Relative location of epitopes involved in synergistic antibody binding using human chorionic gonadotropin as a model
✍ Scribed by Thomas Klonisch; Peter J. Delves; Peter Berger; George Panayotou; Adrian J. Lapthorn; Neil W. Isaacs; Georg Wick; Torben Lund; Ivan M. Roitt
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 965 KB
- Volume
- 26
- Category
- Article
- ISSN
- 0014-2980
No coin nor oath required. For personal study only.
✦ Synopsis
Relative location of epitopes involved in synergistic antibody binding using human chorionic gonadotropin as a model
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 12'I-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 a2/(33/5r a2/ hCGPcrp (C-terminal peptide) and P315kCGPCTP showed the strongest enhancement, with binding more than 10-fold greater than the sum of I2'I-hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups PI/p3,s, c1,2/P3/s, Pl/a2, and a2/a3/' (3-9-fold). The greater frequency of synergy obtained with the linear epitopes of the hCGPcrp can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCGa and core-hCGP (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.