Cancer treatment using ultra-high concentration gaseous nitric oxide and checkpoint inhibitors

Abstract

Cancer treatment using ultra-high concentration gaseous nitric oxide (UNO) and checkpoint inhibitors, optionally with an immune adjuvant, is provided. Additionally, a sensitization treatment for checkpoint inhibitors is provided. Accordingly, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of UNO and a checkpoint inhibitor, optionally with an immune adjuvant.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 358,540, filed Jul. 6, 2022; U.S. Provisional Patent Application No. 63 / 358,542, filed Jul. 6, 2022; U.S. Provisional Patent Application No. 63 / 358,547, filed Jul. 6, 2022; U.S. Provisional Patent Application No. 63 / 439,435, filed Jan. 17, 2023; U.S. Provisional Patent Application No. 63 / 451,783, filed Mar. 13, 2023; and U.S. Provisional Patent Application No. 63 / 460,187, filed Apr. 18, 2023. The entire contents of the applications referenced above are incorporated herein by reference as part of this specification.

[0002] Technical Field In some of its aspects, the present invention relates to cancer treatment using ultra-high concentration gaseous nitric oxide and checkpoint inhibitors.

[0003] Background Art Cancer immunotherapies, including cell-based therapies, antibody therapies, cytokine therapies or adjuvant therapies, and vaccines, have emerged in recent years as promising strategies for treating various types of cancer. Thus, for example, durable remissions have been achieved across a wide variety of different tumor types through modulation of the immune system of existing patients via checkpoint inhibitors such as anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies (e.g., Kamir J. et al. Nat Rev Cancer; volume 21 346-359).

[0004] To date, the ability to successfully treat cancer using immune checkpoint antibodies is very limited. The art has cited numerous reasons, and generally speaking, the use of this tool by physicians is very limited (De Miguel and Calvo Cancer Cell (2020) Sep 14;38(3):326-333).

[0005] Concurrently, tumor ablation is a minimally invasive technique used in the treatment of solid tumors. There are several ablation methods, such as radiofrequency, microwave ablation, focused high-intensity ultrasound ablation, laser ablation, and cryoablation (Knavel, E.M and Brace, C.L. Tech Vasc Interv Radiol 16(4), 192 - 200, 2013). Image-guided tumor ablation for early hepatocellular carcinoma (HCC) is an approved non-surgical treatment that provides local tumor control and favorable survival benefits (Kang, T.W and Rhim, H. Liver Cancer 4(3), 176 - 187, 2015). Local and in situ tumor ablation methods have been shown to enhance the anti-tumor immune response, resulting in the destruction of malignant cells remaining in the primary tumor and distant metastases [Keisari, Y. et al. Cancer Immunol. Immunother. 63, 1 - 9 (2014); Confino et al. Cancer Immunol Immunother 64(2) 191 - 199, 2015]. Notably, in contrast to surgical resection, even if most of the tumor has been destroyed using ablation, antigenic remnants continue to exist at the tumor site / body. This aspect of ablation is responsible for its ability to trigger a systemic anti-tumor immune response.

[0006] Nitric oxide (NO) is a short-lived, endogenously produced gas that acts as a signaling molecule in the body (Thomas, D.D. Redox Biol 5, 225 - 233, 2015). Increasing evidence highlights its wide range of actions in different pathologies, including its involvement in immune cell signaling against cancer (Huerta, S. Futur. Sci. OA 1, FSO44, 2015) and pathogens (Schairer et al. Virulence 3, 271 - 279, 2012).

[0007] Preclinical trials testing the effects of nitric oxide (NO) administered ex vivo have demonstrated its anticancer properties, suggesting that NO could serve as a potent antitumor ablation agent. While low doses of NO have pro-oncogenic properties, at high doses, NO can play a role in cancer therapy either as a single agent or in combination with other anti-cancer compounds (e.g., Huerta S. Future Sci OA. 2015 Aug 1;1(1):FSO44; Vannini F. et al. Redox Biol. 2015 Dec;6:334-343; Seabra AB et al. Eur J Pharmacol. 2018 May 5;826:158-168; Alimoradi H. et al. Pharm Nanotechnol. 2019;7(4):279-303; and Ning S. et al. Biochem Biophys Res Commun. 2014 May 9;447(3):537-42). More specifically, high doses of NO have been shown to promote oxidative / nitrosative stress and DNA damage. Through the generation of reactive nitrogen species including peroxynitrite, DNA can be oxidized and single-strand breaks induced. In addition, NO can induce cell death via both i) necrosis and ii) apoptosis (Seabra AB and Duran N. Eur J Pharmacol. 2018 May 5;826:158-168; Vannini F. et al. Redox Biol. 2015 Dec;6:334-343).

[0008] The use of gaseous NO (gNO) in the treatment of cancer has been previously described in WO 2021 / 105901, WO 2021 / 105900 and WO 2022 / 043931.

[0009] Further background art is Aizhang Xu et al. Cellular & Molecular Immunology (2019) 16:820-832, Wang Shu, et al. PNAS (2016), October 31, 2016 including Stefania Cuzzubbo, et al. Front. Immunol. (2021) 17 February

[0010] Summary of the Invention In one aspect of some embodiments of the present invention, a method for treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of ultra-high concentration gaseous nitric oxide (UNO) at, for example, about 10,000 ppm to about 1,000,000 ppm and a checkpoint inhibitor, thereby treating the cancer in the subject. In one aspect of some embodiments of the present invention, UNO is used as a sensitizing treatment for the checkpoint inhibitor, provided that UNO is used to upregulate the expression of the target immune checkpoint protein prior to the administration of the checkpoint inhibitor.

[0011] In one aspect of some embodiments of the present invention, the method further comprises administering an immune adjuvant.

[0012] In one aspect of some embodiments of the present invention, a combination of UNO and a checkpoint inhibitor for use in treating cancer in a subject in need thereof is provided.

[0013] In one aspect of some embodiments of the present invention, the combination further comprises an immune adjuvant.

[0014] In one aspect of some embodiments of the present invention, at least one checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, and an anti-LAG-3 antibody.

[0015] In one aspect of some embodiments of the present invention, the anti-PD-1 antibody is one of pembrolizumab, nivolumab, semipramab, spartalizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, JTX-4014, INCMGA00012, AMP-224, and AMP-514.

[0016] In one aspect of some embodiments of the present invention, the anti-PD-L1 antibody is one of atezolizumab, durvalumab, avelumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189.

[0017] In one aspect of some embodiments of the present invention, the anti-CTLA-4 antibody is one of ipilimumab and tremelimumab.

[0018] In one aspect of some embodiments of the present invention, the anti-LAG-3 antibody is relatlimab.

[0019] In some embodiments of the present invention, UNO is administered locally.

[0020] In some embodiments of the present invention, local administration includes intratumoral administration.

[0021] In some embodiments of the present invention, UNO is administered at a dose of about 10,000 ppm to about 1,000,000 ppm for a time of about 1 second to about 60 seconds at a volumetric flow rate of about 0.00001 LPM to about 1 LPM.

[0022] In some embodiments of the present invention, UNO is administered at a dose of about 20,000 ppm to about 200,000 ppm or at a dose of about 20,000 ppm to about 100,000 ppm.

[0023] In some embodiments of the present invention, UNO is administered for a time in the range of about 1 second to about 10 minutes.

[0024] In some embodiments of the present invention, UNO is administered at a volumetric flow rate of from about 0.001 LPM to about 0.5 LPM.

[0025] In some embodiments of the present invention, the checkpoint inhibitor is administered prior to UNO.

[0026] In some embodiments of the present invention, UNO is administered prior to the checkpoint inhibitor.

[0027] In some embodiments of the present invention, after administering an effective amount of UNO, cancer cells express an immune checkpoint protein or its binding partner.

[0028] In some embodiments of the present invention, the checkpoint inhibitor is administered every 2 to 7 days.

[0029] In some embodiments of the present invention, the checkpoint inhibitor is administered at least twice.

[0030] In some embodiments of the present invention, the cancer is refractory to treatment with a checkpoint inhibitor.

[0031] In some embodiments of the present invention, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, B7H2, B7H4, CTLA-4, CD80, CD86, LAG-3, TIM-3, KIR, IDO, CD19, OX40, 4-1BB (CD137), CD27, CD70, CD40, GITR, CD28 and ICOS (CD278).

[0032] In some embodiments of the present invention, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, CTLA-4 and LAG-3.

[0033] In some embodiments of the present invention, the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody and an anti-LAG-3 antibody.

[0034] In some embodiments of the present invention, the immune adjuvant is selected from the group consisting of inorganic salts, aluminum salts, organic adjuvants, emulsions, microparticles, liposomes, saponins, cytokines, microbial components, and nucleic acid adjuvants.

[0035] In some embodiments of the present invention, the immune adjuvant comprises a nucleic acid adjuvant.

[0036] In some embodiments of the present invention, the nucleic acid adjuvant comprises CpG oligodeoxynucleotide (CpG ODN).

[0037] In some embodiments of the present invention, the checkpoint inhibitor is administered before UNO.

[0038] In some embodiments of the present invention, the checkpoint inhibitor is administered before the immune adjuvant.

[0039] In some embodiments of the present invention, the immune adjuvant is administered following UNO.

[0040] In some embodiments of the present invention, the cancer is refractory to treatment with a PD-1 inhibitor.

[0041] In some embodiments of the present invention, the cancer is refractory to treatment with a PD-L1 inhibitor.

[0042] In some embodiments of the present invention, the cancer is refractory to treatment with a CTLA-4 inhibitor.

[0043] In some embodiments of the present invention, the cancer is refractory to treatment with a LAG-3 inhibitor.

[0044] In some aspects of the present invention, UNO primes cancer for treatment with a PD-1 inhibitor by upregulating the expression of PD-1 or PD-L1 before administration of the PD-1 inhibitor.

[0045] In some aspects of the present invention, UNO primes cancer for treatment with a PD-L1 inhibitor by upregulating the expression of PD-L1 before administration of the PD-L1 inhibitor.

[0046] In some aspects of the present invention, UNO primes cancer for treatment with a CTLA-4 inhibitor by upregulating the expression of CTLA-4 before administration of the CTLA-4 inhibitor.

[0047] In some aspects of the present invention, administration of UNO elicits a higher tumor-specific immune cell response. In some aspects, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0048] In some aspects of the present invention, the combination of UNO and a checkpoint inhibitor elicits a higher tumor-specific immune cell response. In some aspects of the present invention, the combination of UNO and a checkpoint inhibitor elicits a higher tumor-specific immune cell response synergistically. In some aspects, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0049] In some aspects of the present invention, the combination of UNO and a PD-1 inhibitor elicits a higher tumor-specific immune cell response. In some aspects, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells. In some aspects of the present invention, the combination with UNO and a PD-1 inhibitor elicits a higher tumor-specific immune cell response synergistically. In some aspects, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0050] In some embodiments of the present invention, the combination of UNO and a PD-L1 inhibitor elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells. In some embodiments of the present invention, the combination of UNO and a PD-L1 inhibitor synergistically elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0051] In some embodiments of the present invention, the combination of UNO and a CTLA-4 inhibitor elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells. In some embodiments of the present invention, the combination of UNO and a CTLA-4 inhibitor synergistically elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0052] In some embodiments of the present invention, the combination of UNO and a LAG-3 inhibitor elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells. In some embodiments of the present invention, the combination of UNO and a LAG-3 inhibitor synergistically elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0053] In some embodiments of the present invention, the cancer is positive for microsatellite instability (MSI) and / or mismatch repair deficient (dMMR) markers.

[0054] In some embodiments of the present invention, the cancer is negative for microsatellite instability (MSI) markers and / or mismatch repair deficient (dMMR) markers.

[0055] In some embodiments of the present invention, the cancer is selected from the group consisting of colon cancer, breast cancer, melanoma (e.g., BRAF-positive malignant melanoma), lung cancer (e.g., Non-Small Cell Lung Cancer (NSCLC)), Head and Neck Squamous Cell Cancer (HNSCC), Classical Hodgkin Lymphoma (cHL), Primary Mediastinal Large B-Cell Lymphoma (PMBCL), urothelial cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel Cell Carcinoma (MCC), Renal Cell Carcinoma (RCC), endometrial cancer, Tumor Mutational Burden-High (TMB-H) cancer, Cutaneous Squamous Cell Carcinoma (cSCC), Triple-Negative Breast Cancer (TNBC), low microsatellite instability cancer, high microsatellite instability cancer or mismatch repair-deficient cancer, and low microsatellite instability colorectal cancer (CRC), high microsatellite instability colorectal cancer or mismatch repair-deficient colorectal cancer (CRC).

[0056] In some embodiments, UNO can be administered to treat solid tumors at a dose of 10,000 ppm, 15,000 ppm, 20,000 ppm, 25,000 ppm, 50,000 ppm or 100,000 ppm via local administration such as intratumoral injection. In some embodiments, UNO can be administered at a flow rate of 0.2 L / min for a period of 5 minutes. In some embodiments, UNO can be administered in multiple cycles. In some embodiments, the cycle is 3 weeks (21 days), provided that UNO can be administered on the first day and / or the eighth day of one or more 21-day cycles. In some embodiments, the cycle is 4 weeks (28 days), provided that UNO can be administered on the first day and / or the eighth day and / or the fifteenth day of one or more 28-day cycles. In some embodiments, UNO can be administered to multiple tumor sites. In some embodiments, UNO can be administered in combination with a checkpoint inhibitor, provided that the checkpoint inhibitor is one of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor and a LAG-3 inhibitor. In some embodiments, the checkpoint inhibitor can be administered intravenously following the administration of UNO.

[0057] In some embodiments, UNO can be administered to treat primary or metastatic tumors at a dose of 50,000 ppm via local administration such as intratumoral injection. In some embodiments, UNO can be administered at a flow rate of 2 L / min for a period of 5 minutes. In some embodiments, UNO can be administered over multiple 21-day cycles, provided that UNO can be administered on the first day of one or more 21-day cycles.

[0058] In some embodiments, UNO can be administered to treat triple-negative breast cancer (TNBC) at a dose of 50,000 ppm via local administration such as intratumoral injection. In some embodiments, UNO can be administered at a flow rate of 2 L / min for a period of 5 minutes. In some embodiments, UNO can be administered over multiple 21-day cycles, provided that UNO can be administered on the first day of one or more 21-day cycles. In some embodiments, UNO can be administered in combination with a checkpoint inhibitor, provided that the checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, pembrolizumab can be administered intravenously following the administration of UNO. In some embodiments, pembrolizumab is administered at a dose of 200 mg for a period of 30 minutes every 3 weeks. In some embodiments, pembrolizumab is administered at a dose of 400 mg for a period of 30 minutes every 6 weeks.

[0059] In some embodiments, UNO can be administered to treat advanced cutaneous malignant melanoma at a dose of 25,000 ppm or 50,000 ppm via local administration such as intratumoral injection. In some embodiments, UNO can be administered at a flow rate of 2 L / min for a period of 5 minutes. In some embodiments, UNO can be administered over multiple 21-day cycles, provided that UNO can be administered on the first day of one or more 21-day cycles. In some embodiments, UNO can be administered in combination with a checkpoint inhibitor, provided that the checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is ipilimumab. In some embodiments, ipilimumab is administered intravenously following the administration of UNO, provided that ipilimumab is administered at a dose of 3 mg / kg for a period of 30 minutes. In some embodiments, ipilimumab is administered for a maximum of 4 cycles.

[0060] Unless otherwise defined, all technical and / or chemical terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the embodiments of this invention, representative methods and / or materials are described below. In case of conflict, the patent specification including the definitions shall control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting necessarily.

[0061] The patent or application documents contain at least one drawing created in color. Copies of this patent or patent application containing one or more color drawings are provided by the Patent Office upon request and payment of the necessary fees.

[0062] Some embodiments of the present invention are described herein. By way of example only, reference is made to the accompanying drawings. Referring particularly to the drawings in detail, the details shown are by way of example and for the purpose of illustrative description of embodiments of the present invention. In this regard, the description by the drawings will make it apparent to those skilled in the art how embodiments of the present invention can be practiced.

Brief Description of the Drawings

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[0064] Description of Specific Embodiments of the Invention In some aspects, the present invention relates to the treatment of cancer using UNO and checkpoint inhibitors. In some aspects, the treatment further includes the use of an immune adjuvant. In some aspects, the present invention relates to UNO as a sensitizing treatment for checkpoint inhibitors.

[0065] Before describing at least one aspect of the present invention in detail, it is to be understood that the present invention is not necessarily limited to the details as set forth in the following description or exemplified by the details illustrated in the following examples with respect to its application. The present invention is capable of being practiced or carried out in other aspects or in various ways.

[0066] Cancer immunotherapies, including cell-based therapies, antibody therapies, cytokine therapies or adjuvant therapies, and vaccines, have emerged in recent years as promising strategies for treating various types of cancer. In parallel, tumor ablation is a minimally invasive technique commonly used in the treatment of solid tumors. In situ tumor ablation of solid tumors via delivery of exogenous UNO has been described in recent years.

[0067] While practicing certain aspects of the present invention, the inventors of the present invention have now discovered that treatment using a combination of UNO and a checkpoint inhibitor has a combination of improved anti-cancer effects (Examples 1-2 in the Examples section below).

[0068] The inventors of the present invention have also discovered that treatment using a combination of UNO, a checkpoint inhibitor and an immune adjuvant has a combination of improved anti-cancer effects (Examples 4-6 in the Examples section below).

[0069] As a result, the teachings of the present invention suggest the use of a combination of UNO, a checkpoint inhibitor and optionally an immune adjuvant for the treatment of cancer.

[0070] In some aspects, the teachings of the present invention suggest the use of UNO as a priming treatment for checkpoint inhibitors in cancer treatment, provided that administration of UNO upregulates the expression of one or more immune checkpoint proteins.

[0071] Accordingly, in one aspect of the present invention, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of UNO and a checkpoint inhibitor, thereby treating cancer in the subject. In some embodiments, the method further comprises administering an immune adjuvant. In some embodiments, UNO is administered prior to the checkpoint inhibitor.

[0072] In a further or alternative aspect of the present invention, there is provided a combination of UNO and a checkpoint inhibitor for use in treating cancer in a subject in need thereof. In some embodiments, the combination further comprises an immune adjuvant. In some embodiments, UNO is provided to the subject prior to the checkpoint inhibitor.

[0073] As used herein, the term "treating" refers to curing, reversing, attenuating, reducing, minimizing, suppressing or halting the deleterious effects of a disease or disorder (e.g., cancer). One of ordinary skill in the art can evaluate the development of a pathological condition using various methodologies and assays, and similarly, can evaluate the reduction, remission or regression of a pathological condition (e.g., malignancy) using various methodologies and assays as described below.

[0074] As used throughout this specification, the terms "subject" and "patient" are used interchangeably herein and refer to both human and non-human organisms (i.e., animals). The term "non-human animal" of the present disclosure encompasses all vertebrates for medical and / or laboratory research purposes, such as mammals and non-mammals including non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians. Preferably, the subject is a human subject. More preferably, the subject is a human patient diagnosed with cancer (e.g., a pre-malignant or malignant tumor).

[0075] As used throughout this specification, the term "tumor" describes a tissue composed of multiple cells that is characterized by abnormal cell proliferation and does not perform a physiological function.

[0076] "Abnormal cell proliferation" means the progressive growth of cells that is no longer under the control of the normal body and is uncontrollable. The growth of tumor tissue typically exceeds and does not cooperate with the growth of normal cells or the surrounding tissue.

[0077] "Abnormal cell proliferation" also includes, for example, (1) cancerous (or cancer) cells that proliferate by expressing mutant tyrosine kinases or by overexpressing receptor tyrosine kinases; (2) benign and malignant cells of other proliferative diseases in which abnormal tyrosine kinase activation occurs; (3) any tumor that proliferates by receptor tyrosine kinases; (4) any tumor that proliferates by abnormal serine / threonine kinase activation, and (5) abnormal proliferation of benign and / or malignant cells of other proliferative diseases in which abnormal serine / threonine kinase activation occurs, which describes cell proliferation that is independent of the normal control mechanism (e.g., loss of contact inhibition).

[0078] The tumors as described herein can be primary tumors or secondary tumors.

[0079] The term "malignant tumor" describes a tumor that does not naturally terminate in its growth, is infiltrative of adjacent tissues, and can spread to distant tissues (metastatic). The term "benign tumor" describes a tumor that is not malignant (i.e., does not grow in a highly malignant manner without limit, does not infiltrate surrounding tissues, and does not metastasize).

[0080] The term "primary tumor" describes a tumor that is present at the original site where it first occurred.

[0081] The term "secondary tumor" describes a tumor that has spread from its original (primary) site of growth to another site, a tumor that is adjacent to or distant from the primary site, which is referred to herein and in the art as a metastasis or metastatic tumor. As used herein, the term "secondary tumor" also describes a recurrent tumor, which may originally be present at the original site as a primary tumor and / or may be present at another site as a metastatic tumor.

[0082] In some of any of the aspects described herein, the tumor is a malignant tumor, such as a malignant cancerous tumor, and the tumor cells are cancer cells or cancerous cells.

[0083] In these aspects, the methods and uses as described herein for any of each aspect are for treating cancer or a cancerous tumor in a subject in need thereof.

[0084] The methods and uses as described herein relate to subjects having a primary cancerous tumor, metastatic cancer and / or recurrent cancer as described herein.

[0085] The term "cancer" as used herein encompasses malignant and benign tumors, and disease states arising from primary or secondary tumors, as described herein.

[0086] Examples of benign tumors include, but are not limited to, lipomas, chondromas, adenomas, trichoblastomas, teratomas and hamartomas.

[0087] Cancers treatable according to aspects of the present invention include, but are not limited to, carcinomas, sarcomas, blastomas and germ cell tumors. Carcinomas include, but are not limited to, adenocarcinomas (e.g., small cell lung cancer, renal adenocarcinoma, uterine adenocarcinoma, prostate adenocarcinoma, bladder adenocarcinoma, ovarian adenocarcinoma and / or colorectal adenocarcinoma) and epithelial adenocarcinomas.

[0088] Examples of cancers treatable according to aspects of the present invention include adenocarcinoma, adrenal tumors (e.g., hereditary adrenocortical carcinoma), biliary tract tumors, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., ductal carcinoma, invasive ductal carcinoma, sporadic breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer - 1, breast cancer - 3 and / or breast cancer - ovarian cancer), bronchogenic large cell carcinoma, cervical cancer (e.g., cervical adenocarcinoma), carcinosarcoma, choriocarcinoma, cystadenocarcinoma, dermatofibrosarcoma protuberans, ductal adenocarcinoma, Ehrlich's ascites tumor, embryonal rhabdomyosarcoma, endocrine tumors, endometrial cancer (e.g., endometrial adenocarcinoma), epithelioblastoma, squamous cell carcinoma, epithelial adult tumors, epithelial tumors, erythroleukemia (e.g., Friend type and lymphoblastic), extraskeletal myxoid chondrosarcoma, fibrosarcoma, gallbladder cancer, ganglioneuroma, gastrointestinal tract tumors (e.g., colon carcinoma, rectal carcinoma, colorectal adenocarcinoma, colorectal cancer, colorectal adenoma, hereditary non - polyposis colorectal cancer type 1, hereditary non - polyposis colorectal cancer type 2, hereditary non - polyposis colorectal cancer type 3, hereditary non - polyposis colorectal cancer type 6, hereditary non - polyposis colorectal cancer type 7, small intestine adenocarcinoma and / or colorectal adenocarcinoma, esophageal cancer, esophageal cancer with tylosis, gastric adenocarcinoma, pancreatic adenocarcinoma and / or pancreatic endocrine tumors), germ cell tumors (male germ cell tumors, and / or testicular undifferentiated germ cell tumors and / or ovarian undifferentiated germ cell tumors), giant cell tumors, glioma tumors, gliomas, glioblastoma (e.g., glioblastoma multiforme, astrocytoma), head and neck cancer, heterologous hybridomas, heterologous myelomas, histiocytomas, hybridomas (e.g., B cells), Grawitz tumors, insulinomas, islet tumors, keratosis, large cell adenocarcinoma, leiomyoblastoma, leiomyosarcoma, leukemia (e.g., acute lymphocytic leukemia, acute lymphoblastic leukemia, acute lymphoblastic B - progenitor cell type leukemia, acute lymphoblastic T - cell leukemia, acute megakaryoblastic leukemia, monocytic leukemia, acute myelogenous leukemia, acute myeloidleukemia), acute myeloid leukemia with eosinophilia, B-cell leukemia, eosinophilic leukemia, Friend leukemia, granulocytic leukemia or myeloblastic leukemia, hairy cell leukemia, lymphocytic leukemia, mast cell leukemia, megakaryoblast leukemia, monocytic leukemia, monocytic-macrophage leukemia, myeloblastic leukemia, myeloid leukemia, myelomonocytic leukemia, plasmacytic leukemia, B-precursor cell leukemia, promyelocytic leukemia, subacute leukemia, T-cell leukemia, lymphoid tumors, predisposition to myeloid malignancies, and / or acute non-lymphocytic acute leukemia), Li-Fraumeni syndrome, liposarcoma, liver cancer (e.g., hepatoblastoma, hepatocellular adenocarcinoma, hepatocellular carcinoma, and / or hepatocytoma), lung cancer (e.g., Lewis lung adenocarcinoma, small cell adenocarcinoma and / or non-small cell adenocarcinoma), lymphoma (e.g., Hodgkin disease, non-Hodgkin lymphoma, B-cell lymphoma, diffuse large B-celllymphoma, DLBCL), Burkitt lymphoma, cutaneous T-cell lymphoma, histiocytic lymphoma, lymphoblastic lymphoma, T-cell lymphoma and / or thymic lymphoma), lymphosarcoma, Lynch cancer familial syndrome II, breast carcinoma tumor, mastocytoma, medulloblastoma, medullary carcinoma, melanoma, mesothelioma, metastatic tumor, monocytic tumor, mucinous epidermal cell tumor, multiple glomus tumors, multiple meningiomas, myelodysplastic syndrome, myeloma (e.g., multiple myeloma), nasopharyngeal carcinoma, nephroblastoma, nerve tissue glioma, nerve tissue neuron tumor, neurilemmoma, neuroblastoma, neurogenic tumor, non-melanoma skin cancer, oat cell carcinoma, anaplastic glioma, osteochondroma, myeloma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian cell carcinoma, serous ovarian cancer, and / or ovarian sex cord tumor), papillary cell carcinoma, papilloma, paraganglioma (e.g., familial non-chromaffin paraganglioma), pheochromocytoma, pituitary tumor (invasive), placental trophoblast tumor, plasmacytoma, prostate cancer (e.g., prostatic adenocarcinoma), renal cancer (e.g., Wilms tumor type 2 or type 1), retinoblastoma, rhabdoid tumor (e.g., rhabdoid tumor predisposition syndrome), rhabdomyosarcoma, sacrococcygeal tumor, sarcoma (e.g., Ewing sarcoma, histiocytic cell sarcoma, rat sarcoma, myxosarcoma, osteosarcoma, reticulosarcoma, soft tissue sarcoma and / or synovial sarcoma), schwannoma, small cell carcinoma, spindle cell carcinoma, spinous cell cell tumor, squamous cell carcinoma (e.g., head and neck), subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma (e.g., immature teratoma of the ovary), testicular cancer (e.g., testicular germ cell tumor), transitional cell carcinoma, Turcot syndrome with glioblastoma, thymoma, thyroid cancer (e.g., thyroid follicular carcinoma, thyroid medullary carcinoma and / or papillary thyroid carcinoma), hair follicle epithelioma, choriocarcinoma, undifferentiated carcinoma, uterine cancer, cervical cell carcinoma, including but not limited to these.

[0089] The methods and uses of this aspect can be used to treat one or more solid tumors.

[0090] As used herein, the term "solid tumor" refers to a condition such as cancer that forms abnormal tumor masses such as sarcomas, carcinomas, and lymphomas. For example, solid tumors include, but are not limited to, ovarian tumors, prostate tumors, skin tumors, lung tumors, breast tumors, liver tumors, brain tumors, CNS tumors, kidney tumors, colon tumors, bladder tumors, intestinal tumors, melanomas, gliomas, epitheliomas, anaplastic gliomas, astrocytomas, glioblastomas, and medulloblastomas. Preferred examples of solid tumor diseases include, but are not limited to, non-small cell lung cancer (NSCLC), neuroendocrine tumors, thymomas, fibrous tumors, metastatic colorectal cancer (mCRC) with unresectable metastases, etc. In certain embodiments, the solid tumor disease is adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.

[0091] In some embodiments, the cancer is a solid tumor or includes a solid tumor, for example, adenocarcinoma, adrenal tumor (e.g., hereditary adrenocortical carcinoma), biliary tract tumor, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., ductal breast cancer, invasive ductal carcinoma in situ, sporadic breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer-1, breast cancer-3 and / or breast cancer-ovarian cancer), bronchogenic large cell carcinoma, cervical cancer (e.g., cervical adenocarcinoma), carcinosarcoma, choriocarcinoma, cystadenocarcinoma, dermatofibrosarcoma protuberans, ductal adenocarcinoma, Ehrlich's ascites tumor, embryonal rhabdomyosarcoma, endocrine tumor, endometrial cancer (e.g., endometrial adenocarcinoma), ependymoblastoma, squamous cell carcinoma, epithelial adult tumor, epithelial tumor, extraskeletal myxoid chondrosarcoma, fibrosarcoma, gallbladder cancer, ganglioneuroma, gastrointestinal tract tumor (e.g., colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, type 1 hereditary nonpolyposis colorectal cancer, type 2 hereditary nonpolyposis colorectal cancer, type 3 hereditary nonpolyposis colorectal cancer, type 6 hereditary nonpolyposis colorectal cancer, type 7 hereditary nonpolyposis colorectal cancer, small intestine adenocarcinoma and / or colorectal adenocarcinoma, esophageal cancer, esophageal cancer associated with tylosis, gastric adenocarcinoma, pancreatic adenocarcinoma and / or pancreatic endocrine tumor), germ cell tumor (male germ cell tumor, and / or seminoma undifferentiated and / or ovarian undifferentiated germ cell tumor), giant cell tumor, glioma, glioblastoma (e.g., glioblastoma multiforme, astrocytoma), head and neck cancer, heterologous hybridoma, heterologous myeloma, histiocytoma, hybridoma (e.g., B cell), Grawitz tumor, insulinoma, islet tumor, keratosis, large cell adenocarcinoma, leiomyoblastoma, liposarcoma, liver cancer (e.g., hepatoblastoma, hepatocellular adenocarcinoma, hepatocellular carcinoma, and / or hepatoma), lung cancer (e.g., Lewis lung adenocarcinoma, small cell adenocarcinoma and / or non-small cell adenocarcinoma), lymphoma (e.g., Hodgkin's disease, non-Hodgkin lymphoma, B cell lymphoma, diffuse large B-celllymphoma, DLBCL), Burkitt lymphoma, cutaneous T-cell lymphoma, histiocytic lymphoma, lymphoblastic lymphoma, T-cell lymphoma and / or thymic lymphoma), lymphosarcoma, Lynch cancer family syndrome II, breast tumors, mast cell tumors, medulloblastoma, medullary carcinoma, melanoma, mesothelioma, metastatic tumors, monocytic tumors, mucinous epidermal carcinomas, multiple glomus tumors, multiple meningiomas, myelodysplastic syndromes, myelomas (e.g., multiple myeloma), nasopharyngeal carcinoma, nephroblastoma, neuroglioma of nervous tissue, neurocytoma of nervous tissue, neurilemmoma, neuroblastoma, neurogenic tumors, non-melanoma skin cancer, oat cell carcinoma, anaplastic glioma, osteochondroma, myeloma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian cell carcinoma, serous ovarian cancer, and / or ovarian sex cord tumors), papillary cell carcinoma, papilloma, paraganglioma (e.g., familial non-chromaffin paraganglioma), pheochromocytoma, pituitary tumor (invasive), placental trophoblast tumor, plasmacytoma, prostate cancer (e.g., prostatic adenocarcinoma), renal cancer (e.g., Wilms tumor type 2 or type 1), retinoblastoma, rhabdoid tumor (e.g., rhabdoid tumor predisposition syndrome), rhabdomyosarcoma, sacrococcygeal tumors, sarcomas (e.g., Ewing sarcoma, histiocytic cell sarcoma, rat sarcoma, myxosarcoma, osteosarcoma, reticulosarcoma, soft tissue sarcoma and / or synovial sarcoma), schwannoma, small cell carcinoma, spindle cell carcinoma, spinous cell carcinoma, squamous cell carcinoma (e.g., head and neck), subcutaneous tumors, teratocarcinoma (e.g., pluripotent), teratoma (e.g., immature teratoma of the ovary), testicular cancer (e.g., testicular germ cell tumor), transitional cell carcinoma, Turcot syndrome with glioblastoma, thymoma, thyroid cancer (e.g., thyroid follicular carcinoma, thyroid medullary carcinoma and / or papillary thyroid carcinoma), hair follicle epithelioma, choriocarcinoma, undifferentiated carcinoma, uterine cancer, cervical cell carcinoma.

[0092] In certain embodiments, the cancer is selected from the group consisting of colon cancer, breast cancer, melanoma, lung cancer, head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial cancer, high tumor mutational burden (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), high microsatellite instability cancer or mismatch repair-deficient cancer, and high microsatellite instability colorectal cancer (CRC).

[0093] In certain embodiments, the cancer is colon cancer.

[0094] In certain embodiments, the cancer is a colon tumor.

[0095] In certain embodiments, the cancer is breast cancer.

[0096] In certain embodiments, the cancer is an airway tumor.

[0097] In other certain embodiments, the cancer does not include an airway tumor.

[0098] In certain embodiments, the cancer is resistant to treatment with a checkpoint inhibitor. This can be presented as innate resistance (also known as primary resistance, meaning the patient does not respond at all to the treatment) or acquired resistance (also known as secondary resistance, meaning the patient initially responds to the treatment but later develops resistance).

[0099] As used herein, the term "checkpoint inhibitor" refers to a molecule that inhibits the activity of one or more immune checkpoint proteins and results in the activation of immune cells.

[0100] As used herein, the term "immune checkpoint protein" refers to an antigen-independent protein that controls immune cell activation or function in response to an antigen. An immune checkpoint protein can be either a costimulatory protein (i.e., one that transmits a stimulatory signal that results in activation of an immune cell) or an inhibitory protein (i.e., one that transmits an inhibitory signal that results in suppression of the activity of an immune cell). In some embodiments, the immune checkpoint protein controls the activation or function of T cells. A number of checkpoint proteins are known in the art, including, but not limited to, PD-1, PD-L1, A2aR, B7-H2, B7-H3, B7-H4, CTLA-4, CD73, CD80, CD86, LAG-3, TIM-3, NKG2A, PVRIG / PVRL2, CEACAM1, CEACAM5 / 6, FAK, CCL2 / CCR2, LIF, KIR, IDO, CD19, OX40, 4-1BB (CD137), CD27, CD47 / SIRPα, CD70, CD40, CSF-1, GITR, CD28, IL-1, IL-1R3, IL-8, SEMA4D, Ang-2, CLEVER-1, Axl, phosphatidylserine, and ICOS (CD278).

[0101] In certain embodiments, the immune checkpoint protein is presented on an immune cell, such as a T cell or an antigen-presenting cell.

[0102] In certain embodiments, the immune checkpoint protein is presented on a cancerous cell.

[0103] In certain embodiments, cancer cells present the immune checkpoint protein or its binding partner (i.e., receptor or ligand) after administration of the UNO.

[0104] Thus, in certain embodiments, the method includes determining the presentation of an immune checkpoint protein or binding pair in a biological sample obtained from a subject. Methods for determining expression and / or presentation are well known in the art and include PCR, Western blot, immunostaining, flow cytometry, and the like.

[0105] In certain embodiments, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, CTLA-4, and LAG-3. In certain embodiments, the immune checkpoint protein is PD-1. In certain embodiments, the immune checkpoint protein is PD-L1. In certain embodiments, the immune checkpoint protein is CTLA-4. In certain embodiments, the immune checkpoint protein is LAG-3.

[0106] In certain embodiments, the checkpoint inhibitor includes an antibody.

[0107] Non-limiting examples of PD-L1 inhibitors include atezolizumab, durvalumab, avelumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189, but are not limited thereto. Non-limiting examples of PD-1 inhibitors include pembrolizumab, nivolumab, semipramab, spartalizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, JTX-4014, INCMGA00012, AMP-224, and AMP-514, but are not limited thereto. Non-limiting examples of CTLA-4 inhibitors include ipilimumab, tremelimumab, but are not limited thereto. Non-limiting examples of LAG-3 inhibitors include relatlimab, but are not limited thereto.

[0108] In certain embodiments, the cancer is refractory to treatment with a PD-1 inhibitor.

[0109] In certain embodiments, the cancer is refractory to treatment with a PD-L1 inhibitor.

[0110] In certain embodiments, the cancer is refractory to treatment with a CTLA-4 inhibitor.

[0111] In certain embodiments, the cancer is refractory to treatment with a LAG-3 inhibitor.

[0112] As used herein, the term “immune adjuvant” refers to a substance that increases the antigen-specific activity of an immune response. As used herein, “adjuvant” does not refer to a modulator of immune checkpoint molecules as described herein. Non-limiting examples of immune adjuvants that can be used with certain embodiments of the present invention include inorganic salts, aluminum salts, AS04, MF59, AS01 B , organic adjuvants, emulsions (e.g., Freund’s Complete Adjuvant (FCA), Freund’s Incomplete Adjuvant (FIA), liposomal adjuvant systems), microparticles, liposomes, saponins, cytokines, microbial components, and nucleic acid adjuvants.

[0113] In certain embodiments, the immune adjuvant is a nucleic acid adjuvant (e.g., a DNA molecule, an RNA molecule, a cDNA molecule). Such nucleic acid adjuvants are well known in the art and are disclosed, for example, in Tmizoz et al. Current Opinion in Pharmacology (2018) 41:104-113, the content of which is hereby incorporated by reference in its entirety as part of this specification.

[0114] Non-limiting examples of nucleic acid adjuvants that can be used with certain aspects of the present invention include cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs), cytokine expression vectors, nucleic acid sequences of microorganisms, nucleic acid sequences of viruses [e.g., double stranded RNA (dsRNA) of viral origin, single stranded RNA (ssRNA) derived from viruses], polyinosinic-polycytidylic acid (Poly I:C), PIKA, imiquimod, and cyclic dinucleotides (CDNs).

[0115] In certain aspects, the immune adjuvant is a CpG ODN.

[0116] CpG ODNs are short single-stranded synthetic DNA molecules that contain a cytosine nucleotide (“C”) followed by a guanine nucleotide (“G”) motif. “p” refers to the phosphodiester bond between consecutive nucleotides, although some ODNs have a modified phosphorothioate (PS) backbone instead. When these CpG motifs are not methylated, they act as immune adjuvants. Such CpG ODNs can be readily designed by those skilled in the art or can be commercially available, for example, from IDT, InvivoGen, Novus Biologicals, Creative Biogene, and others.

[0117] In certain aspects, the cancer is positive for a microsatellite instability (MSI) [e.g., high microsatellite instability (MSI-H)] marker and / or a mismatch repair deficiency (dMMR) marker.

[0118] In other certain aspects, the cancer is negative for a microsatellite instability (MSI) marker and / or a mismatch repair deficiency (dMMR) marker.

[0119] Methods for determining MSI / dMMR are well known in the art and include next-generation sequencing (NGS), fluorescent multiplex PCR and C, immunohistochemistry, single-molecule molecular inversion probes (smMIP).

[0120] One of ordinary skill in the art will understand that all of the methods and uses provided herein for inhibiting abnormal growth of tumor cells or tissues and for treating cancer may generally be applicable to all known and future-discovered cancerous cell phenotypes and cancerous growths.

[0121] In certain embodiments, cancer cells present PD-L1 and / or CTLA-4 and / or LAG-3 on their cell membranes.

[0122] Thus, in certain embodiments, the method includes determining the presentation of PD-L1, CTLA-4, and / or LAG-3 in a biological sample obtained from a subject. Methods for determining expression and / or presentation are well known in the art and include PCR, Western blot, immunostaining, flow cytometry, and the like.

[0123] This embodiment relates to tumors of any size and shape, including large, diffuse, amorphous cancerous growths.

[0124] The methods and uses provided herein may be particularly useful for the treatment, control, and / or prevention of tumors (e.g., cancerous tumors) at local sites, including inoperable tumors, tumors for which local treatment is beneficial, and solid tumors.

[0125] In some of any of the embodiments described herein, the methods and uses of this embodiment are for inhibiting the growth of cells of a primary tumor.

[0126] The present invention provides a method for treating cancer by a combination treatment including administration of UNO, preferably for a sufficient period under high pressure, as will be described in more detail below.

[0127] In certain embodiments, effective treatment with UNO is considered when about 10% to about 100% (e.g., 10% - 100%, 15% - 100%, 20% - 100%, 25% - 100%, 30% - 100%, 35% - 100%, 40% - 100%, 45% - 100%, 50% - 100%, 55% - 100%, 60% - 100%, 65% - 100%, 70% - 100%, 75% - 100%, 80% - 100%, 85% - 100%, 90% - 100%) of cancerous cells can be killed by gaseous nitric oxide over the course of one or more administrations, as described herein in every aspect of each embodiment.

[0128] UNO is defined as the delivery of gaseous nitric oxide in a preferably inert gas such as N2, at a concentration of about 10,000 ppm to about 1,000,000 ppm, as will be described in more detail below.

[0129] The method of delivering UNO may include administration of UNO in a continuous mode or a pulsed manner.

[0130] In some embodiments of the present invention, the method affects by administering UNO locally.

[0131] "Locally administering UNO" means bringing UNO into direct contact with tumor cells or tissue, such that UNO is applied directly to the tumor and / or in its vicinity. In some embodiments, local administration of UNO affects the tumor by applying UNO directly to the tumor cells or tissue. In some embodiments, local administration (intratumor) of UNO affects the tumor by applying UNO to the surface of the tumor tissue, e.g., by contacting the surface of the tumor with UNO. In some embodiments, local administration affects the tumor, e.g., by carrying UNO in the vicinity of at least one surface, preferably all tumor surfaces, directly to, or from at least one surface, preferably all tumor surfaces, up to 2 cm or up to 1 cm from the tumor cells or tissue.

[0132] In some of any of the embodiments described herein, UNO is locally administered to a primary tumor and / or a metastatic tumor.

[0133] In some of each of the embodiments described herein, representative organs in which UNO can be locally administered include, but are not limited to, the adrenal gland, bladder, bone, brain, breast, cervix, colon, colorectum, esophagus, gastrointestinal tract, heart, kidney, liver, large intestine, lung, oral cavity, ovary, pancreas, parathyroid gland, pituitary gland, prostate, salivary gland, skin, small intestine, spleen, stomach, thymus, thyroid gland, testis, urinary tract, uterus or vagina. In some embodiments, UNO is locally administered to the liver in the case of primary liver cancer or liver metastases.

[0134] In some embodiments, UNO is locally administered to the colon in the case of primary colon cancer or colon metastases.

[0135] In some of any of the embodiments described herein, local administration affects the tumor such that UNO is injected into the tumor or otherwise delivered to the tumor.

[0136] In some of the various aspects described herein, local administration is effected by spraying UNO onto at least one of the tumor surfaces, or in other cases by applying UNO onto at least one of the tumor surfaces. Then at least some of the UNO enters the tumor, for example, via diffusion.

[0137] In some of the various aspects described herein, local administration is effected by exposing the tumor to UNO in a sealed container such that the UNO contacts the tumor and enters the tumor, for example, via diffusion. The container can be in an open or closed state and can be sized to conform to the contour of the tumor.

[0138] In some of the various aspects described herein, local administration is effected by delivering UNO to a physiological space or cavity containing at least a portion of the tumor tissue such that the tumor contacts the UNO and the UNO enters the tumor, for example, via diffusion.

[0139] In some of the various aspects described herein, as described herein, UNO is administered locally.

[0140] In some of the various aspects described herein, a high dose of UNO is administered locally to the tumor and is reflected by a relatively high concentration in the total amount of gas expressed in ppm (parts per million).

[0141] In some of the various aspects described herein, a high dose of UNO is expressed in ppm as its proportion in a gas carrier. The gas carrier can be air, preferably an inert gas such as N2 or argon (Ar), preferably N2.

[0142] In some of the various aspects described herein, a high dose of UNO is reflected by the mass of UNO administered locally to the tumor per unit volume of the tumor.

[0143] In some of the aspects described herein, UNO is administered at a concentration of from about 10,000 ppm to about 1,000,000 ppm (1% to 100%), including any intermediate values and sub-ranges therebetween, as described herein in each of the aspects. For example, from about 10,000 ppm to about 200,000 ppm, or from about 10,000 ppm to about 100,000 ppm, or from about 15,000 ppm to about 100,000 ppm, or from about 20,000 ppm to about 100,000 ppm, or from about 25,000 ppm to about 100,000 ppm, or from about 25,000 ppm to about 75,000 ppm, or from about 10,000 ppm to about 50,000 ppm, or from about 50,000 ppm to about 100,000 ppm, or about 50,000 ppm, including any intermediate values and sub-ranges between any of the foregoing values.

[0144] In some of the aspects described herein regarding high doses of UNO, the high dose can be obtained by locally administering UNO at a dose of at least 10,000 ppm, or at least 20,000 ppm, or at least 50,000 ppm, optionally up to about 1,000,000 ppm.

[0145] In a representative aspect, UNO is administered at a concentration of from about 20,000 to 200,000 ppm, as described herein in each of the aspects.

[0146] In a representative aspect, UNO is administered at a concentration of from about 20,000 to 100,000 ppm, as described herein in each of the aspects.

[0147] In a representative aspect, UNO is administered at a concentration of about 50,000 ppm, as described herein in each of the aspects.

[0148] In a representative embodiment, UNO is administered at a concentration of about 25,000 ppm, as described herein, in any of the respective embodiments.

[0149] In a representative embodiment, UNO is administered at a concentration of about 20,000 ppm, as described herein, in any of the respective embodiments.

[0150] In a representative embodiment, UNO is administered at a concentration of about 10,000 ppm, as described herein, in any of the respective embodiments.

[0151] In a representative embodiment, UNO is administered at a concentration of about 100,000 ppm, as described herein, in any of the respective embodiments.

[0152] In a representative embodiment, UNO is administered at a concentration of about 100,000 ppm to about 200,000 ppm, or about 200,000 ppm to about 500,000 ppm, or about 500,000 ppm to about 1,000,000 ppm, or about 50,000 ppm or about 100,000 ppm or about 200,000 ppm or about 500,000 ppm or about 1,000,000 ppm, as described herein, in any of the respective embodiments.

[0153] In some of all aspects described herein, UNO is administered at a volumetric flow rate of from about 0.00001 LPM to about 10 LPM, preferably from about 0.0001 LPM to about 1 LPM, or from about 0.001 LPM to 0.5 LPM, including any intermediate values and sub-ranges therebetween, as described herein for any of the respective aspects. For example, the volumetric flow rate can be from about 0.001 LPM to about 0.01 LPM, or from about 0.01 LPM to about 0.1 LPM, or from about 0.1 LPM to about 0.25 LPM, or from about 0.25 LPM to about 0.5 LPM, or from about 0.5 LPM to about 1 LPM, or from about 1 LPM to about 2 LPM, or from about 2 LPM to about 3 LPM, or from about 3 LPM to about 4 LPM, or from about 4 LPM to about 5 LPM, or from about 5 LPM to about 6 LPM, or from about 7 LPM to about 8 LPM, or from about 8 LPM to about 9 LPM, or from about 9 LPM to about 10 LPM, including any intermediate values and sub-ranges therebetween, or for example, can be about 0.0001 LPM, or about 0.001 LPM, or about 0.01 LPM, or about 0.1 LPM, or about 1 LPM, or about 10 LPM.

[0154] In some of all aspects described herein, UNO is administered at a volumetric flow rate of from about 0.001 LPM to about 0.5 LPM, as described herein for any of the respective aspects.

[0155] In some of all aspects described herein, UNO is administered at a volumetric flow rate of about 0.2 LPM, as described herein for any of the respective aspects.

[0156] In some of the aspects described herein, UNO is administered for a period ranging from about 0.1 seconds to about 10 hours, including any intermediate values and sub-ranges therebetween, per administration. For example, the time can be about 0.1 seconds to about 1 hour, or about 1 second to about 10 minutes, or about 1 minute to about 10 minutes, or about 10 seconds to about 10 minutes, or about 0.1 seconds to about 10 minutes, or about 30 seconds to about 3 minutes, or about 1 minute to about 30 minutes, or about 10 minutes to about 60 minutes, or about 60 minutes to about 180 minutes, or about 180 minutes to about 600 minutes, including any intermediate values and sub-ranges therebetween for any of the aforementioned values, or it can be about 30 seconds, about 10 minutes, about 30 minutes or about 60 minutes.

[0157] In some of the aspects described herein, UNO is administered for a time ranging from about 30 seconds to about 10 minutes.

[0158] In some of the aspects described herein, UNO is administered for about 5 minutes.

[0159] In some of the aspects described herein, UNO is administered intermittently, i.e., more than 2 times per day, such that in any of the aspects described herein, the administration period per day is the sum of two or more separate administration periods, which may be of the same length or different lengths.

[0160] In some of all aspects, UNO can be administered at a dose of at least 10,000 ppm, optionally up to about 1,000,000 ppm, or up to about 500,000 ppm, or up to about 200,000 ppm, or up to about 100,000 ppm, including any intermediate values and sub-ranges therebetween for any of the aforementioned values, for a time ranging from about 1 second to about 60 minutes, at a volumetric flow rate (flow rate) of about 0.0001 liters per minute (LPM) to about 1 LPM, as described herein in any of the aspects.

[0161] In some of all aspects, UNO can be administered at a dosage of at least 10,000 ppm, or at least 20,000 ppm, or at least 50,000 ppm, optionally up to about 1,000,000 ppm, for a time of at least 1 second, or at least 10 seconds, or at least 30 seconds, or at least 1 minute, optionally up to about 60 minutes, at a volumetric flow rate (flow rate) of at least 0.0001 liter per minute (LPM), or at least 0.001 LPM, or at least 0.01 LPM, optionally up to about 1 LPM, including any intermediate values and sub - ranges between any of the aforementioned values.

[0162] Alternatively, or in addition, the amount of UNO administered to the tumor is in the range of about 0.1 mg to about 300 mg per tumor cm 3 per administration.

[0163] The UNO concentration (ppm), volumetric flow rate (LPM) and time parameters to achieve the desired mass of UNO, and conversely, the UNO mass achieved by administering UNO in terms of concentration, volumetric flow rate and time, are calculated using the well - known ideal gas equation PV = nRT, where P is pressure, V is volume, n is the number of moles, R is the gas constant, and T is temperature.

[0164] More specifically, these relationships can be calculated or converted to others using the following equations.

Number

[0165] y is the concentration in ppm units, X is the concentration in mole fraction units, and Equation 1 represents the relationship between mole fraction and ppm (y). V is the volume,

Number

[0166] Therefore, for example, at a volumetric flow rate of 0.1 LPM, over a time of 1 minute, and at a concentration of 50,000 ppm, Equation 5 is used to administer approximately 6.1 - approximately 6.2 mg of UNO.

[0167] In some of any of the aspects described herein, UNO is, in any of each of the aspects, as described herein, per administration, at a concentration of about 10,000 ppm - about 1,000,000 ppm (1% - 100%), preferably about 10,000 ppm - about 500,000 ppm, or about 10,000 ppm - about 100,000 ppm, or about 50,000 ppm, at a volumetric flow rate of about 0.0001 LPM - about 10 LPM, preferably about 0.001 LPM - 1 LPM, and is administered over a period in the range of about 1 second - about 30 minutes.

[0168] In some of any of the aspects described herein, UNO is, in any of each of the aspects, as described herein, per administration, at a concentration (dosage) of about 20,000 ppm - about 100,000 ppm, or about 20,000 ppm - about 50,000 ppm, over a period in the range of about 30 seconds - about 10 minutes, and at a volumetric flow rate (flow rate) of about 0.001 LPM - about 0.5 LPM.

[0169] In some of the aspects described herein, UNO is, in any of the respective aspects, as described herein, per administration, 100 mm 3 of UNO at a quantity of 1 mg or less per tumor volume of 3 , and is administered to avoid possible damage to healthy tissues adjacent to or surrounding the tumor being treated.

[0170] In some of the aspects described herein, UNO is, in any of the respective aspects, as described herein, per administration, 1 cm 3 administered at a quantity of about 250 mg per tumor of 3 .

[0171] In some of the aspects described herein, UNO is, in any of the respective aspects, as described herein, administered at a quantity of about 0.01 mg to about 100 mg, or about 0.1 mg to about 10 mg per tumor of 20 mm 3 or less, including any intermediate values and sub - ranges therebetween.

[0172] In some of the aspects described herein, UNO is, in any of the respective aspects, as described herein, per administration, 1 cm 3 administered at a quantity of about 0.1 mg to about 300 mg per tumor of 3 , including any intermediate values and sub - ranges therebetween. For example, UNO is administered at a quantity of about 0.1 mg to about 250 mg, or 0.1 mg to about 100 mg, or 1 mg to about 50 mg, or about 1 mg to about 100 mg, or about 1 mg to about 300 mg, or about 50 mg to about 100 mg, or about 50 mg to about 300 mg, or about 100 mg to about 150 mg, or about 100 mg to about 300 mg, or about 10 mg to about 100 mg, or about 10 mg to about 250 mg, or about 0.1 mg to about 10 mg, or about 10 mg to 200 mg per tumor of 1 cm 3 each administration including any intermediate values and sub - ranges between any of the aforementioned values.

[0173] In some of all aspects described herein, UNO, in any of the aspects, as described herein, is administered to tumors having a volume of up to 20 mm 3 and the amount of UNO administered, in any of the aspects, as described herein, per administration, includes any intermediate value and sub-range between any of the aforementioned values, from about 0.001 mg to about 10 mg, or from about 0.01 mg to about 20 mg, or from about 0.01 mg to about 2 mg, or from about 0.1 mg to about 1.0 mg, or from about 0.2 mg to about 0.8 mg. In other aspects, the tumors have a volume greater than 20 mm 3 in size.

[0174] Without being bound by any particular theory, high doses (concentration or amount) as described herein in any of the aspects, as described herein, inhibit the growth of tumor cells, reduce tumor volume, and / or stimulate an anti-tumor immune response without causing a harmful effect on healthy tissue near the tumor.

[0175] For any of the aspects described herein for the administration of UNO, the administration can be either continuous or pulsed, and for each administration, the indicated dose of UNO is administered either continuously or in a pulsed manner. When the dose is referred to in ppm units, each pulse is the indicated dose concentration as described herein in any of the aspects. When the dose is referred to as the total mass per administration, the indicated dose is divided into pulses.

[0176] In some of all aspects described herein, UNO is pulsed from about 2 to about 50 times, or from about 2 to about 30 times, or from about 2 to about 20 times, or from about 2 to about 15 times, or from about 5 to about 15 times, or about 10 times per administration, including any intermediate value and sub-range therebetween.

[0177] In some of the various aspects described herein, each pulse has a volumetric flow rate (flow rate) of from about 0.00001 LPM to about 0.5 LPM and a UNO of from 10,000 ppm to about 1,000,000 ppm, provided that each pulse is independently from about 0.1 second to about 10 minutes per pulse and has an interruption of from about 0.1 second to about 10 minutes between pulses.

[0178] In some of the various aspects described herein, the UNO of each pulse is independently from about 10 seconds per pulse to about 45 seconds per pulse, including any intermediate value and sub-range therebetween.

[0179] In some of the various aspects described herein, the UNO of each pulse is about 30 seconds per pulse.

[0180] In some of the various aspects described herein, UNO is not administered between pulses, and the time between each two pulses is independently from about 1 second to about 300 seconds, or from about 1 second to about 200 seconds, or from about 1 second to about 100 seconds, or from about 1 second to about 50 seconds, or from about 10 seconds to about 50 seconds, or about 20 seconds, including any intermediate value and sub-range therebetween.

[0181] In some of all of these aspects, the ratio of the time of UNO pulse administration to the rest time between pulses is in the range of 1:2 to 1:5. For example, for each pulse of 5 seconds of UNO administration, the subsequent rest time is independently from 10 to 50 seconds. Preferably, UNO is pulsed such that UNO is delivered about 33% of the time and the rest time or waiting time between pulses is 66% of the time.

[0182] In some of all aspects described herein, UNO is administered (depending on the administration mode) at two or more administration sites in or on the tumor as described herein for any of the respective aspects. In some of these aspects, the distance between the two administration sites is, independently, about 2.5 mm to about 1 cm, or about 0.25 cm to about 0.5 cm, including any intermediate value and sub-range therebetween.

[0183] When UNO is administered to two or more tumor sites, each administration is at the ppm dosage or mass shown herein for any of the respective aspects, or the total mass (amount) administered to all major sites is as shown herein for any of the respective aspects.

[0184] In some of all aspects described herein, the method further includes scavenging excess UNO from one or more administration sites, as described in more detail below. In a representative aspect, scavenging includes applying a reduced pressure (vacuum) around one or more administration sites.

[0185] In some of all aspects described herein regarding high-dose administration of UNO, the administration is performed one or more times per treatment session.

[0186] In some aspects, this is performed once during the treatment session. In some aspects, this is performed 2, 3, or 4 or more times during the treatment session. In some of these aspects, the administration is performed once during the treatment session. Preferably, the administration is performed during the treatment session such that the interval between two administrations is at least 1 day, or at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 1 week.

[0187] The duration of the treatment session can be determined by a person skilled in the art, such as a physician, according to the response of the subject to the treatment, i.e., according to the effect of the treatment on the growth of cells of the primary tumor and / or secondary tumors, as described herein.

[0188] In some aspects of the high-dose administration of UNO described herein, the administration is performed once a day, but administration two or more times a day is also contemplated.

[0189] In some aspects, local administration of UNO includes injecting a high dose of UNO into the outer layer of the tumor, or exposing the outer layer of the tumor to a high dose of UNO, or filling the space or cavity containing the tumor with a high dose of UNO. In some aspects, UNO is at a concentration in the range of about 10,000 ppm to about 1,000,000 ppm, preferably 25,000 ppm or 50,000 ppm, or 100,000 ppm, as described herein in any of the aspects. Local administration to the tumor is at a very low volume of up to 0.1 LPM, preferably about 0.01 LPM, for a period of about 1 second to about 3 hours, depending on the size and location of the tumor.

[0190] In some of the aspects described herein, if the tumor is covered by skin, peritoneum, eschar, or any other thick layer, this layer can be removed before or during exposure of the tumor to local UNO administration.

[0191] In any of the aspects of UNO administration described herein, UNO may be provided by an external source, such as a reservoir of UNO or a chemical generator of UNO. In some aspects, UNO is provided by a reservoir of UNO, preferably a small volume, such as a reservoir of a single dose of UNO (i.e., the dose of UNO used per single administration, as described herein in any of the aspects). Such reservoirs are described in more detail below.

[0192] UNO is preferably of medical purity, i.e., preferably at least about 95%, more preferably at least about 99%, even more preferably at least about 99.5% pure UNO. UNO is preferably provided as a mixture with air, N2, oxygen (O2), and other gases, preferably an inert gas such as N2, and its ppm concentration is within the range of the gases being mixed.

[0193] Aspects of the present invention further relate to a system, also interchangeably referred to herein as a "device", which is configured to locally administer UNO to a tumor as described herein in any of the respective aspects. Such a system is also referred to herein as a delivery system. Such a system is also described in WO 2021 / 105900, the contents of which are hereby incorporated by reference in their entirety as part of this specification.

[0194] Generally, although not mandatory, a system for locally administering UNO can include a pressure controller, a flow meter, optionally an exposure box or container, optionally one or more delivery lines terminated in or connected to a delivery device or configuration to which UNO is administered, and further optionally a NO and / or NOx (defined below) detector. It may be desirable to purge the UNO delivery system with an inert gas such as N2.

[0195] The volume and / or flow rate of the gas administered (delivered) follows any of the respective aspects as described herein, per 1 cm of tissue 3It can be controlled by a digital flow controller designed to deliver a low volume or low flow rate of gas of less than 0.1 LPM. Purging the UNO delivery system, which includes purging the pressure controller, flow meter, and delivery line, can be performed before and / or after UNO topical administration. The gas purge can preferably continue for at least 1 minute or until the NO and NOx (described below) detectors no longer read a signal. In a representative embodiment, N2 is used as the purge gas at a flow rate of at least 0.5 LPM.

[0196] In some of any of the aspects described herein, the delivery device is inserted into the body and advances over or near tumor cells or tissue adjacent to the administration site. When the delivery device or configuration is properly positioned, UNO is supplied to the device and exits the device and enters inside the tumor or on or above the surface of the tumor, depending on the nature of the device and the mode of topical administration selected. In some aspects, the delivery device extends into the tumor to form a seal, thereby further reducing the damage that can be caused by UNO to adjacent normal cells that are present outside the region sealed by the device.

[0197] The delivery device as described herein is meant to describe the components or configuration of the delivery system where gas exits the delivery system and contacts the administration site (e.g., a tumor or its vicinity).

[0198] In some of the aspects described herein, administering UNO to a tumor as described herein in any of the aspects can be achieved by delivery device means such as one or more needles, including, for example, a piercing needle, a piercing spray needle, a non-piercing needle and a non-spray needle, an umbrella needle, a closed-end needle or other needles. The needle can optionally be a nano-sized, micron-sized or macro-sized needle (having a diameter of 1 mm or more). Other delivery devices are described below. The aspect in which a needle is used is typically used by intratumoral injection, for example, when UNO is locally administered into the tumor.

[0199] In some of the aspects described herein regarding the delivery system, the opening through which UNO is delivered can be sized to the tumor size to prevent damage to the area around the cancer. In some aspects, the opening does not exceed the size of the tumor.

[0200] A method of locally administering UNO to a tumor can include contacting at least a portion of the tumor with gaseous nitric oxide, followed by removing the gaseous nitric oxide and NOx gas molecules from the treated site during or after the administration step. NOx includes NO when x is 1 and the oxidized form of NO that can be formed when UNO contacts the physiological environment and / or the environment of the subject, provided that x can be, for example, 2. Aspirating the gas can be performed in a pulsed or continuous mode, preferably synchronized with the administration mode of UNO.

[0201] The delivery system can include a full-body or different-sized chemical hood designed to discharge excess UNO or NOx in a pulsed or continuous mode during treatment, preferably synchronized with the administration mode of UNO.

[0202] The delivery system can include a discharge cylinder directly connected to the controller of the UNO tank. To safely purge the controller, the discharge cylinder can be filled with the gas accumulated in the controller.

[0203] Another aspect of controlling the UNO involves locking the valve by removing the controller or flow meter from the cylinder, thereby including the use of a one-way valve that prevents the release of gas from the gas tank.

[0204] In some of any of the aspects described herein, the methods, uses, and delivery systems as described herein optionally utilize a UNO cylinder with a gas controller and one or more valves, and further optionally, the cylinder further includes a delivery device for performing local administration. The delivery device is preferably in fluid communication with the cylinder via a valve and a gas controller (e.g., a flow controller). Alternatively, the cylinder includes means for connecting the delivery device to the cylinder to obtain fluid communication. The delivery device can be, for example, a scope having an annular shape, or a needle, or a device configured to spray a tumor or otherwise, as described in more detail below.

[0205] In some of any of the aspects described herein, the UNO cylinder is a small cylinder or at least a portable cylinder.

[0206] In some aspects, the cylinder has a volume of less than 1 liter, or less than 0.8 liters, or less than 0.75 liters, or less than 0.5 liters, or less than 0.3 liters.

[0207] The cylinder is preferably under low pressure, such as less than about 40 bar (about 600 psi). Therefore, the cylinder can deliver about 10 liters of UNO. The controller can optionally limit the output pressure, for example, to about 50 psi and can be connected to an emergency on / off valve. The gas flow can be, for example, from about 0.1 LPM to less than about 0.05 LPM in the case of a delivery system configured for injection. Using such a low flow rate during local administration of UNO can help limit exposure to tumors or cancerous cells and can protect surrounding areas or healthy cells and tissues. In other embodiments, the cylinder can be under high pressure greater than 40 bar, such as about 60 bar (about 870 psi) or about 80 bar (about 1160 psi) or about 100 bar (1450 psi). In other embodiments, the cylinder can be under pressure higher than 100 bar.

[0208] In some of any of the aspects described herein, the methods and uses relate to scavenging UNO and optionally other gases, and scavenging can be accomplished by applying a vacuum to remove UNO and other gases from the administration site. Delivery systems as described herein are configured in some aspects to be able to scavenge UNO from the administration site.

[0209] Delivery systems according to some of this aspect additionally or alternatively include one or more vacuum devices, preferably two vacuum devices, for scavenging gaseous nitric oxide and other gases that may be formed during local administration. The vacuum devices can be positioned or held, for example, about 15 cm away during administration, proximal or distal to the tumor or delivery device. The vacuum devices can evacuate all gases from the area at a rate of at least about 50 liters per minute. The gas suction can be performed in a pulsed or continuous mode, preferably synchronized with UNO administration. Before and / or after, the UNO delivery system can also be purged, for example, with N2. Purging can also be performed intermittently during the process.

[0210] In some embodiments, the chemical hood is placed over the top of the tumor or tumor mass and used to apply a vacuum to scavenge UNO. The chemical hood can be placed about 15 cm above the tumor. The chemical hood can evacuate gas at a flow rate of at least about 50 LPM.

[0211] A full-body chemical hood that can accommodate the patient's body can also be used. The patient's head can be placed outside the hood to minimize the risk of inhaling NOx molecules. UNO can be delivered as described herein. The vacuum system can exchange gas at a flow rate of at least 50 liters per minute.

[0212] In each of the embodiments regarding vacuum application, the NO and NOx filtration pumps can be placed in the exhaust line. A soda lime filter such as a Sofnolime filter, or a similar filter capable of absorbing NOx molecules can be used. In each example, the gas suction can be performed in a pulsed or continuous mode, preferably synchronized or not synchronized with UNO administration.

[0213] In some of any of the embodiments described herein, the delivery system is configured to deliver UNO to one or more administration sites by a positive pressure gradient and scavenge UNO from one or more administration sites by a negative pressure gradient. In this way, the delivery system can deliver UNO to one or more administration sites where damage to the accompanying host cells is reduced or nullified.

[0214] Note that a particular level of damage to the accompanying cells can be tolerated and the conditions under which the UNO gas is administered can be optimized to reduce damage to the accompanying cells while also providing the therapeutic effects described herein.

[0215] In some of all aspects described herein, the delivery system includes a gas supply line in fluid communication with a gas supply port. The delivery system can further include an exhaust line in fluid communication with an exhaust port. The delivery system can include one or more gas supply ports and / or one or more exhaust ports, and / or one or more gas supply lines and / or one or more exhaust lines.

[0216] A representative system for local administration and scavenging of gaseous nitric oxide is exemplified, for example, in FIG. 22 of WO 2021 / 105901 pamphlet and includes a container or box that can be filled with at least 0.5 liters per minute (LPM) of UNO supplied from a tank (gas reservoir), as will be described in more detail below. The container can have two or more holes or ports. The first hole or port can be an inlet hole sized to allow insertion of at least a portion of a tumor or a body organ containing a tumor. The discharge hole can be connected to an exhaust pipe or pipe that can remove or discharge excess NOx gas from the box to the outside air, avoiding or minimizing the risk of contaminating the room and the risk of overexposure of staff and the subject (patient) being treated. The application of vacuum can be in a pulsed or continuous mode and is preferably synchronized with UNO administration.

[0217] A representative delivery system can include a small-bore inner cannula that delivers an appropriate dose of UNO to a target site, such as a target site sized 1 mm 2 to about 2 cm 2 The delivery system can further include an outer lumen to which a vacuum can be applied to scavenge excess UNO from surrounding tumor cells and the tumor site and adjacent tissue. Such a configuration enables local administration of UNO to the target site (tumor) without unduly damaging healthy host tissue.

[0218] In a representative method, the main part or a portion thereof is inserted into a hole that substantially matches the diameter of the tumor. The pressure can be reduced by further discharge holes in the box. Excess gas is removed from the box through the discharge holes as described above. For example, the gas can flow into the box containing the tumor for 2 seconds and then the gas can be inhaled for 2 seconds.

[0219] Representative delivery systems include an outer lumen or cannula, trocar, tube, etc. An inner lumen or cannula, tube, etc. can be coaxially disposed inside the outer lumen. The inner lumen is disposed substantially in the center of the outer lumen, although other arrangements are also contemplated.

[0220] In some embodiments of such representative delivery systems, a space, preferably an exhaust space, exists between the outer lumen and the inner lumen. The exhaust space may be annular or may take any other configuration and / or geometric shape. The tip can be attached to the inner lumen at the distal end and is in fluid communication with the inner lumen. In some embodiments, the tip includes a wire mesh or screen that accesses the space inside the inner lumen. The delivery device may be advanced to the administration site in a stored configuration. In some embodiments, the tip is rounded and seals the outer lumen when in the stored position, thus facilitating insertion into the body adjacent to the administration site and where the tip is carried to the administration site.

[0221] When the tip of the system is carried adjacent to the administration site, the system is adjusted to its deployed configuration to affect the administration of UNO. In the deployed configuration, the exhaust passage opens between the distal end of the outer lumen and the tip. UNO is delivered through the inner lumen. UNO exits the inner lumen at the tip that is in fluid communication with the inner lumen. The wire mesh or screen at the distal end of the tip can assist in the diffusion of UNO gas when it is present in the device. The exhausted UNO gas returns to the device through the exhaust passage. In an aspect, a vacuum is applied to the exhaust space between the outer lumen and the inner lumen to draw the discarded UNO. The then-exhausted UNO is carried through the exhaust space, exits the body, and is appropriately disposed of. In this way, the device is capable of scavenging UNO from the administration site.

[0222] In an alternative configuration, the flow of UNO can be reversed such that UNO is delivered through the space between the outer lumen and the inner lumen and removed from the administration site through the inner lumen. In this alternative configuration, a vacuum can be applied to the inner lumen and a positive pressure of UNO can be applied to the space defined by the inner lumen and the outer lumen. In another alternative configuration, the tip is fixed to the outer lumen and the inner lumen and the tip does not have a retracted configuration and a deployed configuration. In this alternative configuration, a standing path is provided in the outer lumen such that UNO is either released from the device or drawn into the device by a vacuum. For example, the standing path can be small holes or slits radially disposed near the distal end of the outer lumen such that they are present around the outer lumen, preferably near the tip of the device.

[0223] In some of the aspects described herein, for some of the aspects described herein, the delivery device can be visually observed by an operator or a person working in cooperation with the operator as it is inserted into the body, advanced towards the administration site, and withdrawn from the body, or otherwise can be made known to the operator or a person working in cooperation with the operator. It is attached to the end of an endoscope or bronchoscope (e.g., a blue fluorescence endoscope or a blue fluorescence bronchoscope). Alternatively, the delivery device can be attached with a guide wire for more effectively inserting, advancing, and withdrawing the device. Additionally, the device can be coated with a fluoroscopic material or can have one or more fluoroscopic tags attached to the device so that its insertion, advancement, and withdrawal can be observed by fluoroscopy.

[0224] In some aspects, a system for delivering UNO to one or more administration sites and scavenging NOx from one or more administration sites is intended to be adapted to the distal end or tip of an endoscope or bronchoscope. The device can be, for example, in an annular shape having a hole in or around its center, and the shape is generally circular. The hole is preferably sized to accommodate the distal end of the endoscope or bronchoscope. For example, the hole is about 0.5 cm to about 10 cm in diameter. The hole can be sized to fit snugly against the distal end of the endoscope or bronchoscope.

[0225] In some of the various aspects described herein, the delivery system includes a dual-needle system where the aspiration needle is disposed adjacent to or in proximity to (e.g., at a distance of 3 mm to 1 cm) the gas delivery needle. The aspiration needle can reduce or maintain the pressure within the tumor. Further, two or more needles can be spaced at least about 2 mm apart. UNO can be delivered at a flow rate of at least about 0.01 LPM as described herein. The needles can be designed to have holes along the length of the needles. In an example, the diameter of the holes is about 1 mm and they are spaced every 2 mm. The needles can be disposed within the lumen and outside the tumor mass, although the shaft of the needle can be disposed within the tissue. The length of the needle can be selected to be at least half of the longest dimension of the tumor. The vacuuming of gas through one or more aspiration needles or holes can be performed in a pulsed or continuous mode, preferably synchronized with the UNO administration. For example, UNO can be flowed into the tumor for 2 seconds, followed by applying vacuum or aspiration for 2 seconds. One or more needles such as multiple needles or an array of needles such as nano-sized or micron-sized needles can be used. In some aspects, the gas can be injected into the tumor by an array of needles spaced at about 0.5 cm intervals. The ratio of aspiration needles to delivery needles can be from 1:10 to 10:1, preferably 1:1. The aspiration needle can be designed to remove less than about 1 liter / minute of gas or fluid per centimeter.

[0226] In some of the various aspects described herein, UNO is applied to the targeted tumor via one or more intratumoral channels. For example, channels can be formed such that gas can be delivered directly to the tumor mass by needles disposed at the center of each channel, each having a width of 2 mm every 4 mm of tissue, at a flow rate higher than, for example, 0.01 LPM. On the other hand, the gas is removed from the scavenging channels. The flow rate in the scavenging channels can be lower than that in the delivery channels, such as at least 0.001 LPM less than the UNO delivery flow rate. The vacuuming can be achieved through the aspiration needle or hose, and can be pulsed or continuous, preferably synchronized with the UNO administration. For example, UNO can be flowed into the tumor for 2 seconds, followed by aspirating gas for 2 seconds.

[0227] In some of any of the aspects described herein, UNO is optionally used in combination with a surgical procedure (e.g., tumor resection) and / or an intratumoral injection of UNO, and / or when the tumor is inoperable, flat, or amorphous, and is sprayed onto the targeted tumor when used in combination with a method as described herein.

[0228] The present invention provides a method of treating cancer by a combination treatment comprising UNO and a checkpoint inhibitor. In some aspects, the method further comprises an immune adjuvant.

[0229] In certain aspects, treatment with UNO and a checkpoint inhibitor has a combination of improved anti-cancer and anti-tumor activities. In certain aspects, treatment with UNO, a checkpoint inhibitor, and an immune adjuvant has a combination of improved anti-cancer and anti-tumor activities.

[0230] As used herein, the phrase "improved anti-cancer or anti-tumor activity" refers to anti-cancer or anti-tumor activity that is at least additive but synergistically improved compared to treatment with each of the agents when administered as a single agent or in combination of two of the agents. This can be determined, for example, by the size of the tumor, tumor regression, symptoms of the disorder, or the effect on the survival of the subject.

[0231] The checkpoint inhibitor of some aspects of the present invention can be administered to a living being in a pharmaceutical composition that is administered to the living being by itself or mixed with a suitable carrier or excipient.

[0232] The immune adjuvant of some aspects of the present invention can be administered to a living being in a pharmaceutical composition that is administered to the living being by itself or mixed with a suitable carrier or excipient.

[0233] As used herein, "pharmaceutical composition" refers to a formulation of one or more of the active ingredients described herein, including other chemical components such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to a living being.

[0234] As used herein, the term "active ingredient" refers to a checkpoint inhibitor and / or an immune adjuvant that can account for a biological effect.

[0235] As used interchangeably herein, the terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" refer to a carrier or diluent that does not cause significant irritation to a living being and does not inhibit the biological activity and properties of the administered compound.

[0236] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0237] Techniques for drug formulation and administration can be found in "Remington’s Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is hereby incorporated by reference as part of this specification.

[0238] Suitable routes of administration can include, for example, oral delivery, rectal delivery, transmucosal delivery, particularly nasal delivery, intestinal delivery, or intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal injection, direct intraventricular, for example, into the right or left ventricle, intracardiac injection into the common coronary artery, intravenous injection, intraperitoneal injection, intranasal injection, or intraocular injection, including parenteral delivery.

[0239] In certain embodiments, the pharmaceutical composition is administered intravenously.

[0240] In certain embodiments, the pharmaceutical composition is administered as an intravenous infusion over 20 - 60 minutes after dilution, such as 30 minutes after dilution.

[0241] Conventional approaches for drug delivery to the central nervous system (CNS) include neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion); molecular manipulation of drugs in attempts to utilize one of the endogenous transport pathways of the BBB (e.g., production of chimeric fusion proteins containing transport peptides with affinity for endothelial cell surface molecules, combined with a drug that is itself unable to cross the BBB); pharmacological strategies designed to increase the lipid solubility of drugs (e.g., conjugation of a water - soluble drug with a lipid or cholesterol carrier); and transient disruption of the integrity of the BBB by osmotic disruption (resulting from injection of a mannitol solution into the carotid artery or use of a bioactive agent such as an angiotensin peptide). However, each of these strategies has limitations such as the inherent risks associated with invasive surgical procedures, size limitations imposed by the constraints inherent in endogenous transport systems, potentially unwanted biological side effects associated with systemic administration of chimeric molecules composed of carrier motifs that may be active outside the CNS, and the risk of potential brain damage in the brain regions where the BBB is disrupted, which result in sub - optimal delivery methods.

[0242] Alternatively, the pharmaceutical composition can be administered in a local, rather than a systemic, manner, for example, via direct injection of the pharmaceutical composition into a tissue region of the patient.

[0243] In certain embodiments, the pharmaceutical composition is administered intratumorally or in the vicinity of the tumor.

[0244] The pharmaceutical compositions of some embodiments of the present invention can be manufactured by methods well - known in the art, such as conventional mixing, dissolving, granulating, tablet - coating, wet - milling, emulsifying, encapsulating, entrapping, or lyophilizing methods.

[0245] Pharmaceutical compositions for use in accordance with some aspects of the present invention can thus be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate the easy processing of the active ingredient into a pharmaceutically usable formulation. Appropriate formulations depend on the chosen route of administration.

[0246] For parenteral administration, the active ingredient of the pharmaceutical composition may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or a physiological saline buffer. For transmucosal administration, permeation enhancers appropriate for the permeated barrier are used in the formulation. Such permeation enhancers are generally known in the art.

[0247] For oral administration, the pharmaceutical composition can be readily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. Such carriers enable the formulation of the pharmaceutical composition into tablets, pills, dragees, capsules, liquids, gels, slurries, suspensions, etc. for oral ingestion by the patient. Pharmacological formulations for oral use are prepared using solid excipients, optionally grinding the resulting mixture to process it into a mixture of granules, and, if desired, adding suitable adjuvants to obtain tablets or dragee cores. Suitable excipients are, in particular, saccharides including lactose, sucrose, mannitol or sorbitol; fillers such as cellulose formulations including, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl cellulose and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar or alginic acid, or salts thereof such as sodium alginate, may be added.

[0248] The sugar-coated tablet core is provided with a suitable coating. For this purpose, optionally, an enriched sugar solution which may contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution and a suitable organic solvent or solvent mixture may be used. Dyes or pigments may be added to the tablets or sugar-coated tablet cores for identification or to characterize different combinations of dosages of the active compounds.

[0249] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, and sealed capsules made of gelatin, and also include plasticizers such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient in a state of being mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin or liquid polyethylene glycol. In addition, a stabilizer may be added. All formulations for oral administration must be in a dosage suitable for the selected route of administration.

[0250] For buccal administration, the composition may take the form of tablets or troches formulated in a conventional manner.

[0251] For administration by nasal inhalation, the active ingredient for use according to some aspects of the present invention is conveniently delivered in the form of an aerosol spray from a pressurized pack or a nebulizer involving the use of a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve that delivers a metered amount. Capsules and cartridges, for example made of gelatin, for use in a dispenser may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0252] The pharmaceutical compositions described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers optionally containing added preservatives. The composition may be a suspension, solution or emulsion in an oily vehicle or an aqueous vehicle, and may contain formulatory agents such as suspending agents, stabilizers and / or dispersing agents.

[0253] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredient in water-soluble form. Additionally, suspensions of the active ingredient may be prepared as appropriate oily or aqueous suspension injections. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous suspension injections may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or suitable agents that increase the solubility of the active ingredient to enable the preparation of highly concentrated solutions.

[0254] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, a sterile pyrogen-free aqueous solution, before use.

[0255] The pharmaceutical compositions of some aspects of the present invention may also be formulated into rectal compositions such as suppositories or retention enemas, using conventional suppository bases such as cocoa butter or other glycerides.

[0256] Pharmaceutical compositions suitable for use in some aspects of the present invention include compositions containing the active ingredient in an amount sufficient to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient that is important for preventing, alleviating or mitigating the symptoms of a disorder (e.g., cancer) or for prolonging the survival of the subject being treated.

[0257] In some of the various aspects described herein, co - administration of UNO and a checkpoint inhibitor allows for administration of UNO and / or the checkpoint inhibitor at sub - therapeutic doses, which can, for example, reduce the harmful effects of the treatment. In some aspects, UNO is administered prior to the checkpoint inhibitor.

[0258] In some of the various aspects described herein, co - administration of a checkpoint inhibitor, an immune adjuvant, and UNO allows for administration of UNO, the checkpoint inhibitor, and / or the immune adjuvant at sub - therapeutic doses, which can, for example, reduce the harmful effects of the treatment.

[0259] As used herein, the term "sub - therapeutic" or "sub - therapeutic dose" refers to a dose of an agent that is lower than the dose of the agent that is effective (when administered alone) to prevent, reduce, or alleviate symptoms of a disorder (e.g., cancer) or to extend the survival of a subject being treated, e.g., lower than the dose of an agent recognized in the art as being effective (when administered alone) for such purposes, or lower than the dose shown to be effective (when administered alone) to prevent, reduce, or alleviate symptoms of a disorder (e.g., tumor) or to extend the survival of a particular subject.

[0260] In other words, the term "sub - therapeutic" or "sub - therapeutic dose" refers to a dose of an agent that is lower than the therapeutically effective amount of the agent (when administered alone) as defined herein for a subject.

[0261] Determination of a therapeutically effective amount is well within the ability of one of ordinary skill in the art, even in view of the detailed disclosure provided herein.

[0262] For any formulation used in the method of the present invention, a therapeutically effective amount or dosage can first be estimated from in vitro assays or cell culture assays. For example, the dosage can be formulated in an animal model to achieve the desired concentration or titer. Such information can be used to more accurately determine useful dosages in humans.

[0263] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro, in cell cultures or in experimental animals using standard pharmaceutical procedures. Data obtained from these in vitro assays, cell culture assays and animal tests can be used to formulate a range of dosages for use in humans. The dosage may vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be selected by each physician in view of the patient's condition. (See, for example, Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Ch. 1, p. 1).

[0264] Dosage and dosing interval may be adjusted individually to provide a level of the active ingredient (minimal effective concentration, MEC) that is sufficient to induce or suppress a biological effect. The MEC varies for each formulation but can be estimated from in vitro data. The dosage required to achieve the MEC will vary depending on individual characteristics and route of administration. Detection assays can be used to determine plasma concentration.

[0265] Depending on the severity and responsiveness of the condition being treated, administration can be a single dose or multiple doses, and a series of treatments can continue for several days to several weeks, or until healing is affected, or until alleviation of the disease state is achieved.

[0266] The amount of the composition administered will, of course, depend on the subject being treated, the severity of the affliction, the mode of administration, the judgment of the prescribing physician, and the like.

[0267] In certain embodiments, the checkpoint inhibitor is administered at a dose of 1-10 mg / kg.

[0268] In certain embodiments, the checkpoint inhibitor is administered at a dose of 100-500 mg (e.g., 200 mg or 400 mg).

[0269] The following are non-limiting, known representative protocols for the administration of checkpoint inhibitors, such as pembrolizumab, that may be used in certain embodiments of the present invention: Melanoma: 200 mg every 3 weeks or 400 mg every 6 weeks; in pediatric patients, 2 mg / kg every 3 weeks (maximum 200 mg); NSCLC: 200 mg every 3 weeks or 400 mg every 6 weeks; HNSCC: 200 mg every 3 weeks or 400 mg every 6 weeks; cHL or PMBCL: in adults, 200 mg every 3 weeks or 400 mg every 6 weeks; in pediatric patients, 2 mg / kg every 3 weeks (maximum 200 mg); urothelial carcinoma: 200 mg every 3 weeks or 400 mg every 6 weeks; MSI-H or dMMR cancer: in adults, 200 mg every 3 weeks or 400 mg every 6 weeks; in pediatric patients, 2 mg / kg every 3 weeks (maximum 200 mg); MSI-H or dMMR CRC: 200 mg every 3 weeks or 400 mg every 6 weeks; MSI-H or dMMR endometrial cancer: 200 mg every 3 weeks or 400 mg every 6 weeks; gastric cancer: 200 mg every 3 weeks or 400 mg every 6 weeks; esophageal cancer: 200 mg every 3 weeks or 400 mg every 6 weeks; cervical cancer: 200 mg every 3 weeks or 400 mg every 6 weeks; HCC: 200 mg every 3 weeks or 400 mg every 6 weeks; MCC: in adults, 200 mg every 3 weeks or 400 mg every 6 weeks; in pediatric patients, 2 mg / kg every 3 weeks (maximum 200 mg); RCC: as a single agent in the adjuvant setting or, in the advanced setting, 200 mg every 3 weeks or 400 mg every 6 weeks in combination with either axitinib 5 mg orally twice daily or lenvatinib 20 mg orally once daily; endometrial cancer: 200 mg every 3 weeks or 400 mg every 6 weeks in combination with lenvatinib 20 mg orally once daily; TMB-H cancer: in adults, 200 mg every 3 weeks or 400 mg every 6 weeks; in pediatric patients, 2 mg / kg every 3 weeks (maximum 200 mg); cSCC: 200 mg every 3 weeks or 400 mg every 6 weeks; TNBC: 200 mg every 3 weeks or 400 mg every 6 weeks.

[0270] In some of any of the aspects described herein, administration of UNO may optionally be performed before, following, and / or concurrently with administration of a checkpoint inhibitor.

[0271] In some of the various aspects described herein, administration of UNO may optionally be carried out before, following, and / or concurrently with administration of a checkpoint inhibitor and / or an immune adjuvant.

[0272] In certain aspects, the checkpoint inhibitor is administered before UNO.

[0273] In certain aspects, the checkpoint inhibitor is administered at least once before UNO and subsequently concurrently with and / or following UNO.

[0274] In certain aspects, the checkpoint inhibitor is administered before the immune adjuvant.

[0275] In certain aspects, the checkpoint inhibitor is administered at least once before the immune adjuvant and subsequently concurrently with and / or following the immune adjuvant.

[0276] In certain aspects, administration of UNO is carried out before administration of the checkpoint inhibitor.

[0277] In certain aspects, UNO is administered at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 48 hours, at least 56 hours, at least 64 hours, at least 72 hours before administration of the checkpoint inhibitor.

[0278] In certain aspects, UNO is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days before administration of the checkpoint inhibitor.

[0279] In certain embodiments, the checkpoint inhibitor is administered at least 2 times, at least 3 times, at least 4 times, or at least 5 times.

[0280] In certain embodiments, the checkpoint inhibitor is administered every 1 - 50 days, every 1 - 30 days, every 1 - 21 days, every 2 - 10 days, or every 2 - 7 days.

[0281] In certain embodiments, the checkpoint inhibitor is administered daily or every 2 days.

[0282] In certain embodiments, the checkpoint inhibitor is administered every 2 days.

[0283] In certain embodiments, the checkpoint inhibitor is administered every 1 - 10 weeks, every 2 - 8 weeks, or every 3 - 6 weeks.

[0284] In certain embodiments, the checkpoint inhibitor is administered weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks.

[0285] In some embodiments, UNO is administered in one or more cycles, alone or in combination with a checkpoint inhibitor. A cycle is defined as a period of 7 - 28 days (e.g., 7 days or 14 days or 21 days or 28 days). In some embodiments, an immune adjuvant is further administered to the subject.

[0286] In some embodiments, the cycle is repeated until the subject experiences untreatable toxicity or disease progression. The subject can then continue subsequent cycles until a clinical benefit is obtained and an objective tumor response can be demonstrated.

[0287] In some embodiments of every cycle, an immune adjuvant is further administered.

[0288] In certain embodiments, administering comprises (i) Administering a checkpoint inhibitor, followed by (ii) Administering UNO, and the like.

[0289] In certain embodiments, administering comprises (i) Administering UNO, followed by (ii) Administering a checkpoint inhibitor, and the like.

[0290] Non-limiting representative protocols for administration can be used in certain embodiments of the invention and are presented in FIGS. 1 and 20.

[0291] In certain embodiments, when an immune adjuvant is used, the checkpoint inhibitor and the immune adjuvant are administered daily for consecutive days.

[0292] In certain embodiments, administering comprises (i) Administering a checkpoint inhibitor, followed by (ii) Administering UNO, followed by (iii) Administering an immune adjuvant, and the like.

[0293] In certain embodiments, administering comprises (i) Administering a checkpoint inhibitor, followed by (ii) Administering a checkpoint inhibitor and UNO, followed by (iii) Sequentially administering a checkpoint inhibitor and an immune adjuvant, and the like.

[0294] In certain embodiments, administering comprises (i) Administering UNO, followed by (ii) Administering a checkpoint inhibitor, followed by (iii) Administering an immune adjuvant, and the like.

[0295] In certain embodiments, administering comprises (i) Administering UNO, followed by (ii) administering a checkpoint inhibitor and UNO, followed by (iii) sequentially administering a checkpoint inhibitor and an immune adjuvant.

[0296] In certain embodiments, UNO is used as a priming treatment for a checkpoint inhibitor, provided that UNO is used to upregulate the expression of a target immune checkpoint protein prior to administration of the checkpoint inhibitor.

[0297] In some embodiments of the invention, UNO primes cancer for treatment with a PD-1 inhibitor by upregulating the expression of PD-1 or PD-L1 prior to administration of the PD-1 inhibitor.

[0298] In some embodiments of the invention, UNO primes cancer for treatment with a PD-L1 inhibitor by upregulating the expression of PD-L1 prior to administration of the PD-L1 inhibitor.

[0299] In some embodiments of the invention, UNO primes cancer for treatment with a CTLA-4 inhibitor by upregulating the expression of CTLA-4 prior to administration of the CTLA-4 inhibitor.

[0300] In some embodiments of the invention, administration of UNO elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0301] In some embodiments of the invention, the combination of UNO and a checkpoint inhibitor elicits a higher tumor-specific immune cell response. In some embodiments of the invention, the combination of UNO and a checkpoint inhibitor elicits a higher tumor-specific immune cell response synergistically. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0302] In some embodiments of the present invention, the combination with UNO and a PD-1 inhibitor elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells. In some embodiments of the present invention, the combination with UNO and a PD-1 inhibitor synergistically elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0303] In some embodiments of the present invention, the combination of UNO and a PD-L1 inhibitor elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells. In some embodiments of the present invention, the combination of UNO and a PD-L1 inhibitor synergistically elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0304] In some embodiments of the present invention, the combination of UNO and a CTLA-4 inhibitor elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells. In some embodiments of the present invention, the combination of UNO and a CTLA-4 inhibitor synergistically elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0305] In some embodiments of the present invention, the combination of UNO and a LAG-3 inhibitor elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells. In some embodiments of the present invention, the combination of UNO and a LAG-3 inhibitor synergistically elicits a higher tumor-specific immune cell response. In some embodiments, the higher tumor-specific immune cell response is an increase in tumor antigen-specific CD8+ T cells.

[0306] In some embodiments, UNO can be administered to treat solid tumors at a dose of one of 10,000 ppm, 15,000 ppm, 20,000 ppm, 25,000 ppm, 50,000 ppm, and 100,000 ppm via local administration such as intratumoral injection. In some embodiments, UNO can be administered at a flow rate of 0.2 L / min for a period of 5 minutes. In some embodiments, UNO can be administered in multiple cycles. In some embodiments, the cycle is 3 weeks (21 days), provided that UNO can be administered on the first day and / or the eighth day of one or more 21-day cycles. In some embodiments, the cycle is 4 weeks (28 days), provided that UNO can be administered on the first day and / or the eighth day and / or the fifteenth day of one or more 28-day cycles. In some embodiments, UNO can be administered in combination with a checkpoint inhibitor, provided that the checkpoint inhibitor is one of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor. In some embodiments, the checkpoint inhibitor can be administered intravenously following the administration of UNO.

[0307] In some embodiments, UNO can be administered to treat a primary tumor or a metastatic tumor at a dose of 50,000 ppm via local administration such as intratumoral injection. In some embodiments, UNO can be administered at a flow rate of 2 L / min for a period of 5 minutes. In some embodiments, UNO can be administered over multiple 21-day cycles, provided that UNO can be administered on the first day of one or more 21-day cycles.

[0308] In some embodiments, UNO can be administered at a dose of 50,000 ppm via local administration such as intratumoral injection to treat triple-negative breast cancer (TNBC). In some embodiments, UNO can be administered at a flow rate of 2 L / min for a period of 5 minutes. In some embodiments, UNO can be administered over multiple 21-day cycles, provided that UNO can be administered on the first day of one or more 21-day cycles. In some embodiments, UNO can be administered in combination with a checkpoint inhibitor, provided that the checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, pembrolizumab can be administered intravenously following the administration of UNO. In some embodiments, pembrolizumab is administered at a dose of 200 mg for a period of 30 minutes every 3 weeks. In some embodiments, pembrolizumab is administered at a dose of 400 mg for a period of 30 minutes every 6 weeks.

[0309] In some embodiments, UNO can be administered at a dose of 25,000 ppm or 50,000 ppm via local administration such as intratumoral injection to treat advanced cutaneous melanoma. In some embodiments, UNO can be administered at a flow rate of 2 L / min for a period of 5 minutes. In some embodiments, UNO can be administered over multiple 21-day cycles, provided that UNO can be administered on the first day of one or more 21-day cycles. In some embodiments, UNO can be administered in combination with a checkpoint inhibitor, provided that the checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is ipilimumab. In some embodiments, ipilimumab is administered intravenously following the administration of UNO, provided that ipilimumab is administered at a dose of 3 mg / kg for a period of 30 minutes. In some embodiments, ipilimumab is administered for a maximum of 4 cycles.

[0310] The compositions of some aspects of the present invention may be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more dosage forms containing the active ingredient if desired. The pack may include a metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be adapted to the notice associated with the container in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, and this notice reflects the approval by the agency of the composition or of the form of human or veterinary administration. Such notice may be, for example, the label approved by the United States Food and Drug Administration for a prescription drug, or the label of an approved product insert. A composition comprising a formulation of the present invention formulated with a compatible pharmaceutical carrier is also prepared, placed in a suitable container, and labeled for the indicated state of treatment, as further detailed above.

[0311] When referring to measurable values such as amounts, including weight, time, dosage, etc., the term "about" as used herein throughout means to include a variation of + / - 20% or + / - 10% from a particular amount, and thus such variations are appropriate in performing the disclosed methods. In aspects, the term "about" means to include a variation of + / - 5%. In aspects, the term "about" means to include a variation of + / - 1%. In aspects, the term "about" means to include a variation of + / - 0.1%.

[0312] The terms "comprise", "comprising", "include", "including", "having" and their conjugations mean "including but not limited to".

[0313] The term "consisting of" means "including and limiting".

[0314] The term "consisting essentially of" means that a composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially change the basic and novel characteristics of the claimed composition, method or structure.

[0315] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "compound" or "at least one compound" may include multiple compositions, including mixtures thereof.

[0316] Throughout this application, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a description of a range should be considered to have specifically disclosed all the possible sub-ranges within that range and individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0317] Whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integral) within the indicated range. The phrases "ranging / ranges between" the first indicated number and the second indicated number and "ranging / ranges from" the first indicated number "to" the second indicated number are used interchangeably herein and are meant to include the first and second indicated numbers and all the fractions and integers therebetween.

[0318] As used herein, the term "method" refers to a manner, means, technique and process known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or a manner, means, technique and process developed from known manners, means, techniques and processes by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, including but not limited to, a manner, means, technique and process for accomplishing a given task.

[0319] It is understood that certain features of the invention, which are described in separate aspects for clarity, may also be provided in combination in a single aspect. Conversely, various features of the invention that are described in a single aspect for brevity may also be provided separately, or in any suitable partial combination, or in any other described aspect of the invention as may be suitable. Specific features described in connection with various aspects should not be considered essential features of those aspects unless the aspect would not function without those elements.

[0320] The various aspects and embodiments of the invention shown above and claimed in the following claims sections are found experimentally demonstrated in the following examples.

[0321] Example Reference is now made to the following examples, which illustrate some aspects of the invention in a non-limiting manner together with the above description. Generally, the nomenclature used herein and the experimental procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques.

[0322] Colorectal Cancer Model Materials and Methods Preparation of Injectable Cancer Cells - 5.0×10 6Mouse colon cancer CT26WT cells (ATCC, catalog number CRL-2638) suspended in Hanks’ Balanced Salt Solution (HBSS) (Biological Industries, Israel) at a concentration of cells / mL were freshly prepared on the day of cancer model induction. The cells were grown to 70% confluence, harvested using trypsin (Biological Industries, Israel), and counted using a cell counting device. The cells were then centrifuged at 1,200 rpm (241 x g) for 8 minutes, and the pellet was resuspended in ice-cold HBSS at a concentration of 5.0 x 10 6 (5.0 x 10 5 cells in 100 μL solution). The cell viability was greater than 90% as determined by trypan blue staining prior to inoculation into mice. At the time of inoculation, the cells were at a maximum of 10 passages.

[0323] Inoculation of CT26 cancer cells into mice - 100 μL / mouse of the CT26 cancer cell suspension (at a concentration of 5.0 x 10 6 cells / mL) was subcutaneously inoculated into the right flank of 9-week-old male BALB / c mice (Envigo, Israel). Five days later, the same dose of CT26 cells was subcutaneously inoculated into the left flank of each mouse. On the 8th day after primary tumor inoculation, the tumor-bearing mice were divided into groups (-2 days) as shown in Table 1 below and treated according to the time series shown in Figure 1.

Table 1

[0324] On the 14th day, a CT26 cancer cell suspension (at a concentration of 5.0 x 10 6 cells / mL) was inoculated under the right upper arm of all animals at a dose volume of 0.1 mL (a total of 500,000 cells per mouse) (challenge tumor).

[0325] Mice were observed for a total of 100 days. During the experiment, mice were evaluated for eight clinical signs and the primary and secondary tumors were measured. Survival tests for mortality and morbidity were performed 2 - 3 times a week on normal working days. Observation of clinical signs included changes in the skin, hair, eyes, mucosa, and the occurrence of secretions and excretions. The presence of abnormal behavior was also confirmed and recorded. Complete regression was defined as mice in which the primary tumor had completely regressed (disappeared), and partial regression was defined as mice in which the volume of the primary tumor was at least 80% smaller than the average tumor volume of the untreated group.

[0326] If the mice showed signs of severe pain and severe distress, or if the weight loss was greater than 20% from the initial weight, or if the total tumor volume of the mice was greater than 1500 mm 3 more, the mice were sacrificed and excluded from the study. When indicated, data obtained from previous experiments using the same tumor model (also referred to as historical data) were compared to treatment with pembrolizumab alone, as obtained from untreated mice and the literature (see https: / / pubmed(dot)ncbi(dot)nlm(dot)nih(dot)gov / 27799536 / ).

[0327] UNO treatment - On the day of treatment (day 0), mice were anesthetized by intraperitoneal (i.p.) injection of 100 mg / kg ketamine and 10 mg / kg xylazine hydrochloride solution. Tumor-bearing mice were treated with 50,000 ppm UNO delivered to the tumor at 0.2 LPM for 5 minutes with a 23G needle. After treatment, all mice were warmed and closely monitored until complete recovery was observed.

[0328] N2 treatment - On the day of treatment (day 0), mice were anesthetized by intraperitoneal (i.p.) injection of 100 mg / kg ketamine and 10 mg / kg xylazine hydrochloride solution. Tumor-bearing mice were treated with N2 delivered to the tumor at 0.2 LPM for 5 minutes with a 23G needle. After treatment, all mice were warmed and closely monitored until complete recovery was observed.

[0329] Pembrolizumab treatment - Pembrolizumab (Keytruda, Trilogy catalog number 7006873300) was administered intraperitoneally (i.p.) at a dose of 10 mg / kg every other day from day - 2 for 4 - 6 times. The injections were made at approximately the same position each time.

[0330] Example 1 Effect of combined treatment with UNO and pembrolizumab in primary and secondary tumors The primary and secondary tumors of mice treated with UNO in combination with pembrolizumab (see Figure 1) were significantly smaller than those of untreated mice or mice treated with N2 in combination with pembrolizumab (Figure 2C and Figures 3 - 4). Furthermore, the combined treatment with UNO and pembrolizumab increased the rejection rate of secondary tumors compared to treatment with N2 in combination with pembrolizumab (Figure 5). In addition, the combined treatment with UNO and pembrolizumab showed the highest percentage of complete and partial regression of primary and secondary tumors (Figures 6A - 6C).

[0331] Example 2 Effect of combined treatment with UNO and pembrolizumab on mouse survival As shown in Figure 7, the combined treatment with UNO and pembrolizumab had a significant effect on mouse survival compared to untreated mice or mice treated with N2 and pembrolizumab. Mice treated with UNO + pembrolizumab survived for 100 days after primary tumor induction (40%), in contrast to the N2 + pembrolizumab group in which all mice died by day 70 after primary tumor induction.

[0332] Example 3 NO gas: UNO at 25,000 ppm to 100,000 ppm was administered from a 2.9 L cylinder where N2 served as the stabilizing gas (Gordon Gas and Chemical, Tel Aviv, Israel). All procedures were carried out inside a chemical hood. The gas was delivered through a PVC hose (International Biomedical, USA) via a pressure controller. The flow rate was set using a manual flow meter to 0.2 (for in vivo tests) to 1.0 (for in vitro tests) liters per minute (LPM).

[0333] Tumor cell line: The mouse CT26WT colon cell line was purchased from Sartorius (Beit Haemek, Israel), a local distributor of the American Type Culture Collection (ATCC). The CT26 cell line was grown in RPMI-based medium (ATCC) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Sartorius).

[0334] Preparation of tumor cells: The tumor cell suspension was prepared at a concentration of 1×10 5 cells / mL for in vitro tests or 5.0×10 6 cells / mL for in vivo tests in cell culture medium or Hank's balanced salt solution (HBSS, Sartorius). The newly prepared cells were grown to 70% confluence, harvested using trypsin (Sartorius), and counted using a hemocytometer.

[0335] In vivo experiment: CT26 cells at a concentration of 5.0×10 5 in 100 μL of HBSS were subcutaneously inoculated (s.c.) into the right flank of 8 - 10 week-old female and male Balb / c mice. The experiment started after the tumors reached an average volume of approximately 80 mm 3 . Mice were evaluated for tumor volume using a digital caliper.

[0336] In Vivo UNO Treatment - Before each treatment, mice were anesthetized by intraperitoneal (i.p.) injection of 100 mg / kg ketamine (Zoetis) and 10 mg / kg - 20 mg / kg xylazine hydrochloride solution (Abic). After 10 minutes, the mice were treated with intratumoral delivery of 50,000 ppm UNO. The needle was inserted horizontally into the tumor and placed approximately at the center (varying according to about half of the tumor diameter, tumor size and shape). A 23G needle was used and the gas was injected for 5 or 10 minutes. A pressure controller connected to the cylinder was used to set the output pressure to about 2 bar. A PTFE-coated stainless steel hose was connected to the pressure controller and a manual flow meter was connected to the other end. A PVC hose was connected to the flow controller and a 23G injection needle. The treatment regimen was 50,000 ppm UNO at a rate of 0.2 liter / min for 5 or 10 minutes using a manual flow meter.

[0337] Anti-PD-1 Treatment Regimen: Up to 5 doses of 5 - 10 mg / kg of mouse anti-PD-1 or anti-mPD-1 (BioXcell, RMP1-14 BP0146, lot - 810421N1) were administered intraperitoneally every 2 days starting from the second day before treatment with UNO.

[0338] Anti-CTLA-4 Treatment Regimen: Up to 5 doses of 5 mg / kg of mouse anti-CTLA-4 or anti-mCTLA-4 (BioXcell, BE0131-100) were injected every 3 days starting from day 0 or 1 after UNO treatment. Each mouse weighed approximately 20 g, and a 1 mL syringe equipped with a 27G needle was used to administer approximately 0.1 mg of anti-mCTLA-4 with a volume of 0.2 mL (0.10 mg × 5 injections = total 0.5 mg).

[0339] Calculation of Tumor Volume: Local tumor growth was determined by measuring three mutually perpendicular tumor diameters 2 - 3 times per week according to the following formula:

Equation

[0340] Challenge tumor inoculation: Two days before the UNO treatment of the primary tumor, an appropriate cancer cell suspension was prepared, and s.c. cell inoculation was repeatedly performed on the contralateral (left) flank. The appearance of the second induced tumor (challenge tumor) was monitored 2-3 times a week by visual and tactile observation.

[0341] Statistical analysis: Statistical analysis was performed using Excel (Microsoft, USA) or GraphPad Prism 9.3.1 (GraphPad Software, USA), and P<0.05 was considered statistically significant unless otherwise specified.

[0342] Combining UNO with anti-PD-1 delays CT26 primary tumor growth in vivo In vitro findings showing upregulation of PD-L1 in CT26 cells (see below) by high-concentration UNO suggest that these cells are more susceptible to the effects of anti-PD-L1 checkpoint inhibitors or anti-PD-1 checkpoint inhibitors here. To test this hypothesis, an in vivo study combining UNO with anti-PD-1 antibody treatment was conducted. CT26 cells were injected into the flanks of immunocompetent mice. When the tumors reached an average size of 50-100 mm 3 , the tumors were injected intratumorally with 50,000 ppm of UNO (n = 15-16 for each group) for 5 or 10 minutes, and the tumor size was monitored (Figure 8A). Anti-mPD-1 administration was started 2 days before the UNO treatment. As a control, mice were treated with each therapy alone. Mice treated with 5 or 10 minutes of UNO gas exposure in combination with anti-mPD-1 showed slower tumor growth than mice treated with each therapy alone. In addition, the average tumor volume of mice treated with 50,000 ppm of UNO + anti-mPD-1 for 10 minutes was significantly smaller than the average tumor volume of animals treated with anti-mPD-1 alone for 9 days after treatment (Figure 8B and Figure 8C, p = 0.0005).

[0343] These results indicate that the combination of short-term intratumoral UNO administration and systemic anti-PD-1 administration resulted in significant primary tumor growth compared to each agent alone.

[0344] Primary tumor regression and reduced susceptibility to secondary tumors after intratumoral UNO treatment combined with systemic anti-PD-1 administration In addition to the significant short-term local effect of UNO treatment in the primary tumor, UNO treatment reduced the growth of both primary and secondary tumors over up to 100 days. Two days before UNO treatment of the primary tumor (50,000 ppm), a second inoculation of CT26 cells was applied to the contralateral flank and anti-mPD-1 treatment was initiated (Figure 9A). Importantly, secondary tumors were induced before gas treatment, thereby mimicking the development of distant metastases and allowing testing of the combination of UNO and anti-mPD-1 treatment for potential abscopal effects.

[0345] Primary tumor regression was observed in 53% of mice treated with UNO and anti-mPD-1. Furthermore, these mice also had no secondary tumors, and the effect was maintained for up to 100 days after UNO treatment (Figure 9B).

[0346] Mouse survival is substantially extended at 1 / 10 the time of treatment with UNO and anti-PD-1 for up to 100 days after UNO treatment Mouse survival was monitored over 100 days after UNO treatment (see Figure 10A). Median survival was significantly extended in mice treated with UNO + anti-PD-1 compared to mice treated with anti-PD-1 (P = 0.065, Figure 10B).

[0347] Breast cancer model For the majority of patients with metastatic triple-negative breast cancer (mTNBC), chemotherapy is the standard first-line treatment, but responses in this population are poor. Anti-PD-1 treatment has certain antitumor activity in patients with mTNBC. The ability of UNO to improve the efficacy of anti-PD-1 / anti-CTLA-4 antibody treatment in the highly aggressive murine breast cancer model 4T1 was investigated.

[0348] Materials and Methods Preparation of 4T1 cells: A cancer cell suspension in Hank's balanced salt solution (HBSS) (Biological Industries, Israel) at a concentration of 5.0×10 6 cells / mL was freshly prepared on the day of cancer model induction. The cells were grown to 70% - 80% confluence and harvested using trypsin. The cells were centrifuged at 1,200 revolutions per minute (rpm) - 241G for 8 minutes, and then the pellet was resuspended in 1 mL of HBSS. The cells were counted using an automatic cell counter, and a suspension with a concentration of 5.0×10 6 cells / mL was prepared. The cell viability had to be greater than 90% before cell inoculation into mice; otherwise, new cells needed to be prepared. At the time of inoculation, the cells were at a maximum of passage 8. Then, the cell suspension was aspirated into a 1 mL syringe for subcutaneous (s.c.) injection using a 27G needle. All injection volumes were used and not resuspended in a stock solution.

[0349] Inoculation of 4T1 cancer cells into mice - 5.0×10 6 A cancer cell suspension at a concentration of 5.0×10 cells / mL was inoculated into the right flank of the animals at a volume of 100 μL per mouse. Female Balb / c mice were inoculated with 100 μL of 5.0×10 6 4T1 cells / mL. Administration was performed as soon as possible after manual shaking after cell preparation and before withdrawal of the cell suspension. The injection was performed using a 1 mL syringe and a 27G needle. In this case, the withdrawal was performed without a needle.

[0350] UNO treatment and N2 treatment - On the day of treatment, the mice were anesthetized by injecting 100 mg / kg of ketamine and 10 mg / kg of xylazine hydrochloride solution intraperitoneally (i.p.). The tumor-bearing mice were treated with either 50,000 ppm or 100,000 ppm of either N2 or UNO. 50,000 ppm of UNO was administered into the tumor with a 23G needle for 10 minutes at an outlet pressure of 0.2 LPM and 2 bar. 100,000 ppm of UNO was administered into the tumor with a 23G needle for 2 minutes at an outlet pressure of 0.2 LPM and 2 bar. After treatment, all the mice were warmed and closely monitored until complete recovery was observed.

[0351] Anti-PD-1 injection - Anti-PD-1 was administered intraperitoneally (i.p.) 5 times at a dose of 10 mg / kg, and i.p. was performed using a 1 mL syringe equipped with a 27G needle. The injections were made at approximately the same position each time.

[0352] Anti-CTLA-4 injection - was administered intraperitoneally (i.p.) up to 5 times at a dose of 5 mg / kg, and i.p. was performed using a 1 mL syringe equipped with a 27G needle. The injections were made at approximately the same position each time.

[0353] UNO / anti-PD-1 single-dose study - Balb / c mice (48) were inoculated with a murine breast cancer carcinoma (4T1) that was subcutaneously injected (s.c.) into the right abdomen. When the average tumor volume reached 200 - 250 mm 3 , the mice were randomized into groups, stabilized with N2, and treated with 5% UNO (50,000 rpm) injected intratumorally (i.t.) at a flow rate of 0.2 liters per minute (LPM) for 10 minutes, or treated as a control with 1 ppm N2 injected intratumorally (i.t.) at a flow rate of 0.2 LPM for 10 minutes. The mice were given a single gas treatment, such as UNO or N2. Mice assigned to the combination treatment group (i.e., gas + anti-mPD-1) were given anti-mPD-1 injections starting on the day of the first gas treatment, administered every 3 days, and the final dose of anti-mPD-1 was injected 12 days after the first gas treatment.

[0354] Anti-mPD-1 administration was initiated 1 day after UNO treatment. The antibody was injected a total of 5 times every 3 days. The primary tumor volume was evaluated using a standard method and a digital caliper. In addition, mouse survival was evaluated. The mice were weighed daily, and their health status was monitored using a mouse pain scoring. Mice that reached a tumor volume of 1500 mm3 or more and / or a score of 12 were humanely euthanized, and the time of death was recorded.

[0355] In the UNO / anti-PD-1 repeated administration test - Balb / c mice (96), mouse breast cancer carcinoma (4T1) to be subcutaneously injected (s.c.) into the right abdomen was inoculated. When the average tumor volume reached 50 - 150 mm 3 , the mice assigned to the gas treatment group were stabilized with N2 and treated with 5% UNO (50,000 rpm) injected intratumorally (i.t.) at a flow rate of 0.2 liters per minute (LPM) for 10 minutes, or treated with N2 injected intratumorally (i.t.) at a flow rate of 0.2 LPM for 10 minutes as a control. The mice were given gas treatments, such as UNO or N2, twice, for example, on the 9th day after tumor cell inoculation and on the 14th day after tumor inoculation 5 days later. The mice assigned to the combination treatment group (i.e., gas + anti-mPD-1) were given anti-mPD-1 injections starting from the day of the first gas treatment, administered every 3 days, and then the final dose of anti-mPD-1 was injected 12 days after the first gas treatment.

[0356] The progression of the primary tumor volume was

Number

[0357] The mice that received two UNO treatments were divided into two groups. In the first group, the primary tumor was surgically removed 15 days after the first UNO treatment, while in the second group, the tumor was not resected. Mouse survival was evaluated. 1500 mm 3Mice that reached the above tumor volume and / or score 12 were humanely euthanized and the time to death was recorded. The occurrence of metastasis was evaluated postmortem.

[0358] Results - Treating 4T1 tumors with single-dose UNO and repeated-dose UNO improved the results compared to anti-PD-1 alone (see Figures 11A - 13B). The mean tumor volume was significantly smaller 14 days after the first UNO treatment, and the Kaplan - Meyer curves revealed a tendency for long-term survival. These results suggest that local short-term treatment with UNO can serve as a treatment option for cancer patients with tumors that are not suitable for checkpoint inhibitor treatment.

[0359] UNO / anti-CTLA-4 repeated dosing study - Sixty Balb / c mice were inoculated with a murine breast cancer carcinoma (4T1) subcutaneously (s.c.) in the right flank. When the mean tumor volume reached 150 - 200 mm 3 mice assigned to the gas treatment group were stabilized with N2 and treated with 10% UNO (100,000 rpm) injected intratumorally (i.t.) at a flow rate of 0.2 liters per minute (LPM) for 2 minutes, or treated with N2 injected intratumorally (i.t.) at a flow rate of 0.2 LPM for 2 minutes as a control. Mice were given gas treatments, such as UNO or N2, twice, for example, 11 days after tumor cell inoculation and 14 days after tumor inoculation, 3 days later. Mice assigned to the combination treatment group (i.e., gas + anti-mCTLA-4) were given anti-mCTLA-4 injections starting 1 day after the first gas treatment, administered every 3 days, and then the final dose of anti-mCTLA-4 was injected for 13 days after the first gas treatment.

[0360] The progression of the primary tumor volume was

Number

[0361] Mouse survival was evaluated. Mice that reached a tumor volume of 1500 mm 3 or greater and / or a score of 12 were humanely euthanized, and the time to death was recorded. The occurrence of metastasis was evaluated postmortem.

[0362] Results - Treating 4T1 tumors with repeated doses of UNO improved the results compared to anti-CTLA-4 alone (see Figure 14). Regardless of treatment, the average tumor volume of mice treated with UNO was smaller on day 13 after the first UNO treatment (10 days after the second treatment) compared to anti-mCTLA-4 alone. The difference in tumor volume between mice receiving combination treatment and those treated with antibody alone was significant. The Kaplan–Meier curve revealed long-term survival. These results suggest that local short-term treatment with UNO can serve as a treatment option for cancer patients with tumors that are not suitable for checkpoint inhibitor treatment.

[0363] Effect of combination treatment with UNO, checkpoint inhibitor, and immune adjuvant Materials and methods Preparation of injectable cancer cells - Mouse colon cancer CT26WT cells (ATCC, catalog number CRL-2638) suspended in Hanks’ Balanced Salt Solution (HBSS) (Biological Industries, Israel) at a concentration of 5.0×10 6 cells / mL were freshly prepared on the day of cancer model induction. The cells were grown to 70% confluence, harvested using trypsin (Biological Industries, Israel), and counted using a cell counting device. The cells were then centrifuged at 1,200 rpm (241xg) for 8 minutes, and the pellet was resuspended in 1 mL at 5.0×10 6 (5.0×10 in 100 μL solution 5Cells) and resuspended in ice-cold HBSS. The cell viability was greater than 90% as determined by trypan blue staining prior to inoculation into mice. At the time of inoculation, the cells were at a maximum of passage 8.

[0364] Inoculation of CT26 cancer cells into mice - 100 μL / mouse (5.0 × 10 6 cells / mL concentration) of the CT26 cancer cell suspension was subcutaneously inoculated into the right flank of 10-week-old male BALB / c mice (Envigo, Netherlands). Five days later, the same dose of CT26 cells was subcutaneously inoculated into the left flank of each mouse. On day 10 after the primary tumor inoculation, the tumor-bearing mice were divided into groups (day 0) as shown in Table 2 below and treated according to the time course shown in Figure 20.

Table 2

[0365] The mice were followed for a total of 52 days. Tumor uptake was monitored by palpation of the flank, and tumor growth was monitored by measuring the tumor diameter using digital calipers. If the total tumor volume of the mice was greater than 1500 mm 3 or if the mouse pain scoring was greater than 12 as approved according to IACUC IL-2112-105-5, the mice were sacrificed and excluded from the study.

[0366] UNO treatment - On the day of treatment, the mice were anesthetized by intraperitoneal (i.p.) injection of 100 mg / kg ketamine and 10 mg / kg xylazine hydrochloride solution. The tumor-bearing mice were treated with 50,000 ppm UNO delivered to the tumor at 0.2 LPM for 5 minutes with a 23G needle. After treatment, all mice were warmed and closely monitored until complete recovery was observed.

[0367] Immune checkpoint inhibitor treatment - Mouse anti-PD-1 or anti-mPD-1 (BioXcell, catalog number BP0146) was intraperitoneally (i.p.) administered 5 times at a dose of 10 mg / kg. The injections were made at approximately the same location each time.

[0368] Injection of class B CpG oligodeoxynucleotides (CpG-B) - CpG-B ODN1826 (IDT, catalog number 230860406) was administered s.c. three times at a dose of 50 μg / 0.1 mL in physiological saline. The injections were made at approximately the same location each time.

[0369] Example 4 Effect of combined treatment with UNO, anti-PD-1, and CpG-B in primary tumors The primary tumors of mice treated with UNO combined with anti-mPD-1 and CpG-B were significantly smaller than those of untreated mice, or mice treated with UNO alone, or mice treated with UNO + anti-mPD-1 (Figure 15). Furthermore, the triple combination treatment group showed the highest rate of complete regression of primary tumors (Figure 16): 6 out of 9 mice (67%) compared to untreated mice (0%), UNO-alone treated mice (10%), and UNO + anti-mPD-1 (20%) treated mice.

[0370] Example 5 Effect of combined treatment with UNO, anti-PD-1, and CpG-B on distant tumor rejection and secondary tumors The triple combination treatment with UNO + anti-mPD-1 + CpG-B had a significant effect on distant tumor rejection at 42 days after treatment (62%) compared to untreated mice (0%), UNO-alone treated mice (11%), and UNO + anti-mPD-1 (25%) treated mice (Figure 17). Furthermore, the secondary tumors of mice treated with UNO combined with anti-mPD-1 and CpG-B were significantly smaller than those of untreated mice, or mice treated with UNO alone, or mice treated with UNO + anti-mPD-1 (Figure 18).

[0371] Example 6 Effect of combined treatment with UNO, anti-PD-1, and CpG-B on mouse survival As shown in Fig. 19, the triple combination treatment with UNO + anti-mPD-1 + CpG-B had a significant effect on mouse survival compared to untreated mice, UNO alone-treated mice, and UNO + anti-mPD-1-treated mice. Specifically, although the last untreated mouse was sacrificed 45 days after the primary tumor inoculation, 10% of the UNO-only group, 20% of the UNO + anti-mPD-1 group, and 67% of the UNO + anti-mPD-1 + CpG-B group survived for 52 days after the primary tumor inoculation.

[0372] Example 7 UNO treatment in vitro increases the presentation of PD-L1 in cancer cells Materials and Methods Cell preparation - Mouse colon cancer CT26WT cells (ATCC number: CRL-2638) were grown to 70% - 80% confluence and harvested using trypsin. Subsequently, the cells were centrifuged at 1,200 revolutions per minute (rpm) - 241G for 8 minutes, and then the pellet was resuspended in 1 mL of cell culture medium (RPMI-1640 supplemented with 10% FBS and 1% Pen-Strep). The cells were counted using an automated cell counter, and a total of 2×10 6 cells were seeded into each 10 mm dish containing 9 mL of cell culture medium. Then the plates were placed in an incubator at 37 °C and 5% CO2 for 24 hours.

[0373] UNO treatment - After 24 hours of incubation, the cell culture medium was removed, and the plates were placed in an acrylic box located in a working chemical fume hood. Before the start of the treatment, the box was exposed to UV for 1 hour to reduce the risk of contamination. The box was closed and sealed with parafilm. A PVC delivery line was inserted into the designated hole at the top of the box and lowered it approximately 5 cm into the box. The flow meter was set to 1 LPM, and the cells were exposed for the periods and concentrations described in Table 3 (control cells were untreated).

Table 3

[0374] After treatment, 9 mL of cell culture medium was added to each well, and the plate was placed in an incubator at 37 °C and 5% CO2 for 24 hours.

[0375] Flow cytometry - The cell culture medium was removed to a 50 mL centrifuge tube. Each well was washed with 2 mL of trypsin and then the trypsin was collected into the previously described 50 mL centrifuge tube. Then, 3 mL of trypsin was added and the dish was placed in an incubator at 37 °C and 5% CO2 for 3 minutes. The remaining cells were detached using a cell scraper. Then 5 mL of cell culture medium was added to the dish and all of its volume (8 mL) was collected into the centrifuge tube. Subsequently, the cells were centrifuged at 1200 rpm (241G) for 8 minutes, the supernatant was discarded, and the cells were resuspended in 1 mL of cell culture medium and stained with an Annexin V-FITC kit (Miltenyi Biotec, catalog number 130-092-052) to detect apoptotic, necrotic, and dead cells, or stained with anti-PD-L1 (Biolegend, Brilliant Violet 421™ anti-mouse CD274 (B7-H1, PD-L1) antibody, catalog number 124315).

[0376] - Cell count (MACSQuant VYB): For cell counting purposes, 100 μL was transferred from each sample to a 96-well plate. 10 6 cells were dispensed into a designated Eppendorf tube for labeling purposes.

[0377] - Anti-mouse CD274 (B7-H1, PD-L1) BV421 antibody (BioLegend, catalog number 124315) and rat IgG2b, isotype control antibody (BioLegend, catalog number 400639): Each sample was stained with 5 μL of anti-mouse CD274 antibody in 95 μL of FACS buffer (total labeling volume of 100 μL). Each isotype control sample was stained with 5 μL of rat IgG2b, isotype control antibody in 95 μL of FACS buffer. The unstained sample was resuspended in 100 μL of FACS buffer. The cells were incubated at 4 °C for 20 minutes covered with aluminum foil. The cells were washed by adding 500 μL of FACS buffer and centrifuged at 300 xg for 7 minutes.

[0378] - Cell staining with the Annexin V FITC kit (catalog number 130-092-052) from Miltenyi: The supernatant was discarded, and the cells were washed with 1.5 mL of 1-fold binding buffer by centrifuging at 300 xg for 7 minutes. An Annexin V staining solution was prepared by diluting the Annexin V FITC reagent 1:11 with 1-fold binding buffer. The cells were resuspended in 110 μL of Annexin V FITC solution (except for the PI single-stained samples and unstained samples resuspended in 110 μL of 1-fold binding buffer). The samples were incubated at room temperature for 15 minutes while protecting from light. The cells were washed by adding 1 mL of 1-fold binding buffer and centrifuged at 300 xg for 7 minutes at room temperature. The samples were resuspended in 500 μL of 1-fold binding buffer.

[0379] - Acquisition with a MACSQuant VYB flow cytometer: 5 μL of PI was added immediately before analysis with the MACSQuant VYB instrument.

[0380] The effect of exposure to UNO on the viability of CT26 cancer cells was evaluated 24 hours after exposure. As shown in Figure 21, exposure to UNO decreased viability and induced apoptosis and necrosis of cancer cells in a dose- and time-dependent manner.

[0381] Interestingly, after exposure to UNO, approximately 70% to approximately 90% of the PI-negative cells expressed PD-L1 depending on the dose and exposure time (Figure 22). Furthermore, the mean fluorescence intensity (MFI) of PD-L1 expression increased in a UNO dose- and time-dependent manner. Thus, by upregulating PD-L1 expression in tumors, the tumors may become more responsive to anti-PD-1 and / or anti-PD-L1 treatment. Therefore, UNO can be used to prime tumors prior to anti-PD-1 treatment or anti-PD-L1 treatment.

[0382] Example 8 Effect of UNO and anti-CTLA-4 on the systemic levels of tumor antigen-specific CD8+ T cells 7 days after treatment Materials and methods: Preparation of injectable cancer cells - 5.0×10 6 Mouse colon cancer CT26WT cells (ATCC, catalog number CRL-2638) suspended in Hanks’ Balanced Salt Solution (HBSS) (Biological Industries, Israel) at a concentration of 5.0×10 cells / mL were freshly prepared on the day of cancer model induction. The cells were grown to 70% confluence, harvested using trypsin (Biological Industries, Israel), and counted using a cell counting device. The cells were then centrifuged at 1,200 rpm (241 x g) for 8 minutes, and the pellet was resuspended in ice-cold HBSS at 5.0×10 6 (5.0×10 cells in 100 μL solution) 5 The cell viability was greater than 90% as determined by trypan blue staining prior to inoculation into mice. At the time of inoculation, the cells were at a maximum of passage 8.

[0383] Inoculation of CT26 cancer cells into mice - 100 μL / mouse of the CT26 cancer cell suspension (5.0×10 6The concentration (cells / mL) was subcutaneously inoculated into the right flank of 10-week-old male BALB / c mice (Envigo, Netherlands). Three days later, the same dose of CT26 cells was subcutaneously inoculated into the left flank of each mouse. For 10 days after the primary tumor inoculation, the tumor-bearing mice were divided into groups (day 0) as shown in Table 4 below.

Table 4

[0384] UNO treatment - On the day of treatment, the mice were anesthetized by intraperitoneal (i.p.) injection of 100 mg / kg ketamine and 10 mg / kg xylazine hydrochloride solution. The tumor-bearing mice were treated with 100,000 ppm UNO delivered to the tumor at 0.2 LPM for 5 minutes with a 23G needle. After treatment, all mice were warmed and closely monitored until complete recovery was observed.

[0385] Immune checkpoint inhibitor treatment - Mouse anti-CTLA-4 or anti-mCTLA-4 (BioXcell, catalog number BE-0131) was administered intraperitoneally (i.p.) twice at a dose of 5 mg / kg. The injections were made at approximately the same location each time.

[0386] Flow cytometry for tumor antigen-specific CD8+ T cells - On day 7 after UNO treatment, 100 μL of peripheral blood was collected from each mouse by submandibular bleeding into a tube containing 0.5 M EDTA (pH 8.0). Red blood cells were removed by incubating in ACK lysis solution for 5 minutes, and the samples were centrifuged at 1500 RPM (300 g) for 5 minutes. Subsequently, the samples were stained with 1 μL / mL of Ghost dye Red710 (TONBO biosciences, catalog number 13-0871) for 15 minutes at 4°C and washed twice with FACS buffer containing 2% fetal bovine serum in PBS. Then the samples were stained with a mixture of 0.5 μg / mL of CD8-FITC antibody (Invitrogen, Clone KT15, catalog number MA516759), 0.66 μg / mL of CD4-APC / Fire810 antibody (Biolegend, Clone GK1.5, catalog number 100480), 3 μg / mL of CD3-PerCP-Vio700 antibody (Miltenyi Biotec, Clone REA641, catalog number 130-120-826), 0.75 μg / mL of CD44-PE-vio770 antibody (Miltenyi Biotec, Clone REA664, catalog number 130-119-127) and 0.75 μg / mL of CD62L-APC antibody (Miltenyi Biotec, Clone REA828, catalog number 130-112-837) and 4 μL / sample of H-2LdMuLVgp70 tetramer (MBL Life science, catalog number TB-M521-1) for 30 minutes at room temperature. Then the samples were washed twice with FACS buffer and acquired on an Attune NxT flow cytometer. The data was analyzed using FlowJo software.

[0387] Results: AH-1+CD8+ T cells (tumor antigen-specific CD8+ T cells) were detected in the blood of all mice treated with UNO, but not in mice treated with anti-mCTLA-4, untreated mice, or naive mice (Figure 24). Furthermore, the systemic level of AH-1+CD8+ T cell levels was higher in mice treated with UNO compared to untreated or naive mice that exhibited AH-1+CD8+ T cells. In addition, AH-1+CD8+ T cells were found in the blood of all mice treated with a combination of UNO and anti-mCTLA-4, and were at higher levels compared to mice treated with anti-mCTLA-4 or UNO alone.

[0388] Example 9 Test the treatment of cutaneous or subcutaneous primary or metastatic tumors using UNO at a dose of 50,000 ppm administered intratumorally. UNO is administered at a flow rate of 2 L / min for a period of 5 minutes. UNO is administered in multiple 21-day cycles. UNO is administered on day 1 of each cycle, for example, every 21 days.

[0389] Example 10 Test the treatment of triple-negative breast cancer (TNBC) using a combination of (i) UNO administered intratumorally at a dose of 50,000 ppm for a period of 5 minutes at a flow rate of 2 L / min, and (ii) pembrolizumab, a PD-1 inhibitor, administered intravenously at a dose of 200 mg to 400 mg for a period of 30 seconds, where UNO is administered immediately prior to the PD-1 inhibitor. UNO is administered in multiple 21-day cycles. UNO is administered on day 1 of each cycle, for example, every 21 days.

[0390] Example 11 (i) A treatment for advanced cutaneous malignant melanoma using a combination of UNO administered intratumorally at a flow rate of 2 L / min for a period of 5 minutes at a dose of 25,000 ppm or 50,000 ppm, and (ii) ipilimumab, a CTLA-4 inhibitor, administered intravenously at a dose of 3 mg / kg for a period of 30 seconds, wherein UNO is administered immediately before the CTLA-4 inhibitor, is tested. The combination is administered in multiple 21-day cycles. The combination is administered, for example, every 21 days on day 1 of each cycle.

[0391] The present invention has been specifically shown and described with reference to preferred embodiments of the invention, but it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as defined in the appended claims.

[0392] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually stated to be incorporated by reference as part of this specification. In addition, any citation or identification of a reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, these should not necessarily be construed as limiting. In addition, any one or more priority documents of this application are hereby incorporated by reference in their entirety as part of this specification.

Claims

**Claim 1** A method for treating cancer in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of ultra-high concentration gaseous nitric oxide (UNO) and a checkpoint inhibitor, and the UNO is administered intratumorally. **Claim 2** A combination for use in treating cancer in a subject in need thereof with ultra-high concentration gaseous nitric oxide (UNO) and a checkpoint inhibitor, provided that the UNO is administered intratumorally. **Claim 3** The method according to claim 1 or the combination for use according to claim 2, wherein the UNO is administered at a dose of about 10,000 ppm to about 1,000,000 ppm for a time of about 1 second to about 60 minutes at a volumetric flow rate of about 0.00001 LPM to about 1 LPM. **Claim 4** The method according to claim 1 or 3 or the combination for use according to claim 2 or 3, wherein the UNO is administered at a dose of about 20,000 ppm to about 200,000 ppm or a dose of about 20,000 ppm to about 100,000 ppm. **Claim 5** The method according to claim 1, 3 or 4 or the combination for use according to any one of claims 2 to 4, wherein the UNO is administered for a time in the range of about 30 seconds to about 10 minutes. **Claim 6** The method according to any one of claims 1, 3 to 5 or the combination for use according to any one of claims 2 to 5, wherein the UNO is administered at a volumetric flow rate of about 0.001 LPM to about 0.5 LPM. **Claim 7** The method according to any one of claims 1, 3 to 6 or the combination for use according to any one of claims 2 to 6, wherein the checkpoint inhibitor is administered before the UNO. **Claim 8** The method according to any one of claims 1, 3 to 7 or the combination for use according to any one of claims 2 to 7, wherein the checkpoint inhibitor is administered every 2 to 7 days. **Claim 9** The method according to any one of claims 1, 3 to 8 or the combination for use according to any one of claims 2 to 8, wherein the checkpoint inhibitor is administered at least twice. **Claim 10** The method according to any one of claims 1, 3 to 9 or the combination for use according to any one of claims 2 to 9, wherein the UNO is administered before the checkpoint inhibitor. **Claim 11** The method according to any one of claims 1, 3 to 10 or the combination for use according to any one of claims 2 to 10, provided that the administration of UNO primes cancer for treatment with the checkpoint inhibitor by upregulating the expression of the target immune checkpoint protein prior to said administration of the checkpoint inhibitor, and the target immune checkpoint protein is one of PD-1, PD-L1 and CTLA-4.

12. The method according to any one of claims 1, 3 to 11 or the combination for use according to any one of claims 2 to 11, wherein the combination of UNO and a checkpoint inhibitor increases tumor-specific immune cells. The combination for use according to any one of claims 2 to 11.

13. The method according to any one of claims 1, 3 to 12 or the combination for use according to any one of claims 2 to 12, wherein the checkpoint inhibitor is one of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

14. The method according to any one of claims 1, 3 to 13 or the combination for use according to any one of claims 2 to 13, wherein the cancer is refractory to treatment with a checkpoint inhibitor.

15. The method according to any one of claims 1, 3 to 14 or the combination for use according to any one of claims 2 to 14, further comprising administering an immune adjuvant.

16. The method according to claim 15 or the combination for use according to claim 15, wherein the immune adjuvant is selected from the group consisting of inorganic salts, aluminum salts, organic adjuvants, emulsions, microparticles, liposomes, saponins, cytokines, microbial components, and nucleic acid adjuvants.

17. The method according to claim 16 or the combination for use according to claim 16, wherein the immune adjuvant is a nucleic acid adjuvant.

18. The method according to claim 17 or the combination for use according to claim 17, wherein the nucleic acid adjuvant comprises cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN).

19. The method according to claim 17 or 18 or the combination for use according to claim 17 or 18, wherein the checkpoint inhibitor is administered before the immune adjuvant.

20. The method according to any one of claims 17 to 19 or the combination for use according to any one of claims 17 to 19, wherein the immune adjuvant is administered subsequent to UNO.

21. The method according to any one of claims 1, 3 to 20 or the combination for use according to any one of claims 2 to 20, wherein the cancer is positive for a microsatellite instability (MSI) marker and / or a mismatch repair deficiency (dMMR) marker.

22. The method according to any one of claims 1, 3 to 21 or the combination for use according to any one of claims 2 to 21, wherein the cancer is negative for a microsatellite instability (MSI) marker and / or a mismatch repair deficiency (dMMR) marker.

23. The method according to any one of claims 1, 3 to 22 or the combination for use according to any one of claims 2 to 22, wherein the cancer is selected from the group consisting of colon cancer, breast cancer, melanoma, lung cancer, head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial cancer, high tumor mutational burden (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), high microsatellite instability cancer or mismatch repair deficient cancer, and high microsatellite instability cancer or mismatch repair deficient colorectal cancer (CRC).

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