Improved antibody production in Chinese hamster ovary cells by ATF4 overexpression

Abstract

To improve antibody production in Chinese hamster ovary (CHO) cells, the humanized antibody-producing CHO DP-12-SF cell line was transfected with the gene encoding activating transcription factor 4 (ATF4), a central factor in the unfolded protein response. Overexpression of ATF4 significantly enhanced the production of antibody in the CHO DP-12-SF cell line. The specific IgG production rate of in the ATF4-overexpressing CHO-ATF4-16 cells was approximately 2.4 times that of the parental host cell line. Clone CHO-ATF4-16 did not show any change in growth rate compared with the parental cells or mock-transfected CHO-DP12-SF cells. The expression levels of mRNAs encoding both the antibody heavy and light chains in the CHO-ATF4-16 clone were analyzed. This analysis showed that ATF4 overexpression improved the total production and specific production rate of antibody without affecting the mRNA transcription level. These results indicate that ATF4 overexpression is a promising method for improving recombinant IgG production in CHO cells.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…d) Constitutive (stable) co-expression of stably integrated genes encoding r-protein and effector genes in clonally derived cell lines. Examples: (Dreesen and Fussenegger, 2011;Haredy et al, 2013;Tastanova et al, 2015). e) Features regarded as advantages are highlighted in boldface type.…”

Use of a protein engineering strategy to overcome limitations in the production of “Difficult to Express” recombinant proteins

“…Attempts to improve the overall capacity of cells to express greater amounts of secreted recombinant proteins have involved host cell engineering targeted at endoplasmic reticulum (ER) chaperones (Chung et al, ; Dorner et al, ; Johari et al, ), protein disulphide isomerases (Borth et al, ; Mohan and Lee, ) as well as unfolded protein response mediators (Becker et al, ; Cain et al, ; Haredy et al, ; KU et al, , ; Omasa et al, ; Tigges and Fussenegger, ). Other sites of engineering have included overexpression of machinery related to the import of nascent polypeptide chains into the ER (Le Fourn et al, ) and vesicular transport components (Peng and Fussenegger, ; Peng et al, , ).…”

Transcriptional and post-transcriptional targeting for enhanced transient gene expression in CHO cells

“…The UPR is a coordinated response to restore ER homeostasis and acts by inhibiting protein synthesis and up-regulating the capacity of cells to fold proteins. UPR mechanisms can also lead to active degradation of misfolded proteins (Haredy et al 2013;Hussain et al 2014).…”

“…Many UPR genes enhance protein expression in stable cell lines and some genes have a positive impact on transient expression albeit under low expression conditions (Tigges and Fussenegger 2006;Ku et al 2008;Ohya et al 2008;Nishimiya et al 2013;Cain et al 2013;Haredy et al 2013). The goal of this study was to improve transient protein expression in CHO cells without resorting to host cell line or plasmid vector engineering.…”

“…We have previously shown that DMA addition can lead to improved expression Rajendra et al 2014). In this study, we identified four genes in UPR that may improve expression, namely XBP1S, ATF4, CHOP and GRP78 (Tigges and Fussenegger 2006;Ohya et al 2008;Nishimiya et al 2013;Cain et al 2013;Haredy et al 2013) and evaluated them in combination with DMA for expression of multiple proteins including human and mouse monoclonal antibodies, human bispecific antibodies and Fc-fusion proteins.…”