Generation and Next-Generation Sequencing-Based Characterization of a Large Human Combinatorial Antibody Library

Abstract

Antibody phage display is a key technology for the discovery and development of target-specific monoclonal antibodies (mAbs) for use in research, diagnostics, and therapy. The construction of a high-quality antibody library, with larger and more diverse antibody repertoires, is essential for the successful development of phage display-derived mAbs. In this study, a large human combinatorial single-chain variable fragment library (1.5 × 1011 colonies) was constructed from Epstein–Barr virus-infected human peripheral blood mononuclear cells stimulated with a combination of two of the activators of human B cells, the Toll-like receptor 7/8 agonist R848 and interleukin-2. Next-generation sequencing analysis with approximately 1.9 × 106 and 2.7 × 106 full-length sequences of heavy chain variable (VH) and κ light chain variable (Vκ) domains, respectively, revealed that the library consists of unique VH (approximately 94%) and Vκ (approximately 91%) sequences with greater diversity than germline sequences. Lastly, multiple unique mAbs with high affinity and broad cross-species reactivity could be isolated from the library against two therapeutically relevant target antigens, validating the library quality. These findings suggest that the novel antibody library we have developed may be useful for the rapid development of target-specific phage display-derived recombinant human mAbs for use in therapeutic and diagnostic applications.

Empowering SARS‐CoV‐2 variant neutralization with a bifunctional antibody engineered with tandem heptad repeat 2 peptides

“…Biopanning was performed to select human antibodies specific to SARS‐CoV‐2 BA.2 RBD from our previously constructed human combinatorial single‐chain variable fragment (scFv) antibody library 23 . Escherichia coli strain ER2738 cells harboring phagemids encoding the scFv genes from the constructed human combinatorial scFv library were cultured in 1.6 L of super broth (SB) supplemented with 50 μg/mL of ampicillin and incubated at 37°C with constant agitation until an OD 600 of 0.6 was achieved.…”

“…Biopanning was performed to select human antibodies specific to SARS-CoV-2 BA.2 RBD from our previously constructed human combinatorial single-chain variable fragment (scFv) antibody library. 23 Escherichia coli strain ER2738 cells harboring phagemids encoding the scFv genes from the constructed human combinatorial scFv library were cultured in 1.6 L of super broth (SB) supplemented with 50 μg/mL of ampicillin and incubated at 37°C with constant agitation until an OD 600 of 0.6 was achieved. Then, 1 × 10 13 plaque-forming units of VCSM13 helper phage (Agilent) were added and incubated at 37°C for 2 h. Subsequently, kanamycin was added to the media at a concentration of 70 μg/mL and incubated at 37°C overnight with constant agitation.…”

A dual-targeting approach using a human bispecific antibody against the receptor-binding domain of the Middle East Respiratory Syndrome Coronavirus

“…After biopanning, 96 phage clones were randomly selected from the final output colonies. The reactivity of these clones to MERS-CoV RBD-His was evaluated using a phage enzyme-linked immunosorbent assay (ELISA), following established methods ( Choi et al, 2023 ). Subsequent DNA sequencing of these clones led to the identification of four distinct scFv clones, each characterized by unique complementarity-determining region (CDR) sequences.…”