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A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer
Published on: September 13, 2022
High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display
Valencio Salema1, Carmen Mañas1, Lidia Cerdán1
1a Department of Microbial Biotechnology , Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus UAM Cantoblanco , Madrid , Spain.
Abstract:
Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.
Insights
This study presents an efficient E. coli display method for isolating high-affinity nanobodies targeting cell surface proteins like EGFR. This bacterial display system overcomes challenges associated with phage display for antibody selection on live cells.
Area of Science:
- Biotechnology
- Immunology
- Molecular Biology
Background:
- Therapeutic antibodies typically target cell surface proteins on tumor and immune cells.
- Phage display is common for antibody selection but challenging for cell surface targets due to phage stickiness.
- Previous work established E. coli display for VHH (nanobody) selection via magnetic cell sorting.
Purpose of the Study:
- To demonstrate the efficacy of E. coli display for isolating nanobodies against cell surface antigens.
- To develop a whole cell-based strategy for antibody library selection on live cells.
- To isolate and characterize high-affinity nanobodies targeting the epidermal growth factor receptor (EGFR).
Main Methods:
- Utilized an E. coli display system for VHH library expression on bacterial surfaces.
- Employed a whole cell-based selection strategy using EGFR-expressing cells and untransfected cells for depletion.
- Applied flow cytometry and surface plasmon resonance for nanobody characterization and binding affinity assessment.
Main Results:
- Successfully isolated high-affinity nanobodies targeting distinct epitopes of EGFR.
- Identified nanobodies that compete with the natural ligand, EGF.
- Demonstrated efficient isolation and characterization of nanobodies against cell surface antigens using E. coli display.
Conclusions:
- E. coli display is an effective alternative to phage display for selecting antibodies against cell surface targets.
- The developed whole cell-based strategy enables efficient isolation of specific, high-affinity nanobodies.
- This method facilitates the discovery of novel nanobodies for therapeutic applications targeting cell surface proteins.
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