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

David Fiebig; Jan P Bogen; Stefania C Carrara; Lukas Deweid; Stefan Zielonka; Julius Grzeschik; Björn Hock; Harald Kolmar

doi:10.3389/fbioe.2022.794389

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

Front Bioeng Biotechnol. 2022 May 10:10:794389. doi: 10.3389/fbioe.2022.794389. eCollection 2022.

Authors

David Fiebig^{1

2}, Jan P Bogen^{1

2}, Stefania C Carrara^{1

2}, Lukas Deweid^{1

2}, Stefan Zielonka¹, Julius Grzeschik³, Björn Hock³, Harald Kolmar^{1

4}

Affiliations

¹ Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.
² Ferring Darmstadt Laboratories, Darmstadt, Germany.
³ Ferring Biologics Innovation Centre, Epalinges, Switzerland.
⁴ Centre for Synthetic Biology, Technical University of Darmstadt, Darmstadt, Germany.

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.

Keywords: antibody hit discovery; bidirectional promoter; golden gate cloning; monoclonal antibodies; reformatting; yeast surface display.