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Updated: Aug 6, 2026
Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells
Published on: May 9, 2025
Selection and characterization of an internalizing epidermal-growth-factor-receptor antibody
Xiaorong Zhao1, Wentao Dai, Limin Cao
1Laboratory of Molecular and Immuno-Pharmacology, Department of Immunology Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People's Republic of China.
Abstract:
Antibody-therapeutic agent conjugates to be delivered directly into the cytosol of tumour cells is required for many target-based therapeutic strategies. For this work, a large non-immune phage-display library was used to select internalizing scFv (single chain variable fragment) directed against EGFR (epidermal growth factor receptor), a tyrosine kinase receptor that is overexpressed in a wide range of tumour cells. The CHO-EGFR-GFP1 (where CHO is Chinese-hamster ovary) cell line, a transfected cell line expressing EGFR-GFP (green fluorescent protein) fusion protein on membranes, and the untransfected cell line CHO-K1 were used as EGFR-positive cells and -negative cells respectively in the subtractive selection procedure. A novel human anti-EGFR scFv (F4-scFv) was isolated. F4-scFv bound native EGFR-bearing cell lines and could be internalized, but did not bind EGFR-negative cell lines. The K(D) value of F4-scFv was 472 nM as determined on A431 cells. F4-scFv could be used to target therapeutic agents into tumour cells and was expected to be non-immunogenic in humans. Use of a transfected cell line expressing GFP-tagged receptors allows selection and characterization of antibodies to native receptors without the need for protein expression and purification, significantly speeding up the generation of targeting antibodies.
Insights
Researchers developed a novel human antibody fragment (F4-scFv) targeting epidermal growth factor receptor (EGFR) on tumor cells. This antibody internalizes into cancer cells, offering a promising new tool for targeted cancer therapies.
Area of Science:
- Biotechnology
- Molecular Biology
- Oncology
Background:
- Targeted cancer therapies require agents that can be delivered directly into tumor cells.
- Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor overexpressed in many tumor types, making it a key therapeutic target.
Purpose of the Study:
- To select and characterize a novel human antibody fragment (scFv) that internalizes into EGFR-expressing tumor cells.
- To develop a new targeting antibody for antibody-drug conjugates (ADCs) for cancer treatment.
Main Methods:
- Utilized a large non-immune phage-display library for antibody selection.
- Employed subtractive selection using EGFR-transfected (CHO-EGFR-GFP1) and untransfected (CHO-K1) cell lines.
- Characterized the selected antibody (F4-scFv) for binding affinity (K(D)) and internalization into EGFR-positive cells.
Main Results:
- Isolated a novel human anti-EGFR scFv, designated F4-scFv.
- F4-scFv demonstrated specific binding to native EGFR on tumor cell lines and exhibited internalization.
- The antibody did not bind to EGFR-negative cell lines, confirming specificity.
- Determined the K(D) value of F4-scFv to be 472 nM on A431 cells.
Conclusions:
- F4-scFv is a promising candidate for targeting therapeutic agents into EGFR-expressing tumor cells.
- The developed antibody is expected to be non-immunogenic in humans.
- Using GFP-tagged receptors in cell lines accelerates antibody generation for targeted therapies.
