Delineation of the Amino Acid Residues Involved in Transcytosis and Catabolism of Mouse IgG1

The MHC class I-related receptor, FcRn, is involved in both the transcytosis of serum γ-globulins (IgGs) and in regulating their serum persistence. The interaction site of FcRn on the Fc region of rodent IgG has been mapped to residues at the CH2-CH3 domain interface using site-directed mutagenesis and x-ray crystallographic analyses. In the current study, the role of individual residues (H310, H433, and N434) at this interface in mediating the Fc-FcRn interaction has been investigated using recombinant, mutated Fc hinge fragments derived from mouse IgG1. In addition, two highly conserved Fc histidines (H435 and H436) have been mutated to alanine, and the resulting mutated Fc hinge fragments were analyzed in both transcytosis and pharmacokinetic studies in mice and in competition binding assays using recombinant, soluble FcRn. The analyses indicate that mutation of H310, H435, and, to a lesser extent, H436 to alanine results in reduced activity of the Fc hinge fragments in both in vivo and in vitro assays. Thus, in addition to the previously defined role of I253 in the FcRn-IgG interaction, these histidines play a key role in mediating the functions conducted by this Fc receptor. The effects of these mutations on binding of Fc hinge fragments to staphylococcal protein A have also been analyzed and demonstrate a partial, but not complete, overlap of the FcRn and staphylococcal protein A interaction sites on mouse IgG1.</p

Increasing the serum persistence of an IgG fragment by random mutagenesis

The major histocompatibility complex (MHC) class l-related receptor FcRn is involved in regulating serum gammaglobulin (IgG) levels in mice. With the aim of increasing the serum half-life of a recombinant murine Fcγ1 fragment, the affinity for binding to FcRn at pH 6,0 has been increased by random mutagenesis of Thr252, Thr254, and Thr256 followed by selection using bacteriophage display. These residues were chosen as they are in proximity to the FcRn-IgG (Fc) interaction site. Two mutants with higher affinity (due to lower off-rates) than the wild-type Fc have been isolated and analyzed in pharmacokinetic studies in mice. The mutant with the highest affinity has a significantly longer serum half-life than the wild type fragment, despite its lower off-rate from FcRn at pH 7.4. The results provide support for the involvement of FcRn in the home-ostasis of serum IgGs in mice. The indications that a homologous FcRn regulates IgG levels in humans suggest that this approach has implications for increasing the serum persistence of therapeutic antibodies.</p