Clustered H chain somatic mutations shared by anti-p-azophenylarsonate antibodies confer enhanced affinity and ablate the cross-reactive idiotype.
A cluster of four consecutive CDR2 somatic mutations are shared by the VH regions of two independently isolated hybridoma antibodies with specificity for p-azophenylarsonate (Ars). The mutations appear to be derived by a series of independent events. To assess the influence of these shared somatic mutations on antibody affinity for Ars and on idiotypy, we introduced them, via site-directed mutagenesis, into the V region of an anti-Ars antibody that was otherwise unmutated and we eliminated them from the mutated context of one of the two antibodies in which they were originally found. Results of affinity measurements by fluorescence quenching and of idiotypic binding assays performed on these engineered mutants demonstrated that the shared mutations increased affinity for Ars and eliminated the predominant Id associated with strain A anti-Ars antibodies and four of five idiotypes defined by anti-idiotypic mAb. These results support the interpretation that a strong affinity-based selection pressure has favored the clonal expansion of B cells with receptors containing these mutations despite the loss of a predominant Id. Thus, in producing antibodies containing V regions conferring high affinity for Ag, the combined processes of somatic mutation and clonal selection have generated a common structural solution through parallel repeats.
Parhami-Seren
B
B
Department of Surgery, Massachusetts General Hospital, Boston 02114.
Wysocki
L J
LJ
Margolies
M N
MN
Sharon
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000915:Antibody Affinity; D001391:Azo Compounds / Q000276:immunology*; D001483:Base Sequence; D003429:Cross Reactions; D004252:DNA Mutational Analysis; D005803:Genes, Immunoglobulin; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007130:Immunoglobulin Idiotypes / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides; D014162:Transfection; D010132:p-Azobenzenearsonate / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001391:Azo Compounds; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D009841:Oligonucleotides; D010132:p-Azobenzenearsonate