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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 third and final step of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library construction, (2) filtered library (FL) construction, and (3) secondary library (SL) construction. In the third step, described here, the nucleotide sequences encoding the single-chain variable fragments (scFvs) of FLs are amplified by PCR and combined with the heavy- chain CDR3 region (HCDR3) and joining fragments (H3J) obtained from a pool of donors to maximize diversity ("natural H3J fragments"). These natural H3J fragments are amplified with a set of primers designed to capture >95% of the natural H3J repertoire. The resultant fragments replace the neutral H3J fragments of the FLs, resulting in the final semisynthetic secondary libraries. The quality of these libraries is assessed by sequencing clones chosen at random from the libraries, typically 96 clones. These libraries are then ready to be used for phage selections on targets of interest, providing a robust antibody discovery platform., (© 2024 Cold Spring Harbor Laboratory Press.)

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., (© 2024 Cold Spring Harbor Laboratory Press.)

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 second of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Gold Plus Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library (FL) construction, and (3) secondary library construction. The second step, described here, involves display of the PLs as single-chain variable fragment (scFv) fusions to protein pIII of the M13 phage, as well as heat shock treatment and subsequent selection of well-folded and thermostable scFvs via protein L binding, whereas unstable and defective scFvs are removed by washing steps and centrifugation. The quality of the filtration process is assessed by sequencing clones chosen at random from the FLs. These libraries, enriched with thermostable antibodies, are then ready to be used for the third and final step of the process: generation of secondary libraries., (© 2024 Cold Spring Harbor Laboratory Press.)

Phage display is a versatile and effective platform for the identification and engineering of biologic-based therapeutics. Using standard molecular biology laboratory techniques, one can create a highly diverse and functional antibody phage-displayed library, and rapidly identify antibody fragments that bind to a target of interest with exquisite specificity and high affinity. Here, we discuss key aspects for the development of an antibody discovery strategy to harness the power of phage display technology to obtain molecules that can successfully be developed into therapeutics, including target validation, antibody design goals, and considerations for preparing and executing phage panning campaigns. Careful design and implementation of discovery campaigns-regardless of the target-provides the best chance of identifying desirable antibody fragments for further therapeutic development, so these principles can be applied to any new discovery project., (© 2024 Cold Spring Harbor Laboratory Press.)