Characterization of scFv libraries derived from mice differently immunized with claudin

Abstract

Claudins (CLs), a family of tetratransmembrane proteins consisting of 27 members, are potential targets for enhancing drug absorption, treating cancer, and vaccination. The small extracellular loop regions of CL proteins have made it difficult to develop CL ligands, including antibodies, and to generate recombinant CL proteins, except for CL‐4. Here, we optimized immunization procedures for developing antibody phage display libraries in a single‐chain antibody fragment (scFv) format. CL‐1‐displaying budded baculovirus (CL‐1‐BV) and plasmid DNA encoding the CL‐1 gene were the antigens. BXSB mice, which spontaneously develop a lupus‐like syndrome, were immunized with CL‐1‐BV. The scFv library comprised 7.95 × 105 colony formation units (CFU); however, most clones bound to both mock‐BV and CL‐1‐BV, indicating that the clones may recognize gp64 derived from BV. To overcome this immune response, gp64 transgenic mice were immunized with CL‐1‐BV and used to generate a scFv library up to 2.6 × 105 CFU. We isolated 6 phage clones that bound to CL‐1, ‐2, ‐4 and ‐5, but not to mock‐BV. Furthermore, we got a specific CL‐1‐bound clone. We also prepared a scFv library up to 1.1 × 106 CFU from mice immunized with CL‐1 DNA, but a phage clone that bound to CL‐1, ‐2, ‐4 and ‐5 was isolated. These findings indicate that the gp64 library might be the most promising library for preparing CL‐binding scFv fragments.