A SINGLE ENGINEERED AMINO-ACID SUBSTITUTION CHANGES ANTIBODY FINE SPECIFICITY

During the acquisition of humoral immunity, the process of somatic hyp ermutation introduces nucleotide substitutions into expressed antibody (Ab) V region genes. Studies employing in vitro mutagenesis have show n that recurrent mutations observed in vivo often enhance the affinity of the target Ab for Ag. Here we show that a single amino acid replac ement at position 35 in the H chain of an unmutated Ab with specificit y for p-azophenylarsonate (Ars) confers specificity for the structural ly related hapten p-azophenylsulfonate (Sulf) while abolishing specifi city for Ars. The mutant Ab binds Sulf with an affinity characteristic of Ab produced by memory B cells. The same mutation in the somaticall y mutated anti-Ars Ab 36-71, for which the Fab crystal structure is kn own, resulted in a significant shift in fine specificity from Ars to S ulf. Examination of the crystal structure suggests that the specificit y change is caused by a decrease in binding site size and/or new hydro gen bond geometry. Because the mutation at position 35 had been observ ed in somatically mutated Ab elicited by immunization with Ars followe d by Sulf, the results confirm that somatic mutation in vivo can alter Ab specificity. The results also support the potential of Ab engineer ing to alter antigenic specificity.