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Updated: Aug 21, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
[Eukaryotic expression and biological activities of anti-p185 erbB2 mouse/human chimeric antibody]
Bei-hai Jiang1, Wen-bin Liu, Lin Meng
1Department of Biochemistry and Molecular Biology, Peking University School of Oncology; Beijing Institute for Cancer Research; Beijing 100034, China.
Objective:
Overexpression of the HER2/neu oncogene is a frequent molecular event in multiple human cancers. Being a cancer antigen, p185(erbB2) is an ideal target for immunotherapy. In order to decrease the immunogenicity of mouse anti-p185(erbB2) monoclonal antibody in human cancer therapy, we constructed the eukaryotic expression vector of anti-p185(erbB2) chimeric monoclonal antibody and verified expression of the chimeric antibody in CHO-dhfr(-) cell.
Methods:
The variable regions of light chain and heavy chain were amplified with RT-PCR and inserted into the chimeric antibody vector pWSD2. After CHO-dhfr(-) cells were transfected with recombination plasmid by lipofectAMINE, the chimeric antibody expressing level was identified with RT-PCR, indirect-ELISA, and Western blot. The specificity of the anti-p185(erbB2) chimeric antibody was testified with ELISA assay and immunoprecipitation. Moreover, the effects of chimeric antibody on the proliferation of breast cancer cell line SKBR3, which is overexpressing p185(erbB2), were measured with MTT assay in vitro.
Results:
The anti-p185(erbB2) chimeric antibody eukaryotic expression vector was constructed successfully and the expression of the chimeric antibody in CHO-dhfr(-) was verified by RT-PCR, indirect-ELISA, and Western blot. ELISA assay showed that chimeric antibody reacted with cells overexpressing p185(erbB2) specifically, but did not react with that non-overexpressing p185(erbB2). Immunoprecipitation test confirmed that the chimeric antibody could bind to p185(erbB2) specifically. The MTT assay demonstrated that the chimeric antibody could inhibit the growth of SKBR3 cells overexpressing p185(erbB2) .
Conclusion:
The anti-p185(erbB2) mouse/human chimeric antibody that was expressed in CHO-dhfr(-) cells can bind to p185(erbB2) specifically and inhibit proliferation of SKBR3 cells overexpressing p185(erbB2) . It has a potential application in biotherapy of cancer.
Insights
A novel chimeric antibody targeting HER2/neu (p185erbB2) was successfully developed and expressed. This engineered antibody specifically binds to cancer cells and inhibits their proliferation, offering potential for cancer biotherapy.
Area of Science:
- Oncology
- Immunology
- Molecular Biology
Context:
- HER2/neu (p185erbB2) oncogene overexpression is common in many human cancers.
- p185erbB2 serves as a cancer antigen, making it a promising target for immunotherapy.
- Mouse anti-p185erbB2 monoclonal antibodies have immunogenicity issues in human therapy.
Purpose:
- To construct a eukaryotic expression vector for an anti-p185erbB2 chimeric monoclonal antibody.
- To reduce the immunogenicity of mouse anti-p185erbB2 antibodies for cancer therapy.
- To verify the expression and functionality of the chimeric antibody.
Summary:
- A chimeric antibody targeting p185erbB2 was constructed using variable regions from a mouse antibody and human constant regions.
- Expression in CHO-dhfr(-) cells was confirmed via RT-PCR, ELISA, and Western blot.
- The chimeric antibody demonstrated specific binding to p185erbB2-overexpressing cells and inhibited SKBR3 breast cancer cell proliferation in vitro.
Impact:
- The developed anti-p185erbB2 chimeric antibody specifically targets cancer cells overexpressing the HER2/neu oncogene.
- This engineered antibody inhibits the proliferation of relevant cancer cell lines, such as SKBR3.
- The study indicates potential for this chimeric antibody in the biotherapy of HER2/neu-positive cancers.
