Structural and functional characterization of disulfide isoforms of the human IgG2 subclass.
In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205 ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1beta ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 >> IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2lambda composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.
Dillon
Thomas M
TM
Department of Pharmaceutics, Amgen Inc., Thousand Oaks, California 91320, USA. Tdillon@amgen.com
Ricci
Margaret Speed
MS
Vezina
Chris
C
Flynn
Gregory C
GC
Liu
Yaoqing Diana
YD
Rehder
Douglas S
DS
Plant
Matthew
M
Henkle
Brad
B
Li
Yu
Y
Deechongkit
Songpon
S
Varnum
Brian
B
Wypych
Jette
J
Balland
Alain
A
Bondarenko
Pavel V
PV
D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D007146:Immunoglobulin lambda-Chains / Q000737:chemistry*; D010084:Oxidation-Reduction; D020033:Protein Isoforms / Q000737:chemistry; D020836:Protein Structure, Quaternary / Q000502:physiology; D013329:Structure-Activity Relationship
D004220:Disulfides; D007074:Immunoglobulin G; D007146:Immunoglobulin lambda-Chains; D020033:Protein Isoforms