CD4⁺ CD25⁺ regulatory T cells suppress the immune system and help to maintain self tolerance and prevent autoimmune disease in healthy patients.  However, their suppressive activity also has undesirable consequences during cancer.  In many cancer patients, there exist effector T cells that recognize tumor antigens, but their ability to attack and eradicate the cancer is hindered by regulatory T cells in the body.  Therefore, if the suppressive function of regulatory T cells can be inhibited, this could give rise to a more potent anti-tumor immune response, which could have significant benefits in cancer immunotherapy.

Regulatory T cells require IL-2 for their expansion, maintenance and suppressive abilities.  Here, we propose the use of IL-2 antagonists as a novel approach to inhibiting regulatory T cells.  Using recently published crystal structures, we identified candidate mutations to disrupt binding to the β or γ subunits of the IL-2 receptor.  Inserting these mutations on an IL-2 variant with high affinity binding to CD25 (equivalent to IL-2Rα, the α subunit of the IL-2 receptor) would create a signaling deficient IL-2 analogue that sequesters IL-2Rα from binding wild type IL-2, thus antagonizing the IL-2 receptor. We have expressed and purified two variants with residue substitutions that disrupt the β and γ subunit binding interfaces and characterized them in a non-regulatory T cell line that expresses the IL-2 receptor.  These mutants retain their high affinity binding to IL-2Rα, and do not agonize the IL-2 receptor.  The two mutants also antagonize the IL-2 receptor with equal efficacy.  We believe that these mutants and this general approach of engineering IL-2 antagonists show promise for the inhibition of regulatory T cells for cancer immunotherapy.