William C. Dougall

Molecular Pathways: Osteoclast-Dependent and Osteoclast-Independent Roles of the RANKL/RANK/OPG Pathway in Tumorigenesis and Metastasis

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a TNF ligand superfamily member that is essential for the formation, activation, and function of osteoclasts. RANKL functions via its cognate receptor RANK, and it is inhibited by the soluble decoy receptor osteoprotegerin (OPG). In skeletal metastases, the ratio of RANKL to OPG is upregulated, which leads to increased osteoclast-mediated bone destruction. These changes in the bone microenvironment not only compromise the structural integrity of bone, leading to severe clinical morbidities, but have also been implicated in establishment of de novo bone metastasis and the progression of existing skeletal tumors. Evaluation of RANKL inhibitors, including the fully human anti-RANKL antibody denosumab, in patients with cancer has shown reductions in tumor-induced bone resorption activity and successful management of skeletal complications of bone metastases. RANKL also functions as a major paracrine effector of the mitogenic action of progesterone in mouse mammary epithelium, and it has a role in ovarian hormone-dependent expansion and regenerative potential of mammary stem cells. RANKL inhibition attenuates mammary tumorigenesis and pulmonary metastases in mouse models. These data suggest that the contribution of progesterone to increased mammary cancer incidence is mediated, at least in part, by RANKL-dependent changes in the mammary epithelium; RANKL also directly promotes distant metastases. In summary, the antitumor and antimetastatic effects of RANKL inhibition can occur by at least 2 distinct mechanisms, one in the bone via osteoclast-dependent effects, and the second via direct effects on the tumor cells of various origins and/or mammary epithelium. Clin Cancer Res; 18(2); 326–35. ©2011 AACR.

Molecular Pathways: Targeting CD96 and TIGIT for Cancer Immunotherapy.

The receptors CD96 and TIGIT are expressed on the surface of T and natural killer (NK) cells, and recent studies suggest both play important inhibitory roles in immune function. CD96 has been shown to modulate immune cell activity in mice, with Cd96−/− mice displaying hypersensitive NK-cell responses to immune challenge and significant tumor resistance. TIGIT overexpression has been shown to reduce NK-cell–mediated cytotoxicity. TIGIT is also upregulated on T cells during cancer and chronic viral infection, with expression associated with effector T-cell exhaustion and increased regulatory T-cell suppression. The counterbalance between the putative inhibitory CD96 and TIGIT receptors and the activating receptor, CD226, offers unique strategies for immuno-oncology drug development. Blocking CD96 or TIGIT with mAbs has been shown to improve tumor control in mice, in particular when used in combination with PD-1/PD-L1 blockade. These results have highlighted these pathways as promising new targets for immune modulation. This review will examine the rationale behind targeting CD96 and TIGIT, and discuss the potential approaches in translating these preclinical findings into novel clinical agents. Clin Cancer Res; 22(21); 5183–8. ©2016 AACR.

TIGIT and CD96: new checkpoint receptor targets for cancer immunotherapy

While therapies targeting the co‐inhibitory or immune checkpoint receptors PD‐1 and CTLA‐4 have shown remarkable success in many cancers, not all patients benefit from these therapies. This has catalyzed enormous interest in the targeting of other immune checkpoint receptors. In this regard, TIGIT and CD96 have recently entered the limelight as novel immune checkpoint receptor targets. TIGIT and CD96 together with the co‐stimulatory receptor CD226 form a pathway that is analogous to the CD28/CTLA‐4 pathway, in which shared ligands and differential receptor:ligand affinities fine‐tune the immune response. Although the roles of TIGIT and CD96 as immune checkpoint receptors in T cell and natural killer cell biology are just beginning to be uncovered, accumulating data support the targeting of these receptors for improving anti‐tumor immune responses. A clear understanding of the immune cell populations regulated by TIGIT and CD96 is key to the design of immunotherapies that target these receptors in combination with other existing immune checkpoint blockade therapies.

Co-administration of RANKL and CTLA4 Antibodies Enhances Lymphocyte-Mediated Antitumor Immunity in Mice

Purpose: Novel partners for established immune checkpoint inhibitors in the treatment of cancer are needed to address the problems of primary and acquired resistance. The efficacy of combination RANKL and CTLA4 blockade in antitumor immunity has been suggested by recent case reports in melanoma. Here, we provide a rationale for this combination in mouse models of cancer. Experimental Design: The efficacy and mechanism of a combination of RANKL and CTLA4 blockade was examined by tumor-infiltrating lymphocyte analysis, tumor growth, and metastasis using a variety of neutralizing antibodies and gene-targeted mice. Results: RANKL blockade improved the efficacy of anti-CTLA4 mAbs against solid tumors and experimental metastases, with regulatory T-cell (Treg)–depleting anti-CTLA4 mAbs of the mouse IgG2a isotype showing greatest combinatorial activity. The optimal combination depended on the presence of activating Fc receptors and lymphocytes (NK cells for metastatic disease and predominantly CD8+ T cells for subcutaneous tumor control), whereas anti-RANKL alone did not require FcR. The significantly higher T-cell infiltration into solid tumors post anti-RANKL and anti-CTLA4 was accompanied by increased T-cell effector function (cytokine polyfunctionality), and anti-RANKL activity occurred independently of Treg depletion. The majority of RANKL expression in tumors was on T cells whereas RANK-expressing cells were mostly tumor-associated macrophages (TAM), with some expression also observed on dendritic cells (DC) and myeloid-derived suppressor cells (MDSC). Conclusions: These results provide a rationale for the further investigation of RANKL–RANK interactions in tumor immunity and a basis for development of translational markers of interest in human clinical trials. Clin Cancer Res; 23(19); 5789–801. ©2017 AACR.

CD155 loss enhances tumor suppression via combined host and tumor-intrinsic mechanisms

Critical immune-suppressive pathways beyond programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) require greater attention. Nectins and nectin-like molecules might be promising targets for immunotherapy, since they play critical roles in cell proliferation and migration and exert immunomodulatory functions in pathophysiological conditions. Here, we show CD155 expression in both malignant cells and tumor-infiltrating myeloid cells in humans and mice. Cd155–/– mice displayed reduced tumor growth and metastasis via DNAM-1 upregulation and enhanced effector function of CD8+ T and NK cells, respectively. CD155-deleted tumor cells also displayed slower tumor growth and reduced metastases, demonstrating the importance of a tumor-intrinsic role of CD155. CD155 absence on host and tumor cells exerted an even greater inhibition of tumor growth and metastasis. Blockade of PD-1 or both PD-1 and CTLA4 was more effective in settings in which CD155 was limiting, suggesting the clinical potential of cotargeting PD-L1 and CD155 function.

RANKL blockade improves efficacy of PD1-PD-L1 blockade or dual PD1-PD-L1 and CTLA4 blockade in mouse models of cancer

ABSTRACT Receptor activator of NF-κB ligand (RANKL) and its receptor RANK, are members of the tumor necrosis factor and receptor superfamilies, respectively. Antibodies targeting RANKL have recently been evaluated in combination with anti-CTLA4 in case reports of human melanoma and mouse models of cancer. However, the efficacy of anti-RANKL in combination with antibodies targeting other immune checkpoint receptors such as PD1 has not been reported. In this study, we demonstrated that blockade of RANKL improves anti-metastatic activity of antibodies targeting PD1/PD-L1 and improves subcutaneous growth suppression in mouse models of melanoma, prostate and colon cancer. Suppression of experimental lung metastasis following combination anti-RANKL with anti-PD1 requires NK cells and IFN-γ, whereas subcutaneous tumor growth suppression with this combination therapy is attenuated in the absence of T cells and IFN-γ. Furthermore, addition of anti-RANKL to anti-PD1 and anti-CTLA4 resulted in superior anti-tumor responses, irrespective of the ability of anti-CTLA4 isotype to engage activating FcR, and concurrent or delayed RANKL blockade was most effective. Early-during-treatment assessment reveals this triple combination therapy compared to dual anti-PD1 and anti-CTLA4 combination therapy further increased the proportion of tumor-infiltrating CD4+ and CD8+ T cells that can produce both IFN-γ and TNF. Finally, RANKL expression appears to identify tumor-specific CD8+ T cells expressing higher levels of PD1 which can be modulated by anti-PD1. These data set the scene for clinical evaluation of denosumab use in patients receiving contemporary immune checkpoint blockade.

CD96 targeted antibodies need not block CD96-CD155 interactions to promote NK cell anti-metastatic activity

ABSTRACT CD96 is a transmembrane glycoprotein Ig superfamily receptor, expressed on various T cell subsets and NK cells, that interacts with nectin and nectin-like proteins, including CD155/polio virus receptor (PVR). Here, we have compared three rat anti-mouse CD96 mAbs, including two that block CD96-CD155 (3.3 and 6A6) and one that does not block CD96-CD155 (8B10). Using flow cytometry, we demonstrated that both mAbs 3.3 and 6A6 bind to the first Ig domain of mouse CD96 and compete with CD155 binding, while mAb 8B10 binds to the second Ig domain and does not block CD155. While Fc isotype was irrelevant concerning the anti-metastatic activity of 3.3 mAb, in four different experimental metastases models and one spontaneous metastasis model, the relative order of anti-metastatic potency was 6A6 > 3.3 > 8B10. The metastatic burden control of all of the anti-CD96 clones was highly dependent on NK cells and IFN-γ. Consistent with its inability to block CD96-CD155 interactions, 8B10 retained anti-metastatic activity in CD155-deficient mice, whereas 3.3 and 6A6 lost potency in CD155-deficient mice. Furthermore, 8B10 retained most of its anti-metastatic activity in IL-12p35-deficient mice whereas the activity of 3.3 and 6A6 were partially lost. All three mAbs were inactive in CD226-deficient mice. Altogether, these data demonstrate anti-CD96 need not block CD96-CD155 interactions (ie. immune checkpoint blockade) to promote NK cell anti-metastatic activity.

TIGIT immune checkpoint blockade restores CD8+ T cell immunity against multiple myeloma

Immune-based therapies hold promise for the treatment of multiple myeloma (MM), but so far, immune checkpoint blockade targeting programmed cell death protein 1 has not proven effective as single agent in this disease. T-cell immunoglobulin and ITIM domains (TIGIT) is another immune checkpoint receptor known to negatively regulate T-cell functions. In this study, we investigated the therapeutic potential of TIGIT blockade to unleash immune responses against MM. We observed that, in both mice and humans, MM progression was associated with high levels of TIGIT expression on CD8+ T cells. TIGIT+ CD8+ T cells from MM patients exhibited a dysfunctional phenotype characterized by decreased proliferation and inability to produce cytokines in response to anti-CD3/CD28/CD2 or myeloma antigen stimulation. Moreover, when challenged with Vk*MYC mouse MM cells, TIGIT-deficient mice showed decreased serum monoclonal immunoglobulin protein levels associated with reduced tumor burden and prolonged survival, indicating that TIGIT limits antimyeloma immune responses. Importantly, blocking TIGIT using monoclonal antibodies increased the effector function of MM patient CD8+ T cells and suppressed MM development. Altogether our data provide evidence for an immune-inhibitory role of TIGIT in MM and support the development of TIGIT-blocking strategies for the treatment of MM patients.

Deficiency of host CD96 and PD-1 or TIGIT enhances tumor immunity without significantly compromising immune homeostasis

ABSTRACT Multiple non-redundant immunosuppressive pathways co-exist in the tumor microenvironment and their co-targeting can increase clinical responses. Indeed, concurrent blockade of CTLA-4 and PD-1 in patients with advanced melanoma increased clinical responses over monotherapy alone although the frequency and severity of immune related adverse events (irAEs) also increased. Nevertheless, a substantial number of patients still display an innate resistance phenotype and are unresponsive to current approved immunotherapies even when utilized in combination. In this study, we generated Pdcd1−/−CD96−/− and Tigit−/−CD96−/− mice to investigate how loss of CD96 in combination with PD-1 or TIGIT impacts on immune homeostasis and hence the potential of inducing immune related toxicities following co-targeting of these pairs of receptors. The ability of Pdcd1−/−CD96−/− and Tigit−/−CD96−/− mice to suppress primary tumor growth was also assessed using the MC38 colon carcinoma and SM1WT1 BRAF-mutated melanoma tumor models. Both Pdcd1−/−CD96−/− or Tigit−/−CD96−/− mice displayed no overt perturbations in immune homeostasis over what was previously reported with Pdcd1−/− or Tigit−/− mice even when aged for 22 months. Interestingly, increased suppression of subcutaneous tumor growth and complete responses was seen in Pdcd1−/−CD96−/− mice compared to Pdcd1−/− or CD96−/− mice depending upon the tumor model. In contrast, in these models, growth suppression in Tigit−/−CD96−/− were similar to Tigit−/− or CD96−/− . This enhanced anti-tumor efficacy of Pdcd1−/−CD96−/− appeared to be due to favorable changes in the ratio of CD8+ T cells to T regulatory cells or CD11b+GR-1hi myeloid cells in the tumor microenvironment. Co-targeting CD96 and PD-1 may increase anti-tumor immunity over targeting PD-1 alone and potentially not induce serious immune-related toxicities and thus appears a promising strategy for clinical development.

Roles of the RANKL–RANK axis in antitumour immunity — implications for therapy

Recognizing that the transformative effects of immunotherapy are currently limited to a minority of patients with cancer, research efforts are increasingly focused on expanding and enhancing clinical responses by combining immunotherapies; the repurposing of existing drugs is an attractive approach, given their well-characterized safety and pharmacokinetic profiles. Receptor activator of nuclear factor-κB (RANK) and the RANK ligand (RANKL) were initially described in the context of T cell–dendritic cell interactions; however, the discovery of an obligate role of RANK signalling in osteoclastogenesis led to the development of the anti-RANKL antibody denosumab for antiresorptive indications, including bone metastases. Randomized clinical trials and post-marketing surveillance studies have established the acceptable safety profile of denosumab. More recently, several case reports involving patients with advanced-stage melanoma have described remarkable responses following concurrent treatment with denosumab and immune-checkpoint inhibitors. Randomized trials assessing similar combinations in patients with melanoma or renal cell carcinoma are now underway. Herein, we discuss the hallmark clinical trials of denosumab in light of possible immunological effects of this agent. We highlight the role of immune cells as sources of RANK and RANKL in the tumour microenvironment and review data on RANKL inhibition in mouse models of cancer. Finally, we describe hypothetical immune-related mechanisms of action, which could be assessed in clinical trials of immune-checkpoint inhibitors and denosumab in patients with cancer.Intriguing evidence suggests that expression of RANK or RANKL by various cells of the tumour microenvironment modulates the anticancer immune response. Herein, the authors review this evidence, discuss the current preclinical and clinical data supporting a potential of RANKL inhibition to improve anticancer immunotherapy and describe hypothetical immune-related mechanisms of action.Key pointsReceptor activator of nuclear factor-κB ligand (RANKL) and its cognate receptor, RANK, are expressed by distinct immune cells in the tumour microenvironment (TME) and might also be expressed in tumour cells, the stroma, and non-malignant tissues.Accumulating observational and preclinical evidence suggests that RANKL–RANK interactions between cells in the TME have immunosuppressive effects.Endogenous inhibitors of RANKL include soluble osteoprotegerin (OPG) and leucine-rich repeat-containing G protein-coupled receptor 4; pharmacological inhibitors include denosumab (a monoclonal antibody) and an OPG–Fc fusion protein (discontinued from clinical development after phase I trials).Denosumab is FDA approved for indications including the prevention of skeletal-related events arising from bone metastases in cancer; however, several trials are now testing the immune anticancer activities of denosumab, including as a partner to immune-checkpoint inhibitors.Among immune cells infiltrating human or mouse cancers, RANK can be expressed on immature dendritic cells, immunosuppressive M2-type macrophages, myeloid-derived suppressor cells, and natural killer cells, whereas CD8+ and CD4+ T cells (including regulatory T cells) can express RANKL.Possible mechanisms whereby RANKL inhibition could improve the effects of immune-checkpoint inhibition in cancer include interruption of an immunosuppressive myeloid–lymphocyte axis, cross-modulation of the TME, and interruption of central tolerance.