Soluble Monomeric IgG1 Fc

Abstract

Background:The Fc region of an antibody is a homodimer of two CH2-CH3 chains. Results: Monomeric IgG1 Fcs (mFcs) were generated by using a novel panning/screening procedure. Conclusion: The mFcs are highly soluble and retain binding to human FcRn comparable with that of Fc. Significance: The mFcs are promising for the development of novel therapeutic antibodies of small size and long half-lives.

Engineered Soluble Monomeric IgG1 CH3 Domain

“…Generation of Soluble mCH3-We previously reported the generation of three mFc proteins using a novel multiple panning/screening procedure (15). A combination of six or seven specific mutations on the CH3 dimerization interface caused mFcs to be highly soluble and monomeric.…”

“…Protein Expression and Purification-mCH3, mCH3 fusion proteins, mCH3cc, and other antibody domains were expressed in E. coli HB2151 by using a procedure similar to that described previously (15). Protein purity was judged by SDS-PAGE, and protein concentration was measured spectrophotometrically (NanoVue, GE Healthcare).…”

“…We have previously generated isolated single CH2 domains and monomeric Fc (mFc) and characterized their interactions with FcRn (13)(14)(15). Here, we report for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3).…”

“…Here, we report for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3). We found that the engineering of CH3 by structurebased mutagenesis, which resulted in soluble mFcs (15), was not effective in the generation of soluble mCH3. This was likely due to the absence of the highly soluble CH2.…”

“…In this current study, we found that a specific combination of four mutations is essential in generating soluble mCH3. In contrast to the wildtype dimeric CH3 (CH3), the mCH3 exhibited pH-dependent binding to a human single-chain soluble FcRn (sFcRn) (15,16), which resembled that of bacterially expressed Fc but with lower affinity (K D ϭ 940 nM) at pH 6. The free energy of mCH3 bind-ing to sFcRn was higher than that of isolated CH2 and dimeric CH3 (which did not bind FcRn) but lower than that of mFc.…”