Antibody repertoire development in camelids

Genst, Erwin De; Saerens, Dirk; Muyldermans, Serge; Conrath, Katja

doi:10.1016/j.dci.2005.06.010

Cited by 166 publications

(135 citation statements)

References 42 publications

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“…A promising alternative to these conventional antibodies are the heavy-chain antibodies of the Camelidae, which naturally lack light chains (17) and have a very small (15-kDa) antigen-binding fragments (VHH) that are extremely stable (18) and can be expressed at high levels in heterologous systems. Unlike normal VH domains, VHH is soluble and displays long surface loops, often larger than the ones for conventional murine and human antibodies, and are able to penetrate cavities in target antigens, such as enzyme active sites (19,20). These characteristics make them strong intracellular proteinneutralizing reagents suitable for loss-of-function screenings.…”

Section: Resultsmentioning

confidence: 99%

Loss-of-function screening by randomized intracellular antibodies: Identification of hnRNP-K as a potential target for metastasis

Inoue

Sawata

Taira

et al. 2007

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

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We have developed a loss-of-function screening system on the basis of intracellular expression of single domain antibodies. We demonstrate its use in identification of potential targets of metastasis of human cancerous cells. Randomized intracellular antibodies were expressed in highly metastatic cells, and a derivative pool of cells with loss of migration phenotype in chemotaxis assay was isolated. Isolation of antibodies from cells with loss of migration phenotype and identification of their target proteins revealed the involvement of the heterogeneous nuclear ribonucleoprotein K (hnRNP-K), a multifunctional signaling protein, in metastasis. Furthermore, we found that the cytoplasmic accumulation of hnRNP-K is crucial for its role in metastasis. The results demonstrate (i) the advantages of our functional interference screening over the gene-knockouts and genesilencing, (ii) hnRNP-K as a potential target of metastasis, and (iii) a potential anti-metastasis peptide validated in in vitro cell migration assays.heterogeneous nuclear ribonucleoprotein K ͉ randomized antibodies ͉ functional screening ͉ cytoplasmic accumulation ͉ antimetastasis M etastasis is a complex phenomenon that involves an array of genetic changes and coordination of many positive and negative factors leading to motile cells that can penetrate into and travel through the bloodstream forming secondary tumors elsewhere in the body. Despite the advances in cancer therapies and the understanding that metastasis is a selective, nonrandom and inefficient process, it significantly contributes to morbidity and mortality of cancer patients and is a major challenge yet to be met in cancer therapeutics (1-4). Increased cell motility is one of the early and essential phenotype of metastatic cancers. Although it has been demonstrated to involve concerted reorganization of the actin cytoskeleton that regulates cell shape, and motility through spatiotemporal activation of Rho-family GTPases (5-9), the molecular mechanisms and its upstream regulators, the potential targets of metastasis, have not been sufficiently defined (10). Amongst a large variety of approaches that can be adopted to identify ''metastasis genes,'' we chose to employ a functional interference of proteins by expression of intracellular antibodies, also called as ''specific functional ablators'' and ''functional therapeutical molecules'' (11-13). We anticipated that the functional interference may serve as a milder and finer approach to reveal biological complexities and pathways that cannot be resolved by gene-knockouts and genesilencing strategies that challenge structural integrity of cells.We constructed a randomized intracellular antibody library (iAb-lib) on the basis of the single heavy-chain small antigenbinding fragments (VHH) of camel antibody. By employing iAb-lib against cell migration of highly malignant human fibrosarcoma, we obtained a derivative cell population in which cell migration was compromised. Cloning and characterization of antibodies from the derivative nonmigrating...

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Section: Resultsmentioning

confidence: 99%

Loss-of-function screening by randomized intracellular antibodies: Identification of hnRNP-K as a potential target for metastasis

Inoue

Sawata

Taira

et al. 2007

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

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“…In addition, our analysis suggests that relative to murine/human CDR-H3s, camelid CDR-H3 regions are enriched in Cys, positively charged (Arg and Lys), and hydrophilic (Gly, Ser, Thr, and Asn) residues, whereas the level of hydrophobic residues (Phe, Val, and Ile) is decreased. These amino acid preferences likely contribute to the additional disulfide bonds in camelid antibodies and, in general, to the integrity and improved solubility of the CDR-H3 loops (31,32).…”

Section: Significancementioning

confidence: 99%

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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“…cDNA from lymphocytes was taken from immunised Cammellidae [11][12][13]. The cDNA can be sourced from lymphocytes isolated from blood taken from the host, avoiding animal sacrifice and contributing to the 3R's of animal use in research.…”

Section: Introductionmentioning

confidence: 99%

The development and optimisation of nanobody based electrochemical immunosensors for IgG

Goode

Dillon

Millner

2016

Sensors and Actuators B: Chemical

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Article:Goode, J orcid.org/0000-0002-3642-1291, Dillon, G and Millner, PA orcid.org/0000-0002-4813-4302 (2016 ReuseUnless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. This has led to the development of alternative immuno-reagents such as non-antibody binding proteins (NABPs). These are low molecular weight proteins which largely avoid the aforementioned advantages of antibodies. They are commonly produced by bacteria enabling the use of DNA technology to manipulate bioreceptors at the molecular level.Single chain VHHs (commonly known as nanobodies) are an antibody derived NABP adapted from camelid heavy chain antibodies which are the isolated binding domain. Whilst nanobodies have been used for diagnostic and therapeutic applications, they have limited demonstration in biosensors.In this study, both antibodies and nanobodies were used to construct a biosensor. In addition nanobody performance was optimised by introducing a novel peptide spacer. The role of nanobody orientation and spacing was thus investigated and spacer length was optimised, leading to an increase in the sensitivity of the biosensor. Highlights: Nanobodies have been used on impedimetric immunosensors to detect rabbit IgG  Unmodified nanobody sensors displayed a decrease in resistance upon analyte recognition.  Nanobodies were modified with a peptide spacer, effectively reversing this phenomena.  The use of a spacer also increased the sensitivity of the immunosensor.

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Antibody repertoire development in camelids

Cited by 166 publications

References 42 publications

Loss-of-function screening by randomized intracellular antibodies: Identification of hnRNP-K as a potential target for metastasis

Loss-of-function screening by randomized intracellular antibodies: Identification of hnRNP-K as a potential target for metastasis

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

The development and optimisation of nanobody based electrochemical immunosensors for IgG

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