Specificity Rescue and Affinity Maturation of a Low-Affinity IgM Antibody against Pro-Gastrin-Releasing Peptide using Phage Display and DNA Shuffling

The objectives of the present study were to use phage display to rescue the specificity of an IgM antibody and by the use of DNA shuffling to construct a sublibrary from which mutants with higher affinity could be selected. As a test system, a hybridoma cell line producing low-affinity IgM against recombinant pro-gastrin-releasing peptide (ProGRP) was chosen as starting material for construction of a single-chain Fv (scFv) phage library. One clone, pGRP5, demonstrating affinity for the antigen, was selected for the study. Random mutations were introduced into the scFv sequence by DNA shuffling, and mutants with raised affinity were selected by biopanning against recombinant ProGRP. Clones with affinities improved by approximately two orders of magnitude were selected after the first round of DNA shuffling. An additional 8- to 9-fold rise in affinity was demonstrated in mutants after the second round of mutagenesis. Sequence analysis demonstrated changes primarily in the complementarity-determining region (CDR) H1, CDR L1 and CDR H3 as compared to the original clone. Thus, by the use of phage display in combination with DNA shuffling, the specificity of an IgM antibody was rescued and the affinity was raised almost three orders of magnitude.