Fibing, David ; Bogen, Jan P. ; Carrara, Stefania C. ; Deweid, Lukas ; Zielonka, Stefan ; Grzeschik, Julius ; Hock, Björn ; Kolmar, Harald (2022)
Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells.
In: Frontiers in Bioengineering and Biotechnology, 2022, 10
doi: 10.26083/tuprints-00021336
Artikel, Zweitveröffentlichung, Verlagsversion
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Kurzbeschreibung (Abstract)
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.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2022 |
| Autor(en): | Fibing, David ; Bogen, Jan P. ; Carrara, Stefania C. ; Deweid, Lukas ; Zielonka, Stefan ; Grzeschik, Julius ; Hock, Björn ; Kolmar, Harald |
| Art des Eintrags: | Zweitveröffentlichung |
| Titel: | Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells |
| Sprache: | Englisch |
| Publikationsjahr: | 2022 |
| Publikationsdatum der Erstveröffentlichung: | 2022 |
| Verlag: | Frontiers |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Frontiers in Bioengineering and Biotechnology |
| Jahrgang/Volume einer Zeitschrift: | 10 |
| Kollation: | 10 Seiten |
| DOI: | 10.26083/tuprints-00021336 |
| URL / URN: | https://tuprints.ulb.tu-darmstadt.de/21336 |
| Zugehörige Links: | |
| Herkunft: | Zweitveröffentlichung aus gefördertem Golden Open Access |
| Kurzbeschreibung (Abstract): | 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. |
| Status: | Verlagsversion |
| URN: | urn:nbn:de:tuda-tuprints-213365 |
| Zusätzliche Informationen: | Keywords: antibody hit discovery, bidirectional promoter, reformatting, golden gate cloning, monoclonal antibodies, yeast surface display |
| Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie |
| Fachbereich(e)/-gebiet(e): | 07 Fachbereich Chemie 07 Fachbereich Chemie > Clemens-Schöpf-Institut > Fachgebiet Biochemie 07 Fachbereich Chemie > Clemens-Schöpf-Institut |
| Hinterlegungsdatum: | 20 Mai 2022 13:06 |
| Letzte Änderung: | 02 Aug 2024 12:41 |
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| Suche nach Titel in: | TUfind oder in Google |
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- Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells. (deposited 20 Mai 2022 13:06) [Gegenwärtig angezeigt]
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