Kosler Stasa, Strukelj Borut, Berlec Ales
Department of Biotechnology, Jozef Stefan Institute, Jamova 39, SI-1000, Ljubljana. Slovenia.
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana. Slovenia.
Curr Pharm Biotechnol. 2017;18(4):318-326. doi: 10.2174/1389201018666170210152218.
Neutralization of proinflammatory cytokines is an established strategy in the treatment of inflammatory bowel disease (IBD). Systemic anti-TNFα antibodies have been used in the clinics for several years, while anti-IL-17/IL-23 antibodies have been less successful so far. We report the development of safe lactic acid bacterium Lb. salivarius with the ability to simultaneously bind IL-17A, IL-23 and TNFα that could be administered orally for the treatment of IBD.
Three different cytokine-binding non-Ig scaffolds (anti-IL-17A fynomer, anti-IL-23-binding adnectin and anti-TNFα-binding affibody) were cloned and expressed in L. lactis in fusion with lysine motif (LysM)-containing surface anchor. Fusion proteins were used for coating of Lb. salivarius. Cytokine- binding ability of bacterial cells was assayed with ELISA. Gastric stability was tested by incubation in simulated gastric juice.
Surface display and functionality of cytokine binding proteins in L. lactis was confirmed by the ability to remove the individual cytokines from the solution. Binding was efficient with different concentrations of cells, different cytokine concentrations and after the incubation in simulated gastric juice. The fusion protein-containing growth media of L. lactis were used for coating of Lb. salivarius in a fast and straightforward manner. Cytokine-binder-coated Lb. salivarius was able to retain individual binders or mixtures of all three binders on its surface, and also provided limited protection from proteolytic and low pH degradation. Cytokine binding capacity of mixture-coated Lb. salivarius was similar to that achieved by coating with individual binders.
Simultaneous binding of IL-17A, IL-23 and TNFα with engineered bacteria was achieved for the first time. Oral administration of such bacteria could exert synergistic effect and thus represents an alternative strategy in the treatment of IBD.
中和促炎细胞因子是治疗炎症性肠病（IBD）的既定策略。全身性抗TNFα抗体已在临床中使用数年，而抗IL-17/IL-23抗体迄今为止效果欠佳。我们报告了具有同时结合IL-17A、IL-23和TNFα能力的安全乳酸菌唾液乳杆菌的研发，该菌可口服用于治疗IBD。
克隆三种不同的细胞因子结合非免疫球蛋白支架（抗IL-17A Fynomer、抗IL-23结合adnectin和抗TNFα结合亲和体），并与含赖氨酸基序（LysM）的表面锚定物融合在乳酸乳球菌中表达。融合蛋白用于包被唾液乳杆菌。用ELISA检测细菌细胞的细胞因子结合能力。通过在模拟胃液中孵育测试胃稳定性。
乳酸乳球菌中细胞因子结合蛋白的表面展示和功能通过从溶液中去除单个细胞因子的能力得以证实。在不同细胞浓度、不同细胞因子浓度以及在模拟胃液中孵育后，结合均有效。乳酸乳球菌含融合蛋白的生长培养基用于快速简便地包被唾液乳杆菌。细胞因子结合剂包被的唾液乳杆菌能够在其表面保留单个结合剂或所有三种结合剂的混合物，并且还提供了有限的抗蛋白水解和低pH降解保护。混合包被的唾液乳杆菌的细胞因子结合能力与用单个结合剂包被所达到的能力相似。
首次实现了工程菌同时结合IL-17A、IL-23和TNFα。口服这种细菌可发挥协同作用，因此代表了一种治疗IBD的替代策略。
