Impact of methionine oxidation on the binding of human IgG1 to Fc Rn and Fc gamma receptors.
Methionine oxidation commonly occurs in the Fc fragment of therapeutic monoclonal antibodies; however, its impact on antibody function has not been addressed. Using surface plasmon resonance and cell binding assays, we examined the impact of methionine oxidation on the binding of two humanized IgG1 antibodies to Fc gamma receptors (Fc gammaR) and to the neonatal Fc receptor (Fc Rn). A panel of Fc gammaRs, including Fc gammaRI, Fc gammaRIIa-131H, Fc gammaRIIa-131R, Fc gammaRIIb/c, Fc gammaRIII ALF, Fc gammaRIII ALV, and Fc gammaRIIIb was evaluated. The binding of oxidized IgG1 molecules to individual receptors remained the same with the exception of Fc gammaRIIa where a subtle decrease in binding to the 131H allele was observed. In contrast, but in agreement with recently reported structural changes associated with Met oxidation, binding to Fc Rn was significantly affected. An increase in K(D) values at pH 6.0 was observed with increasing degree of oxidation, reaching several-fold greater value in highly oxidized samples. To our knowledge this is the first report demonstrating that chemical degradations in the constant region of monoclonal antibodies can impact their function and it highlights the importance of avoiding oxidation in therapeutic antibodies.
Bertolotti-Ciarlet
Andrea
A
Bioprocess Analytical and Formulation Sciences, Merck & Co., Inc., West Point, PA, United States.
Wang
Weirong
W
Lownes
Rebecca
R
Pristatsky
Pavlo
P
Fang
Yulin
Y
McKelvey
Troy
T
Li
Yingzhe
Y
Li
Yunsong
Y
Drummond
James
J
Prueksaritanont
Thomayant
T
Vlasak
Josef
J
D000911:Antibodies, Monoclonal / Q000187:drug effects / Q000378:metabolism; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007074:Immunoglobulin G / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism* / Q000502:physiology*; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011961:Receptors, Fc / Q000378:metabolism*; D017452:Receptors, IgG / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG; D008715:Methionine; D006861:Hydrogen Peroxide; C100688:Fc receptor, neonatal