TOLERANCE TO SINGLE, BUT NOT MULTIPLE, AMINO-ACID REPLACEMENTS IN ANTIBODY V-H CDR2 - A MEANS OF MINIMIZING B-CELL WASTAGE FROM SOMATIC HYPERMUTATION

Mutations in the heavy chain complementarity determining region 2 (CDR 2) of the phosphocholine-specific T15 Ab can have a dramatic effect on the ability of the Ab to bind Ag, A panel of multisite mutants that h ad lost detectable binding to phosphocholine-containing Ags was previo usly created by saturation mutagenesis of the CDR2 region of T15, Base d on the predicted importance of amino acid changes represented in the multisite mutants, we have created single-site mutations, yielding a panel of Abs with which to test 17 of the 19 CDR2 residues. Of the 17 positions examined, only one, Arg(52), is intolerant to change, yieldi ng a nonbinder phenotype even with conservative amino acid replacement , Mutation at two other sites, Ala(50) and Tyr(55), can yield a nonbin der phenotype depending on the amino acid replacement, Single-site mut ations of the remaining 14 positions allowed retention of binding abil ity. Thus, except for positions 50, 52, and 55, multiple mutations mus t be introduced into the CDR2 region to create a nonbinder phenotype. We provide a newly refined model of T15, illustrating the structure an d the interactions of the CDR2 region. Our results imply that introduc tion of point mutations would not normally delete Ag-binding ability u ntil two or more mutations had accumulated. This would minimize potent ially harmful effects of somatic mutation on Ig V region genes and imp rove the chance of survival for an Ab such as T15, which in its unmuta ted form is already well suited to bind Ag.