{
  "abstract": "Background Though proinflammatory cytokines such as Interleukin-2 (IL-2) and Interleukin-12 (IL-12) have been shown to promote initiation and/or amplification of adaptive antitumor immune responses, routine use of the approved HD IL-2 regimen and clinical development of recombinant human IL-12 have been impeded by severe toxicities. We previously reported clinical data from novel tumor-activated INDUKINE molecules for native IL-2 (WTX-124) and IL-12 (WTX-330) demonstrating that each cytokine prodrug showed encouraging monotherapy clinical activity and tolerability in the outpatient setting with evidence of T cell activation and expansion in on-treatment tumor biopsies ( NCT05660384, NCT5678998).Methods Here we used IL-2 and IL-12 INDUKINE molecules to explore whether the mechanisms of action of IL-12 (e.g., generation of de novo antitumor responses through IFNγ-enhanced antigen presentation) and IL-2 (e.g., expansion and enhanced effector function of CD8 T cells) can be safely combined in syngeneic tumor models to improve antitumor activity, especially in poorly immunogenic tumors. We found that concurrent administration of WTX-124 and mouse WTX-330 (mWTX-330) improved antitumor activity over either cytokine alone but was poorly tolerated. Therefore, we explored whether sequential administration of WTX-330 and WTX-124 could similarly enhance antitumor activity but with less toxicity.Results In the EMT6 model, which is resistant to WTX-124, a single dose of mWTX-330 followed by WTX-124 up to one week later was well tolerated and demonstrated enhanced antitumor activity compared to vehicle, WTX-124 alone or a single dose of WTX-330. Multiplex immunofluorescence analysis of tumors revealed that mWTX-330 initially increased infiltration of CD103+ cross presenting dendritic cells (DC) and then subsequently increased the frequency of polyfunctional CD8 T cells and caused an M2 to M1 shift in tumor associated macrophage (TAM) phenotypes. Sequential dosing of WTX-124 further increased the number of polyfunctional CD8 T cells and M1 macrophages. At the end of the study, ELISpot analysis demonstrated that mWTX-330 had primed tumor specific immune responses, which were further amplified by WTX-124.Conclusions We conclude that sequential administration of mWTX-330 and WTX-124 takes advantage of the complementary mechanisms of action of native IL-12 and IL-2, eliciting marked antitumor activity with improved tolerability compared to concurrent dosing regimens. We believe that this approach may be directly translatable to the clinical setting.Ethics Approval All mouse in vivo work was performed in accordance with current regulations and standards of the U.S. Department of Agriculture and the NIH at Charles River Laboratories with the approval of an Institutional Animal Care and Use Committee.",
  "authors": [
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Julie LePrevost"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Kristin Morris"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Sameer S Chopra"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Daniel J Hicklin"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Randi E Isaacs"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Christopher J Nirschl"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Andres Salmeron"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "Kulandayan K Subramanian"
    },
    {
      "affiliations": [
        "Werewolf Therapeutics, Watertown, MA, USA"
      ],
      "name": "William M Winston"
    }
  ],
  "title": "861 Sequential administration of WTX-124 and mWTX-330, IL-2 and IL-12 INDUKINE™ molecules, enhanced antitumor activity in mice bearing poorly immunogenic EMT6 tumors without systemic toxicity",
  "uid": "8c0e3b04-7beb-55b8-8054-ba570bbf8ec7"
}