Investigating the Restraints Upon Recombinant Protein Production in Mammalian Cells via the Manipulation of eEF2, eEF2K and mTOR

Since their first clinical application in the 1980’s recombinant proteins have become an

increasingly larger section of the drug market, growing into a multi-billion dollar global market.

The pursuit of improving the design, production, and application of recombinant proteins for

biotherapeutic uses is a key driver in industry and academia. The majority of the recombinant

biotherapeutic proteins used in the clinic are produced in mammalian cell expression systems

due to their capability to undertake human-like complex post translational modifications. The

currently ‘gold standard’ mammalian cell expression system for the production of recombinant

proteins is the Chinese hamster ovary (CHO) cell line. However, even with advances in

mammalian cell expression technology, there is still a high cost and a long development period

required for a recombinant protein therapeutic to go from design to market. As such,

bottlenecks include the time taken for mammalian cells to grow and divide with slow doubling

times compared to microbial systems and limited capacity to synthesise and secrete

recombinant proteins. One of the cellular processes that underpins both cell growth and

recombinant protein production is the translation of mRNA. Translation consists of three

distinct steps: initiation, elongation and termination. One major cell signalling pathway that is

considered a master regulator of both initiation and elongation of translation is mTOR, also

involved in regulating ribosome biogenesis and cell proliferation. During the process of

polypeptide elongation (mRNA translation), elongation factor 2 (eEF2) is a key control point

that regulates protein synthesis via its de/phosphorylation. Phosphorylation of eEF2 results in

its inactivation; slowing or halting elongation resulting in the attenuating of protein synthesis.

This study set out to establish if manipulation of the mTOR signalling pathway and/or

manipulation of phosphorylation of elongation factor 2 and the kinase that inactivates eEF2,

eEF2K, in CHO cells impacts upon CHO cell growth and recombinant protein production yields.

Transient expression of wild type and a Thr56Ala eEF2 mutant in CHOK1 cells affected the

short term (24-48 hour) phosphorylation of eEF2 but did not appear to have an effect upon

intracellular recombinant protein production and cellular growth in culture over 96 hours.

Stable over expression of the wild type eEF2 construct in CHO cells resulted in a 2-fold increase

in expression of eEF2 and a decrease in phosphorylation of eEF2 at the protein level; but the

there was no change in the levels of total eEF2 mRNA expression. Stable expression of the

Thr56Ala and Thr56Glu eEF2 mutants had a greater effect upon eEF2 expression resulting in a

3-5 fold increase in total eEF2 expression, however the phosphorylation of eEF2 was almost

unchanged in an Ala56 eEF2 cell line, whereas it was reduced in the Glu56 eEF2 mutant cell 20

line. Growth of the CHO cells lines expressing the eEF2 mutants show that over expression of

any of the eEF2 mutants resulted in a change in growth, but the Ala56 eEF2 mutant showed

the largest change in cellular growth. Short term transient expression of recombinant firefly

luciferase in the stable eEF2 cell lines revealed that the Thr56Ala mutant greatly increases the

CHO cells total recombinant protein production. Further, mutation of eEF2 to Ala56 or Glu56

had little effect upon the mis-incorporation of amino acids during translation. The transient

knockdown of eEF2K was achieved and this was shown to prevent eEF2 phosphorylation.

However, CHO cells do not appear to tolerate the knockdown of eEF2K stably or at only very

low levels. This suggests that a sustained, high level of eEF2K knockdown is lethal to the cell;

which would result in the loss of eEF2 regulation for an extended period of time. Transient

expression of the eEF2K shRNA into CHOK1D6 cells stably expressing firefly luciferase, a nonsecreted

protein, resulted in a 5-fold increase in luciferase expression showing that knockdown

of eEF2K increased the short term productivity of these cells. Interestingly, the study of CHO

cell lines with varying recombinant monoclonal antibody protein production capacities

revealed that levels of total and phosphorylated eEF2 did not appear to change in correlation

with the mAb titre. These data suggest that eEF2 activity is tightly regulated across cell lines

and is not directly related to recombinant protein secretion in these industrially used CHO cell

lines The addition of 2 µM of PMA, an activator of mTOR signalling, increased antibody

production in a low producer cell line but had no effect upon the antibody production from a

high producing cell line. Together the data presented here shows that manipulation of eEF2

and eEF2K activity can enhance cellular growth and recombinant protein production from CHO

cells. As such, new engineering approaches that allow the manipulation of both elongation and

polypeptide synthesis combined with the secretory capacity of the cell are likely to yie ld new

CHO host cells with more predictable recombinant protein capacity and further advance our

understanding of the role of mRNA translation in controlling cell proliferation, and both global

and recombinant protein synthesis.