High Expression in CHO Cells and Activity of an Anti-P185(erbB2) Mouse/human Chimeric Antibody.

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.