Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells.

Autor:	Fiebig D; Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.; Ferring Darmstadt Laboratories, Darmstadt, Germany., Bogen JP; Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.; Ferring Darmstadt Laboratories, Darmstadt, Germany., Carrara SC; Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.; Ferring Darmstadt Laboratories, Darmstadt, Germany., Deweid L; Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.; Ferring Darmstadt Laboratories, Darmstadt, Germany., Zielonka S; Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany., Grzeschik J; Ferring Biologics Innovation Centre, Epalinges, Switzerland., Hock B; Ferring Biologics Innovation Centre, Epalinges, Switzerland., Kolmar H; Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.; Centre for Synthetic Biology, Technical University of Darmstadt, Darmstadt, Germany.
Jazyk:	angličtina
Zdroj:	Frontiers in bioengineering and biotechnology [Front Bioeng Biotechnol] 2022 May 10; Vol. 10, pp. 794389. Date of Electronic Publication: 2022 May 10 (Print Publication: 2022).
DOI:	10.3389/fbioe.2022.794389
Abstrakt:	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. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2022 Fiebig, Bogen, Carrara, Deweid, Zielonka, Grzeschik, Hock and Kolmar.)
Databáze:	MEDLINE
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