The Experts below are selected from a list of 8343 Experts worldwide ranked by ideXlab platform
Stefan Dübel - One of the best experts on this subject based on the ideXlab platform.
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Selection of Recombinant antibodies from Antibody gene libraries
Methods in Molecular Biology, 2014Co-Authors: Michael Hust, Anne C. Frenzel, Stefan Dübel, André Frenzel, Thomas SchirrmannAbstract:After the sequencing of the human genome is completed, the research focus shifts toward the analysis of gene products. The human genome encodes more than 30,000 genes. Owing to alternative mRNA splicing and posttranslational modifications, for example, glycosylation, phoshorylation, and so on, the number of different proteins of human proteome is supposed to easily exceed 90,000. Antibodies are key detection reagents for the "postgenomic" analysis of these proteins. Any systematic investigation of the human proteome requires high throughput methods for Antibody generation. In vitro selection systems utilizing Recombinant Antibody repertoires offer this capability and capacity. The most commonly used contemporary in vitro selection system is Antibody phage display, which has already yielded thousands of useful antibodies for therapy, research, and diagnostics. Herein, methods are described for the selection of Recombinant Antibody fragments from naive Antibody gene libraries.
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Construction of Human Naive Antibody Gene Libraries
2012Co-Authors: Michael Hust, Torsten Meyer, Anne C. Frenzel, Thomas Schirrmann, André Frenzel, Stefan DübelAbstract:After the sequencing of the human genome is completed, the research focus shifts toward the analysis of gene products. The human genome encodes more than 30,000 genes. Owing to alternative mRNA splicing and posttranslational modifications, for example, glycosylation, phoshorylation, and so on, the number of different proteins of human proteome is supposed to easily exceed 90,000. Antibodies are key detection reagents for the postgenomic analysis of these proteins. Any systematic investigation of the human proteome requires high throughput methods for Antibody generation. In vitro selection systems utilizing Recombinant Antibody repertoires offer this capability and capacity. The most commonly used contemporary in vitro selection system is Antibody phage display, which has already yielded thousands of useful antibodies for therapy, research, and diagnostics. Herein, methods are described for the selection of Recombinant Antibody fragments from naive Antibody gene libraries.
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A multi-Fc-species system for Recombinant Antibody production
BMC Biotechnology, 2009Co-Authors: Sandrine Moutel, Ahmed El Marjou, Ole Vielemeyer, Philippe Benaroch, Clément Nizak, Stefan Dübel, Franck PerezAbstract: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.
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A multi-Fc-species system for Recombinant Antibody production
BMC Biotechnology, 2009Co-Authors: Sandrine Moutel, Ahmed El Marjou, Ole Vielemeyer, Philippe Benaroch, Clément Nizak, Stefan Dübel, Franck PerezAbstract: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. © 2009 Moutel et al; licensee BioMed Central Ltd.
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Recombinant therapeutic antibodies
Applied Microbiology and Biotechnology, 2007Co-Authors: Stefan DübelAbstract:Recombinant Antibody technology has revolutionized the development of Antibody therapeutics. This minireview offers an overview of enabling technologies and future prospects of this rapidly progressing field.
Michael Eisenhut - One of the best experts on this subject based on the ideXlab platform.
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68Ga-labelled Recombinant Antibody variants for immuno-PET imaging of solid tumours
European Journal of Nuclear Medicine and Molecular Imaging, 2010Co-Authors: Matthias Eder, Fabrice Le Gall, Melvyn Little, Vladimir Rybin, Stefan Knackmuss, Uwe Reusch, Walter Mier, Uwe Haberkorn, Michael EisenhutAbstract:Purpose Recombinant antibodies isolated from human Antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of Recombinant Antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope 68Ga.
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68Ga-labelled Recombinant Antibody variants for immuno-PET imaging of solid tumours
European Journal of Nuclear Medicine and Molecular Imaging, 2010Co-Authors: Matthias Eder, Fabrice Le Gall, Melvyn Little, Vladimir Rybin, Stefan Knackmuss, Uwe Reusch, Walter Mier, Uwe Haberkorn, Michael EisenhutAbstract:Recombinant antibodies isolated from human Antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of Recombinant Antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope (68)Ga.
Franck Perez - One of the best experts on this subject based on the ideXlab platform.
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A multi-Fc-species system for Recombinant Antibody production
BMC Biotechnology, 2009Co-Authors: Sandrine Moutel, Ahmed El Marjou, Ole Vielemeyer, Philippe Benaroch, Clément Nizak, Stefan Dübel, Franck PerezAbstract: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.
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A multi-Fc-species system for Recombinant Antibody production
BMC Biotechnology, 2009Co-Authors: Sandrine Moutel, Ahmed El Marjou, Ole Vielemeyer, Philippe Benaroch, Clément Nizak, Stefan Dübel, Franck PerezAbstract: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. © 2009 Moutel et al; licensee BioMed Central Ltd.
Eva Jordan - One of the best experts on this subject based on the ideXlab platform.
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Antibody Production by the Gram-Positive Bacterium Bacillus megaterium
2009Co-Authors: Eva Jordan, Laila Al-halabi, Thomas Schirrmann, Michael HustAbstract:The increasing demand for Recombinant antibodies as detection reagents in research, diagnostics, and therapy requires appropriate production systems. In contrast to Antibody therapies, small Recombinant Antibody fragments like Fab and scFv are sufficient for most applications in research and diagnostics. These Antibody fragments can also be produced in bacterial hosts. Gram-negative bacteria, particularly Escherichia coli, were extensively studied for the Recombinant Antibody production but they showed only a limited capacity to secrete Antibody fragments into the medium--a prerequisite for easy downstream processing. Gram-positive bacteria are known to efficiently secrete Recombinant proteins into the medium. Recently, we demonstrated the production of scFv and scFab fragments in Bacillus megaterium. Here, we describe the process in detail from transformation of B. megaterium to production and purification of scFv fragments.
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Production of Recombinant Antibody fragments in Bacillus megaterium
Microbial Cell Factories, 2007Co-Authors: Eva Jordan, Rebekka Biedendieck, Thomas Schirrmann, Michael Hust, Andreas Roth, Dieter Jahn, Stefan DübelAbstract:Background Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for Antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete Recombinant proteins into the growth medium. Here we evaluated B. megaterium for the Recombinant production of Antibody fragments. Results The lysozyme specific single chain Fv (scFv) fragment D1.3 was succesfully produced using B. megaterium . The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41°C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium . Per L culture supernatant, more than 400 μg of Recombinant His_6-tagged Antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli . Conclusion High yields of functional scFv Antibody fragments can be produced and secreted into the culture medium by B. megaterium , making this production system a reasonable alternative to E. coli .
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Production of Recombinant Antibody fragments in Bacillus megaterium
Microbial Cell Factories, 2007Co-Authors: Eva Jordan, Rebekka Biedendieck, Thomas Schirrmann, Michael Hust, Andreas Roth, Dieter Jahn, Stefan DübelAbstract:Background Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for Antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete Recombinant proteins into the growth medium. Here we evaluated B. megaterium for the Recombinant production of Antibody fragments.
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Production of Recombinant Antibody fragments in Bacillus megaterium
Microbial Cell Factories, 2007Co-Authors: Eva Jordan, Rebekka Biedendieck, Thomas Schirrmann, Michael Hust, Andreas Roth, Dieter Jahn, Stefan DübelAbstract:BACKGROUND: Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for Antibody synthesis. Due to the outer membrane of gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the gram-positive bacterium Bacillus megaterium was shown to efficiently secrete Recombinant proteins into the growth medium. Here we evaluated B. megaterium for the Recombinant production of Antibody fragments.\n\nRESULTS: The lysozyme specific single chain Fv (scFv) fragment D1.3 was successfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41 degrees C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 microg of Recombinant His6-tagged Antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli.\n\nCONCLUSION: High yields of functional scFv Antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli.
Royston Jefferis - One of the best experts on this subject based on the ideXlab platform.
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Recombinant Antibody therapeutics the impact of glycosylation on mechanisms of action
Trends in Pharmacological Sciences, 2009Co-Authors: Royston JefferisAbstract:More than twenty Recombinant Antibody molecules are now licensed for the treatment of a variety of cancers and chronic diseases. Initially, the attraction of antibodies was their specificity for target antigens; however, it is now appreciated that the downstream consequences of engaging antigen, after the formation of immune complexes, is crucial to clinical outcomes in vivo . This review introduces the structural and functional activities of the IgG class of Recombinant antibodies, in vitro , and criteria that determine choice between the four subclasses. Importantly, we demonstrate that, although accounting for only 2–3% of Antibody mass, glycosylation of the IgG-Fc is essential to the activation of downstream biologic mechanisms (effector functions). Additionally the precise structure of the attached oligosaccharide can influence biologic efficacy. These findings have led to cellular engineering to enable the production of selected glycoforms of Antibody that are considered to be optimal for the disease indication to be treated.
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Recombinant Antibody therapeutics: the impact of glycosylation on mechanisms of action
Trends in Pharmacological Sciences, 2009Co-Authors: Royston JefferisAbstract:More than twenty Recombinant Antibody molecules are now licensed for the treatment of a variety of cancers and chronic diseases. Initially, the attraction of antibodies was their specificity for target antigens; however, it is now appreciated that the downstream consequences of engaging antigen, after the formation of immune complexes, is crucial to clinical outcomes in vivo. This review introduces the structural and functional activities of the IgG class of Recombinant antibodies, in vitro, and criteria that determine choice between the four subclasses. Importantly, we demonstrate that, although accounting for only 2-3% of Antibody mass, glycosylation of the IgG-Fc is essential to the activation of downstream biologic mechanisms (effector functions). Additionally the precise structure of the attached oligosaccharide can influence biologic efficacy. These findings have led to cellular engineering to enable the production of selected glycoforms of Antibody that are considered to be optimal for the disease indication to be treated. © 2009 Elsevier Ltd. All rights reserved.
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Glycosylation of Recombinant Antibody therapeutics
Biotechnology Progress, 2005Co-Authors: Royston JefferisAbstract:The adaptive immune system has the capacity to produce antibodies with a virtually infinite repertoire of specificities. Recombinant antibodies specific for human targets are established in the clinic as therapeutics and represent a major new class of drug. Therapeutic efficacy depends on the formation of complexes with target molecules and subsequent activation of downstream biologic effector mechanisms that result in elimination of the target. The activation of effector mechanisms is dependent on structural characteristics of the Antibody molecule that result from posttranslational modifications, in particular, glycosylation. The production of therapeutic Antibody with a consistent human glycoform profile has been and remains a considerable challenge to the biopharmaceutical industry. Recent research has shown that individual glycoforms of Antibody may provide optimal efficacy for selected outcomes. Thus a further challenge will be the production of a second generation of Antibody therapeutics customized for their clinical indication.