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.