Abstract
Most of the therapeutic antibodies approved for clinical use are full-size IgG1 molecules. The interaction of the IgG1 Fc with the neonatal Fc receptor (FcRn) plays a critical role in maintaining their long half-life. We have hypothesized that isolated Fc domains could be engineered to functionally mimic full-size IgG1 (nanoantibodies) but with decreased (10-fold) size. Here, we report for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3). In contrast to the wild-type dimeric CH3, the mCH3 exhibited pH-dependent binding to FcRn similar to that of Fc. The binding free energy of mCH3 to FcRn was higher than that of isolated CH2 but lower than that of Fc. Therefore, CH3 may contribute a larger portion of the free energy of binding to FcRn than CH2. A fusion protein of mCH3 with an engineered antibody domain (m36.4) also bound to FcRn in a pH-dependent fashion and exhibited significantly higher neutralizing activity against HIV-1 than m36.4-Fc fusion proteins. The m36.4-mCH3 fusion protein was monomeric, stable, soluble, and expressed at a high level in Escherichia coli. We also found that engineering an additional disulfide bond in mCH3 remarkably increased its thermal stability, whereas the FcRn binding was not affected. These data suggest that mCH3 could not only help in the exploration of the dual mechanisms of the CH3 contribution to Fc functions (dimerization and FcRn interactions) but could also be used for the development of candidate therapeutics with optimized half-life, enhanced tissue penetration, access to sterically restricted binding sites, and increased therapeutic efficacy.