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Milking the cow: cattle-derived chimeric ultralong CDR-H3 antibodies and their engineered CDR-H3-only knobbody counterparts targeting epidermal growth factor receptor elicit potent NK cell-mediated cytotoxicity

Pekar, Lukas ; Klewinghaus, Daniel ; Arras, Paul ; Carrara, Stefania C. ; Harwardt, Julia ; Krah, Simon ; Yanakieva, Desislava ; Toleikis, Lars ; Smider, Vaughn V. ; Kolmar, Harald ; Zielonka, Stefan (2021)
Milking the cow: cattle-derived chimeric ultralong CDR-H3 antibodies and their engineered CDR-H3-only knobbody counterparts targeting epidermal growth factor receptor elicit potent NK cell-mediated cytotoxicity.
In: Frontiers in Immunology, 12
doi: 10.3389/fimmu.2021.742418
Article, Bibliographie

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Abstract

In this work, we have generated epidermal growth factor receptor (EGFR)-specific cattle-derived ultralong CDR-H3 antibodies by combining cattle immunization with yeast surface display. After immunization, ultralong CDR-H3 regions were specifically amplified and grafted onto an IGHV1-7 scaffold by homologous recombination to facilitate Fab display. Antigen-specific clones were readily obtained by fluorescence-activated cell sorting (FACS) and reformatted as chimeric antibodies. Binning experiments revealed epitope targeting of domains I, II, and IV of EGFR with none of the generated binders competing with Cetuximab, Matuzumab, or EGF for binding to EGFR. Cattle-derived chimeric antibodies were potent in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against EGFR-overexpressing tumor cells with potencies (EC50 killing) in the picomolar range. Moreover, most of the antibodies were able to significantly inhibit EGFR-mediated downstream signaling. Furthermore, we demonstrate that a minor fraction of CDR-H3 knobs derived from generated antibodies was capable of independently functioning as a paratope facilitating EGFR binding when grafted onto the Fc part of human IgG1. Besides slightly to moderately diminished capacities, these engineered Knobbodies largely retained main properties of their parental antibodies such as cellular binding and triggering of ADCC. Hence, Knobbodies might emerge as promising tools for biotechnological applications upon further optimization.

Item Type: Article
Erschienen: 2021
Creators: Pekar, Lukas ; Klewinghaus, Daniel ; Arras, Paul ; Carrara, Stefania C. ; Harwardt, Julia ; Krah, Simon ; Yanakieva, Desislava ; Toleikis, Lars ; Smider, Vaughn V. ; Kolmar, Harald ; Zielonka, Stefan
Type of entry: Bibliographie
Title: Milking the cow: cattle-derived chimeric ultralong CDR-H3 antibodies and their engineered CDR-H3-only knobbody counterparts targeting epidermal growth factor receptor elicit potent NK cell-mediated cytotoxicity
Language: English
Date: 2021
Place of Publication: Lausanne
Publisher: Frontiers Media S.A.
Journal or Publication Title: Frontiers in Immunology
Volume of the journal: 12
Collation: 17 Seiten
DOI: 10.3389/fimmu.2021.742418
Corresponding Links:
Abstract:

In this work, we have generated epidermal growth factor receptor (EGFR)-specific cattle-derived ultralong CDR-H3 antibodies by combining cattle immunization with yeast surface display. After immunization, ultralong CDR-H3 regions were specifically amplified and grafted onto an IGHV1-7 scaffold by homologous recombination to facilitate Fab display. Antigen-specific clones were readily obtained by fluorescence-activated cell sorting (FACS) and reformatted as chimeric antibodies. Binning experiments revealed epitope targeting of domains I, II, and IV of EGFR with none of the generated binders competing with Cetuximab, Matuzumab, or EGF for binding to EGFR. Cattle-derived chimeric antibodies were potent in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against EGFR-overexpressing tumor cells with potencies (EC50 killing) in the picomolar range. Moreover, most of the antibodies were able to significantly inhibit EGFR-mediated downstream signaling. Furthermore, we demonstrate that a minor fraction of CDR-H3 knobs derived from generated antibodies was capable of independently functioning as a paratope facilitating EGFR binding when grafted onto the Fc part of human IgG1. Besides slightly to moderately diminished capacities, these engineered Knobbodies largely retained main properties of their parental antibodies such as cellular binding and triggering of ADCC. Hence, Knobbodies might emerge as promising tools for biotechnological applications upon further optimization.

Uncontrolled Keywords: antibody display, antibody engineering, cattle antibody, ultralong CDR3, yeast surface display, Knobbody
Additional Information:

This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology

Classification DDC: 500 Science and mathematics > 540 Chemistry
500 Science and mathematics > 570 Life sciences, biology
600 Technology, medicine, applied sciences > 610 Medicine and health
Divisions: 07 Department of Chemistry
07 Department of Chemistry > Clemens-Schöpf-Institut > Fachgebiet Biochemie
Date Deposited: 22 Jan 2024 09:13
Last Modified: 22 Jan 2024 09:13
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