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Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library
1Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
Aim:
To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library.
Methods:
A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation.
Results:
Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells.
Conclusion:
The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.
Insights
Researchers identified specific antibody fragments for hepatocellular carcinoma (HCC) using phage display biopanning. These fragments showed binding activity and inhibited HCC cell growth, offering potential for HCC immunotherapy.
Area of Science:
- Biotechnology
- Immunology
- Oncology
Background:
- Hepatocellular carcinoma (HCC) is a significant global health concern.
- Developing targeted therapies for HCC remains a critical challenge.
Purpose of the Study:
- To identify single-chain variable fragment (scFv) antibody fragments specific for hepatocellular carcinoma using phage display.
- To evaluate the therapeutic potential of these identified scFv fragments.
Main Methods:
- Biopanning of a human naive scFv phage display library against HCC cells (HepG2) and normal liver cells (L02).
- Selection and identification of specific scFv clones using ELISA and flow cytometry.
- Expression, purification, and characterization of scFv antibody fragments in E. coli.
Main Results:
- Two distinct scFv antibody fragments with specific binding to HepG2 cells were identified.
- The expressed scFv fragments demonstrated specific binding and inhibited HepG2 cell proliferation.
- Sequences of identified scFv fragments were deposited in GenBank.
Conclusions:
- Phage library biopanning is an effective method for identifying specific antibody fragments against hepatocellular carcinoma.
- The identified scFv fragments show promise as therapeutic agents for HCC immunotherapy.
