Fc by mutating critical residues located on the IgG1 Fc dimerization interface. Comparing with the wild-type dimeric Fc, monomeric Fc might possess substantial advantages conferred by its smaller size, but also suffers the disadvantage of non-specific binding to some unrelated antigens, raising considerable concerns over its potential clinical development. Here, we describe a phage display-based strategy to examine the effects of multiple mutations of IgG1 monomeric Fc and, simultaneously, to identify new Fc monomers with desired properties. Consequently, we identified a novel monomeric Fc that displayed significantly decreased non-specificity. In addition, it exhibited higher thermal stability and comparable pH-dependent FcRn binding to the previous reported monomeric Fc. These results provide baseline to understand the mechanism underlying the generation of soluble IgG1 Fc monomers and warrant the further clinical development of monomeric Fc-based fusion proteins as well as antigen binders.

The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

“…A monomeric version of L Fc (mFc) was generated by mutating critical residues on the IgG1-Fc dimerization interface located in CH3 domain (T366R, L368H, P395K, and F405R) (Ying et al, 2012;Wang et al, 2017).…”

“…Also, a F405R mutation seems to favor Fc monomer formation (Shan et al, 2016). Later, it was shown that the S351L mutation might contribute to the non-specific binding of mFc to unrelated targets (Ying et al, 2012;Wang et al, 2017).…”

“…Importantly, small monomeric Fc can be exploited to expand the Fc-based therapeutic applications, due to its unique receptor binding pattern. Monomeric Fc (i) binds to FcRn and exhibits a similar in vivo half-life to wild-type dimeric Fc; (ii) lacks binding to FcγRIIIa, which results in the absence of Fc-effector functions; and (iii) possesses high-affinity binding to FcγRI that can be used for toxin targeting (Ying et al, 2012(Ying et al, , 2014Wang et al, 2017).…”

Boosting therapeutic potency of antibodies by taming Fc domain functions

“…However, mFc could nonspecifically bind to several viral or cancer-related proteins, such as gp140 of HIV-1, EDII of Zika virus, mesothelin, 5T4, PD-L1, OX40, and TIM3. Nonspecific bindings have been excluded by phage-display library screening, and the critical role of T366R and L368H on building monomeric status has been identified 117 .…”

Human IgG1 Fc pH-dependent optimization from a constant pH molecular dynamics simulation analysis

“…The simulation study showed that the performance of monomeric Fc with FcRn does not lose the pHdependent binding mechanism, where monomeric Fc was employed for all the simulation systems. [39][40][41][42][43] All the systems showed some degree of conformation rearrangement, with a greater extent observed at pH 7.5 ( Fig. 1 and ESI Fig.…”