Engineering novel designer biologics in plant cells for oral treatment of ulcerative colitis

项目摘要/摘要 炎症性肠病(IBD)，包括两个最常见的亚型：克罗恩病(CD)和 溃疡性结肠炎(UC)，代表一组肠道疾病，导致长期的炎症 消化道。目前的治疗策略，包括传统的抗炎药物和 针对促炎细胞因子肿瘤坏死因子α的新型生物药物 全身用药的疗效和不良反应。结肠靶向口服 对于炎症性肠病的治疗，给予抗肿瘤坏死因子α制剂是非常可取的，因为它提高了药物的疗效，同时 降低全身毒性。植物细胞培养已成为一种安全、经济的生物生产平台 用于治疗蛋白质。植物细胞的一个独特之处在于，它们不仅可以充当“生物工厂”， 也是重组生物制品的口服载体。最近的研究表明，植物细胞 主要由纤维素微纤维制成的管壁可以作为一种极好的天然胶囊，用于口服 生物药物。该项目旨在利用两种独特的翻译后修饰--“糖基-- “磷脂酰肌醇(GPI)锚”和“植物特有的羟脯氨酸(Hyp)-O-糖基化”--战略性地 在植物细胞中设计和设计新型抗肿瘤坏死因子α生物分子以开发新型口服生物药物 用于治疗UC。设计的抗肿瘤坏死因子α生物分子由三个功能结构域组成：N-末端 抗肿瘤坏死因子α抗体的单链片段变量(ScFv)，这是一个专有的Hyp-O-糖基化模块 由“Ser-Pro”基序或(SP)n(n=5至30)的串联重复序列和C-末端GPI锚定组成。而当 Gpi锚点将在植物细胞表面“展示”表达的抗肿瘤坏死因子α生物分子(但仍 封装在细胞壁内)，以推测产生高局部浓度的生物制品，(SP)n 在生物生产和口服给药过程中，糖模将稳定蛋白质的降解。 同时，修饰在(SP)n糖模上的带负电荷的糖链将靶向抗肿瘤坏死因子α 生物分子到发炎的上皮细胞，在那里总是积聚正电荷的蛋白质。设计师反- 将研究由不同大小的(SP)n糖模组成的肿瘤坏死因子α生物分子的蓄积 在烟草BY-2细胞中，生物活性和在模拟胃液中的稳定性，这将确定最佳的 生物分子的设计。为了改善口服植物细胞的药代动力学行为 生物药物，将开发通过填充细胞壁来增强植物细胞壁基质的策略 具有聚合物分子的孔/通道，例如聚乙二醇(PEG)，以增加它们的容量 保护被包裹的蛋白质。最后，口服设计者的治疗效果。 将在葡聚糖硫酸钠中评估抗肿瘤坏死因子α生物制剂(最佳设计)对缓解UC症状的作用 DSS诱导的小鼠结肠炎模型。抗肿瘤坏死因子α生物制品的免疫调节作用将是 通过组织病理学分析和炎性标志物的测定来确定。拟议的研究将 有可能开发一种新的平台来生产治疗UC和其他疾病的有效口服生物药物 结肠中的炎症性疾病。

Project Summary/Abstract Inflammatory bowel disease (IBD), including the two most common subtypes: Crohn’s disease (CD) and ulcerative colitis (UC), represents a group of intestinal disorders that cause prolonged inflammation of the digestive tract. The current therapeutic strategies, including the conventional anti-inflammatory medications and the new biologic drugs targeting the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα), have limited therapeutic efficacy and adverse drug reactions resulting from systemic administration. Colon-targeted oral delivery of anti-TNFα agents is highly desirable for the treatment of IBD, as it improves the drugs’ efficacy while reducing the systemic toxicity. Plant cell culture has emerged as a safe and cost-effective bioproduction platform for therapeutic proteins. A unique feature of the plant cells is that they could serve not only as the “bio-factory,” but also the oral delivery vehicle for recombinant biologics. Recent advances have demonstrated that plant cell walls, made primarily of cellulose microfibrils, can act as an excellent natural capsule for the oral delivery of biologic drugs. This project aims to leverage two unique posttranslational modifications – “glycosyl- phosphatidylinositol (GPI) anchor” and “plant-specific hydroxyproline (Hyp)-O-glycosylation” – to strategically design and engineer novel anti-TNFα biomolecules in plant cells to develop a new class of oral biologic drugs for the treatment of UC. The designer anti-TNFα biomolecules consist of three functional domains: a N-terminal single-chain fragment variable (scFv) of an anti-TNFα antibody, a proprietary Hyp-O-glycosylation module comprised of tandem repeats of the “Ser-Pro” motif, or (SP)n (n= 5 to 30), and a C-terminal GPI anchor. While the GPI anchor will “display” the expressed anti-TNFα biomolecules at the plant cell surface (but still encapsulated within the cell wall) to presumably create a high local concentration of the biologics, the (SP)n glycomodule will stabilize the protein from degradation during both the bioproduction and oral delivery processes. Meanwhile, the negatively charged glycans decorated on the (SP)n glycomodule will target the anti-TNFα biomolecules to the inflamed epithelium where positively charged proteins are always built up. Designer anti- TNFα biomolecules consisting of different sized (SP)n glycomodules will be investigated for their accumulation in tobacco BY-2 cells, biological activity, and stability in a simulated gastric fluid, which will determine an optimal design for the biomolecules. In order to improve the pharmacokinetic behavior of the plant cell produced oral biologic drugs, strategies will be developed to reinforce the plant cell wall matrices by filling in the cell wall pores/channels with polymeric molecules, such as polyethylene glycol (PEG), to increase their capacity for protecting the encapsulated proteins. Finally, the therapeutic effectiveness of the orally administrated designer anti-TNFα biologic (optimal design) in mitigating UC symptoms will be assessed in a dextran sulfate sodium (DSS)-induced colitis mouse model. The immune-modulatory effects of the anti-TNFα biologics will be determined by a histopathological analysis and assay of the inflammatory markers. The proposed research will potentially develop a new platform to produce effective oral biologic drugs for the treatment of UC and other inflammatory diseases in the colon.