The development of nanobodies for therapeutic applications.

Evolution has been continuously honing the design of antibodies to function as specific molecular markers that are able to alert the immune system to the presence of pathogenic antigens, and to recruit complement- and Fc receptor-bearing effector cells. During the past 25 years, the versatility of antibodies has been applied to several therapeutic applications. The development of new technologies, combined with data obtained using a new generation of antibody reagents, have allowed the adaptation of the design of antibodies to better match drug development requirements. Nanobodies are therapeutic proteins derived from the heavy-chain variable (VHH) domains that occur naturally in heavy-chain-only Ig molecules in camelidae. These VHH domains are the smallest known antigen-binding antibody fragments. Nanobodies can be easily produced in prokaryotic or eukaryotic host organisms, and their unique biophysical and pharmacological characteristics render these molecules ideal candidates for drug development. This review describes the structural properties of nanobodies and focuses on their unique features, which distinguishes these molecules from other antibody formats and small-molecule drugs. Possible therapeutic applications of nanobodies are discussed and data from phase I clinical trials of the novel 'first-in-class' anti-thrombotic agent ALX-0081 (Ablynx NV) are presented.

Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis.

OBJECTIVE: The advent of tumor necrosis factor (TNF)-blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin V(H) domains, as TNF antagonists in a mouse model of RA. METHODS: Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNF-specific molecules. To increase the serum half-life and targeting properties, an anti-serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99mTc labeling and gamma camera imaging. RESULTS: The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo. CONCLUSION: These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively.