de Dreu Anne, de Bruin Koen, Hokke Ayla M, Schrijver David P, Beelen Danyel N H, Verhalle Lars M, Clavijo Perez Maria C, Anbergen Tom, Versteeg Iris, Maas Rianne, Zwolsman Robby C, Grao-Roldán Cristina, Bartelet Branca, Trines Mirre M, Hoorn Daniek, Ros Gijs, Toner Yohana C, Kluza Ewelina, Beldman Thijs, Pérez-Medina Carlos, Netea Mihai G, Merkx Maarten, van der Meel Roy, Mulder Willem J M
Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven 5600 MB, the Netherlands.
Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven 5600 MB, the Netherlands.
Bioconjug Chem. 2025 Aug 20;36(8):1698-1708. doi: 10.1021/acs.bioconjchem.5c00182. Epub 2025 Jul 11.
Cytokines play a crucial role in cell communication and immunity, making them interesting potential therapeutics for immune-mediated conditions. However, cytokine therapeutics' clinical translation is hampered by their short blood half-lives and unfavorable biodistribution, resulting in toxicity and poor pharmacokinetics. In this study, we present a strategy to improve cytokines' pharmacokinetic profile by engineering fusions of apolipoproteins and cytokines, which are formulated into apolipoprotein-based nanoparticles (cytokine-aNPs). After establishing chemical and recombinant fusion approaches, we created a small library of diverse proteins, comprising fusions between apolipoprotein A1 or apolipoprotein E with either interleukin 1β, interleukin 2, or interleukin 4. Although chemical conjugation successfully generated biologically active fusion proteins, their yield and purity were insufficient for cytokine-aNP formulation. Using the recombinant method, we expressed and purified the fusion proteins and then incorporated them into cytokine-aNPs. In addition, we show that all cytokine-aNPs remain stable over at least 10 days and are of similar size and shape. We found that the fusion protein's cytokine component remains biologically active after purification and after formulation into cytokine-aNPs. In mice, using zirconium-89 radiolabeling to enable positron emission tomography imaging, we found that the pharmacokinetic profile of the cytokines incorporated into aNPs changed considerably. As compared to the native cytokines, we found the cytokine-aNPs to predominantly accumulate in the spleen, bone marrow, lymph nodes, and liver. Together, our results demonstrate that we can improve cytokines' properties using our fusion protein technology and aNP platform, opening up a translational avenue for nanomedicine-based cytokine therapy.
细胞因子在细胞通讯和免疫中发挥着关键作用，使其成为免疫介导疾病中颇具潜力的治疗手段。然而，细胞因子疗法的临床转化受到其血液半衰期短和生物分布不佳的阻碍，导致毒性和药代动力学较差。在本研究中，我们提出了一种策略，通过构建载脂蛋白与细胞因子的融合体来改善细胞因子的药代动力学特征，这些融合体被制备成基于载脂蛋白的纳米颗粒（细胞因子-aNP）。在建立化学和重组融合方法后，我们创建了一个包含多种蛋白质的小型文库 —— 由载脂蛋白A1或载脂蛋白E与白细胞介素1β、白细胞介素2或白细胞介素4之间的融合体组成。尽管化学偶联成功产生了具有生物活性的融合蛋白，但其产量和纯度不足以用于细胞因子-aNP的制备。使用重组方法，我们表达并纯化了融合蛋白，然后将它们整合到细胞因子-aNP中。此外，我们表明所有细胞因子-aNP在至少10天内保持稳定，并且具有相似的大小和形状。我们发现，融合蛋白中的细胞因子成分在纯化后以及制备成细胞因子-aNP后仍保持生物活性。在小鼠中，我们使用锆-89放射性标记以实现正电子发射断层扫描成像，发现整合到aNP中的细胞因子的药代动力学特征发生了显著变化。与天然细胞因子相比，我们发现细胞因子-aNP主要积聚在脾脏、骨髓、淋巴结和肝脏中。总之，我们的结果表明，我们可以使用我们的融合蛋白技术和aNP平台来改善细胞因子的特性，为基于纳米医学的细胞因子治疗开辟了一条转化途径。
