ABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/genetics , Receptor, ErbB-2/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Base Sequence , Binding Sites , Cricetinae , Flow Cytometry , Gene Expression , Genetic Vectors/genetics , Humans , Immunoglobulin Fc Fragments/genetics , Mice , Molecular Sequence Data , Protein Binding , Protein Engineering , Proto-Oncogene Mas , Receptor, ErbB-2/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies , Tumor Cells, CulturedABSTRACT
AIM: To prepare and identify the antibodies against snake venom C-type lectin like protein Agkisacutacin. METHODS: A BALB/c mouse was immunized with Agkisacutacin and the cells were fused by standard hybridoma technique to prepare mAbs. The stability of the hybridomas secreting mAbs was detected by indirect ELISA. The nuclear type of the hybridomas was analyzed by fluorescent staining and the specificity of mAbs was detected by Western blot. RESULTS: Three cell strains of hybridomas that could steadily secrete anti-Agkisacutacin mAb were obtained. CONCLUSION: The successful preparation of anti-Agkisacutacin mAbs provides an important tool for studying the in vivo metabolism of new anti-thrombus drugs by detecting Agkisacutacin and investigating the mechanism of anti-thrombus of the drugs.
Subject(s)
Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Crotalid Venoms/immunology , Snake Venoms/chemistry , Animals , Antibodies, Monoclonal/metabolism , Antibody Specificity , Crotalid Venoms/biosynthesis , Crotalid Venoms/isolation & purification , Enzyme-Linked Immunosorbent Assay , Female , Hybridomas/metabolism , Mice , Mice, Inbred BALB C , Snake Venoms/immunologyABSTRACT
AIM: To study the cellular metabolism(RIA) in vitro and the biodistribution in vivo of (125)I labeled anti-p185 antibodies(murine mAb A21, mouse-human chimeric antibody A21scFv-Fc and single chain antibody A21 scFv), to provide the basis for clinical application in the diagnosis and treatment of tumor overexpressing p185. METHODS: The specificity of binding of these antibodies to SKOV(3) known to highly express surface antigen p185 was assessd by FACS. The metabolism of the antibodies in SKOV(3) was assayed by cellular RIA. The biodistribution of radiolabeled antibodies was evaluated in nude mice bearing SKOV(3) tumor. RESULTS: Cellular RIA demonstrated these antibodies were internalized after binding to cells, followed by degradation and deiodination in cells, (125)I was then exocytosed. In vivo test showed that these antibodies were concentrated in tumor, but no concentration of control antibody in tumor was found. CONCLUSION: mAb A21, A21 scFv-Fc and A21 scFv can target human ovarian carcinoma cells (SKOV(3)) overexpressing p185 in vitro and in vivo and may be used in the diagnosis and treatment of tumors overexpressing p185.