Targeting receptor activator of NF-kB pathways in combination immunotherapy

Abstract

Immunotherapy has transformed the treatment of various cancers, but the benefits are currently limited to a minority of cancer patients owing mainly to primary immunotherapy resistance. Research efforts to overcome such resistance are increasingly focused on combinations of immunotherapy approaches or the combination of immunotherapy with other anti-cancer treatments. Repurposing existing drugs for this purpose is an attractive approach, given that the safety and pharmacokinetic profile of established drugs are well-characterised. This means that translation to the clinic will be more rapid, with a safety profile usually more predictable. Receptor activator of NF-κB (RANK) and the RANK ligand (RANKL) were discovered in the setting of the immune system, namely T cell–dendritic cell (DC) interactions. However, early research revealed the obligate role of RANK signalling in osteoclastogenesis. This discovery led to the development of the anti-RANKL antibody denosumab for antiresorptive indications. Randomized clinical trials and post-marketing surveillance studies have established the acceptable safety profile of denosumab in both malignant and non-malignant settings, such as bone metastases and osteoporosis. More recently, case reports involving patients with advanced-stage melanoma have described remarkable responses following concurrent treatment with denosumab and immune-checkpoint inhibitors (ICI), suggesting that RANKL inhibition may be an effective combinatorial partner in immunotherapy. However, the broader efficacy and mechanism of action of such combinations in the tumor setting is unknown. Herein, we show that combinations of RANKL blockade with various ICI and ICI combinations result in superior anti-tumor outcomes in mouse models of cancer. The efficacy and mechanism of a combination of RANKL and ICI was examined by tumor infiltrating lymphocyte analysis, and analysis of tumor growth and metastasis using a variety of neutralizing antibodies and gene-targeted mice. In Chapter 3, RANKL inhibition combined with anti-CTLA4 improved the efficacy of anti-CTLA4 in mouse models of prostate cancer and melanoma. This combination required the presence of Fc receptors and lymphocytes (NK cells for metastases and predominantly CD8+ T cells for subcutaneous tumor control). The distribution of RANK and RANKL on tumor-infiltrating lymphocytes (TILs) and immune cells was defined: RANKL was expressed by T cells whereas the majority of RANK expression was by cells of the myeloid compartment such as macrophages and myeloid-derived suppressor cells (MDSCs). Combination RANKL and CTLA4 blockade resulted in increased T-cell influx into tumors and improved polyfunctional cytokine production by those T-cell TILs. In Chapter 4, the efficacy of anti-RANKL in combination with other ICI such as anti-PD1, anti-PD-L1 and dual ICI (anti-PD1 plus anti-CTLA4) was explored in mouse models of melanoma, colon cancer, prostate cancer and lung cancer in metastatic and subcutaneous tumor settings. RANKL blockade improved the efficacy of antibodies targeting PD1/PD-L1 and dual ICI. The combination of RANKL with these ICI required IFNγ and lymphocytes (predominantly NK cells for metastasis and T cells for subcutaneous models). Early-duringtreatment assessment of TILs revealed that combining RANKL blockade with dual ICI increased the polyfunctional cytokine production of T-cell TILs early after the first dose was administered. Furthermore, RANKL expression was shown to identify tumor-specific T-cells with high PD-1 expression which can be modified by anti-PD1 therapy. In Chapter 5, the preclinical research was extended to the development of a novel phase IB/II clinical trial assessing the pharmacodynamics and efficacy of denosumab when added to nivolumab in the neoadjuvant treatment of resectable non-small cell lung cancer (NSCLC). This is an open-label, multicenter randomized two-arm study of two neoadjuvant doses of nivolumab with or without denosumab. The primary endpoint is pharmacodynamics of the dual combination therapy compared with monotherapy (tumor-immune correlates and mechanism of action) with key secondary and exploratory endpoints relating to safety, feasibility and efficacy. In summary, the findings presented in this thesis contribute to knowledge of the role of RANKL/RANK in tumor immunity, and present a rationale and platform for the assessment of combination RANKL inhibition concurrently with ICI in patients with cancer, including NSCLC.