Cancer treatment using NUC-7738

Abstract

The present invention provides NUC-7738 (3 '-deoxyadenosine-5'-O-[phenyl (benzyloxy-L-alanine)] phosphate), a phosphoramidate derivative of 3 '-deoxyadenosine for use in the treatment of proliferative diseases, such as cancer, by reducing the amount of extracellular PD-L1 protein (e.g., soluble PD-L1 or exosome PD L1) and / or increasing the amount of OX40-L. The invention also relates to the use of NUC-7738 in combination with an immune tumor agent, such as an immune checkpoint inhibitor.

Description

Technical Field

[0001] The present invention relates to medical uses and methods for treating proliferative diseases (such as cancer) by reducing the amount of extracellular PD-L1 protein (such as soluble PD-L1 and / or exosomal PD-L1 protein) produced by proliferative disease (such as cancer) cells and / or increasing the amount of OX40L produced by the cells. The inventors found that the protein molecule NUC-7738 reduced the amount of soluble PD-L1 and / or exosomal PD-L1 protein produced by cancer cells and increased the amount of OX40L produced by cancer cells. This observation provides new clinical opportunities for NUC-7738 to treat cancer. The present invention also relates to the use of NUC-7738 in combination with immune checkpoint inhibitors (ICIs) (such as PD-1 or PD-L1 inhibitors) to treat proliferative diseases (such as cancer, such as melanoma and lung cancer). Due to its ability to reduce the amount of PD-L1 protein produced by cancer cells, it opens the ability to use the NUC-7738 / immune checkpoint inhibitor combination in patients who have developed resistance to immune checkpoint blockade, patients who are tolerant to immune checkpoint blockade, patients who have relapsed after immune checkpoint inhibitor therapy, or patients who are not considering immune checkpoint inhibitor therapy due to concerns about non-response to therapy. Suitably, this is possible because the combination with NUC-7738 is expected to allow the use of lower doses of immune checkpoint inhibitors. Background Art

[0002] 3'-deoxyadenosine (3'-dA) is a nucleoside analog of adenosine, lacking a 3'-hydroxyl group on the ribose moiety at the 3' position, and can be produced synthetically from adenosine. References to this synthetic procedure are Robins, JR et al., J. Org. Chem. 1995, 60, 7902-7908 and Aman, S. et al., Organic Process Research & Development 2000, 4, 601-605.

[0003] 3'-dA has been extensively studied as an anticancer agent, but, despite showing potent anticancer activity in preclinical studies, it has not yet been successfully developed or approved as an anticancer agent.

[0004] Due to its structure, 3'-dA and its phosphorylated forms (3'-dA-monophosphate, diphosphate and triphosphate) may interfere with any process that requires adenosine or adenosine monophosphate, diphosphate or triphosphate (AMP, ADP or ATP), respectively. However, after administration, 3'-dA is rapidly deaminated by adenosine deaminase (ADA) in vivo and rapidly metabolized to the inactive metabolite 3'-deoxyinosine. Reference Tsai, YJ et al., J. Agri. Food Chem. 58 4638-43 (2010).

[0005] NUC-7738 (3'-deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alanine)] phosphate) is a phosphoramidate conversion of the monophosphate of the nucleoside 3'-dA. Unlike 3'-dA, it is able to enter cells independently of transporters, and the production of 3'-dAMP is independent of the activation of the enzyme adenosine kinase (AK), which is the rate-limiting process in the formation of the phosphorylated form of 3'-dA. It is also not broken down by the enzyme adenosine deaminase (ADA), thus resulting in higher intracellular 3'-dATP levels. Therefore, NUC-7738 can bypass the transport, activation, and breakdown mechanisms involved in limiting the utilization of 3'-dA.

[0006] Checkpoint inhibitors are a type of cancer immunotherapy that targets immune checkpoints, which are key regulators of the immune system that can suppress immune responses to cancer when stimulated. These immune checkpoints co-evolved with stimulatory immune receptors as a way to control immune responses and prevent potentially dangerous overreactions. Cytotoxic T cells express the immune checkpoint protein PD-1 on their cell surface. If it binds to its ligand PD-L1, this signals to the T cell that it should not be activated, even if the T cell has recognized something it would normally kill.

[0007] In addition to PD-1 and PD-L1, other proteins expressed by immune and non-immune cells (including cancer cells) that are part of the immune checkpoint signaling pathway include CTLA-4, LAG-3, TIGIT, BTLA, OX40 / OX40L, and TIM-3. Cancers can manipulate this system to protect themselves from immune attack by expressing proteins that are part of the immune checkpoint signaling pathway.

[0008] Checkpoint inhibitor therapy can block inhibitory checkpoints, restore immune system function, and lead to immune-mediated cancer cell death. Current approaches focus on using PD-1 / PD-L1 inhibitors as monotherapy or in combination with checkpoint inhibitors that target another part of the checkpoint signaling pathway, such as CTLA-4 inhibitors. Checkpoint inhibitor therapy can also be used in combination with chemotherapy or targeted therapy.

[0009] Resistance to checkpoint inhibitors can either exist before treatment with a checkpoint inhibitor (primary resistance) or develop after treatment (acquired resistance). Patients with primary resistance do not respond to initial treatment and their cancer continues to grow, while patients with acquired resistance initially respond to treatment, but their disease subsequently progresses as resistance mechanisms take effect.

[0010] PD-L1 is expressed on the surface of tumor cells, immune cells, and other cells in the tumor microenvironment, but it is also released from tumor cells and exists in several extracellular forms (see Daassidengr, "The importance of exosomal PDL1 in tumour immune evasion". Nature Reviews Immunology 20(4):209-215, 2020). This includes being found in plasma as a soluble protein (soluble PD-L1 or sPD-L1) or associated with exosomes (a type of extracellular vesicle) (exosomal PD-L1 or xPD-L1). Exosomes are a form of extracellular vesicles that are produced by many cell types, especially at sites of inflammation or cancer. They are characterized by their size and shape, but also by their expression of CD81 on their surface. They are important in transmitting information between cells. Several studies have shown that soluble and exosomal PD-L1 can serve as decoys, effectively neutralizing the benefits of anti-PD-L1 immune checkpoint therapy (see Zhang et al., Cell Biosci 9:19, 2019).Soluble PD-L1 is a circulating programmed death ligand 1 that is measurable in the serum of patients with various types of cancer (Finkelmeier et al., “High levels of the soluble programmed death-ligand (sPD-L1) identify hepatocellular carcinoma patients with a poor prognosis”. Eur J Cancer. 59:152–9, 2016; Okuma et al., “High plasma levels of soluble programmed cell death ligand 1 are prognostic for reduced survival in advanced lung cancer”. Lung Cancer. 104:1–6, 2017; Chang et al., “The correlation and prognostic value of serum levels of soluble programmed death protein 1 (sPD-1) and soluble programmed death-ligand 1 (sPD-L1) in patients with hepatocellular carcinoma.”. Cancer Immunol Immunother. 68:353–63, 2019; Shigemori et al., “Soluble PD-L1 expression in circulation as a predictive marker for recurrence and prognosis in gastric cancer: direct comparison of the clinical burden between tissue and serum PD-L1 expression". Ann Surg Oncol. 26:876–83, 2019). Previous studies have shown that sPD-L1 may impair host immunity and lead to systemic immunosuppression, subsequently leading to cancer progression and poor clinical outcomes (Frigola et al., Clin Cancer Res. (2011) 17:1915–23, 2011). PD-L1 expressed on or released from cancer cells signals cytotoxic T cells in close proximity to them, inactivating them.Therefore, reducing exosomal PD-L1 released from tumors, or decreasing the amount of soluble PD-L1, are new potential targets for concomitant immune checkpoint blockade therapy.

[0011] PD-1 (also known as programmed cell death protein 1 and CD279) is a checkpoint protein on T cells that acts as an "off switch" to help prevent T cells from attacking normal, healthy cells in the body. It does this when it binds to PD-L1 (also known as programmed death ligand 1 and CD274), a protein found on some normal and cancer cells. When PD-1 binds to PD-L1 on a cell, it induces signals that instruct the T cell not to bother the other cell. Some cancer cells produce large amounts of PD-L1, which helps them avoid being recognized by the adaptive immune system and thus evade attack by the host immune system.

[0012] Immune checkpoint proteins [such as CTLA4 (also known as cytotoxic T-lymphocyte-associated protein 4 and CD152), LAG3 (also known as lymphocyte activation gene 3 and CD223), PD-1 and PD-L1, TIGIT, TIM-3, and BTLA] present on immune cells and / or cancer cells are molecular targets that have been found to play an important role in regulating anti-tumor immune responses. Inhibitors of these immune checkpoint proteins (such as CTLA4, LAG3, TIGIT, TIM-3, BTLA, PD-1, and / or PD-L1 inhibitors) promote anti-tumor immune responses and can be used to effectively treat certain forms of cancer.

[0013] OX40L, also known as TNFSF4 and CD252, is expressed on antigen presenting cells but is also found on many other cells, including melanoma cells. It binds to OX40, a member of the TNF receptor superfamily, on T lymphocytes and promotes the activation of effector T cells. Several clinical studies are currently underway investigating the possible role of OX40L as a way to enhance anti-tumor cell T lymphocyte responses, including in combination with drugs that block the PD-L1 / PD-1 axis.

[0014] The connection of OX40L to OX40 in T lymphocytes promotes the maintenance and generation of memory CD8+T cells. In addition, it has been shown that the induced expression of OX40L in tumors leads to the induction of antitumor immunity (Andarini, Sita et al., "Adenovirus vector-mediated in vivo gene transfer of OX40 ligand to tumor cells enhances antitumor immunity of tumor-bearing hosts." Cancer Research 64 (9): 3281-3287, 2004). Summary of the invention

[0015] The data provided in the Examples section herein show that NUC-7738 causes a reduction in mRNA and protein of extracellular forms of PD-L1 (particularly soluble and exosomal PD-L1). It is known that PD-L1 produced by cancer cells is important in helping cancer cells avoid host immune attack. Soluble PD-L1 and exosomal PD-L1 (types of extracellular PD-L1) can be used as bait proteins to block the activation of T cells and reduce their ability to kill tumor cells. Therefore, NUC-7738 can be used to reduce the production of this bait protein, making cancer cells more vulnerable to attack by the host immune system. The data provided in the Examples section show that NUC-7738 also causes an increase in the amount of OX40L produced by cancer cells. By reducing the amount of extracellular PD-L1 (e.g., soluble PD-L1 and / or exosomal PD-L1), and / or increasing the amount of OX40L produced by cancer cells, NUC-7738 exhibits the properties of an immune sensitizer, and can therefore be used alone or to enhance the therapeutic effects of tumor drugs or therapies, which may include immune checkpoint inhibitors (e.g., CTLA4, LAG3, PD-1 or PD-L1 inhibitors), as well as cancer vaccines and adoptive cell therapies, such as chimeric antigen receptor T cells (CAR-T), tumor infiltrating lymphocytes (TIL), and natural killer (NK) cell therapy. NUC-7738 may also be used to treat cancer patients with high levels of soluble PD-L1 and / or exosomal PD-L1 in plasma and / or tumors expressing low levels of OX40L, as well as for the treatment of cancers that are resistant to certain immune checkpoint inhibitors. In addition, by reducing the amount of the decoy protein PD-L1, it is possible to allow the use of lower doses of immune checkpoint inhibitors, and thus not only reduce the toxic side effects of such agents, but also allow patients who have had to stop treatment with immune checkpoint inhibitors due to toxicity, etc. to resume treatment with the or a certain immune checkpoint inhibitor, but with a smaller and less toxic dose. Therefore, NUC-7738 is suitable for use in combination with immune checkpoint inhibitors to treat cancer patients who may have stopped treatment with immune checkpoint inhibitors due to toxicity.

[0016] Therefore, the present disclosure relates in part to a method for treating a proliferative disease such as cancer. Suitably, the method comprises administering a therapeutically effective amount of a composition to a subject in need of such a composition, wherein the composition comprises NUC-7738. Suitably, the therapeutically effective amount is an amount capable of reducing the production of extracellular PD-L1 (such as soluble PD-L1 and / or exosome PD-L1) by cancer cells and / or increasing the production of OX40-L by cancer cells. In a specific embodiment, the NUC-7738 can reduce the level of soluble PD-L1 produced by cancer cells. In a specific embodiment, the NUC-7738 can reduce the level of exosome PD-L1 produced by cancer cells. In a specific embodiment, the NUC-7738 can increase the production of OX40-L by cancer cells. Suitably, the NUC-7738 enhances the adaptive immune system of the host. In a specific embodiment, the NUC-7738 acts as an immune sensitizer. In a specific embodiment, the NUC-7738 can be used to enhance the effect of immunotherapy. Suitably, immunotherapy is an immune checkpoint inhibitor or a cancer vaccine or adoptive cell therapy (also referred to as cellular immunotherapy), such as CAR-T cell therapy. In a specific embodiment, the NUC-7738 is used in combination with an immune checkpoint inhibitor. In a specific embodiment, the NUC-7738 is used in combination with an immune checkpoint inhibitor selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. In a specific embodiment, the NUC-7738 is used in combination with an anti-PD-1 antibody selected from pembrolizumab, cemiplimab, dostarlimab and nivolumab. In a specific embodiment, NUC-7738 is used in combination with pembrolizumab to treat cancer, such as cutaneous melanoma.

[0017] In various embodiments, the proliferative disease is a disease in which high levels of extracellular PD-L1 (e.g., soluble PD-L1 and / or exosomal PD-L1) contribute to the pathogenesis of the proliferative disease (e.g., cancer). In specific embodiments, high levels of soluble PD-L1 and / or exosomal PD-L1 inhibit or block the host immune system from attacking cancer cells. In specific embodiments, high levels of soluble PD-L1 and / or exosomal PD-L1 inhibit or block the effect of immune checkpoint inhibitors administered to patients. Therefore, in specific embodiments, NUC-7738 is used to treat patients whose cancer cells express high levels of extracellular PD-L1 (e.g., soluble PD-L1 and / or exosomal PD-L1) protein.

[0018] In a specific embodiment, the NUC-7738 reduces the expression of soluble PD-L1 and / or exosomal PD-L1 protein. In a specific embodiment, the NUC-7738 reduces the transcription of mRNA encoding soluble PD-L1 and / or exosomal PD-L1 protein.

[0019] In various embodiments, the proliferative disease is a disease in which low levels of OX40-L contribute to the pathogenesis of a proliferative disease (e.g., cancer). In specific embodiments, low levels of OX40-L inhibit or block the host immune system from attacking diseased cells. In specific embodiments, low levels of OX40-L inhibit or block the effect of immune checkpoint inhibitors administered to a patient. Therefore, in specific embodiments, NUC-7738 is used to treat patients whose cancer cells express low levels of OX40-L protein.

[0020] In a specific embodiment, the NUC-7738 increases the expression of OX40-L protein by cancer cells. In a specific embodiment, the NUC-7738 increases the translation of mRNA encoding OX40-L.

[0021] According to one aspect, the invention provides NUC-7738 for treating a proliferative disease such as cancer by reducing the amount of extracellular PD-L1 protein produced by diseased cells and / or increasing the amount of OX40-L protein produced by diseased cells. Suitably, the extracellular PD-L1 is a soluble PD-L1 protein and / or an exosomal PD-L1 protein. Suitably, the treatment is performed by adaptive immunity (e.g., cellular immunity and / or humoral immunity).

[0022] According to another aspect, the present invention provides a method for treating a proliferative disease such as cancer by reducing the amount of extracellular PD-L1 protein produced by proliferative disease cells and / or increasing the amount of OX40-L protein produced by proliferative disease cells in a patient, the method comprising administering a therapeutically effective amount of NUC-7738 to a patient in need thereof. Suitably, the extracellular PD-L1 is a soluble PD-L1 protein and / or an exosomal PD-L1 protein.

[0023] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for use in a method for treating a proliferative disease such as cancer by reducing the amount of extracellular PD-L1 protein produced by diseased cells and / or increasing the amount of OX40-L protein. Suitably, the extracellular PD-L1 is a soluble PD-L1 protein and / or an exosomal PD-L1 protein. Suitably, the proliferative disease is cancer. In a specific embodiment, the amount of extracellular PD-L1 protein, such as soluble PD-L1 protein and / or exosomal PD-L1 protein, produced by cancer cells is reduced, making the cancer cells more easily targeted by the host immune system (such as the patient's cellular immune system).

[0024] According to one aspect, the invention provides NUC-7738 for treating a proliferative disease, such as cancer, by reducing the amount of extracellular PD-L1 protein produced by diseased cells. Suitably, the extracellular PD-L1 is a soluble PD-L1 protein and / or an exosomal PD-L1 protein. Suitably, the treatment is performed through adaptive immunity (e.g., cellular immunity and / or humoral immunity).

[0025] According to another aspect, the present invention provides a method for treating cancer by reducing the amount of extracellular PD-L1 protein (such as soluble PD-L1 protein and / or exosomal PD-L1 protein) produced by cancer cells in a patient, the method comprising administering a therapeutically effective amount of NUC-7738 to a patient in need thereof.

[0026] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a drug for use in a method for treating cancer by reducing the amount of extracellular PD-L1 protein (such as soluble PD-L1 protein and / or exosomal PD-L1 protein) produced by cancer cells. In a specific embodiment, the amount of extracellular PD-L1 protein, such as soluble PD-L1 protein and / or exosomal PD-L1 protein, produced by cancer cells is reduced, making the cancer cells more easily targeted by the host immune system (such as the patient's cellular immune system). The ability of NUC-7738 to cause reduced production and release of extracellular PD-L1 proteins (such as soluble PD-L1 and exosomal PD-L1 proteins) demonstrates that NUC-7738 is an immune sensitizer and can be used as an immune sensitizer. This effect is further supported by the finding that NUC-7738 also causes an increase in cancer cell expression and production of OX40-L, which interacts with OX40 on the surface of T lymphocytes to increase its anti-tumor effectiveness.

[0027] According to another aspect, the present invention provides NUC-7738 for treating a proliferative disease by enhancing a patient's immune response to the proliferative disease. Suitably, the NUC-7738 enhances the patient's adaptive immune response. Suitably, the NUC-7738 inhibits one or more immune blockers, such as extracellular PD-L1 and / or OX40-L

[0028] According to another aspect, the present invention provides NUC-7738 for use as an immunosensitizer. Suitably, the present invention provides NUC-7738 for use as an immunosensitizer in the treatment of cancer.

[0029] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of an immunosensitizer drug. Alternatively, the present invention provides the use of NUC-7738 in the preparation of a drug for treating cancer, wherein the NUC-7738 is an immunosensitizer.

[0030] According to another aspect, the present invention provides a method for enhancing an immune response to a tumor, the method comprising administering a therapeutically effective amount of NUC-7738 alone or in combination with an immuno-oncology agent described herein to a patient in need of such treatment. In a specific embodiment, a method for enhancing the effect of an immuno-oncology agent is provided, the method comprising administering a therapeutically effective amount of an immuno-oncology agent and NUC-7738 simultaneously or sequentially to a patient in need of such treatment.

[0031] According to another aspect, the present invention provides NUC-7738 for use in treating cancer by increasing the amount of OX40-L protein produced by cancer cells. Suitably, the cancer treatment is performed through adaptive immunity (eg, cellular immunity and / or humoral immunity).

[0032] According to another aspect, the present invention provides a method of treating cancer by increasing the amount of OX40-L protein produced by cancer cells in a patient, the method comprising administering a therapeutically effective amount of NUC-7738 to a patient in need thereof.

[0033] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for use in a method of treating cancer by increasing the amount of OX40-L protein produced by cancer cells.

[0034] In a specific embodiment, increasing the amount of OX40-L protein produced by cancer cells makes the cancer cells more easily targeted by the host immune system (eg, the patient's cellular immune system) by promoting effector T cell activation.

[0035] According to another aspect, the present invention provides NUC-7738 for use in treating cancer by reducing the blockade of a patient's immune system targeting cancer cells. Suitably, "reducing the blockade" includes reducing suppression or inhibitors of a patient's immune system.

[0036] According to another aspect, the present invention provides NUC-7738 for use in treating cancer by enhancing a patient's immune response against cancer. Suitably, the NUC-7738 enhances the patient's immune system against cancer.

[0037] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for use in a method of treating cancer by enhancing a patient's immune response to the cancer.

[0038] According to another aspect, the present invention provides a method of treating cancer by enhancing a patient's immune response to the cancer, wherein the method comprises administering a therapeutically effective amount of NUC-7738 to a patient in need thereof.

[0039] According to another aspect, the present invention provides a combination comprising NUC-7738 and an immuno-oncology agent.

[0040] In specific embodiments, the immuno-oncology agent is an immune checkpoint inhibitor, a cancer vaccine, an antibody therapy, or an adoptive cell therapy such as CAR-T cells.

[0041] In a specific embodiment, the immuno-oncology agent is an immune checkpoint inhibitor selected from pembrolizumab, cemiprilimumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. Suitably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, cemiprilimumab, dotalimumab and nivolumab. In a specific embodiment, the immune checkpoint inhibitor is pembrolizumab.

[0042] According to another aspect, the present invention provides a pharmaceutical product comprising NUC-7738 and an immuno-oncology agent.

[0043] In a specific embodiment, the immuno-oncology agent is an immune checkpoint inhibitor. The immune checkpoint inhibitor can be an antibody against an immune checkpoint protein.

[0044] Suitably, the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-TIM-3 antibody, anti-CD40 antibody and anti-CD40L antibody.

[0045] Suitably, the immune checkpoint inhibitor is selected from pembrolizumab, cemiprilimumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. Suitably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, cemiprilimumab, dotalimumab and nivolumab. In a specific embodiment, the immune checkpoint inhibitor is pembrolizumab.

[0046] In one embodiment, the drug product may include a kit of parts, the kit including separate formulations of NUC-7738 and an immune checkpoint inhibitor. The separate formulations of NUC-7738 and an immune checkpoint inhibitor may be administered sequentially and / or simultaneously.

[0047] In another embodiment, the pharmaceutical product is a kit of parts comprising:

[0048] a first container comprising NUC-7738, such as NUC-7738 in combination with a pharmaceutically acceptable adjuvant, diluent or carrier; and

[0049] a second container comprising an immune checkpoint inhibitor, such as an immune checkpoint inhibitor in combination with a pharmaceutically acceptable adjuvant, diluent or carrier, and

[0050] Container means for receiving said first and second containers.

[0051] In one embodiment, the drug product may include one or more unit dosage forms (e.g., vials, tablets, or capsules in blister packs). In one embodiment, each unit dose contains only one drug selected from NUC-7738 and the immune checkpoint inhibitor. In another embodiment, the unit dosage form includes the NUC-7738 compound and the immune checkpoint inhibitor.

[0052] According to another aspect, the present invention provides a combination of NUC-7738 and an immune checkpoint inhibitor as defined herein for the treatment of a proliferative disease. Suitably, the proliferative disease is cancer. In a specific embodiment, the proliferative disease is a disease typically treated with an immune checkpoint inhibitor (such as one selected from the group consisting of pembrolizumab, cemiplizumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab). Table 1 provides a list of diseases that can be treated with specific checkpoint inhibitors.

[0053] The following combinations and diseases are specific embodiments:

[0054] NUC-7738 is used in combination with nivolumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, squamous cell carcinoma, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, and gastroesophageal junction cancer.

[0055] NUC-7738 is used in combination with durvalumab to treat a cancer selected from the group consisting of urothelial carcinoma, non-small cell lung cancer (NSCLC), and extensive-stage small cell lung cancer.

[0056] NUC-7738 is used in combination with pembrolizumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), non-small cell lung cancer, urothelial carcinoma, classical Hodgkin lymphoma, head and neck squamous cell carcinoma, renal cell carcinoma, colorectal cancer, and esophageal cancer.

[0057] NUC-7738 in combination with dotarimab for the treatment of a cancer selected from: mismatch repair deficient (dMMR) recurrent or advanced endometrial cancer.

[0058] NUC-7738 is used in combination with ipilimumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, colorectal cancer, hepatocellular carcinoma, and esophageal cancer.

[0059] NUC-7738 in combination with cemiplimab for the treatment of a cancer selected from the group consisting of squamous cell carcinoma of the skin, basal cell carcinoma, and non-small cell lung cancer.

[0060] NUC-7738 is used in combination with atezolizumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, and hepatocellular carcinoma.

[0061] NUC-7738 in combination with avelumab for the treatment of a cancer selected from the group consisting of Merkel cell carcinoma, urothelial carcinoma, and renal cell carcinoma.

[0062] Suitably, the proliferative disease is cancer and the cancer cells express high levels of soluble or exosomal PD-L1. Suitably, the proliferative disease is cancer and the cancer cells express low levels of OX40L.

[0063] According to another aspect, the present invention provides a method for treating a proliferative disease in a subject in need thereof, comprising administering to the subject a combination of NUC-7738 and an immune checkpoint inhibitor as defined herein. In a specific embodiment, the proliferative disease is a disease typically treated with an immune checkpoint inhibitor (such as one selected from the group consisting of pembrolizumab, cemiplizumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab). Table 1 provides a list of diseases that can be treated with specific checkpoint inhibitors.

[0064] The following combinations and diseases are specific embodiments:

[0065] NUC-7738 is used in combination with nivolumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, squamous cell carcinoma, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, and gastroesophageal junction cancer.

[0066] NUC-7738 is used in combination with durvalumab to treat a cancer selected from the group consisting of urothelial carcinoma, non-small cell lung cancer (NSCLC), and extensive-stage small cell lung cancer.

[0067] NUC-7738 is used in combination with pembrolizumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), non-small cell lung cancer, urothelial carcinoma, classical Hodgkin lymphoma, head and neck squamous cell carcinoma, renal cell carcinoma, colorectal cancer, and esophageal cancer.

[0068] NUC-7738 in combination with dotarimab for the treatment of a cancer selected from: mismatch repair deficient (dMMR) recurrent or advanced endometrial cancer.

[0069] NUC-7738 is used in combination with ipilimumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, colorectal cancer, hepatocellular carcinoma, and esophageal cancer.

[0070] NUC-7738 in combination with cemiplimab for the treatment of a cancer selected from the group consisting of squamous cell carcinoma of the skin, basal cell carcinoma, and non-small cell lung cancer.

[0071] NUC-7738 is used in combination with atezolizumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, and hepatocellular carcinoma.

[0072] NUC-7738 in combination with avelumab for the treatment of a cancer selected from the group consisting of Merkel cell carcinoma, urothelial carcinoma, and renal cell carcinoma.

[0073] Suitably, the immune checkpoint inhibitor is pembrolizumab, cemiplizumab, dotarizumab or nivolumab.

[0074] Suitably, the proliferative disease is cancer and the cancer cells express high levels of soluble or exosomal PD-L1. Suitably, the proliferative disease is cancer and the cancer cells express low levels of OX40L.

[0075] According to another aspect, the present invention provides the use of NUC-7738 in combination with an immune checkpoint inhibitor as defined herein for the preparation of a medicament for the treatment of a proliferative disease.

[0076] In a specific embodiment, the proliferative disease is a disease that is typically treated with an immune checkpoint inhibitor (e.g., one selected from the group consisting of pembrolizumab, cemiplizumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab). Table 1 provides a list of diseases that can be treated with specific checkpoint inhibitors.

[0077] The following combinations and diseases are specific embodiments:

[0078] NUC-7738 is used in combination with nivolumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, squamous cell carcinoma, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, and gastroesophageal junction cancer.

[0079] NUC-7738 is used in combination with durvalumab to treat a cancer selected from the group consisting of urothelial carcinoma, non-small cell lung cancer (NSCLC), and extensive-stage small cell lung cancer.

[0080] NUC-7738 is used in combination with pembrolizumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), non-small cell lung cancer, urothelial carcinoma, classical Hodgkin lymphoma, head and neck squamous cell carcinoma, renal cell carcinoma, colorectal cancer, and esophageal cancer.

[0081] NUC-7738 in combination with dotarimab for the treatment of a cancer selected from: mismatch repair deficient (dMMR) recurrent or advanced endometrial cancer.

[0082] NUC-7738 is used in combination with ipilimumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, colorectal cancer, hepatocellular carcinoma, and esophageal cancer.

[0083] NUC-7738 in combination with cemiplimab for the treatment of a cancer selected from the group consisting of squamous cell carcinoma of the skin, basal cell carcinoma, and non-small cell lung cancer.

[0084] NUC-7738 is used in combination with atezolizumab to treat a cancer selected from the group consisting of melanoma (including cutaneous melanoma), non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, and hepatocellular carcinoma.

[0085] NUC-7738 in combination with avelumab for the treatment of a cancer selected from the group consisting of Merkel cell carcinoma, urothelial carcinoma, and renal cell carcinoma.

[0086] Suitably, the proliferative disease is cancer, and the cancer cells express high levels of soluble or exosomal PD-L1. Suitably, the proliferative disease is cancer, and the cancer cells express low levels of OX40L. Suitably, the immune checkpoint inhibitor is selected from pembrolizumab, cemipril monoclonal antibody, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. Suitably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, cemipril monoclonal antibody, dotalimumab and nivolumab. A particularly suitable immune checkpoint inhibitor for the treatment of cancer (such as cutaneous melanoma) in combination with NUC-7738 is pembrolizumab.

[0087] In specific embodiments of this aspect of the invention, the NUC-7738 and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0088] According to another aspect, the present invention provides NUC-7738 as defined herein for treating a proliferative disease, wherein the NUC-7738 is used for simultaneous or sequential administration with an immune checkpoint inhibitor as defined herein. Suitably, the immune checkpoint inhibitor is selected from the group consisting of: pembrolizumab, cemiprilimumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. Suitably, the proliferative disease is a disease typically treated with an immune checkpoint inhibitor (such as pembrolizumab, cemiprilimumab, dotalimumab or nivolumab).

[0089] According to another aspect, the present invention provides a method for treating a proliferative disease in a subject in need thereof, comprising administering to the subject a combination of NUC-7738 and an immune checkpoint inhibitor as defined herein, wherein the NUC-7738 and the immune checkpoint inhibitor are administered simultaneously or sequentially. Suitably, the immune checkpoint inhibitor is selected from the group consisting of: pembrolizumab, cimiprilimab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. Suitably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, cimiprilimab, dotalimumab and nivolumab. Suitably, the proliferative disease is a disease typically treated with an immune checkpoint inhibitor (such as pembrolizumab, cimiprilimab, dotalimumab or nivolumab).

[0090] According to another aspect, the present invention provides an immune checkpoint inhibitor as defined herein for treating a proliferative disease, wherein the immune checkpoint inhibitor is used to be administered simultaneously or sequentially with NUC-7738 as defined herein. Suitably, the immune checkpoint inhibitor is selected from the group consisting of: pembrolizumab, cimiprilimumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. Suitably, the immune checkpoint inhibitor is pembrolizumab, cimiprilimumab, dotalimumab or nivolumab. Suitably, the proliferative disease is a disease usually treated with an immune checkpoint inhibitor (such as pembrolizumab, cimiprilimumab, dotalimumab or nivolumab).

[0091] According to another aspect, the present invention provides the use of immune checkpoint inhibitors in combination with NUC-7738 defined herein in the preparation of a medicament for treating a proliferative disease. Suitably, the immune checkpoint inhibitor is selected from the group consisting of: pembrolizumab, cimiprilimab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. Suitably, the immune checkpoint inhibitor is pembrolizumab, cimiprilimab, dotalimumab or nivolumab. Suitably, the proliferative disease is a disease typically treated with an immune checkpoint inhibitor (such as pembrolizumab, cimiprilimab, dotalimumab or nivolumab).

[0092] In specific embodiments of this aspect of the invention, the NUC-7738 and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0093] Suitably, the proliferative disease is cancer. In a specific embodiment, the proliferative disease is a disease typically treated with an immune checkpoint inhibitor such as pembrolizumab, cemiplizumab, dotalimumab or nivolumab. According to another aspect, the present invention provides NUC-7738 for treating cancer in a subject whose cancer cells express high levels of soluble or exosomal PD-L1.

[0094] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for treating cancer in a subject whose cancer cells express high levels of extracellular PD-L1 protein, such as soluble or exosomal PD-L1 protein, wherein the treatment comprises administering the NUC-7738 in combination with an immune checkpoint inhibitor as defined herein. Optionally, prior to treatment, the patient is tested to determine whether they have cancer, wherein the patient's cancer cells express high levels of extracellular PD-L1 protein, such as soluble PD-L1 protein or exosomal PD-L1 protein. Suitably, the immune checkpoint inhibitor is pembrolizumab, cemiplizumab, dotalimumab or nivolumab.

[0095] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for use in a method for treating cancer by reducing the amount of extracellular PD-L1 protein, such as soluble protein and / or exosomal PD-L1 protein, produced by cancer cells, wherein the method comprises determining whether a subject has cancer, the cancer cells of the subject expressing high levels of extracellular PD-L1 protein, wherein if the subject has cancer, the cancer cells of the subject expressing high levels of extracellular PD-L1 protein, such as soluble PD-L1 protein or exosomal PD-L1 protein, administering to the subject a combination of NUC-7738 and an immune checkpoint inhibitor as defined herein. Suitably, the immune checkpoint inhibitor is pembrolizumab, cemiplizumab, dotalimumab or nivolumab.

[0096] According to another aspect, the present invention provides a method of treating cancer in a subject whose cancer cells express high levels of extracellular PD-L1 protein, such as soluble PD-L1 protein or exosomal PD-L1 protein, comprising administering to the subject a therapeutically effective amount of NUC-7738 alone or in combination with an immune checkpoint inhibitor. Suitably, the immune checkpoint inhibitor is pembrolizumab, cemiplizumab, dotalimumab or nivolumab.

[0097] According to another aspect, the present invention provides a method of treating cancer in a subject whose cancer cells express high levels of extracellular PD-L1, such as soluble PD-L1 protein or exosomal PD-L1 protein, wherein the cancer cells are contacted with a therapeutically effective amount of NUC-7738. In a specific embodiment of this aspect of the invention, the cancer cells are in the subject or patient. Therefore, suitably, the cancer cells are contacted with a therapeutically effective amount of NUC-7738 by administering a therapeutically effective amount of NUC-7738 to the subject alone or in combination with an immune checkpoint inhibitor. Suitably, the immune checkpoint inhibitor is pembrolizumab, cemiplizumab, dotalimumab or nivolumab.

[0098] Any immune checkpoint inhibitor may be used in the combination therapy defined herein.

[0099] In a specific embodiment, the checkpoint inhibitor is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-TIM-3 antibody, anti-CD40 antibody and anti-CD40L antibody.

[0100] Suitably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, cemiplizumab, dotarizumab or nivolumab.

[0101] In a specific embodiment, the proliferative disease is cancer, characterized in that cancer cells express high levels of extracellular PD-L1 protein, such as soluble PD-L1 or exosomal PD-L1. In a specific embodiment, the proliferative disease is a disease that is typically treated with an immune checkpoint inhibitor (such as pembrolizumab, cemiplizumab, dotalimumab, or nivolumab). Table 1 provides a list of diseases that can be treated with specific checkpoint inhibitors.

[0102] According to another aspect, the present invention provides a method for determining whether a patient will benefit from treatment with NUC-7738, the method comprising: determining the level of soluble PD-L1 and / or exosomal PD-L1 in a biological sample from the patient; wherein if the level of soluble PD-L1 and / or exosomal PD-L1 produced by cancer cells in the biological sample is increased or decreased compared to a reference value, the patient will benefit from treatment with NUC-7738.

[0103] Suitably, the biological sample is a blood sample or a fraction thereof (eg plasma or serum).

[0104] Various aspects of the present invention are based in part on the discovery that NUC-7738 is able to reduce the amount of extracellular PD-L1 protein, including soluble PD-L1 protein and exosomal PD-L1 protein produced by cancer cells. It is well known that PD-L1 masks cancer cells from the immune system, and in particular, elevated soluble PD-L1 is a biomarker for poor prognosis and resistance to immune checkpoint inhibitors. Therefore, the ability of NUC-7738 to reduce the amount of extracellular PD-L1 protein (such as soluble PD-L1 protein and / or exosomal PD-L1 protein) produced by cancer cells provides new clinical opportunities for treating and controlling cancer in patients. For example, by removing or reducing the camouflage of the cancer cells to the immune system, thereby allowing the host immune system to recognize and attack (including killing) cancer cells. The ability of NUC-7738 to reduce the level of extracellular PD-L1 protein (such as soluble PD-L1 protein and / or exosomal PD-L1 protein) will be used to enhance the effectiveness of immuno-oncology agents (including immune checkpoint inhibitors), which provides the ability to treat cancer patients with a combination of NUC-7738 and immuno-oncology agents. When the immuno-oncology agent is an immune checkpoint inhibitor, as described herein, it can also facilitate the use of lower doses of checkpoint inhibitors, thereby reducing the toxic effects of such drugs. Some cancer patients also develop resistance to immune checkpoint inhibitors, which is often associated with an increase in soluble PD-L1 levels. It is expected that the ability of NUC-7738 to target and reduce the amount of soluble or exosomal PD-L1 removes resistance / blockade and thus allows patients to be re-treated with the same or alternative immune checkpoint inhibitors, or treated with immune checkpoint inhibitors that would not be effective without the reduction in soluble PD-L1 induced by NUC-7738. Therefore, NUC-7738 may also be used to treat patients who are developing or have developed resistance to immune checkpoint inhibitors.

[0105] Therefore, in a specific embodiment of any aspect of the present invention, the patient or subject is a patient or subject who is resistant to or has developed resistance to immune checkpoint inhibitors. Such a person does not provide a recognized clinical signal of efficacy for a determined dose of immune checkpoint inhibitors. Suitably, subjects / patients who have developed resistance to immune checkpoint inhibitors will previously show clinical signals of efficacy, such as maintaining or reducing cancer mass or reducing cancer markers, progression-free survival, but the effectiveness of immune checkpoint inhibitors is subsequently reduced or stopped. This indicates that the cancer has developed resistance to the agent. Suitably, subjects / patients who are resistant to (and already resistant to) immune checkpoint inhibitors will have markers, such as low mutation load, microsatellite stability, low tumor infiltrating lymphocytes, high levels of immunosuppressive cytokines, and PTEN loss, which indicates that resistance to immune checkpoint inhibitor treatment is already present (see Liu et al., Am J Clin Dermatol. 20 (1): 41–54, 2019).

[0106] Various aspects of the present invention are also based on the discovery that NUC-7738 is able to increase the amount of OX40L produced by cancer cells. Stimulation of OX40 suggests that it is a candidate for therapeutic immunization strategies in cancer.

[0107] OX40 signaling is important for the initiation and generation of CD4 and CD8 T cell immunity and the development of T cell memory. OX40 is expressed on T cells after the T cell receptor on the surface of the T cells recognizes foreign or abnormal peptides. Binding of OX40 to its ligand OX40L enhances the activation of T cells and has been shown to enhance anti-tumor immune responses (Weinberg et al., IJ Immunol. 164(4):2160-2169, 2000). Therefore, the OX40 / OX40L axis is considered an attractive target for treating autoimmune and inflammatory diseases, as well as for designing vaccination and therapeutic adjuvant strategies for infectious diseases and cancer (see, e.g., Dengdengr, "OX40 (CD14) and OX40 ligand, important immune checkpoints in cancer". Onco Targets Ther. 12: 7347-7353, 2019; Roszik et al., "TNFSF4 (OX40L) expression and survival in locally advanced and metastatic melanoma". Cancer Immunology, Immunotherapy. 68: 1493-1500, 2019; Dannull J, Nair S, Su Z, Boczkowski D, DeBeck C, Yang B. "Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand. Blood. 105: 3206–3213, 2005).

[0108] Reference to NUC-7738 in any aspect or embodiment of the invention includes NUC-7738 as an (S)-phosphate diastereomer or as an (R)-phosphate diastereomer or as a mixture of phosphate diastereomers; it also includes the compound in the form of a free base, or it may be in the form of a pharmaceutically acceptable salt; and, it also includes a pharmaceutical composition comprising said NUC-7738.

[0109] The immune checkpoint inhibitor mentioned in any aspect or embodiment of the present invention includes the drug or a pharmaceutically acceptable salt thereof; it also includes a pharmaceutical composition comprising the immune checkpoint inhibitor.

[0110] The ability of NUC-7738 to reduce the amount / level of soluble PD-L1 protein and / or exosomal PD-L1 protein and / or increase the amount / level of OX40L protein provides new therapeutic opportunities for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] FIG1 shows the concentration of soluble PD-L1 in melanoma (A375) and lung cancer (A549) cell lines before and after treatment with NUC-7738 by ELISA.

[0112] Figure 2 shows the fold change of soluble PD-L1 transcripts in melanoma (A375) and lung cancer (A549) cell lines before and after treatment with NUC-7738.

[0113] Figure 3 Exosomal PD-L1 from four patients before and after treatment with NUC-7738 is shown. Before the first cycle (untreated), 24 hours after the first cycle, and before the second cycle (15 days after the initial cycle). In this case, the cycle refers to each time the patient receives the drug.

[0114] Figure 4 Shown are the expression levels of OX40L mRNA in melanoma (A375) and lung cancer (A549) cell lines treated with NUC-7738. DETAILED DESCRIPTION

[0115] Further features and embodiments of the aspects defined above are described in the heading sections below. Each section may be combined with any of the above aspects in any compatible combination.

[0116] The following definitions may be helpful in understanding the present invention.

[0117] As used herein, "treatment" refers to a clinical intervention that attempts to change the natural course of the treated subject or cell, and can be performed before or during the clinical pathological process. The desired effects of treatment include preventing the occurrence or recurrence of a disease or disorder or its symptoms, alleviating a disease or disorder, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and achieving relief or improved prognosis. In some embodiments, the methods and compositions of the present disclosure can be used to attempt to delay the development of a disease or disorder. The treatment can be used for human treatment and veterinary applications. Unless the context indicates otherwise, the terms "disease" and "disorder" used herein are generally interchangeable.

[0118] "Amelioration" includes alleviation, elimination, reduction and / or alleviation of a disease or condition described herein (or symptoms associated therewith).

[0119] Soluble PD-L1 is secreted by tumor cells and exists in plasma as free soluble protein. Exosomal PD-L1 refers to PD-L1 protein bound to the exosome membrane, which is released from the cell as an extracellular vesicle. As used herein, extracellular PD-L1 refers to PD-L1 protein that is released / shed from the cell and is not bound to the cell. PD-L1 protein released by cells may be circulating PD-L1. Soluble PD-L1 and exosomal PD-L1 are examples of extracellular PD-L1.

[0120] Exosomes are extracellular vesicles (EVs), which are small membrane-enclosed structures that may be shed from the surface of healthy or damaged cells under conditions such as cell activation, growth, and apoptosis. These vesicular structures contain a large number of biologically active proteins, lipids, and nucleic acids obtained from their parent cells. EVs are commonly found in blood (such as plasma), urine, saliva, tears, and many other body fluids. EVs and / or biomarkers present in / on or associated with EVs have been proposed for diagnosis, prognosis, and monitoring of diseases and health conditions.

[0121] Extracellular vesicles can be isolated and purified using a polymer-based resin that is available as a kit ULTRAEV was obtained from Systems Bioscience. The protein content of the isolated EVs can then be quantified using, for example, a bicinchoninic acid (BCA) protein quantification assay. Suitably, quantification of the exosomal protein marker CD81 can be used. In addition, the size distribution of the EVs can be determined using NanoSight.

[0122] The engagement of PD-L1 ligands with PD-1 can alter the tumor microenvironment and inhibit endogenous anti-tumor immune responses mediated by CD8+T cells and other components of the cellular immune system. Kim et al. (Experimental & Molecular Medicine 51:94, 2019) demonstrated that exosomes from non-small cell lung cancer cells expressing PD-L1 play a role in immune escape by reducing T cell activity and promoting tumor growth. They found that the abundance of PD-L1-expressing exosomes isolated from patient plasma correlated with PD-L1 positivity in tumor tissue. They found that exosomes can impair immune function by reducing cytokine production and inducing CD8+T cell apoptosis, suggesting that tumor-derived exosomes expressing PD-L1 may be important mediators of tumor immune escape.

[0123] Soluble PD-L1 (sPD-L1) is a predictive and prognostic biomarker for cancer patients who select or receive immune checkpoint blockade therapy (see Oh, SY, Kim, S., Keam, B. et al., Soluble PD-L1 is a predictive and prognostic biomarker in advanced cancer patients who receive immune checkpoint blockade treatment. Sci Rep 11, 19712 (2021). https: / / doi.org / 10.1038 / s41598-021-99311-y). Similarly, exosomal PD-L1 (xPD-L1) can also be used as a biomarker for cancer patients who select or receive immune checkpoint blockade therapy. (Chen et al., "Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1response". Nature. 560: 382-386, 2018; Li et al., J of Translational Medicine. "Clinical significance of PD-L1expression in serum-derived exosomes in NSCLC patients". 17, 225, 2019) As used herein, the terms "exosomal PD-L1", "xPD-L1", "extravesicular PD-L1" and "EV PD-L1" are used interchangeably.

[0124] The term "high levels of soluble PD-L1 or exosomal PD-L1" refers to an increase above normal levels, usually above clinically relevant reference levels. Thus, high levels of soluble PD-L1 refer to an amount that exceeds the usual / normal amount. This deviation from normal levels can be, for example, an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more.

[0125] Usual or normal levels can be determined by measuring levels in a population of subjects considered healthy or not affected by a disease mediated by or associated with high levels of soluble PD-L1 and applying standard statistical measures. This value can be used to determine an appropriate "reference level" against which to determine whether the level of soluble PD-L1 or exosomal PD-L1 produced by a cell or cell population is normal, high, or low.

[0126] Low levels of OX40L expression are associated with poor prognosis and grade (see Roszik et al., supra). Therefore, OX40L can be used as a biomarker to select suitable patients for treatment with immune checkpoint inhibitors and / or the methods of the present invention. For example, tumors with low OX40L are targets for treatment that increases tumor OX40L expression.

[0127] The term "low level of OX40L" refers to a decrease from a normal level, usually below a clinically relevant reference level. Thus, a low level of OX40L refers to an amount that exceeds the usual / normal amount. This deviation from normal levels can be, for example, a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more.

[0128] A "clinically relevant reference level" is a value determined by appropriate clinical studies, as an amount / level (e.g., a threshold) of, e.g., soluble PD-L1 or exosomal PD-L1 or OX40L levels, used to classify whether the amount is high or not. This is a value that can be used to make clinical decisions. The use of such reference values ​​in diagnostic testing and in making clinical decisions is well known.

[0129] All grammatical forms of the term "administering" refer to administering a therapeutically effective dose of the NUC-7738 to a subject / patient as the sole therapeutic agent or in combination with another therapeutic agent such as an immune checkpoint inhibitor as described herein. It is envisioned that the NUC-7738 is suitably in the form of a composition, preferably a pharmaceutical composition, which can be used in a co-therapeutic method, i.e., co-administered with other agents or drugs, such as other agents for treating the diseases described herein and / or any other therapeutic agent that may be beneficial in the context of the methods of the invention.

[0130] As used herein, "effective amount" refers to an effective amount at a dosage and time required to achieve the desired therapeutic or preventive effect. The exact dosage will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques.

[0131] "Therapeutically effective amount" refers to the amount of an agent (e.g., a compound such as NUC-7738) that produces the effect of administration. The "therapeutically effective amount" of NUC-7738 described herein may vary according to factors such as age, weight, general health, sex, diet, time of administration, drug interactions, and severity of the disease, and may be determined by a person skilled in the art through routine experiments. Sometimes, the term "therapeutically effective amount" is sometimes used interchangeably with the term "pharmaceutically effective amount". The "therapeutically effective amount" of NUC-7738 when used in combination with a specific immune checkpoint inhibitor may be different from the "therapeutically effective amount" when NUC-7738 is administered alone (e.g., as a monotherapy), and it may be different from the "therapeutically effective amount" when used in combination with different immune checkpoint inhibitors.

[0132] As used herein, a therapeutically effective amount is also one in which any toxic or detrimental effects of NUC-7738 are outweighed by the therapeutically beneficial effects.

[0133] Cancer drugs are often administered in treatment cycles. The term "treatment cycle" refers to a period of treatment that is repeated at regular intervals. Any treatment cycle may include a period of rest (no treatment) before receiving the next treatment dose. For example, giving three weeks of treatment followed by a week of rest is a treatment cycle of 28-day cycles. A treatment cycle is usually between 2 and 6 weeks. Multiple treatment cycles can be given one after another in succession so that when you reach the end of a cycle, it starts over again for the next cycle. A series of cycles is called a course of treatment. A course of treatment can take between 3 and 6 months, but it can also be longer or shorter, or it can be indefinite. Patients can receive anywhere from 3 to 10 more cycles of treatment during this period.

[0134] The drug can be administered on every day of the cycle or on specific days of the cycle as part of a treatment regimen that reflects the days or times when the drug is or is not administered. Thus, for example, NUC-7738 can be administered in a 21-day treatment cycle, for example on days 1, 8, and 15, meaning that the patient should receive NUC-7738 once a week because the 7th day after the last administration of cycle 1 (day 15) would be day 1 of cycle 2.

[0135] In some embodiments, the "subject" used in this application is a vertebrate. In certain specific embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (e.g., mice and rats). In certain specific embodiments, the mammalian subject is a human. In some further embodiments, a human subject synonymous with an individual is a particularly preferred subject. As used herein, the term "subject" is used synonymously with the term "patient". Suitably, the subject / patient is a human.

[0136] References herein to administration of NUC-7738 “in combination” with an immunotherapy agent such as a checkpoint inhibitor (e.g., a CTLA4, LAG3, PD-1 or PD-L1 inhibitor), or vice versa, include administration of the agents sequentially or simultaneously with each other, unless otherwise specified.

[0137] As used herein, "simultaneous administration" refers to a treatment in which two agents (e.g., NUC-7738 and an immuno-oncology agent) are administered substantially at the same time. An example of simultaneous administration is mixing the two agents together in the same infusion bag, or administering them simultaneously using a "Y-line" from different infusion bags. An example of simultaneous administration is that the duration of administration of the two agents overlaps at least to some extent.

[0138] "Administering sequentially" as used herein refers to a kind of medicament being administered (i.e., administered separately) after another medicament, however, the time interval between each medicament administration enables two medicaments to play a therapeutic effect simultaneously. Therefore, "administering sequentially" can allow a kind of medicament to be administered within a few seconds, a few minutes, or a few hours after another medicament, provided that the first circulatory half-life of the medicament administered makes them all exist simultaneously with a therapeutically effective amount. The time delay between medicament administration can vary according to the exact nature of medicament, the interaction between them, and their respective half-life. Suitably, the two medicaments are administered within 5, 10, 15, 20, 30, 60, 120 minutes of intervals from each other. The two agents do not need to be administered on the same day, and in fact they can be administered as part of a treatment regimen that includes an administration cycle (e.g., agent 1 is administered on days 1, 8, and 15 of a 21-day cycle, and agent 2 is administered on days 3, 10, and 17 of a 21-day cycle; such a treatment cycle can be repeated one or more times. As another example, agents 1 and 2 can be administered on day 1 of a 21-day cycle, and only agent 2 is administered on one or more other days of the 21-day cycle). Suitably, the two agents are administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days of each other. In a specific embodiment of any aspect of the invention, when NUC-7738 and an immune checkpoint inhibitor are administered sequentially, the NUC-7738 is administered first. In a specific embodiment of any aspect of the invention, when NUC-7738 and an immune checkpoint inhibitor are administered sequentially, the immune checkpoint inhibitor is administered first. "Sequential administration" requires that the two drugs are administered in the same treatment cycle or as part of a course of treatment.

[0139] As used herein, "drug product" refers to a product that contains a drug. For example, examples of drug products include medical devices, pharmaceutical compositions, and kits of parts that suitably include one or more devices, containers, and / or drugs.

[0140] As used herein, the term "immunosensitizer" refers to a drug used to activate or enhance the host immune system. In the case of NUC-7738, it can do this by reducing the amount of soluble or exosomal PD-L1 protein present or by increasing the amount of OX40L protein expressed on cancer cells, which can bind to OX40 on T-lymphocytes and other components of the cellular immune system, resulting in enhanced T-cell activation. Therefore, the immunosensitizer effectively turns on the immune system or removes inhibitors that hinder the effective activation of the immune system, making the immune system sensitive to cancer. As described herein, PD-L1 is an immunosuppressive PD-1 ligand, so by reducing PD-L1, the immune environment is more conducive to cancer cell death mediated by the cellular immune system.

[0141] As used herein, "immuno-oncology agent" refers to any drug that can trigger a host immune response. Such drugs are commonly used in immunotherapy, which is a cancer treatment that helps the subject's immune system fight cancer. Examples of immuno-oncology agents include immune checkpoint inhibitors, particularly anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG3 antibodies, anti-CTLA-4 antibodies, anti-TIM3 antibodies, anti-BTLA antibodies, anti-OX40 antibodies or anti-OX40L antibodies], tumor vaccines and adoptive cell therapy, such as CAR-T cells (CAR-T treatment). In a specific embodiment, the immuno-oncology agent is an immune checkpoint inhibitor, as described herein (e.g., see the section entitled "immune checkpoint inhibitors"), or an adoptive cell therapy, such as a CAR-T cell population or a cancer vaccine, as described herein and known to those skilled in the art. As used herein, the term "immunotherapy agent" is synonymous with "immuno-oncology agent".

[0142] Adoptive cell therapy

[0143] Adoptive cell therapy, also known as cellular immunotherapy, is a form of treatment that uses cells of the host's (patient's) immune system to attack cancer cells.

[0144] Some of these approaches involve isolating immune cells directly from patients and expanding their numbers, while others involve genetically engineering isolated immune cells (via gene therapy) to enhance their ability to recognize and attack cancer cells.

[0145] In a specific embodiment, the adoptive cell therapy is selected from: CAR-T, TIL and NK cell therapy.

[0146] Tumor Infiltrating Lymphocyte (TIL) Therapy

[0147] Most cancer patients have different types of naturally occurring T cells that are able to target cancer cells. In particular, "killer" T cells are able to directly recognize and eliminate cancer cells. However, these cells must be activated before they can effectively kill cancer cells, and then they must be able to maintain this activity long enough to sustain an effective anti-tumor response. In addition, these T cells must be produced in sufficient numbers in order to launch an effective attack.

[0148] One form of adoptive cell therapy that attempts to address these issues is called tumor-infiltrating lymphocyte (TIL) therapy. This approach collects naturally occurring T cells that have infiltrated a patient's tumor, then activates and expands them. Next, large numbers of these activated T cells are infused back into the patient, where they can then find and destroy the tumor.

[0149] et al. reviewed the progress of TIL adoptive cell therapy, including the principles and techniques used (Expert Opin Biol Ther. 22(5):627-641, 2020).

[0150] Engineered T cell receptor (TCR) therapy

[0151] Some patients may not have T cells that already recognize their tumor, or even if they do, these T cells cannot be activated and expanded to sufficient numbers to reject their tumor. For these patients, an approach called engineered T cell receptor (TCR) therapy may be appropriate.

[0152] This approach also involves removing T cells from the patient, but instead of simply activating and expanding available anti-tumor T cells, the T cells are altered so that they present new T cell receptors that enable them to target specific cancer antigens.

[0153] CAR-T cell therapy

[0154] The TIL and TCR therapies mentioned above can only target and eliminate cancer cells that present their antigens in a specific context (when the antigens are bound by the major histocompatibility complex, or MHC).

[0155] Approaches aimed at addressing this limitation include engineering T cells with a synthetic receptor called CAR, which stands for chimeric antigen receptor.

[0156] CAR is a synthetic molecule that includes an extracellular domain and an intracellular domain, the extracellular domain acting as a high-affinity ligand specific for target cell surface antigens (most often derived from antibodies and prepared as single-chain variable fragments-scFv), the intracellular domain ensures that the modified T cells are strongly activated and proliferated in an HLA-independent manner. The basic configuration of the CAR intracellular domain includes one or two costimulatory molecule domains (derived from CD28, 41-BB or OX-40) in series with the CD3z domain.

[0157] A key advantage of CARs is their ability to bind to cancer cells even if their antigens are not presented on the surface via MHC, which can make more cancer cells vulnerable to their attack. However, CAR-T cells can only recognize their own antigens that are naturally expressed on the cell surface, so the range of potential antigen targets is smaller than that of TCRs.

[0158] CAR-T cells (chimeric antigen receptor T cells) are T-cells, usually isolated from the same patient to be treated (autologous), but may also come from a different donor source (allogeneic), which are modified to express a protein called a chimeric antigen receptor (CAR) on their surface. The CAR recognizes and binds to specific antigens on the surface of cancer cells, thereby targeting T cells to cancer cells. The modified CAR-T cells are first expanded in the laboratory and then injected back into the patient, where the CAR-T cells can multiply, target cancer cells and kill them.

[0159] The U.S. Food and Drug Administration (FDA) has approved six CAR-T cell therapies since 2017. All of these CAR-T cell therapies are approved for the treatment of blood cancers, including lymphoma, certain forms of leukemia, and most recently, multiple myeloma.

[0160] The effectiveness of CAR-T cell therapy for solid tumors is limited by the inhibitory effects of immune checkpoints in the solid tumor microenvironment (Ma, S., Li, X., Wang, X., Cheng, L., Li, Z., Zhang, C., et al., Current progress in CAR-T cell therapy for solid tumors. Int. J. Biol. Sci. 15, 2548–2560, 2019; Shi, X., Zhang, D., Li, F., Zhang, Z., Wang, S., Xuan, Y., et al., Targeting glycosylation of PD-1 to enhance CAR-T cell cytotoxicity. J. Hematol. Oncol. 12: 127, 2019).

[0161] Programmed cell death protein-1 (PD-1)-mediated immunosuppression is believed to limit the clinical efficacy of CAR-T cell therapy for solid tumors. It has been proposed that PD-1-mediated immunosuppression is associated with CAR-T cell hypofunction (Moon EK, Wang LC, Dolfi DV, Wilson CB, Ranganathan R, Sun J et al., Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res. 2014; 20:4262–73.), and the combination of CAR-T cell therapy with anti-PD-1 antibody therapy has shown encouraging antitumor activity in patients (Chong EA, Melenhorst JJ, Lacey SF, Ambrose DE, Gonzalez V, Levine BL et al., PD-1 blockade modulates chimeric antigen receptor (CAR)-modified T cells: refueling the CAR. Blood. 129:1039–41, 2017; and Adusumilli PS, Zauderer J et al., PD-1 blockade modulates chimeric antigen receptor (CAR)-modified T cells: refueling the CAR. Blood. 129:1039–41, 2017; and Adusumilli PS, Zauderer J et al., PD-1 blockade modulates chimeric antigen receptor (CAR)-modified T cells: refueling the CAR. Blood. 129:1039–41, 2017). MG, RuschVW, O'Cearbhaill R, Zhu A, Ngai D, et al., Regional delivery of mesothelin-targetedCAR T cells for pleural cancers: safety and preliminary efficacy incombination with anti-PD-1agent. J Clin Oncol. 37:2511, 2019).

[0162] It is believed that reducing the inhibition of immune checkpoints such as PD-1 / PD-L1 will enhance CAR-T cell therapy. The ability of NUC-7738 to reduce the amount of soluble and exosomal PD-L1 is believed to reduce the inhibition of immune checkpoints, therefore enhancing CAR-T cell therapy.

[0163] In addition, the ability of NUC-7738 to increase OX40L expression may benefit CAR-T therapy. For example, Zhang, Huihui et al. ("Achimeric antigen receptor with antigen-independent OX40 signaling mediates potent antitumor activity." Science translational medicine 13.578 (2021): eaba7308.) demonstrated that co-stimulation of CAR-T with OX-40 in tumor cells mediated effective antitumor activity. Therefore, it is believed that the ability of NUC-7738 to increase the amount of OX40L will reduce the inhibitory effects of immune checkpoints, thereby enhancing CAR-T cell therapy.

[0164] Natural Killer (NK) Cell Therapy

[0165] Adoptive cell therapy strategies have begun to introduce other immune cells, such as natural killer (NK) cells. One application being explored in the clinic is to equip these NK cells with cancer-targeting CARs.

[0166] and Rezvani (Int J Hematol. 114(5):554-571, 2021) provided an overview of current trends and evolving concepts to genetically engineer next-generation CAR-NK therapies.

[0167] In a specific embodiment, NUC-7738 is provided for use in combination with adoptive cell therapy. In a specific embodiment, NUC-7738 is provided for enhancing the effect of adoptive cell therapy. Suitably, the adoptive cell therapy is selected from CAR-T, NK and TIL.

[0168] Cancer vaccines

[0169] Cancer vaccines involve the administration of a treatment that results in the presentation of cancer antigens to the immune system, enhancing the immune system's ability to find cancer cells expressing these antigens and eliminate them. Cancer vaccines can be viral, RNA, DNA, or peptide-based and specific for proteins expressed on specific cancer cells (e.g., Sipuleucel-T for prostate cancer or Talimogene laherparepvec for advanced melanoma skin cancer).

[0170] Preclinical studies combining cancer vaccines with immune checkpoint blockade are considered promising. For example, van Elsas et al. (van Elsas A, Hurwitz AA, Allison JP. Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte / macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med. 190:355–66, 1999) examined the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade (alone or in combination with a tumor cell vaccine expressing granulocyte / macrophage colony-stimulating factor (GM-CSF)) in rejecting the highly tumorigenic, poorly immunogenic murine melanoma B16-BL6, and found that in cases using combined treatment, 80% (68 / 85) of the tumors could be eradicated, while each treatment itself showed little or no effect.

[0171] It is believed that reducing the inhibitory effects of immune checkpoints (such as PD-1 / PD-L1) will enhance cancer vaccine therapy. The ability of NUC-7738 to reduce the amount of soluble and exosomal PD-L1 is believed to reduce the inhibitory effects of immune checkpoints, thereby enhancing cancer vaccine therapy. In addition, OX40 stimulation of immune cells has been proposed as a strategy to enhance vaccine effectiveness (Panagioti et al., Front Immunol. 20(8):144, 2017), so the ability of NUC-7738 to increase OX40L expression can enhance the effectiveness of cancer vaccines.

[0172] In a specific embodiment, NUC-7738 is provided for use in combination with a cancer vaccine. In a specific embodiment, NUC-7738 is provided for use in enhancing the effect of a cancer vaccine.

[0173] "NUC-7738"

[0174] The present invention relates to the medical use of NUC-7738 alone or in combination with an immuno-oncology agent, in particular for the treatment of proliferative diseases such as cancer by reducing the level of extracellular PD-L1 protein produced by cancer cells or increasing the level of OX40-L produced by cancer cells. Suitably, the extracellular PD-L1 protein is soluble PD-L1 or exosomal PD-L1. The reduction in the level of soluble PD-L1 or exosomal PD-L1 is believed to be due to NUC-7738 reducing the translation of soluble / exosomal PD-L1. The increase in OX40-L levels is believed to be due to NUC-7738 enhancing the transcription of OX40-L.

[0175] Compound 3'-deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alanyl)] phosphate (also known as NUC-7738) is a phosphoramidate derivative of 3'-deoxyadenosine. The NUC-7738 compound, including its synthesis, is disclosed in WO2016 / 083830 (Nucana). Pharmaceutical compositions comprising phosphoramidate molecules (including NUC-7738) are disclosed in WO2017 / 109491 (Nucana). Specific synthetic routes for NUC-7738 are disclosed in WO2018229493 (Nucana) and WO2018229495 (Nucana).

[0176] NUC-7738 has the structure shown in Formula 1.

[0177]

[0178] NUC-7738 contains one chiral center at the phosphorus atom. The NUC-7738 may exist as a mixture of phosphate diastereomers, as an (S)-epimer in a substantially diastereomerically pure form at the phosphorus atom, or as an (R)-epimer in a substantially diastereomerically pure form at the phosphorus atom. For purposes of the present invention, "substantially diastereomerically pure" is defined as a diastereomeric purity of greater than about 90%. If present in a substantially diastereomerically pure form, the NUC-7738 may have a diastereomeric purity of greater than 95%, 98%, 99% or even 99.5%. Alternatively, the NUC-7738 may exist as a mixture of phosphate diastereomers.

[0179] The (R)- and / or (S)-phosphate diastereomers of NUC-7738 can be obtained in substantially diastereomerically pure form by chromatography, such as HPLC optionally using a chiral column. Alternatively, the (R)- and / or (S)-phosphate diastereomers of NUC-7738 can be obtained in substantially diastereomerically pure form by crystallization from a suitable solvent or solvent system. In a further alternative, the (R)- and / or (S)-phosphate diastereomers of NUC-7738 can be synthesized into diastereomerically pure form using a diastereoselective synthesis method. Any combination of these techniques can be used to provide a diastereomerically pure form, such as a diastereoselective synthesis followed by crystallization or chromatography. It may be more convenient to prepare the diastereomeric mixture of NUC-7738 in protected form, separate the NUC-7738 diastereoisomers in protected form (eg, using chromatography or crystallization), and then remove the protecting groups to provide substantially diastereomerically pure NUC-7738.

[0180] The NUC-7738 used herein may be in the form of a free base or in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include, but are not limited to, pharmaceutically acceptable inorganic acid salts such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, aminosulfonic acid and hydrobromic acid, or pharmaceutically acceptable organic acid salts such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, malic acid, citric acid, lactic acid, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, ethylenediaminetetraacetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid and valine. Suitable base salts are formed from bases that form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases can also be formed, for example hemisulphate and hemicalcium salts.

[0181] The NUC-7738 may be present in a single crystalline form or a mixture of crystalline forms, or it may be amorphous. Thus, the NUC-7738 compound for medical use according to the invention may be administered as a crystalline or amorphous product. For example, it may be obtained in the form of a solid plug, powder or film by methods such as precipitation, crystallization, freeze drying or spray drying or evaporative drying. Microwave or radio frequency drying may be used for this purpose.

[0182] Unless the context indicates otherwise, reference to NUC-7738 in any aspect or embodiment of the invention includes NUC-7738 as the (S)-phosphate diastereomer or as the (R)-phosphate diastereomer or as a mixture of phosphate diastereomers; it also includes the compound in the form of a free base, or it may be in the form of a pharmaceutically acceptable salt; and, it also includes pharmaceutical compositions (e.g., NUC-7738 formulations) comprising said NUC-7738.

[0183] "NUC-7738" preparation

[0184] NUC-7738, including its pharmaceutically acceptable salts, can be used alone, but is generally administered in the form of a pharmaceutical composition in which NUC-7738 is combined with one or more pharmaceutically acceptable excipients such as adjuvants, diluents or carriers. Pharmaceutical excipients are substances other than pharmacologically active drugs or prodrugs that are included in the production process or contained in the finished drug product dosage form. Conventional methods for selecting and preparing suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals-The Science of Dosage Form Designs", ME Aulton, Churchill Livingstone, 1988.

[0185] Suitably, the use of "NUC-7738" mentioned herein may also refer to the use of a pharmaceutical composition comprising NUC-7738.

[0186] Depending on the mode of administration of NUC-7738, the pharmaceutical composition for administering NUC-7738 (including any pharmaceutically acceptable salt thereof) will preferably contain 0.05 to 99% w (weight percent) NUC-7738 or a pharmaceutically acceptable salt thereof, more preferably 0.05 to 80% w NUC-7738 or a pharmaceutically acceptable salt thereof, still more preferably 0.10 to 70% w NUC-7738, and even more preferably 0.10 to 50% w NUC-7738, all weight percentages being based on the total composition.

[0187] NUC-7738 can be administered orally. For oral administration, NUC-7738 can be mixed with an adjuvant or carrier, such as lactose, sucrose, sorbitol, mannitol; starch, such as potato starch, corn starch or pullulan; cellulose derivatives; binders, such as gelatin or polyvinyl pyrrolidone; and / or lubricants, such as magnesium stearate, calcium stearate, polyethylene glycol, wax, paraffin, etc., and then compressed into tablets. If coated tablets are required, the core prepared as described above can be coated with a concentrated sugar solution, which can contain, for example, gum arabic, gelatin, talc and titanium dioxide. Alternatively, the tablet can be coated with a suitable polymer dissolved in a volatile organic solvent.

[0188] In a specific embodiment, the NUC-7738 or a composition comprising NUC-7738 is administered parenterally, particularly intravenously. Parenteral administration methods include, for example, intradermal, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal or intravenous injection and infusion techniques, for example in the form of injection solutions, infusion solutions or tinctures. For parenteral (e.g., intravenous) administration, NUC-7738 can be administered as a sterile aqueous solution or oily solution. Aqueous preparations for intravenous administration, particularly those of NUC-7738 free base, may also contain a pharmaceutically acceptable polar organic solvent, such as dimethylacetamide, and one or more solubilizers or other additives as excipients.

[0189] NUC-7738 or a formulation comprising NUC-7738 according to or used in the present invention may be used to treat humans or other animals, for example to treat commercial animals such as livestock or companion animals such as cats, dogs, etc. Suitably, NUC-7738 or a formulation comprising NUC-7738 according to or used in the present invention is used or may be used to treat humans.

[0190] NUC-7738 according to or for use in the present invention can be obtained, stored and / or administered in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include, but are not limited to, pharmaceutically acceptable inorganic acid salts such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, aminosulfonic acid and hydrobromic acid, or pharmaceutically acceptable organic acid salts such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, malic acid, citric acid, lactic acid, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, ethylenediaminetetraacetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid and valine. Suitable base salts are formed from bases that form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases may also be formed, such as hemisulphates, hemioxalates, and hemicalcium salts. Preferably, the NUC-7738 compound is not in the form of a salt, i.e. it is in the form of a free base / free acid.

[0191] When administered to a subject / patient, the dose administered will of course vary depending on the precise mode of administration, the desired treatment, and the disease / disorder indicated. The dose level, frequency of administration, and duration of treatment will also vary depending on the formulation and the clinical indication, age, and comorbid medical conditions of the patient. The dose size of the NUC-7738 or a pharmaceutical formulation containing it for therapeutic purposes may also vary depending on the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration, in accordance with well-known medical principles.

[0192] Pharmaceutical preparations generally take the form of compositions in which the active compound or a pharmaceutically acceptable salt thereof is combined with one or more pharmaceutically acceptable excipients. One such pharmaceutically acceptable excipient in the formulations of the present invention is a polar aprotic solvent. Conventional methods for selecting and preparing suitable pharmaceutical preparations are described in, for example, "Pharmaceuticals-The Science of Dosage Form Designs", ME Aulton, Churchill Livingstone, 1988.

[0193] The formulations may be suitable for topical application (eg, to the skin), oral administration, or parenteral administration (eg, intravenous administration).

[0194] Any solvents used in the pharmaceutical formulations of the invention should be of pharmaceutical grade, meaning that they have an impurity profile that renders them suitable for administration to humans (eg, intravenous administration).

[0195] For oral administration, the formulations of the invention may comprise the active compound mixed with an adjuvant or carrier, such as lactose, sucrose, sorbitol, mannitol; starch, such as potato starch, corn starch or pullulan; cellulose derivatives; binders, such as gelatin or polyvinyl pyrrolidone; and / or lubricants, such as magnesium stearate, calcium stearate, polyethylene glycol, wax, paraffin, etc., and then compressed into tablets. If coated tablets are required, the core prepared as described above may be coated with a concentrated sugar solution, which may contain, for example, gum arabic, gelatin, talc and titanium dioxide. Alternatively, the tablets may be coated with a suitable polymer dissolved in a volatile organic solvent.

[0196] For the preparation of soft gelatin capsules, the active compound can be mixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatin capsules can contain compound granules using the above-mentioned tablet excipients. In addition, liquid or semisolid preparations of the active compound can be filled into hard gelatin capsules.

[0197] Liquid preparations for oral use can be in the form of syrup or suspension, for example containing a solution of the compounds of this invention, with the remainder being a mixture of sugar and ethanol, water, glycerol and propylene glycol. Optionally, this liquid preparation can contain coloring agents, flavoring agents, sweeteners (such as saccharin), preservatives and / or carboxymethyl cellulose as thickeners or other excipients known to those skilled in the art.

[0198] However, preferably, the formulations comprising NUC-7738 according to or for use in the present invention are for parenteral (e.g., intravenous) administration or for dilution to form a formulation for parenteral (e.g., intravenous) administration. For parenteral (e.g., intravenous) administration, the NUC-7738 compound may be administered as a sterile aqueous solution or an oily solution. Preferably, the active NUC-7738 compound is administered as a sterile aqueous solution.

[0199] Pharmaceutical compositions comprising NUC-7738 according to or for use in the methods of the present invention will preferably contain 0.05 to 99% w (weight percent) NUC-7738, more preferably 0.05 to 80% w NUC-7738, still more preferably 0.10 to 70% w NUC-7738, even more preferably 0.10 to 50% w NUC-7738, all weight percentages being based on the total composition.

[0200] Suitably, the NUC-7738 may be administered in a pharmaceutically effective amount or an amount acceptable for in vivo treatment in a patient. Suitably, the patient is a human, although the patient may be another animal.

[0201] Suitably, the pharmaceutical composition comprising said NUC-7738 can be administered to humans or other animals in an amount sufficient to produce a therapeutic effect according to the above-mentioned methods of treatment / medical use.

[0202] Reference to NUC-7738 in the context of a pharmaceutical composition (e.g., a pharmaceutical composition comprising NUC-7738) includes NUC-7738 as the (S)-phosphate diastereomer or as the (R)-phosphate diastereomer or as a mixture of phosphate diastereomers; it also includes the compound in free base form, or it may be in the form of a pharmaceutically acceptable salt.

[0203] Therapeutically Effective Dose of NUC-7738

[0204] A therapeutically effective amount of NUC-7738 can be an amount sufficient to induce cancer cell death.

[0205] There are a variety of different ways to calculate and express the amount of a therapeutically effective compound, such as NUC-7738, to be administered to a patient. One method that is considered particularly relevant to the dosage of drugs used to prevent or treat cancer is the amount of drug administered per unit of body surface area of ​​the patient. This dosage is usually expressed in m2 (meters per square meter). 2 ) surface area (which can be determined by mass).

[0206] NUC-7738 can be used to treat cancer at a dose of 300 mg / m 2 Up to 1600 mg / m 2 This treatment may be, for example, with 500 mg / m 2 Up to 1150 mg / m 2 Weekly dose, or 900 mg / m 2 Up to 1350 mg / m 2 The nominal dose can be selected from: 900mg / m 2 , 1100mg / m 2 , 1125mg / m 2 and 1350 mg / m 2 NUC-7738 may be used to treat cancer with a reduced dosing frequency, such as Q2W, Q3W, or Q4W, and the dose may be adjusted accordingly.

[0207] The selected weekly dose of NUC-7738 used according to the present invention can be provided in a single administration, or multiple administrations within a week. For example, a weekly dose of the compound of the present invention can be provided in two administrations, three administrations, or more. Thus, at a weekly dose of 900 mg / m 2 In the case of 2Or 450 mg / m2 twice a week 2 Similarly, at a weekly dose of 1350 mg / m 2 In the case of 2 Or 675 mg / m2 twice over the course of one week 2 If the dose is Q2W, Q3W or Q4W, the dose administered can be determined based on the selected weekly dose.

[0208] A suitable amount of NUC-7738 for use according to the present invention may be about 900 mg / m 2 Up to 1350 mg / m 2 , to provide the desired dosage of the compound over the course of one week.

[0209] The weekly dose of NUC-7738 used according to the present invention may be reduced during the course of treatment. For example, treatment may be at about 1350 mg / m 2 , 1125mg / m 2 , 1100mg / m 2 , 900mg / m 2 or 750 mg / m 2 Weekly doses of 10 mg / m are started, and during treatment, the required dose can be reduced to about 750 mg / m 2 (in case the initial dose is higher than this amount), to about 625 mg / m 2 , or 500 mg / m 2 , or even about 375 mg / m 2 .

[0210] The dosage of NUC-7738 used according to the present invention may of course be presented in other ways. The most common of these is the amount of active agent provided per unit body weight. It has been calculated that for an average human patient, 1 mg / m 2 The dosage is equivalent to about 0.025 mg / kg body weight. Therefore, the data show that the compounds of the present invention are effective for treating relapsed or refractory cancers in a dosage range of about 6.25 mg / kg to about 25 mg / kg. Suitable dosages can be, for example, about 9.5 mg / kg and 22.5 mg / kg. In a suitable embodiment, when a weekly dose of about 12.5 mg / kg to 20.5 mg / kg is provided to the patient, the compounds of the present invention achieve effective treatment of relapsed or refractory cancers.

[0211] The NUC-7738 can be administered in a series of treatment cycles, each cycle can last any time, such as 1, 2, 3, 4, 5, 6 weeks, or 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 35 or 42 days, or longer. In specific embodiments, the NUC-7738 is administered to a patient in a series of 21-day treatment cycles, the series being 2, 3, 4, 5, 6, 7, 8, 9, 10 or more treatment cycles. In other specific embodiments, the NUC-7738 is administered to a patient in a series of 42-day treatment cycles, the series being 2, 3, 4, 5, 6 or more treatment cycles.

[0212] In a 21-day treatment cycle, the NUC-7738 can be administered once, twice, or three times. In one embodiment, in each treatment cycle, the NUC-7738 is administered on day 1 of the 21-day treatment cycle. In another embodiment, in each treatment cycle, the NUC-7738 is administered on day 1 and day 8 of the 21-day treatment cycle. In another embodiment, in each treatment cycle, the NUC-7738 is administered on day 1, day 8, and day 15 of the 21-day treatment cycle.

[0213] In a 42-day treatment cycle, the NUC-7738 may be administered 1-6 times. In one embodiment, in each treatment cycle, the NUC-7738 is administered on day 1 of a 42-day treatment cycle. In another embodiment, in each treatment cycle, the NUC-7738 is administered on day 1 and day 8 of a 42-day treatment cycle. In another embodiment, in each treatment cycle, the NUC-7738 is administered on day 1, day 8, and day 15 of a 42-day treatment cycle. In another embodiment, in each treatment cycle, the NUC-7738 is administered on day 1, day 8, day 15, and day 22 of a 42-day treatment cycle. In another embodiment, in each treatment cycle, the NUC-7738 is administered on day 1, day 8, day 15, day 22, and day 29 of a 42-day treatment cycle. In another embodiment, in each treatment cycle, the NUC-7738 is administered on Day 1, Day 8, Day 15, Day 22, Day 29, and Day 35 of a 42-day treatment cycle.

[0214] Considerations regarding NUC-7738 formulations suitable for the treatment methods and medical uses of the present invention are described elsewhere in this disclosure. In the case of injectable formulations of the compounds of the invention, these formulations can be administered intravenously. Intravenous administration can be accomplished over any suitable time frame, such as a 10 minute injection, etc.

[0215] Therapeutically effective doses of immuno-oncology agents such as immune checkpoint inhibitors

[0216] A therapeutically effective amount of an immuno-oncology agent can be determined by one skilled in the art using routine and existing knowledge of dosages of such drugs approved by health authorities as monotherapy or standard clinical studies. A therapeutically effective amount of a checkpoint inhibitor can be an amount sufficient to induce cancer cell death.

[0217] For example, the following table (Table 1) is a non-exhaustive list of approved immune checkpoint inhibitors that may be suitable for the treatment methods and medical uses of the present invention. In certain embodiments, one or more diseases listed in Table 1 are treated in any of the various aspects of the present invention described herein.

[0218]

[0219]

[0220] Appropriate dosages of cancer vaccines and CAR-T cell compositions can be determined by those skilled in the art using routine and existing knowledge of dosages for such agents approved by health authorities as monotherapy or from standard clinical studies.

[0221] Examples of approved CAR-T therapies, diseases treated, and dosages are included in the table below (Table 2):

[0222]

[0223]

[0224] "NUC-7738 medical use"

[0225] According to one aspect, the present invention provides a method for treating a proliferative disease such as cancer. Suitably, the method comprises administering a therapeutically effective amount of a composition to a subject in need of such a composition, wherein the composition comprises NUC-7738. Suitably, the therapeutically effective amount is an amount capable of reducing the production of extracellular PD-L1 protein, such as soluble PD-L1 protein and / or exosomal PD-L1, by cancer cells, and / or increasing the production of OX40-L by cancer cells. In a specific embodiment, the proliferative disease is a disease in which high levels of soluble PD-L1 and / or exosomal PD-L1 contribute to the pathogenesis of the disease. For example, cancer.

[0226] According to one aspect, the present invention provides NUC-7738 for treating cancer by reducing the amount of extracellular PD-L1 protein produced by cancer cells. Suitably, the extracellular PD-L1 protein is soluble PD-L1 protein and / or exosomal PD-L1 protein. Suitably, the treatment of cancer is produced by humoral immunity.

[0227] According to another aspect, the present invention provides a method for treating cancer by reducing the amount of extracellular PD-L1 protein (such as soluble PD-L1 protein and / or exosomal PD-L1 protein) produced by cancer cells in a patient, the method comprising administering a therapeutically effective amount of NUC-7738 to a patient in need thereof.

[0228] According to another aspect, the present invention provides a use of NUC-7738 in the preparation of a medicament for use in a method for treating cancer by reducing the amount of extracellular PD-L1 protein, such as soluble PD-L1 protein and / or exosomal PD-L1 protein, produced by cancer cells.

[0229] According to another aspect, the present invention provides NUC-7738 for use in treating cancer by increasing the amount of OX40-L protein produced by cancer cells.

[0230] According to another aspect, the present invention provides a method of treating cancer by increasing the amount of OX40-L protein produced by cancer cells in a patient, the method comprising administering a therapeutically effective amount of NUC-7738 to a patient in need thereof.

[0231] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for use in a method of treating cancer by increasing the amount of OX40-L protein produced by cancer cells.

[0232] In a specific embodiment, increasing the amount of OX40-L protein produced by cancer cells makes the cancer cells more easily targeted by the host immune system, such as the patient's humoral immune system.

[0233] The ability of NUC-7738 to cause a decrease in the production and release of both soluble PD-L1 and exosomal PD-L1 demonstrates that NUC-7738 is an immune sensitizer and can be used as an immune sensitizer. This effect is further supported by the finding that NUC-7738 also causes an increase in the expression and production of OX-40L by cancer cells.

[0234] Thus, according to another aspect, the present invention provides NUC-7738 for use as an immunosensitizer. Suitably, the present invention provides NUC-7738 for use as an immunosensitizer in the treatment of cancer.

[0235] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament as an immunosensitizer. Alternatively, the present invention provides the use of NUC-7738 in the preparation of a medicament for treating cancer, wherein the NUC-7738 is an immunosensitizer.

[0236] According to another aspect, the invention provides a method of enhancing an immune response to a tumor, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of NUC-7738, alone or in combination with an immuno-oncology agent described herein.

[0237] According to another aspect, the present invention provides NUC-7738 for treating cancer by reducing the blockade of the patient's immune system targeting cancer cells. Suitably, reducing the blockade is removing an inhibitor or suppressor of the patient's immune system. As described herein, the suppressor may be an extracellular PD-L1 protein, such as a soluble PD-L1 protein and / or an exosomal PD-L1 protein.

[0238] According to another aspect, the present invention provides NUC-7738 for use in treating cancer by enhancing a patient's immune response against the cancer.

[0239] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for use in a method of treating cancer by enhancing a patient's immune response to the cancer.

[0240] According to another aspect, the present invention provides a method of treating cancer by enhancing a patient's immune response to the cancer, wherein the method comprises administering a therapeutically effective amount of NUC-7738 to a patient in need thereof.

[0241] In specific embodiments of any of these aspects of the invention, administering NUC-7738 to a patient enhances the patient's immune response to the proliferative disease.

[0242] In specific embodiments of any of these aspects of the invention, administration of NUC-7738 to the patient reduces exhaustion of T cells.

[0243] In specific embodiments of any of these aspects of the invention, administration of NUC-7738 counteracts the effects of extracellular PD-L1 protein.

[0244] In specific embodiments of any of these aspects of the invention, administration of NUC-7738 counteracts the effects of soluble PD-L1 protein or exosomal PD-L1 protein.

[0245] In specific embodiments of any of these aspects of the invention, treatment of cancer is achieved through humoral immunity.

[0246] In specific embodiments of any of these aspects of the invention, said NUC-7738 is capable of reducing the level of soluble PD-L1 produced by cancer cells.

[0247] In specific embodiments of any of these aspects of the invention, said NUC-7738 is capable of reducing the level of exosomal PD-L1 produced by cancer cells.

[0248] In specific embodiments of any of these aspects of the invention, said NUC-7738 is capable of increasing the production of OX40-L by cancer cells.

[0249] In specific embodiments of any of these aspects of the invention, treatment with NUC-7738 results in a decrease in extracellular PD-L1 protein levels.

[0250] In specific embodiments of any of these aspects of the invention, treatment with NUC-7738 results in a decrease in the levels of soluble PD-L1 protein or exosomal PD-L1 protein.

[0251] In specific embodiments, the NUC-7738 reduces the amount of extracellular PD-L1 produced by affected cells (e.g., cancer cells) by at least 30%, such as at least 50%, at least 70%, at least 80%, or at least 90%, relative to pre-treatment levels.

[0252] In specific embodiments, NUC-7738 reduces the amount of soluble PD-L1 protein and / or exosomal PD-L1 protein produced by affected cells (e.g., cancer cells) by at least 30%, such as at least 50%, at least 70%, at least 80% or at least 90%, relative to pre-treatment levels.

[0253] In specific embodiments of any of these aspects of the invention, treatment with NUC-7738 results in increased levels of OX40-L protein produced by proliferating (eg, cancer) cells.

[0254] In a specific embodiment of any one of these aspects of the invention, the NUC-7738 acts as an immune sensitizer. In a specific embodiment, the NUC-7738 can be used to enhance the effect of immunotherapy. In a specific embodiment, the NUC-7738 can be used to enhance the effect of immuno-oncology agents. Suitably, the immune checkpoint inhibitor is an anti-PD-L1 or anti-PD-1 antibody. Suitably, the immune checkpoint inhibitor is selected from pembrolizumab, cemipril monoclonal antibody, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. Suitably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, cemipril monoclonal antibody, dotalimumab and nivolumab. In a specific embodiment for any one of these aspects of the invention, the immune checkpoint inhibitor is pembrolizumab.

[0255] Suitably, the immunotherapy or immuno-oncology agent is an immune checkpoint inhibitor or a cancer vaccine or adoptive cell therapy, such as the CAR-T cell therapy described herein.

[0256] As further described herein, in specific embodiments, the treatment or method of treatment may include administering NUC-7738 in combination with an immunotherapy agent (such as an immune checkpoint inhibitor), adoptive cell therapy (such as CAR-T cells), or a cancer vaccine as described herein.

[0257] In specific embodiments, high levels of extracellular PD-L1 protein inhibit or block the host immune system from attacking cancer cells.

[0258] In specific embodiments, high levels of extracellular PD-L1 protein inhibit or block the effect of an immune checkpoint inhibitor administered to the patient.

[0259] In specific embodiments, high levels of soluble PD-L1 and / or exosomal PD-L1 inhibit or block the host immune system from attacking cancer cells.

[0260] In specific embodiments, high levels of soluble PD-L1 and / or exosomal PD-L1 inhibit or block the effect of an immune checkpoint inhibitor administered to a patient.

[0261] Thus, in specific embodiments, NUC-7738 is used to treat patients whose cancer cells express high levels of extracellular PD-L1 protein (such as soluble PD-L1 and / or exosomal PD-L1 protein).

[0262] In a specific embodiment, the NUC-7738 reduces the expression of soluble PD-L1 and / or exosomal PD-L1 protein. In a specific embodiment, the NUC-7738 reduces the transcription of mRNA encoding soluble PD-L1 and / or exosomal PD-L1 protein.

[0263] In specific embodiments, low levels of OX40-L protein inhibit or block the host immune system from attacking cancer cells.

[0264] In specific embodiments, low levels of OX40-L protein inhibit or block the effect of an immune checkpoint inhibitor administered to the patient.

[0265] In a specific embodiment, the NUC-7738 increases the expression of OX40-L protein by cancer cells. In a specific embodiment, the NUC-7738 increases the transcription of mRNA encoding OX40-L.

[0266] In a specific embodiment, the patient to be treated has a cancer whose cancer cells express high levels of soluble PD-L1 and / or exosomal PD-L1 protein.

[0267] In a specific embodiment, the patient to be treated has a cancer whose cancer cells express low levels of OX40-L protein.

[0268] In a specific embodiment, the patient has previously received treatment with an immune checkpoint inhibitor, optionally wherein the treatment has been stopped. Suitably, the previous treatment with the immune checkpoint inhibitor was stopped due to toxicity, recurrence, or resistance of the cancer to the previous treatment. Suitably, such a patient may be treated with NUC-7738 and / or an immune checkpoint inhibitor, but wherein the immune checkpoint inhibitor is administered at a dose lower than the approved monotherapy dose.

[0269] In specific embodiments, the patient to be treated has a cancer that is resistant to treatment with an immune checkpoint inhibitor.

[0270] In specific embodiments of any of these "NUC-7738 medical uses", the NUC-7738 can be administered in one or more treatment cycles, such as 1, 2, 3, 4, 5, 6, 7, 8 or more treatment cycles.

[0271] "NUC-7738 Combo"

[0272] Administration of NUC-7738 can reduce the amount of immune blockade of cancer cells, so NUC-7738 can be used in combination with one or more immuno-oncology agents, such as immune checkpoint inhibitors, antibody therapies, cancer vaccines (such as therapeutic cancer vaccines), or adoptive cell therapies, such as CAR-T.

[0273] According to another aspect, the invention provides a method of enhancing an immune response to a tumor, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of NUC-7738, alone or in combination with an immuno-oncology agent described herein.

[0274] According to another aspect, the present invention provides a method for enhancing the effect of an immuno-oncology agent, the method comprising administering simultaneously or sequentially a therapeutically effective amount of the immuno-oncology agent and NUC-7738 to a patient in need of such treatment.

[0275] According to another aspect, the present invention provides a combination comprising NUC-7738 and an immuno-oncology agent.

[0276] In specific embodiments, the immuno-oncology agent as described herein is an immune checkpoint inhibitor, a cancer vaccine, or an adoptive cell therapy (such as CAR-T).

[0277] In a specific embodiment, the immuno-oncology agent is an immune checkpoint inhibitor as described herein. Suitably, the immune checkpoint inhibitor is selected from pembrolizumab, cemiplizumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab.

[0278] According to another aspect, the present invention provides a combination comprising NUC-7738 as defined herein and an immune checkpoint inhibitor for use in the treatment of a proliferative disease. Suitably, the proliferative disease is a cancer as described herein.

[0279] Because NUC-7738 has an enhancing effect on immune checkpoint inhibitors, it is expected that immune checkpoint inhibitors can be administered at a dose lower than the standard / approved monotherapy dose of the agent. Suitably, the dose of the immune checkpoint inhibitor can be 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the approved monotherapy dose of the agent.

[0280] In a specific embodiment, the immune checkpoint inhibitor used in combination with the NUC-7738 is administered at a dose of 75% or less of the standard or approved monotherapy dose of the agent. In a specific embodiment, the immune checkpoint inhibitor used in combination with the NUC-7738 is administered at a dose of 50% or less of the standard or approved monotherapy dose of the agent. The approved dose refers to a dose approved / authorized by the appropriate health agency of the country or region (such as the U.S. Food and Drug Administration (FDA) for the United States, the European Medicines Agency (EMA) for Europe, and the Medicines and Healthcare Products Regulatory Agency (MHRA) for the United Kingdom) for a monotherapy agent for the relevant disease. For example, the MHRA approved the anti-PD-1 antibody nivolumab for monotherapy treatment of adult patients with melanoma at a dose of 240 mg every 2 weeks or 480 mg every 4 weeks by intravenous infusion. Therefore, 50% of the approved monotherapy dose is 120 mg every 2 weeks or 240 mg every 4 weeks by intravenous infusion.

[0281] As described herein and understood in the art, some patients develop resistance to administered immune checkpoint inhibitors. Other patients suffer significant toxicities and / or infusion-related reactions that may require delayed dosing or discontinuation of the drug. The ability of NUC-7738 to enhance the effects of immune checkpoint inhibitors should allow the use of lower doses of immune checkpoint inhibitors, which would allow patients to restart treatment that was discontinued due to resistance, or for patients who discontinued the original treatment due to toxicities or infusion-related reactions, restart treatment but at a lower dose.

[0282] "Combination use"

[0283] According to another aspect, the present invention provides NUC-7738 for combined treatment of proliferative diseases, wherein the NUC-7738 is administered in combination with an immuno-oncology agent as described herein. Suitably, the immuno-oncology agent is an immune checkpoint inhibitor as defined herein. Suitably, the immune checkpoint inhibitor is selected from pembrolizumab, cimiprilimab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. Suitably, the immune checkpoint inhibitor is selected from pembrolizumab, cimiprilimab, dotalimumab or nivolumab. Suitably, the immune checkpoint inhibitor is selected from nivolumab and pembrolizumab. Suitably, the immune checkpoint inhibitor is pembrolizumab. Suitably, the proliferative disease is melanoma. Suitably, the melanoma is a cutaneous melanoma. Suitably, the subject with melanoma (e.g., skin melanoma) has received at least one prior treatment for the disease and optionally has made progress. Suitably, the subject with melanoma (e.g., skin melanoma) has received at least one prior immunotherapy treatment for the disease and optionally has made progress. Suitably, the immuno-oncology agent is adoptive cell therapy. Suitably, the adoptive cell therapy is CAR-T cell therapy.

[0284] Therefore, in a specific aspect, the present invention provides NUC-7738 for combined cancer therapy, wherein the NUC-7738 is administered in combination with an immune checkpoint inhibitor selected from pembrolizumab, cemiprilimab, dotalimab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. In a specific embodiment, the NUC-7738 is administered in combination with nivolumab, cemiprilimab, dotalimab and / or pembrolizumab. In a specific embodiment, the NUC-7738 is administered in combination with nivolumab and / or pembrolizumab. In a specific embodiment, the NUC-7738 is administered in combination with pembrolizumab. In a specific embodiment, the cancer is a solid tumor. In a specific embodiment, the cancer is a melanoma. In a specific embodiment, the cancer is a cutaneous melanoma. In a specific embodiment, the cancer is an advanced cutaneous melanoma. In a specific embodiment, the cancer patient suffers from advanced (including metastatic) skin melanoma, and has received at least one, such as 2, 3 or more previous skin melanoma treatments, optionally, wherein the disease has developed after the previous treatment. In this case, the combination therapy (eg, NUC-7738+nivolumab and / or pembrolizumab) is a second-line, third-line or further treatment. In a specific embodiment, the cancer patient suffers from advanced (including metastatic) skin melanoma, and has been treated with immunotherapy. In a specific embodiment, a patient with cancer (eg, skin melanoma) has been treated with an immune checkpoint inhibitor, such as one selected from pembrolizumab, cemiplizumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab.

[0285] Therefore, in a specific aspect, the present invention provides NUC-7738 for combined treatment of melanoma (such as skin melanoma), wherein the NUC-7738 is administered in combination with an immune checkpoint inhibitor selected from pembrolizumab, cimiprilimab, dotalimab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab. In a specific embodiment, the NUC-7738 is administered in combination with nivolumab and / or cimiprilimab and / or dotalimab and / or pembrolizumab. In a specific embodiment, the NUC-7738 is administered in combination with pembrolizumab. In a specific embodiment, the NUC-7738 is administered in combination with pembrolizumab to a patient with skin melanoma. In a specific embodiment, the NUC-7738 is administered in combination with pembrolizumab to a patient with advanced skin melanoma. In a specific embodiment, NUC-7738 is administered in combination with pembrolizumab to a patient with advanced cutaneous melanoma who has received at least one prior cycle of treatment, optionally having received prior immunotherapy.

[0286] According to another aspect, the present invention provides a method for treating a proliferative disease in a subject in need thereof, comprising administering to the subject a combination comprising NUC-7738 and an immuno-oncology agent (such as an immune checkpoint inhibitor as defined herein). Suitably, the proliferative disease is cancer. Suitably, the cancer is selected from the group consisting of: melanoma (including skin melanoma), lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, lymphoma, ovarian cancer, cervical cancer and endometrial cancer. In a specific embodiment, the proliferative disease is a disease that is usually treated with an immune checkpoint inhibitor (such as pembrolizumab). Table 1 provides a list of diseases that can be treated with specific checkpoint inhibitors. Suitably, the proliferative disease is cancer, and the patient or tumor and its microenvironment show high levels of soluble or exosomal PD-L1. Suitably, the proliferative disease is cancer, and the cancer cells express low levels of OX40L.

[0287] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for treating a proliferative disease in combination with an immuno-oncology agent (such as an immune checkpoint inhibitor as defined herein). Suitably, the proliferative disease is cancer.

[0288] In specific embodiments of these aspects of the invention, NUC-7738 and an immuno-oncology agent (such as an immune checkpoint inhibitor) are administered to the subject simultaneously or sequentially.

[0289] In specific embodiments of these aspects of the invention, the NUC-7738 and an immuno-oncology agent (such as an immune checkpoint inhibitor) are administered to the subject in one or more treatment cycles, for example, one or more 21-day or 42-day treatment cycles. The two agents can be administered on the same day or on different days of each treatment cycle. For example, when combining NUC-7738 and pembrolizumab, pembrolizumab can be administered on the first day of a 21-day treatment cycle, and the NUC-7738 can be administered on the first, 8, and 15th days of a 21-day treatment cycle; in another example, pembrolizumab can be administered on the first day of a 42-day treatment cycle, and the NUC-7738 can be administered on the first, 8, 15, 22, 29, and 35th days of a 42-day treatment cycle. In a specific embodiment, the treatment cycle is repeated so that the patient can receive 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more treatment cycles during treatment. When NUC-7738 is combined with an immune checkpoint inhibitor (e.g., pembrolizumab), NUC-7738 may be administered first or second on days when both agents are administered to the patient. Historically, antibodies are typically administered prior to administration of cytotoxic agents, so in a preferred embodiment, an immune checkpoint inhibitor antibody, such as pembrolizumab, is administered prior to administration of the NUC-7738.

[0290] In a specific embodiment, when the combination is NUC-7738 and pembrolizumab, NUC-7738 is administered on days 1, 8, and 15 of a 21-day cycle at a dose selected from: 1125 mg / m 2 , 900mg / m 2 or 1350 mg / m 2 , and 200 mg of pembrolizumab is administered on day 1 of a 21-day cycle. It is possible to administer pembrolizumab at 400 mg (Q6w), in which case NUC-7738 can be administered on days 1, 8, 15, 22, 29, and 35 of a 42-day cycle, and 400 mg of pembrolizumab is administered on day 1.

[0291] When an immune checkpoint inhibitor other than pembrolizumab is used, for example, one selected from cemiplizumab, dotalimumab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab, the implementation scheme for pembrolizumab is adjusted accordingly.

[0292] According to another aspect, the present invention provides NUC-7738 for use in treating a proliferative disease, wherein the NUC-7738 is for simultaneous or sequential administration with an immuno-oncology agent (such as an immune checkpoint inhibitor as defined herein).

[0293] According to another aspect, the present invention provides a method of treating a proliferative disease in a subject in need thereof, comprising administering to the subject a combination comprising NUC-7738 and an immuno-oncology agent (such as an immune checkpoint inhibitor as defined herein), wherein NUC-7738 and the immuno-oncology agent are administered simultaneously or sequentially.

[0294] According to another aspect, the present invention provides an immune checkpoint inhibitor as defined herein for use in the treatment of a proliferative disease, wherein the immune checkpoint inhibitor is for simultaneous or sequential administration with NUC-7738 as defined herein.

[0295] According to another aspect, the present invention provides the use of an immune checkpoint inhibitor in combination with NUC-7738 as defined herein for the preparation of a medicament for the treatment of a proliferative disease.

[0296] In a specific embodiment, the cancer is selected from: skin cancer (such as melanoma Merkel cell carcinoma or skin melanoma), lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, kidney cancer, bladder cancer, lymphoma (such as Hodgkin lymphoma), ovarian cancer, cervical cancer, endometrial cancer. In a specific embodiment, the cancer is a disease that is usually treated with immune checkpoint inhibitors (such as pembrolizumab). Table 1 provides a list of diseases that can be treated with specific checkpoint inhibitors. Suitably, the cancer includes cancer cells expressing high levels of soluble or exosomal PD-L1. Suitably, the cancer includes cancer cells expressing low levels of OX40L.

[0297] “Drug products containing NUC-7738”

[0298] According to another aspect, the present invention provides a pharmaceutical product comprising NUC-7738 and an immuno-oncology agent.

[0299] In a specific embodiment, the immuno-oncology agent is an immune checkpoint inhibitor as described herein.

[0300] In one embodiment, the drug product may include a kit of parts comprising separate formulations of NUC-7738 and an immune checkpoint inhibitor.

[0301] Separate formulations of NUC-7738 and the immune checkpoint inhibitor may be administered sequentially and / or simultaneously or may be adapted for sequential and / or simultaneous administration or may be adjusted for sequential and / or simultaneous administration.

[0302] In another embodiment, the pharmaceutical product is a kit of parts comprising:

[0303] a first container comprising NUC-7738, such as NUC-7738 in combination with a pharmaceutically acceptable adjuvant, diluent or carrier; and

[0304] a second container comprising an immune checkpoint inhibitor, such as an immune checkpoint inhibitor in combination with a pharmaceutically acceptable adjuvant, diluent or carrier, and

[0305] Container means for receiving said first and second containers.

[0306] In a specific embodiment, the drug product may comprise one or more unit dosage forms (e.g., vials, tablets, or capsules in blister packs). In one embodiment, each unit dose comprises only one drug selected from NUC-7738 and the immune checkpoint inhibitor. In another embodiment, the unit dosage form comprises the NUC-7738 compound and the immune checkpoint inhibitor.

[0307] “Immune checkpoint inhibitors”

[0308] Immune checkpoint proteins present on immune cells and / or cancer cells [e.g., PD1 (also known as programmed cell death protein 1 and CD279), PD-L1 (also known as programmed death ligand 1 and CD274), CTLA4 (also known as cytotoxic T-lymphocyte-associated protein 4 and CD152), LAG3 (also known as lymphocyte activation gene 3 and CD223), TIM-3 (also known as T-cell immunoglobulin mucin-3), and TIGIT (also known as T-cell immunoreceptor with Ig and ITIM domains)] are molecular targets that have been found to play an important role in regulating anti-tumor immune responses. Inhibitors of these immune checkpoint proteins (e.g., CTLA4, LAG3, PD1, PD-L1, TIM-3, BTLA, OX40, OX40L, and / or TIGIT inhibitors) promote anti-tumor immune responses and can be used to effectively treat certain forms of cancer.

[0309] The immune checkpoint inhibitors mentioned herein refer to any immune checkpoint inhibitors, including any immune checkpoint inhibitors disclosed herein. Therefore, any immune checkpoint inhibitor can be used in the combination therapy defined herein.

[0310] In one embodiment, the immune checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, LAG3 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, BTLA inhibitors, OX40 inhibitors, OX40L inhibitors and / or TIGIT inhibitors. In a specific embodiment, the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. In a specific embodiment, the immune checkpoint inhibitor is an antibody against immune checkpoint proteins, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG3 antibodies, anti-CTLA-4 antibodies, anti-TIM3 antibodies, anti-BTLA antibodies, anti-OX40 antibodies or anti-OX40L antibodies. As used herein, the term "antibody" also includes fragments of complete antibodies that can bind to target antigens, including Fab, scFV, etc.

[0311] PD-1 is a cell surface receptor protein present on immune cells such as T cells. PD-1 plays an important role in downregulating the immune system and promoting self-tolerance by inhibiting T cell activation. The PD-1 protein is an immune checkpoint that prevents autoimmunity through a dual mechanism of promoting apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes while reducing apoptosis of regulatory T cells (anti-inflammatory suppressor T cells).

[0312] PD-1 thus suppresses the immune system. This prevents autoimmune diseases, but can also stop the immune system from killing cancer cells.

[0313] PD-1 binds two ligands, PD-L1 and PD-L2. PD-L1 is of particular interest because it is highly expressed in several cancers, and thus the role of PD-1 in cancer immune evasion is clear. Monoclonal antibodies targeting the immune-boosting PD-1 have been approved or are in development for the treatment of many types of cancer. Many tumor cells express PD-L1, an immunosuppressive PD-1 ligand; there is good evidence that inhibiting the interaction between PD-1 and PD-L1 can enhance T cell responses in vitro and mediate preclinical anti-tumor activity. This is known as immune checkpoint blockade.

[0314] Examples of drugs that target PD-1 include pembrolizumab (Keytruda TM ), Opdivo TM ) and cemiplimab (Libtayo TM). These drugs have been shown to be effective in treating several types of cancer, including melanoma of the skin, squamous cell carcinoma of the skin, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck cancer, and Hodgkin lymphoma. They are also being studied against many other types of cancer. Examples of drugs in development include BMS-936559 (Bristol Myers Squibb), MGA012 (MacroGenics), and MEDI-0680 (MedImmune).

[0315] Examples of drugs that inhibit PD-L1 include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). These drugs have also been shown to help treat different types of cancer, including bladder cancer, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, and Merkel cell skin cancer (Merkel cell carcinoma). They are also being studied for use against other types of cancer.

[0316] Examples of LAG3 inhibitors include BMS-986016 / relatlimab, TSR-033, REGN3767, MGD013 (a bispecific DART that binds PD-1 and LAG-3), GSK2831781, and LAG525.

[0317] Examples of CTLA-4 inhibitors include MDX-010 / ipilimumab, AGEN 1884, and CP-675,206 / tremelimumab.

[0318] Examples of TIM-3 inhibitors include sabatolimab (also known as MBG453 (Novartis)), cobolimab (also known as TSR-022 (Tesaro / GlaxoSmithKline)), LY3321367 (Lilly), and BMS-986258 (Bristol-Myers Squibb).

[0319] Examples of TIGIT inhibitors include tiragolumab (MTIG7192A; RG6058; Genentech / Roche), AB154 (Arcus Biosciences), MK-7684 (Merck), BMS-986207 (Bristol-Myers Squibb), ASP8374 (Astellas Pharma; Potenza Therapeutics).

[0320] Examples of OX40 inhibitors include: MEDI6469 (AstraZeneca) and BMS-986178 (Bristol-Myers Squib).

[0321] Examples of OX40-L inhibitors include: SL-279252 (PD1-Fc-OX40L - Shattuck Labs).

[0322] Examples of BTLA inhibitors include: INBRX-106 (InhinRX), cudarolimab (also known as IBI 101 (Innovent Biologics)).

[0323] In a specific embodiment, the immune checkpoint inhibitor is selected from BMS-986016 / relalizumab, TSR-033, REGN3767, MGD013 (a bispecific DART that binds PD-1 and LAG-3), GSK2831781 and LAG525, MDX-010 / ipilimumab, AGEN1884 and CP-675, 206 / tesimumab, pembrolizumab, nivolumab, atezolizumab, avi Lulumab, Durvalumab, MBG453, TSR-022, LY3321367, Tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106 and Cudarolimab, or a pharmaceutically acceptable salt or solvate thereof.

[0324] In one embodiment, the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0325] In a specific embodiment, the immune checkpoint inhibitor is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0326] In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0327] In a specific embodiment, the immune checkpoint inhibitor is an anti-PD-L1 antibody selected from the group consisting of nivolumab, pembrolizumab, dotarizumab, and cemiplizumab.

[0328] In a specific embodiment, the immune checkpoint inhibitor is the anti-PD-1 antibody pembrolizumab.

[0329] In a specific embodiment, the immune checkpoint inhibitor is the anti-PD-1 antibody nivolumab.

[0330] In a specific embodiment, the immune checkpoint inhibitor is the anti-PD-1 antibody cemiplimab.

[0331] In a specific embodiment, the immune checkpoint inhibitor is the anti-PD-1 antibody dotarizumab.

[0332] In one embodiment, the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[0333] In a specific embodiment, the immune checkpoint inhibitor is an anti-CTLA-4 antibody selected from ipilimumab or tesimumab.

[0334] In one embodiment, the immune checkpoint inhibitor is an anti-LAG-3 antibody.

[0335] In a specific embodiment, the anti-LAG-3 antibody is relalizumab.

[0336] In one embodiment, the immune checkpoint inhibitor is an anti-TIGIT antibody.

[0337] In a specific embodiment, the anti-TIGIT antibody is tiragolumab.

[0338] In one embodiment, the immune checkpoint inhibitor is an anti-OX40 antibody.

[0339] In a specific embodiment, the anti-OX40 antibody is selected from MEDI6469 and BMS-986178.

[0340] In one embodiment, the immune checkpoint inhibitor is an anti-OX40-L antibody.

[0341] In a specific embodiment, the anti-OX40-L antibody is SL-279252.

[0342] In one embodiment, the immune checkpoint inhibitor is an anti-BTLA antibody.

[0343] In a specific embodiment, the anti-BTLA antibody is selected from INBRX-106 and Cudarolimab.

[0344] Antibodies (including antigen-binding fragments of antibodies)

[0345] Antibodies are immunoglobulin molecules that can specifically bind to targets, such as proteins, polypeptides, peptides, carbohydrates, polynucleotides, lipids, or combinations of the above substances, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In particular, as used herein, the term "antibody" includes complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies such as bispecific antibodies produced by at least two complete antibodies, chimeric antibodies, humanized antibodies, human antibodies, any other modified immunoglobulin molecules, and any fragments thereof containing antigen binding sites, as long as the antibody exhibits the desired biological activity. The term includes complete antibodies (such as IgG1, IgG4, etc.) and antigen binding fragments. The antibody can be from any species. Suitably, the antibody is a human antibody.

[0346] Antigen binding site refers to the part of the molecule that binds to all or part of the target antigen. In an antibody molecule, it can be referred to as an antibody antigen binding site and comprises the antibody portion that specifically binds to all or part of the target antigen. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which is called an epitope. The antibody antigen binding site can be provided by one or more antibody variable domains. Preferably, the antibody antigen binding site comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). The present invention also includes antibody fragments comprising antigen binding sites. Therefore, when referring to antibodies, the term "antigen binding fragment thereof" refers to antibody fragments, such as Fab, Fab', F(ab')2, diabodies, Fv fragments and single-chain Fv (scFv) mutants, which have antigen recognition sites and therefore have the ability to bind antigens. Antigens that bind to immunoglobulin (antibody) fragments are well known in the art. Such fragments do not need to have functional Fc receptor binding sites.

[0347] "Treatment of proliferative diseases"

[0348] The term "proliferative disease" is used herein to refer to unwanted, uncontrolled and abnormal cell proliferation, such as tumor formation or proliferative growth, whether in vitro or in vivo. Examples of proliferative conditions include, but are not limited to, benign, pre-malignant and malignant cell proliferation, including but not limited to malignant tumors, cancer, leukemia, psoriasis, bone disease, (e.g., connective tissue) fibroproliferative diseases and atherosclerosis. Any type of cell can be treated, including but not limited to lung, colon, breast, ovary, prostate, liver, pancreas, brain, bladder, kidney, bone, nerve and skin (including melanoma such as skin melanoma). Various aspects of the present invention have specific applications in the treatment of cancer, especially human cancer. Suitably, the proliferative disease is a disease that is usually treated with immune checkpoint inhibitors (such as pembrolizumab). Table 1 provides a list of diseases that can be treated with specific checkpoint inhibitors. The section entitled "immune checkpoint inhibitors" also identifies cancer types that are recommended to be treated with various immune checkpoint inhibitors. Suitably, the proliferative disease is a cancer comprising cancer cells expressing high levels of soluble or exosomal PD-L1. Suitably, the proliferative disease is cancer comprising cancer cells expressing low levels of OX40L.

[0349] As can be seen in Figure 1, NUC-7738 reduced soluble PD-L1 in both lung and melanoma cell lines, supporting the use of NUC-7738 in the treatment of any proliferative disease mediated by or associated with high levels of sPD-L1 or in which sPD-L1 is implicated (e.g., due to its ability to suppress the host immune system). This includes all cancers.

[0350] In a specific embodiment, the cancer is selected from: skin cancer (such as melanoma Merkel cell carcinoma), lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, kidney cancer, bladder cancer, lymphoma (such as Hodgkin lymphoma), ovarian cancer, cervical cancer, endometrial cancer.

[0351] Melanoma is particularly amenable to treatment with NUC-7738 alone or in combination with immune checkpoint inhibitors such as anti-PD-1 antibodies.

[0352] Cutaneous melanoma is particularly amenable to treatment with NUC-7738 alone or in combination with immune checkpoint inhibitors such as anti-PD-1 antibodies (e.g., pembrolizumab).

[0353] "Diseases or conditions mediated by or associated with high levels of PD-L1 or low levels of OX40L"

[0354] Suitably, NUC-7738 may be used to treat patients with proliferative diseases who generally have high levels of PD-L1, but particularly soluble PD-L1 and / or exosomal PD-L1.

[0355] Suitably, NUC-7738 can be used to treat patients with proliferative diseases with low OX40-L protein levels. Suitably, sPD-L1, xPD-L1 and / or OX40-L levels are levels produced by cancer cells. Suitably, the sPD-L1, xPD-L1 and / or OX40-L levels are detected in a blood sample or fraction thereof (e.g., plasma, serum) from a patient. Suitably, the proliferative disease is cancer, and the patient to be treated can be identified by the level of PD-L1 (e.g., soluble or exosomal PD-L1) or OX40-L in the relevant cells or biological fluids, and therefore the present invention provides the use of NUC-7738 in the treatment of proliferative diseases such as cancer, which are associated with the expression of high levels of PD-L1 (e.g., soluble or exosomal PD-L1) and / or low levels of OX40-L.

[0356] Thus, in part, the present invention relates to the medical use of NUC-7738 for treating a proliferative disease mediated by or associated with high levels of soluble PD-L1 and / or exosomal PD-L1 and / or low levels of OX40-L. Suitably, the proliferative disease is cancer.

[0357] In a specific embodiment, the proliferative disease is a cancer whose cells express high levels of soluble PD-L1 and / or exosomal PD-L1 and / or low levels of OX40-L. In a specific embodiment, the cancer whose cells express high levels of soluble PD-L1 and / or exosomal PD-L1 and / or low levels of OX40-L is selected from: skin cancer (such as melanoma Merkel cell carcinoma), lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, kidney cancer, bladder cancer, lymphoma (such as Hodgkin lymphoma), ovarian cancer, cervical cancer, endometrial cancer.

[0358] According to one aspect, the invention provides NUC-7738 for use in treating cancer in a subject whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1 and / or low levels of OX40-L.

[0359] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for treating cancer in a subject whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1 and / or low levels of OX40-L, wherein the treatment comprises administering NUC-7738 as defined herein in combination with an immune checkpoint inhibitor. Optionally, prior to treatment, the patient is tested to determine whether they have cancer whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1 and / or low levels of OX40-L.

[0360] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for use in a method for treating cancer by reducing the amount of soluble PD-L1 or exosomal PD-L1 produced by cancer cells, wherein the method comprises determining whether a subject has cancer, the cancer cells of the subject expressing high levels of soluble PD-L1 or exosomal PD-L1, wherein if the subject has cancer, the cancer cells of the subject express high levels of soluble or exosomal PD-L1, then administering to the subject a combination of NUC-7738 and an immune checkpoint inhibitor as defined herein.

[0361] According to another aspect, the present invention provides a method of treating cancer in a subject whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1, the method comprising administering to the subject a therapeutically effective amount of NUC-7738 alone or in combination with an immune checkpoint inhibitor.

[0362] According to another aspect, the present invention provides a method of treating cancer in a subject whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1, wherein the cancer cells are contacted with a therapeutically effective amount of NUC-7738. In a specific embodiment of this aspect of the invention, the cancer cells are in a subject or patient. Thus, suitably, the cancer cells are contacted with a therapeutically effective amount of NUC-7738 by administering a therapeutically effective amount of NUC-7738 to the subject alone or in combination with an immune checkpoint inhibitor.

[0363] According to another aspect, the present invention provides the use of NUC-7738 in the preparation of a medicament for use in a method for treating cancer by increasing the amount of OX40-L produced by cancer cells, wherein the method comprises determining whether a subject has cancer and the cancer cells of the subject express low levels of OX40-L, wherein if the subject has cancer and the cancer cells of the subject express low levels of OX40-L, then administering to the subject a combination of NUC-7738 and an immune checkpoint inhibitor as defined herein.

[0364] According to another aspect, the present invention provides a method of treating cancer in a subject whose cancer cells express low levels of OX40-L, the method comprising administering to the subject a therapeutically effective amount of NUC-7738 alone or in combination with an immune checkpoint inhibitor.

[0365] According to another aspect, the present invention provides a method of treating cancer in a subject whose cancer cells express low levels of OX40-L, the method contacting the cancer cells with a therapeutically effective amount of NUC-7738. In a specific embodiment of this aspect of the invention, the cancer cells are in a subject or patient. Thus, suitably, the cancer cells are contacted with a therapeutically effective amount of NUC-7738 by administering a therapeutically effective amount of NUC-7738 to the subject alone or in combination with an immune checkpoint inhibitor.

[0366] In a specific embodiment, the level of soluble PD-L1 and / or exosomal PD-L1 in the serum of a cancer patient whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1 is at least 30% higher than a reference level, such as at least 50%, at least 75%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 750%, at least 1000%, at least 2000% higher than a reference level. The reference level may be a level assigned to a healthy individual, i.e., an individual without a disease (e.g., cancer). Such an individual may be referred to as having normal plasma.

[0367] The level of sPD-L1 in normal plasma is about 10 pg / mL, while in subjects whose cancer cells express high levels of sPD-L1, the level of sPD-L1 in plasma may be about 40 pg / mL to about 200 pg / mL. In specific embodiments, the uses and methods of the present invention are used to treat cancer patients having about 40 pg / mL to 200 pg / mL sPD-L1 in their serum, such as about 60 pg / mL to 150 pg / mL or about 70 pg / mL to 120 pg / mL sPD-L1.

[0368] In specific embodiments, the level of OX40-L in a sample from a cancer patient whose cancer cells express low levels of OX40-L is at least 30% above a reference level, such as at least 50%, at least 75%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 750%, at least 1000%, at least 2000% above a reference level. The reference level may be a level assigned to a healthy individual, i.e., an individual without a disease (e.g., cancer). Such an individual may be referred to as having normal plasma.

[0369] Reference Level

[0370] The reference levels can be determined by parallel measurements of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in normal cells of the patient, or they can be values ​​determined from historical measurements of wild-type / normal cells, such as the average wild-type level determined with relevant statistical significance from multiple samples. In view of the widely recognized inter-patient variability, the reference wild-type level can be a "normal range", such as the lowest value within the normal range. Thus, for example, if the normal range of total protein in the blood is 6 to 8.3 grams per deciliter (g / dL). The reference wild-type value of protein in the blood can be taken as 6g / dL or 8.3g / dL, depending on whether the value to be detected is below or above the normal value. In a similar manner, the normal range of the amount of soluble PD-L1 and / or exosomal PD-L1 or OX40-L can be determined using routine assessment of any specific cell type and used to identify a "reference value" or "reference level" that can be used to determine whether a cell expresses normal, low or high levels of soluble PD-L1, and / or exosomal PD-L1, and / or OX40-L, and therefore whether a patient will benefit from NUC-7738 treatment according to the methods of the present invention. Those skilled in the art will be able to identify appropriate reference values ​​for determining whether a cancer expresses high levels of soluble PD-L1 and / or high levels of exosomal PD-L1 and / or low levels of OX40-L. In specific embodiments, the wild-type value or level can be used as a reference value or level. In other embodiments, the reference value or level is a value or level established from a statistical evaluation of multiple samples (e.g., from healthy individuals and / or individuals with a disease such as cancer) that can be used to assign a test subject to a category (e.g., healthy or having a high level of sPDL-1) with a certain degree of statistical confidence (e.g., 95% confidence level). As described above, one skilled in the art is able to determine a reference / cutoff value or level using established standard techniques.

[0371] In specific embodiments, the NUC-7738 reduces the amount of soluble PD-L1 and / or exosomal PD-L1 produced by affected cells (e.g., cancer cells) by at least 25%, such as at least 30%, at least 40%, at least 50%, at least 70%, at least 80% or at least 90%, relative to pre-treatment levels.

[0372] In a specific embodiment, treatment with NUC-7738 reduces soluble PD-L1 or exosomal PD-L1 levels by at least about 25% compared to levels achieved in the absence of NUC-7738 treatment.

[0373] In specific embodiments, treatment with NUC-7738 reduces soluble PD-L1 or exosomal PD-L1 levels by about 10 to 80% compared to levels achieved in the absence of NUC-7738 treatment.

[0374] In a specific embodiment, the NUC-7738 reduces the amount of soluble PD-L1 and / or exosomal PD-L1 present in serum to a level substantially the same as a normal / reference level. By substantially the same we mean ±10%.

[0375] In specific embodiments, treatment with NUC-7738 reduces soluble PD-L1 or exosomal PD-L1 levels by 2- to 10-fold compared to levels achieved in the absence of NUC-7738 treatment.

[0376] In specific embodiments, NUC-7738 increases the amount of OX40-L produced by affected cells (e.g., cancer cells) by at least 25%, such as at least 30%, at least 40%, at least 50%, at least 70%, at least 80% or at least 90%, relative to pre-treatment levels.

[0377] In a specific embodiment, treatment with NUC-7738 increases OX40-L levels by at least about 25% compared to levels achieved in the absence of NUC-7738 treatment.

[0378] In specific embodiments, treatment with NUC-7738 increases OX40-L levels by about 10 to 80% compared to levels achieved in the absence of NUC-7738 treatment.

[0379] In a specific embodiment, the NUC-7738 increases the level of OX40-L present in serum to a level that is substantially the same as a normal / reference level. By substantially the same we mean ± 10%.

[0380] In specific embodiments, treatment with NUC-7738 increases OX40-L levels by about 2 to 10 fold compared to levels achieved in the absence of NUC-7738 treatment.

[0381] Various aspects of the present invention are based on the discovery that NUC-7738 can reduce the amount of soluble PD-L1 and / or exosomal PD-L1 produced by cancer cells and / or increase the amount of OX40-L produced by cancer cells. It is well known that PD-L1 and OX40-L mask cancer cells from the immune system, and in particular soluble PD-L1 is a biomarker for poor prognosis and resistance to immune checkpoint inhibitors. Therefore, the ability of NUC-7738 to reduce the amount of soluble PD-L1 and / or exosomal PD-L1 produced by cancer cells and / or increase the level of OX40-L produced by cancer cells provides new clinical opportunities for the treatment and management of cancer in patients. For example, by removing or reducing the camouflage of the cancer cells to the immune system, thereby allowing the host immune system to recognize and attack (including killing) cancer cells. The ability of NUC-7738 to reduce the level of soluble PD-L1 and / or exosomal PD-L1 produced by cancer cells and / or to increase the level of OX40-L produced by cancer cells will be used to enhance the effectiveness of immuno-oncology agents (including immune checkpoint inhibitors), which provides the ability to treat cancer patients with a combination of NUC-7738 and immuno-oncology agents. When the immuno-oncology agent is an immune checkpoint inhibitor, as described herein, it can also help to use a lower dose of checkpoint inhibitors, thereby reducing the toxic effects of such drugs. Some cancer patients also develop resistance to immune checkpoint inhibitors, which is often associated with an increase in soluble PD-L1 levels. It is expected that the ability of NUC-7738 to target and reduce the amount of soluble PD-L1 or exosomal PD-L1 removes resistance / blocking, and thus allows patients to be treated again with the same or alternative immune checkpoint inhibitors. Therefore, NUC-7738 can also be used to treat patients who are or have developed resistance to immune checkpoint inhibitors.

[0382] Therefore, in a specific embodiment of any aspect of the invention, the patient or subject is a patient or subject who has developed resistance to an immune checkpoint inhibitor. Suitably, such a person does not provide a recognized clinical signal of efficacy to a determined dose of an immune checkpoint inhibitor.

[0383] Suitably, the subject / patient will have previously shown clinical signs of efficacy, such as maintenance or reduction of cancer mass or reduction of cancer markers, progression-free survival, but the effectiveness of the immune checkpoint inhibitor subsequently decreases or ceases. This indicates that the cancer cells have developed resistance to the agent.

[0384] Suitably, a subject / patient who is resistant to (has developed resistance to) an immune checkpoint inhibitor will have markers that indicate resistance to treatment with an immune checkpoint inhibitor. Various markers that indicate resistance to immune checkpoint therapy have been identified, including: low tumor mutational burden / MSS tumors, low tumor infiltration, high exo-PD-L2 / sPD-L1, and low OX40L.

[0385] Patient selection

[0386] The inventors' discovery that NUC-7738 is able to alter the expression of mRNA encoding soluble / exosomal PD-L1, and to reduce the amount of soluble PD-L1 and exosomal PD-L1, and / or to increase the amount of OX40L, enables a number of methods by which it can be determined whether a particular patient may benefit from receiving NUC-7738 alone or in combination with the immune checkpoint inhibitors described herein.

[0387] Therefore, according to another aspect, the present invention provides a method for determining whether a patient will benefit from treatment with NUC-7738, the method comprising: determining the level of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in a biological sample from the patient; wherein if the level of soluble PD-L1 and / or exosomal PD-L1 produced by cancer cells in the biological sample is increased and / or the level of OX40-L L1 produced by cancer cells in the biological sample is decreased compared to a wild-type or reference value, the patient will benefit from treatment with NUC-7738.

[0388] Suitably, the biological sample is a blood sample or a fraction thereof (eg plasma or serum).

[0389] In specific embodiments of this aspect, the amount or level of soluble PD-L1 or exosomal PD-L1 is determined, and if such amount or level is increased compared to a wild-type or reference value, the patient will benefit from treatment with NUC-7738.

[0390] In a specific embodiment, the level of soluble PD-L1 is determined.

[0391] In a specific embodiment, the level of exosomal PD-L1 is determined.

[0392] In another specific embodiment, the levels of soluble PD-L1 and exosomal PD-L1 are determined.

[0393] In specific embodiments of this aspect, the amount or level of OX40-L is determined, and if such amount or level is reduced compared to a wild-type or reference value, the patient will benefit from treatment with NUC-7738.

[0394] In a specific embodiment, the levels of soluble PD-L1 and OX-40-L are determined.

[0395] In another specific embodiment, the levels of exosomal PD-L1 and OX-40-L are determined.

[0396] In another specific embodiment, the levels of soluble PD-L1 and exosomal PD-L1 and OX-40-L are determined.

[0397] Methods for determining appropriate treatment options

[0398] According to another aspect, the present invention provides a method for determining an appropriate treatment regimen for a patient suffering from a proliferative disease such as cancer, the method comprising: analyzing the levels of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in a biological sample from the patient; wherein the presence of an increased level of soluble PD-L1 determined and / or an increased level of exosomal PD-L1 determined and / or a decreased level of OX40-1 determined in the biological sample relative to a wild-type or reference value indicates that an appropriate treatment regimen will include treating the patient with NUC-7738.

[0399] Those skilled in the art will appreciate that methods of determining an appropriate treatment regimen for a patient include methods of selecting patients for treatment.

[0400] According to another aspect, the present invention provides a method of determining whether a patient with cancer will benefit from treatment with NUC-7738, the method comprising: determining the level of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in a biological sample from the patient; wherein if the level of soluble PD-L1 and / or exosomal PD-L1 in the biological sample is increased and / or the level of OX40-L is decreased compared to wild-type or reference values, the patient will benefit from treatment with NUC-7738. Suitably, the soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in the biological sample has been produced by cancer cells.

[0401] According to another aspect, the present invention provides a method of treating a patient suffering from a proliferative disease such as cancer, the method comprising: analyzing the level of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in a biological sample from the patient; wherein if the determined level of soluble PD-L1 and / or exosomal PD-L1 is higher than a wild-type or reference value, and / or if the determined level of OX40-L is lower than a wild-type or reference value, administering an effective amount of NUC-7738 to the patient.

[0402] Suitably, the levels of PD-L1 (soluble and / or exosomal) and / or OX40-L may be quantitatively determined using a suitable immunoassay.

[0403] Suitably, the biological sample is a blood sample or a fraction thereof (eg plasma or serum).

[0404] Those skilled in the art will appreciate that there are many examples of suitable biological samples that can be used in embodiments of the present invention, such as those listed above. Suitably, such samples may include cancer cells or liquid samples (such as blood or fractions thereof) from diseased tissues or organs (e.g., skin, kidney, brain, liver). Suitable biological samples may be tissue samples, such as samples for histology. The levels of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L as described above in cells in these samples may be directly assessed. Particularly suitable samples are blood samples or blood fractions thereof, such as plasma or serum.

[0405] Techniques for studying biomarker levels are frequently used in clinical assessments (e.g., for diagnostic or prognostic purposes), and their use will be familiar to the skilled artisan required to implement them in the context of the present invention. By way of example only, in a sample containing protein, the presence of soluble PD-L1 and / or exosomal PD-L1 and / or OX-40-L can be assessed by suitable techniques using antibodies reactive with the markers (e.g., PD-L1 or OX40-L, as the case may be). Examples of suitable samples include histological samples (where the presence of the markers can be observed by suitable immunocytochemical techniques).

[0406] Specific implementation plan:

[0407] 1. NUC-7738, for treating a proliferative disease by reducing the amount of extracellular PD-L1 protein produced by proliferative disease cells and / or increasing the amount of OX40-L protein produced by proliferative disease cells.

[0408] 2. NUC-7738 according to embodiment 1, wherein the extracellular PD-L1 protein is soluble PD-L1 and / or exosomal PD-L1.

[0409] 3. NUC-7738 according to embodiment 2, wherein treatment with NUC-7738 results in decreased levels of soluble PD-L1 and / or exosomal PD-L1 and / or increased levels of OX40-L protein produced by the proliferative disease cells.

[0410] 4. NUC-7738 according to any one of embodiments 1 to 3, wherein administration of NUC-7738 to a patient enhances the patient's immune response to a proliferative disease.

[0411] 5. NUC-7738 according to any one of embodiments 1 to 4, wherein

[0412] The treatment of the proliferative disease is effected through adaptive immunity (eg, cellular immunity and / or humoral immunity).

[0413] 6. NUC-7738 according to any of the preceding embodiments, wherein the proliferative disease is cancer.

[0414] 7. NUC-7738, used as an immune sensitizer in cancer treatment.

[0415] 8. NUC-7738 according to embodiment 6 or 7, wherein the cancer is selected from: melanoma (including cutaneous melanoma), lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, lymphoma, ovarian cancer, cervical cancer and endometrial cancer.

[0416] 9. NUC-7738 according to any of the preceding embodiments, wherein the treatment comprises administering NUC-7738 in combination with an immuno-oncology agent, such as an immune checkpoint inhibitor, an antibody therapy, an adoptive cell therapy such as CAR-T therapy, or a cancer vaccine.

[0417] 10. NUC-7738 according to embodiment 6, wherein the treatment comprises administering NUC-7738 in combination with an immune checkpoint inhibitor.

[0418] 11. NUC-7738 according to embodiment 6, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a TIGIT inhibitor, an OX40 inhibitor, an OX40-L inhibitor or a BTLA inhibitor.

[0419] 12. NUC-7738 according to any of the preceding embodiments, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-OX40-L antibody, or an anti-BTLA antibody.

[0420] 13. NUC-7738 according to embodiment 12, wherein the anti-PD-L1 antibody is selected from the group consisting of: atezolizumab, avelumab, and durvalumab.

[0421] 14. NUC-7738 according to embodiment 12, wherein the anti-PD-1 antibody is selected from the group consisting of: nivolumab, pembrolizumab, dotarizumab, and cemiplizumab.

[0422] 15. NUC-7738 according to embodiment 12, wherein the anti-PD-1 antibody is pembrolizumab.

[0423] 16. NUC-7738 according to embodiment 12, wherein the anti-CTLA-4 antibody is ipilimumab or tesimumab.

[0424] 17. NUC-7738 according to embodiment 12, wherein the anti-LAG-3 antibody is relalizumab.

[0425] 18. NUC-7738 according to embodiment 12, wherein the anti-TIGIT antibody is tiragolumab.

[0426] 19. NUC-7738 according to embodiment 12, wherein the anti-BTLA antibody is cudarolimab.

[0427] 20. NUC-7738 according to any one of the preceding embodiments, wherein the proliferative disease is cancer, and the cancer cells express high levels of soluble PD-L1 protein and / or high levels of exosomal PD-L1 protein and / or low levels of OX40L protein.

[0428] 21. NUC-7738 according to embodiment 20, wherein treatment with NUC-7738 reduces the amount of extracellular PD-L1 protein produced by the cancer cells.

[0429] 22. NUC-7738 according to embodiment 21, wherein the extracellular PD-L1 protein is selected from soluble PD-L1 protein or exosomal PD-L1 protein.

[0430] 23. NUC-7738 according to embodiment 20, wherein treatment with NUC-7738 increases the amount of OX40-L protein produced by the cancer cells.

[0431] 24. NUC-7738 according to any of the preceding embodiments, wherein the patient has previously received treatment with an immune checkpoint inhibitor, optionally wherein the treatment has been stopped.

[0432] 25. NUC-7738 according to embodiment 24, wherein previous treatment with an immune checkpoint inhibitor was stopped due to toxicity, relapse, or resistance of the cancer to the previous treatment.

[0433] 26. NUC-7738 according to any one of the embodiments, wherein treatment with NUC-7738 reduces soluble PD- or exosomal PD-L1 levels by at least about 25% compared to levels achieved without NUC-7738 treatment.

[0434] 27. NUC-7738 according to any of the preceding embodiments, wherein the NUC-7738 is administered at about 300 mg / m 2 Up to 1600 mg / m 2 Weekly doses of 500 mg / m 2 Up to 1150 mg / m 2 Weekly dose, or 900 mg / m 2 Up to 1350 mg / m 2 Weekly dose administration.

[0435] 28. NUC-7738 according to any one of embodiments 10 to 27, wherein the immune checkpoint inhibitor used in combination with the NUC-7738 is administered at a dose of 50% or less of the standard monotherapy dose of the agent.

[0436] 298. A method for treating a proliferative disease by reducing the amount of extracellular PD-L1 protein produced by diseased cells in a patient and / or increasing the amount of OX40-L protein produced by diseased cells in the patient, the method comprising administering a therapeutically effective amount of NUC-7738 to a patient in need thereof.

[0437] 30. The method of embodiment 29, wherein the proliferative disease is cancer.

[0438] 31. A method of enhancing an immune response to cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of NUC-7738, alone or in combination with an immuno-oncology agent.

[0439] 33. A method according to embodiment 30 or 31, wherein the cancer is selected from: melanoma (including cutaneous melanoma), lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, lymphoma, ovarian cancer, cervical cancer and endometrial cancer.

[0440] 34. The method according to any one of embodiments 29 to 32, wherein the extracellular PD-L1 is soluble PD-L1 protein and / or exosomal PD-L1 protein.

[0441] 34. The method of any one of embodiments 30 to 33, wherein administration of NUC-7738 reduces the amount of extracellular PD-L1 protein produced by cancer cells and / or increases the amount of OX40-L protein produced by cancer cells.

[0442] 35. A method according to any one of embodiments 31 to 34, wherein the immuno-oncology agent is an immune checkpoint inhibitor, an antibody therapy, an adoptive cell therapy, or a cancer vaccine.

[0443] 36. The method of embodiment 35, wherein the immuno-oncology agent is an immune checkpoint inhibitor.

[0444] 37. A method according to embodiment 36, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a TIGIT inhibitor, an OX40 inhibitor, an OX40-L inhibitor or a BTLA inhibitor.

[0445] 38. The method according to embodiment 37, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-OX40-L antibody or an anti-BTLA antibody.

[0446] 39. The method of embodiment 38, wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0447] 40. The method of embodiment 38, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, dotarizumab, and cemiplizumab.

[0448] 41. The method of embodiment 38, wherein the anti-PD-1 antibody is pembrolizumab.

[0449] 42. The method of embodiment 38, wherein the anti-CTLA-4 antibody is ipilimumab or tesimumab.

[0450] 43. The method of embodiment 38, wherein the anti-LAG-3 antibody is relalizumab.

[0451] 44. According to the method according to embodiment 38, the anti-TIGIT antibody is tiragolumab.

[0452] 45. The method of embodiment 38, wherein the anti-BTLA antibody is cudarolimab.

[0453] 46. ​​The method of any one of embodiments 29 to 45, wherein the proliferative disease is cancer, and the cancer cells express high levels of soluble PD-L1 protein and / or high levels of exosomal PD-L1 protein and / or low levels of OX40L protein.

[0454] 47. A method according to any one of embodiments 29 to 46, wherein the patient has previously received treatment with an immune checkpoint inhibitor, optionally wherein the treatment has been stopped.

[0455] 468. The method of embodiment 47, wherein prior treatment with an immune checkpoint inhibitor was discontinued due to toxicity, recurrence, or resistance of the cancer to prior treatment.

[0456] 49. The method of any one of embodiments 29 to 48, wherein treatment with NUC-7738 reduces soluble PD- or exosomal PD-L1 levels by at least about 25% compared to levels achieved without NUC-7738 treatment.

[0457] 50. The method of any one of embodiments 29 to 48, wherein the NUC-7738 is administered at about 300 mg / m 2 Up to 1600 mg / m 2 Weekly doses of 500 mg / m 2 Up to 1150 mg / m 2 Weekly dose, or 900 mg / m 2 Up to 1350 mg / m 2 Weekly dose administration.

[0458] 51. The method of any one of embodiments 29 to 48, wherein the immune checkpoint inhibitor used in combination with the NUC-7738 is administered at a dose that is 50% or less of the standard monotherapy dose of that agent.

[0459] 520. A combination comprising NUC-7738 and an immuno-oncology agent.

[0460] 53. The combination according to embodiment 52, wherein the immuno-oncology agent is an immune checkpoint inhibitor, an antibody therapy, a cancer vaccine, or an adoptive cell therapy such as CAR-T cells.

[0461] 54. The combination according to embodiment 53, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-OX40-L antibody or an anti-BTLA antibody.

[0462] 55. The combination according to embodiment 53 or 54, wherein the immune checkpoint inhibitor is selected from the group consisting of: BMS-986016 / relalizumab, TSR-033, REGN3767, MGD013 (a bispecific DART that binds PD-1 and LAG-3), GSK2831781 and LAG525, MDX-010 / ipilimumab, AGEN1884 and CP-675, 206 / tesimumab, pembrolizumab, nivolumab , atezolizumab, avelumab, durvalumab, MBG453, TSR-022, LY3321367, tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106 and cudarolimab, or a pharmaceutically acceptable salt or solvate thereof.

[0463] 56. The combination according to any one of embodiments 52 to 55, for use in the treatment of a proliferative disease, such as cancer.

[0464] 57. A kit of parts comprising:

[0465] a first container comprising NUC-7738, such as NUC-7738 in combination with a pharmaceutically acceptable adjuvant, diluent or carrier; and

[0466] a second container comprising an immuno-oncology agent, such as an immune checkpoint inhibitor, in combination with a pharmaceutically acceptable adjuvant, diluent or carrier, and

[0467] Container means for receiving said first and second containers.

[0468] 58. The kit of parts according to embodiment 57, wherein the immune checkpoint inhibitor is selected from the group consisting of: BMS-986016 / relalizumab, TSR-033, REGN3767, MGD013 (a bispecific DART that binds PD-1 and LAG-3), GSK2831781 and LAG525, MDX-010 / ipilimumab, AGEN1884 and CP-675, 206 / tesimumab, pembrolizumab, nivolumab , atezolizumab, avelumab, durvalumab, MBG453, TSR-022, LY3321367, tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106 and cudarolimab, or a pharmaceutically acceptable salt or solvate thereof.

[0469] 59. An immune checkpoint inhibitor for use in treating a proliferative disease such as cancer, wherein the immune checkpoint inhibitor is for administration in combination with NUC-7738, optionally wherein the immune checkpoint inhibitor and NUC-3378 are administered simultaneously or sequentially.

[0470] 60. An immune checkpoint inhibitor according to embodiment 59, wherein the immune checkpoint inhibitor is selected from the group consisting of: BMS-986016 / relalizumab, TSR-033, REGN3767, MGD013 (a bispecific DART that binds PD-1 and LAG-3), GSK2831781 and LAG525, MDX-010 / ipilimumab, AGEN1884 and CP-675, 206 / tesimumab, pembrolizumab, nivolumab Anti-, atezolizumab, avelumab, durvalumab, MBG453, TSR-022, LY3321367, tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106 and cudarolimab, or a pharmaceutically acceptable salt or solvate thereof.

[0471] 61. A method for determining whether a patient will benefit from treatment with NUC-7738, the method comprising: determining the level of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in a biological sample from the patient; wherein if the level of soluble PD-L1 and / or exosomal PD-L1 in the biological sample is increased and / or the level of OX40-L protein in the biological sample is decreased compared to a reference value, then the patient will benefit from treatment with NUC-7738.

[0472] 62. A method according to embodiment 61, wherein the biological sample is a blood sample or a fraction thereof (e.g., plasma or serum).

[0473] 63. The method of embodiment 61 or 62, wherein the patient has a cancer selected from the group consisting of melanoma, lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, lymphoma, ovarian cancer, cervical cancer and endometrial cancer.

[0474] 64. The method of any one of embodiments 61 to 63, wherein the patient is a patient or subject who is resistant or has developed resistance to an immune checkpoint inhibitor.

[0475] 65.NUC-7738 for the treatment of cancer in combination with an immuno-oncology agent, such as an immune checkpoint inhibitor.

[0476] 66. NUC-7738 according to embodiment 65, wherein the cancer is a solid tumor.

[0477] 67. NUC-7738 according to embodiment 65 or 65, wherein the cancer is melanoma, such as cutaneous melanoma.

[0478] 68. NUC-7738 according to any one of embodiments 65 to 67, wherein the cancer is a disease typically treated with an immune checkpoint inhibitor (such as pembrolizumab).

[0479] 69. NUC-7738 according to any one of embodiments 65 to 68, wherein the cancer comprises cancer cells expressing high levels of soluble PD-L1 protein and / or high levels of exosomal PD-L1 protein and / or low levels of OX40L protein.

[0480] 70. NUC-7738 according to any one of embodiments 65 to 69, wherein the treatment is performed on a patient having cancer and the patient has received one or more prior cancer treatments, optionally wherein the treatment has been stopped.

[0481] 71. NUC-7738 according to embodiment 70, wherein the treatment is stopped due to toxicity, relapse, or the cancer becomes resistant to the previous treatment.

[0482] 72. NUC-7738 according to any one of embodiments 65 to 71, wherein the immuno-oncology agent is pembrolizumab, wherein the NUC-7738 is administered on days 1, 8, and 15 of a 21-day cycle, optionally at 1125 mg / m 2 administration, and the pembrolizumab was administered at a dose of 200 mg on day 1 of a 21-day cycle.

[0483] 73. NUC-7738 according to any one of embodiments 65 to 72, wherein the immuno-oncology agent is selected from pembrolizumab, cemiplizumab, dotarizumab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab.

[0484] 74. NUC-7738 according to any one of embodiments 65 to 73, wherein the immuno-oncology agent is pembrolizumab.

[0485] 75. NUC-7738 according to any one of embodiments 65 to 74, wherein the NUC-7738 and the immuno-oncology agent are administered in a 21-day or 42-day treatment cycle.

[0486] 76. NUC-7738 according to any one of embodiments 65 to 75, wherein the NUC-7738 is administered at about 300 mg / m 2 Up to 1600 mg / m 2 Weekly doses of 500 mg / m 2 Up to 1150 mg / m 2 Weekly dose, or 900 mg / m 2 Up to 1350 mg / m 2 Weekly dose administration.

[0487] 77. NUC-7738 according to embodiment 76, wherein the NUC-7738 is administered at a weekly dose selected from: 900 mg / m 2 , 1125mg / m 2 and 1350 mg / m 2 .

[0488] 78. NUC-7738 according to any one of embodiments 65 to 77, wherein the pembrolizumab is administered at a dose of 200 mg once every three weeks (Q3W).

[0489] 79. NUC-7738 according to any one of embodiments 65 to 78, wherein the immuno-oncology agent is pembrolizumab, wherein the NUC-7738 is administered on days 1, 8, and 15 of a 21-day cycle, optionally at 1125 mg / m 2 administration, and the pembrolizumab was administered at a dose of 200 mg on day 1 of a 21-day cycle.

[0490] 80. NUC-7738 according to any one of embodiments 65 to 77, wherein the pembrolizumab is administered at a dose of 400 mg once every six weeks (Q6W).

[0491] 81. NUC-7738 according to embodiment 80, wherein the immuno-oncology agent is pembrolizumab, wherein the NUC-7738 is administered on days 1, 8, 15, 22, 29, and 35 of a 42-day cycle, optionally at 1125 mg / m 2 administration, and the pembrolizumab was administered at a dose of 400 mg on day 1 of a 42-day cycle.

[0492] 82. NUC-7738 according to any one of embodiments 65 to 81, wherein the treatment is performed on a patient having cancer and the patient has previously received treatment with an immune checkpoint inhibitor, optionally wherein the treatment has been stopped.

[0493] 83. NUC-7738 according to embodiment 82, wherein the prior treatment was administration of an immune checkpoint inhibitor and the treatment was stopped due to toxicity, relapse, or resistance of the cancer to the prior treatment.

[0494] The present invention will now be further described with reference to the following examples.

[0495] Example

[0496] Two sets of experiments are described in Examples 1-4. In the first case, plasma samples were collected from patients before and after treatment with NUC-7738 and then prepared for analysis as described below. In the second case, A375 melanoma or A549 lung cancer cell lines were cultured and then exposed to NUC-7738 in vitro at doses and times as described below. Thereafter, proteins and RNA were extracted and analyzed.

[0497] method:

[0498] Patients were treated with NUC-7738 on days 1 and 8 of a 14-day cycle. Plasma samples were collected from patients before dosing, 24 hours after NUC-7738 administration, and 24 hours after cycle 2 (which is when patients received treatment for the second time).

[0499] For extracellular vesicle (EV, exosome) preparation, in T175 Nunc TM EasYFlask TM Cells were cultured in cell culture flasks (ThermoFisher catalog number 159910), approximately 50 x 10 6 cells. Cells were cultured in DMEM, high glucose, GlutaMAX TM The cells were grown in 4% FBS (ThermoFisher Cat. No. 10566016) supplemented with 5% exosome-free FBS and 1% penicillin-streptomycin (ThermoFisher Cat. No. 15070063).

[0500] For sPDL1 analysis, a similar process was performed. Grow in 100 mm TC-treated dishes containing DMEM, high glucose, GlutaMAX TM Supplement (ThermoFisher Catalog No. 10566016), which is supplemented with 10% FBS and 1% penicillin-streptomycin (ThermoFisher Catalog No. 15070063), with approximately 2×10 6 cells.

[0501] A375 melanoma cell line and A549 lung cancer cell line were treated with solvent control DMSO or NUC-7738

[0502] (10 μM) for 72 h, after which culture supernatants were collected for soluble PD-L1 analysis or EV isolation. In addition, adherent cells were collected for RNA and protein isolation.

[0503] Sample preparation for PD-L1 solubility analysis

[0504] Patient plasma samples were centrifuged at 3000 × g for 15 min to remove cell debris. The supernatant was aliquoted into 250 μL of measurements and stored in a refrigerator at -80 ° C. In vitro soluble PD-L1 samples were prepared by collecting the culture supernatant, and at the end of the time point, they were centrifuged at 3000 × g for 15 min to remove cells. In addition, the culture medium was concentrated using a Vivaspin protein concentrator spin column with a 3kDa molecular weight cutoff (Cytiva catalog number 28932358). After optimization, an 8-fold concentration factor was obtained by centrifugation at 4000 x g for 60 min. The concentrated samples were aliquoted and stored at -80 ° C.

[0505] Isolation of exosomes from in vitro and plasma samples

[0506] Patient plasma samples were centrifuged at 3000 × g for 15 min to remove cell debris. Subsequently, the supernatant was aliquoted into 250 μL aliquots and stored in a −80 °C freezer. ULTRAEV Serum and Plasma Separation Kit (System Biosciences catalog number EQULTRA-20A-1). Briefly, 250 μL of plasma sample free of cell debris was used, 67 μL of ExoQuick solution was added, and then incubated at 4°C for 30 min. Subsequently, the plasma sample and ExoQuick solution mixture was centrifuged at 3000x g for 10 min. Then, carefully without disturbing the pellet, all supernatant was aspirated to ensure that no residual traces remained. Next, the pellet was resuspended in the provided buffer B and then buffer A was added. The sample was homogenized in the buffer by carefully pipetting up and down until no lumps of pellet were seen. The resin purification column was conditioned by removing the storage buffer and washing with buffer B. The sample was added to the conditioned purification column and centrifuged at 1000x g for 30 s. Using Qubit TM The protein concentration of the eluted samples was quantified using the Protein and Protein Broad Range (BR) Assay Kit (Thermofisher Cat. No. A50668). The samples were then aliquoted according to the amount required for downstream applications and stored at -80°C.

[0507] Isolation of exosomes from in vitro samples included 5 independent T175 Nunc TM EasYFlask TM Cells were cultured in cell culture flasks (ThermoFisher catalog number 159910), approximately 50 x 10 6 cells. Cells were cultured in DMEM, high glucose, GlutaMAX TM Supplement (ThermoFisher catalog number 10566016). In addition, FBS without exosomes was supplemented. Each T175 flask contained 40 mL of medium, which was combined for each treatment, a total of 200 mL, and the supernatant medium was centrifuged at 4000×g for 15 min to remove any cell debris. In addition, the medium was concentrated to 5 mL using a Centricon Plus-70 centrifugal filter with a cutoff of 100 kDa (Merck catalog number UFC710008). The culture medium was then concentrated using a Centricon Plus-70 centrifugal filter for tissue culture according to the manufacturer's protocol. Exosomes were isolated using the ULTRAEV isolation kit (System Biosciences catalog number EQULTRA-20TC-1). The ExoQuick Ultra exosome isolation protocol for tissue culture remains similar to that for plasma. 1 mL of ExoQuick solution was added to 5 mL of culture medium concentrated sample and then incubated overnight.

[0508] Quantification of soluble PD-L1 by sandwich ELISA

[0509] For the quantitative determination of soluble PD-L1 in patients and in vitro samples, use the ELISA human / cynomolgus monkey PD-L1 / B7-H1 immunoassay (R&D Systems catalog number DB7H10). Samples were prepared as described above; 100 μL of patient plasma sample and 100 μL of concentrated culture medium sample from in vitro were used for each well. Two technical replicates were performed for plasma samples, and three biological replicates and two technical replicates were performed for in vitro samples.

[0510] Briefly, the protocol involves preparing a standard curve ranging from 0-1600 pg / mL from human / cynomolgus PD-L1 / B7-H1 protein standards. Serial dilutions were performed using the provided calibrator diluent RD5-33 (1:3 dilution). 50 μL of assay diluent RD1-41 was added to each well, followed by 100 μL of standard, control, or sample. The samples were incubated for 2 h in a room temperature orbital microplate shaker set at 500 rpm. At the end of the incubation, each well was washed four times using the provided wash buffer. The samples were further incubated with 200 μL of human / cynomolgus B7-H1 conjugate in a room temperature orbital microplate shaker set at 500 rpm for 2 h. The wash step was repeated, and then 200 μL of substrate solution was added to each well. After incubation for 30 min, protected from light, 50 μL of stop solution was added to each well. Wells were read using a microplate reader set to 450 nm and 540 nm for wavelength correction to determine optical density.

[0511] Analysis of the results included subtracting the 540 nm reading from the 450 nm reading and subtracting the blank, diluent only, and sample readings. A standard curve was generated using a four-parameter logistic (4-PL) curve fit. The standard curve was then used to interpolate the PD-L1 concentrations in the samples. For in vitro samples, an 8-fold concentration factor was considered; therefore, the final concentration was divided by 8.

[0512] The expression of PD-L1 protein in isolated exosomes was measured using capillary-based immunoprobing.

[0513] The expression of PD-L1 in exosomes isolated from plasma samples was analyzed using the automated Western blot JESS system (BioTechne, ProteinSimples). The 12-230 kDa separation module of the 25 capillary kit (ProteinSimple, catalog number SM-W004) was used according to the production protocol. In brief, the samples were prepared at appropriate concentrations using the kit master mix and sample dilution buffer containing DT, and then the samples were denatured at 95 ° C for 5 minutes. All samples, primary antibodies, and secondary antibodies were loaded into the provided plates. Anti-CD81 antibody [M38] (Abcam, catalog number ab79559) diluted 1:100 and PD-L1 diluted 1:50 were used. Rabbit mAb (Cell Signaling, catalog number 13684). The secondary antibodies used were from anti-mouse chemiluminescent (ProteinSimple, catalog number DM002) and anti-rabbit near infrared (NIR) (ProteinSimple, catalog number DM007) detection modules, which were used at recommended dilutions. The prepared plates and capillaries were then loaded onto JESS and the samples were run under default settings. Expression was determined by area under the curve using Compass software version 6.1 (ProteinSimple).

[0514] In vitro analysis of soluble PD-L1 transcripts using RT-qPCR

[0515] To determine RNA expression of soluble transcripts, cells were lysed and RNA was isolated using the RNAeasy kit (Qiagen, catalog number 74136). RNA was then reverse transcribed into cDNA using the QuantiNova reverse transcription kit (Qiagen, catalog number 205413). TM Primers for each gene were designed by IDT (Integrated DNA Technologies) and ordered at 100 μM concentration from IDT. Soluble PD-L1 transcript primers were designed to produce an amplicon of approximately 100 bp in size spanning the junction of exon 4 and intron 4. The primer sequences are shown in the following table (Table 3):

[0516]

[0517] RT-qPCR was performed using the qPCRBIO SyGreen MasterMix kit (PCR biosystems catalog number PB20.14-50) and the Rotor qene Q qPCR machine (Qiagen). Ct values ​​were analyzed using the Rotor qene Q software. Fold changes were calculated using the delta delta Ct values.

[0518] result

[0519] In cell line studies, NUC-7738 was converted to the active anticancer metabolite 3'-dATP within 6 hours of treatment, with an average concentration of 80 pmol / 10 6 NUC-7738 reduced sPD-L1 expression in the culture supernatant by 40%. NUC-7738 also reduced ExoPD-L1 protein by 50%. NUC-7738 did not alter cell surface PD-L1 expression.

[0520] A preliminary study of sera from four patients treated with NUC-7738 showed a ≤50% reduction in Exo-PD-L1 compared to pre-treatment levels.

[0521] Example 1

[0522] Figure 1 shows that NUC-7738 reduces the levels of soluble PD-L1 in lung and melanoma cell lines, supporting the notion that NUC-7738 reduces the amount of soluble PD-L1 present in the tumor microenvironment: reduction in sPDL1 levels is known to enhance anti-PD1 and other immune checkpoint therapies.

[0523] Example 2

[0524] Figure 2 shows that NUC-7738 reduces the transcript amounts of soluble PD-L1 transcript isoforms in lung and melanoma cell lines, supporting the hypothesis that this agent acts on tumor cells that produce soluble PD-L1 protein.

[0525] Example 3

[0526] Table 4 (below) and Figure 3 NUC-7738 was shown to reduce exosomal PD-L1 protein levels in plasma.

[0527]

[0528] In three of the four patients, the ratio of PD-L1 and the exosomal marker CD81 decreased 15 days after the initial administration of NUC-7738. In patient 001-019, this reduction was seen within 24 hours. This suggests that NUC-7738 is able to reduce the levels of exosomal PD-L1 in plasma.

[0529] Example 4

[0530] Figure 4showed that NUC-7738 increased the levels of mRNA encoding OX40L in melanoma and lung cell lines.

[0531] in conclusion :

[0532] NUC-7738 reduced the secreted form of PD-L1 while having no effect on cell surface protein levels. The in vitro data were validated in a clinical setting, with a reduction in ExoPD-L1 in plasma samples from patients treated with NUC-7738. These findings suggest that NUC-7738 has the potential to act as an immune sensitizer and restore T-cell function to promote anti-cancer immune responses. The combination of NUC-7738 with PD-(L)1 pathway inhibitors may provide a promising treatment option even in patients who have developed therapeutic resistance to immune checkpoint inhibitor therapy or for other reasons that led to failure of prior immune checkpoint inhibitor therapy (e.g., intolerance or toxicity).

[0533] Example 5- Clinical study of NUC-7738 in combination with pembrolizumab in patients with cutaneous melanoma Research

[0534] A Phase II study (NuTide:701 Extension) will be conducted to evaluate the safety, pharmacokinetics, and clinical activity of NUC-7738 administered on Days 1, 8, and 15 of a 21-day cycle and pembrolizumab administered on Day 1 of a 21-day cycle to subjects with cutaneous melanoma. Patients will be eligible to receive multiple cycles until disease progression or unacceptable toxicity. Safety, tolerability, and efficacy will be evaluated. Extracellular PD-L1 levels will be assessed before and after treatment. 6-12 patients with cutaneous melanoma will be treated initially. NUC-7738 and pembrolizumab will be administered by intravenous infusion.

[0535] NUC-7738 Infusion

[0536] From the NUC-7738 reservoir vial, add the required volume of NUC-7738 to a 500 ml standard saline infusion bag; slowly invert the bag or use a similar method to produce a homogenous, transparent solution. Continue to mix until the solution is thoroughly mixed. Slowly infuse the contents of the infusion bag containing the saline-based NUC-7738 formulation into the patient over a period of approximately 120 minutes. The infusion can be administered by standard means, such as using a central venous administration device or through a cannula.

[0537] Pembrolizumab infusion

[0538] Withdraw the required volume from the KEYTRUDA vial and transfer to an intravenous (IV) bag containing 0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, USP. Mix the diluted solution by gently inverting. Do not vortex. The final concentration of the diluted solution should be between 1 mg / mL and 10 mg / mL.

[0539] The diluted solution is administered intravenously over approximately 30 minutes through an intravenous line containing a sterile, nonpyrogenic, low protein binding 0.2 micron to 5 micron filter in one row or attached (usually using an infusion pump).

[0540] On day 1, pembrolizumab was administered directly prior to NUC-7738 (although the order was not important).

[0541] Subjects eligible for treatment will have histologically confirmed cutaneous melanoma per RECIST v1.1 criteria and measurable disease. Most subjects have progressed on ≤2 prior lines of therapy for advanced / metastatic cutaneous melanoma, which may include 1 prior line of immunotherapy-containing therapy (monotherapy or in combination with chemotherapy). Patients who have not progressed but for whom the addition of NUC-7738 to standard pembrolizumab monotherapy may be appropriate are also eligible.

[0542] Given the ability of NUC-7738 to reduce the amount of soluble and exosomal PD-L1, the inventors anticipate that the combination of NUC-7738 and pembrolizumab will be more effective in treating cutaneous melanoma than pembrolizumab administered as a monotherapy.

Claims

1. NUC-7738 for use in treating a patient suffering from a proliferative disease by enhancing the patient's immune response against the proliferative disease.

2. NUC-7738 according to claim 1, wherein NUC-7738 reduces the amount of extracellular PD-L1 protein produced by the proliferative disease cells and / or increases the amount of OX40-L protein produced by the proliferative disease cells.

3. NUC-7738 according to claim 2, wherein the extracellular PD-L1 protein is soluble PD-L1 and / or exosomal PD-L1.

4. NUC-7738 according to any one of claims 1 to 3, wherein the treatment of the proliferative disease is produced by adaptive immunity (e.g., cellular immunity and / or humoral immunity).

5. NUC-7738 according to any of the preceding claims, wherein the proliferative disease is cancer.

6. NUC-7738, which is used as an immunosensitizer in cancer treatment.

7. NUC-7738 according to claim 5 or 6, wherein the cancer is selected from: melanoma (including cutaneous melanoma), lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, lymphoma, ovarian cancer, cervical cancer and endometrial cancer.

8. NUC-7738 according to any of the preceding claims, wherein the treatment comprises administering NUC-7738 in combination with an immuno-oncology agent, such as an immune checkpoint inhibitor, an antibody therapy, an adoptive cell therapy such as CAR-T therapy, or a cancer vaccine.

9. NUC-7738 according to claim 8, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a TIGIT inhibitor, an OX40 inhibitor, an OX40-L inhibitor or a BTLA inhibitor.

10. NUC-7738 according to claim 8, wherein the immune checkpoint inhibitor is selected from the group consisting of: BMS-986016 / relalizumab, TSR-033, REGN3767, MGD013 (a bispecific DART that binds PD-1 and LAG-3), GSK2831781, LAG525, MDX-010 / ipilimumab, AGEN1884 and CP-675,206 / tesimumab, pembrolizumab, nivolumab, atezolizumab Anti-, avelumab, durvalumab, cimiprilimab, dotalimab, MBG453, TSR-022, LY3321367, tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106 and cudarolimab, or a pharmaceutically acceptable salt or solvate thereof.

11. NUC-7738 according to any of the preceding claims, wherein the patient has previously received treatment with an immune checkpoint inhibitor, optionally wherein the treatment has been stopped due to toxicity, relapse, or the cancer developing resistance to the previous treatment.

12. NUC-7738 according to any one of claims 1 to 3, wherein treatment with NUC-7738 reduces soluble or exosomal PD-L1 levels by at least about 25% compared to levels achieved in the absence of NUC-7738 treatment.

13. A method for treating a proliferative disease such as cancer by reducing the amount of extracellular PD-L1 protein produced by diseased cells and / or increasing the amount of OX40-L protein produced by diseased cells in a patient, the method comprising administering a therapeutically effective amount of NUC-7738 to a patient in need thereof.

14. A method of enhancing an immune response to cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of NUC-7738, alone or in combination with an immuno-oncology agent.

15. The method of claim 13 or 14, wherein the cancer is selected from the group consisting of melanoma (including cutaneous melanoma), lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, lymphoma, ovarian cancer, cervical cancer and endometrial cancer.

16. The method of any one of claims 13 to 15, wherein administration of NUC-7738 reduces the amount of extracellular PD-L1 protein produced by cancer cells and / or increases the amount of OX40-L protein produced by cancer cells.

17. The method of claim 16, wherein the extracellular PD-L1 is soluble PD-L1 protein and / or exosomal PD-L1 protein.

18. The method of any one of claims 14 to 17, wherein the immuno-oncology agent is an immune checkpoint inhibitor, an antibody therapy, an adoptive cell therapy, or a cancer vaccine.

19. The method of claim 18, wherein the immuno-oncology agent is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a TIGIT inhibitor, an OX40 inhibitor, an OX40-L inhibitor or a BTLA inhibitor.

20. The method of claim 18 or 19, wherein the immune checkpoint inhibitor is selected from the group consisting of: BMS-986016 / relalizumab, TSR-033, REGN3767, MGD013 (a bispecific DART that binds PD-1 and LAG-3), GSK2831781 and LAG525, MDX-010 / ipilimumab, AGEN1884 and CP-675, 206 / tesimumab, pembrolizumab, nivolumab, atezolizumab , avelumab, durvalumab, cemiplizumab, dotalimab, MBG453, TSR-022, LY3321367, tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106 and cudarolimab, or a pharmaceutically acceptable salt or solvate thereof.

21. The method of any one of claims 13 to 20, wherein the patient has previously received treatment with an immune checkpoint inhibitor, optionally wherein the treatment has been stopped due to toxicity, relapse, or the cancer developing resistance to the previous treatment.

22. A kit of parts comprising: a first container comprising NUC-7738, such as NUC-7738 in combination with a pharmaceutically acceptable adjuvant, diluent, or carrier; and a second container comprising an immuno-oncology agent, such as an immune checkpoint inhibitor, in combination with a pharmaceutically acceptable adjuvant, diluent or carrier, and Container means for receiving said first and second containers.

23. The kit of parts according to claim 22, wherein the immune checkpoint inhibitor is selected from the group consisting of: BMS-986016 / relalizumab, TSR-033, REGN3767, MGD013 (a bispecific DART that binds PD-1 and LAG-3), GSK2831781 and LAG525, MDX-010 / ipilimumab, AGEN1884 and CP-675, 206 / tesimumab, pembrolizumab, nivolumab, atezolizumab , avelumab, durvalumab, cemiplizumab, dotalimab, MBG453, TSR-022, LY3321367, tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106 and cudarolimab, or a pharmaceutically acceptable salt or solvate thereof.

24. A method for determining whether a patient will benefit from treatment with NUC-7738, the method comprising: determining the level of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in a biological sample from the patient; wherein if the level of soluble PD-L1 and / or exosomal PD-L1 in the biological sample is increased and / or the level of OX40-L protein in the biological sample is decreased compared to a reference value, then the patient will benefit from treatment with NUC-7738.

25. The method of claim 24, wherein the biological sample is a blood sample or a fraction thereof (eg, plasma or serum).

26. The method of claim 24 or 25, wherein the patient has a cancer selected from the group consisting of melanoma (including cutaneous melanoma), lung cancer (including NSCLC), breast cancer, colorectal cancer, kidney cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, lymphoma, ovarian cancer, cervical cancer, and endometrial cancer.

27. An immune checkpoint inhibitor for use in treating a proliferative disease such as cancer, wherein the immune checkpoint inhibitor is for administration in combination with NUC-7738, optionally, wherein the immune checkpoint inhibitor and NUC-3378 are administered simultaneously or sequentially.

28. The immune checkpoint inhibitor of claim 27, wherein the immune checkpoint inhibitor is selected from the group consisting of: BMS-986016 / relalizumab, TSR-033, REGN3767, MGD013 (a bispecific DART that binds PD-1 and LAG-3), GSK2831781 and LAG525, MDX-010 / ipilimumab, AGEN1884 and CP-675, 206 / tesimumab, pembrolizumab, nivolumab Anti-, atezolizumab, avelumab, durvalumab, MBG453, TSR-022, LY3321367, tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106 and cudarolimab, or a pharmaceutically acceptable salt or solvate thereof.

29. NUC-7738 for use in treating cancer in combination with an immuno-oncology agent such as an immune checkpoint inhibitor.

30. NUC-7738 according to claim 29, wherein the immuno-oncology agent is selected from pembrolizumab, cemiplizumab, dotarizumab, nivolumab, durvalumab, ipilimumab, atezolizumab and avelumab.

31. NUC-7738 according to claim 29 or 30, wherein the treatment is performed on a patient having cancer and the patient has previously received treatment with an immune checkpoint inhibitor, optionally wherein the treatment has been stopped.

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