Since the discovery of camelid heavy-chain antibodies in 1993, there has been tremendous excitement for these antibody domains (VHHs/sdAbs/nanobodies) as research tools, diagnostics, and therapeutics. Commercially, several patents were granted to pioneering research groups in Belgium and the Netherlands between 1996–2001. Ablynx was established in 2001 with the aim of exploring the therapeutic applications and development of nanobody drugs. Extensive efforts over two decades at Ablynx led to the first approved nanobody drug, caplacizumab (Cablivi) by the EMA and FDA (2018–2019) for the treatment of rare blood clotting disorders in adults with acquired thrombotic thrombocytopenic purpura (TPP). The relatively long development time between camelid sdAb discovery and their entry into the market reflects the novelty of the approach, together with intellectual property restrictions and freedom-to-operate issues. The approval of the first sdAb drug, together with the expiration of key patents, may open a new horizon for the emergence of camelid sdAbs as mainstream biotherapeutics in the years to come. It remains to be seen if nanobody-based drugs will be cheaper than traditional antibodies. In this review, I provide critical perspectives on camelid sdAbs and present the promises and challenges to their widespread adoption as diagnostic and therapeutic agents.

Campylobacter jejuni is the leading cause of bacterial foodborne illness in the world, with symptoms ranging from acute diarrhea to severe neurological disorders. Contaminated poultry meat is a major source of C. jejuni infection, and therefore, strategies to reduce this organism in poultry, are expected to reduce the incidence of Campylobacter-associated diseases. We have investigated whether oral administration of C. jejuni-specific single-domain antibodies would reduce bacterial colonization levels in chickens. Llama single-domain antibodies specific for C. jejuni were isolated from a phage display library generated from the heavy chain IgG variable domain repertoire of a llama immunized with C. jejuni flagella. Two flagella-specific single-domain antibodies were pentamerized to yield high avidity antibodies capable of multivalent binding to the target antigen. When administered orally to C. jejuni-infected two-day old chicks, the pentabodies significantly reduced C. jejuni colonization in the ceca. In vitro, the motility of the bacteria was also reduced in the presence of the flagella-specific pentabodies, suggesting the mechanism of action is through either direct interference with flagellar motility or antibody-mediated aggregation. Fluorescent microscopy and Western blot analyses revealed specific binding of the anti-flagella pentabodies to the C. jejuni flagellin.

Camelid heavy-chain antibody variable domains (V_HHs) are emerging as potential antimicrobial reagents. We have engineered a previously isolated V_HH (FlagV1M), which binds Campylobacter jejuni flagella, for greater thermal and proteolytic stability. Mutants of FlagV1M were obtained from an error-prone polymerase chain reaction library that was panned in the presence of gastrointestinal (GI) proteases. Additional FlagV1M mutants were obtained through disulfide-bond engineering. Each approach produced V_HHs with enhanced thermal stability and protease resistance. When the beneficial mutations from both approaches were combined, a hyperstabilized V_HH was created with superior stability. The hyperstabilized V_HH bound C. jejuni flagella with wild-type affinity and was capable of potently inhibiting C. jejuni motility in assays performed after sequential digestion with three major GI proteases, demonstrating the remarkable stability imparted to the V_HH by combining our engineering approaches.

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid V_HH single-domain Abs (sdAbs or V_HHs) are not bound by SpG and only sporadically bound by SpA. Currently, V_HHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding V_HHs. We show that SpA binding of V_HHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five V_HHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary V_HH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid V_HHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.