Fabilane Charina S, Stephenson A Carson, Leonard Elissa K, VanDyke Derek, Spangler Jamie B
Program in Molecular Biophysics, Johns Hopkins University, Baltimore, Maryland.
Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Curr Protoc. 2024 May;4(5):e1061. doi: 10.1002/cpz1.1061.
Cytokines constitute a class of secreted proteins that activate transmembrane receptors to coordinate a vast array of physiological processes, particularly those related to immune activity. Due to their vital role in immune regulation, cytokines have garnered great interest as potential therapeutic agents. Unfortunately, the clinical success of cytokine drugs has been limited by their multifunctional activities, which hinder therapeutic performance and lead to harmful toxicities. In addition, the strikingly short circulation half-life of cytokines further hampers their efficacy as drugs. To overcome the translational challenges associated with natural cytokines, significant efforts have focused on engineering cytokines to target their activities and improve their pharmacological properties. One such strategy is the design of fusion proteins that tether a cytokine to an anti-cytokine antibody that selectively biases its functions and extends its serum half-life. These cytokine/antibody fusion proteins (termed immunocytokines) assemble intramolecularly to bias cytokine signaling behavior through multi-layered structural and molecular effects. Here, we present a detailed workflow for the design, production, and functional validation of intramolecularly assembled immunocytokines. In-depth procedures are presented for gene manipulation, mammalian cell-based expression and purification, binding analysis via bio-layer interferometry, and interrogation of cytokine signaling activity on human primary cells. In contrast with immunocytokines in which the tethered cytokine and antibody do not bind one another, intramolecularly assembled immunocytokines require special considerations with respect to their production to avoid oligomerization and/or aggregation. The protocol herein was developed based on experience with immunocytokines that incorporate interleukin-2 (IL-2); however, this modular approach can be extended to any cytokine of interest for a broad range of biomedical applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design and generation of immunocytokine genes Basic Protocol 2: Immunocytokine expression and purification Basic Protocol 3: Validation of immunocytokine assembly and binding by bio-layer interferometry Basic Protocol 4: Analysis of immunocytokine signaling on human primary cells.
细胞因子是一类分泌蛋白，可激活跨膜受体以协调大量生理过程，尤其是与免疫活动相关的过程。由于其在免疫调节中的关键作用，细胞因子作为潜在治疗剂备受关注。不幸的是，细胞因子药物的临床成功受到其多功能活性的限制，这会妨碍治疗效果并导致有害毒性。此外，细胞因子显著短的循环半衰期进一步阻碍了它们作为药物的疗效。为了克服与天然细胞因子相关的转化挑战，大量努力集中在对细胞因子进行工程改造，以靶向其活性并改善其药理特性。一种这样的策略是设计融合蛋白，将细胞因子与抗细胞因子抗体连接，该抗体可选择性地偏向其功能并延长其血清半衰期。这些细胞因子/抗体融合蛋白（称为免疫细胞因子）在分子内组装，通过多层结构和分子效应偏向细胞因子信号传导行为。在这里，我们展示了分子内组装免疫细胞因子的设计、生产和功能验证的详细工作流程。介绍了基因操作、基于哺乳动物细胞的表达和纯化、通过生物层干涉术进行结合分析以及对人原代细胞上细胞因子信号传导活性的研究的深入程序。与其中连接的细胞因子和抗体不相互结合的免疫细胞因子不同，分子内组装的免疫细胞因子在其生产方面需要特殊考虑，以避免寡聚化和/或聚集。本文的方案是基于对包含白细胞介素 -2（IL -2）的免疫细胞因子的经验开发的；然而，这种模块化方法可扩展到任何感兴趣的细胞因子，用于广泛的生物医学应用。© 2024威利期刊有限责任公司。基本方案1：免疫细胞因子基因的设计与生成 基本方案2：免疫细胞因子的表达与纯化 基本方案3：通过生物层干涉术验证免疫细胞因子的组装与结合 基本方案4：人原代细胞上免疫细胞因子信号传导的分析。
