It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

and are conserved during evolution (Kabat et al., 1991). The CDR3 of the V H H is longer on average than that of The antigen-binding site of the camel heavy-chain a V H domain (Vu et al., 1997) et al., 1998). This is surprising, sequences were identified, encoded by 42 and 50 since the active site of enzymes has a low antigenicity for different genes, respectively. Sequence comparison conventional Abs (Novotny, 1991). Thus, the HCAbs indicates that the V H Hs evolved within the V H subgroup recognize a broad range of epitopes, some of which differ III. Nevertheless, the V H H germline segments are from those for conventional Abs. highly diverse, leading to a broad structural repertoire Previously, we identified germline V H and V H H segments of the antigen-binding loops. Seven V H H subfamilies indicating that the variable domain of the HCAbs is were recognized, of which five were confirmed to be encoded by a distinct set of V genes (Nguyen et al., expressed in vivo. Comparison of germline and cDNA 1998). In this study, we investigate the potential V H H sequences demonstrates that the rearranged V H Hs are germline repertoire to gain insight into the ways by which extensively diversified by somatic mutation processes, the dromedary HCAbs acquire a complex repertoire of leading to an additional hypervariable region and a Ag-binding sites. In conventional Abs, the diversity of high incidence of nucleotide insertions or deletions.the Ag-binding site is generated at multiple levels. The These diversification processes are driven by hyper-V H is generated by assembling variable (V), diversity (D) mutation and recombination hotspots embedded in and joining (J) elements (Tonegawa, 1983), in which the the V H H germline genes at the regions affecting the V-gene segment encodes the CDR1 and CDR2; the CDR3 structure of the antigen-binding loops.is generated by the V-D-J joining. In this joining process, Keywords: antigen-binding repertoire/camel heavy-chain great sequence variation is introduced by non-template antibody/gene replacement/germline V H /hypermutation addition of nucleotides at the V-D and D-J junctions hotspots (junctional diversity). Random association of a V H and a V L (combinatorial diversity) generates an immensely diverse Ag-binding repertoire. Additional diversification

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