Generation and analysis of random point mutations in an antibody CDR2 sequence: many mutated antibodies lose their ability to bind antigen.

Abstract

We have investigated the impact of mutations on the binding functions of the phosphocholine (PC)-specific T15 antibody in the absence of antigen selection pressure. The H chain complementarity determining region 2 (CDR2) sequence of T15 antibody was saturated with point mutations by in vitro random mutagenesis. From the mutant library, 289 clones were screened by direct DNA sequencing. The point mutations generated by this method were randomly distributed throughout the CDR2 region and included all kinds of substitutions. 46 unique mutant antibodies, containing one to four point mutations each, were expressed in SP2/0 myeloma cells. Functional analysis on these antibodies has provided insights into several aspects of somatic mutation. (a) The majority (26/46) of mutant antibodies either lost (20/46) or had reduced (6/46) ability to bind PC-protein conjugates or R36a, a PC-expressing strain of Streptococcus pneumoniae. In contrast, none of the mutant antibodies displayed increased binding for these PC antigens. Taken together with calculations of destructive mutations elsewhere in the V region, the data suggest that somatic mutation may cause extensive wastage among B cells during clonal expansion after antigen stimulation. (b) The frequency of binding-loss mutants increased sharply when a second mutation was introduced into the CDR2 sequence; it appears that, in some cases, two or more mutations are needed to destroy binding. (c) The mutant antibodies were tested for their reactivity to 11 non-PC antigens as well as to three PC analogues. None of the mutants gained new reactivity or changed their ability to discriminate structural analogues, supporting the notion that the major role of somatic mutation is to increase or decrease affinity rather than to create new specificities. (d) Mutations in at least five different positions in CDR2 were deleterious, suggesting that these residues may be essential for antigen binding. Three of these positions are novel in that they had not been identified to be important for binding PC by previous crystallographic analysis. (e) Introduction of mutations into two highly conserved residues in CDR2 did not alter the overall conformation of the V region as judged by antiidiotypic analysis, and, in some cases, did not affect the antigen binding function. The results thus indicate that even nonconservative substitutions of invariant residues need not be deleterious, suggesting that their conservation may be due to reasons other than maintaining antibody structure or specificity.