REGENERATION OF ANTIDIGOXIN BINDING-ACTIVITY IN AN ANTIBODY BY CHANGES SURROUNDING THE ORIGINAL BINDING DEFECT

A panel of antibodies which differ in their L chain structures and whi ch bind to structurally defined haptens, would be useful in investigat ing L chain structure and function. In a previous study, chain recombi nant antibody CR24 (26-10 H, 45-20 lambda) was produced by hybridoma-h ybridoma fusion. Although both parental antibodies bound digoxin with high affinity, CR24 lacked detectable digoxin-binding activity. Hybrid oma CR24 was subsequently fused with H chain-loss hybridomas in order to produce a panel of antibodies composed of 26-10 H chains and 26-10 ''like'' L chains. Two antibodies produced were CR260 which demonstrat ed digoxin-binding activity and CR256 which did not. CR260 and CR256 e xpressed only one amino acid difference (Pro to Leu at L-96). This dif ference resulted in the CR256 binding defect. In this report, two new antidigoxin antibodies are described. One, SR2E7, contained the Pro to Leu (L-96) defect, but still bound digoxin. Binding affinities and bi nding specificity patterns, as well as complete V(L) DNA sequence and corresponding protein sequence of the new digoxin binding antibody L c hains (SR2E7 and SR1C7) are presented. Both kappa L chains are highly homologous to the 26-10 kappa L chain as well as the BALB/c germline g ene K5. 1. These results suggest that antibodies which are initially d efective in binding activity can be cured by changing specific amino a cids involved in determining the binding-site structure. Molecular mod elling studies of the binding-site region were completed to address L chain structural changes induced by specific amino acid substitutions.