Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases

Abstract

The IgG1 Fc is a dimeric protein that mediates important antibody effector functions by interacting with Fcg receptors (FcgRs) and the neonatal Fc receptor (FcRn). Here, we report the discovery of a monomeric IgG1 Fc (mFc) that bound to FcgRI with very high affinity, but not to FcgRIIIa, in contrast to wild-type (dimeric) Fc. The binding of mFc to FcRn was the same as that of dimeric Fc. To test whether the high-affinity binding to FcgRI can be used for targeting of toxins, a fusion protein of mFc with a 38 kDa Pseudomonas exotoxin A fragment (PE38), was generated. This fusion protein killed FcgRI-positive macrophage-like U937 cells but not FcgRI-negative cells, and mFc or PE38 alone had no killing activity. The lack of binding to FcgRIIIa resulted in the absence of Fc-mediated cytotoxicity of a scFv-mFc fusion protein targeting mesothelin. The pharmacokinetics of mFc in mice was very similar to that of dimeric Fc. The mFc's unique FcgRs binding pattern and related functionality, combined with its small size, monovalency and the preservation of FcRn binding which results in relatively long half-life in vivo, suggests that mFc has great potential as a component of therapeutics targeting inflammation mediated by activated macrophages overexpressing FcgRI and related diseases, including cancer. Significance StatementFc fusions are a well-established class of therapeutics, but their uses have been limited by several factors, such as the relatively large size and unwanted cytotoxic effects. Here, we describe a small monomeric Fc that can be exploited to greatly expand the Fc-related therapeutic applications, owing to its unique receptor binding pattern and related functionality. The mFc 1) binds to FcRn and exhibits a similar in vivo half-life to that of dimeric Fc; 2) lacks binding to FcgRIIIa which results in the absence of Fcmediated cytotoxicity; 3) possesses high-affinity binding to FcgRI and can be used for FcgRI targeting. The mFc thus has potential as a component of therapeutics targeting inflammation mediated by activated macrophages overexpressing FcgRI.

Engineered antibody domains with significantly increased transcytosis and half-life in macaques mediated by FcRn

“…Additionally, a2b and 2C10 exhibit significantly higher thermal stability (76.9 C and 80.3 C) compared to the isolated CH2 domain; it is comparable to or higher than that of an isolated Fc fragment. 33 They are also highly expressed in E. coli, with yields of over 10 mg per liter of bacterial culture. Although immunogenicity is a potential problem for such engineered proteins, it is likely that the immunogenicity of a2b and 2C10 could be relatively low compared with similar foreign proteins, considering that both the engineered CH2 domain and the FcRn binding motif are of human origin, and that they have been engineered to mimic the native conformation of IgG1 Fc to effectively bind FcRn.…”

Boosting therapeutic potency of antibodies by taming Fc domain functions

“…Fc engineering efforts for better therapeutic efficacy, other than for modulating affinity profiles to Fc receptors, are also being extensively pursued. One example is an engineered Fc that does not form a homodimer but remains as a soluble monomer, mFc, with half size, displaying (i) high affinity for FcγRI, (ii) no detectable binding to FcγRIIIa, and (iii) similar pH-dependent FcRn binding 115,116 . This engineering was focused on downsizing the molecular weight of Fc for therapeutic simplicity.…”

Comprehensive elucidation of the structural and functional roles of engineered disulfide bonds in antibody Fc fragment

“…Although the positions of mutated cysteines were not involved in Fc␥ receptor (Fc␥R) binding (13), it might affect the interaction by conformational change. Therefore, U937, a high Fc␥RI-expression cell line, was used here for measurement of binding of Fc and its mutants to Fc␥RI, which could be a representative for estimation of binding to Fc receptors (14). The obvious fluorescence intensity shift was observed when wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-swere added into U937 cells, indicating their binding to Fc␥RI (Fig.…”