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Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library
T Heitner1, A Moor, J L Garrison
1Department of Anesthesia, University of California, San Francisco, Room 3C-38, San Francisco General Hospital, 1001 Potrero Avenue, San Francisco, CA 94110, USA. marksj@anesthesia.ucsf.edu
Abstract:
The first step in developing a targeted cancer therapeutic is generating a ligand that binds to a receptor which is either tumor specific or sufficiently overexpressed in tumors to provide targeting specificity. For this work, we generated human monoclonal antibodies to the EGF receptor (EGFR), an antigen overexpressed on many solid tumors. Single chain Fv (scFv) antibody fragments were directly selected by panning a phage display library on tumor cells (A431) overexpressing EGFR or Chinese hamster ovary cells (CHO/EGFR cells) transfected with the EGFR gene and recovering endocytosed phage from within the cell. Three unique scFvs were isolated, two from selections on A431 cells and two from selections on CHO/EGFR cells. All three scFv bound native receptor as expressed on a panel of tumor cells and did not bind EGFR negative cells. Phage antibodies and multivalent immunoliposomes constructed from scFv were endocytosed by EGFR expressing cells as shown by confocal microscopy. Native scFv primarily stained the cell surface, with less staining intracellularly. The results demonstrate how phage antibodies binding native cell surface receptors can be directly selected on overexpressing cell lines or transfected cells. Use of a transfected cell line allows selection of antibodies to native receptors without the need for protein expression and purification, significantly speeding the generation of targeting antibodies to genomic sequences. Depending upon the format used, the antibodies can be used to deliver molecules to the cell surface or intracellularly.
Insights
Researchers developed novel human monoclonal antibodies targeting the epidermal growth factor receptor (EGFR) for cancer therapy. These antibodies can be selected using engineered cells, accelerating the creation of targeted cancer treatments.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Targeted cancer therapeutics require ligands binding tumor-specific or overexpressed receptors.
- Epidermal growth factor receptor (EGFR) is overexpressed on many solid tumors, making it a target for cancer therapy.
Purpose of the Study:
- To generate human monoclonal antibodies targeting the epidermal growth factor receptor (EGFR).
- To demonstrate a method for selecting antibodies that bind native receptors on cell surfaces, including using transfected cell lines.
Main Methods:
- Panning a phage display library against A431 tumor cells and Chinese hamster ovary (CHO) cells transfected with EGFR.
- Selection and isolation of single-chain variable fragment (scFv) antibody fragments.
- Validation of scFv binding to native EGFR on various tumor cells using confocal microscopy.
Main Results:
- Three unique scFv antibodies were isolated that specifically bind native EGFR on tumor cells.
- EGFR-expressing cells internalized phage antibodies and immunoliposomes constructed from scFv.
- The method allows selection of antibodies against native receptors without prior protein purification.
Conclusions:
- Phage antibodies binding native cell surface receptors can be directly selected on overexpressing or transfected cell lines.
- Using transfected cell lines speeds up the generation of targeted antibodies.
- Generated antibodies can be utilized for cell surface or intracellular delivery of therapeutic payloads.