3.3 Inducible expression of scFv-Fc antibody gene in
CHO/GEV_scFv-Fc cells
The functionality of our artificial gene expression system mediated by a
transcription factor containing the estrogen-binding domain of the
estrogen receptor can be confirmed using a reporter gene. Thus, we
attempted to induce the production of a model antibody by adding E2 or
4-OHT (Figure 3). Plasmids containing the UAS/scFv-Fc expression unit
were linearized and introduced into CHO/GEV cells. After selection with
appropriate drugs, we obtained a pool of transformed cells and performed
cloning using the limiting dilution method. Among the 22 clones
obtained, we evaluated six clones that functioned correctly in response
to 0.1 µM 4-OHT and selected the one with the best cell proliferation
and scFv-Fc production responsiveness. This cell line, designated as
CHO/GEV_scFv-Fc cells, was further investigated for proliferation and
production induction analysis.
Evaluation of CHO/GEV_scFv-Fc cell proliferation in the presence of
1.25–5 μM E2 revealed no significant impact compared with the control
(Figure 3A). Measurement of scFv-Fc production showed a maximum of 1.0
μg/mL at 5 μM (Figure 3B), with a specific productivity of 5 pg
cell–1 day–1 (Figure 3C). Next, we
attempted to induce antibody production using 4-OHT. As 4-OHT
effectively induced reporter gene expression and protein production
levels, we expected a high induction effect for scFv-Fc production as
well. Addition of 4-OHT at concentrations of 0.01–0.1 μM resulted in
proliferation comparable with the control (Figure 3D). Evaluation of
scFv-Fc production reveals that scFv-Fc gene expression was induced with
0.05 μM or higher 4-OHT concentrations (Figure 3E), and the
concentration of scFv-Fc increased over the culture period. Following
the addition of 0.5 μM or 1.0 μM 4-OHT, the scFv-Fc concentration and
specific productivity reached 4.9 μg/mL and 24 pg
cell–1 day–1, respectively (Figure
3F), which is 5-fold and 4-fold higher compared with E2. We next
examined the combined effect of E2 and 4-OHT addition. With a fixed E2
concentration of 20 μM, we attempted induction of both the reporter gene
and scFv-Fc gene with various 4-OHT concentrations under conditions of
cell growth inhibition (Supplementary Figure S3A and S3D). Overall
induction levels were lower compared with conditions with 4-OHT alone
(Supplementary Figure S3B-C and S3E-F). Therefore, the addition of 4-OHT
alone to induce production is suitable for antibody production in this
artificial gene expression system.