title: Identification of IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn: comparison of soluble receptor-based and cell-based binding assays.
abstract: Clinical response to the anti-CD20 antibody rituximab has been demonstrated to correlate with the polymorphism in the FcγRIIIa receptor where patients homozygous for the higher affinity V158 allotype showed a better response rate. This finding suggests that engineering of anti-CD20 for increased FcγRIIIa affinity could result in improved clinical outcome. To identify variants with increased affinity to FcγRIIIa, we developed quantitative assays using soluble receptors as well as engineered cell lines expressing FcγRI or FcγRIIIa on the cell surface. We assayed a set of anti-CD20 IgG(1) variants that had identical Fab regions, but alterations in the Fc regions, in both the soluble receptor-based and cell-based FcγRIIIa binding assays. We obtained similar relative binding affinity increases and assay precisions. The increase in affinity for FcγRIIIa correlated with the increase in activity in the antibody-dependent cellular cytotoxicity assay. These variants had unaltered FcγRI binding. In addition to Fcγ receptors, IgG also binds to FcRn, the receptor responsible for the long circulating half-life of IgG. The mutations in the anti-CD20 variants were previously found not to affect FcRn binding in the soluble receptor-based assays; consequently, we used anti-Her2 variants with different binding affinities to FcRn to study FcRn binding assays. We generated a cell line expressing FcRn on the cell surface to measure IgG binding and obtained similar ranking of these anti-Her2 variants in the cell-based and the soluble receptor-based FcRn binding assays. In conclusion, both the soluble receptor-based and cell-based binding assays can be used to identify IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn.
authors.affiliation: Assay and Automation Technology, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Lu
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Vernes
authors.forename: Nancy
authors.initials: N
authors.lastname: Chiang
authors.forename: Qinglin
authors.initials: Q
authors.lastname: Ou
authors.forename: Jiabing
authors.initials: J
authors.lastname: Ding
authors.forename: Camellia
authors.initials: C
authors.lastname: Adams
authors.forename: Kyu
authors.initials: K
authors.lastname: Hong
authors.forename: Bao-Tran
authors.initials: BT
authors.lastname: Truong
authors.forename: Domingos
authors.initials: D
authors.lastname: Ng
authors.forename: Amy
authors.initials: A
authors.lastname: Shen
authors.forename: Gerald
authors.initials: G
authors.lastname: Nakamura
authors.forename: Qian
authors.initials: Q
authors.lastname: Gong
authors.forename: Leonard G
authors.initials: LG
authors.lastname: Presta
authors.forename: Maureen
authors.initials: M
authors.lastname: Beresini
authors.forename: Bob
authors.initials: B
authors.lastname: Kelley
authors.forename: Henry
authors.initials: H
authors.lastname: Lowman
authors.forename: Wai Lee
authors.initials: WL
authors.lastname: Wong
authors.forename: Y Gloria
authors.initials: YG
authors.lastname: Meng
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000235:genetics / Q000378:metabolism; D000915:Antibody Affinity; D000920:Antibody-Dependent Cell Cytotoxicity; D018951:Antigens, CD20 / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D016466:CHO Cells; D002462:Cell Membrane / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics* / Q000378:metabolism*; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000276:immunology; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D011485:Protein Binding; D015202:Protein Engineering; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000276:immunology; D011961:Receptors, Fc / Q000235:genetics / Q000378:metabolism*; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000069283:Rituximab; D012995:Solubility
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D018719:Erb-b2 Receptor Tyrosine Kinases; D011961:Receptors, Fc; D017452:Receptors, IgG; D011994:Recombinant Proteins; D000069283:Rituximab; C489017:FCGR1A protein, human; C496124:FCGR3A protein, human; C508053:ERBB2 protein, human; C100688:Fc receptor, neonatal