Modularity and application of synthetic cytokine receptors

细胞因子在包括自身免疫和癌症发展在内的大量病理生理过程中起关键作用。信号转导是通过生物开关实现的，所述生物开关由甘氨酸诱导的单体跨膜受体向多聚体跨膜受体的转化形成。细胞因子信号传导的结构和功能知识使得能够开发完全合成的细胞因子/细胞因子受体系统（SyCyR），其基于合成的同源和异源聚体GFP-和mCherry-细胞因子以及合适的合成纳米抗体受体。在这里，我们将使用我们的SyCyR系统解决合成细胞因子生物学中的三个目标：1。近年来，我们通过SyCyR系统对中枢肿瘤坏死因子受体超家族（TNFRSF）成员Fas和TNFR 1/2进行调控，诱导细胞凋亡和/或NF κ B B活化。为了阐明TNFRSF内的组合受体串扰，我们将自由地组合联合收割机Fas、TNFR 1/2、LT EGFR、CD 27、RANK和BAFFR的合成细胞因子受体。使用这些SyCyR，全面的单核苷酸变体（SNV）筛选将导致鉴定疾病相关的功能丧失突变体。为了简化SyCyR诱导的NF κ B B信号传导的分析，我们将基于NF κ B诱导的合成白细胞介素6基因在模型细胞系Ba/F3-gp 130中的表达，开发NF κ B-容易读出（NERO）系统。值得注意的是，通过合成IL-6激活gp 130受体随后诱导STAT 3/ERK依赖性细胞增殖。2.我们的初步数据显示，内部开发的抗体：抗独特型纳米抗体对能够实现合成的gp 130信号转导，这为我们的SyCyR的临床开发铺平了道路。与合成的GFP-和mCherry-细胞因子相反，批准的抗体帕利珠单抗和筛选的抗独特型纳米抗体是非免疫原性的。首先，我们将对筛选的抗体：抗独特型纳米抗体对进行效率优化。在产生优化的gp 130和Fas-SyCyR后，我们将评估体外和体内原代T和NK细胞的活化和凋亡潜力。3.我们将通过合成自主信号回路开发细胞电路。在最简单的形式中，我们将产生Ba/F3细胞，其携带用于自我维持增殖的两个合成遗传元件：合成gp 130受体基因和在最小STAT 3启动子控制下的合成细胞因子基因。细胞因子表达和受体活化的细胞自主正反馈回路将诱导细胞增殖。这一原理将被用于具有两个和三个通信Ba/F3细胞系的复杂网络。该项目被嵌入到自体嵌合抗原受体（CAR）T细胞疗法和治疗不确定性细胞系的最新发展中，并将补充这些干预措施，作为靶向调节增殖和凋亡的新工具。

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.