Nur77 agonism for natural killer cell-mediated immunity in diseases

Abstract

There is provided a Nur77 agonist, or analogue or derivative thereof, for use in activating a natural killer cell. Also disclosed is a Nur77 agonist, or analogue or derivative thereof, for use in treating a disease; a method of treating or preventing a disease in a subject in need thereof; and use of Nur77 agonist or analogue or derivative thereof in the manufacture of a medicament for treating or preventing a disease.

Description

[0001] Nur77 agonism for natural killer cell-mediated immunity in diseases

[0002] TECHNICAL FIELD

[0003] The present disclosure relates broadly to an agonist and its use in activating an immune cell.

[0004] BACKGROUND

[0005] A key feature of NK cells is their ability to recognize and eliminate a wide range of cells in distress, particularly tumor cells or cells infected with viruses. NK cells exert direct effector functions against cellular targets and participate in the generation, and maintenance of a multicellular immune response. The ability of NK cells to identify and target stressed cells offers expansive therapeutic possibilities in areas such as cancer, infectious diseases, autoimmune disease and inflammatory diseases.

[0006] In cancer patients, NK cell therapy / immunotherapy aims to boost levels of enhanced NK cells. Immunocompromised patients such as patients suffering from cancer or hematopoietic stem cell transplant (HSCT) recipients have a significantly increased risk of infectious complications due to viral, bacterial, or fungal pathogens. It is believed that NK cells are also involved in the host response against such infectious pathogens. In autoimmune disease and inflammatory disease, NK cells have been used to regulate overactive immune responses.

[0007] However, activity of NK cells may be suppressed under conditions of chronic inflammation such as in autoimmune disease, inflammatory disease and / or cancer. Furthermore, despite rapid advancements in emerging cancer immunotherapeutics, the success of adoptive NK cell therapy remains largely limited to the treatment of hematological cancers. This is predominantly due to cellular exhaustion arising from immune suppression in the repressive tumor microenvironment.

[0008] Hence, the ability to stimulate or reinvigorate NK cells for anti-tumor activity such as in solid tumor is an urgent therapeutic need. Till date, it remains unclear how NK cell functionality could be boosted for anti-tumor activity against cancers such as solid tumors. Therefore, there is a need to provide an alternative product to activate NK cell. SUMMARY

[0009] In one aspect, there is provided a Nur77 (NR4A1) agonist, or analogue or derivative thereof, for use in activating a natural killer (NK) cell.

[0010] In another aspect, there is provided a Nur77 (NR4A1) agonist, or analogue or derivative thereof, for use in treating a disease, wherein the agonist activates a NK cell.

[0011] In yet another aspect, there is provided a method of treating or preventing a disease in a subject in need thereof, the method comprising administering a Nur77 (NR4A1) agonist, or analogue or derivative thereof, to the subject, wherein the agonist activates a NK cell.

[0012] In yet another aspect, there is provided use of a Nur77 (NR4A1) agonist or analogue or derivative thereof in the manufacture of a medicament for treating or preventing a disease.

[0013] In some examples, the Nur77 agonist confers NK cell resistance to lipid-mediated immune suppression.

[0014] In some examples, the Nur77 agonist, or analogue or derivative thereof, improves NK cell effector functions.

[0015] In some examples, the Nur77 agonist, or analogue or derivative thereof, reverses lipid-mediated immune suppression of NK cell killing capacity and / or perforin production.

[0016] In some examples, the Nur77 agonist, or analogue or derivative thereof, activates NK cell killing capacity that is independent of T cell responses.

[0017] In some examples, the NK cell is a suppressed NK cell.

[0018] In some examples, the Nur77 agonist, or analogue or derivative thereof, is Cytosporone B (CsnB) or derivatives thereof.

[0019] In some examples, the Nur77 agonist, or analogue or derivative thereof, reduces NK cells’ ability to uptake lipid.

[0020] In some examples, the Nur77 agonist, or analogue or derivative thereof, down- regulates the surface expression of CD36.

[0021] In some examples, the Nur77 agonist, or analogue or derivative thereof, reverses the oxidized low-density lipoprotein (oxLDL)-mediated suppression of perforin.

[0022] In some examples, the Nur77 agonist, or analogue or derivative thereof, represses lipid uptake and fatty acid oxidation in NK cells.

[0023] In some examples, the NK cell is from a subject suffering from a disease.

[0024] In some examples, the NK cell is from a subject suffering from cancer. DEFINITIONS

[0025] The term “agonist” as used herein refers to a substance / compound (such as a drug, a hormone, a neurotransmitter) that binds and activates a specific receptor to produce a biological response. An agonist binds to a receptor inside a cell or on its surface and causes the same action as the substance that normally binds to the receptor.

[0026] The term “agonism” as used herein refers to the process or mechanism by which an agonist binds to a receptor and activates it to produce a biological response.

[0027] The term “analogue” as used herein refers to a substance / compound that is structurally similar to another substance I compound but may have slight modifications in its chemical structure or differ from it in respect to a certain component. It can differ in one or more atoms, functional groups, or substructures, which are replaced with other atoms, groups, or substructures.

[0028] The term “derivative” as used herein refers to a substance / compound that is derived from a parent substance / compound either directly or by modification, partial substitution, replacing, adding or removing functional groups. A derivative is directly obtained from a chemical modification of the parent substance / compound.

[0029] The term “activating / activation” as used herein refers to the process by which an immune cell (i.e., NK cell) is stimulated by signals that switch it from a resting / inactive state to an active functional state. The term “activating / activation” can be used interchangeably with “invigorate” or “stimulate”. NK cell activation is the process by which NK cells transition from a resting state into an effector state. Once activated NK cells can kill infected or cancerous cells and secrete cytokines to regulate immune responses.

[0030] The term “killing capacity I NK cell killing capacity” as used herein refers to the NK cell effector function, which includes NK cells’ ability to recognize and destroy cells (such as virus-infected cells, tumor cells, stressed cells) without prior sensitization. The NK cell effector function or mechanisms of NK cell killing includes i) cytotoxic granule release where perforin and granzymes are released to trigger apoptosis in the target cell, II) activating the death receptor pathway where NK cells which express Fas ligand or TRAIL bind to death receptors on target cells to activate caspase cascades to trigger apoptosis and Hi) antibody-dependent cellular cytoxicity (ADCC) where NK cells express CD16 to recognize antibodies bound to infected / tumor cells to trigger degranulation and apoptosis of antibody-coated cells.

[0031] The term “suppression / immune suppression” as used herein refers to the act or process of suppressing immune function. The suppression of immune function refers to the reduction or decreased or inhibition of the immune system’s ability to respond to threats such as pathogens, cancer cells, or foreign antigens. Examples of suppressed immune function may include but is not limited to reduced immune cell activation, decreased cytokine production, lower antibody or cytotoxic responses, impaired ability to fight infections or recognize abnormal cells.

[0032] In some examples, suppression / immune suppression may be through external factors or natural exhaustion.

[0033] In some examples, suppression / immune suppression through external factors may include lipid-mediated immune suppression, receptor inhibition, immunosuppressive or inhibitory cytokines, metabolic suppression, and the like.

[0034] In some examples, suppression / immune suppression through natural exhaustion may include NK cells that were chronically exposed to persistent antigen or inflammatory stimulation. Natural exhaustion represents a dysfunctional state often maintained even when external factors are withdrawn.

[0035] The term “immunosuppression / immunosuppressive condition” refers to the state or condition in which the immune system’s activity is reduced or inhibited. It is the reduction of the immune system’s ability to mount a normal immune response, either through drugs, disease or other factors.

[0036] The term “suppression of natural killer (NK) cell” as used herein refers to the process or state in which NK cells have reduced activity, function or responsiveness.

[0037] The term “suppressed natural killer (NK) cell” as used herein refers to a NK cell whose activity, cytotoxic function, or cytokine production is reduced. A suppressed NK cell may have reduced cytotoxicity, decreased cytokine production or impaired proliferation or activation. A suppressed NK cell leads to a weakened innate immune response.

[0038] The term “reverse / reverse suppression / reinvigorate suppressed immune cell” as used herein refers to the process of restoring or enhancing the function of immune cells that were previously suppressed.

[0039] The term “downregulation I downregulates expression” as used herein refers to decrease or reduction in the level of gene or protein expression in a cell. Downregulation happens when the process of gene or protein expression is slowed, suppressed, or inhibited, leading to fewer RNA transcripts or proteins being produced.

[0040] The term “desensitize” as used herein refers to the process by which immune cell (such as NK cell) become less affected by signals or conditions that normally suppress their activity, allowing them to maintain or restore their function despite an immunosuppressive environment. The term “cellular exhaustion / immune exhaustion” as used herein refers to a state in which immune cells (i.e., NK cells) become dysfunctional after prolonged or chronic exposure to antigen or stimulation. In some examples, a dysfunctional immune system may exhibit characteristics including, but not limited to, reduced or suppressed function, reduced effector function, varying degree of reduced motility, and the like.

[0041] The terms “treating", "treat" and “therapy”, and synonyms thereof refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a medical condition, which includes but is not limited to diseases, symptoms and disorders. A medical condition also includes a body’s response to a disease or disorder, e.g. inflammation, cancer, or autoimmune disease. Those in need of such treatment include those already with a medical condition as well as those prone to getting the medical condition or those in whom a medical condition is to be prevented.

[0042] The term “immunotherapy” as used herein refers to therapy / treatment of disease that may include systemic or selective activating or suppressing the immune system (as a whole or parts thereof). Immunotherapies designed to amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies. For example, cancer immunotherapy may stimulate the immune system (whole or part thereof) to destroy tumors.

[0043] The term “subject” as used herein includes patients and non-patients. The term “patient” refers to individuals suffering or are likely to suffer from a medical condition, while “non-patients” refer to individuals not suffering and are likely to not suffer from the medical condition. “Non-patients” include healthy individuals, non-diseased individuals and / or an individual free from the medical condition. The term “subject’ includes humans and animals. Animals may include, but are not limited to, mammals (for example nonhuman primates, canine, murine, and the like), and the like. “Murine” refers to any mammal from the family Muridae and I or Leporidae, such as mouse, rat, rabbit, and the like.

[0044] As used herein, the term “biological sample” or “sample” is intended to include any sampling of cells, cell extracts, tissues, organs, or bodily fluids isolated from a subject.

[0045] The term “preventing” and / or “reducing the severity of symptoms” as used herein refers to process of delaying the onset, reducing the severity of symptoms, reducing and / or preventing weight loss, preventing death, inhibiting deterioration, inhibiting further deterioration, and / or ameliorating at least one sign or symptom of a disease.

[0046] The term “ex vivo” as used herein refers to experiments or procedures performed on biological material (such as cells, tissues, or organs) taken from a living organism, outside the organism under controlled laboratory conditions.

[0047] The term “in vivo” as used herein refers to experiments or processes performed within a living organism.

[0048] The term “in vitro” as used herein refers to experiment or processes performed or taking place in a test tube, culture dish or elsewhere outside of a living organism.

[0049] The term “adoptive cell therapy” as used herein refers to a therapy that involves activating patients’ own immune cells ex vivo and then transferring them back to the patients to recognize and eliminate aberrant cells (e.g., cancer cells).

[0050] The term "associated with", used herein when referring to two elements refers to a broad relationship between the two elements. In genomics, “associated” typically refers to entities that are found in a correlative relationship (e.g., when A is upregulated I downregulated, B is also upregulated / downregulated). “Associated” is used when one cannot establish the causal or functional relationship based on correlation.

[0051] The term "and / or", e.g., "X and / or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

[0052] Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements / components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of + / - 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1% of the disclosed value. The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof, is meant to encompass the items listed thereafter and additional items. Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed. Ordinal terms are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term), to distinguish the claim elements. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0053] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0054] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0055] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0056] Exemplary embodiments of the present invention are provided in the following examples. While the exemplary embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, it should be understood that the present invention is not limited to these examples.

[0057] Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1% to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1% to 5% is intended to have specifically disclosed the ranges 1 .00% to 5.00% and also 1 .0% to 5.0% and all their intermediate values (such as 1 .01 %, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1%, 1.2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth / breadth of a range.

[0058] Additionally, when describing some embodiments, the disclosure may have disclosed a method and / or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and / or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

[0059] Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

[0060] DESCRIPTION OF EMBODIMENTS

[0061] The present disclosure discloses a composition, compound, or substance that activates NK cells for use in therapy or immunotherapy. In recent years, NK cell therapy has become an important modality in immune therapy.

[0062] For example, in the treatment of cancer, NK cells are used to target tumors resistant to existing therapy such as chemotherapy, radiation or CAR-T therapy. NK cells have been found to have the ability to selectively kill abnormal cell via activating / inhibitory receptor balance and advantageously sparing most normal cells whilst reduces systemic toxicity that may be found in cancer therapy. NK cell therapy has also been found to have the ability to detect and effective against minimal residual disease that may escape existing therapy.

[0063] In acute infections, NK cells have also been found to lower severe immune reactions compared to other therapies such as T cell therapy. Furthermore, in chronic immune conditions such as autoimmune and inflammatory diseases, NK cells suppress autoreactive immune cells.

[0064] Having said the above, studies in the art have shown that an immunosuppressive tumor environment disadvantageously reduces NK cell function and impairs NK cell cytotoxicity. At the same time, NK cell exhaustion may also occur in chronic infections via persistent antigen exposure and inhibitory cytokines. Therefore, the inventors of the present disclosure are set to provide an alternative composition, compound, or substance that is capable of improving NK cell activation.

[0065] Agonist to activate NK cell

[0066] It is known in the art that NK cells can be activated via multiple pathways, such as, but are not limited to, cytokines, co-culture with feeder cells, genetic engineering, and / or in vivo activation.

[0067] Cytokine stimulation is the most common method in the art of activating NK cells. However, high doses of cytokine stimulation can cause toxicity and induce apoptosis of NK cells. NK cells can also be expanded and activated by co-culture with feeder cells expressing membrane-bound cytokines. However, feeder cells may introduce variability or contamination risk.

[0068] Using genetic engineering, NK cells may be engineered to express chimeric antigen receptors targeting specific tumor antigens; or NK cells may be engineered to produce activating cytokines. However, genetic engineering requires complex manufacturing with safety concerns and high cost.

[0069] The present disclosure provides an alternative composition, compound, or substance to activate NK cells with an agonist.

[0070] In one aspect, there is provided an agonist, or analogue or derivative thereof, for use in activating a NK cell.

[0071] In some examples, there is provided an agonist, or analogue or derivative thereof, for use in activating n NK cell killing capacity.

[0072] In some examples, there is provided an agonist, or analogue or derivative thereof that targets a nuclear receptor / a transcription factor of a NK cell, for use in activating a NK cell / NK cell killing capacity.

[0073] Nur77 agonist to activate NK cell

[0074] In one aspect, there is provided a Nur77 (NR4A1) agonist, or analogue or derivative thereof, for use in activating a NK cell.

[0075] In some examples, there is provided a Nur77 (NR4A1) agonist, or analogue or derivative thereof, for use in activating a NK cell killing capacity.

[0076] In one aspect, there is provided a Nur77 (NR4A1) agonist, or analogue or derivative thereof, for use in activating a NK cell killing capacity, optionally wherein the activating NK cell killing capacity is performed ex vivo or in vivo.

[0077] Without wishing to be bound by theory, Nur77 (nuclear receptor 4A1 (NR4A1)) is a nuclear receptor and a transcription factor that plays a key role in regulating immune cell function, apoptosis, cancer, metabolism and inflammation. The present disclosure identifies Nur77, which is a transcriptional regulator of cellular metabolism and immune functions. The inventors of the present disclosure highlighted the potential of Nur77 as a target for therapeutic intervention to activate and reinvigorate natural killer cells for superior anti-tumor activity. The present disclosure provides a Nur77 agonist for natural killer cell-mediated anti-tumor immunity in a disease (such as cancer). Use of Nur77 agonist for treating / preventing a disease

[0078] In one aspect, there is provided a Nur77 (NR4A1) agonist, or analogue or derivative thereof, for use in treating a disease, wherein the agonist activates an NK cell killing capacity, optionally wherein the activating NK cell killing capacity is performed ex vivo or in vivo.

[0079] In some examples, the activating NK cell killing capacity is performed in vivo, ex vivo and / or in vitro.

[0080] In some examples, the Nur77 agonist, or analogue or derivative thereof activates NK cell killing capacity ex vivo prior to adoptive cell therapy. In some examples, NK cells are treated with Nur77 agonist, or analogue or derivative thereof ex vivo prior to adoptive cell therapy. In some examples, NK cells are treated with Nur77 agonist, or analogue or derivative thereof ex vivo prior to adoptive cell therapy, activating NK cell capacity ex vivo.

[0081] In one aspect, there is provided a method of treating or preventing a disease in a subject in need thereof, the method comprising administering a Nur77 (NR4A) agonist, or analogue or derivative thereof, to the subject, wherein the agonist activates a NK cell.

[0082] In one aspect, there is provided a method of treating or preventing a disease in a subject in need thereof, the method comprising administering a Nur77 (NR4A1) agonist, or analogue or derivative thereof, to the subject, wherein the agonist activates an NK cell killing capacity.

[0083] In some examples, the agonist or analogue or derivative thereof of the present disclosure may be administered orally, parenterally, and the like. In some examples, parenteral administration may include intravenous injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, and the like.

[0084] In one aspect, there is provided use of a Nur77 (NR4A1) agonist or analogue or derivative thereof in the manufacture of a medicament for treating or preventing a disease.

[0085] Obtaining NK cell

[0086] In some examples, the NK cell may be obtained from one or more biological samples / samples from one or more subjects. In some examples, the NK cell may be obtained from one or more biological samples / samples from a subject using isolation methods in the art. Isolation of NK cells may involve separating NK cells from peripheral blood, cord blood, or other tissues and the methods in the art that may be used to isolate NK cell may include, but is not limited to, density gradient centrifugation, fluorescence activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and the like.

[0087] In some examples, the NK cell may be obtained from one or more biological samples / samples from one or more human or animal subjects. In some examples, the NK cell is a human NK cell. In some examples, the NK cell may be obtained from the subject in need of treatment.

[0088] In some examples, the NK cell may be obtained from the peripheral blood (such as peripheral blood NK cell), the tumor microenvironment (such as tumor-infiltrating NK cell), bone marrow, and the like.

[0089] Cytosporone B

[0090] In some examples, the Nur77 agonist, or analogue or derivative thereof, is Cytosporone B (CsnB) or derivatives thereof.

[0091] In some examples, the Nur77 agonist, or analogue or derivative thereof, is Cytosporone B (CsnB) or analogue or derivatives thereof.

[0092] The inventors of the present disclosure further demonstrated that the use of Cytosporone B as a Nur77 agonist enhances NK cell-mediated tumor immunity.

[0093] In some examples, the Cytosporone B has a chemical structure:

[0094] CAS: 321661-62-5.

[0095] Disease

[0096] In some examples, the NK cell is from a subject suffering from a disease.

[0097] In some examples, the NK cell is from a subject suffering from a disease, optionally a proliferative disease. In some examples, the inflammatory disease may include, but is not limited to, arthritis, sarcoidosis, and the like.

[0098] In some examples, the disease may be a proliferative disease such as a tumor / cancer, an inflammatory disease, an infectious disease, an autoimmune disease, an autoimmune disease / disorder, and the like.

[0099] In some examples, the disease may be a tumor / cancer.

[0100] In some examples, the autoimmune disease may include, but is not limited to, systemic lupus erythematosus, adult-onset Still's disease, and the like.

[0101] In some examples, the infectious disease may include, but is not limited to, parasitic infections (such as toxoplasmosis, trypanosomiasis, leishmaniasis, malaria, and the like), viral infections (such as HIV infection, Hepatitis B infection, and the like).

[0102] In some examples, the NK cell is from a subject suffering from cancer.

[0103] In some examples, the cancer may be acute granulocytic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adenocarcinoma, adenosarcoma, adrenal cancer, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, b-cell lymphoma), bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumors, general, germ cell tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin's disease, Hodgkin's lymphoma, hypopharyngeal cancer, infiltrating ductal carcinoma. Infiltrating lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, invasive / infiltrating breast cancer, islet cell cancer, jaw cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastases, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, meddiobiastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymous, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed gliomas, mouth cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin lymphoma, nonHodgkin’s lymphoma, non-small cell lung cancer, oat cell cancer, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian primary peritoneal carcinoma, ovarian sex cord stromal tumor, Paget’s disease, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pine blastoma, pituitary gland cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, sarcoma, bone, sarcoma, soft tissue, sarcoma, uterine, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal column cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer, synovial sarcoma, t-cell lymphoma, testicular cancer, throat cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell cancer, transitional cell cancer, transitional cell cancer, triple-negative breast cancer, tubal cancer, tubular carcinoma, ureteral cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0104] In some examples, the cancer may be a lymphoma, a melanoma, a hepatocellular carcinoma, a liver cancer, and the like.

[0105] In some examples, the cancer may include a solid tumor. In some examples, the solid tumor may include but is not limited to a melanoma, a hepatocellular carcinoma, a liver cancer, and the like.

[0106] Here, the inventors of the present disclosure identified Nur77 as a target for therapeutic intervention to enhance NK cell-mediated immune response within solid tumors.

[0107] Direct activation of NK cell

[0108] In some examples, the agonist, or analogue or derivative thereof, activates NK cell killing capacity that is independent of T cell responses.

[0109] In some examples, the Nur77 agonist (such as CsnB), or analogue or derivative thereof, activates NK cell killing capacity that is independent of T cell responses. As shown in the experimental data of the present disclosure, CsnB elicited antitumor response in major histocompatibility complex class-1 molecules (MHCI)-deficient lymphoma tumor model (such as RMA-S model) and melanoma model expressing low / negative MHCI (such as B16F10) with the growth of tumors suppressed (FIG. 2E and 2F). RMA-S model and B16F10 expressing low / negative MHCI ImpairT cell recognition. The findings from the RMA-S model which is a gold standard for NK cell studies in vivo supports that the Nur77 agonist activates NK cells I NK cell killing capacity directly (i.e. , independent of T cells).

[0110] NK cell resistance to immune suppression

[0111] In some examples, the agonist, or analogue or derivative thereof, confers NK cell resistance to immune suppression.

[0112] In some examples, the Nur77 agonist, or analogue or derivative thereof, confers NK cell resistance to immune suppression.

[0113] In some examples, immune suppression may be due to events, such as, but are not limited to, immune exhaustion, lipid-mediated immune suppression, chronic infection, receptor inhibition, immunosuppressive cytokines / metabolic stress in a tumor environment, and the like.

[0114] In some examples, the Nur77 agonist, or analogue or derivative thereof, confers NK cell resistance to immune suppression and therefore enhances NK cell function. In some examples, the Nur77 agonist, or analogue or derivative thereof, confers NK cell resistance to lipid-mediated immune suppression. NK cell

[0115] In some examples, the NK cell is a suppressed / exhausted NK cell. In some examples, the NK cell is a suppressed NK cell.

[0116] In some examples, the NK cell may be suppressed by events, such as, but are not limited, to immune exhaustion, lipid-mediated immune suppression, chronic infection, receptor inhibition, immunosuppressive cytokines / metabolic stress in a tumor environment, and the like.

[0117] In some examples, NK cell / NK cell killing capacity is suppressed by lipid- mediated immune suppression.

[0118] In some examples, lipid-mediated immune suppression may occur due to an increase amount of lipids, such as, but are not limited to, saturated fatty acids (such as palmitate), oxidized lipids (such as oxidized low-density lipoprotein (LDL)), lipid metabolites (such as prostaglandin E2 (PGE2)), and the like.

[0119] Without wishing to be bound by theory, lipid-mediated immune suppression refers to the dampening of immune cell function e.g., in cancer, chronic infection or metabolic disease by lipids or lipid derivatives. The availability of lipids such as palmitate and alterations in lipid metabolism plays crucial roles in influencing the growth and progression of metastatic cancer cells. However, studies in the art have shown increased lipid accumulation to have negative effects on NK cell-mediated tumor recognition and removal.

[0120] Mechanism of agonist i) Reduced lipid uptake

[0121] In some examples, the agonist, or analogue or derivative thereof, reduces NK cells’ ability to uptake lipid.

[0122] In some examples, the Nur77 agonist (such as CsnB), or analogue or derivative thereof, reduces NK cells’ ability to uptake lipid.

[0123] In some examples, the agonist, or analogue or derivative thereof, reduces / inhibits / decreases NK cells’ ability to uptake lipid boron-dipyrromethene (BODIDY-C12).

[0124] In some examples, the Nur77 agonist, or analogue or derivative thereof, reduces / inhibits / decreases NK cells’ ability to uptake lipid boron-dipyrromethene (BODIDY- C12).

[0125] The reduction in NK cells’ ability to uptake lipid in Nur77 agonist treated cells (such as in CsnB treated cells) can be observed when compared to a control, for example, untreated cells (such as untreated NK cells). The reduction in NK cells’ ability can be observed when compared to a control, for example, untreated NK cells.

[0126] Without wishing to be bound by theory, accumulation of lipids was observed to impair natural killer cytotoxicity and tumor control. Surprisingly, the inventors confirmed the ability of the agonist of the present disclosure in reducing / targeting lipid accumulation in NK cells. This may provide future opportunities to improve prognosis of patients with cancer. ii) Downregulate CD36 surface expression

[0127] In some examples, the agonist, or analogue or derivative thereof, down-regulates the surface expression of CD36. In some examples, the Nur77 agonist (such as CsnB), or analogue or derivative thereof, down-regulates the surface expression of CD36.

[0128] In some examples, the downregulation can be observed in vitro.

[0129] Without wishing to be bound by theory, CD36 acts as a significant regulator of lipid homeostasis. The abnormal expression of CD36 facilitates lipid accumulation, inflammation, endothelial apoptosis, thrombosis etc. CD36, a lipid transporter, is highly expressed in certain types of cancer cells. Higher expressions of CD36 in tumor cells triggers lipid accumulation, promoting rapid tumor growth and initiating metastasis. Therefore, downregulating CD36 expression has broad clinical implications such as enhancing NK cell anti-tumor activity, reducing fatty acid uptake and improving insulin sensitivity in metabolic disease, reducing pathogen uptake in certain infectious diseases, etc.

[0130] Hi) Reverse lipid mediated suppression

[0131] In some examples, the agonist, or analogue or derivative thereof, reverses immune suppression.

[0132] In some examples, the Nur77 agonist (such as CsnB), or analogue or derivative thereof, reverses immune suppression.

[0133] In some examples, the agonist, or analogue or derivative thereof, reverses the lipid mediated suppression of NK cell effectors.

[0134] In some examples, the Nur77 agonist, or analogue or derivative thereof, reverses the lipid-mediated suppression of NK cell effectors.

[0135] In some examples, the agonist (such as CsnB), or analogue or derivative thereof, reverses the oxLDL-mediated suppression of perforin.

[0136] In some examples, the Nur77 agonist (such as CsnB), or analogue or derivative thereof, reverse the oxLDL-mediated suppression of perforin.

[0137] In some examples, the agonist, or analogue or derivative thereof, represses lipid uptake and fatty acid oxidation in NK cells.

[0138] In some examples, the Nur77 agonist (such as CsnB), or analogue or derivative thereof, represses lipid uptake and fatty acid oxidation in NK cells.

[0139] In some examples, the agonist, or analogue or derivative thereof, desensitises NK cells to lipid-mediated immune suppression.

[0140] In some examples, the Nur77 agonist (such as CsnB), or analogue or derivative thereof, desensitizes NK cells to lipid-mediated immune suppression. As shown in the experimental data of the present disclosure, palmitate suppresses NK cell killing capacity and perforin production, which was advantageously reversed by a Nur77 agonist (such as CsnB) treatment (FIG. 3D and 3E). The Nur77 agonist (such as CsnB) treatment of the present disclosure could also reverse oxidized LDL-mediated expression of perforin (FIG. 3F). As such, the inventors of the present disclosure clearly demonstrate the link between Nur77 activity and lipid metabolism in NK cells. iv) Improve NK cell effector functions

[0141] In some examples, the agonist, or analogue or derivative thereof, improves NK cell effector functions.

[0142] In some examples, the Nur77 agonist (such as CsnB), or analogue or derivative thereof, improves NK cell effector functions.

[0143] In some examples, NK cell effector functions may include, but are not limited to, cytotoxicity, expression of perforin, expression of granzymes, expression of FasL, expression of granulysin, secretion of cytokines (e.g. IFN-y, TNF, and the like), expression of death receptors (such as TNF-a, TRAIL - tumor necrosis-factor-related apoptosis-inducing ligand, FasL / CD178, and the like).

[0144] In some examples, the agonist, or analogue or derivative thereof, reverses suppression of NK cell killing capacity and / or perforin production.

[0145] In some examples, the Nur77 agonist (such as CsnB), or analogue or derivative thereof, reverse suppression of NK cell killing capacity and / or perforin production.

[0146] Co-administration with other therapy

[0147] In some examples, the agonist, or analogue or derivative thereof, may be coadministered with an additional immunotherapy.

[0148] In some examples, the agonist, or analogue or derivative thereof, may be coadministered with an additional NK cell-based immunotherapy.

[0149] In some examples, the Nur77 agonist, or analogue or derivative thereof, may be co-administered with an additional immunotherapy (such as NK cell-based immunotherapy).

[0150] Examples of NK cell-based cancer immunotherapy may include but is not limited to adoptive NK cell transfer, CAR-NK cells, NK cell engagers, and the like. Without wishing to be bound by theory, it is believed that Nur77 agonism could improve the performance of conventional NK cell-based cancer immunotherapy by reinvigorating NK cells to strive in the tumor microenvironment.

[0151] In vivo / Ex vivo / In vitro models

[0152] Using both in vitro and in vivo experimental models, the study of the present disclosure uncovered the therapeutic potential of developing Nur77 agonist as a treatment to complement conventional cancer immunotherapy.

[0153] In some examples, the present disclosure may include NK-cell sensitive models. In some examples, the NK-cell sensitive models are NK-cell sensitive tumor models.

[0154] NK cell-sensitive model refers to in vitro or in vivo models that are particularly susceptible to NK-cell mediated cytotoxicity. NK-sensitive models have low / absent MHC I therefore leading them to be useful for testing NK cell recognition and killing.

[0155] In some examples, the murine NK-cell sensitive models may include but are not limited to RMA-S, B16F10, YAC-1 , and the like.

[0156] In some examples, the human NK-cell sensitive models may include but are not limited to chronic myelogenous leukemia (K562), EBV-transformed B-cell line (721.221). Burkitt’s lymphoma (Daudi), and the like.

[0157] In some examples, the in vivo models may include but are not limited to a hepatocarcinoma model (such as Hepa 1-6), a lymphoma model, a major histocompatibility complex I molecule (MHCI)-deficient lymphoma model (such as RMA- S model), a melanoma model, a melanoma model with tumor cells expressing low / negative MHCI (such as B16F10), and the like.

[0158] In some examples, the in vivo models may include a control with the presence of Nur77 in NK cells and an experimental group with conditional deletion of Nur77 in NK cells.

[0159] Adoptive NK cell therapy

[0160] In some examples, present disclosure may include adoptive NK cell therapy.

[0161] In some examples, adoptive NK cell therapy is performed with non-obese diabetic background with severe combined immunodeficiency mutation (NOD SCID model) as the host for a in vivo human xenograft model. In some examples, adoptive NK cell therapy is performed with hepatocyte-derived carcinoma cell line (such as Huh-7) injected into a NOD SCID model. In some examples, adoptive NK cell therapy of the present disclosure may include NK cells pretreated with an agonist ex vivo prior to introduction / infusion into in vivo models.

[0162] In some examples, NK cells may be pretreated with an Nur77 agonist (such as CsnB), or analogue or derivative thereof, ex vivo prior to introduction I infusion into in vivo models.

[0163] In some examples, the control NK cells used in the adoptive NK cell therapy may include such as but are not limited to no NK cell (i.e., no NK cells introduced / infused into in vivo models), untreated NK cell (i.e., NK cell that is not treated with any substance / Nur77 agonist, or analogue or derivative thereof; prior to introduction / infusion into in vivo models), and the like.

[0164] In some examples, NK cells that are used in adoptive NK cell therapy may include but are not limited to CD45.1 NK cells, human NK cells, and the like.

[0165] The NK cells that are used in adoptive NK cell therapy samples such as CD45.1 NK cells allow precise tracking in the hosts (such as CD45.2 hosts) during adoptive transfer studies. Some of the benefits of adoptive transfer therapy include the possibility of synergy with other therapies (such as cancer therapies), bypassing of immunosuppressive tumor microenvironment, etc.

[0166] Advantageously, the present disclosure showed that no mice died in the group which CsnB pre-treated CD45.1 murine NK cells were administered. This is in contrast to the hepatocellular cancer-specific fatalities that were observed in no NK cell control and untreated CD45.1 murine NK cell control groups. Tumor formation in the livers was also reduced in the CsnB pre-treated NK cell group (FIG. 4B and 4C).

[0167] In addition, in human NK cells, only infusion of CsnB pre-treated NK cells was shown to dampen tumor growth (FIG. 4H and 4I). In contrast to untreated NK cells, the expression of Nur77, CD16 and perforin was better sustained by CsnB pre-treated NK cells within the tumors (FIG. 4L and 4N).

[0168] In summary, the inventors of the present disclosure demonstrated that Nur77 activation transcriptionally represses lipid uptake and fatty acid oxidation and desensitizes NK cells to lipid-mediated immune suppression. The findings of the present disclosure support the use of Nur77 agonist such as Cytosporone B or its potential derivatives to improve NK cell effector functions.

[0169] The key advantage here is that Nur77 agonism could further improve the performance of conventional NK cell-based cancer immunotherapy by reinvigorating NK cells to strive in the tumor microenvironment. The study of the present disclosure has demonstrated the following:

[0170] 1. Nur77 agonism via Cytosporone B elicits NK cell dependent anti-tumor responses in syngeneic tumor mouse models, demonstrating the potential of combining Nur77 agonism with NK cell-based immunotherapy.

[0171] 2. Cytosporone B (Nur77 agonist) confers resistance to lipid-mediated immune suppression to enhance NK cell activity.

[0172] RESULTS

[0173] NR4A1 (Nur77) was identified to regulate monocytes inflammatory responses against cancer while on the other hand, it is known to be a potent driver of T cell immune tolerance. Using conditional gene knockout mice and repurposing Cytosporone B (CsnB), the study of the present disclosure revealed Nur77 to transcriptionally reprogram NK cells to be more resilient within the tumor microenvironment for enhanced anti-tumor immunity (FIG. 1).

[0174] The data of the present disclosure demonstrates that Cytosporone B dampens tumor progression in NK cell-sensitive mouse models. FIG. 2A to 2F show Nur77 agonism via Cytosporone B eliciting anti-tumor responses in NK cell-sensitive tumor models. All experiments in FIG. 2A to 2F were performed in wild type C57BL / 6 mice. The inventors of the present disclosure first used a simplified orthotopic hepatocellular carcinoma (HCC) (Hepa1-6) model to demonstrate that Nur77 agonism dampens tumor progression (FIG. 2A to 2D). Next, the inventors of the present disclosure investigated if Cytosporone B could elicit an anti-tumor immunity in the RMA-S model which is a MHCl-deficient lymphoma tumor well-recognized as the gold standard for NK cell studies in vivo. Using the same CsnB treatment regime, the growth of subcutaneous RMA-S tumors was similarly suppressed, suggesting that Nur77-mediated immunity could be independent of T cell responses (FIG. 2E). Likewise, CsnB also elicited anti-tumor response in the B16F10 melanoma model in which the tumor cells expressed low / negative MHCI (FIG. 2F). These data indicate that development of an agonist to activate Nur77 in cancer is highly logical and feasible, providing sound justification for further therapeutic purposes.

[0175] To establish mechanistic insights into Nur77 agonism, the inventors of the present disclosure explore the link between Nur77 activity with lipid metabolism in NK cells. FIG. 3A to 3F shows Nur77 agonism conferring resistance to lipid-mediated immune suppression to enhance NK cell function. The inventors of the present disclosure first demonstrate that CsnB decreases NK cells’ ability to uptake lipid (BODIDY-C12) and at the same time, downregulate the surface expression of CD36 in vitro (FIG. 3A to 3C). Importantly, the inventors of the present disclosure observed that palmitate suppresses NK cell killing capacity and perforin production which could be reversed with CsnB treatment (FIG. 3D and 3E). Likewise, CsnB is able to reverse the oxLDL-mediated expression of perforin (FIG. 3F). Here, the in vitro experiments of the present disclosure were performed on human NK cells to highlight the relevance and translational potential of Nur77 agonism for improving conventional cancer immunotherapy in patients. Overall, the inventors of the present disclosure demonstrated a link between Nur77 activity and lipid uptake in NK cells.

[0176] FIG. 4A to 4N shows Nur77 agonism improving the efficacy of adoptive NK cell therapy to hinder hepatocellular carcinoma (HCC) tumor progression. To determine if Nur77 agonism can yield therapeutic benefits in the adoptive cell therapy setting, the inventors of the present disclosure pre-treated CD45.1 murine NK cells with CsnB ex vivo prior to infusion into NCR1iCRE, NR4A1fl / flmice bearing HCC (FIG. 4A). Prior to intended experimental endpoint, HCC-specific fatalities were observed in the no NK control (2 out of 9) and untreated NK (2 of 6) groups while no mice died in the group which CsnB pre-treated NK cells were administered. Moreover, tumor formation in the livers was also indeed reduced in the group with CsnB pre-treated NK cells infused (FIG. 4B and 4C). At experimental endpoint, FACS profiling further showed differential phenotypes of the infused CD45.1 NK cells particularly in the liver of the tumor-bearing host, whereby the frequencies of CD11b+ and perforin+ NK cells was higher in CsnB pre-treated cells as compared to untreated NK cells (FIG. 4D to 4F). To explore if similar efficacies can be obtained in human NK cells, the inventors of the present disclosure first subcutaneously engrafted Huh7 human HCC cells into NOD SCID Gamma (NSG) mice as xenografts in which the inventors of the present disclosure found to be highly vascularised over 25 days of tumor progression (FIG. 4G and 4H). Similarly, only the infusion of CsnB pre-treated NK cells was able to dampen tumor growth (FIG. 4H and 4I). Importantly, CsnB pre-treated NK cells were found to better infiltrate these subcutaneous xenografts (FIG. 4J). As expected, Nur77 was downregulated together with Tbet and other activation markers while the expression of CD56 and CD36 was increased when comparing intratumoral NK cells to peripheral blood NK cells (FIG. 4K). In contrast to untreated NK cells, the expression of Nur77, CD16 and perforin was better sustained by CsnB pre-treated NK cells within the tumors (FIG. 4L to 4N). Altogether, the findings of the present disclosure suggested that CsnB pre-treatment of NK cells was necessary to obtain desired therapeutic outcomes, highlighting the potential of Nur77 agonism as a means of improving conventional NK cell therapy.

[0177] Materials and Methods

[0178] 1 . In vivo experiments

[0179] C57BL / 6 Wildtype mice (male, 6-8 weeks old) were used for all experiments.

[0180] 1.1 Orthotopic liver cancer model

[0181] Anesthesia is first administered by placing mice in an isoflurane induction chamber. Hair is then removed from the surgical area using an electrical clipper / hair removal cream, with a border of at least 1 cm around the incision site. The incision site is then disinfected with 70% ethanol, followed by 3 alternating scrubs of betadine and 70% ethanol. To gain access to the liver, a small transverse incision was made below the sternum to expose the liver. Hepa-1-6 tumor cells were suspended in 25 ul of PBS, were then slowly injected into the upper left lobe of the liver using a 29G needle. The incision site was then sewn up with 2 or 3 stitches of absorbable vicryl 4-0 sutures. 320ug Cytosporone B (CsnB) is given in 100uL of DMSO via intraperitoneal injection according to the treatment regime illustrated in FIG. 2A. Tumor-bearing mice were euthanized after 14 days post tumor inoculation, and the livers are harvested for physical measurements (FIG. 2B to 2D).

[0182] 1.2 Lymphoma and melanoma model

[0183] Anesthesia is first administered by placing mice in an isoflurane induction chamber. Either 106 RMAS (lymphoma, FIG. 2E) or 105 B16F10 (melanoma, FIG. 2F) tumor cells suspended in 100ul of PBS were suspended with 100ul Matrigel before subcutaneously injected into the right flank of the mice. Cytosporone B (320ug / 100uL DMSO) was then administered into these tumor-bearing mice every 2 days post injection. At the same time, tumor volume was determined every 2 days by physical measurement of the length and width of tumor and calculated using the formula (0.5X length Xwidth2). Mice were euthanized after 12 days post tumor inoculation.

[0184] 2. In vitro assays

[0185] 2.1 NK cell isolation and culture from human peripheral blood Human PBMCs were collected through ficoll density gradient centrifugation (GE Healthcare). Primary NK cells were isolated by negative selection using a human NK cells isolation kit, consistently yielding 95 to 99% purity based on flow cytometry analysis of CD3-negative and CD56-positive cells. Isolated NK cells were cultured in AIMV media supplemented with IL-2 (100 lU / ml) for 48 hours together with additional treatments such as Cytosporone B (CsnB, 2uM), Palmitate (100uM) or oxidised LDL (1% by volume). Intracellular staining of perforin was subsequently performed and measured by flow cytometry (FIG. 3E and 3F).

[0186] 2.2 Measurement of intracellular Lipid uptake

[0187] To determine lipid uptake by NK cells (untreated versus Cytosporone B treated), 1 uM of fluorescence conjugated lipid (BODIPY FL C12) was incubated with NK cells for 40minutes. Fluorescent lipid uptake by NK cells were then measured by flow cytometry (FIG. 3A and 3B). The surface expression of lipid receptor, CD36 was also determined by flow cytometry on NK cells (untreated versus Cytosporone B treated) in a separate experiment (FIG. 3C).

[0188] 2.3 HUH7 killing assay

[0189] NK cells pretreated with Cytosporone B alone or in combination with palmitate were mixed with liver cancer cells (HUH7 cell line) at a tumor-NK ratio of 5:1. After 4 hours, cell suspensions were stained with anti-CD45 and live / dead viability dye to be measured by flow cytometry. Dead HUH7 cells were defined as CD45-negative cells with high uptake of the viability dye (FIG. 3D).

[0190] 2.4 Data analysis

[0191] All flow cytometry data were analysed on Flowjo software. All statistical testing and graphical presentations were performed on Prism Graphpad software.

[0192] DETAILED DESCRIPTION OF FIGURES

[0193] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure. FIG. 1 shows diagrams with the overview of the present study which identifies key effects of Nur77 on NK cell metabolism and function in both in vitro and in vivo experimental model.

[0194] FIG. 2A and 2B shows representative schematics and images of orthotopic-im- planted HCC tumors after in vivo treatment of Cytosporone B (CsnB).

[0195] FIG. 2C shows a bar graph with reduced tumor size at day 15 post tumor inoculation comparing mice treated with either CsnB or vehicle control.

[0196] FIG. 2D shows a bar graph with liver weight measured at day 15 post tumor inoculation comparing mice treated with either CsnB or vehicle control.

[0197] FIG. 2E shows a line graph with tumor progression of RM AS over 12 days of CsnB treatment.

[0198] FIG. 2F shows a line graph with tumor progression of B16F10 over 12 days of CsnB treatment.

[0199] FIG. 3A shows a flow cytometric histogram for the uptake of fluorescent BODIPY- C12 by NK cells treated with CsnB.

[0200] FIG. 3B shows a bar graph with relative quantification for the uptake of fluorescent BODIPY-C12 by NK cells treated with CsnB.

[0201] FIG. 3C shows a line graph with the frequencies of CD36+ NK cells upon CsnB treatment.

[0202] FIG. 3D shows a dot plot with the percentage of HUH-7 tumor cell killing by NK cells treated with CsnB ± palmitate-mediated immunosuppression.

[0203] FIG. 3E shows a dot plot with the relative expression of perforin (PRF1) by NK cells treated with CsnB under palmitate.

[0204] FIG. 3F shows a dot plot with the relative expression of perforin (PRF1) by NK cells treated with CsnB oxidized LDL (oxLDL)-mediated immunosuppression.

[0205] FIG. 4A shows schematics of experimental design in which CD45.1 NK cells were pre-activated prior to adoptive transfers into NCR1iCreNR4A1fl / flmice.

[0206] FIG. 4B shows representative photographs of HCC-containing livers and harvested at experimental endpoint. Tumor formations are marked in black outline.

[0207] FIG. 4C shows a box plot with the weight of HCC-bearing livers at experimental endpoint comparing mice that received different NK cell infusions (n=5 per group) to no NK control (n=9). One-way ANOVA was used to test for significance. *p<0.05, **p<0.01 , ****p<0.0001 and ns= non-significant.

[0208] FIG. 4D shows a heatmap with median frequencies of CD45.1 NK cells expressing various phenotypic markers in blood, spleen and liver. FIG. 4E shows a dot plot with the frequencies of CD11b+ NK cells in blood, spleen and liver to compare CsnB pre-treated NK cells (n=5) to the untreated group (n=3). One-way ANOVA was used to test for significance. *p<0.05, **p<0.01, ****p<0.0001 and ns= non-significant.

[0209] FIG. 4F shows a dot plot with the frequencies of perforin (Prf+) NK cells in blood, spleen and liver to compare CsnB pre-treated NK cells (n=5) to the untreated group (n=3). One-way ANOVA was used to test for significance. *p<0.05, **p<0.01, ****p<0.0001 and ns= non-significant.

[0210] FIG. 4G shows the schematics of an experimental design in which human NK cells were pre-activated prior to adoptive transfers into NSG mice bearing subcutaneous Huh7 xenograft tumors.

[0211] FIG. 4H shows representative photographs of Huh7 xenograft tumors at experimental endpoint.

[0212] FIG. 4I shows a line graph of the progression of tumor growth over 25 days post tumor inoculation. Black arrows marked day 15 and 18 in which NK cells were infused. Two-way ANOVA was used for significance testing to compare different NK cell treatments (n=5) and no NK control (n=3).

[0213] FIG. 4J shows a box plot of the frequencies of infused NK cells (co-expressing CD45, CD56 and CD16) in every millions of total live tissue cells as determined by flow cytometry comparing different NK cell treatments (n=5) and no NK control (n=3). Oneway ANOVA was used to test for significance. *p<0.05, **p<0.01 , ****p<0.0001 and ns= non-significant.

[0214] FIG. 4K shows a heatmap with normalised Z scores for the relative expression of various phenotypic markers in human NK cells isolated from peripheral blood and xenograft tumors comparing CsnB-pretreated NK group to the untreated control (n=5 per group).

[0215] FIG. 4L shows a box plot with the mean fluorescence intensity (MFI) of Nur77 expressed by peripheral blood and tumor-infiltrating NK cells comparing CsnB- pretreated NK group to the untreated control (n=5 per group). One-way ANOVA was used to test for significance. *p<0.05, **p<0.01, ****p<0.0001 and ns= non-significant.

[0216] FIG. 4M shows a box plot with the mean fluorescence intensity (MFI) of CD16 expressed by peripheral blood and tumor-infiltrating NK cells comparing CsnB- pretreated NK group to the untreated control (n=5 per group). One-way ANOVA was used to test for significance. *p<0.05, **p<0.01 , ****p<0.0001 and ns= non-significant. FIG. 4N shows a box plot with the mean fluorescence intensity (MFI) of perforin (Prf) expressed by peripheral blood and tumor-infiltrating NK cells comparing CsnB- pretreated NK group to the untreated control (n=5 per group). One-way ANOVA was used to test for significance. *p<0.05, **p<0.01, ****p<0.0001 and ns= non-significant.

[0217] FIG. 5 shows a schematic illustration of the key effects of Nur77 on NK cell metabolism and function.

[0218] APPLICATIONS

[0219] Embodiments of the methods disclosed herein provide a Nur77 agonist, or analogue or derivative thereof, for use in activating a natural killer cell.

[0220] Advantageously, the present disclosure shows that the Nur77 agonist confers NK cell resistance to lipid-mediated immune suppression.

[0221] Even more advantageously, the present disclosure shows that the Nur77 agonist reverses lipid-mediated immune suppression of NK cell killing capacity and / or perforin production.

[0222] Even more advantageously, the present disclosure shows that the Nur77 agonist can directly activate NK cell killing capacity independent of T cell responses.

[0223] Even more advantageously, the Nur77 agonist identified in the present disclosure provides future opportunities for compound lead optimization, synthesis of derivatives, and clinical drug development.

[0224] It will be appreciated by a person skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

CLAIMS1 . A Nur77 (NR4A1) agonist, or analogue or derivative thereof, for use in activating a natural killer (NK) cell.

2. A Nur77 (NR4A1) agonist, or analogue or derivative thereof, for use in treating a disease, wherein the agonist activates a NK cell.

3. A method of treating or preventing a disease in a subject in need thereof, the method comprising administering a Nur77 (NR4A1) agonist, or analogue or derivative thereof, to the subject, wherein the agonist activates a NK cell.

4. Use of a Nur77 (NR4A1) agonist or analogue or derivative thereof in the manufacture of a medicament for treating or preventing a disease.

5. The agonist, or analogue or derivative thereof, or method or use of any one of the preceding claims, wherein the Nur77 agonist confers NK cell resistance to lipid-mediated immune suppression.

6. The agonist, or analogue or derivative thereof, or method or use of any one of the preceding claims, wherein the Nur77 agonist, or analogue or derivative thereof, improves NK cell effector functions.

7. The agonist, or analogue or derivative thereof, or method or use of any one of the preceding claims, wherein the Nur77 agonist, or analogue or derivative thereof, reverses lipid-mediated immune suppression of NK cell killing capacity and / or perforin production.

8. The agonist, or analogue or derivative thereof, or method or use of any one of the preceding claims, wherein the Nur77 agonist, or analogue or derivative thereof, activates NK cell killing capacity that is independent of T cell responses.

9. The agonist, or analogue or derivative thereof, or method or use of any one of the preceding claims, wherein the NK cell is a suppressed NK cell.

10. The agonist, or analogue or derivative thereof, or method or use of any one of the preceding claims, wherein the Nur77 agonist, or analogue or derivative thereof, is Cytosporone B (CsnB) or derivatives thereof.

11. The agonist, or analogue or derivative thereof, or method of use of any one of the preceding claims, wherein the Nur77 agonist, or analogue or derivative thereof, reduces NK cells’ ability to uptake lipid.

12. The agonist, or analogue or derivative thereof, or method or use of any one of the preceding claims, wherein the Nur77 agonist, or analogue or derivative thereof, down-regulates the surface expression of CD36.

13. The agonist, or analogue or derivative thereof, or method or use of any one of the preceding claims, wherein the Nur77 agonist, or analogue or derivative thereof, reverses the oxidized low-density lipoprotein (oxLDL)-mediated suppression of perforin.

14. The agonist, or analogue or derivative thereof, or method or use of any one of the preceding claims, wherein the Nur77 agonist, or analogue or derivative thereof, represses lipid uptake and fatty acid oxidation in NK cells.

15. The agonist, or analogue or derivative thereof, or method of use of any one of the preceding claims, wherein the NK cell is from a subject suffering from a disease.

16. The product or method of use of any one of the preceding claims, wherein the NK cell is from a subject suffering from cancer.

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