Low-dose human interleukin-2 (hIL-2) treatment is used clinically to treat autoimmune disorders due to the cytokine's preferential expansion of immunosuppressive regulatory T cells (Tregs). However, off-target immune cell activation and short serum half-life limit the clinical potential of IL-2 treatment. Recent work showed that complexes comprising hIL-2 and the anti-hIL-2 antibody F5111 overcome these limitations by preferentially stimulating Tregs over immune effector cells. Although promising, therapeutic translation of this approach is complicated by the need to optimize dosing ratios and by the instability of the cytokine/antibody complex. We leverage structural insights to engineer a single-chain hIL-2/F5111 antibody fusion protein, termed F5111 immunocytokine (IC), which potently and selectively activates and expands Tregs. F5111 IC confers protection in mouse models of colitis and checkpoint inhibitor-induced diabetes mellitus. These results provide a roadmap for IC design and establish a Treg-biased immunotherapy that could be clinically translated for autoimmune disease treatment.

Bloomberg Kimmel Institute for Cancer Immunotherapy Johns Hopkins University Baltimore MD 21231 USA; Sidney Kimmel Comprehensive Cancer Center Johns Hopkins University Baltimore MD 21231 USA

Department of Bioengineering Jonsson Comprehensive Cancer Center Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research University of California Los Angeles Los Angeles CA 90095 USA

Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore MD 21218 USA; Translational Tissue Engineering Center Johns Hopkins University School of Medicine Baltimore MD 21231 USA

Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore MD 21218 USA; Translational Tissue Engineering Center Johns Hopkins University School of Medicine Baltimore MD 21231 USA; Bloomberg Kimmel Institute for Cancer Immunotherapy Johns Hopkins University Baltimore MD 21231 USA; Sidney Kimmel Comprehensive Cancer Center Johns Hopkins University Baltimore MD 21231 USA; Department of Biomedical Engineering Johns Hopkins University School of Medicine Baltimore MD 21205 USA; Department of Oncology Johns Hopkins University School of Medicine Baltimore MD 21231 USA; Department of Ophthalmology Johns Hopkins University School of Medicine Baltimore MD 21287 USA

Department of Pathobiology University of Pennsylvania Philadelphia PA 19104 USA

Diabetes Center University of California San Francisco San Francisco CA 94143 USA

Diabetes Center University of California San Francisco San Francisco CA 94143 USA; Sean N Parker Autoimmune Research Laboratory University of California San Francisco San Francisco CA 94143 USA; Huntsman Cancer Institute University of Utah Health Sciences Center Salt Lake City UT 84112 USA; Department of Pathology University of Utah School of Medicine Salt Lake City UT 84112 USA

Diabetes Center University of California San Francisco San Francisco CA 94143 USA; Sean N Parker Autoimmune Research Laboratory University of California San Francisco San Francisco CA 94143 USA; Sonoma Biotherapeutics South San Francisco CA 94080 USA

Institute of Biotechnology of the Academy of Sciences of the Czech Republic Vestec 252 50 Czech Republic

Institute of Microbiology of the Academy of Sciences of the Czech Republic Prague 142 20 Czech Republic

Translational Research Immunology Group Nuffield Department of Surgical Sciences University of Oxford Oxford OX3 9DU UK

Translational Tissue Engineering Center Johns Hopkins University School of Medicine Baltimore MD 21231 USA; Department of Molecular Microbiology and Immunology Johns Hopkins Bloomberg School of Public Health Baltimore MD 21205 USA

Vascularized Composite Allotransplantation Laboratory Department of Plastic and Reconstructive Surgery Johns Hopkins University School of Medicine Baltimore MD 21205 USA

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Temporal optimization of CD25-biased IL-2 agonists and immune checkpoint blockade leads to synergistic anticancer activity despite robust regulatory T cell expansion

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