mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells

Abstract

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.

mTORC1 induces eukaryotic translation initiation factor 4E interaction with TOS-S6 kinase 1 and its activation

“…Herein, we identify phosphorylated eIF4E as a mediator that transduces mTORC1 signals through its interaction with S6K1-TOS motif. The findings report eIF4E as an important determinant of cellular response to rapamycin in conformity with a long-standing contention that associates stoichiometric abundance of eIF4E with rapamycin resistance 23,24 . Our earlier reports that identify eIF4E as a bonafide mTORC1 substrate 17 and factor responsible to propagate cellular response to rapamycin, serves to reinforce its prospect as an intermediate for S6K1 activation.…”

“…The overexpression of eIF4E and S6K1 corresponded with a concomitant increase in phosphorylation at Ser 209 and Thr 412 respectively, both in cancer tissues and cell lines ( Fig 1C, D). Based on these observations and some earlier studies that have aimed to establish a relationship between eIF4E stoichiometry and mTORC1 response 23,24 , we explored whether eIF4E abundance/phosphorylation relates with the activation status of mTORC1. Accordingly, we transfected myc tagged eIF4E in HEK293 and NIH3T3 cells to assess the impact on the state of S6K1 activity which is normally used as a functional readout for mTORC1 activation 25 .…”

Low concentrations of cadmium chloride promotes protein translation and improve cell line productivity

“…Because eIF4E is the rate‐limiting translation factor for protein synthesis and its availability for protein translation is carefully controlled by interaction with 4EBP1, the expression level of these proteins was investigated in high and low yield clones. Remarkably, the ratio of eIF4E to 4EBP1 was higher in the high producers, supporting the hypothesis that translation is the major bottleneck for productivity (Josse, Xie, Proud, & Smales, ). The main factor that regulates the interaction of eIF4E and 4EBP1 is the mechanistic target of rapamycin (mTOR), a serine/threonine kinase central for integrating growth factor signaling and nutrient availability for anabolic processes (Ben‐Sahra & Manning, ).…”

Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis

“…C). At this point in culture (day 4), higher levels of translation are required to support efficient cellular growth, division and normal cellular processes . On day 4 of batch culture, cell line 3 had the highest P : M ratio of approximately 1.2 whilst the other two recombinant cell lines and the host had approximately equal amounts of polysomes and sub‐polysomes with a ratio of approximately 1 (Fig.…”