{
  "abstract": "Background Cytokine prodrugs represent a promising strategy in the field of cancer immunotherapy, aiming to bolster antitumor immunity while minimizing systemic toxicity. WTX-124 is an engineered IL-2 prodrug designed to be inactive in the periphery and activated solely in the tumor microenvironment (TME) by dysregulated proteases to improve safety without compromising the efficacy of high-dose IL-2. Although the potent anti-tumor effect of WTX-124 has been confirmed in patients and preclinical models, traditional preclinical assessments have relied heavily on static tumor homogenate analysis, hindering the ability to understand the spatiotemporal dynamics of intact (inactive) and cleaved (active/free or released IL-2) drug concentrations and their effects at the tumor site.Methods To address this limitation, we employed a large pore microdialysis technique in a syngeneic murine tumor model, enabling real-time monitoring of IL-2 INDUKINE pharmacology directly at the site of action. Tumor-bearing mice received either vehicle, a single dose of WTX-124, or its non-cleavable analog (NC WTX-124) intravenously (IV). Tumor interstitial fluid (TIF) and plasma were collected at various time points, and tumor tissues were harvested at the end of treatment for immunoassay and multiplex immunofluorescence (IF) analysis.Results A single IV dose of WTX-124 or NC WTX-124 led to consistently low and comparable levels of active IL-2 in plasma, suggesting that prodrug conversion to active cytokine does not occur systemically. Both the intact prodrug and non-cleavable variant exhibited continuous uptake and accumulation in the tumor; however, only treatment with INDUKINE resulted in a significant, time-dependent increase in tumor active IL-2 exposure. In stark contrast, the NC WTX-124 did not yield detectable active IL-2 levels, highlighting the importance of linker cleavage for effective payload release. Additionally, cytokine profiling of the TIF showed a notable increase in IFNg production that correlated with free IL-2 exposure, indicating robust immune activation associated with WTX-124. Ex vivo multiplex IF analysis of tumor tissues revealed an elevation in the density of infiltrating immune cells, including natural killer cells and CD8+ T cells, as well as enhanced expression of key effector molecules such as IFNg, Granzyme B, and Perforin in response to active IL-2 derived from WTX-124. Importantly, this vigorous immune response was not observed in tumors treated with NC WTX-124, suggesting that effective immune modulation is driven by localized IL-2 release.Conclusions These preclinical data clearly demonstrate tumor-selective activation of WTX-124, further substantiating the INDUKINE prodrug design capable of harnessing enzymatic cleavage mechanisms for robust immune modulation.Ethics Approval All mouse in vivo work was performed in accordance with current regulations and standards of the U.S Department of Health and Human Services, Public Health Service (PHS), and the NIH Office of Laboratory Animal Welfare (OLAW). All animal studies were conducted at Charles River Laboratories Den Bosch B.V. (location Groningen, the Netherlands) with approval of the Charles River Laboratories Institutional Animal Care and Use Committees (IACUC).",
  "authors": [
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Celesztina Nagy-Domonkos"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Heather R Brodkin"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Daniel J Hicklin"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Jennifer Ngo"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Christopher J Nirschl"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Kulandayan K Subramanian"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Andres Salmeron"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "William M Winston"
    }
  ],
  "title": "862 Pharmacokinetic insight into the IL-2 INDUKINE prodrug WTX-124: Real-time assessment of tumor-specific activation and immune modulation",
  "uid": "2982cf0b-52bb-5176-8e95-8eeaa1ed741d"
}