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.