A fully human scFv phage display library for rapid antibody fragment reformatting

Abstract

Phage display libraries of human single-chain variable fragments (scFvs) are a reliable source of fully human antibodies for scientific and clinical applications. Frequently, scFvs form the basis of larger, bivalent formats to increase valency and avidity. A small and versatile bivalent antibody fragment is the diabody, a cross-paired scFv dimer (∼55 kDa). However, generation of diabodies from selected scFvs requires decreasing the length of the interdomain scFv linker, typically by overlap PCR. To simplify this process, we designed two scFv linkers with integrated restriction sites for easy linker length reduction (17-residue to 7-residue or 18-residue to 5-residue, respectively) and generated two fully human scFv phage display libraries. The larger library (9 × 10(9) functional members) was employed for selection against a model antigen, human N-cadherin, yielding novel scFv clones with low nanomolar monovalent affinities. ScFv clones from both libraries were reformatted into diabodies by restriction enzyme digestion and re-ligation. Size-exclusion chromatography analysis confirmed the proper dimerization of most of the diabodies. In conclusion, these specially designed scFv phage display libraries allow us to rapidly reformat the selected scFvs into diabodies, which can greatly accelerate early stage antibody development when bivalent fragments are needed for candidate screening.

Keywords: Antibody fragment; N-cadherin; diabody; phage display; scFv.

Fig. 1

Reformatting selected scFvs from common phage libraries. In most conventional scFv phage display libraries, the flanking restriction sites (I and II as shown here) can be utilized to rapidly make minibody and scFv-Fc constructs. However, to reformat an scFv into a diabody, the long linker in an scFv has to be shortened in order to induce dimerization. This is usually accomplished by a series of PCRs, which is far more complicated and time consuming, requiring careful design of multiple sets of primers.

Fig. 2

(A) The map of major components of the pHEN1 phagemid vector. V_H and V_L genes are in the same reading frame with the PelB leader, the linker, the Myc tag, the amber stop codon and the phage minor coat protein gene III. Restriction sites NcoI and NotI can be used for subcloning of the scFv gene. (B) DNA and protein sequences of the linkers. Additional restriction sites were engineered into the 17aa-SSA linker and 18aa-SX linker for easier linker length reduction and library construction.

Fig. 3

Schematic outline of the construction of the V_H and V_L libraries. The library construction begins with two existing V gene libraries (Sheets et al., 1998). The V genes were PCR-amplified and cloned into 17aa-SSA-linker-pHEN1 and 18aa-SX-linker-pHEN1 vectors to build the V_H and V_L libraries. CIP stands for calf intestinal alkaline phosphatase.

Fig. 4

Upon the completion of the V_H and V_L libraries, the scFv libraries were constructed via V gene shuffling. The V_L gene repertoire was cut out from the V_L libraries using NotI enzyme in combination with either AscI or SalI enzyme, and then cloned into the V_H library to build the scFv libraries with the two different linkers. CIP stands for calf intestinal alkaline phosphatase.

Fig. 5

V gene usage of the 17aa-SSA-scFv library and the 18aa-SX-scFv library. Thirty functional clones from each scFv library were analyzed, and the germline genes and subgroups were assigned. The frequency of specific V_H and V_L genes is listed. The V genes are classified into subgroups based on the IMGT (http://www.imgt.org) classification. (V_H subgroups: IGHV1, IGHV3, IGHV5, IGHV6; V_κ subgroups: IGKV1, IGKV4; V_λ subgroups: IGLV1, IGLV2, IGLV3, IGLV6.)

Fig. 6

The affinities of the anti-Ncad scFvs were estimated by ELISA on immobilized Ncad recombinant protein. Each concentration was performed in duplicate. The binding curves were analyzed in Graphpad Prism to calculate dissociation constants using the ‘one site-specific binding’ model.

Fig. 7

(A) Four pairs of anti-Ncad antibody fragments were analyzed by Superdex 75 size-exclusion chromatography. The 17aa-linker and the 7aa-linker worked best for the A1 and G6 clones. For the A6 clone, the 7aa-linker diabody formed a mixture of monomer and dimer. For the D6 clone, the 17aa-linker scFv formed a mixture of monomer and dimer. (B) Four random clones from the 18aa-SX-scFv library were reformatted, expressed and purified. The 5aa-linker successfully induced dimerization for all these clones.