Abstract A074: Discovery of WTX-330, a clinical stage conditionally activated IL-12 INDUKINETM therapeutic with potent antitumor activity in murine syngeneic tumor models resistant to checkpoint blockade
Authors: Nirschl Christopher J , Brodkin Heather R , Chopra Sameer S , Dwyer Connor J , Hicklin Daniel J , Isaacs Randi , Ismail Nesreen , Seidel-Dugan Cynthia , Steuert Zoe , Sullivan Jenna M , Winston William M , Salmeron Andres 
Published: December 2023

Abstract
Background: IL-12 is a pleotropic inflammatory cytokine with broad stimulatory effects on immune cells and robust antitumor activity in syngeneic murine tumor models, making it an attractive target for cancer immunotherapy. Unfortunately, systemic administration of recombinant human IL-12 causes severe toxicity in patients. With the goal of improving the therapeutic index for IL-12 therapy, we have engineered an IL-12 prodrug referred to as an INDUKINETM molecule that is dosed systemically but designed to be selectively activated in tumors.
Methods: Mice bearing MC38, CT26, B16F10, and EMT-6 syngeneic tumors were treated with surrogate mWTX-330 (a chimeric IL-12 containing INDUKINETM molecule), and tumor growth and body weight were monitored over time. Tumor tissues were harvested at various timepoints and analyzed by flow cytometry, NanoString GeoMx Digital Spatial Profiling (DSP), and high-plex immunofluorescence to characterize the mechanisms associated with response.
Results: mWTX-330 is comprised of a half-life extension domain and an inactivation domain tethered to chimeric IL-12 by tumor protease-sensitive linkers. These linkers were chosen for their selective processing by a majority of human tumors in vitro as well as mouse tumors in vitro and in vivo. The linkers have been shown in preclinical studies to remain stable in normal human tissues including serum. Systemic administration of mWTX-330 in mice was well tolerated, resulted in robust antitumor immunity in multiple tumor models, including those resistant to checkpoint blockade, and preferentially activated tumor-infiltrating immune cells versus peripheral cells. Antitumor activity was dependent on in vivo processing of the protease cleavable linkers and required CD8+ T cells for full efficacy, although NK and CD4+ T cells also contributed to tumor growth inhibition. Within the tumor, mWTX-330 increased the frequency of cross-presenting dendritic cells (DCs), activated natural killer (NK) cells, skewed conventional CD4+ T cells toward a T helper 1 (TH1) phenotype, drove regulatory T cells (Treg) fragility, and increased the frequency of polyfunctional CD8+ T cells. mWTX-330 treatment also increased the clonality of tumor-infiltrating T cells by expanding underrepresented T-cell receptor (TCR) clones, drove CD8+ T and NK cells towards increased mitochondrial respiration and fitness, and decreased the frequency of TOX+ exhausted CD8+ T cells within the tumor.
Conclusion: Based on the strong biological rational and preclinical studies of surrogate mWTX-330, we created WTX-330, a fully human IL-12 INDUKINETM molecule that is stable in human serum and is reliably and selectively processed by human tumor samples in vitro. A first-in-human clinical trial to evaluate the safety profile, pharmacokinetics, and preliminary antitumor activity of WTX-330 in patients with relapsed/refractory advanced or metastatic solid tumors or non-Hodgkin lymphoma is now underway (NCT05678998).
Citation Format: Christopher J Nirschl, Heather R Brodkin, Sameer S Chopra, Connor J Dwyer, Daniel J Hicklin, Randi Isaacs, Nesreen Ismail, Cynthia Seidel-Dugan, Zoe Steuert, Jenna M Sullivan, William M Winston, Andres Salmeron. Discovery of WTX-330, a clinical stage conditionally activated IL-12 INDUKINETM therapeutic with potent antitumor activity in murine syngeneic tumor models resistant to checkpoint blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A074.