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

Abstract

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

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…19 V H H 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 V H Hs; 21 in some V H Hs, the long CDR3 loop serves a dual purpose, folding over the former V L interface as well as interacting with cognate antigen. The rearranged V H H 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 CDR4 24 ). V H Hs 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.…”

“…The rearranged V H H 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 CDR4 24 ). V H Hs 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 V H H genes encode non-canonical disulfide linkages formed between cysteine residue pairs (CDR1-CDR3, FR2-CDR3, CDR2-CDR3 or CDR3-CDR3; see Box 2).…”

“…V H Hs 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 V H H genes encode non-canonical disulfide linkages formed between cysteine residue pairs (CDR1-CDR3, FR2-CDR3, CDR2-CDR3 or CDR3-CDR3; see Box 2). V NAR s, the variable domains of cartilaginous fish Ig new antigen receptors, share sequence homology with T-cell receptor and Ig light chain genes 4 and may be descended from Ig-superfamily cell-surface receptors.…”

Strong in Vivo Maturation Compensates for Structurally Restricted H3 Loops in Antibody Repertoires

“…Up to three loops (H1-H3) of the variable domain of these heavy-chain antibodies (VHH) constitute the antigen-binding site. The VHH, like VH, is generated after V(D)J gene rearrangements (10). Despite the absence of the light chain, the heavy-chain antibodies recognize, via their VHHs, a wide range of antigens with affinity constants that are comparable with those found for conven-* This work was supported by the Onderzoeksraad VUB (GOA), the Vlaams Instituut voor Biotechnologie, and the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.…”

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…This disulphide bond probably restricts the flexibility of long CDRs, which is expected to be entropically counterproductive for binding, and therefore allows a strong interaction [53]. Moreover, it may also enable CDRs to adopt new conformations enabling V H Hs to recognise an increased variety of epitopes [40,54].…”