Department of Biotechnology, Jožef Stefan Institute, Jamova 39, Ljubljana SI-1000, Slovenia; Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, Ljubljana SI-1000, Slovenia.
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, Ljubljana SI-1000, Slovenia.
Eur J Pharm Sci. 2023 Nov 1;190:106568. doi: 10.1016/j.ejps.2023.106568. Epub 2023 Aug 22.
The cytokine IL-23 activates the IL-23 receptor (IL-23R) and stimulates the differentiation of naïve T helper (Th) cells into a Th17 cell population that secretes inflammatory cytokines and chemokines. This IL-23/Th17 proinflammatory axis drives inflammation in Crohn's disease and ulcerative colitis and represents a therapeutic target of monoclonal antibodies. Non-immunoglobulin binding proteins based on the Streptococcus albumin-binding domain (ABD) provide a small protein alternative to monoclonal antibodies. They can be readily expressed in bacteria. Lactococcus lactis is a safe lactic acid bacterium that has previously been engineered as a vector for the delivery of recombinant therapeutic proteins to mucosal surfaces. Here, L. lactis was engineered to display or secrete ABD-variants against the IL-17 receptor (IL-17R). Its expression and functionality were confirmed with flow cytometry using specific antibody and recombinant IL-17R, respectively. In addition, L. lactis were engineered into multifunctional bacteria that simultaneously express two binders from pNBBX plasmid. First, binders of IL-17R were combined with binder of IL-17. Second, binders of IL-23R were combined with binders of IL-23. The dual functionality of the bacteria was confirmed by flow cytometry using corresponding targets, namely the recombinant receptors IL-17R and IL-23R or the p19 subunit of IL-23. Binding of IL-17 was confirmed by ELISA. With the latter, 97% of IL-17 was removed from solution by 2 × 10 recombinant bacteria. Moreover, multifunctional bacteria targeting IL-17/IL-17R prevented IL-17A-mediated activation of downstream signaling pathways in HEK-Blue IL-17 cell model. Thus, we have developed several multifunctional L. lactis capable of targeting multiple factors of the IL-23/Th17 proinflammatory axis. This represents a novel therapeutic strategy with synergistic potential for the treatment of intestinal inflammations.
细胞因子 IL-23 激活 IL-23 受体 (IL-23R)，并刺激初始 T 辅助 (Th) 细胞分化为 Th17 细胞群，后者分泌炎症细胞因子和趋化因子。这种 IL-23/Th17 促炎轴驱动克罗恩病和溃疡性结肠炎的炎症反应，是单克隆抗体的治疗靶点。基于链球菌白蛋白结合域 (ABD) 的非免疫球蛋白结合蛋白为单克隆抗体提供了一种小蛋白替代物。它们可以在细菌中容易地表达。乳酸乳球菌是一种安全的乳酸菌，之前已被工程化为将重组治疗蛋白递送到粘膜表面的载体。在这里，通过流式细胞术分别使用特异性抗体和重组 IL-17R ，对表达或分泌针对 IL-17 受体 (IL-17R) 的 ABD 变体的乳酸乳球菌进行了工程改造。证实了其表达和功能。此外，将乳酸乳球菌工程改造为多功能细菌，这些多功能细菌同时从 pNBBX 质粒表达两种结合物。首先，将 IL-17R 的结合物与 IL-17 的结合物结合。其次，将 IL-23R 的结合物与 IL-23 的结合物结合。通过使用相应的靶标，即重组受体 IL-17R 和 IL-23R 或 IL-23 的 p19 亚基，通过流式细胞术证实了细菌的双重功能。通过 ELISA 证实了 IL-17 的结合。通过 2×10 个重组菌，有 97%的 IL-17 从溶液中被去除。此外，靶向 IL-17/IL-17R 的多功能细菌可防止 IL-17A 在 HEK-Blue IL-17 细胞模型中激活下游信号通路。因此，我们开发了几种能够靶向 IL-23/Th17 促炎轴多个因素的多功能乳酸乳球菌。这代表了一种具有协同潜力的新型治疗策略，可用于治疗肠道炎症。
