Somatic evolution of diversity among anti-phosphocholine antibodies induced with Proteus morganii.

The variable region sequences of light and heavy chains (VL and VH) were determined for 11 hybridoma antibodies produced in response to the PC moiety on Proteus morganii. These hybridomas were derived from two separate fusions, one obtained from mice early in a secondary response and the other from late in a secondary response. All of these antibodies possessed a cross-reactive idiotype found on anti-PC antibodies in the M603 family, and exhibited preferential specificity for PC in the context of P. morganii. We found that all of the antibodies were derived from a single VH/VL pair. VH was encoded by V1, DFL16.1 and JH1, and VL was encoded by a consensus VK8 gene and JK5. Antibodies differed from each other by somatic point mutations that occurred at a high rate. The mutations in VL were approximately one-third as abundant as those in VH and were randomly distributed throughout the molecule. Mutations in VH were concentrated in CDR 2 and 3 and had a replacement to silent ratio that was three to six times greater than predicted from random accumulation. Based on the sequence data, a single genealogic tree with multiple branches could accommodate all the hybrids from a fusion. We concluded that in both examples the anti-PC response arose by somatic mutation and stepwise selection from a single precursor. Antigen binding studies with these 11 hybridomas and a 12th that had no mutations revealed that the acquisition of preferential specificity for antigen was dependent on somatic mutation of germline genes. Additional binding studies demonstrated that continued selection during clonal expansion was probably antigen driven. An unexpected finding was five independently selected antibodies from one fusion that had identically mutated VH and VL sequences. We suggest that the hypermutation mechanism is not a continuously active process during clonal expansion and that it is regulated, probably during the mid to late phase of the primary response.