Constitutively-active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells

Monoclonal antibodies (mAbs) are high value agents used for disease therapy (‘biologic drugs’) or as diagnostic tools which are widely used in the health care sector. They are generally manufactured in mammalian cells, in particular Chinese hamster ovary (CHO) cells cultured in defined media, and are harvested from the medium. Rheb is a small GTPase which, when bound to GTP, activates mechanistic target of rapamycin complex 1 (mTORC1), a protein kinase that drives anabolic processes including protein synthesis and ribosome biogenesis. Here we show that certain constitutively-active mutants of Rheb drive faster protein synthesis in CHO cells and increase the expression of proteins involved in the processing of secreted proteins in the endoplasmic reticulum, which expands in response to expression of Rheb mutants. Active Rheb mutants, in particular Rheb[T23M], drive increased cell number under serum-free conditions similar to those used in the biotechnology industry. Rheb[T23M] also enhances the expression of the reporter protein luciferase and, especially strongly, the secreted Gaussia luciferase. Moreover, Rheb[T23M] markedly (2-3 fold) enhances the amount of this luciferase and of a model immunoglobulin secreted into the medium. Our data clearly demonstrate that expressing Rheb[T23M] in CHO cells provides a simple approach to promoting their growth in defined medium and the production of secreted proteins of high commercial value

Soluble lymphotoxin is an important effector molecule in GVHD and GVL

Tumor necrosis factor (TNF) is a key cytokine in the effector phase of graft-versus-host disease (GVHD) after bone marrow transplantation, and TNF inhibitors have shown efficacy in clinical and experimental GVHD. TNF signals through the TNF receptors (TNFR), which also bind soluble lymphotoxin (LT alpha 3), a TNF family member with a previously unexamined role in GVHD pathogenesis. We have used preclinical models to investigate the role of LT in GVHD. We confirm that grafts deficient in LT alpha have an attenuated capacity to induce GVHD equal to that seen when grafts lack TNF. This is not associated with other defects in cytokine production or T-cell function, suggesting that LT alpha 3 exerts its pathogenic activity directly via TNFR signaling. We confirm that donor-derived LT alpha is required for graft-versus-leukemia (GVL) effects, with equal impairment in leukemic clearance seen in recipients of LT alpha- and TNF-deficient grafts. Further impairment in tumor clearance was seen using Tnf/Lta(-/-) donors, suggesting that these molecules play nonredundant roles in GVL. Importantly, donor TNF/LT alpha were only required for GVL where the recipient leukemia was susceptible to apoptosis via p55 TNFR signaling. These data suggest that antagonists neutralizing both TNF and LT alpha 3 may be effective for treatment of GVHD, particularly if residual leukemia lacks the p55 TNFR. (Blood. 2010;115:122-132