| Abstract | 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, VHHs, nanobodies) are being increasingly utilized as viable immunotherapeutic modalities. Currently there are multiple VHH-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 VHH, a bivalent humanized VHH (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 VHH-based CAR-T cell product in 2022 (ciltacabtagene autoleucel; CARVYKTI™) and more recently a trivalent antitumor necrosis factor alpha-based VHH 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 VHHs including llama immunization, construction of phage-displayed libraries, phage panning and screening of the soluble VHHs 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 VHHs. This includes protocols for expression, biotinylation, purification, and characterization of the isolated VHHs. To demonstrate the feasibility of the entire strategy, we present examples of VHHs previously isolated and characterized in our laboratory. |
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