Engineering Fusion Proteins for Nanomedicine-Based Cytokine Therapy.

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 in vivo 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' in vivo properties using our fusion protein technology and aNP platform, opening up a translational avenue for nanomedicine-based cytokine therapy.