Design and validation of a synthetic VH repertoire with tailored diversity for protein recognition

Previous studies have indicated differences in the specificity-determining residues (SDRs) of antibodies that recognize haptens, peptides, or proteins. Here, we designed a V-H repertoire based on the human scaffold 3-23/J(H)4 and diversification of high and medium-usage SDRs of anti-protein and anti-peptide antibodies. The repertoire was synthesized by overlapping polymerase chain reaction (PCR) and combined with the V-L chain of the anti-hen egg-white lysozyme (HEL) antibody D1.3. The resulting chimeric single-chain Fv fragments (scFvs) phage-displayed library was panned in HEL-coated immunotubes. After two rounds of selection under non-stringent conditions, that is, trypsinization after 2 h of incubation at room temperature, 63 of 167 clones analyzed (38%) were found to express scFvs specific to HEL. Twenty clones were characterized by DNA sequencing resulting in 10 unique scFvs. Interestingly, the panel of unique scFvs was highly diverse, with V-H sequences differing in 16 of the 17 positions variegated in the repertoire. Thus, diverse chemico-physical and structural solutions were selected from the library, even when the V-H repertoire was constrained by the V-L chain of D1.3 to yield hinders against a definite region of HEL surface. The more often selected scFvs, namely H6-1 and B7-1, which differed in eight SDRs, showed levels of expression in E. coli TG1 strain, 6 and 10 times higher than the parental D1.3 Fv fragment, respectively. Dissociation constants (K-Ds) measured in the BIAcore were I I and 6.6 nM for H6-1 and B7-1, respectively. These values compared well to the K-D of 4.7 nM measured for D1.3, indicating that the V-H repertoire here designed is a valuable source of diverse, well-expressed and high affinity VH domains. Copyright (c) 2006 John Wiley & Sons, Ltd.