Wittich Christoph, Ettich Julia, Hertell Marcel, Ghosh Roy Biswadeep, Xu Haifeng C, Floss Doreen M, Lang Philipp A, Scheller Jürgen
Institute of Biochemistry and Molecular Biology II, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany.
Institue of Molecular Medicine II, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany.
J Biol Chem. 2025 Mar;301(3):108205. doi: 10.1016/j.jbc.2025.108205. Epub 2025 Jan 17.
Recently, we phenocopied interleukin (IL-)6 signaling using the dimerized single-chain variable fragment (scFv) derived from the respiratory syncytial virus IgG1-antibody palivizumab (PLHFc) to activate a palivizumab antiidiotypic nanobody (AIP)-gp130 receptor fusion protein. Palivizumab was unable to activate STAT3 signaling, so we aimed to create a similar ligand capable of triggering this pathway. Here, we created three variants of the ligand called PLH0Fc, PLH4Fc and PLH8Fc by shortening the spacer region connecting PLH and Fc from 23 amino acids in PLHFc to 0 amino acids or expanding it by rigid linkers of four or eight alpha helical loops, respectively. The rigid-linker ligands had completely altered cellular activation patterns via AIPgp130 fusion proteins. Deleting the extracellular stalk region between transmembrane and AIP in the synthetic receptors AIP2gp130Δstalk and AIP3gp130Δstalk to increase rigidity and enhanced the biological activity of the short spacer PFc ligands. Since scFv constructs are less stable than antibodies and have not been Food and Drug Administration approved, we looked for different antibody backbones. Transferring palivizumab's variable region to a more rigid and hence more agonistic IgG2 backbone (P) maintained affinity while improving agonistic properties activating cells expressing AIP2gp130Δstalk and AIP3gp130Δstalk but not their full-length counterparts. Furthermore, we engineered a tetravalent palivizumab variant (PP) capable of inducing higher-order receptor clustering, activating Fas-induced apoptosis. In summary, we engineered a fully-synthetic cytokine/cytokine receptor pair based on the IgG2-variant of palivizumab and the AIPgp130Δstalk variants opening avenues for therapeutic applications using nonphysiological targets in immunotherapy.
最近，我们利用源自呼吸道合胞病毒IgG1抗体帕利珠单抗（PLHFc）的二聚化单链可变片段（scFv）模拟白细胞介素（IL-）6信号传导，以激活帕利珠单抗抗独特型纳米抗体（AIP）-gp130受体融合蛋白。帕利珠单抗无法激活STAT3信号传导，因此我们旨在创建一种能够触发该信号通路的类似配体。在此，我们通过将连接PLH和Fc的间隔区从PLHFc中的23个氨基酸缩短至0个氨基酸，或分别通过四个或八个α螺旋环的刚性接头将其扩展，创建了三种配体变体，称为PLH0Fc、PLH4Fc和PLH8Fc。刚性接头配体通过AIPgp130融合蛋白完全改变了细胞激活模式。在合成受体AIP2gp130Δstalk和AIP3gp130Δstalk中删除跨膜和AIP之间的细胞外柄区以增加刚性，并增强了短间隔区PFc配体的生物活性。由于scFv构建体比抗体稳定性差且未获得美国食品药品监督管理局批准，我们寻找了不同的抗体骨架。将帕利珠单抗的可变区转移到更刚性从而更具激动性的IgG2骨架（P）上，在保持亲和力的同时改善了激活表达AIP2gp130Δstalk和AIP3gp130Δstalk的细胞而非其全长对应物的激动特性。此外，我们设计了一种能够诱导高阶受体聚集、激活Fas诱导凋亡的四价帕利珠单抗变体（PP）。总之，我们基于帕利珠单抗的IgG2变体和AIPgp130Δstalk变体设计了一对完全合成的细胞因子/细胞因子受体，为免疫疗法中使用非生理性靶点的治疗应用开辟了道路。
