962 A novel alpha-active IL-2-Fc has expanded therapeutic index and robust monotherapy efficacy in mouse cancer models and strong synergy with PD-1 blockade

<h3>Background</h3> IL-2 stimulates potent tumor immunity by expanding and activating tumor-infiltrating cytotoxic CD8<i>⁺</i> T cells and NK cells. Aldesleukin is an approved recombinant human IL-2; however, its short half-life, requirement for 5 days of hospitalization per cycle, and high toxicity [eg, cytokine related syndrome (CRS), capillary leak syndrome (CLS)] severely limit its clinical use. To reduce toxicity, IL-2s were created with abolished binding to IL-2Rα, but these non-alpha IL-2s also had diminished clinical efficacy. DF6215 retains IL-2Rα binding and increases IL-2Rβγ stimulation to improve the benefit-to-risk ratio compared to historic IL-2 drugs. In addition, the modified IL-2 is monovalently fused to an Fc protein prolonging elimination half-life and allowing for less frequent dosing. <h3>Methods</h3> DF6215 potency was characterized in mouse, human, and non-human primate (NHP) cell subsets by STAT5 phosphorylation. The ability of DF6215 to expand and activate immune cell populations was evaluated in vitro using human PBMCs, mouse syngeneic tumor models and in NHP studies. <h3>Results</h3> DF6215 showed increased potency on activated CD8⁺ T cells compared to a non-alpha IL-2 and reduced potency on T regulatory cells compared to aldesleukin. In isolated human CD8⁺ T cells, DF6215 yielded more effector memory phenotype cells after 4 days of in vitro T-cell receptor stimulation, relative to a non-alpha IL-2. DF6215 monotherapy dosed 1 mg/kg once weekly demonstrated potent and durable anti-tumor activity resulting in 100% complete responses (CR) in MC38 tumor-bearing mice. Moreover, DF6215 dosed 0.675 mg/kg once weekly showed greater efficacy (50% CR) compared to a non-alpha IL-2 (30% CR) in the CT26 model. Within the tumor microenvironment, DF6215 led to preferential activation and proliferation of cytotoxic CD8⁺ T cells and NK cells, with limited expansion of Tregs, which resulted in an increased effector-to-Treg ratio. When administered with PD-1 blockade therapy, DF6215 demonstrated strong anti-tumor efficacy and synergy (70% CR in combination vs 10% CR as monotherapy) in the immunologically cold, PD-1 refractory B16F10 melanoma model. In NHP studies, DF6215 expanded peripheral lymphocytes, including activated CD25⁺CD8⁺ T cells which increased up to 40-fold from baseline, without CLS or CRS. <h3>Conclusions</h3> DF6215 is a differentiated IL-2 that preferentially stimulates activated cytotoxic CD8⁺ T cells and drives potent anti-tumor activity in mouse models as a monotherapy which is enhanced in combination with PD-1 blockade. DF6215’s extended half-life, tuned IL-2Rα binding and increased IL-2Rβγ agonism expands IL-2’s therapeutic index. DF6215 is currently being evaluated in a Phase 1/1b clinical trial in patients with advanced solid tumors (NCT06108479). <h3>Ethics Approval</h3> All mouse studies were carried out according to protocols established by Explora BioLabs/CRADL IACUC committee.