Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the first of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library construction, and (3) secondary library construction. The first step, described here, entails design, synthesis, and cloning of four PLs. These PLs are designed with specific properties amenable to therapeutic antibody development using one universal variable heavy (V_H) scaffold and four distinct variable light (V_L) scaffolds. The scaffolds are diversified in positions that bind both protein and peptide targets identified in antibody-antigen complexes of known structure using the amino acid frequencies found in those positions in known human antibody sequences, avoiding residues that may lead to developability liabilities. The diversified scaffolds are combined with 90 synthetic neutral HCDR3 sequences designed with developable human diversity genes (IGHD) and joining heavy genes (IGHJ) in germline configuration, and assembled as single-chain variable fragments (scFvs) in a V_L-linker-V_H orientation. The four designed PLs are synthesized using trinucleotide phosphoramidites (TRIMs) and cloned independently into a phagemid vector for M13 pIII display. Quality control of the cloning of the four PLs is also described, which involves sequencing scFvs in each library.