Catabolism of the murine IgG1 molecule: evidence that both CH2-CH3 domain interfaces are required for persistence of IgG1 in the circulation of mice.
Site-directed mutagenesis of a recombinant Fc-hinge fragment has previously been used to identify a region of the murine IgG1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains. In the current study the nature of this 'catabolic site' has been further analysed using recombinant techniques. Fc-hinge, CH2-hinge, CH2 and CH3 fragments have been expressed in Escherichia coli, purified and analysed in pharmacokinetic studies in mice. The CH2-hinge has been analysed as both a monomer and dimer, and the dimer has a longer beta phase half-life (61.6 h) than the monomer (29.1 h). This suggests that two catabolic sites per Fc fragment are required for serum persistence. The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc-hinge fragment comprising a heterodimer of one Fc-hinge with the wild type (WT) IgG1 sequence and a mutant Fc-hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434). This hybrid is cleared with a beta phase half-life of 37.9 h and this is significantly shorter than that of the WT Fc-hinge fragment (82.9 h). In contrast to the CH2-hinge dimer, the CH3 domain is cleared rapidly (beta phase half-life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment. The data extend our earlier observations concerning a region of the murine IgG1 molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy.
Kim
J K
JK
Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas 75235-8576.
Tsen
M F
MF
Ghetie
V
V
Ward
E S
ES
D000818:Animals; D001483:Base Sequence; D001704:Biopolymers / Q000378:metabolism; D004926:Escherichia coli; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism
D001704:Biopolymers; D007074:Immunoglobulin G; D011994:Recombinant Proteins