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Published on: September 30, 2019
[Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble
Wei-Jin Sheng1, Qing-Fang Miao, Yong-Su Zhen
1Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Abstract:
Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.
Insights
Researchers developed single chain variable fragment (scFv) antibodies targeting the epidermal growth factor receptor (EGFR), a key molecule in cancer therapy. These novel anti-EGFR scFv antibodies show specific binding, paving the way for new cancer treatments.
Area of Science:
- Biotechnology
- Immunology
- Oncology
Background:
- Epidermal growth factor receptor (EGFR) is a crucial target in cancer therapy.
- Development of targeted antibody therapies is essential for effective cancer treatment.
Purpose of the Study:
- To prepare single chain variable fragment (scFv) antibodies specifically targeting EGFR.
- To evaluate the binding activity and potential therapeutic applications of the generated anti-EGFR scFv.
Main Methods:
- Immunization of Balb/c mice with human carcinoma A431 cells to generate immune response.
- Construction of a recombinant phage antibody library using RT-PCR, gene fragment joining, and phagemid vector system.
- Enrichment of anti-EGFR phage scFv clones through affinity panning and screening using ELISA.
- Production and characterization of soluble recombinant antibodies from positive clones.
Main Results:
- A high success rate (45 out of 48 clones) in identifying anti-EGFR scFv positive clones.
- Successful production of soluble recombinant antibodies (approximately 27 kD) with specific binding activity to purified EGFR and EGFR on carcinoma cell membranes.
- Sequencing confirmed the scFv gene (768 bp) encoding 256 amino acid residues, homologous to mouse immunoglobulin.
Conclusions:
- The study successfully prepared anti-EGFR scFv antibodies with specific binding capabilities.
- These findings provide a valuable EGFR-targeted molecule for developing antibody-based drugs and biological cancer therapies.
