Design and construction of a phage-displayed Camelid nanobody library using a simple bioinformatics method

Rational design of synthetic phage-displayed libraries requires the identification of the most appropriate positions for randomization using defined amino acid sets to recapitulate the natural occurrence. The present study uses position-specific scoring matrixes (PSSMs) for identifying and randomizing Camelidae nanobody (VHH) CDR3. The functionality of a synthetic VHH repertoire designed by this method was tested for discovering new VHH binders to recombinant coagulation factor VII (rfVII). Based on PSSM analysis, the CDR3 of cAbBCII10 VHH framework was identified, and a set of amino acids for the substitution of each PSSM-CDR3 position was defined. Using the Rosetta design SwiftLib tool, the final repertoire was back-translated to a degenerate nucleotide sequence. A synthetic phage-displayed library was constructed based on this repertoire and screened for anti-rfVII binders. A synthetic phage-displayed VHH library with 1 × 108 variants was constructed. Three VHH binders to rfVII were isolated from this library with estimated dissociation constants (KD) of 1 × 10−8 M, 5.8 × 10−8 M and 2.6 × 10-7 M. PSSM analysis is a simple and efficient way to design synthetic phage-displayed libraries. •Through Position Specific Scoring Matrixes (PSSMs), CDR3 hypervariable region was identified in the VHH scaffold cAbBCII10. The specified hypervariable region was in close conformity with the CDR3 region defined by the IMGT database.•Based on PSSM scores, a set of possible amino acid substitutions was defined for 13 positions at the CDR3 hypervariable region.•Specific VHH binders to recombinant coagulation factor VII were discovered through phage-displayed screening of the obtained library.