LOCALIZATION OF THE SITE OF THE MURINE IGG1 MOLECULE THAT IS INVOLVEDIN BINDING TO THE MURINE INTESTINAL FC RECEPTOR

Site-directed mutagenesis of a recombinant Fc hinge fragment has recen tly been used to localize the site of the murine IgG1 molecule that is involved in the control of catabolism (the ''catabolic site''). In th e current study, the effects of these CH2 and CH3 domain mutations (Il e 253 to kla 253, His 310 to Ala 310, Gin 311 to Asn 311, His 433 to A la 433 and Asn 434 to Gin 434) on intestinal transfer of Fc hinge frag ments in neonatal mice have been analyzed. Studies using direct transf er and competition assays demonstrate that the mutations affect the tr ansmission from intestinal lumen into serum in a way that correlates c losely with the effects of the mutations on pharmacokinetics. Binding studies of several of the Fe hinge fragments to isolated neonatal brus h borders have been used to confirm the in vivo transmission data. The se analyses have resulted in the localization of the binding site for the intestinal transfer receptor, FcRn, to specific residues of the mu rine Fc hinge fragment. These residues are located at the CH2-CH3 doma in interface and overlap with both the catabolic site and staphylococc al protein A (SpA) binding site. The pH dependence of IgG1 or Fc fragm ent binding to FcRn is consistent with the localization of the FcRn in teraction site to a region of the Fc that encompasses two histidine re sidues (His 310 and His 433). To assess whether one or two FcRn bindin g sites per Fc hinge are required for intestinal transfer, a hybrid Fc hinge fragment comprising a heterodimer of one Fc hinge with the wild -type IgG1 sequence and a mutant Fc hinge with a defective catabolic s ite (mutated at His 310, Gin 311, His 433 and Asn 434) has been analyz ed in direct and competition transmission assays. The studies demonstr ate that the Fc hybrid is transferred with significantly reduced effic iency compared to the wild type Fc hinge homodimer and indicate that t he binding to FcRn, and possibly subsequent transfer, is enhanced by t he presence of two FcRn binding sites per Fc hinge fragment.