Mechanisms of self-association of a human monoclonal antibody CNTO607.
Some antibodies have a tendency to self-associate leading to precipitation at relatively low concentrations. CNTO607, a monoclonal antibody, precipitates irreversibly in phosphate-buffered saline at concentrations above 13 mg/ml. Previous mutagenesis work based on the Fab crystal structure pinpointed a three residue fragment in the heavy chain CDR-3, (99)FHW(100a), as an aggregation epitope that is anchored by two salt bridges. Biophysical characterization of variants reveals that F99 and W100a, but not H100, contribute to the intermolecular interaction. A K210T/K215T mutant designed to disrupt the charge interactions in the aggregation model yielded an antibody that does not precipitate but forms reversible aggregates. An isotype change from IgG1 to IgG4 prevents the antibody from precipitating at low concentration yet the solution viscosity is elevated. To further understand the nature of the antibody self-association, studies on the Fab fragment found high solubility but significant self- and cross-interactions remain. Dynamic light scattering data provides evidence for higher order Fab structure at increased concentrations. Our results provide direct support for the aggregation model that CNTO607 precipitation results primarily from the specific interaction of the Fab arms of neighboring antibodies followed by the development of an extensive network of antibodies inducing large-scale aggregation and precipitation.
Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, 145 King of Prussia Road, Radnor, PA 19087,  USA.
Deidra
D
Bethea
Sheng-Jiun
SJ
Wu
Jinquan
J
Luo
Linus
L
Hyun
Eilyn R
ER
Lacy
Alexey
A
Teplyakov
Steven A
SA
Jacobs
Karyn T
KT
O'Neil
Gary L
GL
Gilliland
Yiqing
Y
Feng
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D018793:Interleukin-13 / Q000276:immunology*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D012995:Solubility
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D018793:Interleukin-13