High Expression in CHO
Cells and Activity of an Anti-P185erbB2 Mouse/human Chimeric
Antibody
YANG Guang, RAN Yu-Liang, SUN Li-Xin, LIU Jun, YU Long, YANG
Zhi-Hua*
( Department of Cellular and Molecular Biology, Cancer Institute, Chinese
Academy of Medical Sciences, Beijing 100021, China )
Abstract The McAb
C25 against human P185erbB2 specifically inhibits proliferation of
cancer cells overexpressing P185erbB2. 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-P185erbB2 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 (1×10－8～2.5×10－7 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 P185erbB2
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 SKBR3 and SKOV3 cells at
the same inhibiting rate asMcAb C25. In conclusion, a mouse/human chimeric
antibody against human P185erbB2 with potential of usage in clinical
therapy of human cancer was constructed and highly expressed.
Key words erbB2；chimeric antibody；expression；engineering antibody；CHO cell
*Corresponding author：Tel,86-10-67781331 ext 8439；Fax,86-10-67713359；e-mail,
[email protected]