Modularity and application of synthetic cytokine receptors

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Cytokines are critically involved in a plethora of pathophysiological processes including autoimmunity and cancer development. Signal transduction is enabled by biological switches formed by cytokine-induced conversion of monomeric to multimeric transmembrane receptors. Structural and functional knowledge of cytokine signalling enabled the development of a fully synthetic cytokine/cytokine receptor system (SyCyR) which is based on synthetic homo- and heteromeric GFP- and mCherry-cytokines and suitable synthetic nanobody-receptors. Here, we will address three objectives in synthetic cytokine biology using our SyCyR system: 1. Recently, we have adopted the SyCyR system to central tumor necrosis factor receptor superfamily (TNFRSF) members Fas and TNFR1/2 to induced apoptosis and/or NFB activation. To elucidate the combinatorial receptor cross-talk within the TNFRSF we will freely combine synthetic cytokine receptors for Fas, TNFR1/2, LTR, CD27, RANK and BAFFR. Using these SyCyRs, a comprehensive single nucleotide variants (SNV) screen will result in the identification of disease-associated loss-of-function mutants. To ease the analysis of SyCyR-induced NFB signaling, we will develop a NFB-easy-read-out (NERO) system based on the NFB-induced expression of a synthetic Interleukin 6 gene in the model cell line Ba/F3-gp130. Of note, activation of gp130 receptors by synthetic IL-6 subsequently induced STAT3/ERK-dependent cellular proliferation. 2. Our preliminary data show that an in-house developed antibody:anti-idiotypic nanobody pair enables synthetic gp130 signal transduction, which pave the way for clinical development of our SyCyRs. In opposite to synthetic GFP- and mCherry-cytokines, the approved antibody Palivizumab and the screened anti-idiotypic nanobody are non-immunogenic. First of all, we will perform an efficiency optimization of the screened antibody:anti-idiotypic nanobody pair. After the generation of optimized gp130 and Fas-SyCyRs, we will assess the activation and apoptosis potential in primary T and NK cells in vitro and in vivo. 3. We will develop cellular circuits via synthetic autonomous signaling loops. In the simplest form, we will generate Ba/F3 cells which carry two synthetic genetic elements for self-sustained proliferation: a synthetic gp130 receptor gene and a synthetic cytokine gene under control of a minimal STAT3 promoter. The cell-autonomous positive feedback loop of cytokine expression and receptor activation will induce cellular proliferation. This principle will be adopted to complex networks with two and three communicating Ba/F3 cell lines. The project is embedded into recent developments of autologous chimeric antigen receptor (CAR) T cell therapy and theragnostic cell lines and will complement these interventions as a new tool for the target-oriented regulation of proliferation and apoptosis.