Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species

Griffin, Laura; Snowden, James; Lawson, Alastair D. G.; Wernery, Ulrich; Kinne, Jörg; Baker, Terry

doi:10.1016/j.jim.2014.01.003

Cited by 41 publications

(38 citation statements)

References 28 publications

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“…inhibition is mediated by the extended CDR-H3s coding for the convex-shaped paratopes that penetrate into the enzyme catalytic cleft (23,26). Consistent with the studies by others (30), our in-depth analysis of over 950 individual camelid antibodies available from the international ImMunoGeneTics (IMGT), Kabat, and abYsis databases determined that there is a bimodal distribution in their CDR-H3 length with peaks at 12 and 19 residues (Fig. S2A).…”

Section: Significancesupporting

confidence: 87%

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Nam

Rodríguez

Remacle

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

110

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Proteases are frequent pharmacological targets, and their inhibitors are valuable drugs in multiple pathologies. The catalytic mechanism and the active-site fold, however, are largely conserved among the protease classes, making the development of the selective inhibitors exceedingly challenging. In our departure from the conventional strategies, we reviewed the structure of known camelid inhibitory antibodies, which block enzyme activities via their unusually long, convex-shaped paratopes. We synthesized the human Fab antibody library (over 1.25 × 10 9 individual variants) that carried the extended, 23-to 27-residue, complementarity-determining region (CDR)-H3 segments. As a proof of principle, we used the catalytic domain of matrix metalloproteinase-14 (MMP-14), a promalignant protease and a drug target in cancer, as bait. In our screens, we identified 20 binders, of which 14 performed as potent and selective inhibitors of MMP-14 rather than as broad-specificity antagonists. Specifically, Fab 3A2 bound to MMP-14 in the vicinity of the active pocket with a high 4.8 nM affinity and was similarly efficient (9.7 nM) in inhibiting the protease cleavage activity. We suggest that the convex paratope antibody libraries described here could be readily generalized to facilitate the design of the antibody inhibitors to many additional enzymes. family members control various physiological and pathological processes. Multiple diseases are associated with altered MMP expression and aberrant proteolysis, including cancer (1), wound healing (2), inflammatory diseases (3, 4), neurological pain (5, 6), and hypertension (7). There is consensus among researchers that the individual MMPs are promising drug targets in diversified pathologies and that inhibitor specificity is required for selective and successful MMP therapies (8-10).However, achieving target specificity and selectivity in small-molecule MMP inhibitors is remarkably challenging (11,12). Because the catalytic mechanism and catalytic domain fold are conserved among the MMP/ADAM (a disintegrin and metalloproteinase)/ ADAMTS (ADAM with thrombospondin motifs) superfamily members, the available small-molecule inhibitors (most frequently, active-site zinc-chelating hydroxamates) target multiple proteinases, resulting in off-target side effects (8,(12)(13)(14). This aspect is problematic, given that some MMPs (e.g., MMP-14) are always protumorigenic, whereas some other MMPs are antitumorigenic in certain cancer microenvironments (15, 16). As a result, broad-spectrum hydroxamates failed in cancer clinical trials due to their low overall efficacy and side effects (13). Alternatively, antibody-based MMP inhibitors are emerging as both research tools and potential therapeutic agents (10, 17-21) because of (i) high affinity and specificity due to the large antigen-antibody interaction area and multiple complementarity-determining regions (CDRs), (ii) long half-life and well-defined action mechanisms, (iii) low immunogenicity and toxicity, and (iv) multiple MMPs potentially targe...

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Section: Significancesupporting

confidence: 87%

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Nam

Rodríguez

Remacle

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

110

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“…Surprisingly, these turned out to have CDR1 and CDR2 canonical structure combinations identical to those of the corresponding human germline orthologs. In addition and in agreement with previous analysis, 20,21 we identified an IGHV5 and IGHV7 gene family, adding to the existing knowledge of IGHV genes in camelids. Using X-ray crystallography we confirmed the presence of predicted canonical structures similar to human canonical folds on 2 selected camelid antibodies separately directed against the therapeutic targets CD70 and MET.…”

Section: Introductionsupporting

confidence: 90%

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

Klarenbeek

Mazouari²,

Desmyter

et al. 2015

mAbs

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de Haard & Ikbel Achour (2015) Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform, mAbs, 7:4, 693-706, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1b and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelidderived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform.

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“…With hindsight, the notion that sdAbs might preferentially target particular types of antigenic structures may not seem totally unexpected, given their recombination from distinct repertoires of V, D and J genes (see Box 1 ), 8 their potential ontogeny from separate B-cell precursors, 9 and for camelid V H Hs, their specialized constant regions bearing very long hinge regions. 10 However, the specific mechanisms of sdAb antigen recognition ( e.g ., the tertiary structures and physicochemical properties of sdAb:antigen interfaces, which may differ fundamentally from those of conventional antibody:antigen interfaces) remain unclear, although several studies have suggested protein cleft recognition as a general function for both V H Hs 11 and V NAR s. 12 Over time, the idea that sdAbs can target ‘cryptic’ epitopes (so-called because they are inaccessible to conventional antibodies, either for steric reasons or due to their fundamental antigenic properties) has become entrenched, and although several case studies have supported it, its generality and implications are questionable. Several excellent recent reviews and opinion pieces have alluded to the nature of sdAb paratopes and their interactions with antigens, but have either not been rigorous in their approach or have incompletely addressed the topic, analyzing the properties of sdAb paratopes only, with no comparison to those of conventional antibodies.…”

Section: Introductionmentioning

confidence: 99%

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

Henry

MacKenzie

2018

mAbs

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Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional VH:VL antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.

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Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species

Cited by 41 publications

References 28 publications

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

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

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