PO.ET02.07 · 实验与分子治疗
生产功能性重组蛋白以推进自身免疫性疾病治疗的整合策略
Integrated strategies for producing functional recombinant proteins to advance autoimmune disease therapeutics
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摘要 Abstract
中文摘要
自身免疫性疾病源于免疫失调，其特征为自身耐受丧失、慢性炎症和多器官病变。然而，免疫信号通路包含大量复杂的蛋白质结构，包括多聚体细胞因子、跨膜受体和胞内通路介质。每一种都必不可少，但因其结构复杂性(如寡聚化、结构域组织和跨膜区)而具有挑战性。在此，我们提出一种整合策略以生产功能性重组蛋白，从而支持自身免疫性疾病的研究和治疗开发。对于可溶性多聚体靶点，采用结构导向的工程化，包括针对IL-17A/IL-17F异二聚体稳定化的连接子优化。对于诸如IL-2受体亚基组装等其他靶点，掺入蛋白标签是重组蛋白生产的最佳方法。最后，通过靶向诱变，可控制TL1A二硫键以调控寡聚化。结合培养和纯化中的工艺优化，难以表达的重组蛋白的开发有助于保留天然构象和生物活性。这些被封装进三个互补平台中加以实施，以确保溶解度和功能重构：用于结构研究、ELISA和表面等离子体共振的去污剂胶束；用于无去污剂、基于细胞检测的纳米盘；以及提供天然膜环境和高免疫原性的病毒样颗粒。这些蛋白进一步支持抗体发现，包括聚焦表位的免疫原设计，例如IL-23/IL-12嵌合体以减少脱靶反应。通过遵循质量源于设计(Quality-by-Design)原则，这一从结构分析到应用验证的闭环工作流程确保了天然功能、稳定性和均一性的维持。总的来说，这些方法通过为机制研究、筛选和治疗开发生成高质量试剂，加速了自身免疫性疾病的药物发现，克服了结构复杂靶点中长期存在的生产瓶颈。
查看英文原文 English abstract
Autoimmune diseases result from immune dysregulation, characterized by loss of self-tolerance, chronic inflammation, and multiorgan pathology. However, the immune signaling pathway is a host of complex protein architectures, including polymeric cytokines, transmembrane receptors, and intracellular pathway mediators. Each are essential but challenging due to structural intricacies such as oligomerization, domain organization, and transmembrane regions. Herein, we present an integrated strategy to produce functional recombinant proteins to support research and therapeutic development in autoimmune disease. For soluble polymeric targets, structure-guided engineering is employed, encompassing linker optimization for IL-17A/IL-17F heterodimer stabilization. For other targets such as the assembly of IL-2 receptor subunits, the incorporation of protein tags is the optimal methodology for recombinant protein production. Finally, by utilizing targeted mutagenesis, oligomerization can be controlled for TL1A disulfide bonds to control oligomerization. With the incorporation of process optimization in culture and purification, the development of hard-to-express recombinant proteins helps preserve native conformation and bioactivity. These are encapsulated into three complementary platforms are implemented to ensure solubility and functional reconstitution: detergent micelles for structural studies, ELISA, and surface plasmon resonance; nanodiscs for detergent-free, cell-based assays; and virus-like particles providing a native membrane context and high immunogenicity. These proteins further enable antibody discovery, including epitope-focused immunogen design, for example, IL-23/IL-12 chimeras to reduce off-target responses. By adhering to Quality-by-Design principles, this closed-loop workflow from structural analysis through application validation ensures maintenance of native functionality, stability, and homogeneity. Collectively, these approaches accelerate drug discovery in autoimmune disease by generating high-quality reagents for mechanistic studies, screening, and therapeutic development, overcoming longstanding production bottlenecks in structurally complex targets.
利益披露 Disclosure
W. Ren, None..
S. Wang, None..
A. Ouyang, None..
S. Chiang, None..
Y. Sun, None.