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

Sofie Mossner,Jürgen Scheller,Jens M Moll,Saskia Triller,Udo Conrad,Hoang T Phan,Marek Cebecauer

doi:10.1371/journal.pone.0230804

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 (EC50) 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 EC50 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 EC50 values (GFP3: 0.58 ng/ml; mCherrry3: 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.

Highlights

Cytokines control immune responses but are involved in homeostatic processes such as development, differentiation, growth and regeneration
We have designed synthetic cytokine receptors (SyCyRs) which phenocopy IL-6 and IL-23 signaling via homodimeric gp130 and heterodimeric IL-23R/IL-12Rbeta1 receptors [2]
The nanobodies serve as extracellular sensors for homo- and heteromeric green fluorescent protein (GFP)-mCherry fusion proteins which induce receptor dimerization

Summary

Introduction

Cytokines control immune responses but are involved in homeostatic processes such as development, differentiation, growth and regeneration. Signal transduction of cytokines is executed by natural biological switches which among many other functions control immune related processes [1]. Cytokines switch transmembrane receptors from the off-state into the on-state via receptor dimerization or multimerization. We have designed synthetic cytokine receptors (SyCyRs) which phenocopy IL-6 and IL-23 signaling via homodimeric gp130 and heterodimeric IL-23R/IL-12Rbeta receptors [2]. SyCyRs incorporate nanobodies recognizing GFP or mCherry [3, 4] fused to transmembrane and intracellular receptor domains. The nanobodies serve as extracellular sensors for homo- and heteromeric GFP-mCherry fusion proteins which induce receptor dimerization. Synthetic cytokine receptors might become important tools for immunotherapeutic applications [6] with Chimeric Antigen Receptor (CAR) T-cell therapy being the first example which has been approved as gene therapy for the treatment of severe cases of acute lymphatic leukemia [7]

Methods

Results

Conclusion