A modified IL-18 drug in combination with CTLA-4 blockade enhances anti-tumor efficacy in preclinical models of renal cell carcinoma
Authors: Schoenfeld David , Djureinovic Dijana , Zhang Lin , Mann Jacqueline , Huck John , Jilaveanu Lucia , Ring Aaron , Kluger Harriet 
Published: August 2023

Abstract

Background
Cytokine-based drugs are currently being explored as alternative cancer immunotherapies. While the cytokine interleukin-18 (IL-18) has immunostimulatory effects, it is negatively regulated by a secreted high-affinity binding protein, IL-18BP, that functions as an immune checkpoint that limits IL-18’s efficacy as a cancer therapeutic. A modified version of IL-18, termed “decoy-resistant” or DR-18, that can avoid trapping by IL-18BP while still maintaining its immune signaling potential, has recently been developed. DR-18 has shown promising preclinical activity in melanoma and colorectal murine models, including potential synergy with anti-PD-1 therapy, and is currently in Phase I trials. In this study, we aim to test the efficacy and determine the cellular mechanism of action of DR-18 in combination with immune checkpoint inhibitors (ICIs) in immunocompetent preclinical models of renal cell carcinoma (RCC), with the goal of establishing the basis for testing these combinations in early phase clinical trials.

Methods
We engrafted tumors subcutaneously using two different syngeneic, immunocompetent murine RCC models: Renca and RAG. Mice were treated with single-agent DR-18 and combinations of DR-18 with single- and dual-agent anti-PD-1 and anti-CTLA-4. Tumor growth and survival were monitored. In the Renca model, plasma was collected at early time-points and cytokine/chemokine levels were profiled using a 31-plex discovery assay. Single-cell RNA and TCR sequencing was also performed on Renca tumors. Additionally, immune cell depletion studies were conducted in the Renca model with antibodies targeting CD8, CD4, NK cells, and interferon-gamma.

Results
In the Renca model, DR-18 monotherapy modestly inhibited tumor growth and prolonged survival. The effects were comparable to single- and dual-agent ICIs. Adding PD-1 blockade to DR-18 did not enhance efficacy whereas the addition of anti-CTLA-4 to DR-18 significantly increased anti-tumor effects. Triple-therapy (DR-18 plus anti-PD-1 plus anti-CTLA-4) did not further inhibit tumor growth or prolong survival compared to the doublet (DR-18 plus anti-CTLA-4). The RAG model was more sensitive to ICIs but produced similar results, again showing modest anti-tumor activity of single-agent DR-18 and enhanced benefit of combining with anti-CTLA-4 but not anti-PD-1. Cytokine/chemokine profiling revealed significantly elevated levels of IP-10 (CXCL10) and MIG (CXCL9) after one cycle of DR-18 plus anti-CTLA-4 compared to control and single-agent treatments, suggesting that these chemokines may be key early mediators of the anti-tumor immune response. Single-cell transcriptomic analysis demonstrated changes in intra-tumoral T cell, macrophage, and granulocyte populations with DR-18 plus anti-CTLA-4 relative to other regimens, including enrichment of CD8+ precursor and terminally exhausted T cells and a neutrophil population associated with interferon signaling. Additionally, single-cell TCR analysis showed a reduction in intra-tumoral clonotype diversity with DR-18 plus anti-CTLA-4 compared to other treatments. Immune cell depletion studies identified CD8+ T cells, NK cells, and interferon-gamma, but not CD4+ T cells, as equally required for efficacy of DR-18 plus anti-CTLA-4.

Conclusions
In this study, we identify DR-18, an IL-18-based drug engineered with resistance to a secreted decoy-receptor protein, in combination with anti-CTLA-4 as having enhanced anti-tumor activity in preclinical models of RCC. This regimen was associated with a more pro-inflammatory immune microenvironment. Further investigation is ongoing to elucidate the cellular mechanism of action of this regimen more fully and lay the groundwork for clinical testing of DR-18-based combination therapy in RCC. In the future, testing novel partner agents outside of anti-PD-1/CTLA-4 and using RCC models of ICI-resistance could be particularly informative and clinically relevant.