A novel strategy to produce high level and high purity of bioactive IL15 fusion proteins from mammalian cells.

IL15, a member of the common γ chain receptor (γc) cytokine family, is gaining attention in recent years as one of the most promising anti-tumor agents. IL15 regulates T cell activation and proliferation, promotes the survival of CD8+ CD44hi memory T cells and is also essential for NK cell expansion and development. Despite the attraction of developing IL15 as an anti-cancer agent, production of recombinant IL15 has proven to be difficult due to the stringent control of IL15 expression at the transcriptional, translational and the post-translational levels. Furthermore, the bioactivity of IL15 fused to an extra functional domain that is isolated from mammalian cells is generally inferior to recombinant IL15 produced by E. coli. In this study, we report that Lysine 86 in IL15 is responsible for the instability in mammalian cells when its C-terminus is fused to the albumin binding scFv (IL15-A10m3). We demonstrate that K86A or K86R mutants increased the expression of the fusion protein from HEK293 cells. When the wild type IL15 is used for the fusion, no recombinant IL15 fusion was detected in the culture media. Additionally, we determined that the residue 112 in IL15 is critical for the bioactivity of IL15-A10m3. Examination of single and double mutants provides a better understanding of how IL15 engages with its receptor complex to achieve full signaling capacity. The results of our experiments were successfully applied to scale up production to levels up to 50 mg/L and >10 mg/L of >95% pure monomeric recombinant fusion proteins after a 2-step purification from culture media. More importantly, the recombinant fusion protein produced is fully active in stimulating T cell proliferation, when compared to the recombinant wild type IL15.

Hemogenic endothelial cell specification requires c-Kit, Notch signaling, and p27-mediated cell-cycle control.

Delineating the mechanism or mechanisms that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic specification, as well as cell-cycle control, of endothelium during embryogenesis. Herein, we define the molecular signals downstream of RA that regulate hemogenic endothelial cell development and demonstrate that cell-cycle control is required for this process. We found that re-expression of c-Kit in RA-deficient (Raldh2(-/-)) primordial endothelium induced Notch signaling and p27 expression, which restored cell-cycle control and rescued hemogenic endothelial cell specification and function. Re-expression of p27 in RA-deficient and Notch-inactivated primordial endothelial cells was sufficient to correct their defects in cell-cycle regulation and hemogenic endothelial cell development. Thus, RA regulation of hemogenic endothelial cell specification requires c-Kit, notch signaling, and p27-mediated cell-cycle control.