Progress in cytokine engineering is driving therapeutic translation by overcoming the inherent limitations of these proteins as drugs. The interleukin-2 (IL-2) cytokine harbors great promise as an immune stimulant for cancer treatment. However, the cytokine's concurrent activation of both pro-inflammatory immune effector cells and anti-inflammatory regulatory T cells, its toxicity at high doses, and its short serum half-life have limited clinical application. One promising approach to improve the selectivity, safety, and longevity of IL-2 is complexation with anti-IL-2 antibodies that bias the cytokine towards the activation of immune effector cells (i.e., effector T cells and natural killer cells). Although this strategy shows therapeutic potential in preclinical cancer models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multi-protein drug and concerns about complex stability. Here, we introduce a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokine's activities towards immune effector cells. We establish the optimal IC construction and further engineer the cytokine/antibody affinity to improve immune biasing function. We demonstrate that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared to natural IL-2 without inducing toxicities associated with IL-2 administration. Collectively, this work presents a roadmap for the design and translation of immunomodulatory cytokine/antibody fusion proteins.

Bloomberg Kimmel Institute for Cancer Immunotherapy Johns Hopkins University School of Medicine; Baltimore USA

Department of Biochemistry Faculty of Science Charles University; Prague Czech Republic

Department of Biology Johns Hopkins University; Baltimore USA

Department of Biomedical Engineering Johns Hopkins University School of Medicine; Baltimore USA

Department of Chemical and Biomolecular Engineering Johns Hopkins University School of Engineering; Baltimore USA

Department of Chemistry Johns Hopkins University; Baltimore USA

Department of Oncology Johns Hopkins University School of Medicine; Baltimore USA

Department of Ophthalmology Johns Hopkins University School of Medicine; Baltimore USA

Institute of Biotechnology of the Academy of Sciences of the Czech Republic; Vestec Czech Republic

Laboratory of Molecular Immunology National Heart Lung and Blood Institute National Institutes of Health; Bethesda USA

Laboratory of Tumor Immunology Institute of Microbiology of the Academy of Sciences of the Czech Republic; Prague Czech Republic

Sidney Kimmel Comprehensive Cancer Center Johns Hopkins University School of Medicine; Baltimore USA

Translational Tissue Engineering Center Johns Hopkins University School of Medicine Baltimore USA

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JCI Insight. 2024 Sep 24;9(18):e173469. doi: 10.1172/jci.insight.173469. PubMed

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