Dimers and multimers of monoclonal IgG1 exhibit higher in vitro binding affinities to Fcgamma receptors.
The in vitro binding of monomeric, dimeric and multimeric forms of monoclonal IgG1 molecules, designated mAb1 and mAb2, to the extracellular domains of Fcgamma receptors RI, RIIA and RIIIB were investigated using a surface plasmon resonance (SPR) based biosensor technique. Stable noncovalent and covalent dimers of mAb1 and mAb2, respectively, were isolated from CHO cell expressed materials. The dissociation constants of monomeric mAb1 and mAb2 were determined to be 1 nM for the FcgammaRI-binding and 6-12 microM for the FcgammaRIIA- and FcgammaRIIIB-binding. Dimeric mAb1 and mAb2 exhibited increased affinities, by 2-3 fold for FcgammaRI and 200-800 fold for FcgammaRIIA and FcgammaRIIIB. Further increases in binding were observed when the antibodies formed large immune complexes with multivalent antigens, but not in a linear relation with size. The binding properties of monomeric mAb2 were identical with and without a bound monovalent antigen, indicating that antigen-binding alone does not induce measurable change in binding of antibodies to Fcgamma receptors. Dimerization is sufficient to show enhancement in the receptor binding. Given the wide distribution of the low-affinity Fcgamma receptors on immune effector cells, the increased affinities to aggregated IgG may lead to some biological consequences, depending on the subsequent signal transduction events. The SPR-based in vitro binding assay is useful in evaluating Fcgamma receptor binding of various species in antibody-based biotherapeutics.
Luo
Yin
Y
Characterization & Analytical Development, Wyeth BioPharma, Andover, MA, USA. yluo@wyeth.com
Lu
Zhaojiang
Z
Raso
Stephen W
SW
Entrican
Clifford
C
Tangarone
Bruce
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D000915:Antibody Affinity; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D015374:Biosensing Techniques; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D055503:Protein Multimerization; D017452:Receptors, IgG / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D017452:Receptors, IgG