(228ca) Selection of CDR-Mimic Peptide By Using Phage Display Method

Biological medicines, such as peptides and antibodies, have been expected to substitute for chemical medicines, because biomolecules have higher specificity for target molecules than chemical compounds. Peptides as drugs with target specificity are more easily selected by in vitro evolution technology than antibody, as well as low production costs that can be synthesized with a general chemical synthesis. However they tend to be readily degraded in vivo and have a limited specificity for target molecules. Although antibody recognizes target molecules with high binding affinities and specificities and shows long biological half-life, development of antibodies for therapy needs for complicated process with a long period. Here, we designed peptide format which can be used not only for peptide medicines, but also for the grafting into antibody: the grafting of the peptide into complementarity determining region (CDR) of camel-type single variable antibody fragment (VHH) to express the function of grafted peptide in antibody.

 To design the peptide format for the grafting into CDR of VHH, we employed the crystal structure of VHH of camel anti-BcII β-lactamase antibody cAbBCII-10. In previous research, the grafting of foreign peptide into CDR1 and CDR3 induced no denaturation of framework, but some trials could not functionalize the grafted VHH. We therefore measured the length of α-carbon atoms between N- and C-terminus of CDR loop based on the crystal structure to make the peptide format that form the same steric structure in CDR as free format has. The measurement gave that the length of α-carbon atoms between N- and C-terminus of CDR3 is 8.70 �. This result showed that CDR3 loop could be mimicked with introduction of disulfide bond.

 Next, we reproduced the CDR mimicked peptide loop on M13 phage surface. To easily construct a large scale library, we applied inverse PCR method to prepare M13 phage libraries. Our method could be expected to reduce loss of library DNA for following electroporation because of 4 steps manipulation procedures. As a result, we constructed M13 phage library with a complexity of ~10⁷ clones.