Generation of IL-2-Fc-antibody conjugates by click chemistry

Abstract

Background: Immunocytokines (ICKs) are antibody directed cytokines produced by genetic fusion of an antibody to a cytokine.

Methods: We now show that antibodies conjugated by click chemistry to interleukin-2 (IL-2)-Fc form fully active conjugates, and in one example, equivalent activity to a genetically produced ICK.

Results: An IL-2-Fc fusion protein was optimized for click chemistry at hinge cysteines using protein stabilizing IL-2 mutations at Lys35 and Cys125 and Fc hinge mutations at Cys142 and Cys148. The IL-2-Fc fusion protein with K35E and C125S mutations with 3 intact hinge cysteines, designated as IL-2-Fc Par, was selected based on its minimal tendency to aggregate. IL-2-Fc-antibody clicked conjugates retained high IL-2 activity and bound target antigens comparable to parent antibodies. An IL-2-Fc-anti-CEA click conjugate showed comparable anti-tumor activity to an anti-CEA-IL-2 ICK in immunocompetent CEA transgenic mice bearing CEA positive orthotopic breast tumors. Significant increases in IFNγ⁺ /CD8⁺ and decreases in FoxP3⁺ /CD4⁺ T-cells were found for the clicked conjugate and ICK therapies, suggesting a common mechanism of tumor reduction.

Conclusion: The production of antibody targeted IL-2 therapy via a click chemistry approach is feasible with comparable activity to genetically produced ICKs with the added advantage of multiplexing with other monoclonal antibodies.

Keywords: IL-2-Fc; carcinoembryonic antigen; click chemistry; immunocytokine; immunotherapy.