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. 2023 Jun 25;12(13):1716. doi: 10.3390/cells12131716

Figure 1.

Figure 1

Optimization of BiKE production. (A) Schematic drawing representing BiKE mediated engagement of NK cells and IL13Rα2-positive glioma cells. The construct consists of an anti-CD16 single-domain antibody (sdCD16), interleukin-15 flanked by flexible protein linkers (IL15), an anti-IL13Rα2 single-chain fragment antibody (scFv IL13Rα2). (B) Optimization of BiKE production regarding the production temperature was conducted at 37 °C and 32 °C. Visualization of protein yields was conducted using Western blot, and binding to IL13Rα2 was demonstrated after production at 32 °C. (C) BiKE optimization for serum conditions was conducted in DMEM media with 10% FBS (10% FBS), DMEM with 1% FBS (1% FBS), and OptiMEM media. BiKE demonstrated the greatest production and maximal binding capacity to its target using the 10% FBS condition. (D) Schematic overview of BiKE production. Production can be efficiently completed in 6-7 days. First, BiKE cDNA was inserted into a pLVX-IRES-zsGreen1 lentiviral vector. A stable 293 T/17 cell line secreting BiKE was generated through lentivirus transfection. 293 T/17 cells producing cells were incubated overnight at 37 °C and transferred to 32 °C for another 2 to 3 days. The supernatant containing BiKE was collected, incubated with cOmplete His-Tag resin overnight at 4 °C, and isolated through His-Tag column purification. Purified BiKE was then dialyzed overnight with PBS serum, concentrated, and stored at −80 °C for future use. ** = p < 0.01, and *** = p < 0.001.