Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands

Abstract

Cytokine signaling is transmitted by cell surface receptors which act as natural biological switches to control cellular functions such as immune reactions. Recently, we have designed synthetic cytokine receptors (SyCyRs) consisting of green fluorescent protein (GFP)-and mCherry-nanobodies fused to the transmembrane and intracellular domains of cytokine receptors. Following stimulation with homo-and heterodimeric GFP-mCherry fusion proteins, the resulting receptors phenocopied signaling induced by physiologically occurring cytokines. GFP and mCherry fusion proteins were produced in E. coli or CHO-K1 cells, but the overall yield and stability was low. Therefore, we applied two alternative multimerization strategies and achieved immunoglobulin Fc-mediated dimeric and coiled-coil GCN4pII-mediated trimeric assemblies. GFP-and/or mCherry-Fc homodimers activated synthetic gp130 cytokine receptors, which naturally respond to Interleukin 6 family cytokines. Activation of these synthetic gp130 receptors resulted in STAT3 and ERK phosphorylation and subsequent proliferation of Ba/F3-gp130 cells. Half-maximal effective concentrations (EC 50) of 8.1 ng/ml and 0.64 ng/ml were determined for dimeric GFP-Fc and mCherry-Fc, respectively. This is well within the expected EC 50 range of the native cytokines. Moreover, we generated tetrameric and hexameric GFP-mCherry-Fc fusion proteins, which were also biologically active. This highlighted the importance of close juxtaposition of two cytokine receptors for efficient receptor activation. Finally, we used a trimeric GCN4pII motif to generate homo-trimeric GFP and mCherry complexes. These synthetic cytokines showed improved EC 50 values (GFP 3 : 0.58 ng/ml; mCherrry 3 : 0.37 ng/ml), over dimeric Fc fused variants. In conclusion, we successfully generated highly effective and stable multimeric synthetic cytokine receptor ligands for activation of synthetic cytokine receptors.

Pro- and anti-apoptotic fate decisions induced by di- and trimeric synthetic cytokine receptors

“…Cell surface expression of G VHH TNFR1, C VHH TNFR1, and C VHH TNFR2 was verified by flow cytometry ( Figure S1 B). Recently, we described Fc-tagged GFP and mCherry fusion proteins as alternative synthetic cytokine ligands, which exhibit increased stability and enable efficient protein purification via Protein A affinity chromatography ( Mossner et al., 2020b ) ( Figure S2 A). In order to assemble receptors in higher-than-dimeric oligomers, trimeric GFP 3 and mCherry 3 ( Mossner et al., 2020b ), and hexameric Fc-tagged fusion proteins of 3xGFP (3xGFP-Fc) or 3xmCherry (3xmCherry-Fc) were generated ( Figures S2 A andS2B).…”

“…The SyCyR system belongs to a new class of fully synthetic cytokine systems combining synthetic ligands and synthetic receptors ( Scheller et al., 2019 ). Until now, the SyCyR system was adopted to classical dimeric cytokine receptor families for IL-6, IL-23, or IL-22 ( Engelowski et al., 2018 ; Mossner et al., 2020a , 2020b ). To date, synthetic systems such as chimeric receptors or synthekines were limited to the analysis of dimeric receptor combinations ( Floss et al., 2017 ; Moraga et al., 2017 ).…”

“…Simultaneous functional integration of pro-and anti-apoptotic synthetic cytokine receptors for IL-6 and Fas-signaling Finally, we combined the apoptotic C VHH Fas with the recently described anti-apoptotic/proliferative synthetic cytokine receptor G VHH gp130 which phenocopied IL-6 signaling (Mossner et al, 2020b) (Figure S9A) to enable the switch from gp130-mediated proliferation into Fas-mediated apoptosis (Figure 4A). Cell surface expression of G VHH gp130 and C VHH Fas in Ba/F3/gp130 cells was shown by flow cytometry (Figure S9B) and proliferation of Ba/F3/G VHH gp130/C VHH Fas was specifically induced by dimeric GFP-Fc (Figure 4B).…”

“…SyCyRs use nanobodies as extracellular ligand binding domain ( Fridy et al., 2014 ; Rothbauer et al., 2008 ), fused to the transmembrane and intracellular domain of the receptor of interest ( Wesolowski et al., 2009 ). Nanobodies serve as sensors for homo- or heterodimeric ligands, e.g., GFP/mCherry fusion proteins ( Mossner et al., 2020b ). Binding of the synthetic ligand to its synthetic receptor then activates the SyCyRs and the endogenous signaling pathway of the intracellular receptor of interest is specifically activated without any background activation ( Engelowski et al., 2018 ; Mossner et al., 2020a ).…”

“…Binding of the synthetic ligand to its synthetic receptor then activates the SyCyRs and the endogenous signaling pathway of the intracellular receptor of interest is specifically activated without any background activation ( Engelowski et al., 2018 ; Mossner et al., 2020a ). We hypothesized that this modular ligand assembly should also allow homo-/heterotrimeric receptor assemblies ( Engelowski et al., 2018 ; Mossner et al., 2020a , 2020b ). The tumor necrosis factor superfamily (TNFSF) consists of 19 ligands and 29 related receptors ( Dostert et al., 2019 ; Vanamee and Faustman, 2018b ).…”