Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein, Additionally, CB4-1 exhibits cross-reactive b inding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides, Crystal structures demonstrate that the epitope pe ptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1, Site directed mutagenesis of the fragme nt variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutatio ns of Ab amino acid side chains, which are in direct contact with the Ag, s how opposite influences on the binding of the two peptides, Whereas the aff inity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr (32) Ala is decreased 250-fold, the binding of the nonhomologous peptide re mains unchanged. In contrast, the mutation light chain variable region Phe( 94)Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep, Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the sc Fv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method al so reveals an inverse compensatory amino acid exchange for the nonhomologou s peptide which increases the affinity to the scFv mutant light chain varia ble region Phe(94)Ala up to the level of the e-pep affinity to the wild-typ e scFv.