Ablynx has expanded its collaborations with large biophama players, such as Merck, Boehringer Ingelheim, Sanofi, and so on, with more than 20 preclinical and clinical programs. company, Ablynx. While entrepreneurial spirit has certainly catalyzed the exploration of nanobodies as marketable products, IP restrictions may be partially responsible for the relatively long time span between the discovery of these biomolecules and their entry into the pharmaceutical market. It is now anticipated that the first VHH-based antibody drug, Caplacizumab, a bivalent anti-vWF antibody for treating rare blood clotting disorders, may be approved and commercialized in 2018 or shortly thereafter. This elusive first approval, along with the expiry of key patents, may substantially alter the scientific and biomedical landscape surrounding camelid sdAbs and pave the way for their emergence as mainstream biotherapeutics. Keywords: camelid Gemigliptin single-domain antibody, heavy chain antibody, VHH, nanobody, antibody engineering, therapeutic antibody Introduction The canonical view of antibodies as molecules Gemigliptin composed of two heavy chains and two light chains was forever changed one day in 1989 following analysis of total and fractionated immunoglobulin G (IgG) molecules in the serum of a dromedary camel in the laboratory of Professor Raymond Hamers at the Vrije Universiteit Brussel (VUB). The serendipitous discovery of antibodies lacking a light chain [heavy chain-only antibodies (HCAbs)] occurred as part of a student-run project aimed at developing a serodiagnostic test for trypanosome infection in camels and water buffalos. The preliminary data showed that besides conventional IgG1 (MW ~150?kDa), two other immunoglobulin fractions (thereafter called IgG2 and IgG3; MW ~90?kDa) were present which contributed up Gemigliptin to 75% of all serum IgGs (1C3). Comparative studies on the sera of new world camelids (and demonstrated strong binding activity in the IgG2 and IgG3 heavy chain-only fractions as shown by radio-immunoprecipitation and blotting experiments (1). In two subsequent reports, phage-display technology and Rabbit Polyclonal to HOXD8 high-resolution crystallography were utilized to (a) build a phage-display library from the lymphocytes of immunized camels and isolate monomeric antigen-specific VHH domains in the absence of the constant regions (11) and (b) solve crystal structures of an unliganded VHH (12) and a VHH:lysozyme complex, reported simultaneously by the VUB team and a DutchCFrench research group (13). The term VHH was originally introduced by the VUB team in 1994 to indicate a VH domain derived from camelid heavy chain antibodies. The feasibility of isolating stable and soluble VHH domains with nanomolar affinities against lysozyme and tetanus toxoid showed very early on the promise of these molecules as high-affinity binding moieties. Crystallography studies revealed additional salient features of an anti-lysozyme VHH, including deep penetration of its long third complementarity-determining region (CDR3) into the active site of the enzyme; this feature had rarely been seen with conventional antibodies and required a fundamental deviation from known human canonical CDR1 structure (13). Further evidence of the unique antigen recognition behavior of VHH domains (including enzyme inhibition) was published over the next several years (11, 14, 15), suggesting that VHHs might probe different sets of epitopes on proteins compared with conventional antibodies. Key proof of concept for producing bivalent/bispecific VHH modalities genetic fusion (using camelid short and long hinge sequences) of anti-lyzozyme and/or anti-tetanus toxin VHHs was also established very early on (14). Molecular Ontogeny of Camelid HCAbs Molecular biology techniques were subsequently applied to decipher Gemigliptin the DNA sequences of HCAbs. The sequencing results showed that nature had designed HCAbs as an additional arm of Gemigliptin the immune systems of camelid ungulates over the course of their evolutionary history. The consensus of these studies suggested camelid HCAbs possessed: (a) no CH1 domain, and therefore, a direct connection of the rearranged VHH exon to the hinge region; (b) one of two types of long (IgG2) and short (IgG3) hinge isotypes; (c) specific conserved amino acid substitutions in framework region 2 (FR2), mainly at VH positions that make contact with the VL in classical antibodies, including Kabat positions.