Optimal design features of camelized human single-domain antibody libraries

We have constructed a human V-H library based on a camelized V-H sequence, The library was constructed with complete randomization of 19 of the 23 CDR 3 residues and was penned against two monoclonal antibody targets to genera te V-H sequences for determination of the antigen contact residue positions . Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the l ibrary showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disu lfide formation was universal. However, surface plasmon resonance and NMR d ata showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the H-1-N-15 HSQC s pectra, indicated that the V-H sequence for library construction was a good choice. These results should be useful in the design of V-H libraries with optimal features.