IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rβ, and common γ [γc]). IL-2Rα, which is highly expressed on regulatory T (TReg) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward TReg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Rα. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating TReg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.

Biodesy Inc South San Francisco CA 94010

Department of Biochemistry Stanford University Stanford CA 94305

Department of Bioengineering Stanford University Stanford CA 94305; and

Department of Biology Stanford University Stanford CA 94305

Department of Molecular and Cellular Physiology Stanford University School of Medicine Stanford CA 94305

Department of Structural Biology Stanford University School of Medicine Stanford CA 94305

Diabetes Center University of California San Francisco San Francisco CA 94143

Howard Hughes Medical Institute Stanford University School of Medicine Stanford CA 94305

Howard Hughes Medical Institute Stanford University School of Medicine Stanford CA 94305;

Laboratory of Tumor Immunology Institute of Microbiology of the Academy of Sciences of the Czech Republic 14220 Prague 4 Krc Czech Republic

Sean N Parker Autoimmune Research Laboratory University of California San Francisco San Francisco CA 94143

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IL-2-driven CD8+ T cell phenotypes: implications for immunotherapy

Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection

IL-2/JES6-1 mAb complexes dramatically increase sensitivity to LPS through IFN-γ production by CD25+Foxp3- T cells