RESUMO
It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.
Assuntos
Camelídeos Americanos/imunologia , Camelus/imunologia , Imunoglobulinas/metabolismo , Nanotecnologia/métodos , Animais , Camelídeos Americanos/metabolismo , Camelus/metabolismo , Engenharia GenéticaRESUMO
Intrabodies offer attractive options for manipulating the protein misfolding that triggers neurodegenerative diseases. In Huntington's disease, where the expanded polyglutamine tract in the extreme N-terminal region of huntingtin exon1 misfolds, two lead intrabodies have been selected against an adjacent peptide, using slightly different approaches. Both are effective at preventing aggregation of a reporter fragment in transient co-transfection assays. However, after intracranial delivery to mutant mouse brains, VL12.3, which is mainly localized to the nucleus, appears to accelerate the mutant phenotype, while C4 scFv, which is largely cytoplasmic, shows partial phenotypic correction. This comparison highlights parameters that could inform intrabody therapeutics for multiple proteostatic diseases.
Assuntos
Núcleo Celular/metabolismo , Doença de Huntington/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/química , Corpo Estriado/química , Corpo Estriado/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Feminino , Masculino , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Dobramento de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , TransfecçãoRESUMO
Whereas antibodies have demonstrated the ability to mimic various compounds, classic heavy/light-chain antibodies may be limited in their applications. First, they tend not to bind enzyme active site clefts. Second, their size and complexity present problems in identifying key elements for binding and in using these elements to produce clinically valuable compounds. We have previously shown how cAb-Lys3, a single variable domain fragment derived from a lysozyme-specific camel antibody naturally lacking light chains, overcomes the first limitation to become the first antibody structure observed penetrating an enzyme active site. We now demonstrate how cAb-Lys3 mimics the oligosaccharide substrate functionally (inhibition constant for lysozyme, 50 nM) and structurally (lysozyme buried surface areas, hydrogen bond partners, and hydrophobic contacts are similar to those seen in sugar-complexed structures). Most striking is the mimicry by the antibody complementary determining region 3 (CDR3) loop, especially Ala104, which mimics the subsite C sugar 2-acetamido group; this group has previously been identified as a key feature in binding lysozyme. Comparative simplicity, high affinity and specificity, potential to reach and interact with active sites, and ability to mimic substrate suggest that camel heavy-chain antibodies present advantages over classic antibodies in the design, production, and application of clinically valuable compounds.
Assuntos
Carboidratos/química , Inibidores Enzimáticos/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Mimetismo Molecular , Animais , Camelus , Configuração de Carboidratos , Carboidratos/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Lisina/imunologia , Micrococcus/imunologia , Modelos MolecularesRESUMO
Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.