工厂提供了一个具有成本效益和可扩展的制药平台,没有宿主来源的污染风险。然而,它们的医疗应用因对外部蛋白质的急性过敏反应而变得复杂。使用非侵入性治疗方式开发用于局部疾病的基于植物的蛋白质治疗剂可以利用植物蛋白的益处,同时避免其固有风险。Dupilumab,它对各种过敏性和自身免疫性疾病有效,但具有全身反应和注射相关的副作用,如果使用小的生物形式局部递送,可能更有益。在这项研究中,我们设计了dupilumab的单链可变片段(scFv),由烟草产生的Dup-scFv,并评价其在气-液界面培养的人鼻上皮细胞(HNECs)中的组织通透性和抗炎功效。尽管在表面等离子体共振测定和细胞结合测定中显示对IL-4Ra的结合亲和力降低了3.67和17倍,分别,Dup-scFv保留了dupilumab的大部分亲和力,最初很高,解离常数(KD)为4.76pM。在气液界面培养的HNECs中,在空气侧施用的Dup-scFv比dupilumab更有效地抑制难以到达的基底细胞中的炎性标志物CCL26。此外,与不可检测水平的dupilumab相比,Dup-scFv具有0.8%的跨细胞层的总体通透性。这些发现表明,植物产生的Dup-scFv可以非侵入性地传递给培养的HNESc以减轻炎症信号,提供了一种实用的方法来利用基于植物的蛋白质进行局部治疗应用。
Plants offer a cost-effective and scalable pharmaceutical platform devoid of host-derived contamination risks. However, their medical application is complicated by the potential for acute allergic reactions to external proteins. Developing plant-based protein therapeutics for localized diseases with non-invasive treatment modalities may capitalize on the benefits of plant proteins while avoiding their inherent risks. Dupilumab, which is effective against a variety of allergic and autoimmune diseases but has systemic responses and injection-related side effects, may be more beneficial if delivered locally using a small biological form. In this study, we engineered a single-chain variable fragment (
scFv) of dupilumab, termed Dup-
scFv produced by Nicotiana benthamiana, and evaluated its tissue permeability and anti-inflammatory efficacy in air-liquid interface cultured human nasal epithelial cells (HNECs). Despite showing 3.67- and 17-fold lower binding affinity for IL-4Ra in surface plasmon resonance assays and cell binding assays, respectively, Dup-
scFv retained most of the affinity of dupilumab, which was originally high, with a dissociation constant (KD) of 4.76 pM. In HNECs cultured at the air-liquid interface, Dup-
scFv administered on the air side inhibited the inflammatory marker CCL26 in hard-to-reach basal cells more effectively than dupilumab. In addition, Dup-
scFv had an overall permeability of 0.8% across cell layers compared to undetectable levels of dupilumab. These findings suggest that plant-produced Dup-
scFv can be delivered non-invasively to cultured HNESc to alleviate inflammatory signaling, providing a practical approach to utilize plant-based proteins for topical therapeutic applications.