Rheb[T23M] and [E40K] drive increased mAb
secretion under conditions similar to those used in industry
While GLuc-CHO cells act as a good reporter system for interrogating the
effect of Rheb mutants on protein secretion, there are several
limitations that make these cells unsuitable for use as an industrial
research tool. First, to be useful in an industrial context, CHO cells
must be grown in suspension and in chemically defined, serum-free media.
Second, while GLuc may be a secreted protein, it is not a model
biotherapeutic protein and lacks glycosylation. To address these issues,
we acquired EXPI-CHO™ cells and modified them to stably express
Rheb[WT], [T23M], [Y35N] or [E40K] (Supplementary Fig.
S3). We then performed SDS-PAGE western blot analysis on lysates
harvested from these cells to confirm the hyperactivity of mTORC1
signalling and ER stress phenotype observed in HEK293 and GLuc-CHO cells
was present in these cells. Consistent with GLuc-CHO cells, in EXPI-CHO
cells stably expressing Rheb mutants or Rheb[WT], [T23M] and
[E40K] drove increased phosphorylation of rpS6 at Ser240/244,
indicative of activated mTORC1, as well as increased phosphorylation of
4E-BP1 at Thr37 and Thr46 suggesting that Rheb[T23M] and [E40K]
drive hyperactive mTORC1 signalling in these cells (Fig. 5a).
In addition, Rheb[T23M] and [E40K] each drove increased
expression of the ATF4 arm of the UPR as well as of ER stress markers
such as BiP (Fig. 5b). Interestingly, we were unable to detect any
differences in protein synthesis via SUnSET assay (Fig. 5c). This is
likely because these cells stably express Rheb mutants and therefore
increased protein synthesis likely results in increased total protein
amounts. As the amounts of cell lysate loaded onto the immunoblot gels
are normalised to total protein, such normalisation will effectively
‘eliminate’ any effect on the rate of protein synthesis. Cells
expressing Rheb[T23M] or [E40K] also showed significantly faster
cell proliferation as assessed by counting cells on a haemocytometer
every 24 h for 7 days (Fig. 5d) as well as in a BrdU assay for DNA
synthesis (Fig. 5e)
To determine the effect of Rheb mutants on mAb production and secretion
in these cells, we transfected cells with a vector encoding the heavy
and light chains of rabbit IgG1 and grew the cells in serum-free,
chemically-defined medium for 10 days with the cells being fed on day 2.
This more closely mimics the type of conditions the biopharmaceutical
industry would use when using a transient expression approach and CHO
cells to produce a recombinant IgG. We found that after 10 days of
growth, Rheb[T23M] and [E40K] promoted a 2-3 fold increase in
IgG1 yield (Fig. 5f). Testing accumulated antibody in the growth medium
every 24 h over the course of the experiment revealed that duration of
antibody secretion was similar in all cases, but that the rate of
release of secreted IgG1 was markedly increased in cells expressing
Rheb[T23M] or [E40K]. This suggests that Rheb[T23M] and
[E40K] promote both increased production and, very importantly,
secretion of a recombinant antibody.