Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries

Naturally occurring heavy chain antibodies (HCAbs) in Camelidae species were a surprise discovery in 1993 by Hamers et al. Since that time, antibody fragments derived from HCAbs have garnered considerable attention by researchers and biotechnology companies. Due to their biophysico-chemical advantages over conventional antibody fragments, camelid single-domain antibodies (sdAbs, V_HHs, nanobodies) are being increasingly utilized as viable immunotherapeutic modalities. Currently there are multiple V_HH-based therapeutic agents in different phases of clinical trials in various formats such as bi- and multivalent, bi- and multi-specific, CAR-T, and antibody-drug conjugates. The first approved V_HH, a bivalent humanized V_HH (caplacizumab), was approved for treating rare blood clotting disorders in 2018 by the EMA and the FDA in 2019. This was followed by the approval of an anti-BCMA V_HH-based CAR-T cell product in 2022 (ciltacabtagene autoleucel; CARVYKTI™) and more recently a trivalent antitumor necrosis factor alpha-based V_HH drug (ozoralizumab; Nanozora^®) in Japan for the treatment of rheumatoid arthritis. In this chapter we provide protocols describing the latest developments in isolating antigen-specific V_HHs including llama immunization, construction of phage-displayed libraries, phage panning and screening of the soluble V_HHs by ELISA, affinity measurements by surface plasmon resonance, functional cell binding by flow cytometry, and additional validation by immunoprecipitation. We present and discuss comprehensive, step-by-step methods for isolating and characterization of antigen-specific V_HHs. This includes protocols for expression, biotinylation, purification, and characterization of the isolated V_HHs. To demonstrate the feasibility of the entire strategy, we present examples of V_HHs previously isolated and characterized in our laboratory.