title: The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.
abstract: Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector functions are desirable. IgG4 Abs are dynamic molecules able to undergo a process known as Fab arm exchange (FAE). This results in functionally monovalent, bispecific antibodies (bsAbs) with unknown specificity and hence, potentially, reduced therapeutic efficacy. IgG4 FAE is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 Abs. To date, the mechanism of FAE is not entirely understood and studies measuring FAE in ex vivo matrices have been hampered by the presence and abundance of endogenous IgG4 wild-type (WT) Abs. Using representative humanized WT IgG4 monoclonal Abs, namely, anti-IL-6 and anti-TNF, and a core-hinge stabilized serine 228 to proline (S228P) anti-IL-6 IgG4 mutant, it is demonstrated for the first time how anti-IgG4 affinity chromatography can be used to prepare physiologically relevant matrices for assessing and quantifying FAE. A novel method for quantifying FAE using a single MSD immunoassay is also reported and confirms previous findings that, dependent on the redox conditions, the S228P mutation can prevent IgG4 FAE to undetectable levels both in vitro and in vivo. Together, the findings and novel methodologies will allow researchers to monitor and quantify FAE of their own IgG4 molecules in physiologically relevant matrices.
authors.affiliation: From the Department of Bioanalytical Sciences, Non-Clinical Development and john.silva@UCB.com.
authors.forename: John-Paul
authors.initials: JP
authors.lastname: Silva
authors.affiliation: From the Department of Bioanalytical Sciences, Non-Clinical Development and.
authors.forename: Olivia
authors.initials: O
authors.lastname: Vetterlein
authors.affiliation: From the Department of Bioanalytical Sciences, Non-Clinical Development and.
authors.forename: Joby
authors.initials: J
authors.lastname: Jose
authors.affiliation: the Department of Antibody Technology and Biology, UCB Pharma, Slough, SL1 3WE United Kingdom.
authors.forename: Shirley
authors.initials: S
authors.lastname: Peters
authors.affiliation: From the Department of Bioanalytical Sciences, Non-Clinical Development and.
authors.forename: Hishani
authors.initials: H
authors.lastname: Kirby
mesh_terms: D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000097:blood / Q000235:genetics* / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood / Q000235:genetics* / Q000276:immunology; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology; D020125:Mutation, Missense / Q000235:genetics* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha
keywords: IgG4; Immunoassay; S228P mutation; affinity chromatography; antibody; antibody engineering; fab arm exchange; immunology; interleukin; tumor necrosis factor (TNF)