[Eukaryotic expression and biological activities of anti-p185 erbB2 mouse/human chimeric antibody].
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) .


CONCLUSIONS
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.
Department of Biochemistry and Molecular Biology, Peking University School of Oncology; Beijing Institute for Cancer Research; Beijing 100034, China.
Bei-hai
BH
Jiang
Wen-bin
WB
Liu
Lin
L
Meng
Jian
J
Wu
Cheng-chao
CC
Shou
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D005057:Eukaryotic Cells / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
D000911:Antibodies, Monoclonal; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins