A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

Abstract

Cytokine receptors are randomly distributed on the cell surface membrane and are activated upon binding of their extracellular ligands to mediate downstream cellular activities. We hypothesized that pharmaceutical clustering of ligand-bound, activated receptors may lead to heretofore unrealized gain-of-function with therapeutically desirable properties. We here describe an engineered bifunctional cytokine borne of the fusion of Granulocyte Macrophage Colony Stimulating Factor (GMCSF) and Interleukin-9 (IL9) (hereafter GIFT9 fusokine) and demonstrate that it chaperones co-clustering of the functionally unrelated GMCSF receptor (GMCSFR) and IL9 receptor (IL9R) on cell surface of target cells. We demonstrate that GIFT9 treatment of MC/9 cells leads to transhyperphosphorylation of IL9R-associated STAT1 by GMCSFR-associated JAK2. We also show that IL9R-associated JAK1 and JAK3 augment phosphorylation of GMCSFR-linked STAT5. The functional relevance of these synergistic JAK/STAT transphosphorylation events translates to an increased mitogenic response by GMCSFR/IL9R-expressing primary marrow mast cells. The notion of inducing heterologous receptor clustering by engineered fusokines such as GIFT9 opens the door to a novel type of biopharmaceutical platform where designer fusokines modulate cell physiology through clustering of targeted receptor complexes.

A GMCSF and IL7 fusion cytokine leads to functional thymic‐dependent T‐cell regeneration in age‐associated immune deficiency

“…Furthermore, STAT5 activation has been shown to play a particularly important role in the expansion and preservation of memory T cells in the context of lymphocytic choriomeningitis virus (LCMV) infection; a result we have recapitulated using GIFT7 to augment STAT5 activity in a mCMV model here 46 GIFT7, while acting biochemically as a partial agonist, ultimately results in a hyperagonistic biological effect by driving an unopposed STAT5 signal. This is presumably due to atypical engagement and clustering of GMCSF and IL7 receptor complexes, an engineered feature of the GIFT fusions well described in our previously published work 24 , 48 . Moreover, our genome‐wide RNA sequence analysis of cytokine‐treated T cells confirms that GIFT7 induces a differential program of gene expression than IL7.…”

“…The notion therefore arises that creating a fusion cytokine borne of the physical linkage of two unrelated cytokines—a ‘fusokine’—will not only possess pharmaceutical properties ascribable to each parental domain, but may also acquire unheralded additive immune features. We have previously demonstrated the novel gain‐of‐function properties of granulocyte‐macrophage colony stimulating factor (GMCSF)‐based common γ c chain fusokines, 17 , 18 including: GIFT2, GIFT15, 19 , 20 , 21 GIFT21, 22 , 23 GIFT9 24 and GIFT4 25 . In this study, we show that engineered fusion GIFT7 delivers hyperagonistic signaling to IL7Rα/γ c ; the unique biological consequence of such is the induction of SPCD8 and CD44 + CD25 − DN1 expansion in thymocyte culture.…”

Engineered Fusokine GIFT4 Licenses the Ability of B Cells to Trigger a Tumoricidal T-cell Response

“…GIFT4 were profiled by Western blot (WB) with anti-GM-CSF or anti-IL-4 antibodies (R&D systems). Murine B-cells (10 6 cells/ml) treated with GIFT4, GM-CSF and IL-4 with or without JAK inhibitors were lysed with buffer as described (21). STAT phosphorylation in B-cells was determined by WB with anti-pSTAT1 (Tyr701, D4A7), anti-pSTAT3 (Tyr705, D3A7), anti-pSTAT5 (Tyr694, D47E7), anti-pSTAT6 (Tyr641, C11A12), or anti-STAT antibodies (Cell Signaling).…”

“…STAT phosphorylation in B-cells was determined by WB with anti-pSTAT1 (Tyr701, D4A7), anti-pSTAT3 (Tyr705, D3A7), anti-pSTAT5 (Tyr694, D47E7), anti-pSTAT6 (Tyr641, C11A12), or anti-STAT antibodies (Cell Signaling). For immunoprecipitation of GMCSFRβ by common γc receptor after GIFT4 or GM-CSF and IL-4 stimulation, the B-cells were lysed with buffer as described (21). One mg of protein per sample was incubated with 2 mg of anti-common γc or anti-GMCSFR antibodies, or IgG isotype control (Santa Cruz Biotechnology, CA) overnight at 4°C.…”

Maltose-Binding Protein Fusion Allows for High Level Bacterial Expression and Purification of Bioactive Mammalian Cytokine Derivatives

“…These GIFTs (GM-CSF and Interleukin Fusion Transgenes) typically lead to STAT hyperphosphorylation in responding lymphomyeloid cells [3], [7], [16]–[18]. The best-characterized member of this family is GIFT15, a fusion of GM-CSF and IL-15 [15], [16], [18].…”