Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis.
Site-directed mutagenesis has been used to change amino acid residues of a recombinant Fc-hinge fragment derived from the murine immunoglobulin (Ig)G1 molecule, and the effects of these mutations on the pharmacokinetics of the Fc-hinge fragment have been determined. Specifically, Ile-253, His-310 and Gln-311 of the CH2 domain and His-433 and Asn-434 of the CH3 domain have been changed. In the three dimensional structure of an antibody, these amino acids are in close proximity to each other at the CH2-CH3 domain interface. The mutated Fc-hinge fragments have been purified from recombinant Escherichia coli cells and their pharmacokinetic parameters determined in mice and compared with those of the wild-type Fc-hinge fragment. The results show that the site of the IgG1 molecule that controls the catabolic rate (the 'catabolic site') is located at the CH2-CH3 domain interface and overlaps with the Staphylococcal protein A binding site.
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
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013205:Staphylococcal Protein A / Q000378:metabolism; D013329:Structure-Activity Relationship
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A