Combination of urolithin and immunotherapy

Abstract

To provide a therapeutic approach that enhances anti-tumor immunity by being used in combination with other medicines.SOLUTION: Provided is a combination of urolithin and immunotherapy for treating a disease state associated with inhibition of T-cell activation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to combinations of urolithins with therapeutic agents, particularly combinations of urolithins with immunotherapy treatments, such as immune checkpoint blockade therapies, e.g., PD-1 antagonists. The invention also relates to pharmaceutical compositions comprising the combinations, processes for preparing the pharmaceutical compositions, and methods of treating diseases with the compositions. [Background technology]

[0002] Urolithins have been proposed as treatments for various conditions associated with insufficient mitochondrial activity, including obesity, reduced metabolic rate, metabolic syndrome, diabetes, cardiovascular disease, hyperlipidemia, neurodegenerative diseases, cognitive disorders, mood disorders, stress, and anxiety disorders, for weight management, or to improve muscle or mental performance (see U.S. Patent No. 5,623,299). The use of urolithins for the treatment of various neoplastic diseases is described in U.S. Patent No. 5,623,299.

[0003] Patent Document 3 discloses a method for increasing autophagy (specifically including mitosis) in cells, the method comprising contacting a cell with an effective amount of a urolithin or a pharmaceutically acceptable salt thereof, thereby increasing autophagy (specifically including mitosis) in the cell. Administration may be to a subject having a disease or condition selected from metabolic stress, cardiovascular disease, endothelial cell dysfunction, sarcopenia, muscle degenerative disease, Duchenne muscular dystrophy, alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver or muscle damage, alpha 1-antitrypsin deficiency, ischemia / reperfusion injury, inflammation, skin aging, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes, hyperlipidemia, osteoarthritis, neurodegenerative disease, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, age-related macular degeneration, mitochondrial disease (including, for example, poor growth, loss of muscle coordination, muscle weakness, visual impairment, hearing impairment, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological disorders, autonomic dysfunction, sometimes learning disabilities, and dementia (as a result of mitochondrial disease)), muscle disease; cancer, cognitive disorders, stress, and mood disorders.

[0004] The immune system is tightly regulated by a network of costimulatory and coinhibitory ligands and receptors. These molecules provide a second signal for T cell activation, providing a balanced network of positive and negative signals to maximize immune responses to infection while limiting self-immunity (see Non-Patent Documents 1 and 2). Examples of costimulatory signals include binding between B7.1 (CD80) and B7.2 (CD86) ligands on antigen-presenting cells (APCs) and CD28 and CTLA-4 receptors on CD4+ T lymphocytes (Non-Patent Documents 3 and 4). Binding of B7.1 or B7.2 to CD28 stimulates T cell activation, whereas binding of B7.1 or B7.2 to CTLA-4 inhibits such activation (Non-Patent Documents 5 and 6). CD28 is constitutively expressed on the surface of T cells (Non-Patent Document 7), whereas CTLA-4 expression is rapidly upregulated after T cell activation (Non-Patent Document 8).

[0005] Other ligands for the CD28 receptor include a group of related B7 molecules, also known as the "B7 superfamily" (Non-Patent Document 9, Non-Patent Document 10, Non-Patent Document 11; Korman et al. (2007) supra). Several members of the B7 superfamily are known, including B7.1 (CD80), B7.2 (CD86), inducible costimulatory ligand (ICOS-L), programmed death-1 ligand (PD-L1, B7-H1), programmed death-2 ligand (PD-L2, B7-DC), B7-H3, B7-H4, and B7-H6 (Non-Patent Document 11).

[0006] The programmed death 1 (PD-1) protein is an inhibitory member of the extended CD28 / CTLA-4 family of T cell regulators (Non-Patent Document 12, Non-Patent Document 13). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. PD-1 has been suggested to exist as a monomer lacking the unpaired cysteine ​​residue characteristic of other CD28 family members. PD-1 is expressed on activated B cells, T cells, and monocytes.

[0007] The PD-1 gene encodes a 55-kDa type I transmembrane protein (Non-Patent Document 14). Although structurally similar to CTLA-4, PD-1 lacks the MYPPY motif, which is important for binding to B7-1 and B7-2. Two ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), have been identified, and they have been shown to downregulate T cell activation upon binding to PD-1 (Non-Patent Document 15, Non-Patent Document 16). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not other CD28 family members. PD-L1 is abundant in various human cancers (Non-Patent Document 17).

[0008] PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signaling (Non-Patent Document 18, Non-Patent Document 19). The interaction between PD-1 and PD-L1 can function as an immune checkpoint, resulting in, for example, a reduction in tumor-infiltrating lymphocytes, a reduction in T cell receptor-mediated proliferation, and / or immune evasion by cancer cells (Non-Patent Document 20, Non-Patent Document 21, Non-Patent Document 22). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2, and the effect is additive when the interaction between PD-1 and PD-L2 is similarly blocked (Non-Patent Document 23, Non-Patent Document 24).

[0009] Antibody inhibitors of immune checkpoints, including PD-1 and PD-L1, are less toxic than broad-spectrum immune activators such as IL-2 and IFN-α and have demonstrated significant antitumor activity in patients with a variety of solid tumors. Two monoclonal antibodies targeting PD-1, pembrolizumab and nivolumab, have demonstrated significant single-agent activity in melanoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), and other solid tumors (NPL 25, NPL 26, NPL 27, NPL 28, NPL 29, NPL 30, NPL 31, NPL 32). In previously treated patients with unresectable melanoma, response rates to pembrolizumab and nivolumab were 34% and 31%, respectively, with progression-free survival of 50 weeks and 9.7 months, respectively (NPL 33, NPL 27). In patients with advanced, previously untreated non-small cell lung cancer, the response rates to pembrolizumab and nivolumab were 26% and 30% (Non-Patent Documents 34 and 35). As can be seen from the response rates detailed above, despite significant activity in some patients, the majority of patients treated with single-agent anti-PD-1 immunotherapy do not benefit from the treatment. [Prior art documents] [Patent documents]

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[0012] It is believed that therapeutic approaches that enhance anti-tumor immunity may work more effectively when used in combination with other agents. Surprisingly, Applicants have found that urolithins significantly enhance the efficacy of immunotherapy treatments, such as PD-1 antagonists.

[0013] According to a first aspect of the invention, there is provided a combination of a urolithin with immunotherapy treatment for the treatment of a disease state associated with the inhibition of T cell activation.

[0014] According to a further aspect of the invention, there is provided a combination of a urolithin with immune checkpoint blockade therapy for the treatment of disease conditions associated with the inhibition of T cell activation.

[0015] According to a further aspect of the invention, there is provided a combination of a urolithin and an immunotherapy treatment for use in the manufacture of a medicament for the treatment of a disease state associated with the inhibition of T cell activation.

[0016] According to a further aspect of the invention, there is provided a combination of a urolithin and immune checkpoint blockade therapy for use in the manufacture of a medicament for the treatment of a disease state associated with the inhibition of T cell activation.

[0017] According to a further aspect of the invention, there is provided a method of treating disease conditions associated with the inhibition of T cell activation using a combination of a urolithin and immunotherapy treatment.

[0018] According to a further aspect of the invention, there is provided a method of treating disease conditions associated with inhibition of T cell activation using a combination of a urolithin and immune checkpoint blockade therapy.

[0019] According to a further aspect of the invention, there is provided a combination of a urolithin and an agent that modulates an immunoinhibitory protein, such as PD-1 or PD-L1, for use in treating a disease state associated with inhibition of T cell activation.

[0020] According to a further aspect of the invention, there is provided a combination of a urolithin and an agent that modulates an immunoinhibitory protein, such as PD-1 or PD-L1, for use in the manufacture of a medicament for the treatment of a disease state associated with the inhibition of T cell activation.

[0021] According to a further aspect of the invention, there is provided a method of treating disease conditions associated with inhibition of T cell activation using a combination of a urolithin and an agent that modulates an immunoinhibitory protein, such as PD-1 or PD-L1.

[0022] The compounds of the invention, or the compounds and treatments, may be administered by separate, sequential or simultaneous administration.

[0023] According to a further aspect of the invention, there is provided a combination of a urolithin and an immunotherapy treatment for use in therapy.

[0024] According to a further aspect of the invention, there is provided a combination of a urolithin and immune checkpoint blockade therapy for use in therapy.

[0025] According to a further aspect of the invention, there is provided a combination of a urolithin and an agent that modulates an immunoinhibitory protein for use in therapy.

[0026] According to a further aspect of the invention, there is provided a composition, e.g., a pharmaceutical composition, comprising a combination of a urolithin and an immunotherapy treatment for use in therapy.

[0027] According to a further aspect of the invention, there is provided a composition, e.g., a pharmaceutical composition, comprising a urolithin in combination with immune checkpoint blockade therapy for use in therapy.

[0028] According to a further aspect of the invention, there is provided a composition, e.g., a pharmaceutical composition, comprising a combination of a urolithin and an agent that modulates an immunoinhibitory protein, for use in therapy.

[0029] According to a further aspect of the invention, there is provided a combination of a urolithin with an immunotherapy treatment.

[0030] According to a further aspect of the invention, there is provided a combination of a urolithin with immune checkpoint blockade therapy.

[0031] According to a further aspect of the invention, there is provided a combination of a urolithin and an agent that modulates an immunoinhibitory protein.

[0032] According to a further aspect of the invention, there is provided a composition, e.g., a pharmaceutical composition, comprising a combination of a urolithin and an immunotherapy treatment.

[0033] According to a further aspect of the invention, there is provided a composition, e.g., a pharmaceutical composition, comprising a urolithin in combination with immune checkpoint blockade therapy.

[0034] According to a further aspect of the invention, there is provided a composition, e.g., a pharmaceutical composition, comprising a combination of a urolithin and an agent that modulates an immunoinhibitory protein.

[0035] In one embodiment, the immune checkpoint blockade therapy is selected from a PD-1 antagonist, an anti-CTLA4 therapy, a CD28 antagonist, a B7 ligand antagonist (e.g., an antagonist of B7-1 (CD80) or B7-2 (CD86)), a CD27 antagonist, a CD40 antagonist, a CD40 ligand, an OX40 antagonist, a GITR antagonist, a CD137 antagonist, and / or a 41-BB-1 antagonist.

[0036] In one embodiment, the immune checkpoint blockade therapy is selected from a PD-1 antagonist, an anti-CTLA4 therapy, and a CD28 antagonist, and / or a B7 ligand antagonist (e.g., an antagonist of B7-1 (CD80), B7-2 (CD86)), a CD27 antagonist, a CD40 antagonist, a CD40 ligand, an OX40 antagonist, a GITR antagonist, a CD137 antagonist, and / or a 41-BB-I antagonist.

[0037] In a further embodiment, the PD-1 antagonist is an anti-PD-1 antibody or a functional portion thereof. Examples of anti-PD-1 antibodies include pembrolizumab, nivolumab (BMS-936558), cemiplimab, and pidilizumab.

[0038] In a further embodiment, the PD-1 antagonist is an anti-PD-L1 antibody or a functional portion thereof. Examples of anti-PD-L1 antibodies include avelumab, atezolizumab (MPDL3280A), and durvalumab.

[0039] In another embodiment, the PD-1 antagonist is a fusion protein such as AMP-224 (a recombinant B7-DC Fc-fusion protein consisting of the extracellular domain of the PD-1 ligand programmed death-ligand 2 (PD-L2, B7-DC) and the Fc region of human immunoglobulin (Ig) G1).

[0040] In a further embodiment, the immune checkpoint blockade therapy is an anti-CTLA4 therapy. Examples of anti-CTLA4 therapies include ipilimumab and tremelimumab.

[0041] The combinations of the present invention are useful in treating diseases in which there is a blockage in the processes leading to T cell activation. Examples of such diseases include cancer and infectious diseases.

[0042] Examples of suitable cancers include solid tumors, including HIV-associated metastatic solid tumors.

[0043] Examples of suitable cancers include bladder cancer, B-cell lymphomas such as Hodgkin's lymphoma, T-cell lymphoma, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chromoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, erythroleukemia, triple-negative breast cancer, breast cancer, ovarian cancer, melanoma including pediatric melanoma, lung cancer such as squamous cell lung cancer and non-small cell lung cancer, pancreatic cancer, glioblastoma, colorectal cancer, head and neck cancer such as head and neck squamous cell carcinoma, cervical cancer, prostate cancer, liver cancer, oral squamous cell carcinoma, skin cancer, medulloblastoma, hepatocellular carcinoma, intrahepatic and extrahepatic bile duct cancer, desmoid tumor, soft tissue sarcoma, adenoid cystic carcinoma, urethral cancer, renal cancer, hepatocellular carcinoma, skin cancer such as Merkel cell carcinoma, gastric cancer, and gastroesophageal cancer.

[0044] In one embodiment, the cancer is colorectal cancer.

[0045] In one embodiment, the preferred cancer is a microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumor.

[0046] Examples of infectious diseases include viral infections, bacterial infections, fungal infections, and parasitic infections.

[0047] Examples of viral infections include lymphocytic choriomeningitis virus (LCMV), HIV, hepatitis B virus (HBV), or hepatitis C virus (HCV).

[0048] Examples of bacterial infections include Helicobacter pylori, Mycobacterium tuberculosis (MTB), sepsis (gram-positive bacteria such as staphylococcus), and nosocomial infections (hospital-acquired infections such as C. difficile).

[0049] Examples of parasitic infections include helminth parasites, including Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Fasciola hepatica, and Heligmosomoides polygyrus; Leishmania, such as Leishmania donovani, Leishmania chagasi, and Leishmania Mexicana; Plasmodium, such as Plasmodium berghei and Plasmodium falciparum; and Toxoplasma, such as Toxoplasma gondii.

[0050] Examples of fungal infections include Candidiasis, Aspergillosis, and Cryptococci.

[0051] Examples of infectious diseases include, but are not limited to, HIV, Hepatitis (A, B, and C), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, and Pseudomonas Aeruginosa.

[0052] Add-on / combination therapy The combination of the present invention may be combined with additional therapies such as radiation therapy and / or one or more therapeutic agents.

[0053] In some embodiments, the composition further comprises one or more therapeutic agents, including an anti-cancer agent, an anti-viral agent, an anti-inflammatory agent, and / or an adjuvant.

[0054] In one embodiment, the additional therapy is radiation therapy.

[0055] In one mode, the radiation therapy is fractionated radiation therapy. In one embodiment, the fractionated radiation therapy comprises 2 to 7 fractions. In another embodiment, the fractionated radiation therapy comprises 3 to 6 fractions. In another embodiment, the fractionated radiation therapy comprises 4 to 5 fractions. In one mode, the fractionated radiation therapy comprises 2, 3, 4, 5, 6, or 7 fractions. In one embodiment, the fractionated radiation therapy comprises 5 fractions.

[0056] In one mode, radiotherapy fractions are administered on consecutive days.In one mode, radiotherapy can comprise two or more doses per day and / or consecutive days.In one mode, radiotherapy fractions are administered on the 1st, 2nd, 3rd, 4th and 5th days.In another mode, radiotherapy comprises about 10Gy in 5 fractions (i.e., 2Gy each day for 5 days).

[0057] Other fractionation schedules may be used, including accelerated fractionation (treatment administered in more daily or weekly doses to reduce the number of weeks of treatment), hyperfractionation (smaller doses of radiation administered two or more times per day), or hypofractionation (larger doses administered once per day or less frequently to reduce the number of treatments).

[0058] Radiation therapy can be X-rays, gamma rays, or charged particles.Radiation therapy can be external or internal radiation therapy (also called brachytherapy).Whole-body radiation therapy using radioactive materials such as radioactive iodine can also be used.

[0059] External beam radiation therapy includes 3D conformational radiation therapy, intensity-modulated radiation therapy, image-guided radiation therapy, tomotherapy, stereotactic radiosurgery, proton therapy, or other charged particle beams.

[0060] In another embodiment, the one or more therapeutic agents include, but are not limited to, small molecules, synthetic drugs, peptides (including cyclic peptides), polypeptides, proteins, nucleic acids (e.g., DNA and RNA nucleotides, including, but not limited to, antisense nucleotide sequences, triple helices, RNAi, and nucleotide sequences encoding biologically active proteins, polypeptides, or peptides), antibodies, synthetic or natural inorganic molecules, mimetics, and synthetic or natural organic molecules.

[0061] Specific examples of such therapeutic agents include immunomodulators (e.g., interferons), anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids, and nonsteroidal anti-inflammatory drugs (e.g., aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), analgesics, leukotriene antagonists (e.g., montelukast, methylxanthines, zafirlukast, and zileuton), beta-2 agonists (e.g., albuterol, biterol, fenoterol, isoetal, metaproterenol, pirbuterol, salbutamol, terbutaline, formoterol), and the like. , salmeterol, and salbutamol terbutaline), anticholinergics (e.g., ipratropium bromide and oxitropium bromide), sulfasalazine, penicillamine, dapsone, antihistamines, antimalarials (e.g., hydroxychloroquine), antivirals (e.g., nucleoside analogs (e.g., remdesivir, zidovudine, acyclovir, ganciclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, and AZT), and antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, erythromycin, penicillin, mithramycin, and anthramycin (AMC)).

[0062] Any therapy known to be useful, or has been used or is currently being used for the treatment of disease conditions associated with the inhibition of T cell activation, can be used with the combination of the present invention.For information on the therapies (such as preventive or therapeutic agents) that have been used or are currently being used for the treatment of disease conditions associated with the inhibition of T cell activation, see, for example, Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 13th ed., McGraw-Hill, New York, 2017; The Merck Manual of Diagnosis and Therapy, Robert S. Porter, MD et al. (eds.), 20th ed., Merck Sharp & Dohme Research Laboratories, Rahway, NJ, 2018; Cecil Textbook of Medicine, 25th ed., Goldman and Schafer (eds.), Elsevier, 2015, and Physicians' Desk Reference (71st ed.2016).

[0063] Non-limiting examples of one or more other therapies that may be used in addition to the combinations of the present invention include, but are not limited to, immunomodulatory agents, such as chemotherapeutic agents and non-chemotherapeutic immunomodulatory agents. Non-limiting examples of chemotherapeutic agents include methotrexate, cyclosporin A, leflunomide, cisplatin, ifosfamide, taxanes such as taxol and paclitaxol, topoisomerase I inhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, temodar, cytochalasin B, glucagon-like growth factor receptor 2 (GFR2), gliomas-like growth factor receptor 3 (GL-2), gliomas-like growth factor receptor 4 (GL-2), gliomas-like growth factor receptor 5 (GL-2), gliomas-like growth factor receptor 6 (GL-2), gliomas-like growth factor receptor 7 (GL-2), gliomas-like growth factor receptor 8 (GL-2), gliomas-like growth factor receptor 9 (GL-2), gliomas-like growth factor receptor 1 ... These include lamicidin D, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin homoglomerate, and cytoxan.

[0064] Urolithin Urolithins are metabolites produced by the action of the intestinal microbiota of mammals, including humans, on ellagitannins and ellagic acid. Ellagitannins and ellagic acid are compounds commonly found in foods such as pomegranates, nuts, and berries. Ellagitannins, by themselves, are minimally absorbed in the intestine. Urolithins are a class of compounds having a representative structure (I) shown below. The structures of some particularly common urolithins are listed in Table 1 below with reference to structure (I). [ka] [Table 1]

[0065] In practice, for commercial-scale production, it is convenient to synthesize urolithins, and synthetic routes are described, for example, in WO 2014 / 004902, WO 2015 / 100213, and WO 2019 / 168972.

[0066] Urolithins of any structure according to structure (I) may be used in the combinations of the invention.

[0067] In one embodiment of the combination of the present invention, suitable compounds are those of formula (I) wherein A, C, D, and Z are independently selected from H and OH, and B, W, X, and Y are all H, and preferably at least one of A, C, D, and Z is OH.

[0068] Particularly suitable compounds are naturally occurring urolithins. Thus, Z is preferably OH, and W, X, and Y are preferably all H. When W, X, and Y are all H, A and B are both H, and C, D, and Z are all OH, the compound is urolithin C. When W, X, and Y are all H, A, B, and C are all H, and D and Z are both OH, the compound is urolithin A. Preferably, the urolithin used in the methods of the disclosure is urolithin A, urolithin B, urolithin C, or urolithin D. Most preferably, the urolithin used is urolithin A. [ka]

[0069] According to one embodiment, there is provided a combination of the invention wherein the compound of formula (I) is urolithin A.

[0070] According to one embodiment, there is provided a combination of the invention wherein the compound of formula (I) is urolithin B.

[0071] According to one embodiment, there is provided a combination of the invention wherein the compound of formula (I) is urolithin C.

[0072] According to one embodiment, there is provided a combination of the invention wherein the compound of formula (I) is urolithin D.

[0073] In one embodiment, urolithin does not include acylated urolithin or optionally substituted acylated urolithin (e.g., acylated urolithin A, acylated urolithin B, acylated urolithin C, acylated urolithin D, acylated urolithin E, or acylated urolithin M5; urolithin C having at least one hydroxyl substituted with a fatty acid-containing group). The term "acyl," as used herein, refers to a chemical substituent of formula -C(0)-R, where R is alkyl, alkenyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl. Optionally substituted acyl is acyl optionally substituted as described herein for each R group. Examples of acyl include fatty acyl (e.g., short-chain fatty acyl (e.g., acetyl)) and benzoyl.

[0074] The present invention also encompasses the use of suitable salts of the compounds of formula (I), such as pharmaceutically acceptable salts. Suitable salts according to the present invention include those formed with organic or inorganic bases. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts, such as potassium and sodium salts, alkaline earth metal salts, such as calcium and magnesium salts, and salts with organic bases, such as dicyclohexylamine, N-methyl-D-glucamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di-, or tri-lower alkylamines, such as ethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl, or dimethylpropylamine, or mono-, di-, or trihydroxylower alkylamines, such as mono-, di-, or triethanolamine.

[0075] immunotherapy treatment Immunotherapeutic treatments for use in the combinations of the present invention include any treatment whose mechanism of action acts partially or primarily through enhancing an individual's immune response, for example, immune checkpoint blockade therapies such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies or fusion proteins, neoadjuvant immunotherapy, and CAR-T immunotherapy (chimeric antigen receptor T-cell therapy);

[0076] Anti-PD-1 antibody PD-1 is a major immune checkpoint receptor expressed by activated T cells and B cells and mediates immunosuppression. PD-1 is a member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), have been identified. These ligands are expressed on antigen-presenting cells and in many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. Inhibition of the PD-1 / PD-L1 interaction mediates potent antitumor activity in preclinical models.

[0077] Human monoclonal antibodies (HuMAbs) that specifically bind to PD-1 with high affinity are disclosed in U.S. Patent Nos. 8,008,449 and 8,779,105. Other anti-PD-1 mAbs are described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, and PCT Publication WO 2012 / 145493. Each of the anti-PD-1 HuMAbs disclosed in U.S. Patent No. 8,008,449 has the following characteristics: (a) 1 × 10 as determined by surface plasmon resonance using a Biacore biosensor system -7 Binds to human PD-1 with a KD of ≤ M (b) does not substantially bind to human CD28, CTLA-4, or ICOS; (c) increasing T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increasing interferon-γ production in an MLR assay; (e) increasing IL-2 secretion in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibiting the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulating antigen-specific memory responses; (i) stimulate an antibody response, and (j) inhibiting tumor cell growth in vivo.

[0078] Anti-PD-1 antibodies useful in the combinations of the present invention include mAbs that specifically bind to human PD-1 and exhibit at least one, at least two, at least three, at least four, or at least five of the aforementioned characteristics. In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as "OPDIVO®" and formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., Cancer Immunol Res. 2(9):846-56 (2014)). In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with nivolumab. In other embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as nivolumab. In certain embodiments, the anti-PD-1 antibody has the same CDRs as nivolumab.

[0079] In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with pembrolizumab. In some embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as pembrolizumab. In certain embodiments, the anti-PD-1 antibody has the same CDRs as pembrolizumab. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as "KEYTRUDA®," lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587; see also http: / / www.cancer.gov / drugdictionary?cdrid=695789 (last accessed July 29, 2019). Pembrolizumab is approved by the FDA for the treatment of recurrent or refractory melanoma. In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with MEDI0608. In yet other embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as MEDI0608. In certain embodiments, the anti-PD-1 antibody has the same CDRs as MEDI0608. In other embodiments, the anti-PD-1 antibody is MEDI0608 (formerly AMP-514), which is a monoclonal antibody. MEDI0608 is described, for example, in U.S. Patent No. 8,609,089(B2) or at https: / / www.cancer.gov / publications / dictionaries / cancer-drug / def / anti-pd-1-monoclonal-antibody-medi0680 (last accessed July 29, 2019).

[0080] In certain embodiments, the first antibody is an anti-PD-1 antagonist. One example of an anti-PD-1 antagonist is AMP-224, which is a B7-DC Fc fusion protein. AMP-224 is discussed in U.S. Application Publication No. 2013 / 0017199, or http: / / www.cancer.gov / publications / dictionaries / cancer-drug?cdrid=700595 (last accessed July 29, 2019).

[0081] In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with BGB-A317. In some embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as BGB-A317. In certain embodiments, the anti-PD-1 antibody has the same CDRs as BGB-A317. In certain embodiments, the anti-PD-1 antibody is BGB-A317, which is a humanized monoclonal antibody. BGB-A317 is described in U.S. Patent Application Publication No. 2015 / 0079109.

[0082] In some embodiments, the antibody is pidilizumab (CT-011), an antibody previously reported to bind to PD-1 but believed to bind to a different target. Pidilizumab is described in U.S. Patent No. 8,686,119 (B2) or WO 2013 / 014668 (A1).

[0083] Anti-PD-1 antibodies useful in the combinations of the present invention also include isolated antibodies that specifically bind to human PD-1 and cross-compete with nivolumab for binding to human PD-1 (see, e.g., U.S. Patent Nos. 8,008,449 and 8,779,105, and WO 2013 / 173223). The ability of antibodies to cross-compete for binding to an antigen indicates that they bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. By binding to the same epitope region of PD-1, these cross-competing antibodies are expected to have functional properties very similar to those of nivolumab. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO 2013 / 173223).

[0084] In certain embodiments, antibodies that cross-compete with nivolumab for binding to human PD-1 or bind to the same epitope region of human PD-1 as nivolumab are mAbs. For administration to human subjects, these cross-competing antibodies can be chimeric, humanized, or human antibodies. Such chimeric, humanized, or human mAbs can be prepared and isolated by methods well known in the art. Anti-PD-1 antibodies useful in the disclosed compositions of the present invention also include antigen-binding portions of the antibodies. It is well documented that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i)V L , V H , C L , and C H1 Fab fragments, which are monovalent fragments consisting of domains; (ii) an F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii)V H and C H1an Fd fragment consisting of the domain, and (iv) V of a single arm of the antibody L and V H Examples of such fragments include Fv fragments consisting of domains.

[0085] Anti-PD-1 antibodies suitable for use in the disclosed compositions are those that bind to PD-1 with high specificity and affinity, block binding of PD-L1 and / or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, the anti-PD-1 antibody comprises an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to those of the whole antibody in inhibiting ligand binding and upregulating the immune system. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1. In other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is a chimeric, humanized, or human monoclonal antibody or portion thereof. In certain embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a human antibody. Antibodies of the IgG1, IgG2, IgG3, or IgG4 isotype can be used. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof comprises a heavy chain constant region of the human IgG1 or IgG4 isotype. In certain other embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody, or antigen-binding portion thereof, contains a S228P mutation, replacing a serine residue in the hinge region with a proline residue normally found at the corresponding position in IgG1 isotype antibodies. This mutation, present in nivolumab, prevents Fab arm exchange with endogenous IgG4 antibodies while retaining the low affinity for activating Fc receptors associated with wild-type IgG4 antibodies (Wang et al. Cancer Immunol Res. 2(9):846-56(2014)). In still other embodiments, the antibody comprises a light chain constant region that is a human kappa or lambda constant region. In other embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is a mAb or antigen-binding portion thereof. In certain embodiments of any of the therapeutic methods described herein that include administration of an anti-PD-1 antibody, the anti-PD-1 antibody is nivolumab. In other embodiments, the anti-PD-1 antibody is pembrolizumab.In other embodiments, the anti-PD-1 antibody is selected from human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in U.S. Patent No. 8,008,449. In yet other embodiments, the anti-PD-1 antibody is MEDI 0608 (formerly AMP-514), AMP-224, or BGB-A317. Because anti-PD-1 and anti-PD-L1 target the same signaling pathway and have been shown in clinical trials to exhibit similar levels of efficacy in a variety of cancers, including renal cell carcinoma (RCC) (see Brahmer et al. (2012) N Engl J Med 366:2455-65; Topalian et al. (2012a) N Engl J Med 366:2443-54; WO 2013 / 173223), an anti-PD-L1 antibody may be substituted for an anti-PD-1 Ab in any of the treatment methods disclosed herein. In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (formerly 12A4 or MDX-1105) (see, e.g., U.S. Patent No. 7,943,743; WO 2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446) (see, e.g., Herbst et al. (2013) J Clin Oncol 31(suppl):3000. Abstract; U.S. Patent No. 8,217,149), or MEDI4736 (Khieif (2013), Proceedings of the European Cancer Congress 2013 (September 27-October 1, 2013, Amsterdam, the Netherlands), Abstract 802). In certain embodiments, the antibody that cross-competes with the above reference PD-L1 antibody for binding to human PD-L1, or that binds to the same epitope region of human PD-L1 as the above reference PD-L1 antibody, is a mAb. For administration to human subjects, these cross-competing antibodies may be chimeric, or humanized or human. Such chimeric, humanized, or human mAbs can be prepared and isolated by methods well known in the art.

[0086] Anti-PD-L1 antibody In certain embodiments, the present application encompasses the use of anti-PD-L1 antibodies in place of anti-PD-1 antibodies. In one embodiment, the anti-PD-L1 antibody inhibits the binding of the PD-L1 receptor, i.e., PD-1, to its ligand, PD-L1. Anti-PD-L1 antibodies useful in the present invention include those disclosed herein. H and / or V L These include engineered antibodies that start with an antibody having one or more of the sequences, and the engineered antibody may have altered properties from the starting antibody. Anti-PD-L1 antibodies may be engineered by various modifications as described above to engineer modified anti-PD-1 antibodies of the invention.

[0087] In some embodiments, anti-PD-L1 antibodies useful in the methods include mAb 28-8, as described in International Patent Application No. 2016 / 176503. In other embodiments, anti-PD-L1 antibodies useful in the combinations of the invention include mAbs 28-1, 28-12, 29-8, and 20-12 (as disclosed in International Patent Application No. 2016 / 176503) or antigen-binding portions thereof, including, for example, Fab, F(ab')2Fd, Fv, and scFv, di-scFv or bi-scFv, and scFv-Fc fragments, diabodies, triabodies, tetrabodies, and isolated CDRs.

[0088] Anti-CTLA-4 antibody The anti-CTLA-4 antibodies of the invention bind to human CTLA-4 in a manner that disrupts the interaction between CTLA-4 and the human B7 receptor. Because the interaction between CTLA-4 and B7 transduces a signal that leads to the inactivation of T cells that bear the CTLA-4 receptor, disruption of the interaction effectively induces, enhances, or prolongs the activation of such T cells, thereby inducing, enhancing, or prolonging an immune response.

[0089] HuMAbs that specifically bind to CTLA-4 with high affinity are disclosed in U.S. Patent Nos. 6,984,720 and 7,605,238. Other anti-PD-1 mAbs are described, for example, in U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121. The anti-PD-1 HuMAbs disclosed in U.S. Patent Nos. 6,984,720 and 7,605,238 have the following characteristics: (a) at least about 10 as determined by Biacore analysis 7 M -1 , or about 10 9 M -1 , or about 10 10 M -1 ~10 11 M -1 The equilibrium association constant (K a ), (b) at least about 10 3 , about 10 4 , or about 10 5 m -1 s -1 The kinetic association constant (k a ), (c) at least about 10 3 , about 10 4 , or about 10 5 m -1 s -1 The kinetic dissociation constant (k d ), and (d) inhibiting the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86).

[0090] Anti-CTLA-4 antibodies useful in the present invention include mAbs that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the aforementioned characteristics.

[0091] An exemplary clinical anti-CTLA-4 antibody is the human mAb 10D1 (now known as ipilimumab and commercially available as YERVOY®), disclosed in U.S. Patent No. 6,984,720. Ipilimumab is an anti-CTLA-4 antibody for use in the methods disclosed herein. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.

[0092] Another anti-CTLA-4 antibody useful in this method is tremelimumab (also known as CP-675,206). Tremelimumab is a human IgG2 monoclonal anti-CTLA-4 antibody. Tremelimumab is described in WO 2012 / 122444, U.S. Patent No. 2012 / 263677, or WO 2007 / 113648(A2).

[0093] Anti-CTLA-4 antibodies useful in the disclosed compositions also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with ipilimumab or tremelimumab for binding to human CTLA-4, or bind to the same epitope region of human CTLA-4 as ipilimumab or tremelimumab. In certain embodiments, antibodies that cross-compete with ipilimumab or tremelimumab for binding to human CTLA-4, or bind to the same epitope region of human CTLA-4 as ipilimumab or tremelimumab, are antibodies that contain a heavy chain of the human IgG1 isotype. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, or humanized or human antibodies. Useful anti-CTLA-4 antibodies also include antigen-binding portions of the above antibodies, such as Fab, F(ab')2, Fd, or Fv fragments.

[0094] Urolithin Administration / Dosage Regime The disclosed combination involves orally administering a urolithin of formula (I) or a salt thereof to a subject at a daily dose ranging from 1.7 to 6.0 millimoles per day, e.g., 1.7 to 2.7 millimoles per day, or 2.8 to 6.0 millimoles per day, for a period of 2 to 16 weeks prior to vaccination. As discussed below, administration of urolithin A in the range of 250 mg to 1000 mg (which corresponds to approximately 1.1 to 4.4 millimoles) is preferred, which results in a surprisingly favorable pharmacokinetic profile compared to the much higher dosage of 2000 mg. In one embodiment, the dose is 250 mg / day; in an alternative embodiment, the dose is 500 mg / day; and in another embodiment, the dose is 1000 mg / day.

[0095] In a further embodiment, the administered dosage is selected from the following: - 250 mg once or twice daily - 500 mg once or twice daily - 750 mg once or twice daily - 1000 mg once or twice daily - 1250 mg once or twice daily, or 1500mg once or twice daily

[0096] The method of the present disclosure involves daily administration of a compound of formula (I) or its salt, or a composition containing the compound or salt. In some embodiments, the compound or composition is administered once a day, i.e., the compound or composition is administered at least once per 24-hour period. In other embodiments, the compound or composition containing the compound is administered multiple times a day, for example, twice a day, or three or four times a day. In such cases, the daily dosage is divided into multiple doses. In one embodiment, administration is once a day, in a second embodiment, administration is twice a day, and in a third embodiment, administration is three times a day.

[0097] The method of the present disclosure usually requires daily administration of a compound of formula (I) or its salt, or a composition containing the compound or salt, for a period of several months.In some embodiments, the method can involve daily administration of a compound of formula (I) or its salt, for example, for at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, 4 months, 6 months, or at least 1 year.In some embodiments, the method includes daily administration of a compound or its salt for up to 3 months, up to 6 months, up to 1 year, up to 2 years, or up to 5 years. In some embodiments, the method comprises administering the compound or salt daily for a period ranging from 21 days to 5 years, 21 days to 2 years, 21 days to 1 year, 21 days to 6 months, 21 days to 12 weeks, 28 days to 5 years, 28 days to 2 years, 28 days to 1 year, 28 days to 6 months, 28 days to 4 months, 28 days to 12 weeks, 6 weeks to 2 years, 6 weeks to 1 year, 8 weeks to 1 year, or 8 weeks to 6 months.

[0098] The disclosed methods involve daily administration of an amount of a compound of Formula (I) or a salt thereof, from 0.7 millimoles per day to a maximum of 2.7 millimoles per day, or from 0.7 millimoles twice per day to a maximum of 2.7 millimoles twice per day. In some embodiments, the amount administered is in the range of 2.0 to 2.5 millimoles. In some embodiments, the amount administered is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, or 2.7 millimoles. In other embodiments, the amount administered is about 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 millimoles. In some preferred embodiments, the method involves administering about 2.2 millimoles of a compound of Formula (I) or a salt thereof (e.g., urolithin A) per day, or two 2.2 millimoles per day. The exact weight of the compound administered will depend on the molecular weight of the compound used. For example, urolithin A has a molecular weight of 228 g / mole (so that 2.20 mmoles is 501.6 mg), and urolithin B has a molecular weight of 212 g / mole (so that 2.20 mmoles is 466.4 mg).

[0099] In further embodiments, the disclosed methods involve daily administration of an amount of a compound of Formula (I) or a salt thereof from 2.8 millimoles per day to up to 6.0 millimoles per day, or twice per day. In some embodiments, the amount administered is in the range of 4.0 to 4.8 millimoles. In some embodiments, the amount administered is about 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 millimoles. In some preferred embodiments, the method involves administering about 4.4 millimoles per day, or twice per day, of a compound of Formula (I) or a salt thereof (e.g., urolithin A). The exact weight of the compound administered will depend on the molecular weight of the compound used. For example, urolithin A has a molecular weight of 228 g / mole (so that 4.40 millimoles is 1003.2 mg), and urolithin B has a molecular weight of 212 g / mole (so that 4.40 millimoles is 932.8 mg).

[0100] In some embodiments, the methods involve administering urolithin A in the range of 400-600 mg per day, or in amounts of 400-600 mg twice per day. In preferred embodiments, the methods involve administering urolithin A in an amount of 450-550 mg per day, or twice per day, more preferably about 500 mg.

[0101] In other embodiments, the method involves administering urolithin A in an amount ranging from 700 to 1300 mg / day twice per day, or in an amount ranging from 750 to 1250 mg per day, or in an amount ranging from 800 to 1200 mg per day, or in an amount ranging from 850 to 1150 mg per day, or in an amount ranging from 900 to 1100 mg per day. In a preferred embodiment, the method involves administering urolithin A in an amount ranging from 950 to 1150 mg per day, or twice per day, more preferably about 1000 mg per day, or twice per day.

[0102] In some preferred embodiments, the methods involve administering urolithin A to the subject in an amount ranging from 4.5 to 11 mg / kg / day, e.g., 4.5 to 8.5 mg / kg / day. In other embodiments, the methods involve administering urolithin A to the subject in an amount ranging from 5 to 9 mg / kg / day. In other embodiments, the methods involve administering urolithin A to the subject in an amount ranging from 6.0 to 8 mg / kg / day.

[0103] In other preferred embodiments, the methods involve administering urolithin A to the subject in an amount ranging from 9 to 18 mg / kg / day, for example, 9 to 17 mg / kg / day. In another embodiment, the methods involve administering urolithin A to the subject in an amount ranging from 10 to 17 mg / kg / day. In another embodiment, the methods involve administering urolithin A to the subject in an amount ranging from 11 to 16 mg / kg / day.

[0104] A dosage regime combining a 500 mg dose and a 1000 mg dose may be advantageous. For example, a twice-daily dosage regime combining a first dose of 1000 mg with a second dose of 500 mg several hours later. The 500 mg dose may be administered 6 to 18 hours after the 1000 mg dose, for example, 8 to 12 hours after the 1000 mg dose. For example, about 12 hours after the 1000 mg dose. Thus, according to a further aspect of the present invention, there is provided a treatment of a disease with a compound of formula (I), comprising a twice-daily dosage regime comprising a first dose of 1000 mg followed by a second dose of 500 mg (the two doses being spaced 6 to 18 hours apart).

[0105] The compound of formula (I) or its salt, or the composition containing the compound salt, can be administered at any suitable time, for example, in the morning or evening after sleep.In some embodiments, it may be preferable to carry out the method at approximately the same time every day, for example, within 15, 30, 60, or 120 minutes from a given time point.

[0106] Immunotherapy Administration / Dosage Regime Appropriate dosages of immunotherapeutic treatments are selected by the treating physician based on clinical indications. Such treatments may involve small molecule compounds or macromolecules such as antibodies.

[0107] For example, the antibody or functional portion thereof is administered in a therapeutically effective amount. Generally, the therapeutically effective amount may vary depending on the age, condition, and sex of the subject, as well as the severity of the subject's medical condition. The therapeutically effective amount of the antibody or functional portion thereof ranges from about 0.001 to about 30 mg / kg body weight, about 0.01 to about 25 mg / kg body weight, about 0.1 to about 20 mg / kg body weight, or about 1 to about 10 mg / kg body weight. Dosage may be adjusted, as needed, to accommodate the observed therapeutic effect.

[0108] In certain embodiments, an antibody or functional portion thereof is administered, wherein the antibody is individually administered at a dosage of at least about 0.1, at least about 0.3, at least about 0.5, at least about 1, at least about 3, at least about 5, at least about 10, or at least about 20 mg / kg, e.g., at least about 1 to at least about 10 mg / kg, e.g., at least about 1 to at least about 3 mg / kg, e.g., at least about 3 mg / kg, e.g., at least about 1 mg / kg. The antibody or functional portion thereof may be administered at a dosing frequency of at least about once per week, at least about once per 2 weeks, at least about once per 3 weeks, or at least about once per 4 weeks, or at least about once per month, for up to 6 to up to 72 doses, or as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs. In some embodiments, the antibody or functional portion thereof is administered at a dosage of about 1 or about 3 mg / kg. In certain embodiments, a continuous regimen comprises administering the antibody or functional portion thereof to a subject at a dosing frequency of about once per week, about once per two weeks, about once per three weeks, about once per four weeks, or about once per month for 6 to 72 doses, or for as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs. In other embodiments, the antibody or functional portion thereof is administered at a dosage of about 1 mg / kg at a dosing frequency of about once per three weeks for up to 48 doses.

[0109] In one embodiment, the compound of formula (I) is administered daily and the immunotherapy treatment, e.g., antibody, is administered every 1 to 4 weeks, e.g., every 2 to 4 weeks, e.g., every 2 or 3 weeks. Generally, the duration of treatment is the number of months until maximum remission.

[0110] The antibody may be administered as a bolus dose to maximize circulating levels of the antibody for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.

[0111] composition The method of the present disclosure preferably involves oral administration of a compound of formula (I) or a salt thereof. Any suitable oral composition containing a compound of formula (I) or a salt thereof can be used. Therefore, the use of various compositions containing a compound of formula (I) and suitable for oral administration is contemplated. Thus, in some embodiments, a compound of formula (I) or a salt thereof is administered in the form of an oral composition containing a compound of formula (I) or a salt thereof and one or more excipients suitable for oral administration. Oral compositions can include compositions in the form of pills, tablets, capsules, caplets, lozenges, troches, granules, powders for suspension, oral solutions, oral suspensions, oral emulsions, syrups, etc.

[0112] In a further embodiment of the invention, the compounds of formula (I) are administered by any means known to those skilled in the art for administering pharmaceuticals, such as intramuscular, sublingual, dermal, inhalation, ocular, and otic.

[0113] Compositions containing a compound of formula (I) can take any physical form suitable for the intended use. For example, they can be in the form of a solid (e.g., a tablet or capsule), a semi-solid (e.g., a softgel), or a liquid (including an emulsion). In some cases, the composition can be in the form of a viscous fluid or paste. Semi-solid forms can also contain conventional excipients in the art. The excipients can provide the desired hardness, shelf life, and flavor, for example, so that the composition has an acceptable taste, an attractive appearance, and good storage stability. Semi-solid forms can be in the form of a paste. If the composition is a softgel, it can be provided, for example, in a capsule with a shell. The shell can be of a conventional type, for example, a soft gelatin-based shell. For example, the composition can also be provided inside a hard capsule-type shell. Liquid compositions can be in the form of a medicine, a dietary supplement, or a beverage for oral ingestion, respectively. Liquid preparations can be solutions, emulsions, slurries, or other semi-liquids. Excipients in liquid compositions can provide, for example, shelf life, appearance, flavor, and texture so that the composition has acceptable taste, attractive appearance, and good storage stability. At certain dilution levels, it may be necessary for the subject to shake the beverage before drinking it to maintain a uniform suspension of the active ingredient.

[0114] In some preferred embodiments, the method includes administering a compound of formula (I) or a salt thereof (e.g., urolithin A) in micronized form. Micronization allows the compound of formula (I) to disperse or dissolve more rapidly. Micronization can be achieved by methods established in the art, such as compressive force milling, hammer milling, universal or pin milling, or jet milling (e.g., spiral jet milling or fluidized bed jet milling). Jet milling is particularly suitable. When a micronized compound is used, the compound preferably has a D of less than 100 μm. 50More preferably, the compound has a particle size of less than 75 μm, such as less than 50 μm, for example less than 25 μm, for example less than 20 μm, for example less than 10 μm. 50 More preferably, the compound has a size in the range of 0.5 to 50 μm, such as 0.5 to 20 μm, for example, 0.5 to 10 μm, for example, 1.0 to 10 μm, for example, 1.5 to 7.5 μm, for example, 2.8 to 5.5 μm. 50 Preferably, the compound has a D of less than 100 μm. 90 More preferably, the compound has a size of less than 75 μm, such as less than 50 μm, for example less than 25 μm, for example less than 20 μm, for example less than 15 μm. 90 The compound preferably has a size D in the range of 5 to 100 μm, for example, 5 to 50 μm, for example, 5 to 20 μm, for example, 7.5 to 15 μm, for example, 8.2 to 16.0 μm. 90 Preferably, the compound has a D in the range of 0.5 to 1.0 μm. 10 Preferably, the compound of formula (I) or a salt thereof (e.g., urolithin A) has a D in the range of 8.2 to 16.0 μm. 90 , D in the range of 2.8 to 5.5 μm 50 , and D in the range of 0.5 to 1.0 μm 10 It has.

[0115] In further embodiments, the compound of formula (I) or salt thereof has a size distribution selected from any one of the following: (i) D in the range of 0.5 to 50 μm 50 size, and D in the range of 5–100 μm 90 size; (ii) The compound has a D in the range of 8.2 to 16.0 μm. 90 Size, D in the range of 2.8 to 5.5 μm 50 size, and D in the range of 0.5–1.0 μm 10 having size; (iii) The compound of formula (I) has a D in the range of 0.5 to 20 μm. 50 size, and D in the range of 5–50 μm90 having size; (iv) Compounds of formula (I) have a D of less than 50 μm 50 size, and D less than 75 μm 90 having size; (v) The compound of formula (I) has a D of less than 25 μm 50 Size, and D less than 50 μm 90 having size; (iv) Compounds of formula (I) have a D of less than 10 μm 50 Size, and D less than 20 μm 90 having size; (v) The compound of formula (I) has a D of less than 10 μm 50 size, and D less than 15 μm 90 having a size; or (vi) The compound of formula (I) has a D of 10 μm 50 size, and D of 20 μm 90 It has a size.

[0116] Compositions Comprising Urolithin or Its Salt and Medium-Chain Triglycerides In some preferred embodiments, the compound of formula (I) or its salt (e.g., urolithin A) is administered in the form of a composition comprising a) a medium chain triglyceride, and b) the compound of formula (I) or its salt. In these embodiments, preferably, the compound of formula (I) (e.g., urolithin A) is in micronized form.

[0117] By selecting suitable medium chain triglycerides and excipients, the physical form of the composition can be tailored to the requirements of the product in question. For example, in some embodiments, the composition can be a pharmaceutical composition. In some embodiments, the composition can be a nutritional composition.

[0118] In many cases, the composition comprising a compound of formula (I) and a medium-chain triglyceride will have the consistency of a viscous liquid or paste and may be provided as a single-serving supplement to a subject's general diet (e.g., in a bar, gel or softgel capsule, hard capsule, or diluted in a beverage), or it may be provided as part or all of a meal.

[0119] When the methods of the present disclosure involve the use of a composition comprising medium-chain triglycerides, the medium-chain triglycerides typically constitute at least 1 wt / w% of the composition, such as at least 5 wt / w%, for example at least 10 wt / w%, for example at least 15 wt / w%. The medium-chain triglycerides preferably constitute 20 wt / w% or more of the composition, such as 25 wt / w% or more by weight of the composition, for example 30 wt / w% or more by weight. For example, the medium-chain triglyceride may constitute 1 to 40 w / w% of the composition, 2 to 40 w / w% of the composition, 5 to 40 w / w% of the composition, 10 to 40 w / w% of the composition, 1 to 99 w / w% of the composition, 5 to 99 w / w% of the composition, 10 to 99 w / w% of the composition, 20 to 99 w / w% of the composition, 5 to 90 w / w% of the composition, 10 to 90 w / w% of the composition, for example, 20 to 90 w / w% of the composition, 20 to 80 w / w% of the composition, for example, 30 to 80 w / w% of the composition, for example, 30 to 70 w / w% of the composition, for example, 30 to 60 w / w% of the composition, for example, 30 to 50 w / w% of the composition, for example, 30 to 40 w / w% of the composition, for example, 30 to 35 w / w% of the composition. For example, the medium chain triglycerides may constitute 40-70 w / w% of the composition, such as 50-70 w / w% of the composition, for example 55-65 w / w% of the composition.

[0120] In such compositions, the compound of formula (I) typically comprises 0.1 to 80 w / w% of the composition, e.g., 0.1 to 60 w / w%, e.g., 0.25 to 50 w / w%. For example, the compound of formula (I) may comprise 0.5 to 50 w / w% of the composition. When the composition is provided as part or all of a meal, the compound of formula (I) may comprise, for example, 0.25 to 5 w / w% of the composition, e.g., 0.3 to 3 w / w% of the composition. When the composition is provided as a single-serving food supplement to a subject's general diet, the urolithin typically comprises 20 to 80 w / w% of the composition, e.g., 20 to 40 w / w%, e.g., 25 to 35 w / w% of the composition. For example, the urolithin may constitute 26-34 w / w% of the composition, such as 28-33 w / w% of the composition, such as 29-32 w / w% of the composition, such as 29-31 w / w% of the composition.

[0121] In such compositions, the weight ratio of the medium-chain triglyceride component to the compound of formula (I) is generally in the range of 0.01:1 to 100:1, such as 0.5:1 to 100:1, for example, 0.5:1 to 50:1, for example, 0.5:1 to 5:1, or, for example, 1:1 to 75:1, for example, 1:1 to 50:1, for example, 1:1 to 20:1, for example, 1:1 to 10:1, for example, 1:1 to 2.5:1, for example, 1:1 to 2:1, for example, 1:1 to 1.5:1. The weight ratio may be 0.01:1 to 10:1, for example, 0.1:1 to 10:1, or 0.01:1 to 5:1, for example, 0.01:1 to 0.1:1.

[0122] In some preferred embodiments, the methods of the present disclosure involve administering a softgel capsule containing a filling, the filling comprising a compound of formula (I) or a salt thereof (e.g., urolithin A) and one or more medium-chain triglycerides. In these embodiments, preferably, the compound of formula (I) or a salt thereof (e.g., urolithin A) is micronized. In embodiments in which a softgel capsule is used, the shell components may be manufactured using conventional ingredients.

[0123] Medium chain triglycerides have the formula CH2(OR1 )-CH(OR 2 )-CH2(OR 3 )(wherein, R 1 , R 2 , and R 3 is a medium chain fatty acid radical), generally compounds of the formula -C(=O)(CH2) n CH3 (where n ranges from 4 to 10, e.g., 6 to 8). Medium-chain fatty acids are fatty acids with an aliphatic tail of 6 to 12 carbon atoms. The aliphatic tail is primarily saturated. Specific medium-chain fatty acids include caproic acid (hexanoic acid, C6:0), caprylic acid (octanoic acid, C8:0), capric acid (decanoic acid, C10:0), and lauric acid (dodecanoic acid, C12:0). Myristic acid (tetradecanoic acid, C14:0) may also be present in small amounts. The most commonly used medium-chain triglycerides generally have a mixture of caprylic and capric triglycerides and contain 95% or more saturated fatty acids. The medium chain triglyceride component present in preferred compositions used in the methods of the present disclosure can consist of a homogeneous single medium chain triglyceride compound type, or more commonly, the medium chain triglyceride component is a mixture of two or more different medium chain triglyceride compounds.

[0124] The European Pharmacopoeia describes medium-chain triglycerides as fixed oils extracted from the hard, dry parts of the endosperm of Cocos nucifera L. (coconut) or from the dried endosperm of Elaeis guineenis Jacq. (African oil palm). Both the European Pharmacopoeia and USPNF have specifications for medium-chain triglycerides that require the presence of specific fatty acids as follows: caproic acid (C6) ≤ 2.0%, caprylic acid (C8) 50.0-80.0%, capric acid (C10) 20.0-50.0%, lauric acid (C12) ≤ 3.0%, and myristic acid (C14) ≤ 1%.

[0125] Medium chain triglycerides for use in preferred compositions include mixtures of triglycerides and fatty acid chains present in the following proportions: C6≦5%, C8 50-70%, C10 30-50%, and C12≦12%, for example, C6≦0.5%, C8 55-65%, C10 35-45%, and C12≦1.5%.

[0126] The medium chain triglycerides used in the preferred compositions can be from any known or suitable source.

[0127] The composition used in the method of the present disclosure can advantageously comprise one or more phospholipids.Particularly preferred phospholipid is phosphatidylcholine.The advantages brought about by phosphatidylcholine can be at least partly due to their amphiphilic nature, for example, due to their emulsifying properties.

[0128] A particularly useful source of phospholipids, especially phosphatidylcholine, is lecithin, and compositions used in the methods of the present disclosure advantageously contain lecithin. When present in a composition, lecithin typically constitutes at least 0.5 wt% of the composition, preferably at least 1 wt% of the composition. Lecithin preferably constitutes 10 wt% or more of the composition, e.g., 20 wt% or more, e.g., 30 wt% or more of the composition by weight. For example, lecithin may constitute 0.5 to 80 wt% of the composition, e.g., 1 to 80 wt% of the composition, e.g., 20 to 80 wt% of the composition, e.g., 40 to 80 wt% of the composition, or, e.g., 0.5 to 75 wt% of the composition, e.g., 1 to 40 wt% of the composition, e.g., 30 to 40 wt% of the composition, e.g., 30 to 35 wt% of the composition, e.g., 30 to 75 wt% of the composition. Alternatively, the lecithin may constitute 0.5-5 w / w% of the composition, such as 1-5 w / w% of the composition, for example, 1-3 w / w% of the composition, for example, 0.5-2 w / w% of the composition, for example, 1-2 w / w% of the composition. The weight ratio between the lecithin (if present) and the urolithin is generally in the range of 0.02:1 to 3:1, such as 0.03:1 to 1.2:1, for example, 1:1 to 1.2:1, for example, 1.1:1 to 1.2:1.

[0129] Commercially produced lecithin that can be used in the compositions described herein typically contains the following major components: 33-35% soybean oil, 20-21% inositol phosphatides, 19-21% phosphatidylcholine, 8-20% phosphatidylethanolamine, 5-11% other phospholipids, 5% free carbohydrates, 2-5% sterols, and 1% water.

[0130] Commercially produced lecithin that may be used in the compositions described herein may, for example, be enriched with phosphatidylcholine, having at least 5 wt. % phosphatidylcholine in the lecithin, for example, at least 10 wt. % phosphatidylcholine in the lecithin, for example, at least 15 wt. % phosphatidylcholine in the lecithin, for example, at least 20 wt. % phosphatidylcholine in the lecithin, for example, at least 25 wt. % phosphatidylcholine in the lecithin, for example, at least 30 wt. % phosphatidylcholine in the lecithin, for example, at least 32 wt. % phosphatidylcholine in the lecithin, for example, at least 40 wt. % phosphatidylcholine in the lecithin.

[0131] Lecithins can also be modified to adjust their properties by one or more of the following processes: alcohol extraction of specific phospholipids to produce lecithins with modified ratios of different phospholipids; acetone extraction to remove oil and obtain powdered or granular phospholipid blends; spray drying onto proteins as carriers; spray chilling with synthetic emulsifiers such as high-melting mono- and diglycerides to produce flaked or powdered products; enzymatic modification (phospholipases, generally and especially phospholipase A2), specifically partial hydrolysis to produce lecithins with pronounced emulsifying behavior; hydrolysis of fatty acid groups with acids and alkalis; acetylation; and hydroxylation of fatty acid chains and amino groups.

[0132] In some embodiments, the methods comprise administering a composition comprising a compound of formula (I) or a salt thereof, a medium chain triglyceride, and an emulsifier (eg, lecithin).

[0133] Pharmaceutical compositions containing a compound of formula (I) or a salt thereof may, for example, contain additional pharmaceutically active compounds.

[0134] The additional components in the composition may be compounds that do not provide a health benefit to the subject, but instead improve the composition in some other way, for example, its taste, texture, or shelf life, as described above. Thus, the composition may further contain one or more compounds selected from emulsifiers, colorants, preservatives, gums, hardening agents, thickeners, sweeteners, and flavoring agents.

[0135] Suitable emulsifiers, stabilizers, colorants, preservatives, gums, hardeners, and thickeners are well known in the art of emulsion and other semi-liquid production. Emulsifiers may include one or more of phosphatidylcholine, lecithin, polysorbates such as polysorbate 60 or polysorbate 80 (Tween-60 and Tween-80), and glycerol monostearate (GMS). Glycerol monostearate is also known as glyceryl monostearate.

[0136] Stabilizers may be used in the compositions described herein. Many compositions are stable suspensions that do not require additional stabilizers. A stable suspension is one that does not undergo phase separation over time. In certain compositions, stability may be improved by including additional stabilizers. Suitable stabilizers for use in the compositions of the present invention include glycerol monostearate (GMS), silicon dioxide, and vegetable shortening. An exemplary stabilizer is GMS, and preferred compositions of the present invention contain GMS. Its properties also make it an excellent solvent for phospholipids, such as those found in lecithin. GMS exists in two polymorphic forms: the α-form, which is dispersible and foamy and useful as an emulsifier or preservative; the β-form, which is suitable for wax matrices; the α-form is converted to the β-form when heated at 50°C.

[0137] GMS is classified into two distinct grades: 40-55 percent monoglyceride and 90 percent monoglyceride. The 40-55 percent monoglyceride, as defined by the European Pharmacopoeia, describes GMS as a mixture of monoacylglycerols, primarily monostearoylglycerol, with some di- and triglycerols. Specifically, the 40-55 grade contains 40-55% monoacylglycerol, 30-45% diacylglycerol, and 5-15% triacylglycerol. The 99 percent grade contains over 90% monoglycerides. The monoglycerides in commercially available GMS products are mixtures of variable ratios of glyceryl monostearate and glyceryl monopalmitate. The European Pharmacopoeia further divides 40-55 percent glyceryl monostearate into three types based on the proportion of stearates in the mixture. Type 1 contains 40.0-60.0% stearic acid, and the sum of palmitic acid and stearic acid is 90% or less. Type 2 contains 60.0-80.0% stearic acid, and the sum of palmitic acid and stearic acid is 90% or less. Type 3 contains 90.0-99.0% stearic acid, and the sum of palmitic acid and stearic acid is 96% or less. Any form of GMS can be used in the composition.

[0138] In some embodiments, the method involves administering a composition comprising a medium chain triglyceride, a compound of formula (I) or a salt thereof (e.g., urolithin A), and a stabilizer, e.g., glycerol monostearate. In some embodiments, the method involves administering a composition comprising an emulsifier and a stabilizer.

[0139] Metal chelating or sequestrant agents, such as the sodium calcium salt of ethylenediaminetetraacetic acid (EDTA), may also be used. Other components that may be included in the formulations of the present invention may include polyethylene glycol, silicon dioxide, vegetable shortening, and beeswax.

[0140] Flavorings can be beneficial in the compositions used in the methods described herein.In liquid or semi-liquid compositions, fruit flavors can be provided, for example, by including fruit sauce or puree.Typical flavors include strawberry, raspberry, blueberry, apricot, pomegranate, peach, pineapple, lemon, orange, and apple.Generally, fruit flavors include fruit extracts, fruit jams, or fruit purees, along with sweeteners, starches, stabilizers, natural and / or artificial flavors, colorants, preservatives, water, and citric acid or other suitable acid combinations for pH control.

[0141] Unit dose compositions used in the methods described herein preferably contain 250 mg or 500 mg of the compound of formula (I), e.g., 250 mg or 500 mg of urolithin A. The unit dose may be, for example, in the form of a tablet or capsule, or in the form of a beverage provided in a container such as a bottle or pouch sufficient to hold a single dose (e.g., 50-500 ml, 100-300 ml, e.g., 250 ml or 500 ml). In a further preferred alternative, the unit dose is in the form of a softgel capsule, e.g., containing 250 mg of urolithin A.

[0142] Representative urolithin compositions are shown in the table below. Representative composition A: [Table 2]

[0143] Immunotherapy treatment composition Provided herein are compositions comprising an antibody or fusion protein for use in immunotherapy treatments, e.g., the combinations of the present invention. Also provided herein are compositions comprising an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein. Compositions include bulk drug compositions useful for manufacturing pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions that can be used to prepare unit dosage forms (i.e., compositions suitable for administration to a subject or patient). The composition (e.g., pharmaceutical composition) includes an effective amount of an immunotherapy treatment, such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein, and a pharmaceutically acceptable carrier. In certain embodiments, the composition (e.g., pharmaceutical composition) includes an effective amount of one or more antibodies or proteins, e.g., an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein.

[0144] Pharmaceutical compositions may be formulated in any conventional manner using one or more pharmaceutically acceptable carriers, eg, adjuvants, or excipients.

[0145] Adjuvants include, but are not limited to, Freund's adjuvant (complete and incomplete) or MF59C.1 adjuvant.

[0146] Suitable pharmaceutical carriers include sterile liquids such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc.). In one embodiment, water is a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0147] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may contain a small amount of wetting or emulsifying agent, or pH buffering agent. These compositions may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0148] In certain embodiments, immunotherapy treatments for use in the combinations of the present invention are administered to a subject according to the methods described herein and are administered as pharmaceutical compositions.

[0149] Generally, the components of a pharmaceutical composition comprising an immunotherapeutic treatment such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When an immunotherapeutic treatment such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein is administered by infusion, it can be dispensed from an infusion bottle containing sterile pharmaceutical-grade water or saline (e.g., PBS). When an immunotherapeutic treatment such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0150] In some embodiments, an immunotherapeutic treatment such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein may be formulated for administration by any method known to those of skill in the art, including, but not limited to, parenteral (e.g., subcutaneous, intravenous, intratumoral, or intramuscular) administration. In one embodiment, an immunotherapeutic treatment such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein is formulated for local or systemic parenteral administration, e.g., intratumoral administration. In a specific embodiment, an immunotherapeutic treatment such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein is formulated for subcutaneous or intravenous administration, respectively. In one embodiment, an immunotherapeutic treatment such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein is formulated in a pharmaceutically compatible solution.

[0151] Immunotherapeutic treatments such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, or fusion proteins can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable preparations can be presented in unit dosage form, for example, in ampoules or multi-dose containers, with added preservatives. The compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use.

[0152] For the avoidance of doubt, the combinations of the present invention may be formulated in the same composition or may be formulated in separate compositions for simultaneous, separate or sequential administration.

[0153] Also within the scope of the present invention are kits, including pharmaceutical kits, containing the combinations of the present invention for therapeutic use. The kits typically include a label indicating the intended use of the contents of the kit and instructions for use. The term "label" includes any writing or recorded material supplied on or with the kit, or otherwise accompanying the kit. Certain embodiments of the pharmaceutical kit include a urolithin, e.g., urolithin A, and an immunotherapy treatment, such as an immune checkpoint blockade therapy, in unit dosage form.

[0154] In one embodiment, there is provided a kit for the treatment of a disease state associated with the inhibition of T cell activation, comprising: (a) urolithin, (b) immunotherapy treatment, e.g., immune checkpoint blockade therapy; (c) a container for containing the agent; and (d) Optionally, a kit is provided containing instructions for simultaneous, separate, or sequential administration.

[0155] The term "antibody or functional portion thereof" is used in the broadest sense. It can be artificial, such as a monoclonal antibody (mAb) produced by conventional hybridoma technology, recombinant technology, and / or a functional fragment thereof. It can include both intact immunoglobulin molecules, e.g., polyclonal antibodies, monoclonal antibodies (mAb), monospecific antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, animal antibodies (e.g., camelid antibodies), chimeric antibodies, and parts, fragments, regions, peptides, and derivatives thereof (provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant technology), such as immunoglobulins lacking light chains, Fab, Fab', F(ab'), Fv, scFv, antibody fragments, diabodies, Fd, CDR regions, or any portion or peptide sequence of an antibody capable of binding to an antigen or epitope. In one embodiment, the functional portion is a single-chain antibody, a single-chain variable fragment (scFv), a Fab fragment, or a F(ab') fragment.

[0156] An antibody or functional portion is said to be "capable of binding" a molecule if it specifically reacts with the molecule, thereby allowing the molecule to bind to the antibody. Antibody fragments or portions may lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and have less nonspecific tissue binding than an intact antibody. Exemplary antibodies can be produced from intact antibodies using methods well known in the art, for example, by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). Antibody portions can be produced by any of the above methods or by expressing portions of recombinant molecules. For example, the CDR regions of a recombinant antibody can be isolated and subcloned into an appropriate expression vector.

[0157] In one embodiment, the antibody or functional portion is a human antibody. The use of human antibodies for human therapy can reduce the possibility of side effects due to immune responses in human individuals to non-human sequences. In another embodiment, the antibody or functional portion is humanized. In another embodiment, the antibody or functional portion is a chimeric antibody. Thus, a sequence of interest, such as a binding site of interest, can be included in the antibody or functional portion.

[0158] In one embodiment, the antibody may have an IgG, IgA, IgM, or IgE isotype, hi one embodiment, the antibody is an IgG.

[0159] The term "cancer" refers to a disease characterized by the rapid and uncontrollable growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Various examples of cancer are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid tumors, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0160] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.

[0161] The term "excipient" refers to a substance that is formulated with an active ingredient of a drug, for example, to bulk up a solid formulation containing a small amount of a potent active ingredient for purposes of long-term stabilization (and thus often referred to as a "bulking agent," "filler," or "diluent"), or to impart a therapeutic enhancement to the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.

[0162] The abbreviation "HumAb" refers to a humanized monoclonal antibody.

[0163] The term "lecithin" refers to any group of fatty substances occurring in animal and plant tissues, including phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). Commercially available lecithin obtained from soybeans and sunflowers contains the phospholipids phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. Lecithin can be obtained by chemical extraction from its source in a nonpolar solvent such as hexane, ethanol, acetone, petroleum ether, or benzene, or by mechanical extraction. Specifically, lecithin can be obtained by extraction from sources including soybeans, eggs, milk, rapeseed, cottonseed, and sunflower. Commercially available lecithin for use in edible preparations can be easily purchased.

[0164] The term "immune checkpoint blockade therapy(ies)" refers to a therapeutic approach that removes inhibitory signals of T cell activation, allowing reactive T cells to overcome control mechanisms and mount an effective immune response, e.g., tumor-reactive T cells to mount an effective anti-tumor response. For an overview of checkpoint blockade therapy, see Wei et al. (2018) Cancer Discovery 8(8), 1-18. Examples of immune checkpoints include PD-1, CTLA-4, lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin and ITIM domain (TIGIT), and T-cell immunoglobulin-3 (TIM-3).

[0165] The term "immunotherapeutic treatment" refers to any treatment whose mechanism of action works partly or primarily through enhancing an individual's immune response.

[0166] The abbreviation "mAb" refers to monoclonal antibody.

[0167] The term "PD-1 antagonist" refers to any agent that blocks the inhibitory effects of PD-1 on the immune system. For example, PD-1 antagonists include agents that directly block the binding of PD-1 to its receptor and agents that have an allosteric effect on the activity of PD-1.

[0168] The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans.

[0169] The term "programmed death-1 (PD-1)" refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is primarily expressed in previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one epitope with hPD-1. The complete hPD-1 sequence can be found in GenBank accession number U64863.

[0170] The term "programmed death-ligand-1 (PD-L1)" refers to one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1," as used herein, includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs that share at least one epitope with hPD-L1. The complete hPD-L1 sequence can be found in GenBank Accession No. Q9NZQ7.

[0171] The term "programmed death-ligand-2 (PD-L2)" refers to one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L2," as used herein, includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, as well as analogs that share at least one epitope with hPD-L2. The complete hPD-L1 sequence can be found in GenBank Accession No. Q9BQ51.

[0172] The term "separate" administration means that each of two or more compounds is administered to a patient simultaneously, substantially simultaneously, or sequentially in any order from non-fixed dose forms. There may or may not be a specified time interval between the administration of each compound.

[0173] The term "sequential" administration means that each of two or more compounds is administered to a patient in separate acts from a non-fixed (separate) dosage form. The acts of administration may or may not be related by a specified time interval. For example, administering the compounds over a specified period of time, such as once every 14 to 21 days.

[0174] The term "concurrent" administration means that each of two or more compounds is administered to a patient in a single action, e.g., in which each compound is administered independently at substantially the same time or separately within a time interval that allows the compounds to exert a "cooperative therapeutic effect."

[0175] The invention will now be described with reference to the following non-limiting examples. [Brief explanation of the drawings]

[0176] [Figure 1] A blood cell lineage derived from hematopoietic stem cells. [Figure 2]AOM model of colorectal cancer. Representative images of H&E staining of colonic mucosa collected in control and UA 200 mpk (equivalent to 2.28 g UA per kg diet)-treated animals. Below are graphs showing quantification of the number of lesions (left) and their average size (right) in animals treated with control, urolithin A (UA) 50 mpk (UA LD), and UA 200 mpk (UA HD). [Figure 3] AOM model of colorectal cancer. Representative images of anti-CD3 immunostaining of colonic mucosa collected from control and UA 200mpk (equivalent to 2.28g UA / kg diet)-treated animals. Below are graphs showing quantification of the number (left) and percentage (right) of CD3-positive cells in control and UA 200mpk (UA HD)-treated animals. [Figure 4] APTK organoid model. Tumor volume of subcutaneously injected APTK organoid xenograft tumors in animals fed a control or UA 200mpk diet. [Figure 5] APTK organoid model. Quantification of the number (left) and percentage (right) of CD3-positive cells in control (top) and UA 200mpk (urolithin A)-treated animals. [Figure 6] APTK organoid model. Representative images of anti-cleaved caspase 3 immunostaining of APTK organoid-derived tumors collected in control and UA 200mpk-treated animals. Below is a graph showing quantification of the percentage of cleaved caspase 3-positive cells in control and UA 200mpk (urolithin A)-treated animals. [Figure 7] APTK organoid model. Tumor volume of subcutaneously injected APTK organoid xenograft tumors in animals fed a control diet, a UA 200mpk diet combined with anti-CD8 injection or its isotype q2d. [Figure 8]APTK organoid model. Tumor volume of subcutaneously injected APTK organoid xenograft tumors in animals fed a control diet, a UA 200mpk diet combined with anti-PD1 injection or its isotype q3d. *P<0.05, **P<0.01 (Mann-Whitney test for tumor size at the end of the experiment). [Example]

[0177] The invention will now be described with reference to the following non-limiting examples.

[0178] Example 1: Urolithin A (UA) reduces the incidence and size of lesions and increases the infiltration of T cytotoxic cells within tumors in a mouse model of colorectal cancer (AOM model). A model of sporadic tumor formation can be obtained by repeatedly administering azoxymethane (AOM) to female wild-type FVB mice without dextran sulfate sodium. Specifically, tumors are induced in wild-type animals by weekly intraperitoneal injection of AOM (10 mg / kg) for 6 weeks. Mice are analyzed after 18–24 weeks. Urolithin A is administered to the diet or control laboratory diet at a dose of 50 or 200 mg / kg body weight (mpk) per day 1 week before the start of the six AOM injections, during the 6 weeks of AOM injections, and approximately 20 weeks after these injections, for a total of approximately 27–30 weeks. At the end of the treatment period, mice are euthanized by cervical dislocation for collection of intestinal tissue. Colon tissue is stained for hematoxylin and eosin to count and measure the number of lesions.

[0179] As can be seen in Figure 2, treatment of animals with 200 mpk UA significantly reduces the number of lesions in the AOM model of colorectal cancer. The lesions in UA-treated animals also appear to be smaller in size.

[0180] To determine whether this effect could be explained, at least in part, by immunogenic effects, colonic mucosa was also examined for CD3-positive cells, a marker of the T cell lineage. Figure 3 shows the results of anti-CD3 immunostaining. The colonic mucosa of animals treated with 200 mpk UA tended to have more CD3-positive cells, providing evidence of active recruitment of T cells for the elimination of cancer cells.

[0181] Example 2: Urolithin A (UA) inhibits the growth of APTK subcutaneous xenograft tumors and increases the infiltration of T cytotoxic cells within the tumor. Colorectal cancer organoids (hereafter referred to as APTK organoids) are generated by selectively introducing tumorigenic mutations in the genes APC (A), p53 (P), Tgf-beta1 (T), and K-ras (K). Because APTK organoids are developed on a C57 / BL6 genetic background, they can be transplanted to generate tumors in wild-type BL6 mice with intact immune systems, allowing for the study of immune responses to developing tumors. One week prior to subcutaneous implantation, mice are fed either a urolithin A-containing diet or a control diet. Tumor growth is assessed by caliper measurement. The resulting tumors are analyzed histologically.

[0182] As shown in Figure 4, animals treated with UA 200mpk exhibited significantly lower tumor volumes compared to animals receiving the control diet, indicating that UA prevents tumor growth.

[0183] To determine whether this effect could be explained, at least in part, by immunogenic effects, APTK-derived subcutaneous tumors were also examined for CD3-positive cells, a marker of T cell lineage. Figure 5 shows the results of anti-CD3 immunostaining. Tumors from animals treated with 200 mpk UA tended to have fewer CD3-positive cells at the invasive periphery but more CD3-positive cells within the tumor core, providing evidence of active recruitment of T cells for the elimination of cancer cells.

[0184] Figure 6 shows tumor staining for cleaved caspase 3, a marker of apoptosis. Treatment with 200 mpk UA resulted in a significant increase in cleaved Casp 3 staining, indicating that it induces apoptosis in APTK organoid-derived tumor cells.

[0185] Example 3: Urolithin A (UA) requires the presence of CD8+ lymphocytes to promote its anti-tumor effect. To demonstrate that the predicted tumor-suppressive inflammatory effects of urolithin A are due to a cytotoxic T cell response, treatment with urolithin A was combined with depletion of CD8+ cytotoxic T cells. In this model, C57 / BL6 mice were maintained on 200 mpk of urolithin A or a control diet. One week after the start of the dietary intervention, mice were subcutaneously implanted with APTK organoids as described above. Starting on day 3, the mice received five intraperitoneal injections (150 μg every two days) of anti-CD8 or isotype control antibody (BioXCell) to deplete CD8+ cytotoxic T cells. As in the previous experiment, tumor growth was measured by caliper measurement.

[0186] Figure 7 shows that the antitumor effect of UA is lost when anti-CD8 treatment is used to remove CD8+ cytotoxic T cells, implying that at least part of the effect of UA in treating tumors is driven by its immunogenic effect.

[0187] Example 4: Urolithin A (UA) acts synergistically with anti-PD1 immunotherapy. C57BL / 6 mice were implanted subcutaneously with APTK organoids as described in Example 2, fed either urolithin A 200mpk or a control diet (starting one week prior to subcutaneous injection), and received four intraperitoneal injections of blocking anti-PD1 or isotype control antibody (BioXCell) (200μg every three days, starting on day 5). As in previous experiments, tumor growth is measured by caliper measurement.

[0188] Figure 8 shows that no difference was observed in the effect of urolithin A (200 mpk) alone (i.e., with an isotype control antibody) treatment versus anti-PD1 antibody treatment with respect to tumor growth. However, when anti-PD-1 and urolithin were co-administered, Applicants observed a dramatic synergistic effect on tumor growth delay, providing clear evidence that urolithin A enhances a much stronger biological anti-tumor response to anti-PD1 therapy than anti-PD1 therapy alone.

[0189] equivalent The present invention has been described broadly and comprehensively herein. Those skilled in the art can readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application(s) for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the scope of the invention, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent. Moreover, each of the narrower species and subgeneric groupings that fall within the scope of the generic disclosure also form part of the invention. This includes the generic description of the invention with a provisio or negative limitation removing any subject matter from the genus, regardless of whether the deleted material is specifically recited herein.

[0190] Incorporation by Reference The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated herein by reference in their entirety as if each individual publication were specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from such articles, patents, patent applications, or other physical and electronic documents.

[0191] Preferred embodiments of the present invention will be described below in detail. Embodiment 1 Combining a urolithin with immunotherapy treatment for the treatment of disease states associated with the inhibition of T cell activation. Embodiment 2 The combination of embodiment 1, wherein said immunotherapy treatment is an immune checkpoint blockade therapy. Embodiment 3 The combination of embodiment 1 or embodiment 2, wherein the disease state associated with T cell activation is selected from cancer and infectious disease. Embodiment 4 4. The combination of embodiment 3, wherein the disease state associated with T cell activation is cancer. Embodiment 5 5. The combination of embodiment 4, wherein the cancer is selected from bladder cancer, melanoma including childhood melanoma, lung cancer, e.g., small cell lung cancer, non-small cell lung cancer, lung squamous cell carcinoma, head and neck cancer, e.g., head and neck squamous cell carcinoma, B cell lymphoma, e.g., Hodgkin's lymphoma, T cell lymphoma, urothelial carcinoma, renal cancer, hepatocellular carcinoma, skin cancer, e.g., Merkel cell carcinoma, gastric cancer, and gastroesophageal cancer. Embodiment 6 5. The combination of embodiment 4, wherein said cancer is selected from microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors. Embodiment 7 The combination of embodiment 4, wherein the cancer is colorectal cancer. Embodiment 8 The combination of embodiment 1 or embodiment 2, wherein the disease state is an infectious disease. Embodiment 9 The combination of embodiment 8, wherein the infectious disease is selected from a viral infection, a bacterial infection, and a parasitic infection. Embodiment 10 10. The combination of any of embodiments 2-9, wherein said immune checkpoint blockade therapy is selected from a PD-1 antagonist, an anti-CTLA4 therapy, a C28 antagonist, a B7-1 (CD80) and / or B7-2 (CD86) ligand antagonist, a CD27 antagonist, a CD40 antagonist, a CD40 ligand, an OX40 antagonist, a GITR antagonist, a CD137 antagonist, and / or a 41-BB-I antagonist. Embodiment 11 11. The combination of embodiment 10, wherein the PD-1 antagonist is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, or a fusion protein. Embodiment 12 The combination of any of the preceding embodiments, further comprising one or more additional therapeutic agents. Embodiment 13 A pharmaceutical composition comprising a combination of a urolithin and an immunotherapy treatment. Embodiment 14 14. The pharmaceutical composition of embodiment 13, wherein said immunotherapy treatment is selected from immune checkpoint blockade therapy, neoadjuvant immunotherapy, and CAR-T immunotherapy. Embodiment 15 1. A kit for the treatment of a disease state associated with the inhibition of T cell activation, comprising: (a) urolithin, (b) immunotherapy treatment, e.g., immune checkpoint blockade therapy; (c) a container(s) for containing said agent; and (d) A kit, optionally containing instructions for simultaneous, separate, or sequential administration.

Claims

[Claim 1] Combining a urolithin with immunotherapy treatment for the treatment of disease states associated with the inhibition of T cell activation.

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