Modulation of antibody affinity by a non‐contact residue

Protein Science - Tập 2 Số 2 - Trang 206-214 - 1993
Joel F. Schildbach1, Richard I. Near2, Robert E. Bruccoleri3, Edgar Haber4, P.D. Jeffrey3, Jiřı́ Novotný3, S. Sheriff3, Michael N. Margolies2,5
1Program on Immunology, Harvard University Graduate School of Arts and Sciences, Cambridge, Massachusetts 02138
2Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
3Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543
4Cardiovascular Biology Laboratory, Division of Biological Sciences, Harvard School of Public Health, Boston, Massachusetts 02115
5Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Tóm tắt

AbstractAntibody LB4, produced by a spontaneous variant of the murine anti‐digoxin monoclonal antibody 26–10, has an affinity for digoxin two orders of magnitude lower than that of the parent antibody due to replacement of serine with phenylalanine at position 52 of the heavy chain variable region (Schildbach, J.F., Panka, D.J., Parks, D.R., et al., 1991, J. Biol. Chem. 266, 4640–4647). To examine the basis for the decreased affinity, a panel of engineered antibodies with substitutions at position 52 was created, and their affinities for digoxin were measured. The antibody affinities decreased concomitantly with increasing size of the substituted side chains, although the shape of the side chains also influenced affinity. The crystal structure of the 26–10 Fab complexed with digoxin (P.D.J., R.K. Strong, L.C. Sieker, C. Chang, R.L. Campbell, G.A. Petsko, E.H., M.N.M., & S.S., submitted for publication) shows that the serine at heavy chain position 52 is not in contact with hapten, but is adjacent to a tyrosine at heavy chain position 33 that is a contact residue. The mutant antibodies were modeled by applying a conformational search procedure to position side chains, using the 26–10 Fab crystal structure as a starting point. The results suggest that each of the substituted side chains may be accommodated within the antibody without substantial structural rearrangement, and that none of these substituted side chains are able to contact hapten. These modeling results are consistent with the substituents at position 52 having only an indirect influence upon antibody affinity. The mutagenesis and modeling results suggest that even conservative replacements of non‐contact residues can alter affinity indirectly through their impact on contact residue placement.

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Protein Science

Tập 2 Số 2

206-214

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