HEAVY-CHAIN POSITION-50 IS A DETERMINANT OF AFFINITY AND SPECIFICITY FOR THE ANTIDIGOXIN ANTIBODY-26-10

Antibody produced by a variant of the murine anti-digoxin hybridoma 26 -10 has reduced afrinity for digoxin but enhanced recognition of the d igoxin 12-hydroxyl due to a Tyr to His substitution at heavy chain pos ition 50 (Schildbach, J. F., Panka, D. J., Parks, D. R., Jager, G. C., Novotny, J., Herzenberg, L. A., Mudgett-Hunter, M., Bruccoleri, R. E. , Haber, E., and Margolies, M. N. (1991) J. Biol. Chem. 266, 4640-4647 ). Consistent with these data, the 26-10 Fab-digoxin x-ray crystal str ucture (Jeffrey, P. D., Strong, R. K., Sieker, L. C., Chang, C. Y., Ca mpbell, R. L., Petsko, G. A., Haber, E., Margolies, M. N., and Sheriff , S. (1993) Proc. Natl. Acad. Sci. U. S. A., in press) reveals that Ty r-50 contacts a region of digoxin that includes the hapten- 12 carbon. To determine the effects of other heavy chain position 50 substitutio ns, mutant antibodies were engineered, and their affinities for digoxi n and digoxin analogues were measured. The affinity of the mutant anti bodies for digoxin roughly correlates with the size of the position 50 side chain. Substitutions of Trp or Phe have no effect on affinity, w hereas substitutions of Asn, His, Leu, Ala, Gly, and Asp confer progre ssively lower affinities. Although Trp and Phe mutants exhibit wild-ty pe specificity, Asn and Asp mutants have improved affinity for digoxin relative to digitoxin (12-deshydroxydigoxin). Leu, Ala, and Gly mutan ts have improved affinity for 12-acetyldigoxin relative to digoxin as compared with 26-10. These results indicate that position 50 is a dete rminant of both antibody affinity and fine specificity for antibody 26 -10 and that single-amino acid substitutions can alter antibody fine s pecificity. Models of the mutants were computationally constructed, an d haptens were docked into the modeled binding sites. The results sugg est that 12-acetyldigoxigenin occupies different orientations in the 2 6-10 and in the Ala mutant binding sites, resulting in altered binding .