CONTRIBUTION OF A SINGLE HEAVY-CHAIN RESIDUE TO SPECIFICITY OF AN ANTIDIGOXIN MONOCLONAL-ANTIBODY

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduce d affinity of surface antibody for antigen. Nucleotide and partial ami no acid sequencing of the variant antibody variable regions revealed t hat 1 variant had a single amino acid substitution: Lys for Asn at hea vy chain position 35. The second variant antibody had 2 heavy chain su bstitutions: Tyr for Asn at position 35, and Met for Arg at position 3 8. Mutagenesis experiments confirmed that the position 35 substitution s were solely responsible for the markedly reduced affinity of both va riant antibodies. Several mutants with more conservative position 35 s ubstitutions were engineered to ascertain the contribution of Asn 35 t o the binding of digoxin to antibody 26-10. Replacement of Asn with Gi n reduced affinity for digoxin 10-fold relative to the wild-type antib ody, but maintained wild-type fine specificity for cardiac glycoside a nalogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduce d affinity by at least 90-fold and caused distinct shifts in fine spec ificity. The Ala mutant demonstrated greatly increased relative affini ties for 16-acetylated haptens and haptens with a saturated lactone. T he X-ray crystal structure of the 26-10 Fab in complex with digoxin (J effrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds wit h 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore perfo rmed molecular modeling experiments which suggested that substitution of Gin or Asp can maintain these hydrogen bonds whereas the other subs tituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in b inding of the wild-type and Gin-mutant antibodies, however, suggest th at these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of thi s antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume als o provides space that can accommodate a congener with a 16-acetyl grou p or saturated lactone, accounting for the altered fine specificity of this antibody.