Engineering CHO cells with an oncogenic KIT improves cells growth, resilience to stress, and productivity

Abstract

An optimized biomanufacturing process in mammalian cells is contingent on the ability of the producing cells to reach high viable cell densities. In addition, at the peak of growth, cells need to continue producing the biological entity at a consistent quality. Thus, engineering cells with robust growth performance and resilience to variable stress conditions is highly desirable. The tyrosine kinase receptor, KIT, plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity, and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form. Here, we investigated the effect of oncogenic D816V KIT on the performance of CHO-K1 cells under conventional tissue culture growth settings and when adapted, to shaking conditions. The onco-KIT promoted global protein synthesis, elevated the expression of a secretable transgene, enhanced proliferation, and improved the overall titers of a model glycoprotein. Moreover, the expression of the onco-KIT endowed the cells with a remarkable resistance to various stress conditions. Our data suggest that the introduction of onco-KIT can serve as a strategy for improving glycoprotein biomanufacturing. Biotechnol. Bioeng. 2017;114: 2560-2570. © 2017 Wiley Periodicals, Inc.

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

“…To assess whether cadmium can also promote the expression of a secreted biological molecule in CHO cells we generated CHO‐K1 cells that stably express GFP fused to the heavy chain of human IgG1 (GFP‐Fc). This molecule is secreted as a dimer and its expression can be estimated by GFP fluorescence, intracellularly and in the media (Mahameed & Tirosh, ). Treatment of CHO‐K1 in full media with 5 µM cadmium resulted in a robust increase in GFP‐Fc intracellular levels as measured by flow cytometry (Figure a).…”

“…(GFP-Fc). This molecule is secreted as a dimer and its expression can be estimated by GFP fluorescence, intracellularly and in the media (Mahameed & Tirosh, 2017). Treatment of CHO-K1 in full media with 5 µM cadmium resulted in a robust increase in GFP-Fc intracellular levels as measured by flow cytometry (Figure 3a).…”

“…With the understanding that mTORC1 activity may promote cell line productivity, several cell engineering strategies have been used to induce mTORC1 in producing cells. This includes the overexpression of mTOR protein itself (Dreesen & Fussenegger, 2011), overexpression of its positive regulators (Mahameed & Tirosh, 2017), and the removal of its inhibitors (McVey et al, 2016). This usually resulted in improved specific productivity.…”

Towards Enhancing Therapeutic Glycoprotein Bioproduction: Interventions in the PI3K/AKT/mTOR Pathway

“…KIT, also known as CD117, is a type-III tyrosine kinase that may possess the D816V mutation found in many cancer types (Longley et al, 2001). Since this oncogenic version of KIT is constitutively active in a ligand-independent manner, it has been suggested that overexpression of this mutant in CHO producing cells may serve as a simple genetic strategy to boost their production capacity (Mahameed and Tirosh, 2017). A significant increase in proliferation rates was observed, when D816V-KIT was stably expressed in producing CHO cells, and the cultured cells were resilient to cultivation-encountered stressors (such ER stress, starvation and hypoxia).…”

“…It has been shown that the D816V mutant resulted in an almost two-fold increase in harvested titers during a batch assay. This oncogenic version of KIT promotes the overall protein translation efficiency through elevation in S6 and 4EBP1 phosphorylation levels, presumably through mTORC1 activation (Mahameed and Tirosh, 2017).…”