title: Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and βklotho agonist antibodies.
abstract: Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or βklotho receptor (βklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for βklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.
authors.affiliation: Amgen British Columbia, 7990 Enterprise Street, Burnaby, British Columbia, Canada.
authors.forename: Ognjen
authors.initials: O
authors.lastname: Grujic
authors.affiliation: Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, CA, United States.
authors.forename: Jennitte
authors.initials: J
authors.lastname: Stevens
authors.affiliation: Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, CA, United States.
authors.forename: Robert Y-T
authors.initials: RY
authors.lastname: Chou
authors.affiliation: Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA, United States.
authors.forename: Jennifer V
authors.initials: JV
authors.lastname: Weiszmann
authors.affiliation: Amgen British Columbia, 7990 Enterprise Street, Burnaby, British Columbia, Canada.
authors.forename: Laura
authors.initials: L
authors.lastname: Sekirov
authors.affiliation: Amgen British Columbia, 7990 Enterprise Street, Burnaby, British Columbia, Canada.
authors.forename: Christy
authors.initials: C
authors.lastname: Thomson
authors.affiliation: Amgen British Columbia, 7990 Enterprise Street, Burnaby, British Columbia, Canada.
authors.forename: Anita
authors.initials: A
authors.lastname: Badh
authors.affiliation: Amgen British Columbia, 7990 Enterprise Street, Burnaby, British Columbia, Canada.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Grauer
authors.affiliation: Amgen British Columbia, 7990 Enterprise Street, Burnaby, British Columbia, Canada.
authors.forename: Brian
authors.initials: B
authors.lastname: Chan
authors.affiliation: Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, CA, United States.
authors.forename: Kevin
authors.initials: K
authors.lastname: Graham
authors.affiliation: Amgen British Columbia, 7990 Enterprise Street, Burnaby, British Columbia, Canada.
authors.forename: Kathy
authors.initials: K
authors.lastname: Manchulenko
authors.affiliation: Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, CA, United States.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Dillon
authors.affiliation: Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Amgen British Columbia, 7990 Enterprise Street, Burnaby, British Columbia, Canada. Electronic address: ifoltz@amgen.com.
authors.forename: Ian N
authors.initials: IN
authors.lastname: Foltz
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D000954:Antigens, Surface / Q000737:chemistry* / Q000276:immunology*; D016466:CHO Cells; D003412:Cricetulus; D004220:Disulfides / Q000737:chemistry / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods; D000090265:Klotho Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology*; D064446:Orexin Receptors; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000276:immunology*; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C477337:CD200R1 protein, human; D004220:Disulfides; C538995:KLB protein, human; D008565:Membrane Proteins; D064446:Orexin Receptors; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D000090265:Klotho Proteins
keywords: Agonist; Antibody isoform; CD200R; Subclass; βklotho