858 TILKine-2: a novel best-in-class tumor infiltrating lymphocyte (TIL) targeting engineered IL-2 with superior pre-clinical efficacy and safety for immunotherapy of cancer

BackgroundHigh-dose Interleukin-2 is the earliest FDA-approved immunotherapy for metastatic melanoma and renal cell carcinoma. Unfortunately, its application is limited due to its short half-life and severe toxicity at the therapeutic dose. To limit systemic toxicity, tumor-targeting antibody-based delivery of IL-2 has been developed, however with poor outcomes. We here deploy a novel strategy to deliver IL-2 to the tumor microenvironment by binding to Tumor-Infiltrating Lymphocytes (TILs). TILKine-2 is a recombinant bifunctional protein comprised of an antibody directed against TILs (TILAb) fused to an engineered IL-2, which simultaneously revives and expands antigen-primed exhausted T cells. The IL-2 portion of TILKine-2 was engineered to have improved tolerability, slower receptor-mediated clearance, and prolonged half-life.MethodsTarget binding of TILKine-2 was evaluated by cell-free and cell-based methods. In vitro functional characterization was performed using human peripheral blood mononuclear cells (PBMCs). Pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor activity of murine TILKine-2 surrogate (TILKine-2s) were evaluated in various syngeneic models. The safety and immune cell activation of TILKine-2 were assessed in non-human primates (NHPs).ResultsStructure-based design and activity-guided fine-tuning resulted in an optimized IL-2 variant that was fused to TILAb to generate TILKine-2. TILKine-2 demonstrated TIL-target antigen binding and blocking activity with sub-nM potency. TILKine-2 has a binding activity abolished to IL-2Rα and fine-tuned to IL-2Rβγ. In PBMCs, TILKine-2 potently induced intracellular signaling and cell proliferation in IL-2Rβγ dominant effector CD8+T and NK cells along with IFN-γ secretion. In vivo, TILKine-2 displayed significantly prolonged half-life with sustained proliferation, expansion, and Granzyme B expression on CD8+T and NK cells. Notably, the effects were more pronounced in the tumor than periphery, leading to massive immune hot tumors. Consequently, TILKine-2s exhibited robust anti-tumor primary and memory response in both cold and hot tumor models (MC38, CT26, B16F10, PAN02). Furthermore, TILKine-2s demonstrated superior and synergistic anti-tumor efficacy compared to TILAb alone, engineered IL-2 alone, or their combination, with 100% tumor regression resulting in ~80% tumor free mice in MC38 and Pan02 models. In NHPs, TILKine-2 preferentially induced memory CD8+T, total CD8+T, and NK cell expansion.