Development of the dual-vector system-III (DVS-III), which facilitates affinity maturation of a Fab antibody via light chain shuffling

Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system

“…Briefly, the pHg3A-3 [16] vector was treated with XhoI and SalI restriction endonucleases (Takara, Japan) to remove the Fd (V H + C H1 ) chain linked to the gIII gene segment, and the resulting 4.3 kb vector moiety was ligated to the Fd gene of the EG-19-11 Fab obtained by PCR amplification (reverse primer: 5 -CTCGAGGCCCAGCCGGCCAT GGCCCAGGTGCAGCT-3 , forward primer; 5 -GTCGACTTAGGATTCCAGATCCTCTTCTGAGATG AGT TTTTGTTCGGATTCTATACTAGTACAAGATTTGGGCTC-3 ) and digested with the same set of restriction endonucleases. Electrocompetent E. coli TG1 cells carrying the recombinant pFdEx plasmid were prepared and transformed with 100 ng of the pLf1T-3 phagemid [16] containing the EG-19-11 light chain. Soluble Fab molecules were then produced from the E. coli TG1 cells by growing them in 2× YT/AT supplemented with 0.02% arabinose and 0.1 mM isopropyl-␤-d-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich).…”

“…A secondary antibody library was constructed using the DVS-III as previously described [16]. Briefly, the 350 bp V H gene utilized for Fab EG-L2-11 was obtained by treating the recombinant pHg3f1-2 phagemid vector with SfiI and then subsequently subcloning it into the pHg3A-3 plasmid of the DVS-III.…”

“…Phage antibodies were prepared by superinfecting E. coli TG1 cells expressing EG-19-11 or an anti-IL-15 Fab 4H10-LP4 [16], and immunofluorescence was conducted as previously described with slight modifications [21]. A431 and NIH3T3 cells were grown on coverslips to 50% confluence in six-well plates and incubated with phage antibody for 2 h at 37 • C. The coverslips were then washed six times with PBS, three times for 10 min with glycine buffer (50 mM glycine (pH 2.8), 500 mM NaCl), neutralized with PBS and fixed with PBS containing 4% (w/v) paraformaldehyde for 5 min at RT.…”

“…It is known that light chain shuffling is one of the easiest ways to achieve affinity maturation of Fab clones [27]. Accordingly, we have previously reported the successful affinity maturation of the Fab 4H10 specific for human IL-15 from K D ≈ 200 ≈ 6 nM using a light chain sublibrary, HuNL-D3, containing 1.4 × 10 8 diversity of human naïve kappa chains [16]. For affinity maturation of EG-L2-11 Fab, thereby, a secondary combinatorial Fab library was created with a diversity generated by combining the heavy chain of EG-L2-11 Fab with the HuNL-D3, and two rounds of biopanning were performed against the EGFR-Fc protein that immobilized on microtiter plates.…”

“…doi:10.1016/j.imlet.2010.08.009 nanomolar range from a naïve Ig repertoire [14], and thus, lengthy antibody gene manipulations are usually required to improve the affinities of antibodies of naïve origin. In this study, we attempted to generate high-affinity anti-EGFR human Fab antibodies of naïve origin using our unique hierarchical antibody library system, which consists of a human naïve heavy chain sublibrary, termed HuNH2, created in the dual-vector system-II (DVS-II) designed for isolating target-specific heavy chains [15] and a human naïve light chain sublibrary, termed HuNL-D3, created in the DVS-III designed for affinity maturation through light chain optimization [16].…”