Humanization of Chicken‐Derived scFv Using Yeast Surface Display and NGS Data Mining. Issue 3 (9th November 2020)

• Record Type: Journal Article
• Authors: Elter, Adrian; Bogen, Jan P.; Hinz, Steffen C.; Fiebig, David; Macarrón Palacios, Arturo; Grzeschik, Julius; Hock, Björn; Kolmar, Harald
• Is Part Of: Biotechnology journal. Volume 16:Issue 3(2021)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-11-09
• Subjects: chicken antibody -- fluorescence‐activated cell sorting -- humanization -- next‐generation sequencing -- yeast surface display; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.202000231
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract: Generation of high‐affinity monoclonal antibodies by immunization of chickens is a valuable strategy, particularly for obtaining antibodies directed against epitopes that are conserved in mammals. A generic procedure is established for the humanization of chicken‐derived antibodies. To this end, high‐affinity binders of the epidermal growth factor receptor extracellular domain are isolated from immunized chickens using yeast surface display. Complementarity determining regions (CDRs) of two high‐affinity binders are grafted onto a human acceptor framework. Simultaneously, Vernier zone residues, responsible for spatial CDR arrangement, are partially randomized. A yeast surface display library comprising ≈300 000 variants is screened for high‐affinity binders in the scFv and Fab formats. Next‐generation sequencing discloses humanized antibody variants with restored affinity and improved protein characteristics compared to the parental chicken antibodies. Furthermore, the sequencing data give new insights into the importance of antibody format, used during the humanization process. Starting from the antibody repertoire of immunized chickens, this work features an effective and fast high‐throughput approach for the generation of multiple humanized antibodies with potential therapeutic relevance. Abstract : By grafting the complementarity determining regions of a chicken‐derived scFv onto a human framework and subsequent randomization of Vernier Residues, yeast surface display libraries are generated, encoding either a humanized Fab or an scFv. Following fluorescence‐activated cell sorting, resulting clones are analyzed by next‐generation sequencing and reformatted into humanized antibodies exhibiting parental affinities and improved biophysical properties.