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1.
Eur J Endocrinol ; 187(6): 797-807, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36206174

RESUMO

Objectives: After bilateral adrenalectomy in Cushing's disease, corticotroph tumor progression occurs in one-third to half of patients. However, progression speed is variable, ranging from slow to rapid. The aim was to explore corticotroph progression speed, its consequences and its risk factors. Design: A retrospective single-center observational study. Methods: In total,103 patients with Cushing's disease who underwent bilateral adrenalectomy between 1990 and 2020 were included. Clinical, biological, histological and MRI features were collected. Median duration of follow-up after bilateral adrenalectomy was 9.31 years. Results: In total,44 patients progressed (43%). Corticotroph tumor progression speed ranged from 1 to 40.7 mm per year. Progression speed was not different before and after bilateral adrenalectomy (P = 0.29). In univariate analyses, predictive factors for rapid corticotroph tumor progression included the severity of Cushing's disease before adrenalectomy as the cause of adrenalectomy, high ACTH in the year following adrenalectomy and high Ki67 immunopositivity in the tumor. During follow-up, early morning ACTH absolute variation was associated with corticotroph tumor progression speed (P-value = 0.001). ACTH measurement after dynamic testing did not improve this association. Conclusion: After adrenalectomy, corticotroph progression speed is highly variable and manageable with MRI and ACTH surveillance. Progression speed does not seem related to bilateral adrenalectomy but rather to intrinsic properties of highly proliferative and secreting tumors.


Assuntos
Hipersecreção Hipofisária de ACTH , Humanos , Hipersecreção Hipofisária de ACTH/diagnóstico por imagem , Hipersecreção Hipofisária de ACTH/cirurgia , Hipersecreção Hipofisária de ACTH/etiologia , Corticotrofos/metabolismo , Adrenalectomia/efeitos adversos , Estudos Retrospectivos , Hormônio Adrenocorticotrópico/metabolismo
2.
Elife ; 102021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34318748

RESUMO

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs, and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell types in vivo. Here, we show that by exploiting either endogenous or synthetic receptor-ligand interactions and leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in Caenorhabditis elegans, with subcellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.


Assuntos
Caenorhabditis elegans/citologia , DNA/química , Nanotecnologia/métodos , Ácidos Nucleicos/química , Animais , Técnicas Biossensoriais/instrumentação , Caenorhabditis elegans/metabolismo , Nanoestruturas/química , Ácidos Nucleicos/metabolismo
3.
Cancers (Basel) ; 12(11)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33182650

RESUMO

The fibroblast growth factor receptor 4 (FGFR4) is overexpressed in rhabdomyosarcoma (RMS) and represents a promising target for treatments based on specific and efficient antibodies. Despite progress, there is an urgent need for targeted treatment options to improve survival rates, and to limit long-term side effects. From phage display libraries we selected FGFR4-specific single-domain antibodies (sdAb) binding to recombinant FGFR4 and validated them by flow cytometry, surface plasmon resonance, and fluorescence microscopy. The specificity of the selected sdAb was verified on FGFR4-wild type and FGFR4-knock out cells. FGFR4-sdAb were used to decorate vincristine-loaded liposomes and to generate chimeric antigen receptor (CAR) T cells. First, incubation of RMS cells with FGFR4-sdAb revealed that FGFR4-sdAb can block FGF19-FGFR4 signaling via the MAPK pathway and could therefore serve as therapeutics for FGFR4-dependent cancers. Second, FGFR4-targeted vincristine-loaded liposomes bound specifically to RMS cells and were internalized by the receptor, demonstrating the potential for active drug delivery to the tumor. Third, FGFR4-CAR T cells, generated with one sdAb candidate, demonstrated strong and specific cytotoxicity against FGFR4 expressing RMS cells. We selected novel FGFR4-sdAb with high specificity and nano- to picomolar affinities for FGFR4 which have the potential to enable multiple FGFR4-targeted cancer therapy approaches.

4.
iScience ; 23(4): 100987, 2020 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-32224433

RESUMO

Human mononuclear phagocytes comprise several subsets of dendritic cells (DCs), monocytes, and macrophages. Distinguishing one population from another is challenging, especially in inflamed tissues, owing to the promiscuous expression of phenotypic markers. Using a synthetic library of humanized llama single domain antibodies, we identified a novel surface marker for human naturally occurring monocyte-derived DCs. Our antibody targets an extra-cellular domain of LSP-1, specifically on monocyte-derived DCs, but not on other leukocytes, in particular monocytes, macrophages, classical DCs, or the recently described blood DC3 population. Our findings will pave the way for a better characterization of human mononuclear phagocytes in pathological settings.

5.
Methods Mol Biol ; 1827: 491-503, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30196513

RESUMO

Intracellularly expressed recombinant antibodies, or intrabodies, are powerful tools for cell biology studies as well as therapeutic applications. Cell biologists use them to either block the intracellular antibody target or to image endogenous target dynamics. We describe here methods to select recombinant antibodies from antibody phage display libraries and to subsequently express them as fluorescent intrabodies.


Assuntos
Anticorpos/isolamento & purificação , Espaço Intracelular/metabolismo , Engenharia de Proteínas/métodos , Imunofluorescência , Humanos , Imageamento Tridimensional
6.
Biochem Biophys Res Commun ; 493(4): 1567-1572, 2017 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-29017919

RESUMO

Antibodies are essential reagents that are increasingly used in diagnostics and therapy. Their specificity and capacity to recognize their native antigen are critical characteristics for their in vivo application. Follicle-stimulating hormone receptor is a GPCR protein regulating ovarian follicular maturation and spermatogenesis. Recently, its potentiality as a cancer biomarker has been demonstrated but no antibody suitable for in vivo tumor targeting and treatment has been characterized so far. In this paper we describe the first successful attempt to recover recombinant antibodies against the FSHR and that: i) are directly panned from a pre-immune library using whole cells expressing the target receptor at their surface; ii) show inhibitory activity towards the FSH-induced cAMP accumulation; iii) do not share the same epitope with the natural binder FSH; iv) can be produced inexpensively as mono- or bivalent functional molecules in the bacterial cytoplasm. We expect that the proposed biopanning strategy will be profitable to identify useful functional antibodies for further members of the GPCR class.


Assuntos
Biblioteca de Peptídeos , Receptores do FSH/antagonistas & inibidores , Receptores do FSH/imunologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Animais , Especificidade de Anticorpos , AMP Cíclico/metabolismo , Feminino , Hormônio Foliculoestimulante/farmacologia , Células HEK293 , Humanos , Imunização , Células L , Masculino , Camundongos , Domínios Proteicos , Receptores do FSH/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transdução de Sinais , Solubilidade
7.
Elife ; 62017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29022874

RESUMO

Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.


Assuntos
Dinamina I/metabolismo , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Polimerização , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Anticorpos de Domínio Único/metabolismo
8.
Pigment Cell Melanoma Res ; 30(3): 317-327, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28140525

RESUMO

Monoclonal antibodies specific for biomarkers expressed on the surface of uveal melanoma (UM) cells would simplify the immune capture and genomic characterization of heterogeneous tumor cells originated from patient-derived xenografts (PDXs). Antibodies against four independent tumor antigens were isolated by panning a nanobody synthetic library. Such antibodies enabled flow cytometry-based sorting of distinct cell subpopulations from UM PDXs and to analyze their genomic features. The complexity and specificity of the biochemical and genomic biomarker combinations mirrored the UM tumor polyclonality. The data showed that MUC18 is highly and universally displayed on the surface of UM cells with different genetic background and consequently represents a reliable pan-biomarker for their identification and purification. In contrast, the other three biomarkers were detected in very variable combinations in UM PDX cells. The availability of the identified nanobodies will be instrumental in developing clone-specific drug evaluation and rational clinical strategies based on accurate genomic profiling.


Assuntos
Biomarcadores Tumorais/metabolismo , Heterogeneidade Genética , Melanoma/genética , Melanoma/metabolismo , Anticorpos de Domínio Único/metabolismo , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Xenoenxertos , Humanos
9.
Elife ; 52016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27434673

RESUMO

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.


Assuntos
Anticorpos Monoclonais Humanizados , Biologia Molecular/métodos , Biblioteca de Peptídeos , Anticorpos de Domínio Único , Animais , Camelídeos Americanos , Humanos
10.
Nat Commun ; 6: 7933, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26260773

RESUMO

Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.


Assuntos
Anticorpos , Simulação por Computador , Microscopia/métodos , Microtúbulos/ultraestrutura , Anticorpos de Domínio Único , Animais , Linhagem Celular , Humanos
11.
Biosci Rep ; 35(4)2015 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-26182430

RESUMO

A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes.


Assuntos
Engenharia de Proteínas/métodos , Cristalografia por Raios X , Proteínas de Fluorescência Verde/química , Ligação Proteica , Estrutura Secundária de Proteína
12.
J Clin Endocrinol Metab ; 100(3): 850-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25478934

RESUMO

CONTEXT: Prader-Willi syndrome (PWS), the most frequent syndrome of obesity, is a model of early fat mass (FM) development, but scarce data exist on adipose tissue characteristics. OBJECTIVE: The objective of the study was to compare metabolic, fat distribution, and transcriptomic signatures of sc adipose tissue (scAT) in PWS adults, with matched obese adults with primary obesities. MAIN OUTCOMES AND MEASURES: Hormonal and metabolic assessments, systemic inflammation, and gene expression in scAT were compared between PWS patients and obese controls (OCs). Each 42nd PWS patient was matched with one randomly paired control with primary obesity. Matching factors were age, gender, fat mass (percentage), and diabetic status. RESULTS: Compared with OCs, the PWS group had a decreased percentage of trunk FM and a better metabolic profile with decreased insulin and homeostasis model assessment, an index of insulin-resistance, and increased concentrations of serum adiponectin and ghrelin. Adipocyte size relative to body fat was significantly higher in PWS vs OCs. scAT in PWS patients was characterized by a transcriptomic functional signature with enrichment of themes related to immunoinflammation, the extracellular matrix, and angiogenesis. A RT-PCR targeted study revealed that candidate genes encoding proinflammatory markers and remodeling molecules, CD68, CD3e, IL-1ß, chemokine (C-C motif) ligand 5, collagen type 4-α, and lysyl oxidase, were down-regulated. CONCLUSION: Matched for FM, PWS subjects have a better metabolic profile, a phenotype that could be linked to changes in scAT remodeling and promotion of adipocyte growth.


Assuntos
Adiposidade/genética , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/metabolismo , Gordura Subcutânea/metabolismo , Transcriptoma , Adolescente , Adulto , Distribuição da Gordura Corporal , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Análise em Microsséries , Obesidade/complicações , Obesidade/genética , Obesidade/metabolismo , Síndrome de Prader-Willi/complicações , Adulto Jovem
13.
Microb Cell Fact ; 13: 140, 2014 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-25223348

RESUMO

BACKGROUND: The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space. RESULTS: We demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF). CONCLUSIONS: The collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.


Assuntos
Camelus/metabolismo , Compartimento Celular , Citoplasma/metabolismo , Escherichia coli/metabolismo , Imunoglobulina G/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Anticorpos de Cadeia Única/biossíntese , Animais , Afinidade de Anticorpos , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Camundongos , Periplasma/metabolismo , Receptor ErbB-2/metabolismo , Reprodutibilidade dos Testes , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Methods Mol Biol ; 907: 667-79, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22907379

RESUMO

Intracellularly expressed recombinant antibodies, or intrabodies, are powerful tools for cell biology studies as well as therapeutic applications. Cell biologists use them to either block the intracellular antibody target or to image endogenous target dynamics. We describe here methods to select recombinant antibodies from antibody phage display libraries and to subsequently express them as fluorescent intrabodies.


Assuntos
Anticorpos/metabolismo , Técnicas de Visualização da Superfície Celular/métodos , Espaço Intracelular/imunologia , Imunofluorescência , Vetores Genéticos/genética , Células HeLa , Humanos , Imageamento Tridimensional , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
15.
J Clin Endocrinol Metab ; 97(9): E1677-85, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22745246

RESUMO

AIMS AND HYPOTHESIS: Mast cells are immune cells known for their role in several inflammatory and fibrotic diseases. Recent works in mice suggest that mast cells could be cellular actors involved in the pathophysiology of obesity, a disease characterized by white adipose tissue (WAT) and systemic inflammation. The aim of the study was to better characterize mast cells in WAT of obese with or without type 2 diabetes and lean subjects as well as to explore the relationship with WAT inflammation and fibrosis. METHODS: Subcutaneous and omental adipose tissue from six lean subjects, 10 obese nondiabetic, and 10 diabetic patients was analyzed by immunohistochemistry and real-time PCR for inflammatory and fibrosis markers. Cytokines secretion of mast cells isolated from WAT and cultured in different conditions was estimated by cytokine array kit. RESULTS: We found that mast cells are activated in human adipose tissue and localized preferentially in fibrosis depots, a local condition that stimulates their inflammatory state. Mast cells with tryptase(+) chymase(+) staining tended to be higher in obese omental adipose tissue. We found positive links between mast cell number and several characteristics of obese WAT including fibrosis, macrophage accumulation, and endothelial cell inflammation. Mast cell number and their inflammatory phenotype are associated with diabetes parameters. CONCLUSION AND INTERPRETATION: Mast cells are cellular actors of WAT inflammation and possibly fibrotic state found in obesity and diabetes. Whether mast cells could be involved in the pathophysiology of diabetes needs additional study as well as the positioning of these cells in driving pathological alterations of WAT in these chronic metabolic diseases.


Assuntos
Tecido Adiposo/patologia , Diabetes Mellitus Tipo 2/patologia , Inflamação/patologia , Mastócitos/patologia , Obesidade Mórbida/patologia , Tecido Adiposo Branco/patologia , Adulto , Biomarcadores/análise , Glicemia/metabolismo , Contagem de Células , Separação Celular , Quimases/química , Células Endoteliais/patologia , Feminino , Fibrose/patologia , Homeostase/fisiologia , Humanos , Imuno-Histoquímica , Lipídeos/sangue , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Obesidade Mórbida/metabolismo , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Triptases/química
17.
Med Sci (Paris) ; 25(12): 1173-6, 2009 Dec.
Artigo em Francês | MEDLINE | ID: mdl-20035701

RESUMO

In the 1980s, progress in molecular biology enabled the manipulation and cloning of antibody fragments as functional scFv (single chain Fv). Because of their small size and relative ease of expression, scFv opened the road for new medical and biotechnological applications. scFvs can be easily expressed and targeted to different cellular compartments (cytosol, nucleus, endoplasmic reticulum, mitochondria, inner surface of the plasma membrane, etc.), using specific signals to target or retain them in a given compartment. Recombinant antibodies can thus be used as intracellular antibodies (intrabody) to neutralize, disrupt or track endogenous antigen. Intrabodies not only represent new tools for fundamental research to study the dynamics of endogenous proteins, but may also bring interesting options for applied research in terms of intracellular immunization for therapeutic use.


Assuntos
Anticorpos de Cadeia Única/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Antivirais/farmacocinética , Antivirais/uso terapêutico , Compartimento Celular , Vias de Administração de Medicamentos , Sistemas de Liberação de Medicamentos , Humanos , Hibridomas/imunologia , Líquido Intracelular , Camundongos , Peso Molecular , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Transporte Proteico , Proteínas/antagonistas & inibidores , Proteínas/imunologia , Proteínas/fisiologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/uso terapêutico , Viroses/tratamento farmacológico , Xenopus laevis
18.
J Biol Chem ; 284(31): 20791-5, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19473967

RESUMO

In the current post-genomic era, large scale efforts are underway to functionally explore the proteome by assembling large antibody libraries. However, because many proteins are modified post-translationally to regulate their function, collections of modification-specific sensors are also needed. Here we applied a novel approach to select monoclonal phosphospecific antibodies directly from the full-length protein and without up-front phosphoamino acid identification. We chose as antigen GRASP65, a well studied Golgi phosphoprotein. Bacterially produced full-length protein was first incubated with mitotic cytosol, thus allowing modification by naturally occurring kinases, and then used directly for affinity-based antibody selection using a single chain variable fragment phagemid library. In less than 1 week, three distinct and highly functional monoclonal phosphospecific antibodies against two GRASP65 epitopes were obtained and subsequently characterized. The presented approach is carried out fully in vitro, requires no prior knowledge of the phosphoamino acid identity, and is fast and inexpensive. It therefore has great potential to be an attractive alternative to classic animal-based protocols for the selection of post-translation modification sensors and thus to become an invaluable tool in our quest to understand the proteome in all its complexity.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Fosfo-Específicos/análise , Anticorpos Fosfo-Específicos/imunologia , Fosfoaminoácidos/análise , Engenharia de Proteínas/métodos , Animais , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Mapeamento de Epitopos , Proteínas da Matriz do Complexo de Golgi , Região Variável de Imunoglobulina/imunologia , Proteínas de Membrana/imunologia , Modelos Imunológicos , Fosforilação , Ratos
19.
BMC Biotechnol ; 9: 14, 2009 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-19245715

RESUMO

BACKGROUND: Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural antibodies. RESULTS: We developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use. This series enables the fusion of single chain Fv antibodies with human, mouse or rabbit Fc so that a given antibody is no longer restricted to a particular species. This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies. We also show that this multi-Fc species production system can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies and we converted the widely used 9E10 mouse anti-Myc-tag antibody into a human and a rabbit antibody. CONCLUSION: Altogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use.


Assuntos
Anticorpos Monoclonais/biossíntese , Fragmentos Fc das Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Expressão Gênica , Vetores Genéticos , Células HeLa , Humanos , Camundongos , Plasmídeos , Coelhos
20.
Biotechnol J ; 4(1): 38-43, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19156724

RESUMO

Antibodies are essential for the identification and characterization of proteins. In the current postgenomic era the need for highly specific antibodies has further increased not only for research applications but also because they represent one of the most promising therapeutic options, especially in the field of cancer treatment. One appealing approach for rapid and inexpensive antibody generation is the use of phage display. This technique allows for a fast and animal-free selection of highly functional alternatives to classical antibodies. However, one strong limitation of this recombinant approach has been the difficulty in producing and purifying antigens. These steps have to be adjusted for each new target, are time consuming and sometimes present an insurmountable obstacle. Here we report the development of new antibody selection approach where antigens are produced through in vitro translation and are used directly and without the need for purification. With this approach we were able to rapidly select recombinant antibodies directed against GFP and the mammalian protein tsg101, respectively. We believe that our method greatly facilitates antigen preparation and thus may broaden the use of the recombinant approach for antibody generation, especially since the technique could in the future be adapted to a high-throughput technology, thus further accelerating antibody selection.


Assuntos
Anticorpos/genética , Região Variável de Imunoglobulina/genética , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Anticorpos/metabolismo , Antígenos/metabolismo , Biotina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Região Variável de Imunoglobulina/metabolismo , Microscopia de Fluorescência , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo
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