Cancer treatment

Abstract

The present invention provides NUC-7738 (3'-deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alaninyl)] 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 exosomal PD-L1) and / or increasing the amount of OX40-L. The present invention also relates to the use of NUC-7738 in combination with cancer immunotherapeutic agents, such as immune checkpoint inhibitors.

Description

Technical Field

[0001] The present invention relates to a medical use and method for treating a proliferative disease (e.g., cancer) by reducing the amount of extracellular PD-L1 protein (e.g., soluble PD-L1 or exosomal PD-L1) produced by cells of the proliferative disease (e.g., cancer) and / or increasing the amount of OX40L produced by said cells. The inventors have discovered that the protide molecule NUC-7738 reduces the amount of soluble PD-L1 or exosomal PD-L1 produced by cancer cells and increases the amount of OX40L produced by cancer cells. This observation provides a new clinical opportunity for NUC-7738 in cancer treatment. The present invention also relates to the use of NUC-7738 in combination with an immune checkpoint inhibitor (ICI), e.g., a PD-1 or PD-L1 inhibitor, for treating a proliferative disease such as cancer (e.g., lung cancer). Due to the ability to reduce the amount of PD-L1 protein produced by cancer cells, the possibility of combining NUC-7738 / immune checkpoint inhibitor has been developed in patients who are resistant to immune checkpoint inhibitors, patients who are refractory to immune checkpoint inhibitors, patients who relapse after immune checkpoint inhibitor therapy, and patients who are considered unsuitable for immune checkpoint inhibitor therapy due to concerns that they will not respond to the therapy. Preferably, this is acceptable because it is expected that the combination with NUC-7738 will enable the use of immune checkpoint inhibitors at a lower dose.

Background Art

[0002] 3'-Deoxyadenosine (3'-dA) is a nucleoside analog of adenosine lacking the 3'-hydroxyl group at the 3'-position of the ribose moiety and is synthetically generated from adenosine. For such synthetic methods, reference can be made to Robins, J. R. et al, J. Org. Chem. 1995, 60, 7902-7908 and Aman, S. et al, Organic Process Research & Development 2000, 4, 601-605.

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

[0004] Due to its structure, 3'-dA and its phosphorylated forms (3'-dA-mono, di, and tri-phosphate) potentially interfere with all processes that require adenosine or adenosine mono, di, or tri-phosphate (AMP, ADP, or ATP), respectively. However, after administration, 3'-dA is rapidly deaminated by adenosine deaminase (ADA) in vivo and is quickly metabolized to the inactive metabolite, 3'-deoxyinosine. See Tsai, Y-J et al, J. Agri. Food Chem. 58 4638-43 (2010).

[0005] NUC-7738 (3'-deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alaninyl)] phosphate) is a phosphoramidate transformation of the monophosphate of the nucleoside 3'-dA. Unlike 3'-dA, it can penetrate into cells independently of transporters, and 3'-dAMP is generated independently of the activating enzyme adenosine kinase (AK) (a rate-limiting way during the formation of the phosphorylated form of 3'-dA). Also, there is no degradation by the enzyme adenosine deaminase (ADA), and thus the intracellular level of 3'-dATP is increased. Thus, NUC-7738 can bypass the mechanisms (including transport, activation, and degradation) that limit the usefulness of 3'-dA.

[0006] Checkpoint inhibitors are one form of cancer immunotherapy that targets immune checkpoints, which are key regulatory factors of the immune system that weaken the immune response to cancer when stimulated. These immune checkpoints have co-evolved with stimulatory immune receptors as one way to control the immune response and prevent dangerous overactive immune reactions. Cytotoxic T cells express the immune checkpoint protein PD-1 on their cell surface. When this binds to its ligand PD-L1, it signals to the T cell that it should not be activated, even if it recognizes something that 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) are part of immune checkpoint signaling pathways including CTLA-4, LAG-3, TIGIT, BTLA, OX40 / OX40L, and TIM-3. Cancers can manipulate this system to protect themselves from immune attack via the expression of proteins that are part of immune checkpoint signaling pathways.

[0008] Checkpoint inhibitor therapy can block inhibitory checkpoints, restore the function of the immune system, and result in immune-mediated cancer cell death. Current approaches focus on the use of PD-1 / PD-L1 inhibitors as monotherapy or in combination with other checkpoint inhibitors targeting other parts of the checkpoint signaling pathway (e.g., CTLA-4 inhibitors). Checkpoint inhibitor therapy may also be combined with chemotherapy or targeted therapy.

[0009] Resistance to checkpoint inhibitors exists prior to treatment with checkpoint inhibitor therapy (primary resistance) or develops after treatment (acquired resistance). Patients with transient resistance do not respond to initial therapy and their cancer continues to grow, while patients with acquired resistance initially respond to therapy but their disease then 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 is also released from tumor cells and exists as several extracellular forms (see Daassi et al., "The importance of exosomal PDL1 in tumor immune evasion," Nature Reviews Immunology 20(4): 209-215, 2020). This includes those found as soluble proteins (soluble PD-L1 or sPD-L1) or extracellular vesicle types associated with exosomes (exosomal PD-L1 or xPD-L1). Exosomes are a type of extracellular vesicle produced by many cell types, particularly at sites of inflammatory cancer. They are characterized by their size and shape but also by the expression of CD81 on their surface. They are important in the transmission of messages between cells. Several studies have shown that soluble and exosomal PD-L1 can act as decoys and effectively nullify the benefits of anti-PD-L1 immune checkpoint therapy (see Zhang et al. Cell Biosci 9: 19, 2019).Soluble PD-L1 is a measurable circulating programmed cell death ligand 1 in the sera of various types of cancer patients (Finkelmeier et al., "High levels of soluble programmed death ligand 1 (sPD-L1) identify hepatocellular carcinoma patients with poor prognosis", Eur J Cancer. 59: 152-9, 2016; Okuma et al., "High blood concentrations of soluble programmed cell death ligand 1 represent a poor survival prognosis in advanced lung cancer", Lung Cancer. 104: 1-6, 2017; Chang et al., "Correlation and prognostic value of blood levels of soluble programmed death protein 1 (sPD-1) and soluble programmed death-ligand 1 (sPD-L1) in hepatocellular carcinoma patients", Cancer Immunol Immunother. 68: 353-63, 2019; Shigemori et al., "Expression of soluble PD-L1 in the bloodstream as a predictive marker for recurrence and prognosis in gastric cancer: a direct comparison of clinical burden between tissue PD-L1 expression and serum PD-L1 expression", Ann Surg Oncol. 26: 876-83, 2019). Previous studies have shown that sPD-L1 impairs host immunity and contributes to systemic immunosuppression, resulting in cancer progression and poor clinical outcomes (Frigola et al., Clin Cancer Res. (2011) 17: 1915-23, 2011). APD-L1 expressed on or released from cancer cells signals to cytotoxic T cells in close proximity to them to inactivate them. Therefore, reducing exosomal PD-L1 released from tumors or reducing the amount of soluble PD-L1 is a novel potential target for therapies involving immune checkpoint inhibitors.

[0011] PD-1 (also known as programmed death protein and CD279) is a checkpoint protein in T cells and acts as a type of "off switch" that assists in preventing T cells from attacking normal healthy cells in the body. PD-1 exerts the said effect when binding to PD-L1 (also known as programmed death ligand 1 and CD274), a protein present in some normal cells and cancer cells. When PD-1 binds to PD-L1 on the cell surface, PD-1 induces a signal that instructs T cells to release only other cells. Some cancer cells produce large amounts of PD-L1, and PD-L1 helps cells avoid identification by the proper immune system, thus avoiding attacks from the host immune system.

[0012] Immune checkpoint proteins (such as CTLA4 (also known as cytotoxic T lymphocyte antigen 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 known to play important roles in the regulation of 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 used to effectively treat certain types of cancer.

[0013] OX40L (also known as TNFSF4 and CD252) is expressed in antigen-presenting cells but not described in many other cells including melanoma cells. OX40L binds to OX40 (a member of the TNF receptor superfamily) on T lymphocytes and promotes the activation of effector T cells. There are several ongoing studies investigating the possible role of OX40L as one means of promoting anti-malignant tumor T lymphocyte responses (including in combination with agents that block the PD-L1 / PD-1 axis).

[0014] Ligation of OX40L with OX40 in T lymphocytes promotes the maintenance and generation of memory CD8+ T cells. Furthermore, induced expression of OX40L in tumors has been shown to lead to the induction of anti-tumor immunity (Andarini, Sita, et al., "Adenovirus vector-mediated in vivo gene transfer of OX40 ligand into tumor cells enhances anti-tumor immunity in tumor-bearing hosts", Cancer Research, 64(9): 3281-3287, 2004). Summary of the Invention Problems to be Solved by the Invention

[0015] In the Examples section of this specification, there is data showing that NUC-7738 results in a decrease in the proteins of mRNA and extracellular PD-L1, particularly soluble and exosomal PD-L1. PD-L1 produced by cancer cells is known to be important in that cancer cells assist in avoiding host immune attacks. Soluble PD-L1 and exosomal PD-L1 (types of extracellular PD-L1) can act as decoy proteins that block T cell activation and reduce the ability of T cells to kill tumor cells. Thus, NUC-7738 is used to reduce the production of this decoy protein and make cancer cells more sensitive to attacks via the host immune system. In the Examples section, there is also data showing that NUC-7738 also results in an increase in the amount of OX40L produced by cancer cells. By decreasing 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 enhancer and, alone or in combination with cancer vaccines and adoptive cell therapies such as chimeric antigen receptor T cell (CAR-T), tumor infiltrating lymphocyte (TIL), and natural killer (NK) cell therapies, is used to enhance the therapeutic effect of anti-cancer agents or therapies (including immune checkpoint inhibitors such as CTLA4, LAG3, PD-1, or PD-L1 inhibitors). NUC-7738 is also suitable for use in the treatment of cancer patients with high levels of soluble PD-L1 and / or exosomal PD-L1 in the serum or patients with tumors expressing low levels of OX40L and in the treatment of cancers that develop resistance to certain immune checkpoint inhibitors. Furthermore, by decreasing the amount of the decoy protein PD-L1, it enables the use of lower doses of immune checkpoint inhibitors, not only reducing the toxic side effects of such agents, but also enabling patients who have had to discontinue immune checkpoint inhibitor treatment due to toxicity, for example, to resume treatment with that or other immune checkpoint inhibitors (at lower, less toxic doses). Therefore, NUC-7738 is suitable for combination with immune checkpoint inhibitors in the treatment of cancer patients in whom treatment with immune checkpoint inhibitors has been discontinued (presumably due to toxicity).

Means for Solving the Problem

[0016] Thus, this specification relates in part to a method for treating a proliferative disease, such as cancer. Preferably, the method comprises administering a therapeutically effective amount of a composition to a patient in need thereof, wherein the composition comprises NUC-7738. Preferably, the therapeutically effective amount is an amount capable of reducing the production of extracellular PD-L1 (e.g., soluble PD-L1 and / or exosomal PD-L1) by cancer cells and / or increasing the production of OX40-L by cancer cells. In a particular embodiment, NUC-7738 can reduce the level of soluble PD-L1 produced by cancer cells. In a particular embodiment, NUC-7738 can reduce the level of exosomal PD-L1 produced by cancer cells. NUC-7738 can increase the production of OX40-L by cancer cells. Preferably, NUC-7738 enhances the host's adaptive immune system. In a particular embodiment, NUC-7738 acts as an immunopotentiator. In a particular embodiment, NUC-7738 is used to enhance the effect of immunotherapy. Preferably, the immunotherapy is an immune checkpoint inhibitor or a cancer vaccine or adoptive cell therapy (also known as cellular immunotherapy), such as CAR-T cell therapy. In a particular embodiment, NUC-7738 is used in combination with an immune checkpoint inhibitor. In a particular embodiment, NUC-7738 is used in combination with an immune checkpoint inhibitor selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. In a particular embodiment, NUC-7738 is used in combination with an anti-PD-1 antibody selected from pembrolizumab, cemiplimab, dostarlimab, and nivolumab. In a particular embodiment, NUC-7738 is used in combination with pembrolizumab to treat cancer such as cutaneous melanoma.

[0017] In various specific examples, a proliferative disease, such as cancer, is a disease in which high levels of extracellular PD-L1 (e.g., soluble PD-L1 and / or exosomal PD-L1) contribute to the onset. In a particular specific example, high levels of soluble PD-L1 and / or exosomal PD-L1 inhibit or block the host immune system from attacking cancer cells. In a particular specific example, 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. Accordingly, in a particular specific example, NUC-7738 is used for the treatment of cancer 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 particular specific example, NUC-7738 reduces the expression of soluble PD-L1 and / or exosomal PD-L1 protein. In a particular specific example, NUC-7738 reduces the transcription of mRNA encoding soluble PD-L1 and / or exosomal PD-L1 protein. In various specific examples, a proliferative disease, such as cancer, is a disease in which low levels of OX40-L contribute to the onset. In a particular specific example, low levels of OX40-L inhibit or block the host immune system from attacking diseased cells. In a particular specific example, low levels of OX40-L inhibit or block the effect of an immune checkpoint inhibitor administered to a patient. Accordingly, in a particular specific example, NUC-7738 is used for the treatment of cancer patients whose cancer cells express low levels of OX40-L protein.

[0019] In a particular specific example, NUC-7738 increases the expression of OX40-L protein by cancer cells. In a particular specific example, NUC-7738 increases the translation of mRNA encoding OX40-L.

[0020] According to one aspect, the present invention provides NUC-7738 for use in the treatment of proliferative diseases, 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. Preferably, the extracellular PD-L1 is a soluble PD-L1 protein and / or an exosomal PD-L1 protein. Preferably, the treatment is effected by adaptive immunity (e.g., cellular immunity and / or humoral immunity).

[0021] According to another aspect, the present invention provides a method of treating a proliferative disease, such as cancer, by reducing the amount of extracellular PD-L1 protein produced by the proliferative disease cells of a patient and / or increasing the amount of OX40-L protein, the method comprising administering to a patient in need thereof a therapeutically effective amount of NUC-7738. Preferably, the extracellular PD-L1 is a soluble PD-L1 protein and / or an exosomal PD-L1 protein.

[0022] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in a method of 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. Preferably, the extracellular PD-L1 is a soluble PD-L1 protein and / or an exosomal PD-L1 protein. Preferably, the proliferative disease is cancer. In a particular embodiment, by reducing the amount of extracellular PD-L1 protein, such as a soluble PD-L1 protein and / or an exosomal PD-L1 protein, produced by cancer cells, the cancer cells are rendered more sensitive to targeting by the host immune system, such as the patient's cellular immune system.

[0023] According to one aspect, the present invention provides NUC-7738 for use in the treatment of proliferative diseases, such as cancer, by reducing the amount of extracellular PD-L1 protein produced by diseased cells. Preferably, the extracellular PD-L1 is a soluble PD-L1 protein and / or an exosomal PD-L1 protein. Preferably, the treatment is effected by the adaptive immune system (e.g., cellular and / or humoral immunity).

[0024] According to another aspect, the present invention provides a method of treating cancer by reducing the amount of extracellular PD-L1 protein (e.g., soluble PD-L1 protein and / or exosomal PD-L1 protein) produced by cancer cells in a patient, the method comprising administering to a patient in need thereof a therapeutically effective amount of NUC-7738.

[0025] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in a method of 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 one particular example, 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, the cancer cells become more sensitive to targeting by the host immune system, e.g., the patient's cellular immune system. The ability of NUC-7738 to produce a reduction in the production and release of extracellular PD-L1 protein, such as soluble PD-L1 protein and / or exosomal PD-L1 protein, supports that NUC-7738 is an immune promoter and is used as an immune promoter. This role is further supported by the finding that NUC-7738 results in an increase in the expression and production of OX40-L (which interacts with OX40 on the surface of T-lymphocytes to enhance its anti-tumor effect) by cancer cells.

[0026] According to another aspect, the present invention provides NUC-7738 for use in the treatment of a proliferative disease by enhancing a patient's immune response to the proliferative disease. Preferably, NUC-7738 enhances the patient's adaptive immune response. Preferably, NUC-7738 inhibits one or more immune blockers, such as extracellular PD-L1 and / or OX40-L.

[0027] According to another aspect, the present invention provides NUC-7738 for use as an immunopotentiator. Preferably, the present invention provides NUC-7738 for use as an immunopotentiator in cancer treatment.

[0028] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of an immunopotentiator drug. Alternatively, the present invention provides the use of NUC-7738 in the manufacture of a medicament for cancer treatment, wherein NUC-7738 is an immunopotentiator.

[0029] According to another aspect, the present invention provides a method of enhancing an immune response against 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 immunotherapeutic agent as described herein. In a particular embodiment, a method of enhancing the effect of an immunotherapeutic agent is provided, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of the immunotherapeutic agent simultaneously with or sequentially to NUC-7738.

[0030] According to another aspect, the present invention provides NUC-7738 for use in cancer treatment by increasing the amount of OX40-L protein produced by cancer cells. Preferably, the cancer treatment is effected by adaptive immunity (e.g., cellular and / or humoral immunity).

[0031] 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 to a patient in need of a therapeutically effective amount of NUC-7738.

[0032] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in a method of treating cancer by increasing the amount of OX40-L protein produced by cancer cells.

[0033] In a particular embodiment, by increasing the amount of OX40-L protein produced by cancer cells, the cancer cells become more sensitive to targeting by the host immune system (e.g., the patient's cellular immune system) by promoting the activation of effector T cells.

[0034] According to another aspect, the present invention provides NUC-7738 for use in the treatment of cancer by reducing the interference with the targeting of cancer cells by the patient's immune system. Preferably, "reducing the interference" includes reducing the suppression or suppressors of the patient's immune system.

[0035] According to another aspect, the present invention provides the use of NUC-7738 for use in the treatment of cancer by enhancing the patient's immune response against cancer. Preferably, NUC-7738 increases the patient's immune system against cancer.

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

[0037] According to an aspect, the present invention provides a method of treating cancer by enhancing the patient's immune response against cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of NUC-7738.

[0038] According to an aspect, the present invention provides a combination comprising NUC-7738 and an immunotherapeutic agent.

[0039] In a specific example, the immunotherapeutic agent is an immune checkpoint inhibitor, a cancer vaccine, an antibody therapeutic agent, or an adoptive cell therapy agent (e.g., CAR-T cells).

[0040] In a specific example, the immunotherapeutic agent is an immune checkpoint inhibitor selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, cemiplimab, dostarlimab, and nivolumab. In a particular specific example, the immune checkpoint inhibitor is pembrolizumab.

[0041] According to another aspect, the present invention provides a pharmaceutical product comprising NUC-7738 and an immunotherapeutic agent.

[0042] In a particular specific example, the immunotherapeutic agent is an immune checkpoint inhibitor. The immune checkpoint inhibitor is an antibody against an immune checkpoint protein.

[0043] Preferably, the immune checkpoint inhibitor is an antibody selected from 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.

[0044] Preferably, the immune checkpoint inhibitor is selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. Preferably, the immune checkpoint inhibitor is an anti-PD-L1 antibody selected from pembrolizumab, cemiplimab, dostarlimab, and nivolumab. In a particular specific example, the immune checkpoint inhibitor is pembrolizumab.

[0045] In a specific example, the pharmaceutical product can consist of a kit of separate formulations of NUC-7738 and an immune checkpoint inhibitor. The separate formulations of NUC-7738 and the immune checkpoint inhibitor are administered sequentially or simultaneously.

[0046] In other specific examples, the pharmaceutical product is a first container containing NUC-7738 such as NUC-7738 with a pharmaceutically acceptable adjuvant, diluent or carrier; and a second container containing an immune checkpoint inhibitor such as an immune checkpoint inhibitor with a pharmaceutically acceptable adjuvant, diluent or carrier; and container means for containing said first and second containers which is a kit comprising.

[0047] In a specific example, the pharmaceutical product can consist of one or more unit dosage forms (e.g., vials, tablets, or capsules within blister packs). In one specific example, each unit dosage contains only one agent selected from NUC-7738 and an immune checkpoint inhibitor. In other specific examples, the unit dosage form contains both the NUC-7738 compound and the immune checkpoint inhibitor.

[0048] According to another aspect, the present invention provides a combination comprising NUC-7738 and an immune checkpoint inhibitor as defined herein for use in the treatment of a proliferative disease. Preferably, the proliferative disease is cancer. In a particular specific example, the proliferative disease is a disease that is normally treated with an immune checkpoint inhibitor (e.g., one selected from the group consisting of pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab). Table 1 provides a list of diseases for which a particular checkpoint inhibitor is used for treatment.

[0049] The following combinations and diseases are particular specific examples: Nivolumab in combination with NUC-7738 for use in the treatment of cancers selected from 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 esophagogastric junction cancer; NUC-7738 in combination with durvalumab for use in the treatment of cancers selected from urothelial carcinoma, non-small cell lung cancer (NSCLC), and advanced small cell lung cancer; NUC-7738 in combination with pembrolizumab for use in the treatment of cancers selected from 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; NUC-7738 in combination with dostarlimab for use in the treatment of cancers selected from mismatch repair proficient (dMMR) recurrent or advanced endometrial cancer; NUC-7738 in combination with ipilimumab for use in the treatment of cancers selected from melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, colorectal cancer, hepatocellular carcinoma, and esophageal cancer; NUC-7738 in combination with semiprimab for use in the treatment of cancers selected from cutaneous squamous cell carcinoma, basal cell carcinoma, and non-small cell lung cancer; NUC-7738 in combination with atezolizumab for use in the treatment of cancers selected from melanoma (including cutaneous melanoma), non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, and hepatocellular carcinoma; NUC-7738 in combination with avelumab for use in the treatment of cancers selected from Merkel cell carcinoma, urothelial carcinoma, and renal cell carcinoma.

[0050] Preferably, the proliferative disorder is cancer, and the cancer cells express high levels of soluble or exosomal PD-L1. Preferably, the proliferative disorder is cancer, and the cancer cells express low levels of OX40L.

[0051] According to another aspect, the present invention provides a method of treating a proliferative disease in a subject in need thereof, the method comprising administering to the subject a combination comprising NUC-7738 and an immune checkpoint inhibitor as defined herein. In particular embodiments, the proliferative disease is typically a disease that is treated by an immune checkpoint inhibitor (e.g., one selected from the group consisting of pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab). Table 1 provides a list of diseases for which a particular checkpoint inhibitor is used for treatment.

[0052] The following combinations and diseases are specific examples: NUC-7738 in combination with nivolumab for use in the treatment of cancers selected from 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; NUC-7738 in combination with durvalumab for use in the treatment of cancers selected from urothelial carcinoma, non-small cell lung cancer (NSCLC), and advanced small cell lung cancer; NUC-7738 in combination with pembrolizumab for use in the treatment of cancers selected from 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; NUC-7738 in combination with dostarlimab for use in the treatment of cancers selected from mismatch repair proficient (dMMR) recurrent or advanced endometrial cancer; NUC-7738 in combination with ipilimumab for use in the treatment of cancers selected from melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, colorectal cancer, hepatocellular carcinoma, and esophageal cancer; NUC-7738 in combination with cemiplimab for use in the treatment of cancers selected from cutaneous squamous cell carcinoma, basal cell carcinoma, and non-small cell lung cancer; NUC-7738 in combination with atezolizumab for use in the treatment of cancers selected from melanoma (including cutaneous melanoma), non-small cell lung cancer, small cell lung cancer, urothelial cancer, and hepatocellular carcinoma; NUC-7738 in combination with avelumab for use in the treatment of cancers selected from Merkel cell carcinoma, urothelial cancer, and renal cell carcinoma.

[0053] Preferably, the immune checkpoint inhibitor is pembrolizumab, cemiplimab, dostarlimab, or nivolumab.

[0054] Preferably, the proliferative disease is cancer, and the cancer cells express high levels of soluble or exosomal PD-L1. Preferably, the proliferative disease is cancer, and the cancer cells express low levels of OX40L.

[0055] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for treating a proliferative disease in combination with an immune checkpoint inhibitor as defined herein.

[0056] In particular specific examples, the proliferative disease is a disease that is typically treated with an immune checkpoint inhibitor (for example, one selected from the group consisting of pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab). Table 1 provides a list of diseases for which a particular checkpoint inhibitor is used for treatment.

[0057] The following combinations and diseases are specific examples: NUC-7738 in combination with nivolumab for use in the treatment of cancers selected from melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, squamous cell carcinoma, urothelial cancer, colorectal cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, and esophagogastric junction cancer; NUC-7738 in combination with durvalumab for use in the treatment of cancers selected from urothelial cancer, non-small cell lung cancer (NSCLC), and advanced small cell lung cancer; NUC-7738 in combination with pembrolizumab for use in the treatment of cancers selected from melanoma (including cutaneous melanoma), non-small cell lung cancer, urothelial cancer, classical Hodgkin lymphoma, head and neck squamous cell carcinoma, renal cell carcinoma, colorectal cancer, and esophageal cancer; NUC-7738 in combination with dostarlimab for use in the treatment of cancers selected from recurrent or advanced endometrial cancer with normal mismatch repair function (dMMR); NUC-7738 in combination with ipilimumab for use in the treatment of cancers selected from melanoma (including cutaneous melanoma), renal cell carcinoma, non-small cell lung cancer, mesothelioma, classical Hodgkin lymphoma, colorectal cancer, hepatocellular carcinoma, and esophageal cancer; NUC-7738 in combination with semiprimab for use in the treatment of cancers selected from cutaneous squamous cell carcinoma, basal cell carcinoma, and non-small cell lung cancer; NUC-7738 in combination with atezolizumab for use in the treatment of cancers selected from melanoma (including cutaneous melanoma), non-small cell lung cancer, small cell lung cancer, urothelial cancer, and hepatocellular carcinoma; NUC-7738 in combination with avelumab for use in the treatment of cancers selected from Merkel cell carcinoma, urothelial cancer, and renal cell carcinoma.

[0058] Preferably, the proliferative disorder is cancer and the cancer cells express high levels of soluble or exosomal PD-L1. Preferably, the proliferative disorder is cancer and the cancer cells express low levels of OX40L. Preferably, the immune checkpoint inhibitor is selected from pembrolizumab, semiprimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. A particularly preferred immune checkpoint inhibitor for combination with NUC-7738 for treating cancers such as cutaneous melanoma is pembrolizumab.

[0059] In a particular embodiment of this aspect of the invention, NUC-7738 and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0060] According to another aspect, the present invention provides NUC-7738 for use in the treatment of a proliferative disease, as defined herein, wherein NUC-7738 is for co-administration or sequential administration with an immune checkpoint inhibitor as defined herein. Preferably, the immune checkpoint inhibitor is selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. Preferably, the proliferative disease is a disease that is typically treated with an immune checkpoint inhibitor, such as pembrolizumab, cemiplimab, dostarlimab, or nivolumab.

[0061] According to another aspect, the present invention provides a method of treating a proliferative disease in a subject, comprising administering to a subject in need of such treatment a combination comprising NUC-7738 and an immune checkpoint inhibitor as defined herein, wherein the NUC-7738 and the immune checkpoint inhibitor are administered simultaneously or sequentially. Preferably, the immune checkpoint inhibitor is selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, cemiplimab, dostarlimab, and nivolumab. Preferably, the proliferative disease is a disease that is typically treated with an immune checkpoint inhibitor, such as pembrolizumab, cemiplimab, dostarlimab, or nivolumab.

[0062] According to another aspect, the present invention provides an immune checkpoint inhibitor for use in the treatment of a proliferative disease as defined herein, wherein the immune checkpoint inhibitor is for co - administration or sequential administration with NUC - 7738 as defined herein. Preferably, the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. Preferably, the immune checkpoint inhibitor is pembrolizumab, cemiplimab, dostarlimab, or nivolumab. Preferably, the proliferative disease is a disease that is typically treated with an immune checkpoint inhibitor, such as pembrolizumab, cemiplimab, dostarlimab, or nivolumab.

[0063] According to another aspect, the present invention provides the use of an immune checkpoint inhibitor in the manufacture of a medicament for the treatment of a proliferative disease in combination with NUC - 7738 as defined herein. Preferably, the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. Preferably, the immune checkpoint inhibitor is pembrolizumab, cemiplimab, dostarlimab, or nivolumab. Preferably, the proliferative disease is a disease that is typically treated with an immune checkpoint inhibitor, such as pembrolizumab, cemiplimab, dostarlimab, or nivolumab.

[0064] In a particular embodiment of this aspect of the invention, NUC - 7738 and the immune checkpoint inhibitor are administered to a subject simultaneously or sequentially.

[0065] Suitably, the proliferative disease is cancer. In a particular specific example, the proliferative disease is a disease that is typically treated with an immune checkpoint inhibitor, such as pembrolizumab, cemiplimab, dostarlimab, or nivolumab. According to another aspect, the present invention provides NUC-7738 for use in the treatment of cancer in a subject in which cancer cells express high levels of soluble or exosomal PD-L1.

[0066] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for the treatment of cancer (the treatment comprising administering NUC-7738 in combination with an immune checkpoint inhibitor as defined herein) in a subject in which cancer cells express high levels of extracellular PD-L1 protein, such as soluble or exosomal PD-L1 protein. Optionally, prior to treatment, a test is performed on the patient to determine whether the patient is a patient with cancer in which 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, cemiplimab, dostarlimab, or nivolumab.

[0067] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in a method of treating cancer by reducing extracellular PD-L1 protein, such as soluble protein and / or exosomal PD-L1 protein, produced by cancer cells, wherein the method comprises determining whether the patient is a patient with cancer in which cancer cells express high levels of extracellular PD-L1 protein, and administering to the patient a combination of NUC-7738 and an immune checkpoint inhibitor as defined herein when the patient is a patient with cancer in which 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, cemiplimab, dostarlimab, or nivolumab.

[0068] According to another aspect, the present invention provides a method for treating cancer in a patient with cancer in which cancer cells express a high level of extracellular PD-L1 protein, such as soluble PD-L1 protein or exosomal PD-L1 protein, comprising administering to the patient a therapeutically effective amount of NUC-7738 alone or in combination with an immune checkpoint inhibitor. Preferably, the immune checkpoint inhibitor is pembrolizumab, semiprimumab, dostarlimab, or nivolumab.

[0069] According to another aspect, the present invention provides a method for treating cancer in a patient with cancer in which cancer cells express a high level of extracellular PD-L1 protein, such as soluble PD-L1 protein or exosomal PD-L1 protein, comprising contacting the cancer cells with a therapeutically effective amount of NUC-7738. In a particular embodiment of this aspect of the invention, the cancer cells are in a subject or patient. Thus, preferably, the cancer cells are contacted with a therapeutically effective amount of NUC-7738 by administering to the patient a therapeutically effective amount of NUC-7738 alone or in combination with an immune checkpoint inhibitor. Preferably, the immune checkpoint inhibitor is pembrolizumab, semiprimumab, dostarlimab, or nivolumab.

[0070] A variety of immune checkpoint inhibitors are used in the combination therapies defined herein.

[0071] In a particular embodiment, the checkpoint inhibitor is selected from 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.

[0072] Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, semiprimumab, dostarlimab, and nivolumab.

[0073] In a particular embodiment, the proliferative disease is cancer characterized by cancer cells that express high levels of extracellular PD-L1 protein, such as soluble PD-L1 or exosomal PD-L1. In a particular embodiment, the proliferative disease is a disease that is typically treated with an immune checkpoint inhibitor, such as pembrolizumab, cemiplimab, dostarlimab, or nivolumab. Table 1 provides a list of diseases for which a particular checkpoint inhibitor is used for treatment.

[0074] 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 measuring 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 elevated compared to a reference value, the patient will benefit from treatment with NUC-7738.

[0075] Preferably, the biological sample is a blood sample or a fraction thereof (e.g., plasma or serum).

[0076] Various aspects of the present invention are based, in part, on the finding that NUC-7738 can reduce the amount of extracellular PD-L1 protein (including soluble PD-L1 protein and exosomal PD-L1 protein) produced by cancer cells. PD-L1 is known to hide cancer cells from the immune system, and in particular, increased soluble PD-L1 is a biomarker for poor prognosis and resistance to immune checkpoint inhibitors. Thus, 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 a novel clinical opportunity for the treatment and management of cancer in patients. For example, by eliminating or reducing the hiding of cancer cells from the immune system, the host immune system is enabled to recognize and attack (including killing) cancer cells. 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, functions to enhance the effect of immunotherapeutic agents (including immune checkpoint inhibitors) and provides the ability to treat cancer patients by the combined use of NUC-7738 and immunotherapeutic agents. When the immunotherapeutic agent is an immune checkpoint inhibitor as described herein, the use of the checkpoint inhibitor at a lower dose is enabled, and in this way, the toxic effects of such an agent can be reduced. Certain cancer patients may sometimes develop resistance to immune checkpoint inhibitors associated with increased soluble PD-L1 levels. The ability of NUC-7738 to target soluble or exosomal PD-L1 and reduce its amount is predicted to remove resistance / obstruction, and in this way, enables patients to be retreated with the same or other immune checkpoint inhibitors or to be treated with immune checkpoint inhibitors that are not effective without the reduction of soluble PD-L1 induced by NUC-7738. Therefore, NUC-7738 is also used to treat patients who develop resistance to immune checkpoint inhibitors or who have resistance to existing immune checkpoint inhibitors.

[0077] Thus, in a particular example of any aspect of the present invention, the patient or subject is one who is resistant to an immune checkpoint inhibitor or has resistance to an immune checkpoint inhibitor. Such a person is one in whom a clinical signal of efficacy recognized with a given dose of an immune checkpoint inhibitor is not provided. Preferably, a subject / patient who has developed resistance to an immune checkpoint inhibitor would already have shown a clinical signal of efficacy, such as maintenance or reduction of a cancer mass or reduction of a cancer marker, progression-free survival, but the efficacy of the immune checkpoint inhibitor has decreased or ceased. This is an indication that the cancer has become resistant to the agent. Preferably, a subject / patient resistant to an immune checkpoint inhibitor (having pre-existing resistance) would have markers indicative of pre-existing resistance to an immune checkpoint inhibitor, such as low mutational burden, microsatellite stability, low tumor infiltrating lymphocytes, high levels of immunosuppressive cytokines, PTEN deficiency (see Liu et al., Am J Clin Dermatol. 20(1): 41-54, 2019).

[0078] The various aspects of the present invention are based on the finding that NUC-7738 can increase the amount of OX40L produced by cancer cells. Stimulating OX40 has been shown to be a candidate for an immunotherapy strategy for cancer.

[0079] OX40 signaling is important for the priming and development of CD4 and CD8 T cell immunity and for the development of T cell memory. OX40 is expressed on T cells and is then recognized on the cell surface by the T cell receptor in the context of recognition of foreign or aberrant proteins. Engagement of OX40 by its ligand, OX40L, has been shown to enhance T cell activation and increase the anti-tumor immune response (Weinberg et al., IJ Immunol. 164(4): 2160-2169, 2000). Thus, the OX40 / OX40L axis has been recognized as an interesting target for the design of vaccine and therapeutic adjuvant strategies for infectious diseases and cancer as well as for the treatment of autoimmune and inflammatory diseases (see, for example, Deng et al., "OX40 (CD14) and OX40 Ligand: Important Immune Checkpoints in Cancer", Onco Targets Ther. 12: 7347-7353, 2019; Roszik et al., "Expression and Survival of TNFSF4 (OX40L) 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., "Enhancement of the Immunostimulatory Function of Dendritic Cells by Gene Transfer Using mRNA Encoding OX40 Ligand", Blood. 105: 3206-3213, 2005).

[0080] NUC-7738 in various embodiments or specific examples of the present invention includes NUC-7738 as the (S)-phosphate diastereomer, as the (R)-phosphate diastereomer, or as a mixture of phosphate diastereomers; also includes the compound in the form of the free base or in the form of a pharmaceutically acceptable salt; and also includes a pharmaceutical composition comprising NUC-7738.

[0081] The immune checkpoint inhibitor in various embodiments or specific examples of the present invention includes the agent or its pharmaceutically acceptable salt; also includes a pharmaceutical composition comprising the immune checkpoint inhibitor.

[0082] 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 a new therapeutic opportunity for treating cancer.

Brief Description of the Drawings

[0083]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0084] Further features and specific examples of the above aspects are described below in the section with headings. Each section can be combined with any of the above aspects in any non-conflicting combination.

[0085] The following definitions may be useful in understanding the present invention. As used herein, "treatment" refers to a clinical intervention in an attempt to alter the natural processes of a subject or cell being treated, and is performed before or during a clinical pathology process. Desirable effects of treatment include preventing the onset or recurrence of a disease or disorder or its symptoms, alleviating the disorder or symptoms of a disease, reducing the direct or indirect pathological consequences of a disease, reducing the rate of disease progression, improving or alleviating the disease state, and achieving remission or improved prognosis. In some specific examples, the methods and compositions disclosed herein are useful in attempts to delay the onset of a disease or disorder. Treatment applies to both human treatment and veterinary use. As used herein, the terms "disease" and "disorder" are generally interchangeable unless the context otherwise supports.

[0086] "Improvement" includes that the disease or disorder (and symptoms associated therewith) described herein is alleviated, reduced, decreased, and / or mitigated.

[0087] Soluble PD-L1 is secreted by cancer cells and exists in plasma as a free protein. Exosomal PD-L1 is a PD-L1 protein bound to the exosomal membrane and is released from cells as extracellular vesicles. As used herein, extracellular PD-L1 is a PD-L1 protein that is released / shed from cells and is not bound to cells. The PD-L1 protein released from cells is circulating PD-L1. Soluble PD-L1 and exosomal PD-L1 are examples of extracellular PD-L1.

[0088] Exosomes (a type of extracellular vesicle (EV)) are small membrane-enclosed structures that are readily shed from the surface of healthy or diseased cells in environments such as cell activation, growth, and apoptosis. These vesicular structures contain significant amounts of biologically active proteins, lipids, and nucleic acids acquired from their parent cells. EVs are generally found in blood (e.g., plasma), urine, saliva, tears, and many other body fluids. EVs and / or biomarkers present in, on, or associated with them have been proposed for use in diagnosis, prognosis, and monitoring of diseases and health states.

[0089] Extracellular vesicles are isolated and purified using a polymer-based resin used as the kit ExoQuick® ULTRA EV (Systems Bioscience). Subsequently, the isolated EVs are quantified for their protein amount, for example, using a bicinchoninic acid (BCA) protein quantification assay. Preferably, quantification of the exosomal protein marker CD81 is used. In addition, the particle size distribution of the EVs is measured using NanoSight.

[0090] The association of PD-1 with the PD-L1 ligand can alter the tumor microenvironment and suppress the intrinsic antitumor immune response 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 derived from non-small cell lung cancer cells expressing PD-L1 play a role in immune evasion by reducing T cell activity and promoting tumor growth. They found that the abundance of exosomes expressing PD-L1 isolated from the plasma of patients correlates with PD-L1 positivity in tumor tissues. They found that exosomes can restore immune function by reducing cytokine production and inducing apoptosis in CD8+ T cells, indicating that tumor-derived exosomes expressing PD-L1 are important mediators of tumor immune evasion.

[0091] Soluble PD-L1 (sPD-L1) is an efficacy prediction and prognostic biomarker for cancer patients selected or receiving immune checkpoint blockade therapy (see Oh, S.Y., Kim, S., Keam, B. et al., "Soluble PD-L1 is an efficacy prediction and prognostic biomarker in advanced cancer patients receiving immune checkpoint blockade therapy", Sci Rep, 11, 19712 (2021): https: / / doi.org / 10.1038 / s41598-021-99311-y). Similarly, exosomal PD-L1 (xPD-L1) is also used as a biomarker for cancer patients selected or receiving immune checkpoint blockade therapy (Chen et al., "Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response", Nature, 560: 382-386, 2018; Li et al., J of Translational Medicine, "Clinical significance of PD-L1 expression in serum-derived exosomes of NSCLC patients", 17, 225, 2019). As used herein, the terms "exosomal PD-L1", "xPD-L1", "extracellular vesicle PD-L1", and "EV PD-L1" are interchangeable.

[0092] The term "high levels of soluble PD-L1 or exosomal PD-L1" refers to an increase above normal levels, typically above a clinically appropriate reference level. Thus, high levels of soluble PD-L1 refer to outside the range of a clinically appropriate normal / usual amount. Such a deviation from normal levels is, for example, an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more.

[0093] The normal or usual level is determined by applying standard statistical means to measure levels from a population of subjects who are healthy or not affected by diseases mediated by high levels of soluble PD-L1 or associated with high levels of soluble PD-L1. Such values are used to determine an appropriate "reference level" 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.

[0094] Low levels of OX40L expression are associated with poor prognosis and grade (see Roszik et al. supra). Accordingly, OX40L is used as a biomarker for selecting patients suitable for treatment with immune checkpoint inhibitors and / or the methods of the present invention. For example, tumors with low OX40L are targets for treatments that increase tumor OX40L expression.

[0095] The term "low levels of OX40L" refers to a decrease from the normal level, typically below a clinically appropriate reference level. Thus, "low levels of OX40L" refers to an amount outside the range of the usual / normal amount. Deviation from the normal level can be, for example, a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more.

[0096] A "clinically appropriate reference level" is a value determined from a clinical trial appropriate as an amount / level (e.g., a threshold) of soluble PD-L1 or exosomal PD-L1 or a level of OX40L to classify whether a value is high or not. It is a value used to make a clinical decision. The use of such reference values is known to be for performing a diagnostic test or a clinical decision.

[0097] The term "administered" or "administering" (in all its grammatical forms) means the administration of a therapeutically effective amount of NUC-7738 to a subject / patient (as a monotherapy agent or in combination with other therapeutic agents, e.g., the immune checkpoint inhibitors described herein). NUC-7738 is preferably in the form of a composition, preferably a pharmaceutical composition, and the pharmaceutical composition is used in combination therapy, i.e., co-administration with other pharmaceuticals or agents, e.g., pharmaceuticals for the treatment of the diseases described herein and / or other therapeutic agents that are considered beneficial in the methods described herein.

[0098] As used herein, "effective amount" means an amount effective for a number of times and periods necessary to achieve the desired therapeutic or physiological effect. The exact dosage varies depending on the therapeutic purpose and is determined by one of ordinary skill in the art using known techniques.

[0099] "Therapeutically effective amount" means the amount of an agent (e.g., a compound such as NUC-7738) that produces an effect upon administration. The "therapeutically effective amount" of NUC-7738 as described herein varies depending on factors such as age, body weight, general health, sex, diet, time of administration, drug interactions, and the severity of the disorder, and is determined by one of ordinary skill in the art by routine experimentation. Sometimes, the term "therapeutically effective amount" is used interchangeably with the term "pharmaceutically effective amount". The "therapeutically effective amount" of NUC-7738 when co-administered with a particular immune checkpoint inhibitor may be different from the "therapeutically effective amount" when NUC-7738 is used alone (monotherapy) and may also be different from the "therapeutically effective amount" when co-administered with other immune checkpoint inhibitors.

[0100] When used in the present invention, a therapeutically effective amount is also an amount where the therapeutically beneficial effect exceeds the toxicity or harmful effects of NUC-7738.

[0101] Anticancer agents are usually administered in treatment cycles. The term "treatment cycle" refers to a period of treatment that is repeated according to a regular schedule. Any treatment cycle can include a rest (non-treatment) period before the next dosing of the treatment. For example, a treatment that includes a one-week rest period after three weeks is a treatment cycle with a 28-day cycle. Treatment cycles are usually 2 to 6 weeks. Multiple treatment cycles are performed sequentially in a series, whereby the next cycle is initiated after the end of one cycle. A series of cycles is called one course of treatment. One course of treatment may take 3 to 6 months, but it may be longer or shorter, or not limited. During that time, the patient receives 3 to 10 or more treatment cycles.

[0102] The agent is administered on any day of the cycle or on a specific day of the cycle as part of a treatment regimen that reflects the day or time when the agent is or is not administered. Thus, for example, NUC-7738 may be administered in a 21-day treatment cycle in which the agent is administered on days 1, 8, and 15, which means that since day 7 (day 15) after the last administration in cycle 1 is day 1 of cycle 2, the patient will receive NUC-7738 once a week.

[0103] In some specific examples, as used in this specification, the "subject" is a vertebrate. In a particular specific example, 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 a particular specific example, the mammalian subject is a human. In some further specific examples, a human patient (synonymous with an individual) is a particular subject. As used herein, the term "subject" is used synonymously with the term "patient". Preferably, the subject / patient is a human.

[0104] When NUC-7738 referred to herein is used in combination with an immunotherapeutic agent, such as a checkpoint inhibitor (e.g., a CTLA4, LAG3, PD-1, or PD-L1 inhibitor), or vice versa, unless otherwise stated, it includes the agent being administered sequentially or simultaneously with another agent.

[0105] As used herein, "co-administration" refers to a therapy in which both agents (e.g., NUC-7738 and an immunotherapy agent) are administered substantially simultaneously. Examples of co-administration are cases where the two agents are mixed in the same infusion bag or are administered simultaneously using a "Y-line" from different infusion bags. An example of co-administration is a case where the administration periods of the two agents at least partially overlap.

[0106] As used herein, "sequential administration" means administering one agent after (i.e., separately from) another agent, where the period between administrations of each agent is such that both agents can act therapeutically simultaneously. Thus, "sequential" administration allows one agent to be administered seconds, minutes, or hours after the administration of another agent, provided that the circulating half-life of the first-administered agent is such that they can both be present in therapeutically effective amounts simultaneously. The time delay between administrations of the agents can vary depending on the exact nature of the agents, their interaction with each other, and their respective half-lives. Preferably, the two agents are administered to each other within 5, 10, 15, 20, 30, 60, 120 minutes. The two agents need not be administered on the same day; indeed, they can be administered as part of a treatment regimen that includes dosing cycles (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; then such a treatment cycle is repeated one or more times. In other specific examples, agents 1 and 2 can be administered on day 1 of a 21-day cycle, and agent 2 alone can be administered on one or more other days of the 21-day cycle). Preferably, the two agents are administered to each other within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In a particular specific example of any aspect of the present invention, when NUC-7738 and an immune checkpoint inhibitor are administered sequentially, NUC-7738 is administered first. In a particular specific example of any aspect of the present 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 agents be administered in the same treatment cycle or as part of a treatment course. As used herein, "pharmaceutical product" refers to a product comprising a medicament. For example, examples of pharmaceutical products include medical devices, pharmaceutical compositions, and preferably kits comprising one or more means, containers, and / or parts comprising a medicament.

[0107] As used herein, the term "immunopotentiator" refers to an agent that acts to activate or enhance the host immune system. In the context of NUC-7738, the immunopotentiator acts by reducing the amount of soluble or exosomal PD-L1 protein present, or by increasing the amount of OX40L protein expressed in cancer cells (which can bind to OX40 on T-lymphocytes and other components in the cellular immune system, resulting in enhanced activation of T cells). In this way, the immunopotentiator switches on the immune system or removes the brakes (inhibitors) that prevent effective activation of the immune system, whereby the immune system becomes sensitized to cancer. As described herein, PD-L1 is an immune-suppressive PD-1 ligand, and by reducing PD-L1, the immune environment more readily promotes cancer cell death mediated by the cellular immune system.

[0108] As used herein, the term "immunotherapeutic agent" refers to various agents that can induce a host immune response. Such agents are typically used in immunotherapy, a type of cancer treatment that aids the attack of cancer by the subject's immune system. Examples of immunotherapeutic 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 agents such as CAR-T cells (CAR-T therapy agents). In particular specific examples, the immunotherapeutic agent is, as described herein (see, for example, the section on "immune checkpoint inhibitors"), an immune checkpoint inhibitor, or, as described herein and known to those skilled in the art, an adoptive cell therapy agent such as a CAR-T cell population, or a cancer vaccine. As used herein, the term "immunotherapeutic agent" is synonymous with "cancer immunotherapeutic agent".

[0109] "Adoptive cell therapy" Adoptive cell therapy (also known as cellular immunotherapy) is one form of treatment that uses cells of the host (patient)'s immune system to attack cancer cells.

[0110] Some of these approaches involve directly isolating immune cells from a patient and expanding their numbers, while others involve genetically engineering the isolated immune cells (by gene therapy) to enhance their ability to identify and attack cancer cells. In particular examples, adoptive cell therapy is selected from CAR-T, TIL, and NK cell therapy.

[0111] "Tumor Infiltrating Lymphocyte (TIL) Therapy" Most cancer patients have different types of naturally occurring T cells that can target cancer cells. In particular, "killer" T cells can recognize and directly eliminate cancer cells. However, such cells must be activated before they can effectively kill cancer cells and then must be able to maintain their activity for a long enough time to sustain an effective anti-tumor response. Furthermore, a sufficient number of these cells must be produced to mount an effective attack.

[0112] One form of adoptive cell therapy that attempts to address these issues is called Tumor Infiltrating Lymphocyte (TIL) therapy. In this approach, naturally occurring T cells that have already infiltrated the patient's tumor are harvested and then activated and expanded. Subsequently, large numbers of these activated T cells are reinfused into the patient, where the cells seek out and destroy the tumor.

[0113] Granhoj et al. have considered the progress in adoptive cell therapy (including the principles and techniques used) (Expert Opin Biol Ther. 22(5): 627-641, 2020).

[0114] "Genetically Recombinant T Cell Receptor (TCR) Therapy" Certain patients either do not already have T cells that recognize their tumors or, if they do, these T cells cannot be expanded to an activated and sufficient number to enable tumor rejection. For these patients, an approach known as genetically engineered T cell receptor (TCR) therapy may be appropriate.

[0115] This approach involves collecting T cells from the patient, but rather than simply, activating and expanding available anti-tumor T cells, the T cells are modified to provide a novel T cell receptor that can target specific cancer antigens.

[0116] "CAR-T cell therapy" The TIL and TCR therapies described above target and can only eliminate cancer cells that present those antigens in certain situations (when the antigen is bound by the major histocompatibility complex (MHC)).

[0117] An approach designed to address this limitation involves genetically engineering T cells with a synthetic receptor known as a CAR (which stands for chimeric antigen receptor).

[0118] A CAR is a synthetic molecule that comprises an extracellular domain that functions as a specific anti-affinity ligand for a target cell surface antigen (most often derived from an antibody and produced as a single-chain variable fragment - scFv) and an intracellular domain that ensures potent activation and proliferation of modified T cells in an HLA-independent manner. The basic arrangement of the CAR extracellular domain comprises one or two co-stimulatory molecule domains (derived from CD28, 4-1BB, or OX-40) arranged in series with the CD3z domain.

[0119] An important advantage of CARs is their ability to bind to cancer cells, even when their antigens are not present on the surface via MHC (making cancer cells vulnerable to their attack). However, CAR-T cells can only recognize antigens that are naturally expressed on the cell surface by themselves, so the range of potential antigen targets is smaller than that by TCR.

[0120] CAR-T cells (chimeric antigen receptor T cells) are usually isolated from the same patient (autologous) receiving treatment, but can also be isolated from different donor sources (allogeneic) and genetically engineered to express proteins on their surfaces (called chimeric antigen receptors (CARs)). CARs recognize and bind to specific antigens on the surface of cancer cells, thus directing T cells towards cancer cells. The genetically engineered CAR-T cells are first expanded in the laboratory and then reinjected into the patient, where the CAR-T cells proliferate, are directed towards cancer cells, and kill the cancer cells.

[0121] Since 2017, six CAR-T cell therapies have been approved by the US Food and Drug Administration (FDA). All are approved for the treatment of blood cancers (including lymphoma, several types of leukemia, and more recently multiple myeloma).

[0122] The efficacy of CAR-T cell therapy for solid tumors is inhibited by the inhibitory effects of immune checkpoints in the microenvironment of solid tumors (Ma, S., Li, X., Wang, X., Cheng, L., Li, Z., Zhang, C. et al., "Recent Developments 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 Increase the Cytotoxicity of CAR-T Cells", J. Hematol. Oncol., 12: 127, 2019).

[0123] To contribute to the limited clinical efficacy of CAR-T cell therapy in solid tumors, programmed cell death protein-1 (PD-1)-mediated immunosuppression has been proposed. PD-1-mediated immunosuppression has been proposed to be involved in reduced CAR-T cell function (Moon EK, Wang LC, Dolfi DV, Wilson CB, Ranganathan R, Sun J et al., "Reversible and multifactorial T cell hypofunction 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 and anti-PD-1 antibody therapy has been shown to stimulate 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 cells: replenishment of CAR", Blood. 129: 1039-41, 2017; and Adusumilli PS, Zauderer MG, Rusch VW, O'Cearbhaill R, Zhu A, Ngai D et al., "Local delivery of mesothelin-targeted CAR T cells for pleural cancer: safety and preliminary efficacy in combination with anti-PD-1 agents", J Clin Oncol. 37: 2511, 2019).

[0124] It is believed that reducing the inhibitory effect of immune checkpoints (such as PD-1 / PD-L1) enhances CAR-T cell therapy. The ability of NUC-7738 to reduce the amount of soluble or exosomal PD-L1 is believed to reduce the inhibitory effect of immune checkpoints and increase CAR-T cell therapy.

[0125] Furthermore, the ability of NUC-7738 to increase the expression of OX40L seems to be beneficial for CAR-T therapy. For example, Zhang, Huihui, et al. ("Chimeric antigen receptors with antigen-independent OX40 signaling mediate potent antitumor effects", Science translational medicine, 13.578 (2021): eaba7308) demonstrated that co-stimulation of CAR-T and OX-40 in tumor cells mediates potent antitumor activity. Thus, we are convinced that the ability of NUC-7738 to increase the amount of OX40L reduces the inhibitory effect of immune checkpoint and increases CAR-T therapy.

[0126] "Natural killer (NK) cell therapy" Adoptive cell therapy strategies have been initiated to incorporate other immune cells, such as natural killer (NK) cells. Applications being investigated clinically include endowing these NK cells with cancer-targeting CARs.

[0127] Biederstadt and Rezvani (Int J Hematol. 114(5): 554-571, 2021) provide an overview of current trends and evolving concepts for genetically engineering next-generation CAR-NK therapies.

[0128] In particular specific examples, NUC-7738 is provided for use in combination with adoptive cell therapy. In particular specific examples, NUC-7738 is provided for use in increasing the effect of adoptive cell therapy. Preferably, the adoptive cell therapy is selected from CAR-T, NK, and TIL.

[0129] "Cancer vaccine" Cancer vaccines involve the administration of therapies that enhance the immune system's ability to present cancer antigens to the immune system and to find and eliminate cancer cells that express these antigens. Cancer vaccines can be viral, RNA, DNA, or peptide-based and are specific for proteins expressed on particular cancer cells (e.g., sipuleucel-T for prostate cancer or talimogene laherparepvec for advanced melanoma skin cancer).

[0130] Preclinical trials are expected for combining cancer vaccines with immune checkpoint inhibitors. For example, van Elsas et al. (van Elsas A, Hurwitz AA, Allison JP., "Combined immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) and granulocyte / macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors with autoimmune depigmentation", J Exp Med., 190: 355-66, 1999) tested the efficacy of anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) alone or in combination with granulocyte / macrophage colony-stimulating factor (GM-CSF)-expressing tumor cell vaccines against highly tumorigenic, poorly immunogenic mouse melanoma B16-BL6 and found that tumors were eradicated in 80% (68 / 85) when combined, while individual therapies were either ineffective or had low efficacy.

[0131] Reducing the inhibitory effect of immune checkpoint inhibitors (such as PD-1 / PD-L1) is thought to expand cancer vaccine therapy. The ability of NUC-7738 to reduce the amount of soluble or exosomal PD-L1 is thought to reduce the inhibitory effect of immune checkpoints and expand cancer vaccine therapy. Furthermore, stimulation of OX40 on immune cells has been proposed as a strategy to enhance vaccine efficacy (Panagioti et al., Front Immunol. 20(8): 144, 2017), and thus the ability of NUC-7738 to increase the expression of OX40L will likely enhance the efficacy of cancer vaccines.

[0132] In a particular specific example, NUC-7738 is provided for use in combination with a cancer vaccine. In a particular specific example, NUC-7738 is provided for use in increasing the effect of a cancer vaccine.

[0133] 「NUC-7738」 The present invention relates to the medical use of NUC-7738, either alone or in combination with immunotherapy, particularly for the treatment of proliferative diseases such as cancer by reducing the level of extracellular PD-L1 protein produced by cancer cells or by increasing the level of OX40-L produced by cancer cells. Preferably, the extracellular PD-L1 protein is soluble PD-L1 or exosomal PD-L1. A decrease in the level of soluble PD-L1 or exosomal PD-L1 is thought to occur by NUC-7738 reducing the translation of soluble / exosomal PD-L1. An increase in the level of OX40-L is thought to occur by NUC-7738 enhancing the transcription of OX40-L.

[0134] The compound 3'-deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alaninyl)] phosphate (also referred to as NUC-7738) is a phosphoramidate derivative of 3'-deoxyadenosine. NUC-7738 is disclosed in WO2016 / 083830 (Nucana) including its synthesis method. Pharmaceutical compositions comprising phosphoramidate molecules (including NUC-7738) are disclosed in WO2017 / 109491 (Nucana). WO2018 / 229493 (Nucana) and WO2018 / 229495 (Nucana) disclose specific synthetic routes of NUC-7738.

[0135] NUC-7738 has the structure represented by Formula 1:

Chemical formula

[0136] NUC-7738 has a chiral center at the phosphorus atom. NUC-7738 can exist as a mixture of phosphate diastereomers, as the (S)-epimer at the phosphorus atom in a substantially diastereomerically pure form, or as the (R)-epimer at the phosphorus atom in a substantially diastereomerically pure form. "Substantially diastereomerically pure" is defined for the purposes of the present invention as a diastereomeric purity of about 90% or greater. If it exists in a substantially diastereomerically pure form, NUC-7738 has a diastereomeric purity of 95%, 98%, 99%, or 99.5% or greater. Alternatively, NUC-7738 can also exist as a mixture of phosphate diastereomers.

[0137] The (R)- and / or (S)-phosphates of NUC-7738 can be obtained in a substantially diastereomerically pure form by chromatography, such as HPLC, optionally using a chiral column. Alternatively, the (R)- and / or (S)-phosphates of NUC-7738 can be obtained in a substantially diastereomerically pure form by crystallization from a suitable solvent or solvent system. In yet another alternative, the (R)- and / or (S)-phosphates of NUC-7738 are synthesized in a substantially diastereomerically pure form by diastereoselective synthesis. To provide a diastereomerically pure form, combinations of these techniques can be used, such as diastereoselective synthesis followed by crystallization or chromatography. It is more convenient to generate a protected form of the NUC-7738 diastereomer mixture, separate the protected form of the NUC-7738 diastereomer (e.g., using chromatography or crystallization), and remove the protecting group to provide substantially pure NUC-7738.

[0138] NUC-7738 used herein may be in the form of the free base or in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, sulfamic acid, and hydrobromic acid, or salts of pharmaceutically acceptable organic acids 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, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid, but are not limited thereto.

[0139] NUC-7738 exists in single crystal form or as a mixture of crystal forms, or is amorphous. Thus, the NUC-7738 compound for medical use according to the present invention is administered as a crystalline or amorphous product. NUC-7738 can be obtained, for example, as a solid plug, powder, or film by methods such as precipitation, crystallization, freeze-drying, spray-drying, or evaporation-drying. For this purpose, microwave drying or radio frequency drying can be used.

[0140] Unless otherwise indicated, NUC-7738 referred to in various aspects or embodiments of the present invention includes NUC-7738 as the (S)-phosphate diastereomer or as the (R)-phosphate diastereomer, or as a mixture of phosphate diastereomers. Also included are compounds in the form of the free base or in the form of a pharmaceutically acceptable salt. Also included are pharmaceutical compositions (e.g., NUC-7738 formulations) comprising NUC-7738.

[0141] "NUC-7738 formulation" NUC-7738 (including its pharmaceutically acceptable salts) is used alone, but generally, NUC-7738 is administered in the form of a pharmaceutical composition with one or more pharmaceutically acceptable additives, such as adjuvants, diluents, or carriers. A pharmaceutical additive is a substance other than a pharmacologically active agent or prodrug contained in the manufacturing process or contained in the final pharmaceutical product dosage form. The usual procedures for the selection and preparation of suitable pharmaceutical formulations are described, for example, in "Pharmaceutics - The Science of Dosage Form Design", M. E. Aulton, Churchill Livingstone, 1988.

[0142] Preferably, the "use of NUC-7738" also includes the "use of a pharmaceutical composition comprising NUC-7738".

[0143] Depending on the mode of administration of NUC-7738, the pharmaceutical composition used to administer NUC-7738 (including various pharmaceutically acceptable salts thereof) preferably comprises 0.05 to 99% by mass of NUC-7738 or its pharmaceutically acceptable salt, more preferably 0.05 to 80% by mass of NUC-7738 or its pharmaceutically acceptable salt, even more preferably 0.10 to 70% by mass of NUC-7738, and still more preferably 0.10 to 50% by mass of NUC-7738 (the mass% is based on the total of the composition).

[0144] NUC-7738 is administered orally. For oral administration, NUC-7738 is mixed with adjuvants or carriers such as lactose, sucrose, sorbitol, mannitol; starches such as potato starch, corn starch, or amylopectin; cellulose derivatives; binders such as gelatin or polyvinylpyrrolidone; 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 cores prepared as above can be coated with a concentrated sugar solution (which can contain, for example, gum arabic, gelatin, talc, and titanium dioxide). Alternatively, the tablets may be coated with a suitable polymer dissolved in an easily volatile organic solvent.

[0145] In a particular specific example, NUC-7738 or a composition comprising NUC-7738 is administered parenterally, particularly by intravenous administration. Parenteral administration methods include, for example, intradermal, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal, or intravenous injection, and infusion techniques (e.g., in the form of injection solutions, infusions, or tinctures). For parenteral (e.g., intravenous) administration, NUC-7738 is administered as a sterile aqueous or oily solution. Aqueous formulations for intravenous administration, particularly aqueous formulations of the free base of NUC-7738, can also contain, as additives, pharmaceutically acceptable polar organic solvents such as dimethylacetamide, and one or more solubilizers or other additives.

[0146] NUC-7738 or a composition comprising NUC-7738 according to or for use in the present invention is used for the treatment of humans or other animals, for example, for treating commercial animals such as livestock or companion animals (cats, dogs, etc.). Preferably, NUC-7738 or a composition comprising NUC-7738 according to or for use in the present invention is for human treatment or is used in the treatment of humans.

[0147] NUC-7738 according to or for use in the present invention is obtained, stored and / or administered in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carboxylic acid, boric acid, sulfamic acid, and hydrobromic acid, or pharmaceutically acceptable organic acids 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, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid, but are not limited thereto. Suitable basic salts are formed from bases that form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts such as hemisulfate, hemioxalate, and hemicalcium salts are also formed. Preferably, the NUC-7738 compound is not in the form of a salt, i.e., in the free base / free acid form.

[0148] When administered to a subject / patient, the dosage form will, of course, vary according to the exact mode of administration, the desired treatment, and the disease / disorder. Dosage levels, frequency of administration, and duration of treatment are expected to vary according to the patient's prescription and clinical adaptation, age, and comorbidities. The dosage size for therapeutic purposes of NUC-7738 or a composition comprising NUC-7738 also varies according to known medical principles depending on the nature and severity of the disorder, the age and sex of the animal or patient, and the route of administration.

[0149] Pharmaceutical formulations typically take the form of a composition in which an active compound or a pharmaceutically acceptable salt thereof is accompanied by one or more pharmaceutically acceptable additives. One such pharmaceutically acceptable additive in the formulations of the present invention is a polar aprotic solvent. General procedures for the selection and preparation of suitable pharmaceutical formulations are described, for example, in "Pharmaceutics - The Science of Dosage Form Design", M. E. Aulton, Churchill Livingstone, 1988.

[0150] The formulations are suitable for topical administration (e.g., skin application), oral administration, or parenteral administration (e.g., intravenous administration).

[0151] The various solvents used in the formulations of the present invention must be of pharmaceutical grade (meaning having a purity such as to be suitable for administration to humans (e.g., intravenous administration)).

[0152] For oral administration, the formulations of the present invention comprise an adjuvant or carrier such as lactose, sucrose, sorbitol, mannitol; starch such as potato starch, corn starch, or amylopectin; cellulose derivatives; a binder such as gelatin or polyvinylpyrrolidone; and / or a lubricant such as magnesium stearate, calcium stearate, polyethylene glycol, wax, paraffin, etc., and are then compressed into tablets. If coated tablets are required, the core prepared as above can be coated with a concentrated sugar solution (which can contain, for example, gum arabic, gelatin, talc, and titanium dioxide). Alternatively, the tablets may be coated with a suitable polymer dissolved in an easily volatile organic solvent.

[0153] For the preparation of soft gelatin capsules, the active compound is mixed, for example, with a vegetable oil or polyethylene glycol. Hard gelatin capsules can contain granules of the compound using the above additives for tablets. Liquid or semi-solid formulations of the active compound are also filled into hard gelatin capsules.

[0154] Liquid formulations for oral administration are in the form of syrups or suspensions. For example, the solution contains the compound of the present invention, and the remainder is a mixture of sugar and ethanol, water, glycerol and propylene glycol. Such liquid formulations may optionally contain coloring agents, flavors, sweeteners (such as saccharin), preservatives and / or carboxymethyl cellulose (as a thickening agent) or other additives known to those skilled in the art.

[0155] However, preferably, the formulation comprising NUC-7738 according to or for use in the present invention is for parenteral administration (e.g., intravenous administration) or for dilution to form a formulation for parenteral administration (e.g., intravenous administration). For parenteral administration (e.g., intravenous administration), the NUC-7738 compound is administered as a sterilized aqueous or oily solution. Preferably, the active NUC-7738 compound is administered as a sterilized aqueous solution.

[0156] The pharmaceutical composition comprising NUC-7738 according to or for use in the method of the present invention preferably comprises 0.05 to 99% by mass of NUC-7738, more preferably 0.05 to 80% by mass of NUC-7738, even more preferably 0.10 to 70% by mass of NUC-7738, and still more preferably 0.10 to 50% by mass of NUC-7738 (the % by mass is based on the total of the composition).

[0157] Preferably, NUC-7738 is administered in a pharmaceutically effective or acceptable amount for in vivo treatment of a patient. Preferably, the patient is a human (the patient may also be other animals).

[0158] Preferably, NUC-7738 (including its pharmaceutical composition) is administered to a human or other animal in an amount sufficient to exert a therapeutic effect according to the above-described method of therapeutic / medical use.

[0159] Regarding the pharmaceutical composition of NUC-7738, for example, a pharmaceutical composition comprising NUC-7738 includes NUC-7738 as the (S)-phosphate diastereomer or as the (R)-phosphate diastereomer. It can also contain the free base form of the compound or may be in the form of a pharmaceutically acceptable salt.

[0160] "Therapeutically effective amount of NUC-7738" A therapeutically effective amount of NUC-7738 is an amount sufficient to induce the death of cancer cells. There are various different means for calculating and indicating the amount of a therapeutically effective compound (such as NUC-7738) administered to a patient.

[0161] One method that is considered particularly appropriate for the dosage of an agent for the prevention or treatment of cancer is the amount of the agent administered per unit of the patient's body surface area. Such a dosage is generally expressed as the amount of the agent (measured by mass) per square meter of body surface area (m 2 ).

[0162] In the use of NUC-7738 for the treatment of cancer, a weekly dosage of 300 - 1600 mg / m 2 is used. In such treatment, for example, a weekly dosage of 500 - 1150 mg / m 2 or a weekly dosage of 900 - 1350 mg / m 2 is used. The standard dosages are selected from 900 mg / m 2 , 1100 mg / m 2 , 1125 mg / m 2 , and 1350 mg / m 2 . The use of NUC-7738 for the treatment of cancer can use a reduced dosing frequency, such as Q2W, Q3W, or Q4W, at appropriately adjusted dosages.

[0163] The selected weekly dosage of NUC-7738 for use according to the present invention is provided as a single administration or multiple administrations per week. For example, the weekly dosage of the compound of the present invention is provided with 2 administration opportunities, 3 administration opportunities, or more. Thus, a weekly dosage of 900 mg / m2 In the case of, 300 mg / m within one week 2 each by three administrations or 450 mg / m in one week 2 each by two administrations. Similarly, for a weekly dose of 1350 mg / m 2 in the case of, 450 mg / m within one week 2 each by three administrations or 675 mg / m in one week 2 each by two administrations. If the administration is Q2W, Q3W, or Q4W, the dose to be administered is determined based on the selected weekly dose.

[0164] The preferred amount of NUC-7738 to be administered in a single treatment to provide the desired dose of this compound for use according to the present invention in one week is about 900 - 1350 mg / m 2 is.

[0165] The weekly dose of NUC-7738 for use according to the present invention may be reduced during the course of treatment. For example, the treatment starts at a weekly dose of about 1350 mg / m 2 , 1125 mg / m 2 , 1100 mg / m 2 , 900 mg / m 2 , or 750 mg / m 2 and during the course of treatment, the required dose is reduced to about 750 mg / m 2 (if the initial dose is equal to or more than this amount), about 625 mg / m 2 , 500 mg / m 2 , or about 375 mg / m 2 .

[0166] The dose of NUC-7738 for use according to the present invention may also be presented in other ways. Most commonly, it is the amount of the active agent provided per body weight. For a standard human patient, a dose of 1 mg / m 2It is calculated to be equivalent to about 0.025 mg / kg (body weight). Therefore, the data indicate that the compound of the present invention is effective for the treatment of recurrent or refractory cancer at a dose of about 6.25 to about 25 mg / kg. In a preferred specific example, the compound of the present invention achieves an effective treatment of recurrent or refractory cancer when administered to a patient at a weekly dose in the range of about 12.5 to 20.5 mg / kg.

[0167] NUC-7738 is administered in a series of treatment cycles, and each cycle can be of any duration, for example, 1, 2, 3, 4, 5, 6 weeks in length, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 35, or 42 days in length. In a particular specific example, NUC-7738 is administered to a patient in a series of 21-day treatment cycles, and such a series can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more treatment cycles. In a particular specific example, NUC-7738 is administered to a patient in a series of 42-day treatment cycles, and such a series can be 2, 3, 4, 5, 6, or more treatment cycles.

[0168] NUC-7738 may be administered 1, 2, or 3 times in a 21-day treatment cycle. In one specific example, in each treatment cycle, NUC-7738 is administered on the first day of a 21-day treatment cycle. In other specific examples, in each treatment cycle, NUC-7738 is administered on the first and eighth days of a 21-day treatment cycle. In other specific examples, in each treatment cycle, NUC-7738 is administered on the first, eighth, and fifteenth days of a 21-day treatment cycle. NUC-7738 may be administered 1 to 6 times in a 42-day treatment cycle. In one specific example, in each treatment cycle, NUC-7738 is administered on the first day of a 42-day treatment cycle. In other specific examples, in each treatment cycle, NUC-7738 is administered on the first and eighth days of a 42-day treatment cycle. In other specific examples, in each treatment cycle, NUC-7738 is administered on the first, eighth, fifteenth, and twenty-second days of a 42-day treatment cycle. In other specific examples, in each treatment cycle, NUC-7738 is administered on the first, eighth, fifteenth, twenty-second, and twenty-ninth days of a 42-day treatment cycle. In other specific examples, in each treatment cycle, NUC-7738 is administered on the first, eighth, fifteenth, twenty-second, twenty-ninth, and thirty-fifth days of a 42-day treatment cycle.

[0169] Considerations regarding formulations of NUC-7738 suitable for use in the treatment methods and medical uses of the present invention are described elsewhere in this specification. In the case of injectable formulations of the compounds of the present invention, these are administered intravenously. Intravenous administration is achieved over various suitable time frames, such as infusion over 10 minutes, etc.

[0170] "A therapeutically effective amount of an immunotherapeutic agent, such as an immune checkpoint inhibitor" A therapeutically effective amount of an immunotherapeutic agent is determined using general and existing knowledge regarding dosages approved by health authorities for the agent as a monotherapy, or standard clinical studies. A therapeutically effective amount of a checkpoint inhibitor is an amount sufficient to induce death of cancer cells.

[0171] As an example, the following table (Table 1) is a non-limiting list of approved immune checkpoint inhibitors suitable for use in the treatment methods and medical uses of the present invention. In certain specific examples, one or more of the diseases listed in Table 1 are treated by any of the various aspects of the present invention as presented herein.

[0172] [Table 1]

[0173] Appropriate dosages of cancer vaccines and CAR-T cell compositions are determined using general and existing knowledge regarding dosages approved by health authorities for agents as monotherapies or from standard clinical studies.

[0174] Examples of approved CAR-T therapy agents, diseases to be treated, and dosages are included in the following table (Table 2).

[0175] [Table 2]

[0176] "Medical Use of NUC-7738" According to one aspect, the present invention provides a method for treating a proliferative disease such as cancer. Preferably, the method comprises administering a therapeutically effective amount of a composition to a subject in need thereof, said composition comprising NUC-7738. Preferably, the therapeutically effective amount is an amount capable of reducing the production of extracellular PD-L1 protein by cancer cells, such as soluble PD-L1 protein and / or exosomal PD-L1, and / or increasing the production of OX40-L by cancer cells. In particular specific examples, the proliferative disease is a disease in which high levels of soluble PD-L1 and / or exosomal PD-L1 contribute to the onset of the disease. Such as cancer.

[0177] According to one aspect, the present invention provides NUC-7738 for use in the treatment of cancer by reducing the amount of extracellular PD-L1 protein produced by cancer cells. Preferably, the extracellular PD-L1 protein is soluble PD-L1 protein and / or exosomal PD-L1 protein. Preferably, the cancer treatment is carried out via humoral immunity.

[0178] According to another aspect, the present invention provides a method of 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 of a patient, the method comprising administering to a patient in need thereof a therapeutically effective amount of NUC-7738.

[0179] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in a method of 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.

[0180] According to another aspect, the present invention provides NUC-7738 for use in the treatment of cancer by increasing the amount of OX40-L protein produced by cancer cells.

[0181] 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 of a patient, the method comprising administering to a patient in need thereof a therapeutically effective amount of NUC-7738.

[0182] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in a method of treating cancer by increasing the amount of OX40-L protein produced by cancer cells.

[0183] In a particular example, by increasing the amount of OX40-L protein produced by cancer cells, the cancer cells become more sensitive to targeting by the host immune system, such as the patient's humoral immune system.

[0184] The ability of NUC-7738 to cause an increase in the production and release of soluble PD-L1 or exosomal PD-L1 supports that NUC-7738 is an immunopotentiator and can be used as an immunopotentiator. This role is further supported by the finding that NUC-7738 causes an increase in the expression and production of OX-40L by cancer cells.

[0185] Thus, according to another aspect, the present invention provides NUC-7738 for use as an immunopotentiator. Preferably, the present invention provides NUC-7738 for use as an immunopotentiator in cancer treatment.

[0186] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament as an immunopotentiator. Alternatively, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in cancer treatment, wherein NUC-7738 is an immunopotentiator.

[0187] According to another aspect, the present invention provides a method of enhancing an immune response against a tumor, the method comprising administering to a patient in need of treatment a therapeutically effective amount of NUC-7738 alone or in combination with an immunotherapeutic agent as described herein.

[0188] According to another aspect, the present invention provides NUC-7738 for use in cancer treatment by reducing the interference with the targeting of cancer cells by the patient's immune system. Preferably, reducing the interference is the suppression of the patient's immune system or the removal of an inhibitor. As described herein, the inhibitor is an extracellular PD-L1 protein, such as a soluble PD-L1 protein and / or an exosomal PD-L1 protein.

[0189] According to another aspect, the present invention provides NUC-7738 for use in the treatment of cancer by enhancing a patient's immune response against cancer.

[0190] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in a method of treating cancer by enhancing a patient's immune response against cancer.

[0191] According to another aspect, the present invention provides a method of treating cancer by enhancing a patient's immune response against cancer, the method comprising administering to a patient in need of treatment a therapeutically effective amount of NUC-7738.

[0192] In a particular embodiment of any of these aspects of the present invention, administration of NUC-7738 to a patient enhances the patient's immune response against a proliferative disease.

[0193] In a particular embodiment of any of these aspects of the present invention, administration of NUC-7738 to a patient reduces T cell depletion.

[0194] In a particular embodiment of any of these aspects of the present invention, administration of NUC-7738 to a patient nullifies the effect of extracellular PD-L1 protein.

[0195] In a particular embodiment of any of these aspects of the present invention, administration of NUC-7738 to a patient nullifies the effect of soluble PD-L1 protein or exosomal PD-L1 protein.

[0196] In a particular embodiment of any of these aspects of the present invention, cancer treatment is effected via humoral immunity.

[0197] In a particular embodiment of any of these aspects of the present invention, NUC-7738 can reduce the level of soluble PD-L1 protein produced by cancer cells.

[0198] In certain specific examples of any of these aspects of the present invention, NUC-7738 can reduce the level of exosomal PD-L1 protein produced by cancer cells.

[0199] In certain specific examples of any of these aspects of the present invention, NUC-7738 can increase the production of OX40-L by cancer cells.

[0200] In certain specific examples of any of these aspects of the present invention, treatment with NUC-7738 results in a reduction in the level of extracellular PD-L1 protein.

[0201] In certain specific examples of any of these aspects of the present invention, treatment with NUC-7738 results in a reduction in the level of soluble PD-L1 protein or exosomal PD-L1 protein.

[0202] In certain specific examples, NUC-7738 reduces the amount of extracellular PD-L1 produced by diseased cells (e.g., cancer cells) by at least 30%, such as at least 50%, at least 70%, at least 80%, or at least 90% compared to the level before treatment.

[0203] In certain specific examples, NUC-7738 reduces the amount of soluble PD-L1 protein and / or exosomal PD-L1 produced by diseased cells (e.g., cancer cells) by at least 30%, such as at least 50%, at least 70%, at least 80%, or at least 90% compared to the level before treatment.

[0204] In certain specific examples of any of these aspects of the present invention, treatment with NUC-7738 results in an increase in the level of OX40-L produced by diseased cells (e.g., cancer cells).

[0205] In certain specific examples of any of these aspects of the present invention, NUC-7738 acts as an immunopotentiator. In certain specific examples, NUC-7738 is used to enhance the effect of immunotherapy. In certain specific examples, NUC-7738 is used to enhance the effect of an immunotherapeutic agent. Preferably, the immune checkpoint inhibitor is an anti-PD-L1 or anti-PD-1 antibody. Preferably, the immune checkpoint inhibitor is selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab. In certain specific examples of the use in any of the aspects of the invention described herein, the immune checkpoint inhibitor is pembrolizumab.

[0206] Preferably, the immunotherapy or immunotherapeutic agent is an immune checkpoint inhibitor or a cancer vaccine or adoptive cell therapy, such as CAR-T cell therapy described herein.

[0207] As described herein, in certain specific examples, the treatment or therapy can comprise administration of NUC-7738 in combination with an immunotherapeutic agent, such as an immune checkpoint inhibitor, adoptive cell therapy, such as CAR-T cells, or a cancer vaccine.

[0208] In certain specific examples, high levels of extracellular PD-L1 protein inhibit or interfere with the host immune system's attack on cancer cells.

[0209] In certain specific examples, high levels of extracellular PD-L1 protein inhibit or interfere with the effect of an immune checkpoint inhibitor administered to a patient.

[0210] In certain specific examples, high levels of soluble PD-L1 and / or exosomal PD-L1 inhibit or interfere with the host immune system's attack on cancer cells.

[0211] In particular examples, high levels of soluble PD-L1 or exosomal PD-L1 inhibit or interfere with the effects of immune checkpoint inhibitors administered to a patient.

[0212] Accordingly, in particular examples, NUC-7738 is for use in the treatment of patients whose cancer cells express high levels of extracellular PD-L1 protein, such as soluble PD-L1 and / or exosomal PD-L1 protein.

[0213] In particular examples, NUC-7738 reduces the expression of soluble PD-L1 and / or exosomal PD-L1 protein. In particular examples, NUC-7738 reduces the transcription of mRNA encoding soluble PD-L1 and / or exosomal PD-L1 protein.

[0214] In particular examples, low levels of OX40-L protein inhibit or interfere with the host immune system's attack on cancer cells.

[0215] In particular examples, low levels of OX40-L protein inhibit or interfere with the effects of immune checkpoint inhibitors administered to a patient.

[0216] In particular examples, NUC-7738 increases the expression of OX40-L protein by cancer cells.

[0217] In particular examples, NUC-7738 increases the transcription of mRNA encoding OX40-L.

[0218] In particular examples, the patient to be treated suffers from cancer whose cancer cells express high levels of soluble PD-L1 and / or exosomal PD-L1 protein.

[0219] In particular examples, the patient to be treated suffers from cancer whose cancer cells express low levels of OX40-L protein.

[0220] In certain specific examples, the patient has already received treatment with an immune checkpoint inhibitor (optionally, such treatment has been discontinued). Preferably, the previous treatment with an immune checkpoint inhibitor has been discontinued due to toxicity, recurrence, or the cancer has become resistant to the previous treatment. Preferably, such patients are treated with NUC-7738 and an immune checkpoint inhibitor, provided that the immune checkpoint inhibitor is administered at a dose lower than the approved single-agent dose.

[0221] In certain specific examples, the patient to be treated is a cancer patient who has become resistant to treatment with an immune checkpoint inhibitor.

[0222] In certain specific examples of any of these "medical uses of NUC-7738", NUC-7738 is administered in one or more treatment cycles, for example, 1, 2, 3, 4, 5, 6, 7, 8, or more treatment cycles.

[0223] "Combination of NUC-7738" Administration of NUC-7738 can reduce the amount of immune inhibition by cancer cells, and NUC-7738 is used in combination with one or more immunotherapeutic agents, such as immune checkpoint inhibitors, antibody therapies, cancer vaccines (e.g., therapeutic cancer vaccines), or adoptive cell therapies (e.g., CAR-T).

[0224] In another aspect, the present invention provides a method of enhancing an immune response against a tumor, the method comprising administering to a patient in need of treatment a therapeutically effective amount of NUC-7738 alone or in combination with an immunotherapeutic agent as described herein.

[0225] In another aspect, the present invention provides a method of enhancing the effect of an immunotherapeutic agent, the method comprising administering to a patient in need of treatment a therapeutically effective amount of the immunotherapeutic agent simultaneously or sequentially with NUC-7738.

[0226] In another aspect, the present invention provides a combination comprising NUC-7738 and an immunotherapeutic agent.

[0227] In particular examples, the immunotherapeutic agent is, as described herein, an immune checkpoint inhibitor, antibody therapy, cancer vaccine (e.g., therapeutic cancer vaccine), or adoptive cell therapy (e.g., CAR-T).

[0228] In particular examples, the immunotherapeutic agent is, as described herein, an immune checkpoint inhibitor. Preferably, the immune checkpoint inhibitor is selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab.

[0229] According to another aspect, the present invention provides a combination comprising NUC-7738 and an immune checkpoint inhibitor as described herein for use in the treatment of a proliferative disease. Preferably, the proliferative disease is cancer as described herein.

[0230] Due to the enhancing effect of NUC-773 on the immune checkpoint inhibitor, it is predicted that the immune checkpoint inhibitor will be administered at a dose lower than the monotherapy dose for the standard / approved agent. Preferably, the dose of the immune checkpoint inhibitor is 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the monotherapy dose approved for the agent.

[0231] In a particular embodiment, the immune checkpoint inhibitor for use in combination with NUC-7738 is administered at a dose that is 75% or less of the standard / approved monotherapy dose for the agent. In a particular embodiment, the immune checkpoint inhibitor for use in combination with NUC-7738 is administered at a dose that is 50% or less of the standard / approved monotherapy dose for the agent. The approved dose refers to the dose approved / authorized by the appropriate health authority for the country or region, e.g., the US Food and Drug Administration (FDA) for the US, the European Medicines Agency (EMA) for Europe, and the Medicines and Healthcare products Regulatory Agency (MHRA) for the UK, for use of the agent as a monotherapy agent in the relevant disease. For example, the anti-PD-1 antibody nivolumab has been approved by the MHRA for monotherapy of adult melanoma patients by intravenous infusion at a dose of 240 mg every 2 weeks or 480 mg every 4 weeks. Thus, a dose that is 50% of the approved monotherapy dose is a dose of 120 mg every 2 weeks or 240 mg every 4 weeks by intravenous infusion.

[0232] As described herein and as appreciated in the art, some patients develop resistance to the administered immune checkpoint inhibitor. Other patients suffer from significant toxic side effects and / or infusion-related reactions (requiring delay or interruption of administration). The ability of NUC-7738 to enhance the effect of the immune checkpoint inhibitor allows for the use of lower doses of the immune checkpoint inhibitor, and patients can resume treatment that had been discontinued due to resistance, or resume treatment at a lower dose for patients who had interrupted the original treatment due to toxic side effects or infusion-related reactions.

[0233] "Combination" According to another aspect, the present invention provides NUC-7738 for use in combination therapy of proliferative diseases, wherein NUC-7738 is administered in combination with an immunotherapeutic agent as described herein. Preferably, the immune checkpoint inhibitor is selected from pembrolizumab, cemiplimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. Preferably, the immune checkpoint inhibitor is selected from pembrolizumab, cemiplimab, dostarlimab, or nivolumab. Preferably, the immune checkpoint inhibitor is selected from nivolumab and pembrolizumab. Preferably, the proliferative disease is melanoma. Preferably, the melanoma is cutaneous melanoma. Preferably, a subject with melanoma, such as cutaneous melanoma, has received therapy for the disease and optionally has progressed to at least one prior therapy. Preferably, a subject with melanoma, such as cutaneous melanoma, has received therapy for the disease and optionally has progressed in the disease in at least one immunotherapeutic agent treatment. Preferably, the immunotherapeutic agent is adoptive cell therapy. Preferably, the adoptive cell therapy is CAR-T cell therapy.

[0234] Thus, in particular embodiments, the present invention provides NUC-7738 for use in the combination treatment of cancer, wherein NUC-7738 is administered in combination with an immune checkpoint inhibitor selected from pembrolizumab, semiprilimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and abelumab. In particular embodiments, NUC-7738 is administered in combination with nivolumab, semiprilimab, dostarlimab, and / or pembrolizumab. In particular embodiments, NUC-7738 is administered in combination with nivolumab and / or pembrolizumab. In particular embodiments, NUC-7738 is administered in combination with pembrolizumab. In particular embodiments, the cancer is a solid tumor. In particular embodiments, the cancer is melanoma. In particular embodiments, the cancer is cutaneous melanoma. In particular embodiments, the cancer is advanced cutaneous melanoma. In particular embodiments, the cancer patient is a patient with advanced (including metastatic) cutaneous melanoma. In particular embodiments, the cancer patient is a patient with advanced (including metastatic) cutaneous melanoma and has received prior treatment for cutaneous melanoma at least once, for example, 2, 3, or more times (optionally, is undergoing the prior treatment). In such circumstances, the combination treatment (e.g., NUC-7738 + nivolumab and / or pembrolizumab) is second-line, third-line, or fourth-line treatment. In particular embodiments, the cancer patient is a patient with advanced (metastatic) cutaneous melanoma and has already received immunotherapy. In particular embodiments, a cancer patient, for example, a patient with cutaneous melanoma, has been treated with an immune checkpoint inhibitor selected from immune checkpoint inhibitors such as pembrolizumab, semiprilimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and abelumab.

[0235] Thus, in particular embodiments, the present invention provides NUC-7738 for use in the combined treatment of melanoma, such as cutaneous melanoma, where NUC-7738 is administered in combination with an immune checkpoint inhibitor selected from pembrolizumab, semiprilimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. In particular embodiments, NUC-7738 is administered in combination with nivolumab and / or semiprilimab and / or dostarlimab and / or pembrolizumab. In particular embodiments, NUC-7738 is administered in combination with pembrolizumab. In particular embodiments, NUC-7738 is administered in combination with pembrolizumab to patients with cutaneous melanoma. In particular embodiments, NUC-7738 is administered in combination with pembrolizumab to patients with advanced cutaneous melanoma. In particular embodiments, NUC-7738 is administered in combination with pembrolizumab to patients with advanced cutaneous melanoma who have received at least one prior therapy, optionally a prior immunotherapy.

[0236] According to another aspect, the present invention provides a method of treating a proliferative disease in a subject in need of treatment, the method comprising administering to the subject a composition comprising NUC-7738 and an immunotherapeutic agent, such as an immune checkpoint inhibitor as described herein. Preferably, the proliferative disease is cancer. Preferably, 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. In particular embodiments, the proliferative disease is typically a disease treated with an immune checkpoint inhibitor, such as pembrolizumab. Table 1 provides a list of diseases in which particular checkpoint inhibitors are used in treatment. Preferably, the proliferative disease is cancer and the patient or tumor and its environment exhibit high levels of soluble or exosomal PD-L1. Preferably, the proliferative disease is cancer and the cancer cells express low levels of OX40L.

[0237] According to another aspect, the present invention provides NUC-7738 for use in the manufacture of a medicament for treating a proliferative disease, for example, in combination with an immunotherapeutic agent, such as an immune checkpoint inhibitor as described herein. Preferably, the proliferative disease is cancer.

[0238] In particular examples of these aspects of the invention, NUC-7738 and an immunotherapeutic agent, such as an immune checkpoint inhibitor, are administered to a subject simultaneously or sequentially.

[0239] In particular examples of these aspects of the invention, NUC-7738 and an immunotherapeutic agent, such as an immune checkpoint inhibitor, are administered to a subject in one or more treatment cycles, for example, one or more 24-day or 42-day treatment cycles. The two agents are administered on the same or different days of each treatment cycle. For example, when combining NUC-7738 and pembrolizumab, pembrolizumab is administered on day 1 of a 21-day treatment cycle, and NUC-7738 is administered on days 1, 8, and 15 of the 21-day treatment cycle; in another particular example, pembrolizumab is administered on day 1 of a 42-day treatment cycle, and NUC-7738 is administered on days 1, 8, 15, 22, 29, and 35 of the 42-day treatment cycle. In particular examples, the treatment cycle is repeated such that the patient undergoes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more treatment cycles over the course of treatment. For the days on which both agents are administered to the patient, when NUC-7738 is used in combination with an immune checkpoint inhibitor, such as pembrolizumab, NUC-7738 is administered first or second. It is beneficial for the antibody to be administered before the cytotoxic antigen, and in a preferred particular example, an immune checkpoint inhibitor antibody, such as pembrolizumab, is administered before NUC-7738.

[0240] In a particular example, when NUC-7738 and pembrolizumab are used in combination, NUC-7738 is administered at 1125 mg / m 2 , 900 mg / m 2 , or 1350 mg / m 2Administered at a dosage selected from, pembrolizumab is administered at 200 mg on the first day of a 21-day cycle. In the case where NUC-7738 is administered on the first day, the eighth day, the fifteenth day, the twenty-second day, the twenty-ninth day, and the thirty-fifth day of a 42-day cycle and pembrolizumab 400 mg is administered on the first day, pembrolizumab can be administered at 400 mg (Q6w).

[0241] When using an immune checkpoint inhibitor other than pembrolizumab, for example, one selected from semiprimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab, the specific examples as for pembrolizumab are changed as appropriate where they should be changed and applied.

[0242] According to another aspect, the present invention provides NUC-7738 for use in the treatment of a proliferative disease, wherein NUC-7738 is for simultaneous or sequential administration with an immunotherapeutic agent, for example, an immune checkpoint inhibitor as defined herein.

[0243] According to another aspect, the present invention provides a method for treating a proliferative disease in a subject in need thereof, the method comprising administering to the subject a combination comprising NUC-7738 and an immunotherapeutic agent, for example, an immune checkpoint inhibitor as defined herein, wherein NUC-7738 and the immunotherapeutic agent are administered simultaneously or sequentially.

[0244] 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.

[0245] According to another aspect, the present invention provides the use of an immune checkpoint inhibitor in the manufacture of a medicament for treating a proliferative disease in combination with NUC-7738 as defined herein.

[0246] In particular embodiments, the cancer is selected from the group consisting of skin cancer (e.g., melanoma, Merkel cell carcinoma, or 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, renal cancer, lymphoma (e.g., Hodgkin lymphoma), ovarian cancer, cervical cancer, and endometrial cancer. In particular embodiments, the cancer is typically a cancer that is treated with an immunotherapeutic agent, such as pembrolizumab. Table 1 provides a list of diseases in which particular checkpoint inhibitors are used in treatment. Preferably, the cancer comprises cancer cells that express high levels of soluble or exosomal PD-L1. Preferably, the cancer comprises cancer cells that express low levels of OX40L.

[0247] "Pharmaceutical product comprising NUC-7738" According to another aspect, the present invention provides a pharmaceutical product comprising NUC-7738 and an immunotherapeutic agent.

[0248] In particular embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor as described herein.

[0249] In one embodiment, the pharmaceutical product may be a partial kit comprising separate formulations of NUC-7738 and an immune checkpoint inhibitor.

[0250] The separate formulations of NUC-7738 and the immune checkpoint inhibitor are administered, preferably sequentially and / or simultaneously, or administered with modifications from sequential and / or simultaneous administration.

[0251] According to another aspect, the pharmaceutical product is a first container comprising NUC-7738, for example NUC-7738 with a pharmaceutically acceptable adjuvant, diluent, or carrier; and a second container comprising an immune checkpoint inhibitor, for example an immune checkpoint inhibitor with a pharmaceutically acceptable adjuvant, diluent, or carrier; and container means for containing said first and second containers and is a kit comprising the same.

[0252] In specific examples, the pharmaceutical product comprises one or more unit dosage forms (e.g., vials, tablets, or capsules within a blister pack). In one specific example, each unit dosage form comprises only one agent selected from NUC-7738 and an immune checkpoint inhibitor. In other specific examples, the unit dosage form comprises both NUC-7738 and an immune checkpoint inhibitor.

[0253] "immune checkpoint inhibitor" Immune checkpoint proteins present on immune cells and / or cancer cells [e.g., PD1 (also known as programmed cell death protein 1 or 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 recognized to play important roles in the regulation of 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 that are utilized to effectively treat specific types of cancer.

[0254] The immune checkpoint inhibitor referred to herein is any immune checkpoint inhibitor including the various ones disclosed herein. Thus, any immune checkpoint inhibitor is used in the combination therapy defined herein.

[0255] 1. In a specific example, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, an OX40 inhibitor, an OX40L inhibitor, and / or a TIGIT inhibitor. In a particular specific example, the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. In a particular specific example, the immune checkpoint inhibitor is an antibody against an immune checkpoint protein, for example, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG3 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-BTLA antibody, an anti-OX40 antibody, or an anti-OX40L antibody. As used herein, the term "antibody" includes fragments of all antibodies capable of binding to a target antigen (including Fab, scFV, etc.).

[0256] PD-1 is a cell surface receptor protein present on immune cells, such as T cells. PD-1 plays an important role in the downregulation of the immune system and the promotion of self-tolerance by suppressing T cell activation. The PD-1 protein guards against autoimmunity through a dual mechanism that promotes apoptosis (programmed cell death) of antigen-specific cells in lymph nodes, and at the same time, is an immune checkpoint that reduces apoptosis in regulatory T cells (anti-inflammatory suppressor T cells).

[0257] Therefore, PD-1 inhibits the immune system. This prevents autoimmune diseases, but the immune system also hinders cancer cell death.

[0258] PD-1 binds to two ligands, PD-L1 and PD-L2. PD-L1 is highly expressed in several cancers, which is particularly interesting because it well establishes the role of PD-1 in cancer immune evasion. PD-1-targeted monoclonal antibodies that boost the immune system have been approved or are in development for the treatment of various cancers. Many tumor cells express PD-L1 (the immunosuppressive PD-1 ligand); inhibition of the interaction between PD-1 and PD-L1 can increase in vitro T cell responses and has been well demonstrated to mediate preclinical antitumor activity. This is known as immune checkpoint inhibition.

[0259] Examples of agents that target PD-1 include pembrolizumab (Keytruda (trademark)), nivolumab (Opdivo (trademark)), and cemiplimab (Libtayo (trademark)). These agents have been shown to be effective in the treatment of several types of cancer, including cutaneous melanoma, cutaneous squamous cell carcinoma, non-small cell lung cancer, renal cancer, bladder cancer, head and neck cancer, and Hodgkin lymphoma. They are also being studied for use against many other types of cancer. Examples of agents in development include BMS-936559 (Bristol Myers Squibb), MGA012 (MacroGenics), and MEDI-0680 (MedImmune).

[0260] Examples of agents that inhibit PD-L1 include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). These agents have also been shown to be beneficial in the treatment of different types of cancer, including bladder cancer, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, and Merkel cell carcinoma of the skin (Merkel cell cancer). They are also being studied for use against other types of cancer.

[0261] Examples of LAG3 inhibitors include BMS-986016 / relatlimab, TSR-033, REGN3767, MGD013 (bispecific DART that binds PD-1 and LAG-3), GSK2831781, and LAG525.

[0262] Examples of CTLA-4 inhibitors include MDX-010 / ipilimumab, AGEN1884, and CP-675,206 / tremelimumab.

[0263] 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).

[0264] Examples of TIGIT inhibitors include tiragolumab (MTIG7192A; RG6058; Genentech / Roche), AB154 (Arcus Bioscience), MK-7684 (Merck), BMS-986207 (Bristol-Myers Squibb), ASP8374 (Astellas Pharma; Potenza Therapeutics).

[0265] Examples of OX40 inhibitors include MEDI6469 (AstraZeneca) and BMS-986178 (Bristol-Myers Squib). Examples of OX40-L inhibitors include SL-279252 (PD1-Fc-OX40L - Shattuck Labs).

[0266] Examples of BTLA inhibitors include INBRX-106 (InhinRX), cudarolimab (also known as IBI 101 (Innovent Biologics)).

[0267] In particular examples, the immune checkpoint inhibitor is selected from BMS-986016 / relatlimab, TSR-033, REGN3767, MGD013 (bispecific DART that binds PD-1 and LAG-3), GSK2831781, LAG525, MDX-010 / ipilimumab, AGEN1884, and CP-675,206 / tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, MBG453, TSR-022, LY3321367, tirigolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106, and cudarolimab, or a pharmaceutically acceptable salt or solvate compound thereof.

[0268] In one particular example, the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0269] In particular examples, the immune checkpoint inhibitor is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0270] In one particular example, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0271] In particular examples, the immune checkpoint inhibitor is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, dostarlimab, and semiprimab.

[0272] In particular examples, the immune checkpoint inhibitor is the anti-PD-1 antibody pembrolizumab.

[0273] In particular examples, the immune checkpoint inhibitor is the anti-PD-1 antibody nivolumab.

[0274] In particular examples, the immune checkpoint inhibitor is the anti-PD-1 antibody semiprimab.

[0275] In a particular specific example, the immune checkpoint inhibitor is the anti-PD-1 antibody dostarlimab.

[0276] In one specific example, the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[0277] In a particular specific example, the immune checkpoint inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab or tremelimumab.

[0278] In one specific example, the immune checkpoint inhibitor is an anti-LAG-3 antibody.

[0279] In a particular specific example, the anti-LAG-3 antibody is relatlimab.

[0280] In one specific example, the immune checkpoint inhibitor is an anti-TIGIT antibody.

[0281] In a particular specific example, the anti-TIGIT antibody is tiragolumab.

[0282] In one specific example, the immune checkpoint inhibitor is an anti-OX40 antibody.

[0283] In a particular specific example, the anti-OX40 antibody is selected from MEDI6469 and BMS-986178.

[0284] In one specific example, the immune checkpoint inhibitor is an anti-OX40-L antibody.

[0285] In a particular specific example, the anti-OX40-L antibody is SL-279252.

[0286] In one specific example, the immune checkpoint inhibitor is an anti-BTLA antibody.

[0287] In a particular specific example, the anti-BTLA antibody is selected from INBRX-106 and cudarolimab.

[0288] "Antibody (including antigen-binding fragments of the antibody)" An antibody is an immunoglobulin molecule, and the immunoglobulin molecule can specifically bind to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, through at least one antigen recognition site located in the variable domain of the immunoglobulin molecule. In particular, as used herein, the term "antibody" refers to intact polyclonal antibodies, intact monoclonal antibodies, multispecific antibodies, such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, various other modified immunoglobulin molecules, and various fragments thereof (including antigen-binding sites as long as the antibody exhibits the desired biological activity). The term includes whole antibodies (e.g., IgG1, IgG4, etc.) and antigen-binding fragments. Antibodies are selected from any species. Preferably, the antibody is a human antibody.

[0289] The antigen-binding site refers to the part of a molecule that binds to all or part of a target antigen. In an antibody molecule, the antigen-binding site can also be referred to as the antibody-antigen binding site and includes the part of the antibody that specifically binds to all or part of the target antigen. When the antigen is large, the antibody can bind only to a particular part of the antigen, which is called an epitope. The antibody-antigen binding site is 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 encompasses antibody fragments comprising an antigen-binding site. Thus, the term "its antigen-binding fragment" (with respect to an antibody) refers to an antibody fragment, such as Fab, Fab', F(ab')2, bispecific antibody, Fv fragment, and single-chain Fv (scFv) variants having an antigen-binding site and, as a result, the ability to bind to an antigen. Antibody-binding immunoglobulin (antibody) fragments are known in the art. Such fragments need not have a functional Fc receptor-binding site.

[0290] "Treatment of proliferative diseases" The term "proliferative disorder" is used herein to refer to unwanted, uncontrolled, and abnormal cell proliferation, e.g., neoplastic or hypertrophic growth, whether in vivo or in vitro. Examples of proliferative states include, but are not limited to, benign, pre-malignant, and malignant cell proliferation (including, but not limited to, malignant neoplasms and tumors, cancer, leukemia, psoriasis, bone diseases, fibroproliferative diseases (e.g., of connective tissue), and atherosclerosis). Any type of cell (including, but not limited to, lung, colon, breast, ovary, prostate, liver, pancreas, brain, bladder, kidney, bone, nerve, and skin) may be treated (including melanomas such as cutaneous melanoma). Various aspects of the invention have particular utility in the treatment of cancer, particularly human cancer. Preferably, the proliferative disorder is a disorder that is typically treated with an immune checkpoint inhibitor such as pembrolizumab. Table 1 provides a list of diseases in which particular checkpoint inhibitors are used in treatment. The section entitled "Immune Checkpoint Inhibitors" identifies the types of cancer for which various immune checkpoint inhibitors have been proposed for treatment. Preferably, the proliferative disorder is a cancer comprising cancer cells that express a high level of soluble or exosomal PD-L1. Preferably, the proliferative disorder is a cancer comprising cancer cells that express a low level of OX40L.

[0291] As can be understood from FIG. 1, NUC-7738 decreases soluble PD-L1 in lung and melanoma cell lines, which supports the use of NUC-7738 in the treatment of any proliferative disorder mediated or associated by high levels of sPD-L1 or in which sPD-L1 is involved (e.g., by its ability to weaken the host immune system). This includes all cancers.

[0292] In particular embodiments, the cancer is selected from the group consisting of skin cancer (e.g., melanoma, Merkel cell carcinoma, or 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, renal cancer, lymphoma (e.g., Hodgkin lymphoma), ovarian cancer, cervical cancer, and endometrial cancer.

[0293] Melanoma has been particularly studied for treatment with NUC-7738 alone or in combination with an immune checkpoint inhibitor such as an anti-PD-1 antibody.

[0294] Cutaneous melanoma has been particularly studied for treatment with NUC-7738 alone or in combination with an immune checkpoint inhibitor (e.g., pembrolizumab) such as an anti-PD-1 antibody.

[0295] "Diseases or conditions mediated or associated with high levels of PD-L1 or low levels of OX40L" Preferably, NUC-7738 is used to treat patients with a proliferative disease generally having high levels of PD-L1 (particularly, soluble PD-L1 and / or exosomal PD-L1). Preferably, NUC-7738 is used to treat patients with a proliferative disease having low OX40-L levels. Preferably, sPD-L1, xPD-L1 and / or OX40-L are produced by cancer cells. Preferably, the levels of sPD-L1, xPD-L1 and / or OX40-L are detected in a blood sample or a fragment thereof (e.g., serum, plasma) from a patient. Preferably, the proliferative disease is cancer, and the patients to be treated are identified by the levels of PD-L1 (e.g., soluble or exosomal PD-L1) or OX40-L in a suitable cell or biological fluid, and as a result, the present invention provides the use of NUC-7738 in the treatment of proliferative diseases (e.g., cancer) associated with the expression of high levels of PD-L1 (e.g., soluble or exosomal PD-L1) or low levels of OX40-L.

[0296] Accordingly, the present invention relates to the medical use of NUC-7738 for the treatment of proliferative diseases (e.g., cancer) mediated or associated with high levels of PD-L1 (e.g., soluble PD-L1 and / or exosomal PD-L1) and / or low levels of OX40-L. Preferably, the proliferative disease is cancer.

[0297] In a particular specific example, the proliferative disease is a cancer in which the cells express high levels of soluble PD-L1 and / or exosomal PD-L1 and / or low levels of OX40-L. In a particular specific example, the cancer in which the cells express high levels of soluble PD-L1 and / or exosomal PD-L1 and / or low levels of OX40-L is selected from the group consisting of skin cancer (e.g., 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, bladder cancer, renal cancer, lymphoma (e.g., Hodgkin lymphoma), ovarian cancer, cervical cancer, and endometrial cancer.

[0298] According to one aspect, the present invention provides NUC-7738 for use in the treatment of cancer in a subject afflicted with a cancer in which the cells express high levels of soluble PD-L1 or exosomal PD-L1 and / or low levels of OX40-L.

[0299] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for treating cancer in a subject afflicted with a cancer in which the cells express high levels of soluble PD-L1 or exosomal PD-L1 and / or low levels of OX40-L, said treatment comprising administering NUC-7738 in combination with an immune checkpoint inhibitor as defined herein. Optionally, prior to treatment, a test is performed to determine whether the patient is afflicted with a cancer in which the cells express high levels of soluble PD-L1 and / or high levels of exosomal PD-L1 and / or low levels of OX40-L.

[0300] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in a method of treating cancer by reducing the amount of soluble PD-L1 or exosomal PD-L1 produced by cancer cells, said method comprising determining whether a subject is suffering from a cancer whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1, and administering to the subject a combination of NUC-7738 and an immune checkpoint inhibitor as defined herein if the subject is suffering from a cancer whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1.

[0301] According to another aspect, the present invention provides a method of treating cancer in a subject suffering from a cancer whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1, said method comprising administering a therapeutically effective amount of NUC-7738 alone or in combination with an immune checkpoint inhibitor as defined herein.

[0302] According to another aspect, the present invention provides a method of treating cancer in a subject suffering from a cancer whose cancer cells express high levels of soluble PD-L1 or exosomal PD-L1, said method comprising contacting cancer cells with a therapeutically effective amount of NUC-7738. In a particular embodiment of this aspect of the invention, the cancer cells are present in the body of the subject or patient. Thus, preferably, by administering to the subject or patient a therapeutically effective amount of NUC-7738 alone or in combination with an immune checkpoint inhibitor, the cancer cells are contacted with a therapeutically effective amount of NUC-7738.

[0303] According to another aspect, the present invention provides the use of NUC-7738 in the manufacture of a medicament for use in a method of treating cancer by increasing the amount of OX40-L produced by cancer cells, said method comprising determining whether a subject is suffering from a cancer in which the cancer cells express low levels of OX40-L, and, if the subject is suffering from a cancer in which the cancer cells express low levels of OX40-L, administering to the subject a combination of NUC-7738 and an immune checkpoint inhibitor as defined herein.

[0304] According to another aspect, the present invention provides a method of treating cancer in a subject suffering from a cancer in which the cancer cells express low levels of OX40-L, said method comprising administering to the subject a therapeutically effective amount of NUC-7738 alone or in combination with an immune checkpoint inhibitor.

[0305] According to another aspect, the present invention provides a method of treating cancer in a subject suffering from a cancer in which the cancer cells express low levels of OX40-L, said method comprising contacting the cancer cells with a therapeutically effective amount of NUC-7738. In a particular embodiment of this aspect of the invention, the cancer cells are present in the body of the subject or patient. Thus, preferably, by administering to the subject or patient a therapeutically effective amount of NUC-7738 alone or in combination with an immune checkpoint inhibitor, the cancer cells are contacted with a therapeutically effective amount of NUC-7738.

[0306] In a particular embodiment, the level of soluble PD-L1 or exosomal PD-L1 in the plasma of a cancer patient in which the cancer cells express high levels of soluble PD-L1 or exosomal PD-L1 is at least 30%, for example 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% greater than the standard level. The standard level is the level set for a healthy subject, i.e., a subject not suffering from a disease (e.g., cancer).

[0307] The level of sPD-L1 in normal serum is about 10 pg / ml, while in subjects suffering from cancers in which the cancer cells express high levels of sPD-L1, the level of sPD-L in serum is about 40 - 200 pg / ml. In particular specific examples, the uses and methods of the present invention relate to the treatment of cancer patients having about 40 - 200 pg / ml of sPD-L1 in serum, for example, about 60 - 150 pg / ml or about 70 - 120 pg / ml.

[0308] In particular specific examples, the level of OX40-L in a sample (e.g., cells or biological fluid, etc.) from a cancer patient in which the cancer cells express low levels of OX40-L is at least 30%, for example, 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% greater than the standard level. The standard level is the level set for healthy individuals, i.e., those not suffering from a disease. Such individuals are referred to as those having normal serum.

[0309] "Standard level" The standard level is determined by the parallel measurement of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in the normal cells of the patient, or is a value determined from historically significant measurements of wild-type / normal cells, for example, the average wild-type level measured with appropriate statistical significance from multiple specimens.

[0310] In view of the widely recognized variability among patients, the standard wild-type level is the "normal range", for example, the minimum value of the normal range. Thus, by way of example, if the normal range of total protein in the blood is assumed to be 6 - 8.3 g / dl. The standard wild-type level of protein in the blood is adopted as the value 6 g / dl or 8.3 g / dl depending on whether a value smaller or larger than normal is detected. Similarly, the normal range for the amount of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L is determined using routine evaluations for any particular cell type, and whether the cells express normal, low, or high levels of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L, and thus, is used to identify the "standard value" or "standard level" used to determine whether a patient will benefit from treatment with NUC-773 according to the method of the present invention. One of ordinary skill in the art can identify the suitability used to determine whether cancer expresses high levels of soluble PD-L1 and / or high levels of exosomal PD-L1 and / or low levels of OX40-L to establish the standard level. In a particular specific example, the wild-type value or level is used as the standard value or level. In other specific examples, the standard value or level is a value or level established from a statistical evaluation of multiple specimens (from healthy subjects and / or diseases, such as cancer patients) used to assign subjects to categories (e.g., healthy or those with high levels of sPDL-1) with a particular statistical confidence (e.g., 95% confidence). As described above, one of ordinary skill in the art can determine the standard / cutoff value or level using established or reference techniques

[0311] In a particular specific example, NUC-7738 reduces the amount of soluble PD-L1 or exosomal PD-L1 produced by diseased cells (e.g., cancer cells) by at least 25%, for example, at least 30%, at least 40%, at least 50%, at least 70%, at least 80%, or at least 90% compared to the pre-treatment level.

[0312] In a particular specific example, treatment with NUC-7738 reduces the level of soluble PD-L1 or exosomal PD-L1 by at least about 25% compared to the level obtained in the absence of NUC-7738 treatment.

[0313] In a particular specific example, treatment with NUC-7738 reduces the level of soluble PD-L1 or exosomal PD-L1 by at least about 10 - 80% compared to the level obtained in the absence of NUC-7738 treatment.

[0314] In a particular specific example, NUC-7738 reduces the amount of soluble PD-L1 and / or exosomal PD-L1 present in the serum to a level substantially the same as the normal / standard level. Substantially the same means ±10%.

[0315] In a particular specific example, treatment with NUC-7738 reduces the level of soluble PD-L1 or exosomal PD-L1 by 2 - 10 times compared to the level obtained without NUC-7738 treatment.

[0316] In a particular specific example, NUC-7738 increases the amount of OX40-L produced by diseased cells (such as cancer cells) by at least 25%, for example, at least 30%, at least 40%, at least 50%, at least 70%, at least 80%, or at least 90% compared to the pre-treatment level.

[0317] In a particular specific example, treatment with NUC-7738 increases the level of OX40-L by at least 25% compared to the level obtained without NUC-7738 treatment.

[0318] In a particular specific example, treatment with NUC-7738 increases the level of OX40-L by about 10 - 80% compared to the level obtained without NUC-7738 treatment.

[0319] In a particular specific example, NUC-7738 increases the level of OX40-L present in serum to a level that is substantially the same as the normal / standard level. Substantially the same means ±10%.

[0320] In a particular specific example, treatment with NUC-7738 increases the level of OX40-L by 2 to 10 times compared to the level obtained without NUC-7738 treatment.

[0321] Various aspects of the present invention are based on the finding that NUC-7738 can reduce the amount of soluble PD-L1 or exosomal PD-L1 produced by cancer cells and / or increase the amount of OX40-L produced by cancer cells. 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. Thus, 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 a novel clinical opportunity for treating and managing cancer in patients. For example, by eliminating or reducing the disguise of cancer cells from the immune system, the host immune system can 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 increase the level of OX40-L functions to enhance the efficacy of immunotherapeutic agents (including immune checkpoint inhibitors), providing the possibility of treating cancer patients by the combined use of NUC-7738 and immunotherapeutic agents. When the immunotherapeutic agent as described herein is an immune checkpoint inhibitor, it promotes the use of the checkpoint inhibitor at a lower dose, and as a result, reduces the toxic effects of such an agent. Certain cancer patients often develop resistance to immune checkpoint inhibitors accompanied by an increase in soluble PD-L1 levels. The ability of NUC-7738 to target and reduce the amount of soluble PD-L1 or exosomal PD-L1 is predicted to eliminate resistance / blockade, thus enabling patients to be retreated with the same or other immune checkpoint inhibitors. Therefore, NUC-7738 is also used to treat patients who are resistant to or have developed resistance to immune checkpoint inhibitors.

[0322] Thus, in a particular example of any aspect of the present invention, the patient or subject is one who is resistant to or has developed resistance to an immune checkpoint inhibitor. Preferably, such a person is one in whom an established dose of the immune checkpoint inhibitor does not provide a signal of approved efficacy.

[0323] Preferably, the subject / patient will already have shown a clinical signal of efficacy, such as tumor persistence or reduction, or a reduction in cancer markers, progression-free survival, but the efficacy of the immune checkpoint inhibitor has been reduced or stopped. This is an indication that the cancer cells have become resistant to the agent.

[0324] Preferably, a subject / patient who is resistant to an immune checkpoint inhibitor (resistance already exists) will have a marker indicating that resistance to immune checkpoint inhibitor therapy already exists. Various markers indicating resistance to immune checkpoint inhibitor therapy have been identified and include low tumor gene mutation amount / MSS tumor, low tumor infiltration, high exo-PD-L2 / sPD-L1, high or low OX40L.

[0325] "Patient selection" The inventors' finding that NUC-7738 can alter the expression of mRNA encoding soluble / exosomal PD-L1 and reduce the amount of soluble PD-L1 or exosomal PD-L1 and / or increase the amount of OX40L enables a number of ways by which it is possible to determine whether a particular patient is likely to benefit from receiving NUC-7738 alone or in combination with an immune checkpoint inhibitor as described herein.

[0326] Thus, 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 measuring the levels of soluble PD-L1 and / or exosomal PD-L1 and / or OX40L 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 OX40L produced by cancer cells in the biological sample is reduced compared to the wild-type or standard level, the patient will benefit from treatment with NUC-7738.

[0327] Preferably, the biological sample is a blood sample or a fraction thereof (e.g., serum or plasma).

[0328] In a particular embodiment of this aspect, the amount or level of soluble PD-L1 or exosomal PD-L1 is measured, and if such amount or level is increased compared to the wild-type or standard value, the patient will benefit from treatment with NUC-7738. In a particular embodiment, the level of soluble PD-L1 is measured.

[0329] In a particular embodiment, the level of exosomal PD-L1 is measured.

[0330] In another particular embodiment, the levels of soluble PD-L1 and exosomal PD-L1 are measured.

[0331] In a particular embodiment of this aspect, the amount or level of OX40-L is measured, and if such amount or level is reduced compared to the wild-type or standard value, the patient will benefit from treatment with NUC-7738.

[0332] In a particular embodiment, the levels of soluble PD-L1 and OX-40-L are measured.

[0333] In another particular embodiment, the levels of exosomal PD-L1 and OX-40-L are measured.

[0334] In other specific examples, the levels of soluble PD-L1, exosomal PD-L1, and OX-40-L are measured.

[0335] "Method for determining a suitable treatment regimen" According to another aspect, the present invention provides a method for determining a suitable treatment regimen for a patient with a proliferative disease, such as cancer, the method comprising assaying a biological sample taken from the patient for the levels of soluble PD-L1 and / or exosomal PD-L1 and / or OX-40-L, wherein an increase in the measured level of soluble PD-L1 and / or an increase in the measured exosomal PD-L1 level and / or a decrease in the measured OX-40-L level, as compared to wild type or a standard value, in the biological sample indicates that the suitable treatment regimen comprises treatment of the patient with NUC-7738.

[0336] One skilled in the art will recognize that a method for determining a suitable treatment regimen for a patient includes a method for selecting a patient for treatment.

[0337] According to another aspect, the present invention provides a method for determining whether a cancer patient will benefit from treatment with NUC-7738, the method comprising measuring the levels of soluble PD-L1 and / or exosomal PD-L1 and / or OX40L in a biological sample from the patient, wherein if the levels of soluble PD-L1 and / or exosomal PD-L1 in the biological sample are increased and / or the level of OX40L is decreased as compared to wild type or a standard level, the patient will benefit from treatment with NUC-7738. Preferably, the soluble PD-L1 and / or exosomal PD-L1 and / or OX40L in the biological sample are produced by cancer cells.

[0338] According to another aspect, the present invention provides a method for treating a patient with a proliferative disorder, such as cancer, the method comprising examining a biological sample from the patient for the levels of soluble PD-L1 and / or exosomal PD-L1 and / or OX40L, and administering to the patient a clinically effective amount of NUC-7738 if, and / or if the level of measured soluble PD-L1 and / or exosomal PD-L1 is higher than wild type or a standard value, and / or if the level of OX40L is lower than wild type or a standard value.

[0339] Preferably, the levels of PD-L1 (soluble or exosomal) and / or OX40-L are quantified using an immunoassay.

[0340] Preferably, the biological sample is a blood sample or a fragment thereof (e.g., serum or plasma).

[0341] One skilled in the art will recognize that there are many suitable examples of biological samples for use in the specific examples of the present invention described above. Preferably, such samples will include diseased tissue or organs (e.g., skin, kidney, brain, liver) or fluid samples (e.g., blood or a fraction thereof). Preferred biological samples are tissue samples (e.g., samples used in histology). As described above, the cells in such samples are directly examined for the levels of soluble PD-L1 and / or exosomal PD-L1 and / or OX40L. Particularly preferred samples are blood samples or blood fractions thereof, e.g., serum or plasma.

[0342] Techniques for examining the levels of biomarkers are frequently used in clinical assessments (e.g., for diagnostic or prognostic purposes), and their use will be familiar to those skilled in the art of performing them in the context of the present invention. By way of mere example, in a sample containing protein, the presence of soluble PD-L1 and / or exosomal PD-L1 and / or OX40L is examined by a suitable technique using an antibody that reacts with the biomarker in question (e.g., PD-L1 or OX40-L as required), and the presence of the biomarker is visualized by a suitable immunocytochemical technique.

[0343] "Particular specific examples" 1. NUC-7738 for use in the treatment of a proliferative disease by reducing the amount of extracellular PD-L1 protein produced by proliferative disease cells and / or increasing the amount of OX40L protein.

[0344] 2. NUC-7738 for use according to specific example 1, wherein the extracellular PD-L1 is soluble PD-L1 and / or exosomal PD-L1.

[0345] 3. NUC-7738 for use according to specific example 2, wherein treatment with NUC-7738 results in a decrease in the level of soluble PD-L1 and / or exosomal PD-L1 produced by proliferative disease cells and / or an increase in the level of OX40L protein.

[0346] 4. NUC-7738 for use according to any of specific examples 1 to 3, wherein administration of NUC-7738 to a patient enhances the patient's immune response to a proliferative disease.

[0347] 5. NUC-7738 for use according to any of specific examples 1 to 4, wherein the treatment of the proliferative disease occurs through adaptive immunity (e.g., cellular immunity and / or humoral immunity).

[0348] 6. NUC-7738 for use according to any of the above specific examples, wherein the proliferative disease is cancer.

[0349] NUC-7738 for use as an immune promoter in cancer treatment.

[0350] 8. NUC-7738 for use according to specific example 6 or 7, 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.

[0351] 9. NUC-7738 for use according to any of the above specific examples, wherein the treatment comprises administering NUC-7738 in combination with an immunotherapeutic agent, such as an immune checkpoint inhibitor, antibody therapy, adoptive cell therapy, such as CAR-T therapy, or a cancer vaccine.

[0352] 10. NUC-7738 for use according to specific example 6, wherein the treatment comprises administering NUC-7738 in combination with an immune checkpoint inhibitor.

[0353] 11. NUC-7738 for use according to specific example 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.

[0354] 12. NUC-7738 for use according to any of the above specific examples, wherein the immune checkpoint inhibitor is selected from 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.

[0355] 13. NUC-7738 for use according to specific example 12, wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0356] 14. NUC-7738 for use according to Specific Example 12, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, dostarlimab, and semaprimab.

[0357] 15. NUC-7738 for use according to Specific Example 12, wherein the anti-PD-1 antibody is pembrolizumab.

[0358] 16. NUC-7738 for use according to Specific Example 12, wherein the anti-CTLA-4 antibody is ipilimumab or tremelimumab.

[0359] 17. NUC-7738 for use according to Specific Example 12, wherein the anti-LAG-3 antibody is relatlimab.

[0360] 18. NUC-7738 for use according to Specific Example 12, wherein the anti-TIGIT antibody is tirigolumab.

[0361] 19. NUC-7738 for use according to Specific Example 12, wherein the anti-BTLA antibody is cudaralimab.

[0362] 20. NUC-7738 for use according to any of the above specific examples, 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.

[0363] 21. NUC-7738 for use according to Specific Example 20, wherein the treatment with NUC-7738 reduces the amount of extracellular PD-L1 protein produced by cancer cells.

[0364] 22. NUC-7738 for use according to Specific Example 21, wherein the extracellular PD-L1 is selected from soluble PD-L1 protein or exosomal PD-L1 protein.

[0365] NUC-7738 for use according to Specific Example 20, wherein treatment with NUC-7738 increases the amount of OX40-L protein produced by cancer cells.

[0366] NUC-7738 for use according to any of the above specific examples, wherein the patient has already received treatment with an immune checkpoint inhibitor and optionally, said treatment has been discontinued.

[0367] NUC-7738 for use according to Specific Example 24, wherein prior treatment with an immune checkpoint inhibitor was discontinued due to toxicity, recurrence, or the cancer becoming resistant to the prior treatment.

[0368] NUC-7738 for use according to any of the above specific examples, wherein treatment with NUC-7738 reduces the level of soluble or exosomal PD-L1 by at least 25% compared to the level obtained without NUC-7738 treatment.

[0369] 27. NUC-7738 is administered at a weekly dose of about 300 - 1600 mg / m 2 , for example, at a weekly dose of 500 - 1150 mg / m 2 , or at a weekly dose of 900 - 1350 mg / m 2 for use according to any of the above specific examples.

[0370] NUC-7738 for use according to any of Specific Examples 10 - 27, wherein the immune checkpoint inhibitor used in combination with NUC-7738 is administered at a dose of 50% or less of the standard single-agent therapy dose for the agent.

[0371] A method of treating a proliferative disease by reducing the amount of extracellular PD-L1 protein produced by diseased cells in a patient and / or by increasing the amount of OX40-L protein, the method comprising administering to a patient in need thereof a therapeutically effective amount of NUC-7738.

[0372] 30. The method according to specific example 29, wherein the proliferative disorder is cancer.

[0373] 31. A method for enhancing an immune response against cancer, comprising administering to a patient in need of such treatment a therapeutically effective amount of NUC-7738 alone or in combination with an immunotherapeutic agent.

[0374] 32. The method according to specific example 30 or 31, 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.

[0375] 33. The method according to any one of specific examples 29 to 32, wherein the extracellular PD-L1 comprises a soluble PD-L1 protein or an exosomal PD-L1 protein.

[0376] 34. The method according to any one of specific examples 30 to 33, wherein the 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.

[0377] 35. The method according to any one of specific examples 31 to 34, wherein the immunotherapeutic agent is an immune checkpoint inhibitor, antibody therapy, adoptive cell therapy, or cancer vaccine.

[0378] 36. The method according to specific example 35, wherein the immunotherapeutic agent is an immune checkpoint inhibitor.

[0379] 37. The method according to specific example 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.

[0380] 38. The method according to specific example 37, wherein the immune checkpoint inhibitor is selected from 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.

[0381] 39. The method according to specific example 38, wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0382] 40. The method according to specific example 38, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, dostarlimab, and semaprilimab.

[0383] 41. The method according to specific example 38, wherein the anti-PD-1 antibody is pembrolizumab.

[0384] 42. The method according to specific example 38, wherein the anti-CTLA-4 antibody is ipilimumab or tremelimumab.

[0385] 43. The method according to specific example 38, wherein the anti-LAG-3 antibody is relatlimab.

[0386] 44. The method according to specific example 38, wherein the anti-TIGIT antibody is tiragolumab.

[0387] 45. The method according to specific example 38, wherein the anti-BTLA antibody is cudaralimab.

[0388] 46. The method according to any one of specific examples 29 to 45, wherein the proliferative disease is cancer, and the cancer cells express a high level of soluble PD-L1 protein and / or a high level of exosomal PD-L1 protein and / or a low level of OX40L protein.

[0389] 47. The method according to any one of specific examples 29 to 16, wherein the patient has already received treatment with an immune checkpoint inhibitor, and optionally, the treatment has been discontinued.

[0390] 48. The method according to specific example 47, wherein prior treatment with an immune checkpoint inhibitor was discontinued due to toxicity, recurrence, or resistance to treatment of the cancer seat.

[0391] 49. The method according to any one of specific examples 29 - 48, wherein treatment with NUC-7738 reduces the level of soluble or exosomal PD-L1 by at least 25% compared to the level obtained without NUC-7738 treatment.

[0392] 50. Administer NUC-7738 at a weekly dose of about 300 - 1600 mg / m 2 , for example, at a weekly dose of 500 - 1150 mg / m 2 , or at a weekly dose of 900 - 1350 mg / m 2 according to the method of any one of specific examples 29 - 48.

[0393] 51. The method according to any one of specific examples 29 - 48, wherein an immune checkpoint inhibitor used in combination with NUC-7738 is administered at a dose of 50% or less of the standard monotherapy dose for the agent.

[0394] 52. A combination comprising NUC-7738 and an immunotherapeutic agent.

[0395] 53. The combination according to specific example 52, wherein the immunotherapeutic agent is an immune checkpoint inhibitor, antibody therapy, cancer vaccine, or adoptive cell therapy, such as CAR-T cells.

[0396] 54. The combination according to specific example 53, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-OX40 antibody, anti-OX40-L antibody, or anti-BTLA antibody.

[0397] 55. A combination according to specific example 53 or 54, wherein the immune checkpoint inhibitor is selected from the group consisting of BMS-986016 / relatlimab, TSR-033, REGN3767, MGD013 (bispecific DART that binds PD-1 and LAG-3), GSK2831781, LAG525, MDX-010 / ipilimumab, AGEN1884, and CP-675,206 / tremelimumab, 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.

[0398] 56. A combination according to any one of specific examples 52 to 55 for use in the treatment of a proliferative disease, such as cancer.

[0399] 57. A kit comprising: a first container comprising NUC-7738, such as NUC-7738, with a pharmaceutically acceptable adjuvant, diluent or carrier; a second container comprising an immune checkpoint inhibitor, such as an immune checkpoint inhibitor, with a pharmaceutically acceptable adjuvant, diluent or carrier; and container means for containing the first and second containers.

[0400] 58. A kit according to specific example 57, wherein the immune checkpoint inhibitor is selected from the group consisting of BMS-986016 / relatlimab, TSR-033, REGN3767, MGD013 (bispecific DART that binds PD-1 and LAG-3), GSK2831781, LAG525, MDX-010 / ipilimumab, AGEN1884, and CP-675,206 / tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, MBG453, TSR-022, LY3321367, tirigolumab (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.

[0401] 59. An immune checkpoint inhibitor for use in the treatment of a proliferative disorder, such as cancer, wherein the immune checkpoint inhibitor is for use in combination with NUC-7738, and optionally, the immune checkpoint inhibitor and NUC-7738 are administered simultaneously or sequentially.

[0402] 60. An immune checkpoint inhibitor for use according to specific example 59, wherein the immune checkpoint inhibitor is selected from the group consisting of BMS-986016 / relatlimab, TSR-033, REGN3767, MGD013 (bispecific DART that binds PD-1 and LAG-3), GSK2831781, LAG525, MDX-010 / ipilimumab, AGEN1884, and CP-675,206 / tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, MBG453, TSR-022, LY3321367, tirigolumab (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.

[0403] 61. A method for determining whether a patient will benefit from treatment with NUC-7738, comprising measuring the level of soluble PD-L1 and / or exosomal PD-L1 and / or OX40L in a biological sample from the patient, and wherein if, compared to a wild-type or standard level, the level of soluble PD-L1 and / or exosomal PD-L1 in the biological sample is increased and / or the level of OX40L in the biological sample is reduced, the patient will benefit from treatment with NUC-7738.

[0404] 62. The method according to specific example 61, wherein the biological sample is a blood sample or a fraction thereof (e.g., serum or plasma).

[0405] 63. The method according to specific example 61 or 62, wherein the patient has 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.

[0406] 64. The method according to any one of specific examples 61 to 63, wherein the patient is resistant to or has developed resistance to an immune checkpoint inhibitor.

[0407] 65. NUC-7738 for use in the treatment of cancer in combination with an immunotherapeutic agent, e.g., an immune checkpoint inhibitor.

[0408] 66. NUC-7738 for use according to specific example 65, wherein the cancer is a solid tumor.

[0409] 67. NUC-7738 for use according to specific example 65 or 66, wherein the cancer is melanoma, e.g., cutaneous melanoma.

[0410] 68. NUC-7738 for use according to any one of specific examples 65 to 67, wherein the cancer is a cancer that is typically treated with an immune checkpoint inhibitor, e.g., pembrolizumab.

[0411] 69. NUC-7738 for use according to any of specific examples 65 to 68, wherein the cancer comprises cancer cells that express high levels of soluble PD-L1 protein and / or high levels of exosomal PD-L1 protein and / or low levels of OX40L protein.

[0412] 70. NUC-7738 for use according to any of specific examples 65 to 69, wherein the treatment is of a cancer patient and a patient who has previously received one or more cancer treatments and optionally whose treatment has been discontinued.

[0413] 71. NUC-7738 for use according to specific example 70, wherein the treatment has been discontinued due to toxicity, recurrence, or resistance of the cancer to a previous treatment.

[0414] 72. The immunotherapeutic agent is pembrolizumab, and NUC-7738 is administered optionally at 1125 mg / m 2 on days 1, 8, and 15 of a 21-day cycle, and pembrolizumab is administered at a dose of 200 mg on day 1 of a 21-day cycle. NUC-7738 for use according to any of specific examples 65 to 71.

[0415] 73. The immunotherapeutic agent is selected from pembrolizumab, semiprimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab. NUC-7738 for use according to any of specific examples 65 to 72.

[0416] 74. The immunotherapeutic agent is pembrolizumab. NUC-7738 for use according to any of specific examples 65 to 73.

[0417] 75. NUC-7738 and the immunotherapeutic agent are administered in a 21-day or 42-day treatment cycle. NUC-7738 for use according to any of specific examples 65 to 74.

[0418] 76. NUC-7738 at a weekly dose of about 300 to 1600 mg / m 2 for example, 500 to 1150 mg / m2 weekly dose of, or 900 - 1350 mg / m 2 administered at a weekly dose of, NUC-7738 for use according to any of Specific Examples 65 - 75.

[0419] 77. NUC-7738 is administered at a weekly dose selected from 900 mg / m 2 , 1125 mg / m 2 , and 1350 mg / m 2 for use according to Specific Example 76. NUC-7738 for use according to Specific Example 76.

[0420] 78. Pembrolizumab is administered once every 3 weeks (Q3W) at a dose of 200 mg, NUC-7738 for use according to any of Specific Examples 65 - 77.

[0421] 79. The immunotherapeutic agent is pembrolizumab, and NUC-7738 is optionally administered at 1125 mg / m 2 on the 1st, 8th, and 15th days of a 21-day cycle, and pembrolizumab is administered at a dose of 200 mg on the 1st day of a 21-day cycle, NUC-7738 for use according to any of Specific Examples 65 - 78.

[0422] 80. Pembrolizumab is administered once every 6 weeks (Q6W) at a dose of 400 mg, NUC-7738 for use according to any of Specific Examples 65 - 77.

[0423] 81. The immunotherapeutic agent is pembrolizumab, and NUC-7738 is optionally administered at 1125 mg / m 2 on the 1st, 8th, 15th, 22nd, 29th, and 35th days of a 42-day cycle, and pembrolizumab is administered at a dose of 400 mg on the 1st day of a 42-day cycle, NUC-7738 for use according to Specific Example 80.

[0424] 82. The treatment is for cancer patients and patients who have previously received cancer treatment with an immunotherapeutic agent and, optionally, whose treatment has been discontinued, NUC-7738 for use according to any of Specific Examples 65 - 81.

[0425] NUC-7738 for use according to Specific Example 82, wherein the prior treatment is administration of an immune checkpoint inhibitor and the treatment has been discontinued due to toxicity, recurrence, or the cancer has become resistant to the prior treatment.

[0426] Next, the present invention will be described in more detail with reference to the following examples. [Examples] Two sets of experiments are described in Examples 1 to 4. First, serum samples were collected from patients before and after treatment with NUC-7738 and then prepared for analysis as described below. Second, A375 melanoma or A549 lung cancer cell lines were cultured and then exposed to NUC-7738 in vitro at the doses and frequencies described below. Thereafter, proteins and RNAs were extracted and analyzed.

[0427] Method: Patients were treated with NUC-7738 administered on Days 1 and 8 of a 14-day cycle. Serum samples from the patients were collected before dosing, 24 hours after NUC-7738 dosing, and 24 hours after Cycle 2 (i.e., the second time the patient received treatment). For the preparation of extracellular vesicles (EVs, exosomes), cells were cultured in a T175 Nunc™ EasYFlask™ cell culture flask (ThermoFisher Cat #159910) at a cell number of approximately 50×10 6 and grown in DMEM high glucose GlutaMAX™ Supplement (ThermoFisher Cat #10566016) supplemented with 5% exosome-free FBS and 1% Penicillin-Streptomycin (ThermoFisher Cat #15070063). For sPDL1 analysis, a similar method was attempted. Cells were cultured in a Corning® 100 mm TC-treated culture dish with DMEM high glucose GlutaMAX™ Supplement (ThermoFisher Cat #10566016) supplemented with 10% FBS and 1% Penicillin-Streptomycin (ThermoFisher Cat #15070063) at a cell number of approximately 2×10 6 cells. The A375 melanoma cell line and A549 lung cancer cell line were treated with solvent-control DMSO or NUC-7738 (10 μM) for 72 hours, and then the culture supernatants were collected for soluble PD-L1 analysis or EV isolation. In addition, adherent cells were collected for RNA and protein isolation.

[0428] "Preparation of Samples for Soluble PD-L1 Assay" Patient serum was centrifuged at 3000×g for 15 minutes to remove cell debris. The supernatant was aliquoted with a 250 μl pipette and stored in an -80 °C freezer. In vitro soluble PD-L1 samples were prepared by collecting the culture supernatant and removing cells by centrifugation at 3000 x g for 15 minutes at the end of a predetermined time. Furthermore, the medium was concentrated using a Vivaspin protein concentration spin column with a 3 kDa molecular weight cut-off (Cytiva Cat. #28932358). For optimization, a concentration factor of 8× was achieved by centrifugation at 4000×g for 60 minutes. The concentrated samples were aliquoted and stored at -80 °C.

[0429] "Isolation of Exosomes from In Vitro and Serum Sample Types" The patient's serum samples were centrifuged at 3000×g for 15 minutes to remove cell debris. Subsequently, 250 μl of the supernatant was aliquoted using a measuring instrument and stored in an -80°C freezer. For exosome isolation, the ExoQuick® ULTRA EV Isolation Kit for plasma and serum (System Biosciences Cat # EQULTRA-20A-1) was used according to the manufacturer's protocol. Briefly, 250 μl of the cell debris-free serum sample was used, 67 μl of ExoQuick Solution was added, and then it was incubated at 4°C for 30 minutes. Thereafter, the mixture of the serum sample and ExoQuick Solution was centrifuged at 3000×g for 10 minutes. Then, the entire volume of the supernatant was carefully aspirated without disturbing the pellet to ensure that no traces of residue 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 in and out until no aggregated clumps of the pellet were visible. 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 1000×g for 30 seconds. For the protein concentration, the eluted sample was quantified using the Qubit™ Protein and Protein Broad Range (BR) Assay Kit (Thermofisher Cat # A50668). Then, the sample was aliquoted according to the amount required for downstream applications and stored at -80°C. Isolation of exosomes from in vitro samples was performed using 5 separate T175 Nunc® EasYFlask® cell culture flasks (ThermoFisher Cat #159910) (cells approximately 50×10 6Including growing cells in (accommodating a number of). Cells were grown in DMEM high glucose GlutaMAX™ Supplement (ThermoFisher Cat #10566016) supplemented with exosome-free FBS. Each T175 flask contained 40 ml of medium, which was pooled for each treatment (total volume 200 ml), and the supernatant of the medium was centrifuged at 4000×g for 15 minutes to remove cell debris. Further, the medium was concentrated to 5 l using a 100 kDa cut-off Centricon Plus-70 centrifugal filter (Merck, Cat #UFC710008). Then, exosomes were isolated using the ExoQuick® ULTRA EV Isolation Kit for tissue culture medium (System Biosciences Cat # EQULTRA-20TC-1) according to the manufacturer's protocol. The isolation protocol for ExoQuick Ultra exosomes for tissue culture was the same as that for serum. 1 ml of ExoQuick Solution was added to 5 ml of the medium-concentrated sample, followed by overnight incubation.

[0430] "Quantification of Soluble PD-L1 Using Sandwich ELISA" For the quantitative measurement of soluble PD-L1 in patient and in vitro samples, the Quantikine® ELISA Human / Cynomolgus PD-L1 / B7-H1 Immunoassay (R&D Systems Cat. #DB7H10) was used according to the manufacturer's recommendations. Samples were prepared as described above: 100 μl of patient serum sample and 100 μl of concentrated medium sample from in vitro were used per well. The serum samples were repeated twice, and for the in vitro samples, the number of samples was increased to three and repeated and repeated twice. Briefly, the protocol involves the preparation of a standard curve from human / cynomolgus PD-L1 / B7-H1 protein standards in the range of 0 - 1600 pg / ml. 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 standards, controls, or samples. Samples were incubated at room temperature for 2 hours. At the end of incubation, each well was washed 4 times with the provided wash buffer. Samples were further incubated at room temperature for 2 hours with 200 μl of human / cynomolgus B7-H1 on an orbital microplate shaker set at 500 rpm. The washing step was repeated, and then 200 μl of substrate solution was added to each well. After incubating for 30 minutes in the dark, 50 μl of stop solution was added to each well. To measure the optical density, each well was read using a microplate reader set at 450 nm and 540 nm for wavelength correction. Analysis of the results includes subtracting the 540 nm reading from the 450 nm reading and subtracting the blank, dilution only, and sample readings. The standard curve was generated using the four-parameter logistic (4-PL) curve fitting method. Then, the standard curve was used to interpolate the concentration of PD-L1 in the samples. For in vitro samples, an 8× concentration factor was considered. Therefore, the final concentration was divided by 8.

[0431] "Measurement of PD-L1 Protein Expression in Isolated Exosomes Using Capillary-Based Immunoprobing" The expression of PD-L1 in exosomes isolated from serum samples was analyzed using the automated Western blotting JESS system (BioTechne, ProteinSimples). A 12–230 kDa separation module of the 25 capillary kit (ProteinSimple, Cat #SM-W004) was used as per the manufacturer's protocol. Briefly, samples were prepared at appropriate concentrations using sample dilution buffer containing kit master-mix and DT, and then the samples were denatured at 95 °C for 5 minutes. All samples, primary antibodies, and secondary antibodies were loaded onto the provided plates. Anti-CD81 antibody [M38] (Abcam, Cat #ab79559) diluted 1:100 and PD-L1 (E1L3N® XP rabbit mAb (Cell Signalling, Cat #13684) diluted 1:50 were used. The secondary antibodies used were based on anti-mouse chemiluminescence (ProteinSimple, Cat #DM002) and anti-rabbit near-infrared (NIR) (ProteinSimple, Cat #DM007) detection modules and were used at the recommended dilutions. The prepared plates and capillaries were then loaded onto JESS and the samples were run in the initial settings. Expression was measured by the area under the curve using Compass Software v 6.1 (ProteinSimple).

[0432] "In Vitro Analysis of Transcripts of Soluble PD-L1 Using RT-qPCR" To measure the RNA expression of soluble transcripts, cells were lysed and RNA was isolated using the RNAeasy kit (Qiagen, Cat #74136). Subsequently, reverse transcription of RNA into cDNA was performed using the QuantiNova Reverse Transcription kit (Qiagen, Cat #205413). Primers for each gene were designed using PrimerQuest™ (Integrated DNA Technologies) and ordered from IDT at a concentration of 100 μM. Soluble transcript primers were designed to create an amplicon of approximately 100 bp size that crosses the exon 4 and intron 4 junction. The primer sequences are shown in the table below (Table 3).

[0433]

Table 3

[0434] Result In cell line studies, within 6 hours of treatment in cells maintained at an average concentration of 80 picomoles / 10 6 (cells) (maintained for at least 24 hours and reduced by approximately 50% at 42 hours), NUC-7738 was converted to the active anti-cancer metabolite 3'-dATP. NUC-7738 decreased mRNA by 40% or less and, in addition, decreased the expression of sPD-L1 in the culture supernatant by 3-fold or less over time. NUC-7738 also decreased ExoPD-L1 by 50%. NUC-7738 did not change surface PD-L1 expression. Preliminary studies in plasma collected from 4 patients treated with NUC-7738 showed a decrease in Exo-PD-L1 of ≤50% compared to pre-treatment levels.

Example

[0435] Figure 1 shows a reduction in the level of soluble PD-L1 in both lung cancer cell lines and melanoma cell lines, which supports that NUC-7738 reduces the amount of soluble PD-L1 present in the tumor microenvironment. A reduction in the sPDL1 level is known to enhance anti-PD1 and other immune checkpoint therapies.

Example

[0436] Figure 2 demonstrates a reduction in the amount of transcripts of soluble PD-L1 transcript isoforms in lung cancer cell lines and melanoma cell lines, which supports that the agent acts on the cells required to produce soluble PD-L1.

Example

[0437] Table 4 below and Figure 3 show that NUC-7738 reduces the exosomal PD-L1 level in serum.

[0438]

Table 4

Example

[0439] Figure 4 demonstrates that NUC-7738 increases the level of mRNA encoding OX40L in both melanoma cell lines and lung cancer cell lines. Conclusion NUC-7738 reduces secreted PD-L1 but is ineffective on the level of cell surface proteins. In vitro data was confirmed in a clinical setting, whereby ExoPD-L1 was reduced in plasma samples from patients treated with NUC-7738. These findings indicate that NUC-7738 may act as an immune promoter and restore the function of T cells that promote anti-cancer immune responses. NUC-7738 in combination with PD-(L)1 pathway inhibitors provides a promising treatment option even in patients who have experienced treatment resistance to immune checkpoint inhibitor therapy or have other reasons (e.g., resistance or toxicity) for the failure of previous immune checkpoint inhibitor therapy.

Example

[0440] Clinical study on the combination of NUC-7738 and pembrolizumab in patients with cutaneous melanoma To assay the safety, pharmacokinetics, and clinical activity of NUC-7738 administered on Days 1, 8, and 15 of a 21-day cycle in combination with pembrolizumab administered on Day 1 of a 21-day cycle to subjects with cutaneous melanoma, a Phase II trial, NuTide:701 expansion, is conducted. Patients are eligible to receive multiple cycles until disease progression or unacceptable toxicity. Assessments of safety, tolerance, and efficacy are performed. The levels of extracellular PD-L1 are investigated before and after treatment. Initially, 6 to 12 cutaneous melanoma patients are treated. NUC-7738 and pembrolizumab are administered by intravenous infusion.

[0441] "Infusion of NUC-7738" From the stock vial of NUC-7738, add the desired volume of NUC-7738 to a 500 ml standard saline infusion bag. To produce a homogeneous and clear solution, gently invert the bag or use a similar method. Continue mixing until the solution is well mixed. Infuse the contents of the infusion bag containing the saline-based NUC-7738 formulation slowly into the patient over approximately 120 minutes. The infusion is administered using standard means, e.g., via a central venous access device or via a cannula.

[0442] "Infusion of Pembrolizumab" Withdraw the desired volume from the vial of pembrolizumab (KEYTRUDA) and transfer it to an intravenous (IV) bag containing 0.9% Sodium Chloride Injection USP or 5% Dextrose Injection USP. Gently invert to mix the dilution solution. Do not shake. The final concentration of the dilution solution is 1 - 10 mg / ml. Administer the dilution solution intravenously over approximately 30 minutes through an intravenous line containing a sterile, non - pyrogenic, low - protein - binding 0.2 - 5 micron in - line or add - on filter (generally performed with an infusion pump). On Day 1, administer pembrolizumab directly before NUC - 7738 (however, the order is not necessarily a problem).

[0443] The subjects to be treated will be histologically confirmed to have a disease with a diagnosis of cutaneous melanoma that is measurable according to the RECIST guideline v1.1 criteria. Most subjects will be progressing on ≤ 2 prior treatments for advanced / metastatic cutaneous melanoma (including one prior treatment with an immunotherapy regimen (monotherapy or combination with a chemotherapeutic agent)). Subjects who are not progressing but for whom the addition of NUC - 7738 to standard pembrolizumab monotherapy is appropriate are also eligible.

[0444] In view of the ability of NUC - 7738 to reduce the amount of soluble or exosomal PD - L1, the inventors expect that the combination of NUC - 7738 and pembrolizumab will be more effective than pembrolizumab administered as monotherapy in the treatment of cutaneous melanoma.

Claims

Claim 1 NUC-7738 for use in the treatment of a patient with a proliferative disorder by enhancing the patient's immune response against the proliferative disorder. Claim 2 NUC-7738 for use according to claim 1, wherein NUC-7738 reduces the amount of extracellular PD-L1 protein produced by proliferative disorder cells and / or increases the amount of OX40-L protein. Claim 3 NUC-7738 for use according to claim 2, wherein the extracellular PD-L1 protein is soluble PD-L1 and / or exosomal PD-L1. Claim 4 NUC-7738 for use according to any one of claims 1 to 3, wherein the treatment of the proliferative disorder is effected through adaptive immunity (e.g., cellular immunity and / or humoral immunity). Claim 5 NUC-7738 for use according to claim 1, wherein the proliferative disorder is cancer. Claim 6 NUC-7738 for use as an immune promoter in the treatment of cancer. Claim 7 NUC-7738 for use 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. Claim 8 NUC-7738 for use according to claim 1 or 6, wherein the treatment comprises administration of NUC-7738 in combination with an immunotherapeutic agent such as an immune checkpoint inhibitor, antibody therapy, adoptive cell therapy (such as CAR-T therapy), or cancer vaccine. Claim 9 NUC-7738 for use 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. Claim 10 NUC-7738 for use according to claim 8, wherein the immune checkpoint inhibitor is selected from the group consisting of BMS-986016 / relatlimab, TSR-033, REGN3767, MGD013 (bispecific DART that binds PD-1 and LAG-3), GSK2831781, LAG525, MDX-010 / ipilimumab, AGEN1884, and CP-675,206 / tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, semipilimumab, dostarlimab, MBG453, TSR-022, LY3321367, tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106, and cudarlimab, or a pharmaceutically acceptable salt or solvate thereof.

11. NUC-7738 for use according to claim 1 or 6, wherein the patient has already received treatment with an immune checkpoint inhibitor, and optionally, the treatment has been discontinued due to toxicity, recurrence, or the cancer becoming resistant to the previous treatment.

12. NUC-7738 for use according to claim 1 or 6, wherein treatment with NUC-7738 reduces the level of soluble or exosomal PD-L1 by at least about 25% compared to the level obtained without NUC-7738 treatment.

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

14. A method of enhancing an immune response against cancer, 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 immunotherapeutic agent.

15. The method according to claim 13 or 14, 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.

16. The method according to claim 13 or 14, 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.

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

18. The method according to claim 14, wherein the immunotherapeutic agent is an immune checkpoint inhibitor, antibody therapy, adoptive cell therapy, or cancer vaccine.

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

21. The method according to claim 13 or 14, wherein the patient has already received treatment with an immune checkpoint inhibitor, and optionally, the treatment has been discontinued due to toxicity, recurrence, or the cancer becoming resistant to the previous treatment.

22. A first container containing NUC-7738, for example, NUC-7738 with a pharmaceutically acceptable adjuvant, diluent, or carrier; and A second container containing an immunotherapeutic agent, for example, an immune checkpoint inhibitor with a pharmaceutically acceptable adjuvant, diluent, or carrier; and Container means for containing the first and second containers A kit comprising.

23. The kit according to claim 22, wherein the immune checkpoint inhibitor is selected from the group consisting of BMS-986016 / relatlimab, TSR-033, REGN3767, MGD013 (bispecific DART that binds PD-1 and LAG-3), GSK2831781, LAG525, MDX-010 / ipilimumab, AGEN1884, and CP-675,206 / tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, semaprilumab, dostarlimab, MBG453, TSR-022, LY3321367, tiragolumab (MTIG7192A; RG6058), AB154, MK-7684, BMS-986207, ASP8374, MEDI6469, BMS-986178, SL-279252, INBRX-106, and cudarlimab, 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 measuring the level of soluble PD-L1 and / or exosomal PD-L1 and / or OX40-L in a biological sample from the patient and comparing it to a reference value, and if the level of soluble PD-L1 and / or exosomal PD-L1 in the biological sample is elevated and / or the level of OX40-L protein in the biological sample is decreased, determining that the patient will benefit from treatment with NUC-7738.

25. The method according to claim 24, wherein the biological sample is a blood sample or a fraction thereof (e.g., serum or plasma).

26. The method according to claim 24 or 25, wherein the patient has 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 the treatment of a proliferative disease such as cancer, wherein the immune checkpoint inhibitor is for administration together with NUC-7738, and optionally, the immune checkpoint inhibitor and NUC-3378 are administered simultaneously or sequentially.

28. The immune checkpoint inhibitor according to claim 27, which is selected from the group consisting of BMS-986016 / relatlimab, TSR-033, REGN3767, MGD013 (bispecific DART that binds PD-1 and LAG-3), GSK2831781, LAG525, MDX-010 / ipilimumab, AGEN1884, and CP-675,206 / tremelimumab, 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.

29. NUC-7738 for use in the treatment of cancer in combination with an immunotherapeutic agent such as an immune checkpoint inhibitor.

30. NUC-7738 for use according to claim 29, wherein the immunotherapeutic agent is selected from pembrolizumab, semiprimab, dostarlimab, nivolumab, durvalumab, ipilimumab, atezolizumab, and avelumab.

31. NUC-7738 for use according to claim 29 or 30, wherein the treatment is for cancer patients and patients who have already been treated with an immune checkpoint inhibitor and optionally the treatment has been discontinued.

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