title: Relative location of epitopes involved in synergistic antibody binding using human chorionic gonadotropin as a model.
abstract: We systematically screened a large panel of well-characterized monoclonal antibodies (mAb) directed towards various epitopes on human chorionic gonadotropin (hCG) for synergistic binding of 125I-hCG when they were adsorbed to a solid phase. The epitope locations involved in synergy were then related to the crystal structure of hCG and discussed in accordance with available data on the hCG epitopes. Enhanced binding of hCG was specific for certain pairs of mAb and was reflected in a 3-50-fold increased apparent functional affinity constant for hCG. Surface plasmon resonance revealed that when the mAb were captured by a polyclonal anti-IgG1 coupled to the Biacore chip, the off rates for hCG were significantly slower with synergistic mAb combinations than for the corresponding single mAb or nonsynergistic pairs of mAb, whereas the on rates did not differ appreciably. Each of the two antibodies involved in synergistic binding of hCG (more than 3-fold compared to additive binding of the two mAb) always belonged to a different epitope cluster in a separate antigenic domain on hCG. Synergistic epitope combinations on holo-hCG were located in similar structural planes. Combinations of mAb directed towards the epitope clusters alpha 2/beta 3/5, alpha 2/hCG beta CTP (C-terminal peptide) and beta 3/5/hCG beta CTP showed the strongest enhancement, with binding more than 10-fold greater than the sum of 125I-hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups beta 1/beta 3/5, c 1/2/beta 3/5, beta 1/alpha 2, and alpha 2/alpha 3/5 (3-9-fold). The greater frequency of synergy obtained with the linear epitopes of the hCG beta CTP can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCG alpha and core-hCG beta (residues 1-112). In general, these studies provide a method for rapid screening of synergistic antibody pairs which also helps to identify non-overlapping epitopes that are accessible in similar structural planes. In turn, this facilitates the design of high-affinity bispecific antibodies targetted to a single antigen molecule.
authors.affiliation: Department of Immunology, University College London Medical School, GB.
authors.forename: T
authors.initials: T
authors.lastname: Klonisch
authors.forename: P J
authors.initials: PJ
authors.lastname: Delves
authors.forename: P
authors.initials: P
authors.lastname: Berger
authors.forename: G
authors.initials: G
authors.lastname: Panayotou
authors.forename: A J
authors.initials: AJ
authors.lastname: Lapthorn
authors.forename: N W
authors.initials: NW
authors.lastname: Isaacs
authors.forename: G
authors.initials: G
authors.lastname: Wick
authors.forename: T
authors.initials: T
authors.lastname: Lund
authors.forename: I M
authors.initials: IM
authors.lastname: Roitt
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001666:Binding Sites, Antibody; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D004357:Drug Synergism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D000939:Epitopes; D015292:Glycoprotein Hormones, alpha Subunit