Related Experiment Videos
High Expression in CHO Cells and Activity of an Anti-P185(erbB2) Mouse/human Chimeric Antibody
Guang Yang1, Yu-Liang Ran, Li-Xin Sun
1Department of Cellular and Molecular Biology, Cancer Institute, Chinese Academy of Medical Sciences, Beijing 100021, China. zhyang@public.bta.net.cn
Abstract:
The McAb C25 against human P185(erbB2) specifically inhibits proliferation of cancer cells overexpressing P185(erbB2). In order to decrease HAMA response in clinical therapy of human cancer using McAb, and to express this antibody efficiently in CHO cells, an anti-P185(erbB2) mouse/human chimeric antibody gene containing variable region of C25 gene was constructed. The expression vectors were constructed using genomic DNA of human IgG1 constant region, and using neo and dhfr genes driven by weaker promoters as selectable marker genes. Variable region genes of C25 were cloned by RT-PCR. VL and VH genes of C25 were sequenced and then inserted, respectively, into the light chain and heavy chain expression vectors. The two expression vectors were cotransfected into CHO-dhfr(-) cells with LipofectAMINE. The specificity of the chimeric antibody was verified using cellular-ELISA and immuno-fluorescence techniques. ELISA and RT-PCR were used toconfirm that the chimeric antibody containing both variable region of C25 and human constant region. At 72 h post-transfection, the chimeric antibody could be detected in supernatant of CHO cells by ELISA assay and the yield was 1 mg/L. After the selection by G418, stepwise MTX pressure (1x10(-8) 2.5x10(-7) mol/L) culture was carried out, and the yield of the chimeric antibody was increased up to 100 mg/L. The chimeric antibody was demonstrated to have the antigen specificity to P185(erbB2) and to carry the human antibody constant region by cellular-ELISA, immuno-fluorescence assay, indirect-ELISA and RT-PCR. The chimeric antibody could inhibit proliferation of SKBR(3) and SKOV(3) cells at the same inhibiting rate asMcAb C25. In conclusion, a mouse/human chimeric antibody against human P185(erbB2) with potential of usage in clinical therapy of human cancer was constructed and highly expressed.
Insights
A novel mouse/human chimeric antibody targeting P185(erbB2) was developed to reduce human anti-mouse antibody (HAMA) response in cancer therapy. This engineered antibody demonstrates high expression and specific inhibition of cancer cell proliferation, showing potential for clinical application.
Area of Science:
- Biotechnology
- Immunology
- Oncology
Background:
- Monoclonal antibody (McAb) C25 targets human P185(erbB2), inhibiting cancer cell proliferation.
- Human Anti-Mouse Antibody (HAMA) response limits clinical efficacy of mouse antibodies.
- Efficient expression of chimeric antibodies in host cells is crucial for therapeutic development.
Purpose of the Study:
- To construct a mouse/human chimeric antibody gene targeting P185(erbB2) to reduce HAMA response.
- To achieve efficient expression of the chimeric antibody in Chinese Hamster Ovary (CHO) cells.
- To evaluate the specificity, efficacy, and expression yield of the developed chimeric antibody.
Main Methods:
- Construction of chimeric antibody expression vectors using genomic DNA of human IgG1 constant region.
- Cloning and insertion of C25 variable region genes (VL and VH) into expression vectors.
- Co-transfection into CHO-dhfr(-) cells, selection using neo and dhfr genes, and stepwise methotrexate (MTX) pressure for amplification.
- Verification of antibody specificity and characteristics using cellular-ELISA, immuno-fluorescence, indirect-ELISA, and RT-PCR.
Main Results:
- Chimeric antibody detected in CHO cell supernatant at 72 h post-transfection with an initial yield of 1 mg/L.
- Yield increased to 100 mg/L after G418 selection and stepwise MTX pressure.
- The chimeric antibody exhibited specific binding to P185(erbB2) and inhibited proliferation of SKBR(3) and SKOV(3) cells comparably to McAb C25.
Conclusions:
- A mouse/human chimeric antibody targeting P185(erbB2) was successfully constructed and engineered for high expression in CHO cells.
- The developed antibody retains antigen specificity and therapeutic efficacy, comparable to the original mouse antibody.
- This chimeric antibody holds significant potential for clinical application in human cancer therapy by mitigating HAMA response.