Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185(c-erbB-2).
The c-erbB-2 proto-oncogene encodes a 185kDa protein p185, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p185. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.
Cheng
Lian Sheng
LS
School of Life Science, University of Science and Technology of China, Hefei 230027, China.
Liu
Ai Ping
AP
Yang
Jia Hong
JH
Dong
Yan Qiu
YQ
Li
Liang Wei
LW
Wang
Jing
J
Wang
Chao Chen
CC
Liu
Jing
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D006224:Cricetinae; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D057127:Single-Chain Antibodies; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C000715470:MAS1 protein, human; C479628:monoclonal antibody scFv A21