Background High-dose recombinant human interleukin-2 (aldesleukin) elicits durable anti-tumor immunity and gained FDA approval two decades prior to checkpoint blockers. However, use of aldesleukin is limited due to treatment-related life-threatening toxicities. Second generation efforts to alleviate toxicities have largely focused on eliminating binding to IL-2Rα, often with half-life extension. We have determined that mice and non-human primates (NHPs) treated with a 2nd generation IL-2 surrogate that does not bind IL-2Rα still experience characteristic dose-limiting toxicities, including vascular leak syndrome (VLS), and exhibit dysregulated peripheral immune function due to reduced Treg activation. To overcome these toxicities and improve the therapeutic index of IL-2 as an anti-tumor immunotherapy, we employed protein engineering to generate XTX201, a highly potent 3rd generation IL-2 that is designed to be selectively active in tumors, stimulating cytolytic responses against tumor cells while sparing systemic immune activation.
Methods XTX201 binding interactions were measured with SPR, and bioactivity was measured using STAT-5 phosphorylation in human PBMCs and reporter cell lines. Anti-tumor efficacy and immune activation was evaluated in tumors compared to peripheral organs in syngeneic tumor mouse models. Safety and pharmacokinetics were evaluated in rodents and NHPs.
Results Non-activated XTX201 showed no detectable binding to IL-2Rα or IL-2Rβ, and limited IL-2R-dependent STAT-5 signaling in vitro. Activation of XTX201 resulted in high-affinity binding to IL-2Rβ and no binding to IL-2Rα, leading to a ~1000-fold reduction in Treg activation as compared to WT IL-2, while retaining CD8+ T and NK cell activation. Mice and NHPs treated with a 2nd generation IL-2 surrogate experienced toxicities that are commonly observed in patients treated with aldesleukin, including pulmonary edema, VLS, fever and lethality. However, XTX201 did not induce toxicities at exposures 100-fold higher than the MTD of the activated version, and achieved similar anti-tumor efficacy in mice. Experiments in primary human solid tumors and human plasma indicated that XTX201 is preferentially activated in the tumor microenvironment.
Conclusions Our data demonstrate that 2nd generation IL-2s that are systemically active and lack binding to IL-2Rα exhibit dose-limiting toxicities unless further engineered for selective activity in tumors. XTX201, a 3rd generation, tumor-selective IL-2, exhibits a long half-life and is innocuous outside of tumors. XTX201 is activated within tumors to release an IL-2Rβ/γ biased cytokine that inhibits tumor growth in syngeneic models, and exhibits tumor-specific pharmacodynamic effects without peripheral toxicities. XTX201 has the potential to be a best-in-class IL-2 immunotherapy by expanding the curative anti-tumor activity of aldesleukin while minimizing dose-limiting toxicities.
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