Box 1.

Immunogenetics of sdAbs

VHHs, the variable domains of camelid heavy chain-only antibodies, are recombined during B-cell development from a unique set of germline V genes and common D and J genes (shared with the VH domains of conventional tetrameric antibodies) located within the igh locus on chromosome 4.8 Most camelid VHH and VH genes18 are homologous to human IGHV3-family genes (~75–90% identity) and encode distinctive solubilizing residues in FR2 (Phe/Tyr42, Glu49, Arg50 and Gly 52 using IMGT numbering; these positions map to the VH:VL interface in conventional antibodies), although functional VHHs lacking this consensus have been isolated.19,20 Some camelid V genes may ‘promiscuously’ recombine with both heavy chain-only and conventional antibody constant region genes.19 VHH domains bear unusually long CDR3 loops in comparison with human and murine conventional antibodies,21,22 probably reflecting increased non-templated nucleotide addition, although this may be a feature of only a subset of VHHs;21 in some VHHs, the long CDR3 loop serves a dual purpose, folding over the former VL interface as well as interacting with cognate antigen. The rearranged VHH exon is thought to undergo elevated rates of somatic hypermutation of both CDRs and FRs (e.g., FR1-encoding sequences immediately flanking CDR1;23–25 FR2-encoding sequences which may play a role in structuring the CDR3 loop;20,25 FR3-encoding sequences that form a β-turn which can make contact with antigen, sometimes called CDR424). VHHs may also acquire somatic insertions and deletions at higher rates than conventional antibodies,24 and may under some circumstances undergo secondary rearrangement events using a cryptic recombination signal sequence in FR3.24 Some VHH genes encode non-canonical disulfide linkages formed between cysteine residue pairs (CDR1-CDR3, FR2-CDR3, CDR2-CDR3 or CDR3-CDR3; see Box 2).VNARs, the variable domains of cartilaginous fish Ig new antigen receptors, share sequence homology with T-cell receptor and Ig light chain genes4 and may be descended from Ig-superfamily cell-surface receptors.26 Compared with Ig VH domains, VNARs lack two β strands (C’ and C’’) and consequently CDR2 is absent, although loops connecting the C-D and D-E strands (HV2 and HV4, respectively) can make contact with antigen. During B-cell development, VNAR domains are rearranged from a small number of loci (perhaps only three) distinct from those encoding other types of Ig molecules detectable in serum (IgM, IgW). Each locus contains one V gene, two or three D genes and one J gene and thus primary repertoire diversity is almost entirely CDR3-based:4 since VNAR CDR3 loops are formed through either three or four independent rearrangement events, these tend to be long.27 Unlike the VH domains of IgMs and IgWs, VNARs accrue somatic hypermutations upon encounter with antigen primarily in CDR1 and CDR3 but also in HV2 and HV4.27,28