ID: 162: Split2-cytokine protein-ligation: Non-toxic and background-free induction of Interleukin (IL-)6 Trans-signaling by split-intein mediated protein ligation of biologically inactive split-IL-6 in vivo

Abstract

Interleukin (IL-)6 trans-signaling via the soluble IL-6 receptor (sIL-6R) activates all cells of the body, whereas Interleukin (IL-)6 classic signaling via the membrane-bound IL-6R is limited mainly to hepatocytes and lymphocytes. Accordingly, the phenotype of IL-6:sIL-6R expressing transgenic mice is much severe as compared to single IL-6 transgenic mice. Due to occurrence of free IL-6, classic signaling is also activated during simultaneous expression of IL-6 and sIL-6R complicating the background-free analysis of IL-6 trans-signaling in vivo. IL-6 is physically trapped in Hyper-IL-6, the fusion protein of IL-6 and the sIL-6R, and specifically induces only IL-6 trans-signaling. Interestingly, Hyper-IL-6 transgenic mice have not been described to date, likely due to toxicity issues which prevents establishment of Hyper-IL-6 transgenic mice. Split-intein mediated protein ligation enables the production of toxic proteins. Here, we efficiently adapted this technology to protein ligation of the designer cytokine Hyper-IL-6 (I-H-IL-6) within the cellular secretory compartment using gp41-1 and IMPDH split-inteins. The presence of non-ligated IL-6 did, however, still enable induction of IL-6 classic signaling. To prevent the production of non-ligated biologically active IL-6, biologically inactive IL-6 split-cytokines were developed using structure-guided modeling. Biologically inactive split-IL-6 variants were combined with split-intein mediated protein ligation, resulting in background-free generation of Hyper-IL-6 (Split2-H-IL-6) in vitro and in vivo. The split2-cytokine protein ligation was applied for a second Hyper-cytokine and therefore likely represents a general strategy to generate highly potent and background-free composite Hyper-cytokines in vitro and in vivo.