Pudewell Silke, Heuser Julia, Dorogobed Inna, Lipinski Britta, Tran Thi Hong Hue, Metzenmacher Pia, Kunze Richard, Geyer Felix, Zielonka Stefan, Floss Doreen M, Kolmar Harald, Moll Jens M, Scheller Jürgen
Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany.
Applied Biochemistry, Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.
Front Immunol. 2025 Aug 15;16:1613004. doi: 10.3389/fimmu.2025.1613004. eCollection 2025.
Although Interleukin (IL)-6-type cytokine signaling is critical for maintaining the body's homeostasis, aberrant signaling has been observed in numerous diseases including autoimmunity and cancer. Currently, all approved biologics that inhibit IL-6-type cytokines specifically target the key pro-inflammatory mediator IL-6 or its receptor (IL6R). Historically, direct inhibition of glycoprotein 130 (gp130)-the shared transmembrane receptor for IL-6-type cytokines-was avoided due to concerns that broad suppression might cause more harm than benefit. However, this view is being reconsidered in light of the clinical success of Janus kinase (JAK) inhibitors, which broadly disrupt cytokine signaling, including pathways mediated by gp130.
Here we developed four single domain antibodies (sdAb), consisting out of a camelid-derived nanobody and a human Fc-fragment, and characterized them by direct protein interaction analysis, epitope binding, epitope binning, as well as inhibition of cytokine-induced stimulation and proliferation of appropriate Ba/F3 cell lines and trans-migration in HT-29 cells.
The four sdAb-Fc constructs GP01-, GP11- GP13- and GP20-Fc bind directly to gp130 in the cytokine binding module (CBM) and largely inhibit IL-6-type cytokine signaling by interfering with the high-affinity binding site of IL-6, IL-11, CLCF1, CT1, CNTF, OSM and LIF. Furthermore, we functionally demonstrate the inhibitory effect of the selected nanobodies in cell-based transmigration assays of the human colorectal cancer cell line HT-29.
In summary, our study has identified and characterized four novel inhibitory high-affinity gp130 nanobodies with potential for use in cytokine-dependent autoimmunity or cancer therapy.
尽管白细胞介素（IL）-6 型细胞因子信号传导对于维持机体稳态至关重要，但在包括自身免疫性疾病和癌症在内的多种疾病中都观察到了异常信号传导。目前，所有已获批的抑制 IL-6 型细胞因子的生物制剂都特异性靶向关键的促炎介质 IL-6 或其受体（IL6R）。从历史上看，由于担心广泛抑制可能弊大于利，人们一直避免直接抑制糖蛋白 130（gp130）——IL-6 型细胞因子的共同跨膜受体。然而，鉴于 Janus 激酶（JAK）抑制剂在临床上的成功，这种观点正在被重新审视；JAK 抑制剂广泛破坏细胞因子信号传导，包括由 gp130 介导的信号通路。
在此我们开发了四种单域抗体（sdAb），其由骆驼科动物来源的纳米抗体和人 Fc 片段组成，并通过直接蛋白质相互作用分析、表位结合、表位分组以及抑制细胞因子诱导的适当 Ba/F3 细胞系刺激和增殖以及 HT-29 细胞中的迁移等方法对其进行了表征。
四种 sdAb-Fc 构建体 GP-01-Fc、GP-11-Fc、GP-13-Fc 和 GP-20-Fc 在细胞因子结合模块（CBM）中直接与 gp132 结合，并通过干扰 IL-6、IL-11、CLCF1、CT-1、CNTF、OSM 和 LIF 的高亲和力结合位点，在很大程度上抑制 IL-6 型细胞因子信号传导。此外，我们在人结肠癌细胞系 HT-29 的基于细胞的迁移试验中从功能上证明了所选纳米抗体的抑制作用。
总之，我们的研究鉴定并表征了四种新型抑制性高亲和力 gp130 纳米抗体，它们具有用于细胞因子依赖性自身免疫或癌症治疗的潜力。
