Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells
Yeast-surface display (YSD) is commonly applied to screen Fab immune or naïve libraries for binders of predefined target molecules. However, reformatting of isolated variants represents a time-intensive bottleneck. Herein, we present a novel approach to facilitate a lean transition from antibody scr...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-05-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2022.794389/full |
| _version_ | 1811307796440809472 |
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| author | David Fiebig David Fiebig Jan P. Bogen Jan P. Bogen Stefania C. Carrara Stefania C. Carrara Lukas Deweid Lukas Deweid Stefan Zielonka Julius Grzeschik Björn Hock Harald Kolmar Harald Kolmar |
| author_facet | David Fiebig David Fiebig Jan P. Bogen Jan P. Bogen Stefania C. Carrara Stefania C. Carrara Lukas Deweid Lukas Deweid Stefan Zielonka Julius Grzeschik Björn Hock Harald Kolmar Harald Kolmar |
| author_sort | David Fiebig |
| collection | DOAJ |
| description | Yeast-surface display (YSD) is commonly applied to screen Fab immune or naïve libraries for binders of predefined target molecules. However, reformatting of isolated variants represents a time-intensive bottleneck. Herein, we present a novel approach to facilitate a lean transition from antibody screening using YSD Fab libraries to the production of full-length IgG antibodies in Expi293-F cells. In this study, utilizing Golden Gate Cloning (GGC) and a bidirectional promoter system, an exemplary Fab-displaying YSD library was generated based on immunised transgene rats. After subsequent screening for antigen-specific antibody candidates by fluorescence-activated cell sorting (FACS), the Fab-encoding genes were subcloned into a bidirectional mammalian expression vector, exhibiting CH2-CH3 encoding genes, in a GGC-mediated, PCR-free manner. This novel, straightforward and time-saving workflow allows the VH/VL pairing to be preserved. This study resulted in antibody variants exhibiting suitable biophysical properties and covered a broad VH diversity after two rounds of FACS screening, as revealed by NGS analysis. Ultimately, we demonstrate that the implication of such a gene transfer system streamlines antibody hit discovery efforts, allowing the faster characterisation of antibodies against a plethora of targets that may lead to new therapeutic agents. |
| first_indexed | 2024-04-13T09:10:42Z |
| format | Article |
| id | doaj.art-07962c2c56b2491d9d889fe9fed966c1 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-13T09:10:42Z |
| publishDate | 2022-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-07962c2c56b2491d9d889fe9fed966c12022-12-22T02:52:52ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852022-05-011010.3389/fbioe.2022.794389794389Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian CellsDavid Fiebig0David Fiebig1Jan P. Bogen2Jan P. Bogen3Stefania C. Carrara4Stefania C. Carrara5Lukas Deweid6Lukas Deweid7Stefan Zielonka8Julius Grzeschik9Björn Hock10Harald Kolmar11Harald Kolmar12Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, GermanyFerring Darmstadt Laboratories, Darmstadt, GermanyInstitute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, GermanyFerring Darmstadt Laboratories, Darmstadt, GermanyInstitute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, GermanyFerring Darmstadt Laboratories, Darmstadt, GermanyInstitute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, GermanyFerring Darmstadt Laboratories, Darmstadt, GermanyInstitute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, GermanyFerring Biologics Innovation Centre, Epalinges, SwitzerlandFerring Biologics Innovation Centre, Epalinges, SwitzerlandInstitute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, GermanyCentre for Synthetic Biology, Technical University of Darmstadt, Darmstadt, GermanyYeast-surface display (YSD) is commonly applied to screen Fab immune or naïve libraries for binders of predefined target molecules. However, reformatting of isolated variants represents a time-intensive bottleneck. Herein, we present a novel approach to facilitate a lean transition from antibody screening using YSD Fab libraries to the production of full-length IgG antibodies in Expi293-F cells. In this study, utilizing Golden Gate Cloning (GGC) and a bidirectional promoter system, an exemplary Fab-displaying YSD library was generated based on immunised transgene rats. After subsequent screening for antigen-specific antibody candidates by fluorescence-activated cell sorting (FACS), the Fab-encoding genes were subcloned into a bidirectional mammalian expression vector, exhibiting CH2-CH3 encoding genes, in a GGC-mediated, PCR-free manner. This novel, straightforward and time-saving workflow allows the VH/VL pairing to be preserved. This study resulted in antibody variants exhibiting suitable biophysical properties and covered a broad VH diversity after two rounds of FACS screening, as revealed by NGS analysis. Ultimately, we demonstrate that the implication of such a gene transfer system streamlines antibody hit discovery efforts, allowing the faster characterisation of antibodies against a plethora of targets that may lead to new therapeutic agents.https://www.frontiersin.org/articles/10.3389/fbioe.2022.794389/fullantibody hit discoverybidirectional promoterreformattinggolden gate cloningmonoclonal antibodiesyeast surface display |
| spellingShingle | David Fiebig David Fiebig Jan P. Bogen Jan P. Bogen Stefania C. Carrara Stefania C. Carrara Lukas Deweid Lukas Deweid Stefan Zielonka Julius Grzeschik Björn Hock Harald Kolmar Harald Kolmar Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells Frontiers in Bioengineering and Biotechnology antibody hit discovery bidirectional promoter reformatting golden gate cloning monoclonal antibodies yeast surface display |
| title | Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells |
| title_full | Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells |
| title_fullStr | Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells |
| title_full_unstemmed | Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells |
| title_short | Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells |
| title_sort | streamlining the transition from yeast surface display of antibody fragment immune libraries to the production as igg format in mammalian cells |
| topic | antibody hit discovery bidirectional promoter reformatting golden gate cloning monoclonal antibodies yeast surface display |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2022.794389/full |
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