Copper chelating therapy

Abstract

Disclosed herein is the use of a copper chelating agent or an agent capable of inducing a copper chelating agent for the treatment of cancer, in particular for increasing the efficiency of an immunotherapy for the treatment of cancer. Disclosed herein are methods of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a copper chelating agent or a copper chelating agent inducer in combination with a therapeutically effective amount of an anti-cancer immunotherapeutic agent. Also disclosed herein are kits or combinations and compositions comprising a copper chelating agent or a copper chelating agent inducer and an anti-cancer immunotherapeutic agent, and uses thereof for the treatment of cancer.

Description

Cross-Reference to Related Applications

[0001] This application claims priority from Australian provisional application No. 2023901504, the entire contents of which are incorporated herein by cross-reference in their entirety. TECHNICAL FIELD

[0002] The present invention relates to the use of copper chelators or agents capable of inducing copper chelators for the treatment of cancer, in particular for the use in improving the efficiency of immunotherapies for the treatment of cancer. However, it will be appreciated that the present invention is not limited to this particular field of use. BACKGROUND

[0003] The following discussion of the prior art is provided to present the invention in an appropriate technical context and to enable its advantages to be more fully understood. However, any discussion of the prior art throughout the specification should not be assumed to mean that the described prior art is widely known or is constitutionally part of the common general knowledge in the art.

[0004] Despite the significant investment and advances made, current cancer treatments are still far from ideal. There is a need for further improvements in treatment methods for treating different types of cancer.

[0005] Immunotherapies have improved survival rates for cancer patients; however, the efficacy of immunotherapy treatments can be hindered by immunosuppressive tumour microenvironments Figure 1 ). For example, one mechanism by which cancer cells protect themselves from anti-tumour immune responses is overexpression of Programmed Death Ligand 1 (PD-L1). The immune checkpoint protein Programmed Death Receptor 1 (PD-1), expressed by lymphocytes, negatively regulates T cell effector functions, resulting in decreased cytokine production and cytotoxic activity against target cells, including tumour cells.

[0006] The FDA has approved several therapeutic mAbs targeting PD-L1 / PD-1 for adult melanoma and lung cancer. However, their efficacy, in particular against PD-L1 / PD-1 blockade, is limited in many patients by cancer cell drug resistance and in other patients by immune-related adverse events. There is a need to develop new therapies that are capable of inhibiting the mechanisms by which cancer cells protect themselves from attack by the immune system, to enable improved survival rates for patients undergoing immunotherapeutic tumour treatments. Such immunotherapeutic treatments include, for example, the use of immune checkpoint inhibitors, chimeric antigen receptor (CAR) T cell therapies, the use of bi-specific T cell engagers (BiTEs), (CAR) NK cell therapies, adoptive cell therapies, and antibody-dependent cellular cytotoxicity (ADCC) therapies.

[0007] It is an object of the present invention to overcome or ameliorate one or more of the disadvantages of the prior art, or to provide a useful alternative. SUMMARY

[0008] The inventors of the present application have surprisingly found that the combination of a copper chelator or copper chelation inducer with an anti-cancer immunotherapeutic agent provides a synergistic improvement in the treatment of cancer.

[0009] In a first aspect of the application, there is provided a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a copper chelator or copper chelator inducer in combination with a therapeutically effective amount of an anti-cancer immunotherapeutic agent.

[0010] In a second aspect of the application, there is provided the use of a copper chelator or copper chelator inducer in the manufacture of a medicament for treating cancer in a subject in need thereof, wherein the copper chelator or copper chelator inducer is administered or to be administered to the subject in combination with an anti-cancer immunotherapeutic agent.

[0011] In a third aspect of the application, there is provided the use of an anti-cancer immunotherapeutic agent in the manufacture of a medicament for treating cancer in a subject in need thereof, wherein the anti-cancer immunotherapeutic agent is administered or to be administered to the subject in combination with a copper chelator or copper chelator inducer.

[0012] In a fourth aspect of the application, there is provided the use of a copper chelator or copper chelator inducer and an anti-cancer immunotherapeutic agent in the manufacture of a medicament for treating cancer in a subject in need thereof.

[0013] In a fifth aspect of the application, there is provided a copper chelator or copper chelator inducer for use in treating cancer in a subject in need thereof, wherein the copper chelator or copper chelator inducer is administered or to be administered to the subject in combination with an anti-cancer immunotherapeutic agent.

[0014] In a sixth aspect of the application, there is provided an anti-cancer immunotherapeutic agent for use in treating cancer in a subject in need thereof, wherein the anti-cancer immunotherapeutic agent is administered or to be administered to the subject in combination with a copper chelator or copper chelator inducer.

[0015] In a seventh aspect of the application, there is provided a kit or combination comprising a copper chelator or copper chelator inducer and an anti-cancer immunotherapeutic agent for use in treating cancer in a subject in need thereof, optionally the kit or combination can further comprise instructions for administering the copper chelator or copper chelator inducer and administering the anti-cancer immunotherapeutic agent to treat cancer.

[0016] The following options can be used in conjunction with the first, second, third, fourth, fifth, sixth or seventh aspects, either individually or in any combination.

[0017] In certain embodiments, the copper chelator or copper chelator inducer and the anti-cancer agent are administered or to be administered to the subject separately.

[0018] In certain embodiments, the copper chelator or copper chelator inducer and the anti-cancer agent are administered simultaneously or are to be administered simultaneously to the subject.

[0019] In certain embodiments, the anti-cancer immunotherapeutic agent is selected from the group consisting of an antibody, an immune checkpoint inhibitor (e.g., an ICI targeting a- CTLA4 and / or an ICI targeting a-PDL1), a chimeric antigen receptor (CAR) T cell, a bi-specific T cell engager (BiTE), a (CAR) NK cell, an adoptive immune cell, and an antibody-dependent immune cell. In certain specific embodiments, the anti-cancer immunotherapeutic agent is an anti-GD2 immunotherapeutic agent. In certain specific embodiments, the anti-cancer immunotherapeutic agent is a GD2-directed chimeric antigen receptor (CAR) T cell or an anti-GD2 antibody. In certain specific embodiments, the anti-cancer immunotherapeutic agent is an anti-GD2 antibody. In certain specific embodiments, the anti-cancer immunotherapeutic agent is selected from the group consisting of atezolizumab (Tecentriq), avelumab (Bavencio), dostarlizumab (Jemperli), durvalumab (Imfinzi), ipilimumab (Yervoy), nivolumab (Opdivo), dinutiximab (Unituxin), dinutuxinab beta (Qarziba), and pembrolizumab (Keytruda).

[0020] In certain embodiments, the subject is selected from the group consisting of a human, a pet, and a livestock. In specific embodiments, the subject is a human.

[0021] A skilled artisan will appreciate that all tumors utilize copper and thus the therapies of the present application are applicable to the treatment of all types of cancer. In one embodiment, the cancer is selected from the group consisting of lung cancer; breast cancer; colorectal cancer; anal cancer; pancreatic cancer; eye cancer; prostate cancer; ovarian cancer; liver and bile duct cancer; esophageal cancer; non-Hodgkin lymphoma; bladder cancer; uterine cancer; glioma, glioblastoma, medulloblastoma, and other brain tumors; kidney cancer; myelofibrosis, head and neck cancer; stomach cancer; multiple myeloma; testicular cancer; germ cell tumors; neuroendocrine tumors; cervical cancer; oral cancer; carcinoid tumors of the gastrointestinal, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors including sarcomas, fibrosarcomas, hemangiomas, angiomatosis, hemangiopericytomas, pseudohemangioma-like interstitial hyperplasia, myofibroblastomas, fibromatosis, inflammatory myofibroblastic tumors, lipomas, angiolipomas, granular cell tumors, neurofibromas, schwannomas, angiosarcomas, liposarcomas, rhabdomyosarcomas, osteosarcomas, leiomyomas, or leiomyosarcomas. In further embodiments, the cancer is selected from the group consisting of hepatocellular carcinoma, adenocarcinoma, breast cancer, and prostate cancer. In further embodiments, the cancer is selected from the group consisting of neuroblastoma, breast cancer, glioblastoma, ovarian cancer, lung cancer, prostate cancer, gastric cancer, mesothelioma, liver cancer, cervical cancer, bladder cancer, thyroid cancer, pancreatic cancer, oral cancer, osteosarcoma, head and neck cancer, medulloblastoma, low-grade glioma, DMG (diffuse midline glioma), DIPG (diffuse intrinsic pontine glioma), and leukemia. In one embodiment, the cancer is breast cancer or neuroblastoma. In one embodiment, the cancer is selected from neuroblastoma and mesothelioma. In one embodiment, the cancer is neuroblastoma. In one embodiment, the cancer is treatable by modulation of GD2, for example treatment with an anti-GD2 immunotherapeutic (e.g., an anti-GD2 antibody or a GD2-directed chimeric antigen receptor (CAR) T cell). In certain embodiments, the cancer expresses GD2. In certain embodiments, the cancer is MYCN-mediated.

[0022] In one embodiment, lung cancer includes lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, non-small cell carcinoma, small cell carcinoma, and mesothelioma. In one embodiment, breast cancer includes ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, and mucinous carcinoma. In one embodiment, colorectal cancer includes colon cancer and rectal cancer. In one embodiment, pancreatic cancer includes pancreatic adenocarcinoma, islet cell carcinoma, and neuroendocrine tumors.

[0023] In an embodiment, ovarian cancer includes ovarian epithelial cancer or surface epithelial stromal tumor, including serous tumor, endometrioid tumor, and mucinous cystadenocarcinoma, as well as sex cord-stromal tumor. In an embodiment, liver and bile duct cancer includes hepatocellular carcinoma, cholangiocarcinoma, and hemangioma. In an embodiment, esophageal cancer includes esophageal adenocarcinoma and squamous cell carcinoma. In an embodiment, uterine cancer includes endometrial adenocarcinoma, uterine papillary serous carcinoma, uterine clear cell carcinoma, uterine sarcoma and leiomyosarcoma, and mixed Mullerian tumor. In an embodiment, kidney cancer includes renal cell carcinoma, clear cell carcinoma, and Wilm's tumor. In an embodiment, head and neck cancer includes squamous cell carcinoma. In an embodiment, stomach cancer includes gastric adenocarcinoma and gastrointestinal stromal tumor. In an embodiment, eye cancer includes retinoblastoma and uveal melanoma.

[0024] In certain embodiments, the copper chelator is selected from the group consisting of ethylenediamine, polyethylenimine and derivatives thereof, tetrathiomolybdate; N,N’ - bis(2-aminoethyl)ethane-1,2-diamine (trientine, triethylenetetramine, TETA); penicillamine; D-penicillamine; N’ - (2-aminoethyl)-; N - [2-(2-aminoethylamino)ethyl]ethane-1,2-diamine (tetraethylenepentamine, TEPA); 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid (EDTA); 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA); N, N,N’,N’ - tetra(2-pyridyl-methyl)ethylenediamine (TPEN); 2-[4,8,11-tris(carboxymethyl)- 1,4,8,11-tetraazacyclotetradecan-1-yl]acetic acid; 2-[4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]acetic acid (DOTA); (2S)-2-amino-3-methyl-3- mercaptobutyric acid (D-pen); bis(sulfidyl)molybdenum sulfide (TTM); 5-chloro-7-iodo-8- hydroxyquinoline (Cliquinol); metformin; diethyldithiocarbamic acid (DDC); 2,9-dimethyl- 1,10-phenanthroline (Neocuproine); 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (Bathocuproine); 4-[2,9-dimethyl-7-(4-sulfophenyl)-1,10-phenanthroline-4-yl]benzenesulfonic acid (BCS); and N,N’- bis(cyclohexylideneamino)oxalamide (cuprizone). In certain embodiments, the copper chelator is selected from the group consisting of triethylenetetramine, tetraethylenepentamine, penicillamine, tetrathiomolybdate, and salts thereof. In certain specific embodiments, the copper chelator is selected from the group consisting of tetraethylenepentamine, triethylenetetramine, and salts thereof. In certain specific embodiments, the copper chelator is triethylenetetramine or a salt thereof.

[0025] In certain embodiments, the copper chelator inducer is a zinc salt.

[0026] In certain specific embodiments, the copper chelator is TEPA or TETA, and the anti-cancer immunotherapy agent is an anti-GD2 immunotherapy agent, optionally, the anti-cancer immunotherapy agent is a GD2-directed chimeric antigen receptor (CAR) T cell or an anti-GD2 antibody, optionally, the anti-cancer immunotherapy agent is an anti-GD2 mAb.

[0027] In certain embodiments, the copper chelator is trientine tetrahydrochloride, and the anti-cancer immunotherapy agent comprises daratumumab beta, optionally trientine tetrahydrochloride and daratumumab beta are administered in combination with irinotecan and temozolomide, optionally wherein the cancer is neuroblastoma, optionally wherein the subject to be treated is a child (less than 21 years old, less than 18 years old, or less than 17 years old, 16 years old, 15 years old, 14 years old, 13 years old, or 12 years old).

[0028] In certain embodiments, the copper chelator or copper chelator inducer is administered by a different route than the anti-cancer immunotherapy agent. For example, in certain embodiments, the copper chelator or copper inducer is administered orally, while the anti-cancer immunotherapy agent is administered parenterally, optionally intraperitoneally.

[0029] In certain specific embodiments, the copper chelator is TEPA or TETA administered orally; and the anti-cancer immunotherapy agent is an anti-GD2 immunotherapy agent, e.g., a GD2-directed chimeric antigen receptor (CAR) T cell or an anti-GD2 antibody, administered parenterally, optionally intraperitoneally. In certain specific embodiments, the copper chelator is TEPA or TETA administered orally; and the anti-cancer immunotherapy agent is an anti-GD2 mAb administered parenterally, optionally intraperitoneally.

[0030] In certain embodiments, the copper chelator or copper chelator inducer is administered daily, every two days, every three days, weekly, or every two weeks.

[0031] In certain embodiments, the immunotherapy agent is administered daily, every two days, every three days, weekly, or every two weeks.

[0032] In some specific embodiments, the copper chelator is TEPA or TETA, which is administered orally for at least 3, 4, or 5 days or more per week, and the anticancer immunotherapy agent is antiGD2 mAb, which is administered parenterally (optionally intraperitoneally) for at least 1, 2, or 3 days or more per week.

[0033] In some specific embodiments, the copper chelator is TEPA or TETA, which is administered orally for at least 3, 4, or 5 days or more per week, and the anticancer immunotherapy agent is an antiGD2 immunotherapy agent, which is administered parenterally (optionally intraperitoneally) for at least 1, 2, or 3 days or more per week, and the antiGD2 immunotherapy agent is optionally a GD2-directed chimeric antigen receptor (CAR) T cell or an antiGD2 antibody.

[0034] In certain embodiments, the dose of copper-chelating agent or copper-chelating agent inducing agent per administration can be about 0.5 mg / kg to about 4000 mg / kg, or about 1 mg / kg to about 3000 mg / kg, about 1 mg / kg to about 2000 mg / kg, about 1 mg / kg to about 950 mg / kg, about 1 mg / kg to about 950 mg / kg, about 10 mg / kg to about 950 mg / kg, about 21 mg / kg to about 950 mg / kg, about 41 mg / kg to about 950 mg / kg, about 60 mg / kg to 950 mg / kg, about 80 mg / kg to about 950 mg / kg, about 100 mg / kg to about 950 mg / kg, about 150 mg / kg to about 950 mg / kg, about 200 mg / kg to about 950 mg / kg, about 250 mg / kg to about 950 mg / kg, about 300 mg / kg to about 950 mg / kg, about 350 mg / kg to about 950 mg / kg, about 400 mg / kg to about 950 mg / kg, about 450 mg / kg to about 950 mg / kg, about 500 mg / kg to about 950 mg / kg, about 550 mg / kg to about 950 mg / kg, about 600 mg / kg to about 950 mg / kg, about 1 mg / kg to about 920 mg / kg, about 1 mg / kg to about 880 mg / kg, about 1 mg / kg to about 840 mg / kg, about 1 mg / kg to about 810 mg / kg, about 1 mg / kg to about 780 mg / kg, about 1 mg / kg to about 740 mg / kg, about 1 mg / kg to about 700 mg / kg, about 1 mg / kg to about 670 mg / kg, about 1 mg / kg to about 640 mg / kg, about 1 mg / kg to about 600 mg / kg, about 100 mg / kg to about 600 mg / kg, about 200 mg / kg to about 600 mg / kg, about 300 mg / kg to about 600 mg / kg, about 400 mg / kg to about 600 mg / kg, about 500 mg / kg to about 600 mg / kg, about 100 mg / kg to about 500 mg / kg, about 100 mg / kg to about 400 mg / kg, about 100 mg / kg to about 300 mg / kg, or about 100 mg / kg to about 200 mg / kg.It can be greater than or equal to about 1 mg / kg, 11 mg / kg, 21 mg / kg, 31 mg / kg, 41 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, or 300 mg / kg. It can be less than or equal to about 4000 mg / kg, 3000 mg / kg, 2000 mg / kg, 1000 mg / kg, 950 mg / kg, 920 mg / kg, 880 mg / kg, 840 mg / kg, 810 mg / kg, 780 mg / kg, 740 mg / kg, 700 mg / kg, 670 mg / kg, 640 mg / kg, 600 mg / kg, 550 mg / kg, 500 mg / kg, 450 mg / kg, or 400 mg / kg. In certain embodiments, it can be, for example, about 1 mg / kg, 11 mg / kg, 21 mg / kg, 31 mg / kg, 41 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 120 mg / kg, 150 mg / kg, 180 mg / kg, 200 mg / kg, 220 mg / kg, 250 mg / kg, 280 mg / kg, 300 mg / kg, 320 mg / kg, 350 mg / kg, 380 mg / kg, 400 mg / kg, 420 mg / kg, 450 mg / kg, 480 mg / kg, 500 mg / kg, 520 mg / kg, 550 mg / kg, 580 mg / kg, 600 mg / kg, 670 mg / kg, 700 mg / kg, 740 mg / kg, 780 mg / kg, 810 mg / kg, 840 mg / kg, 880 mg / kg, 920 mg / kg, 950 mg / kg, 1000 mg / kg, 1100 mg / kg, 1200 mg / kg, 1500 mg / kg, 2000 mg / kg, 2500 mg / kg, 3000 mg / kg, 3500 mg / kg, or 4000 mg / kg.

[0035] In certain embodiments, the dose of the anti-cancer immunotherapeutic agent per administration can be about 0.01 mg / m 2 (wherein m 2 is the body surface area of the subject) to about 250 mg / m 2 , or about 4 mg / m 2about 250 mg / m 2 about 8 mg / m 2 about 250 mg / m 2 about 12 mg / m 2 about 250 mg / m 2 about 16 mg / m 2 about 250 mg / m 2 about 20 mg / m 2 about 250 mg / m 2 about 23 mg / m 2 about 250 mg / m 2 about 26 mg / m 2 about 250 mg / m 2 about 29 mg / m 2 about 250 mg / m 2 about 32 mg / m 2 about 250 mg / m 2 about 35 mg / m 2 about 250 mg / m 2 about 38 mg / m 2 about 250 mg / m 2 about 41 mg / m 2 about 250 mg / m 2 about 44 mg / m 2 about 250 mg / m 2 about 47 mg / m 2 about 250 mg / m 2 about 50 mg / m 2 about 250 mg / m 2 about 0.01 mg / m 2 about 230 mg / m 2 about 0.01 mg / m 2 about 210 mg / m 2 about 0.01 mg / m 2 about 190 mg / m 2 about 0.01 mg / m 2 about 170 mg / m 2 about 0.01 mg / m 2 about 150 mg / m 2 about 0.01 mg / m 2 about 130 mg / m 2 about 0.01 mg / m 2 about 110 mg / m 2 about 0.01 mg / m 2Approximately 90 mg / m 2 Approximately 0.01 mg / m 2 Approximately 70 mg / m 2 Approximately 0.01 mg / m 2 Approximately 50 mg / m 2 Approximately 20 mg / m 2 Approximately 50 mg / m 2 Approximately 26 mg / m 2 Approximately 50 mg / m 2 Approximately 32 mg / m 2 Approximately 50 mg / m 2 Approximately 38 mg / m 2 Approximately 50 mg / m 2 about 44 mg / m 2 Approximately 50 mg / m 2 Approximately 20 mg / m 2 Approximately 44 mg / m 2 Approximately 20 mg / m 2 Approximately 38 mg / m 2 Approximately 20 mg / m 2 Approximately 32 mg / m 2 or about 20 mg / m 2 Approximately 26 mg / m 2 It can be greater than or equal to approximately 0.01 mg / m³. 2 2 mg / m 2 4 mg / m 2 6 mg / m 2 8 mg / m 2 10 mg / m 2 12mg / m 2 14 mg / m 2 16 mg / m 2 18 mg / m 2 20 mg / m 2 23 mg / m 2 26 mg / m 2 29 mg / m 2 Or 32 mg / m 2 It can be less than or equal to approximately 250 mg / m³. 2 230 mg / m 2 210 mg / m 2 190 mg / m 2 170 mg / m 2 150 mg / m 2 130 mg / m 2, 110 mg / m 2 , 90 mg / m 2 , 70 mg / m 2 , 50 mg / m 2 , 47 mg / m 2 , 44 mg / m 2 , 41 mg / m 2 , or 38 mg / m 2 . In certain embodiments, it can be, for example, about 0.01 mg / m 2 , 2 mg / m 2 , 4 mg / m 2 , 6 mg / m 2 , 8 mg / m 2 , 10 mg / m 2 , 12 mg / m 2 , 14 mg / m 2 , 16 mg / m 2 , 18 mg / m 2 , 20 mg / m 2 , 22 mg / m 2 , 23 mg / m 2 , 24 mg / m 2 , 26 mg / m 2 , 28 mg / m 2 , 29 mg / m 2 , 30 mg / m 2 , 32 mg / m 2 , 34 mg / m 2 , 35 mg / m 2 , 36 mg / m 2 , 38 mg / m 2 , 40 mg / m 2 , 41 mg / m 2 , 42 mg / m 2 , 44 mg / m 2 , 46 mg / m 2 , 47 mg / m 2 , 48 mg / m 2 , 50 mg / m 2 , 90 mg / m 2 , 110 mg / m 2 , 130 mg / m 2 , 150 mg / m 2 , 170 mg / m 2 , 190 mg / m 2 , 210 mg / m 2 , 230 mg / m2 or 250 mg / m 2 .

[0036] In certain embodiments, the copper chelator, the copper chelator inducer, and / or the anti-cancer immunotherapeutic agent can be in the form of a pharmaceutical composition individually or together (i.e., copper chelator and anti-cancer immunotherapeutic agent; or alternatively, copper chelator inducer and anti-cancer immunotherapeutic agent). pharmaceutical composition

[0037] In one embodiment, there is provided a pharmaceutical composition that can comprise a copper chelator as described above, a copper chelator inducer as described above, an anti-cancer immunotherapeutic agent as described above, a copper chelator as described above and an anti-cancer immunotherapeutic agent as described above, or a copper chelator inducer as described above and an anti-cancer immunotherapeutic agent as described above; and a pharmaceutically acceptable excipient.

[0038] The phrase "pharmaceutically acceptable excipient" includes a pharmaceutically acceptable carrier and diluent. The phrase "pharmaceutically acceptable carrier" refers to any of those carriers known to those skilled in the art to be suitable for the particular mode of administration. Moreover, the components described above can be formulated as the only pharmaceutically active ingredient in the composition, or can be combined with other active ingredients.

[0039] The phrase "pharmaceutically acceptable salt" refers to any salt of a compound that is suitable for use in pharmaceutical applications. Pharmaceutically acceptable salts means those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art and include acid addition salts and base salts. Semi-salts of acids and bases can also be formed. Pharmaceutically acceptable salts include inorganic acid salts (e.g. hydrochlorides, hydrobromides, sulfates, etc.); and organic acid salts (e.g. formates, acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, maleates, butyrates, valerates, fumarates, etc.).

[0040] The components of the pharmaceutical composition (i.e., a copper chelator as described above, a copper chelator inducer as described above, an anti-cancer immunotherapeutic agent as described above, a copper chelator as described above and an anti-cancer immunotherapeutic agent as described above, or a copper chelator inducer as described above and an anti-cancer immunotherapeutic agent as described above) can exist in unsolvated and solvated forms. The term'solvate' is used herein to describe a molecular complex comprising the copper chelator, the copper chelator inducer, and / or the anti-cancer immunotherapeutic agent and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules (e.g. ethanol). The term 'hydrate' is used when the solvent is water.

[0041] In one embodiment, the copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics described herein can be administered in the form of a "prodrug." The term "prodrug" refers to a compound which, upon in vivo administration, is metabolized or otherwise converted into a biologically, pharmaceutically, or therapeutically active form of the copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics described above by one or more steps or processes. Prodrugs can be prepared by modifying functional groups present in the compound in such a way that their modifications allow for their cleavage in vivo to yield the copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics described herein in their biologically, pharmaceutically, or therapeutically active form in conventional operations or by in vivo cleavage.

[0042] The compositions herein comprise one or more of the copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics provided herein. In one embodiment, the copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics are formulated into suitable pharmaceutical preparations, such as solutions, suspensions, tablets, creams, gels, dispersible tablets, pills, capsules, powders, sustained release formulations, or elixirs for oral administration, or sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparations and dry powder inhalers. In one embodiment, the copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel et al. Pharmaceutical Dosage Forms and Drug Delivery Systems (1985, 126). Introduction to Pharmaceutical Dosage Forms, Fourth Edition

[0043] In the compositions, an effective concentration of one or more of the copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics, or pharmaceutically acceptable derivatives thereof, is mixed with a suitable pharmaceutical carrier. The copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics can be derivatized into the corresponding salts, esters, acids, bases, solvates, hydrates, or prodrugs as described above prior to formulation. The concentration of the copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics in the composition is effective to deliver an amount upon administration that treats, prevents, or ameliorates one or more symptoms of the disease or disorder being treated.

[0044] In one embodiment, the compositions are formulated for single dose administration. To formulate the compositions, a weight fraction of the copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier in an effective concentration such that the condition being treated is alleviated, prevented, or one or more symptoms are ameliorated.

[0045] ​The copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent is included in a pharmaceutically acceptable carrier in an amount sufficient to produce a therapeutically useful effect in the patient being treated without adverse side effects. Therapeutically effective concentrations can be determined empirically by testing the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent in the in vitro and in vivo systems described herein, and then extrapolated therefrom for human dosages.

[0046] The concentration of the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent in the pharmaceutical composition will depend on the rate of absorption, distribution, inactivation, and excretion of the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent, the physical-chemical characteristics of the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent, the dosage schedule and amount administered, and other factors known to those of skill in the art.

[0047] In one embodiment, a therapeutically effective dose should produce a serum concentration of active ingredient (i.e., copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent) of from about 0.1 ng / mL to about 50 - 100 µg / mL. In another embodiment, the pharmaceutical composition should provide a dosage of from about 0.001 mg to about 2000 mg of copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent per kilogram body weight per day. Pharmaceutical dosage unit forms are prepared to provide a dosage of from about 0.01 mg, 0.1 mg, or 1 mg to about 500 mg, 1000 mg, or 2000 mg and in one embodiment from about 10 mg to about 500 mg of active ingredient or combination of essential ingredients per dosage unit form.

[0048] Dosing can be performed at intervals of several minutes, hours, days, weeks, months or years, or continuously over any of these time periods. Suitable doses are in the range of about 0.1 ng per kg body weight per dose to 1 g per kg body weight per dose. Doses are preferably in the range of 1 µg to 1 g per kg body weight per dose, for example in the range of 1 mg to 1 g per kg body weight per dose. Suitably, doses are in the range of 1 µg to 500 mg per kg body weight per dose, for example in the range of 1 µg to 200 mg per kg body weight per dose or in the range of 1 µg to 100 mg per kg body weight per dose. Other suitable doses can be in the range of 1 mg to 250 mg per kg body weight per dose (including 1 mg to 10, 20, 50 or 100 mg per kg body weight per dose) or in the range of 10 µg to 100 mg per kg body weight per dose.

[0049] Suitable dosages and administration regimes can be determined by the attending physician, and can depend on the particular condition being treated, the severity of the condition, and the overall health, age and body weight of the subject.

[0050] In cases where the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic exhibits insufficient solubility, methods of solubilizing these substances can be used. These methods are known to those of skill in the art, and include, but are not limited to, the use of cosolvents (e.g., dimethylsulfoxide (DMSO)), the use of surfactants (e.g., TWEEN ® ), dissolution in aqueous sodium bicarbonate, formulation of the substance of interest as a nanoparticle, and the like. Derivatives of these substances, such as prodrugs of these substances, can also be used to formulate effective pharmaceutical compositions.

[0051] After mixing or adding the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic, the resulting mixture can be a solution, suspension, emulsion, or the like. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic in the selected carrier or vehicle. Effective concentrations are sufficient to ameliorate the symptoms of the disease, disorder, or condition being treated, and can be determined empirically.

[0052] Pharmaceutical compositions are provided in unit dosage form comprising suitable amounts of the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic or pharmaceutically acceptable derivatives thereof for administration to humans and animals. In one embodiment, the pharmaceutically therapeutic active substances and their derivatives are formulated and administered in unit-dosage or multiple-dosage forms. The active ingredient can be administered at once, or can be divided into multiple smaller doses to be administered at intervals of time. Unit-dosage form as used herein refers to physically discrete units suitable for single administration to human and animal subjects and as is known in the art. Each unit-dosage contains a predetermined quantity of the therapeutic active copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dosage forms include ampoules and syringes, and individually packaged tablets or capsules. Unit-dosage forms can be administered as fractions or multiples of the amounts in these units. Multiple-dosage forms are multiple unit-dosage forms packaged in a single container, which are administered in their separate unit-dosage forms. Examples of multiple-dosage forms include vials, bottles of tablets or capsules, or bottles of pints or gallons. Thus, multiple-dosage forms are multiple unit-dosage in a single package.

[0053] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975.

[0054] Dosage forms or compositions containing active ingredients in the range of 0.005% to 100% (wt.%) and the remainder consisting of nontoxic carrier can be prepared. Methods of preparing these compositions are known to those of skill in the art. Contemplated compositions can comprise 0.001%-100% (wt.%) active ingredient, in one embodiment 0.195% (wt.%) active ingredient, in another embodiment 75-85% (wt.%) active ingredient. Modes of administration

[0055] Suitable modes of administration include injection (subcutaneous, intravenous, intraperitoneal, etc.), oral administration, inhalation, transdermal application, topical cream or gel or powder, ophthalmic, ocular and nasal dosage forms, vaginal or rectal administration. Depending on the route of administration, the formulation and / or the compound (i.e., copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent) can be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions that can inactivate the therapeutic activity of the compound. The compound can also be administered parenterally or intraperitoneally. Compositions for oral administration

[0056] Oral pharmaceutical dosage forms are solid, gel, or liquid. Solid dosage forms are tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed chewable lozenges and tablets that can be enteric-coated, sugar-coated, sustained release, or film-coated. Capsules can be hard gelatin capsules or soft gelatin capsules, while granules and powders can be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art. Solid compositions for oral administration

[0057] In certain embodiments, the formulation is a solid dosage form, and in one embodiment, the formulation is a capsule or tablet. Tablets, pills, capsules, troches and the like can contain one or more of the following ingredients or compounds of similar nature: binders; lubricants; diluents; glidants; disintegrants; coloring agents; sweetening agents; flavoring agents; wetting agents; emetic coatings; and film coatings. Examples of binders include microcrystalline cellulose, gum tragacanth, dextrose solution, acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidone, povidone, crospovidone, sucrose, and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium, and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrants include croscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methylcellulose, agar, and carboxymethylcellulose. Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate. Sweetening agents include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, as well as any kind of spray-dried flavoring. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds which impart an enjoyable sensation, such as, but not limited to, peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Emetic coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

[0058] The copper-chelator, copper-chelator inducer, and / or anti-cancer immunotherapeutic agent, or a pharmaceutically acceptable derivative thereof, can be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with an antacid or other such ingredients.

[0059] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. Other various materials can be present as coatings or excipients, for example, sugar and other enteric agents. The copper-chelator, copper-chelator inducer, and / or anti-cancer immunotherapeutic agent can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum, or the like. A syrup can contain, in addition to the active compound, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

[0060] The active substances can also be mixed with other active substances which do not impair the desired action or which supplement the desired action, such as antacids, H2 blockers and diuretics. The active ingredient is a copper chelator, a copper chelator inducer and / or an anti-cancer immunotherapeutic agent or a pharmaceutically acceptable derivative thereof as described herein. Higher concentrations of the active ingredient (up to about 98% by weight) can be included.

[0061] In certain embodiments, tablet and capsule formulations can be coated as is known to those of skill in the art to modify or sustain release of the active ingredient. Thus, they may, for example, be coated with conventional enteric agents such as phenyl salicylate, wax and cellulose acetate phthalate. Liquid compositions for oral administration

[0062] Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions can be oil-in-water or water-in-oil.

[0063] For example, a liquid pharmaceutically administrable composition can be prepared by dissolving, dispersing, or otherwise mixing an active compound (i.e., a copper chelator, a copper chelator inducer and / or an anti-cancer immunotherapeutic agent) as defined above and optional pharmaceutical adjuvants in a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, e.g., acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.

[0064] Elixirs are clear, sweetened, aqueous alcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated sugar (e.g., sucrose) solutions into which the active ingredients are dissolved or suspended. Emulsions are two-phase systems in which one liquid is dispersed in the other. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used for non-effervescent granules, which are to be reconstituted prior to administration, include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substances used for effervescent granules, which are to be reconstituted prior to administration, include organic acids and sources of carbon dioxide. Coloring agents and flavoring agents are used in all of the above dosage forms.

[0065] Solvents include glycerin, sorbitol, ethanol, and syrup. Examples of preservatives include glycerin, methyl- and propyl-parabens, benzoic acid, sodium benzoate, and ethanol. Examples of nonaqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum, and acacia. Sweetening agents include sucrose, syrups, glycerin, and artificial sweetening agents such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acids. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents include natural flavors extracted from plants such as fruits, and synthetic blends of compounds which produce a pleasant taste sensation.

[0066] For solid dosage forms, in one embodiment, solutions or suspensions of the active compound, e.g., in propylene carbonate, vegetable oils, or triglycerides, are encapsulated in gelatin capsules. For liquid dosage forms, solutions of the active compound, e.g., in polyethylene glycol, can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, e.g., water, to enable easy measurement in amounts suitable for administration.

[0067] Alternatively, liquid or semisolid oral formulations can be prepared by dissolving or dispersing the active compound (i.e., copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent) or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate), and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those described in U.S. Pat. Nos. RE 28,819 and 4,358,603. Briefly, such formulations include, but are not limited to, those containing a compound provided herein, a dialkylated mono- or polyalkylene glycol, including, but not limited to, 1,2-dimethoxy methane, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether (where 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol), and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarin, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters and dithioaminoo acids.

[0068] Other formulations include, but are not limited to, aqueous alcoholic solutions comprising a pharmaceutically acceptable acetal. The alcohol used in these formulations is any pharmaceutically acceptable water-miscible solvent having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes, such as acetaldehyde diethyl acetal. Injectables, solutions, and emulsions

[0069] Parenteral administration is also contemplated herein, in one embodiment characterized by subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions further comprise one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. Additionally, if desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0070] Implantable slow- or sustained-release systems are also contemplated herein to maintain a constant dosage level. Briefly, the compounds provided herein (i.e., copper chelators, copper chelator inducers, and / or anti-cancer immunotherapeutics) are dispersed in a solid internal matrix, such as polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, organosilicon carbonate copolymer, hydrogels of hydrophilic polymers such as esters of acrylic and methacrylic acids, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate, which is surrounded by an external polymeric membrane that is insoluble in body fluids, such as polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, chlorobutyl rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of chloroethylene with vinyl acetate, partial vinylidene chloride, copolymers of ethylene and propylene, ionomeric polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / ethylene vinyl alcohol copolymer, ethylene / vinyl acetate / ethylene vinyl alcohol terpolymer, and ethylene / ethyleneoxy ethanol copolymer. The compounds diffuse through the external polymeric membrane in a rate-controlling step. The percentage of active compound included in such parenteral compositions depends highly on its particular nature, as well as the activity of the compound and the needs of the subject.

[0071] Parenteral administration of the compositions includes intravenous, subcutaneous, and intramuscular administration. The articles for parenteral administration include sterile solutions of the injectable type ready for use; sterile dry soluble products (e.g., lyophilized powders) which can be combined directly with a solvent immediately before use, including tablets for subcutaneous injection; sterile suspensions of the injectable type ready for use; sterile dry insoluble products which can be combined with a vehicle immediately before use, and sterile emulsions. The solutions can be aqueous or non-aqueous solutions.

[0072] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0073] Pharmaceutically acceptable carriers used in the parenteral articles include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances.

[0074] Examples of aqueous vehicles include sodium chloride injection, Ringers Injection, isotonic dextrose injection, sterile water injection, dextrose in lactated Ringers injection. Non-aqueous parenteral vehicles include non-volatile oils of vegetable origin, olive oil, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents, either preventing or inhibiting the growth of microorganisms, necessary for preventing a microbial growth in the parenteral articles packaged in multiple-dose containers include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN 80). Sequestering or chelating agents include ethylenediaminetetraacetic acid (EDTA). Pharmaceutical carriers further include ethanol, polyethylene glycol, and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric or lactic acid for pH adjustment.

[0075] The concentration of the pharmaceutically active compounds (i.e., copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent) is adjusted so that the injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight, and condition of the patient or animal, as is known in the art.

[0076] Unit-dose parenteral articles can be packaged in ampules, vials, or prefilled syringes. All articles for parenteral administration must be sterile, as is known and practiced in the art.

[0077] For example, intravenous or intraarterial infusion of a sterile aqueous solution containing the active compound (i.e., copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent) is an effective mode of administration. Another embodiment is a sterile aqueous or oleaginous solution or suspension containing the active substance which is injected as needed to produce the desired pharmacological effect.

[0078] Injectables are designed for local as well as systemic administration. In one embodiment, therapeutically effective dosages are formulated to contain concentrations of active compound (i.e., copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent) in the tissue to be treated of at least about 0.1% w / w up to about 90% w / w or more, in certain embodiments, in excess of 1% w / w.

[0079] The copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent can be suspended in micronized or other suitable form, or can be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent in the selected carrier or vehicle. Effective concentrations are sufficient to ameliorate the symptoms of the condition being treated, and can be determined empirically. Lyophilized powders

[0080] Also of interest herein are lyophilized powders, which can be reconstituted into solutions, emulsions, and other mixtures for administration. They can also be reconstituted and formulated as solids or gels.

[0081] A sterile lyophilized powder can be prepared by dissolving a copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. The solvent can contain an excipient that improves the stability of the powder or the reconstituted solution prepared from the powder, or other pharmacological components. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent can also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art, in one embodiment, at a pH of about neutral. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art, provides the desired formulation. In one embodiment, the resulting solution will be aliquoted into vials for lyophilization. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as at about 4°C to room temperature.

[0082] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. To reconstitute, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound chosen. Such amounts can be determined empirically. Topical administration

[0083] The topical mixtures are prepared as described for topical and systemic administration. The resulting mixture can be a solution, suspension, emulsion, or the like, and is formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, enema, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0084] The copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent, or pharmaceutically acceptable derivatives thereof, can be formulated as an aerosol for topical application (e.g., by inhalation). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as microfine powders for insufflation, alone or in combination with an inert carrier such as lactose. In this case, the diameter of the particles of the formulation is, in an embodiment, less than 50 microns, and, in an embodiment, less than 10 microns.

[0085] The copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent can be formulated for topical or external application, for example, for topical application to the skin and mucosa (e.g., in the eye), in the form of gels, creams, and lotions, as well as for ophthalmic applications or for intracisternal or intraspinal applications. Topical administration is contemplated for transdermal delivery, and can also be used for ocular or mucosal administration, or for inhalation therapy. Nasal solutions of the active compounds alone (i.e., the copper chelator, copper chelator inducer, and / or anti-cancer immunotherapeutic agent) or in combination with other pharmaceutically acceptable excipients can also be administered.

[0086] These solutions, particularly those intended for ophthalmic use, can be formulated as 0.01% - 10% (vol.%) isotonic solutions with appropriate salts, at a pH of about 5-7. Compositions for other routes of administration

[0087] Other routes of administration are also contemplated herein, such as transdermal patches, including iontophoresis and electrophoresis devices, vaginal and rectal administration.

[0088] Transdermal patches, including iontophoresis and electrophoresis devices, are well known to those skilled in the art. For example, pharmaceutical dosage forms for rectal administration include rectal suppositories, capsules, and tablets for systemic action. As used herein, a rectal suppository refers to a solid for insertion into the rectum that melts or softens at body temperature, releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are matrices or mediators and reagents that increase the melting point. Examples of matrices include cocoa butter, glycerin gelatin, carbowax (polyethylene glycol), and suitable mixtures of monoglycerides, diglycerides, and triglycerides of fatty acids. Combinations of multiple matrices can be used. Reagents that increase the melting point of suppositories include cetylene and waxes. Rectal suppositories can be prepared by compression or molding. In one embodiment, the rectal suppository weighs about 2-3 grams.

[0089] Tablets and capsules for rectal administration are prepared using the same pharmaceutically acceptable substances and the same methods as those for oral administration. Targeted formulations

[0090] The copper chelating agents, copper chelating agent inducers, and / or anticancer immunotherapeutic agents or their pharmaceutically acceptable derivatives provided herein can also be formulated to target specific tissues, receptors, or other areas of the body of a subject to be treated. Many such targeting methods are well known to those skilled in the art. All of these targeting methods are considered herein for use in the compositions of the present invention.

[0091] In one embodiment, liposome suspensions, including tissue-targeting liposomes such as tumor-targeting liposomes, are also suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in U.S. Patent No. 4,522,811. Briefly, liposomes such as multilayer vesicles (MLVs) can be formed by drying egg phosphatidylcholine and cephalinositol serine (7:3 molar ratio) on the inner wall of a flask. A solution of the compound provided herein in phosphate-buffered saline (PBS) lacking divalent cations is added, and the flask is shaken until the lipid membrane is dispersed. The resulting vesicles are washed to remove unencapsulated compounds, precipitated by centrifugation, and then resuspended in PBS.

[0092] Co-administration with other drugs According to another aspect of the invention, it is contemplated that copper chelators, copper chelator inducers, and / or anticancer immunotherapeutic agents as described herein can be administered in combination with medicines that are considered by those skilled in the art to be current standards of care for the target condition to subjects in need. Such combinations provide subjects with one or more advantages, such as requiring a reduced dose to achieve similar benefits, obtaining the desired remission in a shorter time, etc.

[0093] The copper chelator, copper chelator inducer and / or anti-cancer immunotherapeutic agent according to the application can be administered as part of a therapeutic regimen with other drugs. For example, administration of a combination of active compounds can be desirable in order to treat a particular disease or condition. Thus, two or more pharmaceutical compositions can be combined in a kit suitable for coadministration of the compositions, falling within the scope of the application, wherein at least one of the pharmaceutical compositions comprises a copper chelator, copper chelator inducer and / or anti-cancer immunotherapeutic agent according to the application.

[0094] In one embodiment, the copper chelator, copper chelator inducer and / or anti-cancer immunotherapeutic agent can be administered with a second therapeutic agent. In one embodiment, the second therapeutic agent is an anti-cancer agent.

[0095] When two or more active ingredients are coadministered, the active ingredients can be administered simultaneously, sequentially or separately. In one embodiment, the copper chelator, copper chelator inducer and / or anti-cancer immunotherapeutic agent is coadministered with another therapeutic agent simultaneously. In another embodiment, the copper chelator, copper chelator inducer and / or anti-cancer immunotherapeutic agent is administered sequentially with another therapeutic agent. In a further embodiment, the copper chelator, copper chelator inducer and / or anti-cancer immunotherapeutic agent is administered separately with another therapeutic agent.

[0096] In an eighth aspect of the application, there is provided a pharmaceutical composition comprising a copper chelator or copper chelator inducer and an anti-cancer immunotherapeutic agent.

[0097] The following options can be used in conjunction with the eighth aspect, either individually or in any combination.

[0098] The pharmaceutical composition can comprise a pharmaceutically acceptable carrier or excipient.

[0099] The copper chelator, copper chelator inducer, anti-cancer immunotherapeutic agent and / or pharmaceutical composition can be as described above in relation to the first, second, third, fourth, fifth, sixth or seventh aspect.

[0100] The pharmaceutical composition of the eighth aspect can be used in the method of the first aspect. The method of the first aspect can use the pharmaceutical composition of the eighth aspect.

[0101] The pharmaceutical composition of the eighth aspect can be used in the use of the second aspect. The use of the second aspect can use the pharmaceutical composition of the eighth aspect.

[0102] The pharmaceutical composition of the eighth aspect can be used in the use of the third aspect. The use of the third aspect can use the pharmaceutical composition of the eighth aspect.

[0103] The pharmaceutical composition of the eighth aspect can be used in the uses of the fourth aspect. The uses of the fourth aspect can use the pharmaceutical composition of the eighth aspect.

[0104] The pharmaceutical composition of the eighth aspect can comprise the copper chelator or copper chelator inducer of the fifth aspect. The copper chelator or copper chelator inducer of the fifth aspect can be a component of the pharmaceutical composition of the eighth aspect.

[0105] The pharmaceutical composition of the eighth aspect can comprise the anti-cancer immunotherapeutic of the sixth aspect. The anti-cancer immunotherapeutic of the sixth aspect can be a component of the pharmaceutical composition of the eighth aspect.

[0106] The pharmaceutical composition of the eighth aspect can comprise the kit or combination of the seventh aspect. The kit or combination of the seventh aspect can be a component of the pharmaceutical composition of the eighth aspect.

[0107] Disclosed herein are the following forms: 1. A method of enhancing an anti-cancer immune response and efficacy of an immunotherapy in a subject having a tumor, the method comprising administering to the subject a copper chelator in an amount effective to chelate copper from a tumor microenvironment, thereby reducing copper-mediated immune suppression and enhancing an anti-cancer immune response and efficacy of an immunotherapy against the tumor.

[0108] 2. The method of Form 1, wherein the copper chelator is selected from the group consisting of tetrathiomolybdate, trientine, penicillamine, D-penicillamine, and zinc salts.

[0109] 3. The method of Form 1, wherein the subject has a solid tumor or a hematological malignancy.

[0110] 4. The method of Form 1, wherein the immunotherapy is selected from the group consisting of an immune checkpoint inhibitor (e.g., an ICI targeting a-CTLA4 and / or an ICI targeting a-PDL1), a chimeric antigen receptor (CAR) T cell therapy, a bispecific T cell engager (BiTE), a (CAR) NK cell therapy, an adoptive cell therapy, and antibody-dependent cellular cytotoxicity (ADCC).

[0111] 5. The method of Form 1, wherein the copper chelator is administered prior to, concurrently with, or after the immunotherapy.

[0112] 6. A pharmaceutical composition comprising a copper chelator and a pharmaceutically acceptable carrier or excipient, wherein the copper chelator is present in an amount effective to chelate copper from a tumor microenvironment, thereby reducing copper-mediated immune suppression and enhancing an anti-cancer immune response and efficacy of an immunotherapy against a tumor.

[0113] 7. The pharmaceutical composition of Form 6, wherein the copper-chelating agent is selected from the group consisting of tetrathiomolybdate, trientine, penicillamine, D-penicillamine, and zinc salts.

[0114] 8. The pharmaceutical composition of Form 6, further comprising an immunotherapy selected from the group consisting of immune checkpoint inhibitors (e.g., ICI targeting a-CTLA4 and / or ICI targeting a-PDL1), chimeric antigen receptor (CAR) T cell therapy, bispecific T cell engager (BiTE), (CAR) NK cell therapy, adoptive cell therapy, and antibody-dependent cellular cytotoxicity (ADCC).

[0115] 9. A method of treating a subject having a tumor, the method comprising administering to the subject an amount of the pharmaceutical composition of Form 6 effective to chelate copper from the tumor microenvironment, thereby reducing copper-mediated immune suppression and enhancing an anti-cancer immune response and the efficacy of an immunotherapy against the tumor.

[0116] 10. The method of Form 9, wherein the subject has a solid tumor or a hematological malignancy.

[0117] 11. The method of Form 9, wherein the immunotherapy is selected from the group consisting of immune checkpoint inhibitors (e.g., ICI targeting a-CTLA4 and / or ICI targeting a-PDL1), chimeric antigen receptor (CAR) T cell therapy, bispecific T cell engager (BiTE), (CAR) NK cell therapy, adoptive cell therapy, and antibody-dependent cellular cytotoxicity (ADCC).

[0118] 12. The method of Form 9, wherein the copper-chelating agent is administered prior to, concurrently with, or after the immunotherapy.

[0119] 13. A method of enhancing an anti-cancer immune response and the efficacy of an immunotherapy in a subject having a tumor, the method comprising administering to the subject an amount of a copper-chelating agent effective to chelate copper from the tumor microenvironment, thereby reducing copper-mediated immune suppression, enhancing an anti-cancer immune response, and activating a myeloid cell response against the tumor.

[0120] 14. The method of Form 13, wherein the myeloid cell response is mediated by dendritic cells, macrophages, or neutrophils.

[0121] 15. The method of Form 13, wherein the copper-chelating agent is selected from the group consisting of tetrathiomolybdate, trientine, penicillamine, D-penicillamine, and zinc salts.

[0122] 16. The method of Form 13, wherein the subject has a solid tumor or a hematological malignancy.

[0123] 17. The method of Form 13, wherein the immunotherapy is selected from the group consisting of immune checkpoint inhibitors (e.g., ICI targeting a-CTLA4 and / or ICI targeting a-PDL1), chimeric antigen receptor (CAR) T cell therapy, bispecific T cell engager (BiTE), (CAR) NK cell therapy, adoptive cell therapy, and antibody-dependent cellular cytotoxicity (ADCC).

[0124] 18. The method of Form 13, wherein the copper chelator is administered prior to, concurrently with, or after the immunotherapy.

[0125] 19. A pharmaceutical composition comprising a copper chelator and a pharmaceutically acceptable carrier or excipient, wherein the copper chelator is present in an amount effective to chelate copper from a tumor microenvironment, thereby reducing copper-mediated immune suppression, enhancing an anti-cancer immune response, and activating a myeloid cell response against a tumor.

[0126] 20. The pharmaceutical composition of Form 19, wherein the myeloid cell response is mediated by dendritic cells, macrophages, or neutrophils.

[0127] 21. The pharmaceutical composition of Form 19, wherein the copper chelator is selected from the group consisting of tetrathiomolybdate, trientine, penicillamine, D-penicillamine, and zinc salts.

[0128] 22. The pharmaceutical composition of Form 19, further comprising an immunotherapy selected from the group consisting of immune checkpoint inhibitors (e.g., ICI targeting a-CTLA4 and / or ICI targeting a-PDL1), chimeric antigen receptor (CAR) T cell therapy, bispecific T cell engager (BiTE), (CAR) NK cell therapy, adoptive cell therapy, and antibody-dependent cellular cytotoxicity (ADCC).

[0129] 23. A method of treating a subject having a tumor, the method comprising administering to the subject a pharmaceutical composition of Form 19 in an amount effective to chelate copper from a tumor microenvironment, thereby reducing copper-mediated immune suppression, enhancing an anti-cancer immune response, and activating a myeloid cell response against the tumor.

[0130] 24. The method of Form 23, wherein the myeloid cell response is mediated by dendritic cells, macrophages, or neutrophils.

[0131] 25. The method of Form 23, wherein the subject has a solid tumor or a hematological malignancy.

[0132] 26. The method of Form 23, wherein the immunotherapy is selected from the group consisting of an immune checkpoint inhibitor (e.g., an ICI targeting a-CTLA4 and / or an ICI targeting a-PDLl), a chimeric antigen receptor (CAR) T cell therapy, a bispecific T cell engager (BiTE), a (CAR) NK cell therapy, an adoptive cell therapy, and antibody-dependent cellular cytotoxicity (ADCC).

[0133] 27. The method of Form 23, wherein the copper chelator is administered prior to, concurrently with, or after the immunotherapy.

[0134] 28. A method of enhancing the efficacy of antibody-dependent cellular cytotoxicity (ADCC) in a subject having a tumor, the method comprising administering to the subject a copper chelator in an amount effective to chelate copper from the tumor microenvironment, thereby reducing copper-mediated immunosuppression, enhancing an anti-cancer immune response, and enhancing the efficacy of ADCC.

[0135] 29. The method of Form 28, wherein the copper chelator is selected from the group consisting of tetrathiomolybdate, trientine, penicillamine, D-penicillamine, and a zinc salt.

[0136] 30. The method of Form 28, wherein the subject has a solid tumor or a hematologic malignancy.

[0137] 31. The method of Form 28, wherein ADCC is mediated by natural killer (NK) cells, monocytes, or macrophages.

[0138] 32. The method of Form 28, wherein the copper chelator is administered prior to, concurrently with, or after administration of an antibody used in ADCC.

[0139] 33. A pharmaceutical composition comprising a copper chelator and a pharmaceutically acceptable carrier or excipient, wherein the copper chelator is present in an amount effective to chelate copper from the tumor microenvironment, thereby reducing copper-mediated immunosuppression, enhancing an anti-cancer immune response, and enhancing the efficacy of ADCC.

[0140] 34. The pharmaceutical composition of Form 33, wherein the copper chelator is selected from the group consisting of tetrathiomolybdate, trientine, penicillamine, D-penicillamine, and a zinc salt.

[0141] 35. The pharmaceutical composition of Form 33, further comprising an antibody that mediates ADCC.

[0142] 36. The pharmaceutical composition of Form 33, wherein ADCC is mediated by natural killer (NK) cells, monocytes, or macrophages.

[0143] 37. A method of treating a subject with a tumor, the method comprising administering to the subject an amount of the pharmaceutical composition according to form 33 that effectively chelates copper from the tumor microenvironment, thereby reducing copper-mediated immunosuppression, enhancing anticancer immune responses, and enhancing the efficacy of ADCC.

[0144] 38. The method according to form 37, wherein the copper chelating agent is selected from the group consisting of tetrathiomolybdate, trientine, penicillamine, D-penicillamine and zinc salt.

[0145] 39. The method according to form 37, wherein the subject has a solid tumor or a malignant hematologic disease.

[0146] 40. The method according to form 37, wherein ADCC is mediated by natural killer (NK) cells, monocytes, or macrophages.

[0147] 41. The method according to form 37, wherein the copper chelating agent is applied before, simultaneously with, or after the application of the antibody used in ADCC. Brief description of the attached diagram

[0148] The patent or application document contains at least one color drawing. A copy of the patent or patent application publication and the color drawing will be provided by the patent office upon request and payment of the necessary fees (if appropriate).

[0149] Figure 1 Illustration of the tumor microenvironment.

[0150] Figure 2 TEPA Cu 2+ Formation of complexes.

[0151] Figure 3 Illustration of Embodiment 1 disclosed herein.

[0152] Figure 4 Single-cell RNA sequencing of tumor cells.

[0153] Figure 5 : A graph showing the downregulation of the MYCN oncogene.

[0154] Figure 6 Single-cell RNA sequencing of infiltrating immune cells. The figure also shows that copper chelation induced a nearly 5-fold increase in neutrophil infiltration into the tumor microenvironment (the most abundant type of leukocyte in circulation).

[0155] Figure 7 Heatmap showing that the main gene responsible for copper uptake is highly expressed only in neutrophils.

[0156] Figure 8GSEA analysis indicates that copper chelation polarizes infiltrating neutrophils towards the "N1" phenotype.

[0157] Figure 9 Opal multiplex analysis.

[0158] Figure 10 Opal multiplex analysis summary shows that in addition to increased NCR1+ natural killer cells and CD8+ cytotoxic T cells, there is also increased infiltration of CD11B+ myeloid cells into the tumor microenvironment with copper chelation.

[0159] Figure 11 Single cell RNAseq network.

[0160] Figure 12 Cytokine assay shows that copper chelation increases levels of pro-inflammatory cytokines in the tumor.

[0161] Figure 13 In vitro model of tumor cell (SK-N-BE(2)C) / CD45+ immune cell co-culture. Figure 13 A shows that the copper chelator TEPA removes copper from tumor cells (SK-N-BE(2)C) and it is released into the culture medium where it is completely taken up by CD45+ immune cells. Figure 13 B shows that neutrophils are able to take up copper released by cancer cells in response to copper chelation therapy in the culture medium.

[0162] Figure 14 Tissue sections were stained with copper-specific probes to determine the intracellular copper distribution on a single cell basis. This experiment shows that after copper chelation treatment, copper is decreased on the tumor and increased in immune cells.

[0163] Figure 15 Dosing regimen for TEPA or saline of Example 3 and vehicle, IgG or anti-GD2 moAb. TEPA was first given as a single agent at 400 mg / kg / day for 5 days (pre-treatment); thereafter five 7-day treatment cycles were performed in which TEPA 400 mg / kg / day was administered for five days and anti-GD2 was administered in a combined fashion on the first and fifth day. Treatment was stopped after the fifth 7-day cycle.

[0164] Figure 16 Opal multiplex immunohistochemistry of Example 3.

[0165] Figure 17 Opal multiplex immunohistochemistry shows that CD8 / NCR1 / CD11B infiltration is increased when TEPA is combined with anti-GD2 therapy.

[0166] Figure 18Survival (A) and body weight (B) of mice of Example 3.

[0167] Figure 19 Structural comparison of TEPA and TETA and their complexes with Cu 2+

[0168] Figure 20 Dose regimen of TETA or physiological saline and vehicle, IgG or anti-GD2 moAb of Example 4.

[0169] Figure 21 Opal multiplex immunohistochemistry of Example 4.

[0170] Figure 22 Opal multiplex immunohistochemistry shows increased CD8 / NCR1 / CD11B infiltration when TETA is combined with anti-GD2 therapy.

[0171] Figure 23 Survival (A) and body weight (B) of mice of Example 4.

[0172] Figure 24 Structure of TETA Cu 2+ complexes.

[0173] Figure 25 Equilibrium state of TETA as a function of pH.

[0174] Figure 26 Structure of TEPA Cu 2+ complexes.

[0175] Figure 27 Copper chelation enhances anti-tumor activity of anti-GD2 immunotherapy. a) Experimental design and dosing strategy (IP, intraperitoneal; p.o., oral gavage). b) Th-MYCN Kaplan-Meier survival curves of mice (n = 10 / group). c) Statistical comparison of survival curves given in b using the Mantel-Cox log-rank test (two-tailed). d) Representative images of pooled OPAL multiplex immunofluorescence profiles depicting tumor distribution of NCR1+ natural killer cells (red), CD8+ cytotoxic T cells (yellow) and CD11b+ myeloid cells (white) 14 days post-treatment. Th-MYCN Tumor distribution of NCR1+ natural killer cells (red), CD8+ cytotoxic T cells (yellow) and CD11b+ myeloid cells (white) in neuroblastoma tumor tissue. Scale bar, 100 µm. e) Quantification of immune cells from d as positive counts per 1000 nuclei. Data presented as mean ± SEM (n = 3-4 / group). Significance was calculated using the one-tailed Mann-Whitney U test (*p < 0.005, **p < 0.01, ***p < 0.001, ns = not significant). Abbreviation: moAb, monoclonal antibody.​

[0176] Figure 28 Copper chelation promotes an immune-permissive tumor microenvironment. a) Experimental design and dosing strategy. b) From control and TEPA treatment. Th-MYCN Cytokine levels in tumor lysates obtained from mice. Data are presented as mean ± SEM (n = 4–11 / group). Significance was calculated using the Mann-Whitney U test (*p ≤ 0.05; **p ≤ 0.01). c) Representative images of combined OPAL multiplex immunofluorescence spectra depicting the results after one week of treatment. Th-MYCN Distribution of NCR1+ natural killer cells (red), CD8+ cytotoxic T cells (yellow), and CD11b+ myeloid cells (white) in neuroblastoma tumor tissue. Scale bar, 100 µm. Immunocytocyte quantification in figure e)c is based on counts per 1000 nuclei. Data are presented as mean ± SEM (n = 3–5 / group). Significance was calculated using the Mann-Whitney U test (*p ≤ 0.05).

[0177] Figure 29 Copper chelation restores anti-tumor immunity through pro-inflammatory signaling. a) Control group and TEPA-treated group resected after 7 days. Th-MYCN Representative images of the tumor core, stained with fluorescently conjugated antibodies for PanCK (red), smooth muscle actin (yellow), and CD45 (green), and stained with DAPI nuclear dye. Scale bar, 300 µm. (b) Th-MYCN Sankey plot of the tumor core, depicting the proportion of target regions with no / true CD45 invasion, plotted relative to treatment group and duration. c) Volcano plot of genes upregulated and downregulated in invasive and non-invasive target regions treated with TEPA (log2 fold change (FC) vs. log10 p-value). p, false discovery rate (FDR), adjusted with Benjamini-Hochberg correction. Threshold: p < 0.1; |Log2FC| > 0.5. d) Overexpression analysis of relatively enriched pathways in invasive tumor regions treated with TEPA compared to non-invasive regions, presented as nodes with related genes as branches. e) Bar chart visualization of the data presented in d.

[0178] Figure 30 The neuroblastoma tumor microenvironment is sensitive to copper chelation therapy and promotes neutrophil infiltration. a) Schematic diagram of the experimental design and tumor treatment workflow for single-cell RNA sequencing. b) Uniform manifold approximation and projection (UMAP) representation of tumor compartments (13,544 cells), stained by treatment group. c) Relationship with intracellular copper levels ( Mt1 , Mt2) and neuroblastoma oncogene Mycn Violin plots of relevant gene expression levels, split by treatment group; ***p < 0.001. Horizontal lines represent median of data.d) Features enriched by gene set enrichment analysis MYC Enrichment plot of the Target_V1 gene set.e) Split UMAP representation of immune cell compartments (12,127 cells total) according to treatment group, colored by annotated immune subsets (n = 13).f) Bar plot of the proportion of immune cell subsets shown in e.g) Dot plot of relevant gene expression markers used to classify immune subsets defined in e.

[0179] Figure 31 Neutrophil replacement tumorogenic signaling drives restoration of anti-tumor immunity.a) Overexpression analysis of pathways relatively enriched in immune cell clusters of TEPA-treated compared to control, presented using single-cell RNA sequencing with relevant genes as nodes of branches.b) Circular network plot of significantly cell-cell interaction pathways in control and TEPA-treated tumor data sets. Width of edges represents strength of interaction, and thickness of edges is proportional to signal strength.c) Scatter plot comparing outgoing and incoming interaction strength between control and TEPA-treated samples.

[0180] Figure 32 Copper chelation promotes neutrophil egress and N1 polarization through copper mobilization.a) Heat map comparing mean expression of copper metabolism-related genes within immune cell clusters across treatment groups.b) Heat map comparing mean expression of genes screened to form N1 (anti-tumor) and N2 (pro-tumor) neutrophil phenotype signatures. Data presented in a and b plots were obtained from single-cell RNA sequencing, where relevant cell values were averaged and scaled by row (z-score).c) Gene set enrichment analysis showing top-ranked pathways relatively enriched in TEPA-treated neutrophils. The “N1 anti-tumor neutrophil” signature was constructed using N1 -related genes listed in b.d) Flow cytometry plots comparing neutrophil polarization in control and TEPA-treated mice seven days after treatment. Th- MYCNCount and percentage of circulating neutrophils obtained in mice. Data are expressed as mean ± SEM (n = 4 / group). Significance was calculated using Mann-Whitney U test (*p < 0.05). e) IncuCyte cell imaging (10x objective) of neuroblastoma cell line SK-N-BE(2)-C transfected with plasmid encoding turbo green fluorescent protein (tGFP) tagged MT1X protein after 24h of TEPA treatment. Scale bar, 100 pm. f) Concentration of copper in the conditioned medium before and after incubation with naïve neutrophils isolated from a healthy donor for 30 min. Data are expressed as mean ± SEM (n = 3). Significance was calculated using paired t-test (*p < 0.05).

[0181] Figure 33: Copper chelator TETA synergizes with anti-GD2 therapy to mediate anti-tumor activity in neuroblastoma syngeneic NXS2 model. a) Tumor growth kinetics in a neuroblastoma syngeneic model involving subcutaneous inoculation of A / J mice with NXS2 cells. Animals were treated starting one week post-inoculation (black arrows) and treated with saline (control) or TETA (400 mg / kg / day) for 7 consecutive days by oral gavage, then blood and tumors were collected (n = 4 / group). Significance was calculated using Mann-Whitney U test at each time point (*p < 0.05). Flow cytometry analysis was performed on freshly dissociated tumors, which were gated for b) neutrophil frequency and c) proportion of cells expressing MMP9 (indicative of N1 proinflammatory phenotype). Significance was calculated using Mann-Whitney U test (*p < 0.05). d) Experimental design and immunocombination dosing regimen of the syngeneic NXS2 -> A / J preclinical model. e-g Arrows indicate treatment period. e) Relative body weight change of tumor-bearing mice measured from the inoculation date. f) Relative tumor volume measured from the inoculation date. g) Survival curve of tumor-bearing mice measured from the inoculation date. h) Statistical pairwise comparison of survival curves in g using Mantel-Cox log-rank test (two-tailed). Data are representative of one experiment (n = 8-10 / group). i) Representative images of merged OPAL multiplex immunofluorescence spectra depicting tumor distribution of NCR1+ natural killer cells (red), CD8+ cytotoxic T cells (yellow) and CD11b+ myeloid cells (white) in NXS2 neuroblastoma tumor tissue 14 days post-treatment. Scale bar, 100 pm. j) Quantification of immune cells in i as counts per 1000 nuclei (n = 3-4 / group). Significance was calculated using one-tailed Mann-Whitney U test (*p < 0.005, **p < 0.01, ***p < 0.001, ns: not significant). Abbreviations: IP, intraperitoneal; moAb, monoclonal antibody; p.o., oral gavage.

[0182] Figure 34 : Gene set enrichment analysis of the following immune clusters determined by single-cell RNA sequencing: a) B cells, b) basophils, c) CD4+ naive T cells, d) CD4+ memory T cells, e) CD8+ naive T cells, f) CD8+ NKT-like cells, g) dendritic cells, h) double negative regulatory T cells, i) macrophages, j) eosinophils, and k) natural killer cells. The gamma-delta T cell cluster was excluded from the analysis due to low cell numbers.

[0183] Figure 35The top-ranked pathways were identified through gene ensemble enrichment analysis of immune clusters determined by single-cell RNA sequencing. The bar chart shows the enrichment pathways in infiltrating immune cells in TEPA-treated tumors compared to control tumors. The bars are associated with individual immune clusters, as indicated in the legend.

[0184] Figure 36 Heatmap of the leading signal transduction networks contributing to efferent or afferent signal transduction in tumors of different immune cell clusters in a) control and b) TEPA-treated tumors. Relative signal intensity is indicated by green shading. The horizontal bars at the top represent the amount of efferent (left) and afferent (right) interactions for each immune cell cluster, and the vertical bars on the right represent the relative intensity of a specific signal transduction network.

[0185] Figure 37 The dot plot shows the most significant a) efferent and b) afferent interactions between neutrophils and other immune cell clusters between the control group (grey) and the TEPA-treated group (red), represented as ligand-receptor pairs.

[0186] Figure 38 Tumor immune infiltration is associated with higher transcriptional levels of the copper export protein ATP7A. Box-and-whisker plots of ATP7A per million transcripts (TPM) and their respective correlations with Pediatric Solid Cancer Immunopediatric Characteristic Score (IPASS) status are presented.

[0187] Figure 39 Copper chelator TETA is a safe and non-toxic treatment strategy. One week after treatment, hematological analyses were performed on metabolites associated with a) hepatic, b) renal, and c) systemic toxicity. Data are presented as mean ± SEM (n = 3 / group). Significance (ns, not significant) was calculated using the Mann-Whitney U test.

[0188] Figure 40 a) Used for Th-MYCN a) Flow cytometry gating strategies for single-cell RNA sequencing in tumors to illustrate the selection of surviving NK1.1+ natural killer cells, CD3+ T cells, CD11b+ myeloid cells, and tumor compartments. b) Gating strategies used to determine the frequency of surviving CD45+CD11b+Ly6G+ neutrophils in preclinical NXS2 tumors.

[0189] Figure 41EZH2 inhibition is an exciting emerging pathway to increase GD2 antigen expression in neuroblastoma and Ewing's sarcoma. The copper chelator TEPA strongly downregulated EZH2 in diffuse midline glioma (DMG) invasive pediatric brain cancer (a) and neuroblastoma cell lines (b). Transforming this work in vivo, flow cytometry revealed that, one week after treatment, TEPA significantly reduced EZH2 expression in... Th-MYCN GD2 expression was strongly upregulated in neuroblastoma tumors (c). Interestingly, preliminary data from a glioblastoma xenograft model suggest that one week of TEPA pretreatment was comparable to GD2-targeted CAR T cells (1x10⁻¹²). 6 The combination of intravenous (IV, single-dose) injections per cell had a synergistic effect in reducing tumor growth (d). These data strongly suggest that copper chelation can epigenetically regulate GD2 antigen expression and thereby enhance sensitivity to GD2 CAR T immunotherapy. Importantly, these results support the idea that by reducing intratumoral copper, we can simultaneously reduce immune evasion mechanisms in the tumor microenvironment (TME) while increasing the target of GD2-CAR T cells in cancer cells.

[0190] Figure 42 Dosage regimen for copper chelation therapy in a mesothelioma model. Animals received oral TETA at a daily dose of 800 mg / kg for 2 weeks, followed by administration every other day until the end of the experiment.

[0191] Figure 43 Tumor growth data of the AB1-HA mesothelioma model treated with a combination of immune checkpoint inhibitors (ICIs) and copper chelators (TETAs): a) PBS alone; b) ICIs targeting α-CTLA4 and α-PDL1; c) TETA alone; d) a combination of TETA, an ICI targeting α-CTLA4, and an ICI targeting α-PDL1.

[0192] Figure 44 Animal survival data of the AB1-HA mesothelioma model treated with a combination of immune checkpoint inhibitors (ICIs) and copper chelators (TETAs).

[0193] Figure 45 Tumor growth data of the AE17-OVA mesothelioma model treated with a combination of immune checkpoint inhibitors (ICIs) and copper chelators (TETAs): a) PBS alone; b) ICIs targeting α-CTLA4 and α-PDL1; c) TETA alone; d) a combination of TETA, an ICI targeting α-CTLA4, and an ICI targeting α-PDL1.

[0194] Figure 46Animal survival data for AE17-OVA mesothelioma model treated with immune checkpoint inhibitor (ICI) and copper chelator (TETA) combination.

[0195] Figure 47 Flow cytometry results using PBS or TETA showing the proportion of the following cells as a percentage of live CD45+ve: CD3-ve (top panel) and CD3+ve (bottom panel).

[0196] Figure 48 Flow cytometry results using PBS or TETA showing the proportion of the following cells as a percentage of live CD45+ve: CD4+ve (top panel) and CD8+ve (bottom panel).

[0197] Figure 49 Flow cytometry results using PBS or TETA showing the proportion of the following cells as a percentage of live CD45+ve: CD4+ve (top panel) and FOXP3 CD25+ve (bottom panel).

[0198] Figure 50 Flow cytometry results using PBS or TETA showing the proportion of the following cells as a percentage of live CD45+ve: NKP46+ve (top panel) and Ly6G CD11b+ve (bottom panel).

[0199] Figure 51 Flow cytometry results using PBS or TETA showing the proportion of the following cells as a percentage of live CD45+ve: NKP46+ve (top panel) and NKP46 CD11b+ve (bottom panel). Definitions

[0200] In describing and claiming the present application, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the application only and is not intended to be limiting.

[0201] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0202] Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". As used herein, the terms "comprises", "comprising", or the like can be used to describe a general process, method, or composition which can include, but is not limited to, a number of elements or steps.

[0203] The transitional phrase "composed of..." excludes any unspecified element, step, or component. If present in a claim, this would exclude materials other than those listed, except for impurities typically associated with them. When the phrase "composed of..." appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the elements listed in that clause; other elements are not excluded from the claim as a whole.

[0204] The transitional phrase "consistent primarily of..." is used to define a composition, process, or method that includes additional materials, steps, features, components, or elements beyond those literally disclosed, provided that such additional materials, steps, features, components, or elements do not materially affect the essential and novel features of the claimed invention. The term "consistent primarily of..." falls in the middle between "comprising" and "consisting of...".

[0205] Where the applicant defines the invention or a part thereof using open-ended terms such as “comprising”, it should be readily understood (unless otherwise stated) that the specification should be interpreted as also using the terms “consistently composed of” or “components of” to describe the invention. In other words, regarding the terms “comprising,” “components of,” and “components of”, where one of these three terms is used herein, the subject matter disclosed and claimed in this application may include the use of any of the other two terms. Therefore, in some embodiments not otherwise explicitly described, any instance of “comprising” may be replaced by “components of” or optionally “components of”.

[0206] Except as specified in the claims or operational embodiments, or otherwise indicated, all figures used herein to represent amounts of ingredients or reaction conditions should be understood to be modified by the term "about" in all cases. These examples are not intended to limit the scope of the invention.

[0207] In the following text, or where otherwise stated, "%" means "% by weight", "ratio" means "ratio by weight" and "parts" means "parts by weight".

[0208] Unless otherwise stated, the terms “mainly,” “dominant,” and “substantially” as used herein shall mean comprising more than 50% by weight.

[0209] As used herein, with respect to numbers in a numerical range, the terms“about,”“approximately,” and“substantially” are understood to refer to a range of -10% to +10% of the reference number, preferably -5% to +5% of the reference number, more preferably -1 % to +1 % of the reference number, most preferably -0.1 % to +0.1 % of the reference number. In addition, with respect to numerical ranges, these terms are to be interpreted as providing support for the claims for any number or subset of numbers within that range. For example, a disclosure of 1 to 10 is to be interpreted as supporting ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, 8 to 10, and so on.

[0210] The terms“preferred” and“preferably” refer to embodiments of the application that can provide certain benefits under certain circumstances. However, other embodiments can also be preferred under the same or other circumstances. Additionally, recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude those other embodiments from the scope of the application.

[0211] As used herein, the term“copper chelator” refers to an organic molecule having more than one atom capable of forming a coordinate bond with a copper ion, optionally with Cu 1+ and / or Cu 2+ ions. In certain embodiments, a copper chelator can form a copper complex with a copper ion having a stability constant greater than 2, optionally greater than 5. Exemplary copper chelators known in the art include: ethylenediamine, polyethylenimine and derivatives thereof, tetrathiomolybdate; N,N’ bis(2-aminoethyl)ethane-1,2-diamine (trientine, triethylenetetramine, TETA); penicillamine; D-penicillamine; N’ (2-aminoethyl)- N [2-(2-aminoethylamino)ethyl]ethane-1,2-diamine (tetraethylenepentamine, TEPA); 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid (EDTA); 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA); N, N,N’,N’- Tetra(2-pyridyl-methyl)ethylenediamine (TPEN); 2-[4,8,11-tris(carboxymethyl)- 1,4,8,11-tetraazacyclotetradec-1-yl]acetic acid; 2-[4,7,10-tris(carboxymethyl)-1,4,7,10- tetraazacyclododec-1-yl]acetic acid (DOTA); (2S)-2-amino-3-methyl-3-thiolbutyric acid (D-pen); bis(sulfidyl)molybdenum sulfide (TTM); 5-chloro-7-iodo-8-hydroxyquinoline (Cliquinol); diethyldithiocarbamic acid (DDC); 2,9-dimethyl-1,10-phenanthroline (Neocuproine); 2,9-dimethyl-4,7-diphenyl- 1,10-phenanthroline (Bathocuproine); 4-[2,9-dimethyl-7-(4-sulfophenyl)-1,10-phenanthroline-4- yl]benzenesulfonic acid (BCS); and N,N’ - Bis(cyclohexylideneamino)oxalylamide (Cuprizone).

[0212] As used herein, the term "copper chelator inducer" refers to a substance that, when administered to a subject, causes the subject to produce a copper chelator or increases the production of a copper chelator by the subject. Exemplary copper chelator inducers known in the art include zinc salts, which, when administered to a mammal, induce the production of metallothionein, which is itself a copper chelator.

[0213] As used herein, the term "anti-cancer immunotherapeutic agent" refers to an agent used to treat cancer by stimulating or suppressing the immune system of a subject. Exemplary anti-cancer immunotherapeutic agents known in the art include antibodies, immune checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, bi-specific T cell engagers (BiTEs), (CAR) NK cells, adoptive immune cells, and antibody-dependent immune cells. Abbreviations

[0214] Antibody-dependent cytotoxicity (ADCC); 4-[2,9-dimethyl-7-(4-sulfophenyl)-1,10-phenanthroline-4-yl]benzenesulfonic acid (BCS); Butylated hydroxyanisole (BHA); Butylated hydroxytoluene (BHT); Bispecific T-cell connective (BiTE); Chimeric antigen receptor (CAR); Diethyldithiocarbamate (DDC); Diffuse intrinsic pontine glioma (DIPG); Diffuse midline glioma (DMG); Dimethyl sulfoxide (DMSO); 2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetic acid (DOTA) (2S)-2-amino-3-methyl-3-thiol butyric acid (D-pen); 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA); ethylenediaminetetraacetic acid (EDTA); US Food and Drug Administration (FDA); Gene set enrichment analysis (GSEA); Immune checkpoint inhibitors (ICI); Monoclonal antibodies (mAb); Multilayer vesicles (MLV); Natural killer cells (NK cells); Phosphate-buffered saline (PBS); Programmed death receptor 1 (PD-1); Programmed death ligand 1 (PD-L1); Tetraethylenepentamine (TEPA); Triethylenetetraamine (TETA); N, N,N’,N’ -Tetra(2-pyridyl-methyl)ethylenediamine (TPEN); and bis(thionyl)molybdenum sulfide (TTM).

[0215] Preferred features, embodiments, and variations of the present invention can be seen from the following examples, which provide sufficient information for those skilled in the art to practice the invention. These examples should not be construed as limiting the scope of the foregoing overview of the invention in any way. Example

[0216] Example 1: TEPA in TH-MYCN transgenic mouse model TEPA is a copper chelating agent that can bind with Cu. 2+ Formation of copper chelate complexes ( Figure 2 TEPA was administered orally at a daily dose of 400 mg / kg to TH-MYCN GEMM transgenic mice (which spontaneously develop aggressive tumors with location, histology, biology, and cytogenetic abnormalities very similar to human neuroblastoma) for 7 days. The control group received an equivalent volume of oral saline solution daily for 7 days. Figure 3 ).

[0217] Single-cell RNA sequencing was used to assess changes occurring in different cells that constitute the tumor microenvironment. Tumor cells were observed to have reduced intracellular copper levels, as well as those associated with replacement genes. Mt1 and Mt2 As shown in the expression. Furthermore, copper chelation reduces...Mycn expression of the genes Mycn are key oncogenic drivers of neuroblastoma, often associated with high-risk disease Figure 4 A, 4B, and 5).

[0218] Single-cell RNA sequencing of infiltrating immune cells revealed a diverse repertoire of subpopulations. Importantly, copper chelation induced a nearly 5-fold increase in neutrophil (the most abundant leukocyte type in circulation) infiltration into the tumor microenvironment. Interestingly, neutrophil subpopulations were characterized by the highest average expression of key genes involved in intracellular copper uptake, including Steap4 , Slc31a1 and Atp7a ( Figure 6 and 7 ). Neutrophilia is closely associated with copper deficiency in humans and can be reversed by oral copper supplementation.

[0219] GSEA analysis revealed that copper chelation polarized infiltrating neutrophils towards an “N1” phenotype, which is strongly associated with pro-inflammatory / anti-tumor effector functions Figure 8 .

[0220] Opal multiplex analysis reflected an increase in infiltration of CD11B+ myeloid cells into the tumor microenvironment with copper chelation in addition to an increase in NCR1-positive natural killer cells and CD8-positive cytotoxic T cells Figure 9 and 10 .

[0221] Single-cell RNAseq network analysis revealed key pathways upregulated by copper chelation treatment. Notably, these were related to lymphocyte and myeloid cell chemotaxis, activation, and differentiation. These were complemented by key associated cytokines that promote cell recruitment into the tumor microenvironment and activation. Importantly, NK and myeloid cells are associated with mediating antibody-dependent cellular cytotoxicity.

[0222] N1 polarized neutrophils have a pro-inflammatory phenotype characterized, among other things, by higher levels of intercellular adhesion molecule (ICAM)-1 and high secretion of interferon (IFN) gamma-induced protein 10 (IP-10) / C-X-C motif chemokine 10 (CXCL10) and tumor necrosis factor (TNF). Furthermore, neutrophils incubated in an in vitro environment that mimics tumors showed high cell surface expression of C-X-C motif chemokine receptor 2 (CXCR2) and secreted high levels of interleukin (IL)-8.

[0223] ​Without being bound by theory, the inventors speculate that copper chelation upregulates the migration and activation of lymphocytes, myeloid cells to promote an anti-tumor immune response, including increased inflammation, functional enrichment of interferon gamma and beta pathways in the TME, increased migration of leukocytes to sites of inflammation, differentiation and expansion of lymphocytes and myeloid cells, and formation of ROS to kill tumors.

[0224] Example 2: TEPA transfers copper from tumor cells to immune cells Using an in vitro model of tumor cell (SK-N-BE(2)C) / CD45-positive immune cell co-culture, it was found that TEPA was able to promote the release of copper from tumor cells into the culture medium ( Figure 13 A - grey column). When normal (CD45+) lymphocytes were isolated from whole blood and added ( Figure 13 A - red column), the released copper was completely taken up from the environment during immune cell response and related effector function activation. This demonstrates the sensitivity of the immune system to copper in promoting an anti-tumor immune response.

[0225] Given previous findings regarding neutrophils in the tumor microenvironment, neutrophils were isolated from whole blood using a neutrophil-specific protocol. The results again indicated that when treated with TEPA, copper was released into the culture medium, which was subsequently taken up by neutrophils ( Figure 13 B).

[0226] Furthermore, it was found that tumor cell-specific inhibition of neutrophil activation (SK-N-BE(2)-C tumor cells alone condition), without being bound by theory, the inventors believe this indicates that copper chelation promotes the induction of neutrophil activation by immunomodulatory signals (e.g., immune checkpoint molecules).

[0227] To assess in situ trafficking of copper in mouse neuroblastoma tumors, tissue sections were stained with a copper-specific probe to determine the intracellular copper distribution on a single cell basis ( Figure 14 ). Figure 14 A indicates that TEPA selectively depletes copper from tumor cells (e.g., control shows overall darker than TEPA), however, a strong copper signal is detected in CD45+ infiltrating lymphocytes (black cells indicated by yellow arrows). Altogether, this provides evidence of copper trafficking from tumors to immune cells using in vitro and in vivo techniques.

[0228] Example 3: TEPA and anti-GD2 moAb combination therapy in TH-MYCN transgenic mouse model A gold standard in vivo model of neuroblastoma was used to assess the efficacy of copper chelation therapy to enhance anti-GD2 efficacy. According to Figure 15TH-MYCN GEMM transgenic mice (i.e. dosed on days 0, 2, 3, 4, 5, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, etc. to complete 5 weekly dosing cycles) at a daily dose of 400 mg / kg via oral gavage. Groups of mice that did not receive TEPA received equivalent volumes of oral saline doses at the same time points. Groups of mice also received saline vehicle, IgG, or anti-GD2 ip (20ug) doses on days 7 and 11 (also repeated over 5 weekly cycles, as Figure 15 depicted in FIG. 1.

[0229] Prior to the addition of combination therapy (TEPA + anti-GD2), copper chelation therapy was used to prime the tumor microenvironment to promote infiltration and activation of key cell subsets. In a short-term study, Opal multiplex immunohistochemistry showed increased CD8 / NCR1 / CD11B infiltration when TEPA was combined with anti-GD2 therapy Figure 16 and 17 ). Furthermore, this combination of therapies was observed to significantly improve survival and prevent disease recurrence compared to anti-GD2 alone Figure 18 A) Copper depletion therapy with TEPA was shown to be non-toxic, as evidenced by no change in animal body weight loss Figure 18 B). This synergistic effect of the combination of the copper chelator TEPA with the anti-cancer immunotherapeutic agent anti-GD2 was unexpected and highlights the role of copper chelation in preventing tumor immunosuppression.

[0230] Example 4: TETA and anti-GD2 moAb combination therapy in TH-MYCN transgenic mouse model Given this promising result with TEPA, TETA was also analyzed to see if this effect could be repeated with a different copper chelator. TETA is a clinically approved TEPA analog (see Figure 19 ) that was first approved for use in the treatment of copper overload disorders, i.e. Wilson’s Disease. It is taken orally three times per day and despite being used in a life-long treatment regimen, it has shown an extremely safe toxicity profile in both children and adults.

[0231] Using a second in vivo model of neuroblastoma, the copper chelator TETA was similarly tested according to the dosing regimen depicted in Figure 20 Similar to TEPA, using OPAL multiplex immunohistochemistry, TETA was used to increase CD8 / NCR1 / CD11B infiltration into the tumor microenvironment Figure 21 and 22 ). Again, it was found that priming the TME with copper chelation therapy was able to successfully augment anti-GD2 therapy and prevent relapse in approximately 30% of animals Figure 23A). Importantly, no significant changes were observed in the animals with any treatment regimen, which underscores the safety profile of this therapy. Figure 23 B).

[0232] Unbound by theory, the inventors speculate that the synergistic effects of combination therapy with TEPA and anti-GD2 mAb, or TETA and anti-GD2 mAb, may follow the following possible mechanisms: Due to the concentration gradient of TETA / TEPA, it spreads within cancer cells.

[0233] Complex formation (this is an equilibrium state in which complex formation is dominant - see...) Figure 24 and 26 (The complex structure in the text).

[0234] Due to the concentration gradient, Cu(TETA) 2+ / Cu(TEPA) 2+ The complex diffuses outside the cancer cells (Cu(TETA)). 2+ / Cu(TEPA) 2+ Complex formation is predominant at biological pH levels - see [link to relevant documentation] Figure 25 ).

[0235] Due to the concentration gradient, Cu(TETA) 2+ / Cu(TEPA) 2+ The complex diffuses within immune cells.

[0236] Cu in immune cells 2+ The release of is a result of the altered coordination balance caused by the use of copper ions through cellular pathways (a state of equilibrium in which the dissociation of the complex is dominant).

[0237] Example 5: Copper chelation enhances anti-GD2 antibody treatment efficacy The potential of copper chelation to enhance the efficacy of anti-GD2 antibody therapy in a transgenic Th-MYCN model was tested. Mice with established tumors were administered the copper chelator TEPA as a monotherapy for one week prior to initiating anti-GD2 antibody therapy to stimulate immune activation in the tumor microenvironment. Subsequently, mice were treated daily with TEPA for four weeks in combination with twice-weekly anti-GD2 antibody therapy. Figure 27 a). Then, TEPA and anti-GD2 therapy were reduced to twice-weekly administration until ethical endpoints were met (tumor volume ≥1000 mm). 3 The maximum duration of treatment is 180 days. Importantly, this combination of immune responses was well tolerated, and no side effects were observed.

[0238] Anti-GD2 monotherapy induced modest anti-tumor activity, but this activity was substantially enhanced with the addition of TEPA, resulting in a significant prolongation of survival time in approximately 30% of animals (p=0.043) and durable responses Figure 27 b, 27c). To examine the relevant changes occurring in the immune environment, OPAL multiplex immunohistochemistry was performed on tumors obtained from animals at day 14 post-treatment Figure 27 d). Anti-GD2 therapy alone significantly increased cytotoxic T cell (p=0.023) and myeloid cell (p=0.007) infiltration, and there was a trend for increased NK infiltration, compared to the control isotype group. Nonetheless, the combination of copper chelation with anti-GD2 therapy substantially enhanced the infiltration of NK (p=0.007) and myeloid (p=0.001) cell subsets, without affecting cytotoxic T cell infiltration Figure 27 e). NK cells are considered the main effectors of ADCC in neuroblastoma, eliciting responses through Fc-receptor binding. Unexpectedly, it was observed that copper chelation therapy also increased the frequency of infiltrating CD1 1 b+ myeloid cells, which are associated with immunosuppressive activity in neuroblastoma, yet can also act as effective effectors of ADCC. This immunological induction strategy suggests that copper chelation is an effective adjuvant that, in combination with anti-GD2 antibody therapy, increases immune infiltration and enhances tumor control.

[0239] Example 6: Copper chelation regulates cytokine levels to drive immune cell infiltration Secreted soluble cytokines in the tumor microenvironment induce pleiotropic effects on immune cells. To understand how the immune response is regulated during the induction sequence, the changes occurring in the local cytokine environment of copper chelation were investigated using a multiplex cytokine profiling. Transcriptomic data confirmed that treatment produced a significant increase in cytokine levels associated with immune cell recruitment (RANTES / CCL5, GM-CSF, KC / CXCL1 / IL-8) and activation and effector functions (IL-10, TNF-a, IFN-g, IP-10 / CXCL10, IL-2, IL-6), while decreasing the levels of the immunosuppressive cytokine TGF-b Figure 28 b).

[0240] Given the role of NK cells in promoting anti-tumor responses by modulating the effector activity of monocytes, macrophages, neutrophils, dendritic cells and T cells, they were considered the main source of the above-mentioned cytokines. Therefore, OPAL multiplex immunohistochemistry was performed to simultaneously verify the presence of NCR1+ NK cells, CD8+ cytotoxic T cells and CD1 1 b+ myeloid cells in the tumor microenvironment Figure 28 c). A significant increase in NK and cytotoxic T cell frequency was observed Figure 28d). Interestingly, copper chelation therapy significantly increased myeloid cells, which are known to enhance the function of CD8+ T cell effectors in neuroblastoma.

[0241] These data suggest that copper chelation favorably regulates cytokine levels to promote immune cell infiltration, thereby fostering an immune-permissible neuroblastoma tumor microenvironment.

[0242] Example 7: Copper chelation destabilizes neuroblastoma tumor microenvironment To further characterize the changes occurring in the tumor microenvironment during induction, a [system / mechanism] was constructed. Th-MYCN A tissue microarray of tumors, consisting of untreated controls and TEPA-treated tumors resected 3 and 7 days after treatment. Target regions (ROIs) were selected using the GeoMxNanoString spatial transcriptomics platform based on the ratio of pancytokeratin (PanCK) and the panleukocyte marker CD45, to conditionally assign each region to be immune-infiltrating. Copper chelation therapy with TEPA was observed to be strongly associated with immune infiltration, as determined by CD45 staining of the tumor core. Figure 29 a). When comparing non-invasive / infiltrated partitions, TEPA-treated tumors were observed to show a time-dependent increase in immune infiltration ( Figure 29 b). Next, differential gene expression analysis was performed, comparing TEPA-treated invasive ROIs with TEPA-treated non-invasive ROIs to minimize crosstalk between tumor and immune cell signals. Interestingly, it was observed that... Mgp and Mprip and Znrf1 The upregulation of both neuronal differentiation and neuroblastoma is a key feature associated with a favorable clinical prognosis. Figure 29 c). Gene set enrichment analysis reported upregulation of pro-inflammatory pathways involved in interferon (IFN) and tumor necrosis factor (TNF) signaling, upregulation of the p53 pathway involved in tumor suppression, and downregulation of MYC targets responsible for oncogenic signaling. Figure 29 (d, 29e). In summary, these results strongly suggest the instability of the neuroblastoma tumor microenvironment.

[0243] Example 8: Copper chelation enhances neutrophil infiltration in neuroblastoma tumor microenvironment To characterize functional changes in cells at single-cell resolution, tumors treated with saline or the copper chelate TEPA for 7 days were analyzed using the BD Rhapsody system. Fresh tumor sections were dissected using a protocol that ensured high viability of tumors and immune compartments for subsequent enrichment by flow cytometry sorting. Figure 30 a). After quality control, dimensionality reduction, and clustering, 13,555 tumor cells were obtained (using... MycnGene expression definitions; Figure 30 b). Importantly, tumor cells were highly responsive to copper chelation therapy, where metallothionein Mt1 and Mt2 Gene expression of metallothionein (a surrogate marker for intracellular copper concentration) was significantly decreased Figure 4 c) (21). Unexpectedly, tumor oncogenes Mycn and associated targets were also significantly downregulated, which were reported to decrease tumor cell proliferation, increase immunogenicity and increase immune cell infiltration Figure 30 c, 30d).

[0244] The immune compartment was composed of 12,127 immune cells (using Ptprc Gene expression definitions; Figure 30 e), where a diverse repertoire of 13 unique clusters was identified in both treatment groups Figure 30 e-30g), including lymphoid and myeloid lineages as well as unconventional subpopulations such as CD8+ NKT-like and gamma-delta (gd) T cells. These data support Th-MYCN tumor-associated immune subpopulations in neuroblastoma models and clinical neuroblastoma samples as reviewed by others; however, the proportions can vary due to different analytical techniques and tumor staging. The number of cell clusters remained essentially unchanged by copper chelation treatment, with the notable exception of neutrophils, which showed a significant five-fold increase Figure 30 f) To assign this cluster, recently identified gene markers S100a8 / a9 were used, which together with previously established markers form the heterodimeric calprotectin involved in neutrophil recruitment and activation Figure 30 g).

[0245] Overall, these data show the impact of copper chelation in the neuroblastoma tumor microenvironment, including tumor cell downregulation Mycn of its targets and increased neutrophil infiltration.

[0246] Example 9: Copper chelation restores anti-tumor immune response through neutrophil signaling Next, the impact of copper chelation on the immune compartment was assessed by mapping the biological processes of the treatment and control groups. Enrichment of gene sets associated with CD8+ expansion, hypoxia and autophagy responses related to myeloid cells, lymphocyte and myeloid cell migration, differentiation and activation, and cytokine responses to IFN-g and tumor necrosis factor (TNF) family were observed Figure 31 a). The bar plots and top-ranking pathways resulting from gene set enrichment analysis of each immune cluster following TEPA treatment indicate metabolic changes associated with activation of innate immune responses Figure 34 and 35 ​

[0247] Anti-tumor immune responses involve complex coordination of multiple cell types across innate and adaptive cell subsets. To understand how these processes are facilitated, CellChatDB was used to infer changes in cell-cell communication between immune and tumor clusters in the control and TEPA treated datasets. An overall increase in signaling networks was observed with copper chelation, determined by an increase in the presence and / or strength of cell-cell interactions Figure 31 b). Furthermore, when comparing incoming interaction strengths, treatment led to neutrophils displacing tumor cells, implicating inhibition of tumor-induced immune suppression Figure 31 c; Figure 36 ). Given the increase in infiltrating neutrophils upon TEPA treatment, ligand-receptor expression between this cluster and other subtypes was examined, and it was found that this was primarily driven by the galectin-9 (Lgals9) axis Figure 37 Without being bound by theory, the inventors believe that copper chelation can skew the immunosuppressive tumor microenvironment and restore anti-tumor responses by enhancing neutrophil signaling.

[0248] Example 10: Copper mobilization enhances anti-tumor activity of neutrophils Given the criticality of copper for effective immune function, copper metabolism changes occurring in immune compartments were investigated using single-cell transcriptomic data. Comparing treatment groups, no changes in a selected set of copper-related genes were observed, likely due to tight homeostatic regulation of copper in non-malignant healthy cells Figure 32 a). However, neutrophil-restricted expression of Slc31a1 , Atp7a and Steap4 (the major genes responsible for copper import and export in cells in mammals) was observed.

[0249] Similar to the M1-M2 spectrum used for macrophage polarization states, N1-N2 have recently been proposed for neutrophils to designate anti-tumor and pro-tumor functions, respectively. Using a recently published dataset, N1 and N2 gene signatures were curated, and it was found that tumor-infiltrating neutrophils exhibited a pro-inflammatory N1 phenotype that was independent of copper chelation treatment Figure 32 b). Gene set enrichment analysis comparing treatment groups was then performed, and a significant enrichment of genes associated with curated N1 anti-tumor neutrophils and IFN-g response pathways was observed Figure 32 c). IFN-g stimulation has been shown to enhance N1 -associated properties such as ICAM1 expression, reactive oxygen species and neutrophil extracellular trap formation, direct and antibody-dependent cellular cytotoxicity, and T cell recruitment and activation.

[0250] Because cytokines GM-CSF and KC are effective neutrophil chemical inducers upregulated by copper chelation therapy ( Figure 28 (b) The inventors hypothesized that copper chelation could induce neutrophil mobilization from the bone marrow. To evaluate this, immunophenotyping was performed on peripheral blood from control and TEPA-treated animals, and a significant increase in the abundance and relative percentage of circulating neutrophils was confirmed. Figure 32 d). No other immune subsets or erythrocyte measurements were significantly affected (data not shown), and these results collectively underscore the unique relationship between copper levels and neutrophils.

[0251] Systemic copper deficiency is associated with a decrease in circulating neutrophil numbers and suppression of effector function, which can be rapidly reversed by copper supplementation. Since neuroblastoma cells exhibit elevated copper levels to drive tumor progression (e.g., proliferation, angiogenesis, metastasis), the inventors hypothesize that this sequestration may create a copper-depleting microenvironment that suppresses immune cell function, particularly neutrophils. Therefore, the inventors hypothesize that copper chelators such as TEPA can redirect copper ions from “rich” tumor cells to “poor” immune cells to slow tumor progression and restore anti-tumor immunity.

[0252] To test this hypothesis, by using MT1X-tGFP We constructed a transduced neuroblastoma cell line, SK-N-BE(2)-C, to validate single-cell findings. This allowed for visual confirmation of the fluorescent signal (as opposed to...). MT1X Expressed in proportion, MT1X It is a surrogate marker of intracellular copper and does indeed decrease 24 hours after TEPA treatment. Figure 32 e). This indicates that, in vitro, tumor copper is released extracellularly and therefore the distribution of copper can be inferred by measuring the copper concentration in the culture medium.

[0253] To this end, an interleaved co-culture system was designed to allow for accurate tracking of copper flow using the SK-N-BE(2)-C cell line during a simulated neuroblastoma-neutrophil interaction. Conditioned culture media were collected, and copper concentrations were measured before and after the addition of initial circulating neutrophils isolated from healthy donors. Notably, copper concentrations were slightly lower in the presence of untreated neuroblastoma cell isolates compared to control media, suggesting that cell isolation provides available copper to support their proliferation. Figure 32 f, black column). Consistent with the hypothesis, copper saturation was observed after treatment of tumor cells with TEPA, which was subsequently uptaken by neutrophils after incubation. Figure 32Importantly, this phenomenon did not occur in untreated neuroblastoma cells, suggesting the presence of a secreted soluble factor that hinders neutrophil copper uptake, and is restored by copper chelation therapy.

[0254] To support this phenomenon, elevated levels of copper export protein ATP7A were significantly associated with T-cell infiltrated tumor microenvironments in a broad range of pediatric solid malignancies, including neuroblastoma Figure 38 This suggests that redistribution of copper in the tumor environment is indeed beneficial and can be promoted by using copper chelation therapy.

[0255] These findings show the role of copper chelation in stimulating the recruitment of proinflammatory neutrophils and enhancing N1 function associated with anti-tumor responses.

[0256] Example 11: TETA plus anti-GD2 antibody immunotherapy combination provides a therapeutic strategy for neuroblastoma While Th-MYCN The model is considered a MYCN Standard for expanding preclinical studies of neuroblastoma, manual palpation was used to determine tumor burden. To be able to better monitor tumor growth kinetics, a syngeneic model was exploited that was generated by subcutaneous injection of NXS2 cells (hybrids derived from C1300 neuroblastoma cell line and dorsal root ganglion cells) into immunocompetent A / J mice. This model has been extensively used to study anti-GD2 directed therapies, including combination therapies. After demonstrating the ability of copper chelation therapy, the feasibility of repurposing TETA (analog of TEPA), an FDA-approved copper chelator used as first-line therapy for hepatolenticular degeneration, was evaluated.

[0257] Using the syngeneic NXS2 model, treatment was initiated on day seven post-inoculation Figure 33 a, black arrows), and animals were randomly assigned to receive control (saline) or TETA (400 mg / kg) daily by oral gavage for seven days. Similar to TEPA, TETA slowed tumor growth as a single agent (p=0.028, day 14) Figure 33 a). Subsequently, the neutrophil infiltration and phenotype of dissociated tumors were evaluated by flow cytometry. TETA treatment increased the frequency of CD11b+Ly6G+neutrophils, which exhibited increased MMP9 expression, associated with N1 polarization status Figure 33 b, 33c). This indicates that TETA stimulates the infiltration and activation of proinflammatory N1 neutrophils, which become key effectors of antibody-based immunotherapies.

[0258] In patients with hepatolenticular degeneration, copper chelation therapy can produce surges in free copper levels that often cause toxic side effects. To identify potential adverse reactions that can arise in the context of a tumor, peripheral blood was taken from NXS2 inoculated mice that received a one-week TETA treatment and subjected to blood chemistry analysis. TETA treatment did not affect the concentration of any analyte related to liver, kidney or overall systemic disease compared to the control group Figure 39 ). This is particularly important considering that the liver is the systemic reservoir for copper.

[0259] Overall, these results demonstrate that TETA is a good substitute for TEPA and can well remodel the tumor microenvironment of neuroblastoma.

[0260] This encouraging data prompted the study of the effect of TETA and anti-GD2 antibody as a combination therapy in the NXS2 syngeneic model. The treatment regimen was similar to the TEPA / Th-MYCN regimen used in the model; however, treatment was stopped after four combination therapy cycles to assess the rate of relapse Figure 33 d). After the start of the treatment, mice experienced a slight weight loss due to the introduction of daily gavage as a stressor. After acclimation, all treatments were well tolerated, including the immune combination group, with no adverse events reported Figure 33 e). As a highly aggressive neuroblastoma model, the control group exhibited rapid tumor expansion. However, the immune combination group was observed to effectively inhibit tumor growth Figure 33 f). Notably, treatment with TETA + IgG substantially reduced the tumor burden of an individual animal that died from rapid relapse after treatment was stopped at day 42, likely due to tumor escape. In contrast Th-MYCN to the model Figure 27 b, 27c), anti-GD2 treatment alone did not mediate a substantial anti-tumor effect. Nonetheless, the TETA + anti-GD2 antibody combination produced a significant anti-tumor effect (median survival: 29 days versus 19 days in saline + anti-GD2, p = 0.009), leading to approximately 40% of animals being durably eradicated Figure 33 g, 33h). Notably, these animals showed no signs of relapse until the end of the experiment at 90 days after treatment was stopped. Overall, the addition of TETA to anti-GD2 therapy significantly prolonged survival compared to the respective monotherapy groups.

[0261] To examine changes in immune compartments (NCR1+ NK cells, CD8+ cytotoxic T cells, and CD11b+ myeloid cells) as a result of treatment, OPAL multiplex immunohistochemistry was performed on tumors excised 14 days after treatment Figure 33i). The control groups (saline + mediator; saline + IgG) showed high similarity in tumor growth and survival, indicating that the allotype antibody did not induce an immunogenic effect. In all subgroups examined, TETA and anti-GD2 monotherapy were comparable and significantly promoted immune infiltration compared to the control group. Figure 33 j). The addition of TETA to anti-GD2 therapy primarily enhanced CD11b+ infiltration (p=0.02), presumably neutrophils; no changes in NCR1+ or CD8+ frequencies were observed. Figure 33 j). Notably, the combination group exhibited superior tumor control, suggesting the activation of TETA-mediated antitumor immune responses.

[0262] In summary, these results reinforce the crucial role of copper as a modulator of the neuroblastoma tumor microenvironment. These results demonstrate the ability of copper chelators to successfully circumvent the immune evasion phenotype and elicit a strong anti-tumor immune response. Furthermore, the results confirm that TETA is a highly effective, non-toxic, and specific copper chelator. These findings provide primary evidence for the reuse of the clinically approved copper chelator Cuprior® as an immunomodulator to enhance the efficacy of anti-GD2 immunotherapy and improve responses in neuroblastoma patients.

[0263] Example 12: Combination therapy of copper-chelator TEPA and GD2-targeted CAR T cells EZH2 inhibition is an exciting emerging pathway to increase GD2 antigen expression in neuroblastoma and Ewing's sarcoma. The copper chelator TEPA has been found to be effective in diffuse midline glioma (DMG) and aggressive childhood brain cancer. Figure 41 a) and neuroblastoma cell line ( Figure 41 b) EZH2 was strongly downregulated. This work was translated in vivo, and flow cytometry revealed that after one week of treatment, TEPA significantly reduced EZH2 levels. Th-MYCN GD2 expression is strongly upregulated in neuroblastoma tumors. Figure 41 c). Interestingly, preliminary data from a glioblastoma xenograft model suggest that one week of TEPA pretreatment is comparable to that of GD2-targeted CAR T cells (1x10⁻¹²). 6 The combination of intravenous (IV, single-dose) injections per cell had a synergistic effect in reducing tumor growth. These data strongly suggest that copper chelation can epigenetically regulate GD2 antigen expression, thereby enhancing sensitivity to GD2 CAR T immunotherapy. Importantly, these results support the idea that reducing intratumoral copper can simultaneously reduce immune evasion mechanisms in the tumor microenvironment (TME) and increase the targeting of GD2-CAR T cells in cancer cells.

[0264] Example 13: Copper-chelation therapy in two models of mesothelioma Two murine models (AB1-HA and AE17-OVA) were generated by exposure to asbestos. They exhibit similar mutational burden and loss of key genes associated with human disease. The models have two genetic backgrounds BALB / c and C57BL / 6 and are responsive to checkpoint therapy by ICI targeting a-CTLA4 and ICI targeting a-PDL1. As part of clinical trials, these models were used to develop current treatments for mesothelioma. The AB1-HA model has a higher cure rate in ICI treatment and can be used to determine increased response rates. The AE17-OVA model does not typically cure by ICI treatment alone and can be used to find adjunct therapies that can improve response rates.

[0265] Animals received daily doses of 800 mg / kg oral TETA for 2 weeks, followed by every other day dosing until the end of the experiment. ICI doses were administered according to the dosing schedule depicted in Figure 42 No toxicity or weight loss was observed in mice.

[0266] Tumor growth data ( Figure 43 and 45 ) and survival data ( Figure 44 and 46 ) for each model show that the use of combination therapy increased response rates and increased overall survival in the AB1-HA mesothelioma model and significantly reduced tumor size in the AE17-OVA model. Flow cytometry results ( Figures 47-51 ) for mice treated with 800 mg / kg TETA for 1 week (1 week post inoculation) show that CD8 and NK cells were significantly increased and there was a trend for increased CD45 and neutrophils.

[0267] Based on the results shown in the above examples, the skilled person would reasonably expect that any copper-chelating agent can be used in combination with any anti-cancer immunotherapeutic agent to treat any cancer type.

[0268] Methods In vivo studies All experimental procedures were approved by the Animal Care and Ethics Committee (ACEC) of the University of New South Wales (approval numbers ACEC 20 / 25B, 21 / 96B and 18 / 97B) in accordance with the Animal Research Act 1985 (New South Wales, Australia) and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2013). All animals were housed in a specific pathogen-free facility with temperatures maintained at 22-24 °C, with a 12-hour light / dark cycle. Mice were housed in Ventirack cages (Tecniplast, Italy) and provided with food and water ad libitum and received environmental enrichment.

[0269] For in vivo treatment: the copper chelators tetraethylenepentamine pentahydrochloride (TEPA [C8H23N5·5HCl], Sigma, USA; #357683) and triethylenetetramine tetrahydrochloride (TETA [C6H18N4·4HCl], Sigma, USA; #161969) were freshly dissolved in a saline vehicle and administered at 400 mg / kg by oral gavage. Anti-GD2 monoclonal antibody (clone 14G2a, #BE0318) and IgG isotype control (clone IgG2a, #BE0085) were obtained from BioXCell, USA and freshly diluted in medical grade phosphate buffered saline and administered intraperitoneally at a 100 pg bolus dose.

[0270] Th-MYCN The model was generously provided by Professor Michelle Haber (Children’s Cancer Institute, Australia) and approved for use by the Institutional Biosafety Committee. Th-MYCN Mice were maintained in situ, genotyped and only homozygous mice were used for experiments. For tumour characterisation studies, male and female animals were recruited when small tumours (3-4 mm 3 ) were palpable and treated with saline or TEPA (400 mg / kg) for seven days, then tumours were harvested and sectioned for downstream applications. For immunotherapy combination studies, female and male mice were recruited as above and randomly assigned to the following treatment groups: saline + saline vehicle; saline + IgG; saline + anti-GD2; TEPA + IgG; TEPA + anti-GD2. For survival experiments, animals (n=5 / group / sex) were weighed and palpated regularly to determine tumour progression, regression or recurrence and sacrificed when tumour volume diameters were determined to be >10 mm.

[0271] Female A / J mice were obtained from the Animal Resource Centre (Perth, Australia) and NXS2 cell line was provided by Professor Holger Lode (University of Greifswald, Germany). Six to seven week old animals were injected subcutaneously with 1.5 × 10 6 cells in a 1 : 1 mixture of serum-free Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA, #11995065) and Matrigel (Corning, USA, #354234). Tumours were transplanted for 7 days (reaching 50-100mm 3 ) before treatment with saline or TEPA (400mg / kg) was initiated for 7 days. Mice were assigned to treatment groups to obtain approximately equal average initial tumour size to mitigate bias. Animals were weighed twice weekly and tumour volume was measured using digital callipers (calculated as 0.5 x length x width2), or daily if >800mm3was exceeded. For the immunotherapy combination study, mice were assigned to the following treatment groups: saline + saline vehicle; saline + IgG; saline + anti-GD2; TEPA + IgG; TEPA + anti-GD2. In the survival experiment, animals were sacrificed once tumour volume reached >1000mm 3 .

[0272] OPAL multiplex immunohistochemistry (IHC) Tumour sections were formalin fixed and paraffin embedded (FFPE) by the Katharina Gaus Light Microscopy Facility (KGLMF) at the University of New South Wales. Tumours were cut into 4 pm sections and for preparation of staining, slides were baked at 58 °C for 1 h before deparaffinisation and rehydration using a Gemini AS automated slide stainer (Epredia, USA). Chromogen-based IHC analysis was performed using a BOND-RX automated staining system (Leica Biosystems, USA). Spleens obtained from tumour-bearing Th-MYCN mice were used as controls for monoclonal and OPAL multiplex optimisation. The following antibodies were all obtained from Abeam, UK: rabbit monoclonal NCR1 (clone EPR23097-35, #ab233558, 1 :500, EDTA pH 8-9 antigen retrieval), rabbit monoclonal CD8a (clone EPR20305, #ab209775, 1 :1000, EDTA pH 8-9 antigen retrieval) and rabbit monoclonal CD1 1 b (clone EPR1344, #ab133357, 1 :20000, citrate pH 6 antigen retrieval). Immunofluorescence signals were visualised using OPAL 7 colour automated IHC kits (Akoya Biosciences, USA; #NEL871001KT) using TSA dyes 650, 570 and 520 respectively, and counterstained with spectral DAPI. Optimisation also included the sequencing of antibodies which was determined to obtain the same dynamic range between each fluorophore to avoid signal “cross-talk” known as umbrella effect. Labeled slides were imaged using automated exposure at 20x magnification using a Vectra Polaris system (Akoya Biosciences, USA). Whole slide imaging was performed by multispectral field scanning using Phenochart software vl. l.0 (Akoya Biosciences, USA). Acquired images were unmixing using Inform v2.5.1 (Akoya Biosciences, USA) and subsequently stitched together in HALO Suite v3.6.4134.193 (Indica Labs, USA) to produce multispectral TIFF images of the whole slide. Nuclei segmentation was performed using HALO AI v3.6.4134 which utilises artificial intelligence and machine learning to train the software. Cell phenotype analysis was performed using the HighPlex FL v4.2.5 module using defined thresholds for nuclei detection and minimum fluorescence intensity. The classifier was trained for all slides and necrotic areas were excluded prior to analysis. Analysis was performed on the whole tumour section (excluding necrotic areas) and cells were classified as positive if the fluorescence intensity exceeded a predetermined threshold.Cell density was determined as the number of positive cells per 1000 identified nuclei in each tumor section.

[0273] Single cell RNA sequencing (scRNA-seq) Fresh tumor sections were minced roughly and incubated in a tumor digestion mixture at 37 °C at 130 RPM for 1 h, which consisted of DMEM supplemented with 25 μg / mL DNase I and 20 μg / mL collagenase IV. Single cell dissociation was achieved by passing the mixture through a 70 μm MACS SmartStrainer (Miltenyi Biotec, Germany; #130-110-916). Cells were pelleted at 330×g for 5 min and resuspended in ACK lysis buffer at room temperature to remove contaminating red blood cells and neutralized with staining buffer (BD Biosciences, USA; #554656).

[0274] To reduce non-specific antibody staining of IgG receptors, 1 × 10 6 cells were aliquoted and pre-incubated with mouse BD Fc Block CD16 / CD32 (BD Pharmingen, USA; #553142). Cells were incubated with CD3e-FITC (ThermoFisher; #11-0031-82, clone 145-2C11, 1:100), NK1.1-PE (Thermo Fisher; #12-5941-82, clone PK136, 1:200) and CD11b-BV421 (BioLegend; #101251, clone M1 / 70, 1:200). Using a previously optimized gating strategy, tumor cells were positively selected using the absence of the described markers. Using a BD FACSAria III (BD Biosciences, USA), approximately 50,000 single cells of each subset (CD3−NK1.1−CD11b− tumor cells, CD3+ / NK1.1+ lymphocytes and CD11b+ myeloid cells) were sorted into individual tubes containing fetal bovine serum (FBS; Gibco, USA, #10100-147). A representative gating strategy is presented in Figure 40 a.

[0275] Transcriptome data from approximately 25,000 total cells applied per cartridge were captured using the BD Rhapsody system (BD Biosciences, USA) (1× control; 1× TEPA-treated). Whole transcriptome libraries were constructed according to the BD Rhapsody single-cell whole transcriptome amplification workflow, following the manufacturer's instructions. The libraries were quantified using a high-sensitivity DNA array on a Bioanalyzer 2200 (Agilent, USA; #5067-4626) and the Qubit high-sensitivity double-stranded DNA assay kit (Thermo Fisher Scientific, USA; #Q32851). The resulting DNA libraries were sequenced using the Illumina NovaSeq 6000 S42×150 bp kit, yielding an average of 80,000 reads per cell.

[0276] The BD RhapsodyWTA analysis pipeline provided on the Seven Bridges platform (Seven Bridges Genomics, USA) was used to transform raw sequencing data into gene expression profiles for individual cells. The pipeline included low-quality read removal, read alignment, gene expression quantification, and data normalization. To reduce bias during dimensionality reduction, downstream analyses were performed separately for tumor and immune cell compartments. To separate tumor compartments, Mycn>0 was selected. Ptprc Cells with a value of 0 were retained, excluding those expressing at least one lymphocyte-associated biomarker in the dataset. Cd3d , Cd3e , Cd3g , Cd8a , Cd8b1 This resulted in a total of 13,560 cells across the control and treatment groups within the tumor compartment. To isolate the immune compartment, cells expressing... Ptprc >0; Mycn Cells with a count of 0 were retained, including lymphocytes previously excluded from the tumor compartment. This resulted in a total of 22,182 cells across the control and treatment groups in the immune compartment.

[0277] In the immune cell compartment, 12,127 cells passed filtering conditions with the following thresholds: 200 < nFeature_RNA < 5500 (number of transcripts); nCount_RNA < 3000 (number of counts); percent mt < 25 (percent of mitochondrial counts); and percent ribo < 20 (percent of ribosomal counts). In the tumor cell compartment, 13,544 cells successfully passed filtering conditions of nFeature_RNA > 200; percent mt < 25; and percent ribo < 15. After exploring the empirical distribution of these variables in 2D space, library-specific threshold values were manually assessed. The CCA integration method implemented in Seurat was used to perform integration-based anchor analysis to correct for batch effects. Raw counts were normalized by natural log transformation.

[0278] Separate uniform manifold approximation and projection (UMAP) plots were generated for the tumor and immune cell compartments. Principal component analysis (PCA) was used to reduce the dimensionality of the integrated datasets. For the immune cell compartment, 60 PCs were used to retain > 70% of the variability. Cells were then clustered and sub-clustered by the Louvain algorithm with a resolution ranging from 0.2 to 0.3. For the tumor cell compartment, 80 PCs were used to retain > 86% of the variability. Cells were then clustered by the Louvain algorithm with a resolution of 0.3.

[0279] Cluster annotation of immune cell subsets was initially performed using the scType platform and subsequently manually curated based on different gene markers identified by the MAST algorithm after batch correction. Due to the presence of a minimum number of cells (n < 50), it was not possible to accurately assign cell types to 2 / 15 identified immune clusters and therefore excluded from analysis. Regarding the tumor cell compartment, after integrating control and treated samples, it was not possible to accurately assign cell types or phenotypic states to 4 identified clusters, however this observed change corresponded to differences between control and TEPA treated groups. Final cell annotation was performed using well-established relevant markers in the literature and similar confirmation was performed using previous studies annotations by A / Prof Fabio Luciani.

[0280] Pathway enrichment analysis was performed to gain insight into the biological processes and pathways associated with each independent cell cluster. The analysis consisted of two main steps: gene set enrichment analysis (GSEA) using the fgsea function and over-representation analysis (ORA) using the clusterProfiler package. Gene sets from the Molecular Signature Database (MsigDB) were used, as well as target selected pathways obtained from the Reactome, Gene Ontology (GO) and WikiPathways (WP) databases (GO:0048870, R-MMU-6798695, WP3941, WP4466, WP412). In addition, custom signatures were included: copper-related genes, N1 anti-tumour phenotype and N2 pro-tumour phenotype, as they were not available in public databases.

[0281] For GSEA, an enrichment score was calculated to assess the enrichment of gene sets in the gene expression profiles of each cell type. Gene expression profiles were ordered based on the magnitude of change of significantly differentially expressed genes between control and treatment. To visualize the results, an explanatory bar plot was generated for each cell type, showing the top significantly enriched pathways, each associated with a specific negative enrichment score (NES) value. To further explore the enriched pathways, a network plot was created to highlight the top 10 genes associated with each gene set. Pathways that were not relevant or provided less information were excluded from the visualization to focus on the most relevant findings.

[0282] For ORA analysis, the GO database was used to identify over-represented gene sets in each cell type. Fisher’s exact test was used to identify significantly enriched GO terms associated with biological processes and molecular functions. To visualize the results of the ORA analysis, a selection of enriched pathways was performed for each cell type. A network plot was generated, with each node representing a pathway and colour-coded according to the corresponding cell type. In addition, the genes enriched in each pathway were identified and displayed in the network plot, allowing a comprehensive view of the genes associated with each enriched pathway in each cell type.

[0283] Statistical tests for pathway enrichment analysis were performed according to established methods. Multiple testing correction using the Benjamini-Hochberg procedure was applied to adjust p-values for multiple comparisons. The threshold for determining significance of enriched pathways was determined based on the adjusted p-value and corresponded to p-adj < 0.1.

[0284] Using CellChat (v2), the inference and analysis of cell-cell communication networks was performed using CellChatDB (v2) as the reference library containing approximately 3,300 validated ligand-receptor interactions. The number of inferred signaling networks was reduced to 16 using a 25% cutoff mean, i.e. for a given certain cell population, the mean gene expression of a certain ligand-receptor pair was set to zero if the percentage of cells expressing this cell in the cell population < 25%.

[0285] Tumor cytokine signature analysis Frozen tumor sections were homogenized in RIPA lysis buffer (Roche, USA; #04693159001) supplemented with lx protease and phosphatase inhibitors using a TissueRuptor II (Qiagen, Germany). To normalize cytokine concentrations, total extracted proteins were calculated using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher, USA; #23225) according to the manufacturer’s instructions. Tumor cytokine levels were measured using a 36-Plex mouse ProcartaPlex Panel 1A (Thermo Fisher, USA; #EPX360-26092-901) according to the manufacturer’s instructions. The Luminex MAGPIX system (Luminex Corporation, USA) was calibrated by a MAGPIX calibration and performance verification kit (Millipore, USA) and data was acquired using xPONENT software (Luminex Corporation, USA). Acquired data was analyzed using Multiplex Analyst software v5.1 (Merck, Germany) as median fluorescence intensity (MFI) with a spline curve fit to calculate analyte concentrations in samples. Prior to running, samples were diluted 1:10 with assay diluent and the resulting concentrations were normalized to extracted proteins.

[0286] MindRay blood analysis Peripheral blood was obtained from animals and collected in K2-EDTA tubes (Greiner, Germany; #450532) and analyzed immediately using a Mindray BC-5150 automated hematology analyzer (Mindray, China) according to the manufacturer’s instructions.

[0287] Generation of SK-N-BE(2)-C pCMV6-Ac-GFP cell line The neuroblastoma cell line SK-N-BE(2)-C was obtained from the American Type Culture Collection, with working stocks managed centrally by the Children's Cancer Institute Cell Bank. Both master and working stocks were verified using short tandem repeat profiling and routinely verified as mycoplasma negative. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA, #11995065) supplemented with 10% foetal bovine serum (FBS, Australia origin; Gibco, USA, #10100147). Cells were incubated under standard conditions (37°C, 5% C02, 95% humidity) and routinely passaged when they reached approximately 80% confluence. The neuroblastoma cell line SK-N-BE(2)-C was stably transfected with the plasmid pCMV6-Ac-GFP, which contains the human MT1X (NM_005952) transcript with a C-terminal TurboGFP tag (tGFP) (Origene, USA; #RG207116). Cultures were maintained under positive selection using the geneticin selective antibiotic (G418 [Thermo Fisher, USA; #10131035]) at 1 mg / mL. Cells were plated and treated with 6 mM TEPA for 24 hours and imaged using the IncuCyte Live-Cell Analysis System (Essen BioScience, USA). Images were taken in phase contrast and green fluorescence channels (auto exposure) using a 10x objective.

[0288] Fractionated co-culture copper transfer assay Individual or 0.25 x 10 6 SK-N-BE(2)-C cells were plated in 2% FBS / DMEM and then treated with 1 mM TEPA overnight. The resultant conditioned media was collected immediately after neutrophils were ready for incubation. A portion of the conditioned media was collected as a pre-incubation control, designated: - neutrophils.

[0289] Peripheral blood from healthy donors was collected into K2-EDTA tubes (BD Biosciences, USA; #366643) by venipuncture. Red blood cells were allowed to sediment for 30 min using a 50% volume of dextran solution (6% dextran in double distilled water [Merck, USA; #09184-50G-F]; 0.9% NaCl). White blood cells were collected using 10x PBS (without Ca 2+ / Mg 2+Percoll [Merck, USA; #P4937-100ML] to prepare a Percoll gradient, forming 1 x PBS (without Ca 2+ / Mg 2+ ) in the bottom layer (about 55% Percoll solution), middle layer (about 68%) and top layer (about 81%).

[0290] The bottom layer was added to a new 15 mL Falcon tube, followed by carefully overlaying the middle layer so as not to disturb the interface. The top layer containing the lymphocytes was collected from the separated blood, taking care not to contaminate the red blood cells, and was moved into a new 15 mL Falcon tube. The tube was centrifuged at 350 x g for 20 min at 20 °C (acceleration and brake speeds were both 9). The resulting plasma supernatant was decanted and the pelleted lymphocytes were gently resuspended in the Percoll top layer, which was then carefully overlaid onto the previously prepared gradient. The resulting preparation was centrifuged at 700 x g for 20 min at 20 °C (acceleration and brake speeds were both 0). The resulting Percoll gradient produced a top lymphocyte layer and a bottom neutrophil layer. The neutrophil layer was obtained and resuspended in 2% FBS / DMEM and centrifuged at 250 x g for 6 min at 20 °C (acceleration speed was 5 and brake speed was 9). The supernatant was aspirated and the neutrophils were resuspended in 2% FBS / DMEM for counting.

[0291] 0.25 x 10 6 Neutrophils were aliquoted into 1.5 mL Eppendorf tubes for each type of conditioned medium at 0.25 x 10

[0292] The copper concentration in the medium samples was quantitatively determined using the QuantiChrom Copper Assay Kit (Universal Biologicals, UK; #DICU-250) according to the manufacturer’s instructions. Fresh 2% FBS / DMEM was used as a blank and a 300 pg / dL standard was prepared. Absorbance was determined using a Benchmark Plus plate reader with Microplate Manager v5.2.1 (Bio-Rad, USA) at a wavelength of 356 nm.

[0293] Tissue microarray construction and digital spatial profiling hybridization Tissue microarrays (TMAs) were prepared in duplicate from 20 Th-MYCN tumor samples (10 controls and 10 TEPA-treated samples) (1 mm core). FFPE TMA blocks were cut into 4 µm pieces, transferred to Bond Plus slides (Leica Biosystems, USA; #S21.2113.A) and processed using Nanostring GeoMx DSP technology accessibility program. In short, slides were hybridized to a GeoMx mouse whole transcriptome atlas (approximately 18,000 targets), followed by immunofluorescence staining using: pancytokeratin (PanCK; clone AE1 / AE3, ThermoFisher; 53-9003-82) to identify tumor cells, smooth muscle actin (SMA, clone 1A4, Abcam; #ab184675) to identify the extracellular matrix, CD45 (clone D3F8Q, Cell Signaling Technology; #35154) to identify all hematopoietic cells, and DNA GeoMx nuclear staining agent (Nanostring, #121303303) to identify the nucleus. The stained slides were then mounted on a Nanostring GeoMx instrument and scanned.

[0294] For each tumor core, three target geometric regions (ROIs) were selected (ROI = 120) and binary-defined as either immune-infiltrating or non-infiltrating, determined by the presence or absence of CD45 staining.

[0295] Digital spatial profiling data processing Segment and probe quality control was performed using the Bioconductor software package GeomxTools. Notably, 20 ROIs containing necrotic sections were subsequently excluded. The Seurat software package was used to perform the following downstream analyses. Principal component analysis (PCA) was used to reduce the dimensionality of the dataset, with 50 PCs used to retain >85% of the variability. Unsupervised clustering did not reveal any patterns of variation independent of treatment and invasion. Pathway enrichment analysis was performed on the single-cell sequencing dataset as described above, where three criteria were evaluated to determine differential gene expression and gene set enrichment within ROI groups: treatment vs. control; invasive vs. non-invasive (treated ROIs); and treatment vs. control (invasive ROIs). Gene set enrichment analysis (GSEA) was performed on log2 transformations and normalized gene expression matrices of invasive and non-invasive TEPA-treated ROIs.

[0296] Flow cytometry immunophenotyping Tumor sections were dissociated into single cell suspensions for flow cytometry staining as previously described. Cells were stained in fluorescence-activated cell sorting (FACS) buffer (1 x phosphate-buffered saline [PBS] / 1% FBS / 0.5 mM EDTA) with the following surface antibodies: CD45-BV510 (clone 30-F11, 1:250, BD Biosciences, #563891), CD11b-APC-ef780 (clone M1 / 70, 1:400, Thermo Fisher, #47-0112-82), Ly6G-streptavidin-BUV737 (clone 1A8, 1:300, BioLegend, #127604). Following surface staining, cells were intracellularly stained with MMP9-APC (clone S51-82, 1:1250, StressMarq Biosciences, #SMC-396D-APC) using the BD Cytofix / Cytoperm Fixation / Permeabilization Kit (BD Biosciences, USA; #554714) according to the manufacturer’s instructions. Sample acquisition was performed using a BD FACSAria III (BD Biosciences, USA) and analysis was performed using FlowJo v10 (TreeStar, USA). Representative gating strategies are presented in FIG. 6. Figure 40 b.

[0297] VetScan blood chemistry analysis Peripheral blood was obtained from animals and collected in lithium heparin tubes (Greiner, Germany; #450536) and analyzed immediately using the VetScan VS2 chemistry analyzer (Zoetis, USA) using the Comprehensive Diagnostic Profile rotor (Zoetis, USA; #500-0038) according to the manufacturer’s instructions.

[0298] Statistical analysis All in vitro experiments were repeated at least three times, and data are presented as mean ± standard error of the mean (SEM). Differences between groups were determined using the Mann- Whitney U test (unpaired or paired, as indicated). Kaplan-Meier survival curves were analysed using the two-tailed Mantel-Cox log-rank test. All statistics, except single-cell RNA sequencing data, were performed using GraphPad Prism v10 software (Dotmatics, UK). For all experiments, p-values < 0.05 were considered statistically significant (*p < 0.005, **p < 0.01, ***p < 0.001, ns: not significant).

[0299] The embodiments disclosed herein demonstrate that copper chelators can be potent modulators of the neuroblastoma microenvironment. They can slow MYCN-mediated tumour cell metabolism and progression and can promote infiltration and activation of key subsets involved in ADCC. Importantly, they can enhance the efficacy of anti-GD2 immunotherapy. This effect can improve patient response and outcome in anti-GD2 immunotherapy and can also improve patient response and outcome in other immunotherapies that would benefit from the effect of copper inhibitors to suppress immunosuppression in the tumour microenvironment.

[0300] Further embodiments of the application as described herein are defined in the following paragraphs: 1. A method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a copper chelator or a copper chelator inducer in combination with a therapeutically effective amount of an anti-cancer immunotherapeutic agent.

[0301] 2. Use of a copper chelator or a copper chelator inducer for the manufacture of a medicament for treating cancer in a subject in need thereof, wherein the copper chelator or copper chelator inducer is administered or to be administered to the subject in combination with an anti-cancer immunotherapeutic agent.

[0302] 3. Use of an anti-cancer immunotherapeutic agent for the manufacture of a medicament for treating cancer in a subject in need thereof, wherein the anti-cancer immunotherapeutic agent is administered or to be administered to the subject in combination with a copper chelator or a copper chelator inducer.

[0303] 4. Use of a copper chelator or a copper chelator inducer and an anti-cancer immunotherapeutic agent for the manufacture of a medicament for treating cancer in a subject in need thereof.

[0304] 5. A copper chelator or a copper chelator inducer for use in treating cancer in a subject in need thereof, wherein the copper chelator or copper chelator inducer is administered or to be administered to the subject in combination with an anti-cancer immunotherapeutic agent.

[0305] 6. An anti-cancer immunotherapeutic for use in treating cancer in a subject in need thereof, wherein the anti-cancer immunotherapeutic is administered or to be administered to the subject in combination with a copper-chelator or a copper-chelator inducer.

[0306] 7. A combination comprising a copper-chelator or a copper-chelator inducer and an anti-cancer immunotherapeutic for use in treating cancer in a subject in need thereof.

[0307] 8. The copper-chelator or copper-chelator inducer for use, the anti-cancer immunotherapeutic for use, the combination for use, or the method according to any one or more of the preceding paragraphs, wherein the copper-chelator or copper-chelator inducer and the anti-cancer agent are administered or to be administered to the subject separately.

[0308] 9. The copper-chelator or copper-chelator inducer for use, the anti-cancer immunotherapeutic for use, the combination for use, or the method according to any one or more of the preceding paragraphs, wherein the copper-chelator or copper-chelator inducer and the anti-cancer agent are administered or to be administered to the subject simultaneously.

[0309] 10. The copper-chelator or copper-chelator inducer for use, the anti-cancer immunotherapeutic for use, the combination for use, or the method according to any one or more of the preceding paragraphs, wherein the anti-cancer immunotherapeutic is selected from the group consisting of an antibody, an immune checkpoint inhibitor (e.g. an ICI targeting a-CTLA4 and / or an ICI targeting a-PDL1), a chimeric antigen receptor (CAR) T cell, a bi-specific T cell engager (BiTE), a (CAR) NK cell, an adoptive immune cell, and an antibody-dependent immune cell; optionally, the anti-cancer immunotherapeutic is an anti-GD2 immunotherapeutic; optionally, the anti-cancer immunotherapeutic is a GD2-directed chimeric antigen receptor (CAR) T cell or an anti-GD2 antibody; optionally, the anti-cancer immunotherapeutic is an anti-GD2 antibody; optionally, the anti-cancer immunotherapeutic is selected from the group consisting of atezolizumab (Tecentriq), avelumab (Bavencio), dostarlimab (Jemperli), durvalumab (Imfinzi), ipilimumab (Yervoy), nivolumab (Opdivo), dinutuximab (Unituxin), dinutuximab beta (Qarziba), and pembrolizumab (Keytruda).

[0310] 11. The copper-chelator or copper-chelator inducer for use, the anti-cancer immunotherapeutic for use, the combination for use, or the method according to any one or more of the preceding paragraphs, wherein the anti-cancer immunotherapeutic is an anti-GD2 immunotherapeutic, optionally a GD2-directed chimeric antigen receptor (CAR) T cell or an anti-GD2 antibody; optionally, wherein the anti-cancer immunotherapeutic is an anti-GD2 antibody.

[0311] 12. The method, use, copper-chelating agent or copper-chelating agent inducing agent for use, anticancer immunotherapeutic agent for use or combination according to any one or more of the preceding paragraphs, wherein the anticancer immunotherapeutic agent is selected from the group consisting of atezolizumab (Tecentriq), avelumab (Bavencio), dostarlimab (Jemperli), durvalumab (Imfinzi), ipilimumab (Yervoy), nivolumab (Opdivo), dinutuximab (Unituxin), dinutuximab beta (Qarziba) and pembrolizumab (Keytruda).

[0312] 13. The method, use, copper-chelator or copper-chelator inducing agent for use, anticancer immunotherapeutic agent for use, or combination of any one or more of the preceding paragraphs, wherein the cancer is selected from the group consisting of: lung cancer; breast cancer; colorectal cancer; anal cancer; pancreatic cancer; eye cancer; prostate cancer; ovarian cancer; liver and bile duct cancer; esophageal cancer; non-Hodgkin lymphoma; bladder cancer; uterine cancer; glioma, glioblastoma, medulloblastoma and other brain tumors; kidney cancer; myelofibrosis, head and neck cancer; stomach cancer; multiple myeloma; testicular cancer; germ cell tumors; neuroendocrine tumors; cervical cancer; oral cancer; carcinoid tumors of the gastrointestinal tract, breast and other organs; plasmacytoid carcinoma; mesenchymal tumors, including sarcomas, fibrosarcoma, hemangioma, angiomatosis, hemangiopericytoma, pseudovasculitic mesenchymal proliferation, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma or leiomyosarcoma; optionally neuroblastoma, breast cancer, glioblastoma, ovarian cancer, lung cancer, prostate cancer, stomach cancer, mesothelioma, liver cancer, cervical cancer, bladder cancer, thyroid cancer, pancreatic cancer, oral cancer, osteosarcoma, head and neck cancer, medulloblastoma, low-grade glioma, DMG (diffuse midline glioma), DIPG (diffuse intrinsic pontine glioma), and leukemia; optionally, wherein the cancer is selected from neuroblastoma and mesothelioma; optionally, the cancer is selected from the group consisting of: hepatocellular carcinoma, adenocarcinoma, breast cancer, and prostate cancer; optionally, the cancer is selected from the group consisting of: neuroblastoma, breast cancer, glioblastoma, ovarian cancer, lung cancer, prostate cancer, stomach cancer, mesothelioma, liver cancer, cervical cancer, bladder cancer, thyroid cancer, pancreatic cancer, oral cancer, osteosarcoma, head and neck cancer, medulloblastoma, low-grade glioma, DMG (diffuse midline glioma), DIPG (diffuse intrinsic pontine glioma), and leukemia; optionally, the cancer is breast cancer or neuroblastoma; optionally, the cancer is selected from neuroblastoma and mesothelioma; optionally, the cancer is neuroblastoma; optionally, the cancer is treatable by modulation of GD2, for example treatment with an anti-GD2 immunotherapeutic agent (e.g. an anti-GD2 antibody or a GD2-directed chimeric antigen receptor (CAR) T cell); optionally, the cancer expresses GD2; optionally, the cancer is MYCN-mediated; optionally, wherein the cancer is lung cancer, including lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, non-small cell carcinoma, small cell carcinoma, and mesothelioma; optionally, wherein the cancer is breast cancer, including ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, and mucinous carcinoma; optionally, wherein the cancer is colorectal cancer, including colon cancer and rectal cancer; optionally, wherein the cancer is pancreatic cancer, including pancreatic adenocarcinoma, islet cell carcinoma, and neuroendocrine tumors;Optionally, wherein the cancer is ovarian cancer, the ovarian cancer includes ovarian epithelial cancer or surface epithelial-stromal tumor, including serous tumor, endometrioid tumor, and mucinous cystadenocarcinoma, and sex cord-stromal tumor; optionally, wherein the cancer is liver and bile duct cancer, the liver and bile duct cancer includes hepatocellular carcinoma, cholangiocarcinoma, and hemangioma; optionally, wherein the cancer is esophageal cancer, the esophageal cancer includes esophageal adenocarcinoma and squamous cell carcinoma; optionally, wherein the cancer is uterine cancer, the uterine cancer includes endometrial adenocarcinoma, uterine papillary serous carcinoma, uterine clear cell carcinoma, uterine sarcoma, and leiomyosarcoma, and Mullerian mixed tumor; optionally, wherein the cancer is kidney cancer, the kidney cancer includes renal cell carcinoma, clear cell carcinoma, and Wilms' tumor; optionally, wherein the cancer is head and neck cancer, the head and neck cancer includes squamous cell carcinoma; optionally, wherein the cancer is gastric cancer, the gastric cancer includes gastric adenocarcinoma and gastrointestinal stromal tumor; optionally, wherein the cancer is eye cancer, the eye cancer includes retinoblastoma and uveal melanoma.

[0313] 14. The copper-chelating agent or copper-chelating agent inducer for use, the copper-chelating agent or copper-chelating agent inducer for use, the anti-cancer immunotherapeutic agent or combination for use, the method according to any one or more of the preceding paragraphs, wherein the copper-chelating agent is selected from the group consisting of ethylenediamine, polyethylenimine and derivatives thereof, tetrathiomolybdate; N,N’ bis(2-aminoethyl)ethane-1,2-diamine (trientine, triethylenetetramine, TETA); penicillamine; D-penicillamine; N’ (2-aminoethyl)- N [2-(2-aminoethylamino)ethyl]ethane-1,2-diamine (tetraethylenepentamine, TEPA); 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid (EDTA); 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA); N, N,N’,N’ tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN); 2-[4,8,11-tris(carboxymethyl)- 1,4,8,11-tetraazacyclotetradec-1-yl]acetic acid; 2-[4,7,10-tris(carboxymethyl)-1,4,7,10- tetraazacyclododec-1-yl]acetic acid (DOTA); (2S)-2-amino-3-methyl-3-thiolbutanoic acid (D-pen); bis(sulfidyl)molybdenum sulfide (TTM); 5-chloro-7-iodo-8-hydroxyquinoline (Cliquinol); metformin; diethyldithiocarbamic acid (DDC); 2,9-dimethyl-1,10-phenanthroline (new copper reagent); 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (bathocuproin); 4-[2,9-dimethyl-7-(4-sulfophenyl)-1,10-phenanthroline-4-yl]benzenesulfonic acid (BCS); and N,N’- bis(cyclohexylideneamino)oxalamide (Cuprizone); optionally, the copper chelator is selected from the group consisting of triethylenetetramine, tetraethylenepentamine, penicillamine, tetrathiomolybdate and salts thereof; optionally, the copper chelator is selected from the group consisting of tetraethylenepentamine, triethylenetetramine and salts thereof; optionally, the copper chelator is triethylenetetramine or a salt thereof; optionally, the copper chelator inducer is a zinc salt; optionally, the copper chelator is selected from the group consisting of triethylenetetramine, tetraethylenepentamine, penicillamine, tetrathiomolybdate and salts thereof.

[0314] 15. The method, use, copper chelator or copper chelator inducer for use, anti-cancer immunotherapeutic agent or combination for use according to any one or more of the preceding paragraphs, wherein the copper chelator is triethylenetetramine or a salt thereof.

[0315] 16. The method, use, copper chelator or copper chelator inducer for use, anti-cancer immunotherapeutic agent or combination for use according to any one or more of the preceding paragraphs, wherein the copper chelator inducer is a zinc salt.

[0316] 17. The method, use, copper chelator or copper chelator inducer for use, anti-cancer immunotherapeutic agent or combination for use according to any one or more of the preceding paragraphs, wherein the copper chelator or copper chelator inducer and the anti-cancer immunotherapeutic agent are administered or are to be administered in combination with a further active agent, optionally wherein the further active agent is an anti-cancer agent, optionally wherein the anti-cancer agent is selected from the group consisting of irinotecan (Camptosar) and temozolomide (Temodar).

[0317] 18. A pharmaceutical composition comprising a copper chelator or copper chelator inducer and an anti-cancer immunotherapeutic agent.

[0318] 19. The pharmaceutical composition according to any one or more of the preceding paragraphs, wherein the anti-cancer immunotherapeutic agent is selected from the group consisting of an antibody, an immune checkpoint inhibitor (e.g., an ICI targeting alpha-CTLA4 and / or an ICI targeting alpha-PDL1), a chimeric antigen receptor (CAR) T cell, a bi-specific T cell engager (BiTE), a (CAR) NK cell, an adoptive immune cell, and an antibody-dependent immune cell; optionally, the anti-cancer immunotherapeutic agent is an anti-GD2 immunotherapeutic agent; optionally, the anti-cancer immunotherapeutic agent is a GD2-directed chimeric antigen receptor (CAR) T cell or an anti-GD2 antibody; optionally, the anti-cancer immunotherapeutic agent is an anti-GD2 antibody; optionally, the anti-cancer immunotherapeutic agent is selected from the group consisting of atezolizumab (Tecentriq), avelumab (Bavencio), dostarlimab (Jemperli), durvalumab (Imfinzi), ipilimumab (Yervoy), nivolumab (Opdivo), dinutuximab (Unituxin), dinutuximab beta (Qarziba), and pembrolizumab (Keytruda); optionally, wherein the anti-cancer immunotherapeutic agent is an anti-GD2 immunotherapeutic agent, optionally a GD2-directed chimeric antigen receptor (CAR) T cell or an anti-GD2 antibody; optionally, wherein the anti-cancer immunotherapeutic agent is an anti-GD2 antibody.

[0319] 20. The pharmaceutical composition according to any one or more of the preceding paragraphs, wherein the anti-cancer immunotherapeutic agent is selected from the group consisting of an antibody, an immune checkpoint inhibitor (e.g., an ICI targeting alpha-CTLA4 and / or an ICI targeting alpha-PDL1), a chimeric antigen receptor (CAR) T cell, a bi-specific T cell engager (BiTE), a (CAR) NK cell, an adoptive immune cell, and an antibody-dependent immune cell.

[0320] 21. The pharmaceutical composition according to any one or more of the preceding paragraphs, wherein the copper chelator is selected from the group consisting of ethylenediamine, polyethylenimine and derivatives thereof, tetrathiomolybdate; N,N'-bis(2-aminoethyl)ethane-1,2-diamine (trientine, triethylenetetramine, TETA); penicillamine; D-penicillamine; N'-(2-aminoethyl)-N-[2-(2-aminoethylamino)ethyl]ethane-1,2-diamine (tetraethylenepentamine, TEPA); 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid (EDTA); 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA); N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN); 2-[4,8,11-tris(carboxymethyl)-1,4,8,11-tetraazacyclotetradecan-1-yl]acetic acid; 2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetic acid (DOTA); (2S)-2-amino-3-methyl-3-thiolbutyric acid (D-pen); bis(sulfidyl) molybdenum sulfide (TTM); 5-chloro-7-iodo-8-hydroxyquinoline (Cliquinol); metformin; diethyldithiocarbamic acid (DDC); 2,9-dimethyl-1,10-phenanthroline (neocuproine); 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (bathocuproin); 4-[2,9-dimethyl-7-(4-sulfophenyl)-1,10-phenanthroline-4-yl]benzenesulfonic acid (BCS); and N,N'-bis(cyclohexylideneamino)oxamide (Cuprizone); optionally, the copper chelator is selected from the group consisting of triethylenetetramine, tetraethylenepentamine, penicillamine, tetrathiomolybdate and salts thereof; optionally, the copper chelator is selected from the group consisting of tetraethylenepentamine, triethylenetetramine and salts thereof; optionally, the copper chelator is triethylenetetramine or a salt thereof; optionally, the copper chelator inducer is a zinc salt; optionally, the copper chelator is selected from the group consisting of triethylenetetramine, tetraethylenepentamine, penicillamine, tetrathiomolybdate and salts thereof; optionally, wherein the copper chelator is selected from the group consisting of triethylenetetramine, tetraethylenepentamine, penicillamine, tetrathiomolybdate and salts thereof.

[0321] 22. The pharmaceutical composition according to any one or more of the preceding paragraphs, wherein the copper chelator inducer is a zinc salt.

[0322] While the application has been described by reference to specific examples, it should be understood that the application can be embodied in many other forms. In particular, features of any of the described examples can be provided in any combination or arrangement in any other described example. Various modifications and changes in light thereof will be obvious to those skilled in the art without departing from the scope and spirit of the application. It should be understood that the application is not intended to be limited by the exemplary embodiments and examples set forth herein, and that these examples and embodiments are provided only as examples of the application, the scope of which is to be limited only by the claims set forth in the application appended hereto.

Claims

1. A method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a copper-chelator or a copper-chelator inducer in combination with a therapeutically effective amount of an anti-cancer immunotherapy agent.

2. Use of a copper-chelator or a copper-chelator inducer in the manufacture of a medicament for treating cancer in a subject in need thereof, wherein the copper-chelator or copper-chelator inducer is administered or to be administered to the subject in combination with an anti-cancer immunotherapy agent.

3. Use of an anti-cancer immunotherapy agent in the manufacture of a medicament for treating cancer in a subject in need thereof, wherein the anti-cancer immunotherapy agent is administered or to be administered to the subject in combination with a copper-chelator or a copper-chelator inducer.

4. Use of a copper-chelator or a copper-chelator inducer and an anti-cancer immunotherapy agent in the manufacture of a medicament for treating cancer in a subject in need thereof.

5. A copper-chelator or a copper-chelator inducer for use in treating cancer in a subject in need thereof, wherein the copper-chelator or copper-chelator inducer is administered or to be administered to the subject in combination with an anti-cancer immunotherapy agent.

6. An anti-cancer immunotherapy agent for use in treating cancer in a subject in need thereof, wherein the anti-cancer immunotherapy agent is administered or to be administered to the subject in combination with a copper-chelator or a copper-chelator inducer.

7. A kit or combination comprising a copper-chelator or a copper-chelator inducer and an anti-cancer immunotherapy agent for use in treating cancer in a subject in need thereof.

8. The method according to claim 1; the use according to any one of claims 2 to 4; the copper-chelator or copper-chelator inducer for use according to claim 5; the anti-cancer immunotherapy agent for use according to claim 6; or the kit or combination for use according to claim 7; wherein the copper-chelator or copper-chelator inducer and the anti-cancer agent are administered separately or to be administered separately to the subject.

9. The method according to claim 1 ; the use according to any one of claims 2 to 4; the copper-chelator or copper-chelator inducer for use according to claim 5; the anti-cancer immunotherapy agent for use according to claim 6; or the kit or combination for use according to claim 7; wherein the copper-chelator or copper-chelator inducer and the anti-cancer agent are administered simultaneously or to be administered simultaneously to the subject.

10. The method according to claim 1, 8 or 9; the use according to any one of claims 2 to 4 and 8 to 9; the copper-chelator or copper-chelator inducer for use according to claim 5 and 8 to 9; the anti-cancer immunotherapy agent for use according to claim 6 and 8 to 9; or the kit or combination for use according to claim 7 and 8 to 9; wherein the anti-cancer immunotherapy agent is selected from the group consisting of an antibody, an immune checkpoint inhibitor, a chimeric antigen receptor (CAR) T cell, a bispecific T cell engager (BiTE), a (CAR) NK cell, an adoptive immune cell and an antibody-dependent immune cell.

11. The method of any one of claims 1 and 8-10; the use of any one of claims 2-4 and 8-10; the copper-chelator or copper-chelator inducing agent for use of claims 5 and 8-10; the anti-cancer immunotherapeutic agent for use of claims 6 and 8-10; or the kit or combination for use of claims 7 and 8-10; wherein the anti-cancer immunotherapeutic agent is an anti-GD2 immunotherapeutic agent, optionally a GD2-directed chimeric antigen receptor (CAR) T cell or an anti-GD2 antibody; optionally, wherein the anti-cancer immunotherapeutic agent is an anti-GD2 antibody.

12. The method of any one of claims 1 and 8-10; the use of any one of claims 2-4 and 8-10; the copper-chelator or copper-chelator inducing agent for use of claims 5 and 8-10; the anti-cancer immunotherapeutic agent for use of claims 6 and 8-10; or the kit or combination for use of claims 7 and 8-10; wherein the anti-cancer immunotherapeutic agent is selected from the group consisting of atezolizumab (Tecentriq), avelumab (Bavencio), dostarlimab (Jemperli), durvalumab (Imfinzi), ipilimumab (Yervoy), nivolumab (Opdivo), dinutuximab (Unituxin), dinutuximab beta (Qarziba), and pembrolizumab (Keytruda).

13. The method of any one of claims 1 and 8-12; the use of any one of claims 2-4 and 8-12; the copper-chelator or copper-chelator inducing agent for use of claims 5 and 8-12; the anti-cancer immunotherapeutic agent for use of claims 6 and 8-12; or the kit or combination for use of claims 7 and 8-12; wherein the cancer is selected from the group consisting of neuroblastoma, breast cancer, glioblastoma, ovarian cancer, lung cancer, prostate cancer, gastric cancer, mesothelioma, liver cancer, cervical cancer, bladder cancer, thyroid cancer, pancreatic cancer, oral cancer, osteosarcoma, head and neck cancer, medulloblastoma, low-grade glioma, DMG (diffuse midline glioma), DIPG (diffuse intrinsic pontine glioma), and leukemia; optionally wherein the cancer is selected from the group consisting of neuroblastoma and mesothelioma.

14. The method of any one of claims 1 and 8-13; the use of any one of claims 2-4 and 8-13; the copper-chelator or copper-chelator inducing agent for use of claims 5 and 8-13; the anti-cancer immunotherapeutic agent for use of claims 6 and 8-13; or the kit or combination for use of claims 7 and 8-13; wherein the copper-chelator is selected from the group consisting of triethylenetetramine, tetraethylenepentamine, penicillamine, tetrathiomolybdate, and salts thereof.

15. The method of any one of claims 1 and 8-14; the use of any one of claims 2-4 and 8-14; the copper-chelator or copper-chelator inducing agent for use of claims 5 and 8-14; the anti-cancer immunotherapeutic agent for use of claims 6 and 8-14; or the kit or combination for use of claims 7 and 8-14; wherein the copper-chelator is triethylenetetramine or a salt thereof.

16. The method of any one of claims 1 and 8-13; the use of any one of claims 2-4 and 8-13; the copper-chelator or copper-chelator inducing agent for use of claims 5 and 8-13; the anti-cancer immunotherapeutic agent for use of claims 6 and 8-13; or the kit or combination for use of claims 7 and 8-13; wherein the copper-chelator inducing agent is a zinc salt.

17. The method of any one of claims 1 and 8-16; the use of any one of claims 2-4 and 8-16; the copper-chelator or copper-chelator inducing agent for use of claims 5 and 8-16; the anti-cancer immunotherapeutic agent for use of claims 6 and 8-16; or the kit or combination for use of claims 7 and 8-16; wherein the copper-chelator or copper-chelator inducing agent and the anti-cancer immunotherapeutic agent are administered or to be administered in combination with an additional active agent, optionally wherein the additional active agent is an anti-cancer agent, optionally wherein the anti-cancer agent is selected from irinotecan (Camptosar) and temozolomide (Temodar).

18. A pharmaceutical composition comprising a copper-chelator or copper-chelator inducing agent and an anti-cancer immunotherapeutic agent.

19. The pharmaceutical composition of claim 18, wherein the anti-cancer immunotherapeutic agent is an anti-GD2 immunotherapeutic agent, optionally a GD2-directed chimeric antigen receptor (CAR) T cell or an anti-GD2 antibody; optionally, wherein the anti-cancer immunotherapeutic agent is an anti-GD2 antibody.

20. The pharmaceutical composition of claim 18, wherein the anti-cancer immunotherapeutic agent is selected from an antibody, an immune checkpoint inhibitor (e.g., an ICI targeting a-CTLA4 and / or an ICI targeting a-PDL1), a chimeric antigen receptor (CAR) T cell, a bispecific T cell engager (BiTE), a (CAR) NK cell, an adoptive immune cell, and an antibody-dependent immune cell.

21. The pharmaceutical composition of any one of claims 18-20, wherein the copper-chelator is selected from triethylenetetramine, tetraethylenepentamine, penicillamine, tetrathiomolybdate, and salts thereof.

22. The pharmaceutical composition of any one of claims 18-20, wherein the copper-chelator inducing agent is a zinc salt.

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