Camel heavy-chain antibodies: diverse germline VHH and specific mechanismsenlarge the antigen-binding repertoire

The antigen-binding site of the camel heavy-chain antibodies devoid of ligh t chain consists of a single variable domain (VHH) that obviously lacks the V-H-V-L combinatorial diversity, To evaluate the extent of the VHH antigen -binding repertoire, a germline database was constructed from PCR-amplified THH/ V-H segments of a single specimen of Camelus dromedarius. A total of 33 VHH and 39 VH unique sequences were identified, encoded by 42 and 50 dif ferent genes, respectively. Sequence comparison indicates that the V(H)Hs e volved within the VH subgroup III, Nevertheless, the VHH germline segments are highly diverse, leading to a broad structural repertoire of the antigen -binding loops, Seven VHH subfamilies were recognized, of which five were c onfirmed to be expressed lit vivo. Comparison of germline and cDNA sequence s demonstrates that the rearranged V(H)Hs are extensively diversified by so matic mutation processes, leading to an additional hypervariable region and a high incidence of nucleotide insertions or deletions, These diversificat ion processes are driven by hypermutation and recombination hotspots embedd ed in the VHH germline genes at the regions affecting the structure of the antigen-binding loops.