WO2020033331A1

WO2020033331A1 - Method and compositions for treating colon cancer and breast cancer

Info

Publication number: WO2020033331A1
Application number: PCT/US2019/045161
Authority: WO
Inventors: Anna MARTNER; Hanna Grauers WIKTORIN; Kristoffer Hellstrand
Original assignee: Martner Anna; Wiktorin Hanna Grauers; Kristoffer Hellstrand
Priority date: 2018-08-06
Filing date: 2019-08-05
Publication date: 2020-02-13

Abstract

Some embodiments of the uses and compositions provided herein relate to the amelioration or treatment of a cancer in a subject, such as a colon cancer or a breast cancer. In some, embodiments, a cancer in a subject can be ameliorated or treated by reducing the activity of NOX2 in a cell of the subject. In some embodiments, a cancer in a subject can be ameliorated or treated by reducing the activity of NOX2 in a cell of the subject in combination with reducing the activity of PD-1 in a cell of the subject, and/or reducing the activity of PD-L1 in a cell of the subject.

Description

METHOD AND COMPOSITIONS FOR TREATING COLON CANCER

AND BREAST CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Prov. App. No. 62/714,888 entitled“METHOD AND COMPOSITIONS FOR TREATING COLON CANCER AND BREAST CANCER” filed August 6, 2018, which is incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 20l90805_SEQUENCE_LISTING.txt. The file is about 11.1 KB and was created on August 5, 2019, and is being submitted electronically via EFS-web.

FIELD

[0003] Some embodiments of the methods and compositions provided herein relate to the amelioration or treatment of a cancer in a subject, such as a colon cancer or a breast cancer. In some, embodiments, a cancer in a subject can be ameliorated or treated by reducing the activity of NOX2 in the subject. In some embodiments, a cancer in a subject can be ameliorated or treated by reducing the activity of NOX2 in the subject in combination with reducing the activity of PD-l in the subject, and/or reducing the activity of PD-L1 in the subject.

BACKGROUND

[0004] Colon cancer, also known as bowel cancer and colorectal cancer includes the development of cancer from the colon or rectum. The five-year survival rate in the United States is around 65%. Individual likelihood of survival can depend on how advanced the cancer is, whether or not all the cancer can be removed with surgery and the person's overall health. Globally, colorectal cancer is the third most common type of cancer, making up about 10% of all cases. In 2012, there were 1.4 million new cases and 694,000 deaths from the disease. It is more common in developed countries, where more than 65% of cases are found. Treatments used for colon cancer can include surgery, radiation therapy, chemotherapy and targeted therapy. Cancers that are confined within the wall of the colon may be curable with surgery, while cancer that has spread widely are usually not curable, with management being directed towards improving quality of life and symptoms.

[0005] Breast cancer develops from breast tissue. Outcomes for breast cancer vary depending on the cancer type, extent of disease, and person's age. Worldwide, breast cancer is the leading type of cancer in women, accounting for 25% of all cases. In 2012 it resulted in 1.68 million new cases and 522,000 deaths. In those who have been diagnosed with breast cancer, a number of treatments may be used, including surgery, radiation therapy, chemotherapy, hormonal therapy and targeted therapy. Despite advances in the field there remains a need to develop new and improved therapies to treat such cancers.

SUMMARY

[0006] Some embodiments of the methods and compositions provided herein relate to a method of treating or ameliorating a cancer in a subject, wherein the cancer is a breast cancer or a colon cancer, the method comprising reducing the activity of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject.

[0007] Some embodiments of the methods and compositions provided herein relate to include reducing the activity of programmed cell death protein 1 (PD-l) or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject.

[0008] Some embodiments of the methods and compositions provided herein relate to reducing the activity of programmed cell death protein ligand 1 (PD-L1) or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0009] In some embodiments, reducing the activity of NOX2 comprises administering an effective amount of a NOX2 inhibitor to the subject. In some embodiments, the NOX2 inhibitor is selected from the group consisting of histamine dihydrochloride (HDC), histamine, N-methyl-histamine, 4-methyl-histamine, histamine phosphate, histamine diphosphate, GSK2795039, apocymn, GKT136901, GKT137831, ML171, VAS2870, VAS3947, celastrol, ebselen, perhexiline, grindelic acid, NOX2ds-tat, NOXAlds, fulvene-5, ACD 084, NSC23766, CAS 1177865-17-6, and CAS 1090893-12-1, and shionogi. In some embodiments, the NOX2 inhibitor is HDC.

[0010] In some embodiments, reducing the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell comprises contacting the cell with an isolated nucleic acid that is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme. In some embodiments, the isolated nucleic acid comprises a sequence encoding NOX2 or a fragment thereof, a sequence encoding antisense NOX2 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding NOX2 or a fragment thereof. In some embodiments, the isolated nucleic acid comprises a gRNA comprising a sequence complementary to the sequence of a target gene selected from the group consisting of NOX2, CYBA, NCF1, NCF2, NCF4, RAC1, and RAC2. In some embodiments, the target gene is NOX2.

[0011] In some embodiments, reducing the activity of PD-l comprises administering an effective amount of a PD-l inhibitor to the subject. In some embodiments, the PD-l inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, AMP-224, AMP-514, and PDR001. In some embodiments, the PD- 1 inhibitor is an anti-PD-l antibody or an antigen binding fragment thereof selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, and cemiplimab.

[0012] In some embodiments, reducing the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell comprises contacting the cell with an isolated nucleic acid selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme. In some embodiments, the isolated nucleic acid comprises a sequence encoding PD-l or a fragment thereof, a sequence encoding antisense PD-l or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-l or a fragment thereof.

[0013] In some embodiments, reducing the activity of PD-L1 comprises administering an effective amount of a PD-L1 inhibitor to the subject. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS-936559, and CK-301. In some embodiments, the PD-L1 inhibitor is an anti-PD-Ll antibody or antigen binding fragment thereof selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0014] In some embodiments, reducing the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell comprises contacting the cell with an isolated nucleic acid selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme. In some embodiments, the isolated nucleic acid comprises a sequence encoding PD-L1 or a fragment thereof, a sequence encoding antisense PD-L1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-L1 or a fragment thereof.

[0015] In some embodiments, an agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered concurrently with an agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, and/or an agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0016] In some embodiments, an agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered sequentially with an agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, and/or an agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0017] In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a myeloid cell. In some embodiments, the cell is myeloid-derived suppressor cell (MDSC). In some embodiments, the cell is an intratumoral MDSC. In some embodiments, the cell is a peripheral CDl4+HLA-DR^/low MDSC. In some embodiments, the cell is a GR1+ MDSC. In some embodiments, the cell is a monocytic MDSC. In some embodiments, the cell is a granulocytic MDSC.

[0018] In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 10%. In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 30%. In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 50%. In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 80%. In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 95%.

[0019] In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 50%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 100%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 200%.

[0020] In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cell with an effector phenotype by at least about 5%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cell with an effector phenotype by at least about 10%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cell with an effector phenotype by at least about 20%.

[0021] In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are natural killer (NK) cells by at least about 50%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are NK cells by at least about 100%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are NK cells by at least about 200%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are NK cells by at least about 600%. [0022] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is colon cancer.

[0023] In some embodiments, the subject is mammalian. In some embodiments, the subject is human.

[0024] Some embodiments of the methods and compositions provided herein relate to use of a first agent to treat or ameliorate a cancer in a subject, wherein the cancer is a breast cancer or a colon cancer, wherein the first agent reduces the activity of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject.

[0025] In some embodiments, the use of the first agent is in combination with a second agent which reduces the activity of programmed cell death protein 1 (PD-l) or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject.

[0026] In some embodiments, the use of the first agent is in combination with a third agent which reduces the activity of programmed cell death protein ligand 1 (PD-L1) or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L 1 protein in a cell of the subject.

[0027] In some embodiments, the first agent comprises a NOX2 inhibitor. In some embodiments, the NOX2 inhibitor is selected from the group consisting of histamine dihydrochloride (HDC), histamine, N-methyl-histamine, 4-methyl-histamine, histamine phosphate, histamine diphosphate, GSK2795039, apocynin, GKT136901, GKT137831, ML171, VAS2870, VAS3947, celastrol, ebselen, perhexiline, grindelic acid, NOX2ds-tat, NOXAlds, fulvene-5, ACD 084, NSC23766, CAS 1177865-17-6, CAS 1090893-12-1, and shionogi. In some embodiments, the NOX2 inhibitor is HDC.

[0028] In some embodiments, the first agent comprises an isolated nucleic acid which reduces the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject, wherein the isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme. In some embodiments, the isolated nucleic acid comprises a sequence encoding NOX2 or a fragment thereof, a sequence encoding antisense NOX2 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding NOX2 or a fragment thereof. In some embodiments, the isolated nucleic acid comprises a gRNA comprising a sequence complementary to the sequence of a target gene selected from the group consisting of NOX2, CYBA, NCF1, NCF2, NCF4, RAC1, and RAC2. In some embodiments, the target gene is NOX2.

[0029] In some embodiments, the second agent comprises a PD-l inhibitor. In some embodiments, the PD-l inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, AMP-224, AMP-514, and PDROOl. In some embodiments, the PD-l inhibitor is an anti-PD-l antibody or antigen binding fragment thereof selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, and cemiplimab.

[0030] In some embodiments, the second agent comprises an isolated nucleic acid which reduces the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, wherein the isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme. In some embodiments, the isolated nucleic acid comprises a sequence encoding PD-l or a fragment thereof, a sequence encoding antisense PD-l or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-l or a fragment thereof.

[0031] In some embodiments, the third agent comprises a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS-936559, and CK-301 In some embodiments, the PD-Ll inhibitor is an anti-PD-Ll antibody or an antigen binding fragment thereof selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0032] In some embodiments, the third agent comprises an isolated nucleic acid which reduces the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject, wherein the isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme. In some embodiments, the isolated nucleic acid comprises a sequence encoding PD-L1 or a fragment thereof, a sequence encoding antisense PD-L1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-L1 or a fragment thereof.

[0033] In some embodiments, the first agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered concurrently with the second agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD- 1 protein in a cell of the subject, and/or the third agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0034] In some embodiments, the first agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered sequentially with the second agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, and/or the third agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0035] In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a myeloid cell. In some embodiments, the cell is myeloid-derived suppressor cell (MDSC). In some embodiments, the cell is an intratumoral MDSC. In some embodiments, the cell is a peripheral CDl4+HLA-DR-/lowMDSC. In some embodiments, the cell is a GR1+ MDSC. In some embodiments, the cell is a monocytic MDSC. In some embodiments, the cell is a granulocytic MDSC.

[0036] In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 10%. In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 30%. In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 50%. In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 80%. In some embodiments, the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 100%. [0037] In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 50%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 100%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 200%.

[0038] In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cells with an effector phenotype by at least about 5%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cells with an effector phenotype by at least about 10%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cells with an effector phenotype by at least about 20%.

[0039] In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are natural killer (NK) cells by at least about 50%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are NK cells by at least about 100%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are NK cells by at least about 200%. In some embodiments, the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are NK cells by at least about 600%.

[0040] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is colon cancer.

[0041] In some embodiments, the subject is mammalian. In some embodiments, the subject is human.

[0042] Accordingly, some aspects described herein relate to the following numbered alternatives:

[0043] 1. A method of treating or ameliorating a cancer in a subject, wherein the cancer is a breast cancer or a colon cancer, the method comprising reducing the activity of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject. [0044] 2. The method of alternative 1, further comprising reducing the activity of programmed cell death protein 1 (PD-l) or the expression level of a nucleic acid encoding PD- 1 or the expression level of PD-l protein in a cell of the subject.

[0045] 3. The method of alternative 1 or 2, further comprising reducing the activity of programmed cell death protein ligand 1 (PD-L1) or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0046] 4. The method of any one of alternatives 1-3, wherein reducing the activity of NOX2 comprises administering an effective amount of a NOX2 inhibitor to the subject.

[0047] 5. The method of alternative 4, wherein the NOX2 inhibitor is selected from the group consisting of histamine dihydrochloride (HDC), histamine, N-methyl-histamine, 4- methyl-histamine, histamine phosphate, histamine diphosphate, GSK2795039, apocynin, GKT136901, GKT137831, ML171, VAS2870, VAS3947, celastrol, ebselen, perhexihne, grmdelic acid, NOX2ds-tat, NOXAlds, fulvene-5, ACD 084, NSC23766, CAS 1177865-17- 6, and CAS 1090893-12-1, and shionogi.

[0048] 6. The method of alternative 5, wherein the NOX2 inhibitor is HDC.

[0049] 7. The method of any one of alternatives 1-6, wherein reducing the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell comprises contacting the cell with an isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

[0050] 8. The method of alternative 7, wherein the isolated nucleic acid comprises a sequence encoding NOX2 or a fragment thereof, a sequence encoding antisense NOX2 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding NOX2 or a fragment thereof.

[0051] 9. The method of alternative 7, wherein the isolated nucleic acid comprises a gRNA comprising a sequence complementary to the sequence of a target gene selected from the group consisting of NOX2, CYBA, NCF1, NCF2, NCF4, RAC1, and RAC2.

[0052] 10. The method of alternative 9, wherein the target gene is NOX2.

[0053] 11. The method of any one of alternatives 2-10, wherein reducing the activity of PD-l comprises administering an effective amount of a PD-l inhibitor to the subject. [0054] 12. The method of alternative 11, wherein the PD-l inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, AMP-224, AMP-514, and PDR001.

[0055] 13. The method of alternative 11, wherein the PD-l inhibitor is an anti-PD-

1 antibody or antigen binding fragment thereof selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, and cemiplimab.

[0056] 14. The method of any one of alternatives 2-13, wherein reducing the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell comprises contacting the cell with an isolated nucleic acid selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

[0057] 15. The method of alternative 14, wherein the isolated nucleic acid comprises a sequence encoding PD-l or a fragment thereof, a sequence encoding antisense PD-l or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-l or a fragment thereof.

[0058] 16. The method of any one of alternatives 3-15, wherein reducing the activity of PD-L1 comprises administering an effective amount of a PD-L1 inhibitor to the subject.

[0059] 17. The method of alternative 16, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS-936559, and CK- 301.

[0060] 18. The method of alternative 16, wherein the PD-L1 inhibitor is an anti-

PD-L1 antibody or antigen binding fragment thereof selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0061] 19. The method of any one of alternatives 3-18, wherein reducing the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell comprises contacting the cell with an isolated nucleic acid selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

[0062] 20. The method of alternative 19, wherein the isolated nucleic acid comprises a sequence encoding PD-L1 or a fragment thereof, a sequence encoding antisense PD-L1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-L1 or a fragment thereof.

[0063] 21. The method of any one of alternatives 3-20, wherein an agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered concurrently with an agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, and/or an agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0064] 22. The method of any one of altneratives 3-20, wherein an agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered sequentially with an agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, and/or an agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0065] 23. The method of any one of alternatives 1-22, wherein the cell is a hematopoietic cell.

[0066] 24. The method of any one of alternatives 1 -23, wherein the cell is a myeloid cell.

[0067] 25. The method of any one of alternatives 1-24, wherein the cell is myeloid- derived suppressor cell (MDSC).

[0068] 26. The method of alternative 25, wherein the cell is an intratumoral MDSC.

[0069] 27. The method of alternative 25 or 26, wherein the cell is a peripheral

CD 14⁺HLA-DR ^/low MDSC.

[0070] 28. The method of alternative 25 or 26, wherein the cell is a GRl⁺ MDSC.

[0071] 29. The method of alternative 25 or 26, wherein the cell is a monocytic

MDSC.

[0072] 30. The method of alternative 25 or 26, wherein the cell is a granulocytic

MDSC. [0073] 31. The method of any one of alternatives 1-30, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 10%.

[0074] 32. The method of any one of alternatives 1-31, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 30%.

[0075] 33. The method of any one of alternatives 1-32, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 50%.

[0076] 34. The method of any one of alternatives 1-33, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 80%.

[0077] 35. The method of any one of alternatives 1-34, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 95%.

[0078] 36. The method of any one of alternatives 1-35, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 50%.

[0079] 37. The method of any one of alternatives 1-36, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 100%.

[0080] 38. The method of any one of alternatives 1-37, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 200%.

[0081] 39. The method of any one of alternatives 1-38, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cell with an effector phenotype by at least about 5%.

[0082] 40. The method of any one of alternatives 1-39, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cell with an effector phenotype by at least about 10%.

[0083] 41. The method of any one of alternatives 1-40, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cell with an effector phenotype by at least about 20%.

[0084] 42. The method of any one of alternatives 1-41, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are natural killer (NK) cells by at least about 50%. [0085] 43. The method of any one of alternatives 1-42, wherein the treatment is sufficient to achieve a increase in a fraction of intratumoral lymphocytes which are NK cells by at least about 100%.

[0086] 44. The method of any one of alternatives 1-43, wherein the treatment is sufficient to achieve a increase in a fraction of intratumoral lymphocytes which are NK cells by at least about 200%.

[0087] 45. The method of any one of alternatives 1-44, wherein the treatment is sufficient to achieve a increase in a fraction of intratumoral lymphocytes which are NK cells by at least about 600%.

[0088] 46. The method of any one of alternatives 1-45, wherein the cancer is breast cancer.

[0089] 47. The method of any one of alternatives 1-45, wherein the cancer is colon cancer.

[0090] 48. The method of any one of alternatives 1-47, wherein the subject is mammalian.

[0091] 49. The method of any one of alternatives 1-48, wherein the subject is human.

[0092] 50. Use of a first agent to treat or ameliorate a cancer in a subject, wherein the cancer is a breast cancer or a colon cancer, wherein the first agent reduces the activity of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject.

[0093] 51. The use of the first agent of alternative 50 in combination with a second agent which reduces the activity of programmed cell death protein 1 (PD-l) or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject.

[0094] 52. The use of the first agent of alternative 51 in combination with a third agent which reduces the activity of programmed cell death protein ligand 1 (PD-L1) or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0095] 53. The use of any one of alternatives 50-52, wherein the first agent comprises a NOX2 inhibitor. [0096] 54. The use of alternative 53, wherein the NOX2 inhibitor is selected from the group consisting of histamine dihydrochloride (HDC), histamine, N-methyl-histamine, 4- methyl-histamine, histamine phosphate, histamine diphosphate, GSK2795039, apocynin, GKT136901, GKT137831, ML171, VAS2870, VAS3947, celastrol, ebselen, perhexihne, grmdelic acid, NOX2ds-tat, NOXAlds, fulvene-5, ACD 084, NSC23766, CAS 1177865-17- 6, and CAS 1090893-12-1, and shionogi.

[0097] 55. The use of alternative 54, wherein the NOX2 inhibitor is HDC.

[0098] 56. The use of any one of alternatives 50-55, wherein the first agent comprises an isolated nucleic acid which reduces the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject, wherein the isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

[0099] 57. The use of alternative 56, wherein the isolated nucleic acid comprises a sequence encoding NOX2 or a fragment thereof, a sequence encoding antisense NOX2 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding NOX2 or a fragment thereof.

[0100] 58. The use of alternative 56, wherein the isolated nucleic acid comprises a gRNA comprising a sequence complementary to the sequence of a target gene selected from the group consisting of NOX2, CYBA, NCF1, NCF2, NCF4, RAC1, and RAC2.

[0101] 59. The use of alternative 58, wherein the target gene is NOX2.

[0102] 60. The use of any one of alternatives 51-59, wherein the second agent comprises a PD-l inhibitor.

[0103] 61. The use of alternative 60, wherein the PD-l inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, AMP-224, AMP-514, and PDR001.

[0104] 62. The use of alternative 60, wherein the PD-l inhibitor is an anti-PD-l antibody or antigen binding fragment thereof selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, and cemiplimab.

[0105] 63. The use of any one of alternatives 51-62, wherein the second agent comprises an isolated nucleic acid which reduces the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, wherein the isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

[0106] 64. The use of alternative 63, wherein the isolated nucleic acid comprises a sequence encoding PD-l or a fragment thereof, a sequence encoding antisense PD-l or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-l or a fragment thereof.

[0107] 65. The use of any one of alternatives 52-64, wherein the third agent comprises a PD-L1 inhibitor.

[0108] 66. The use of alternative 65, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS-936559, and CK-301

[0109] 67. The use of alternative 65, wherein the PD-L1 inhibitor is an anti-PD-Ll antibody or antigen binding fragment thereof selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0110] 68. The use of any one of alternatives 52-67, wherein the third agent comprises an isolated nucleic acid which reduces the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject, wherein the isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

[0111] 69. The use of alternative 68, wherein the isolated nucleic acid comprises a sequence encoding PD-L1 or a fragment thereof, a sequence encoding antisense PD-L1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-L1 or a fragment thereof.

[0112] 70. The use of any one of alternatives 52-69, wherein the first agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered concurrently with the second agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, and/or the third agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0113] 71. The use of any one of alternatives 52-69, wherein the first agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered sequentially with the second agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, and/or the third agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

[0114] 72. The use of any one of alternatives 50-71, wherein the cell is a hematopoietic cell.

[0115] 73. The use of any one of alternatives 50-72, wherein the cell is a myeloid cell.

[0116] 74. The use of any one of alternatives 50-73, wherein the cell is myeloid- derived suppressor cell (MDSC).

[0117] 75. The use of alternative 74, wherein the cell is an intratumoral MDSC.

[0118] 76. The use of alternative 73 or 75, wherein the cell is a peripheral

CD 14⁺HLA-DR ^/low MDSC.

[0119] 77. The use of alternative 73 or 75 wherein the cell is a GRl⁺ MDSC.

[0120] 78. The use of alternative 73 or 75, wherein the cell is a monocytic MDSC.

[0121] 79. The use of alternative 73 or 75, wherein the cell is a granulocytic MDSC.

[0122] 80. The use of any one of alternatives 50-79, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 10%.

[0123] 81. The use of any one of alternatives 50-80, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 30%.

[0124] 82. The use of any one of alternatives 50-81, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 50%.

[0125] 83. The use of any one of alternatives 50-82, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 80%.

[0126] 84. The use of any one of alternatives 50-83, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 100%. [0127] 85. The use of any one of alternatives 50-84, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 50%.

[0128] 86. The use of any one of alternatives 50-85, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 100%.

[0129] 87. The use of any one of alternatives 50-86, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 200%.

[0130] 88. The use of any one of alternatives 50-87, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cells with an effector phenotype by at least about 5%.

[0131] 89. The use of any one of alternatives 50-88, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cells with an effector phenotype by at least about 10%.

[0132] 90. The use of any one of alternatives 50-89, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cells with an effector phenotype by at least about 20%.

[0133] 91. The use of any one of alternatives 50-90, wherein the treatment is sufficient to achieve a decrease in a fraction of intratumoral lymphocytes which are natural killer (NK) cells by at least about 50%.

[0134] 92. The use of any one of alternatives 50-91, wherein the treatment is sufficient to achieve a decrease in a fraction of intratumoral lymphocytes which are NK cells by at least about 100%.

[0135] 93. The use of any one of alternatives 50-92, wherein the treatment is sufficient to achieve a decrease in a fraction of intratumoral lymphocytes which are NK cells by at least about 200%.

[0136] 94. The use of any one of alternatives 50-93, wherein the treatment is sufficient to achieve a decrease in a fraction of intratumoral lymphocytes which are NK cells by at least about 600%. [0137] 95. The use of any one of alternatives 50-94, wherein the cancer is breast cancer.

[0138] 96. The use of any one of alternatives 50-94, wherein the cancer is colon cancer.

[0139] 97. The use of any one of alternatives 50-96, wherein the subject is mammalian.

[0140] 98. The use of any one of alternatives 50-97, wherein the subject is human.

[0141] These features, together with other features herein further explained, will become obvious through a reading of the following description of the drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0142] FIG. 1A is a graph of tumor size over time for wild-type mice inoculated with EL-4 cells, and treated with HDC, or untreated (control). The tumor size is normalized against the mean tumor size of untreated mice at the end of each experiment.

[0143] FIG. 1B is a graph of is a graph of tumor size over time for wild-type mice inoculated with 4T1 cells, and treated with HDC, or untreated (control). The tumor size is normalized against the mean tumor size of untreated mice at the end of each experiment.

[0144] FIG.1C is a graph of tumor size over time for wild-type mice depleted of GR1+ cells, inoculated with EL-4 cells, and treated with HDC, or untreated (control). The tumor size is normalized against the mean tumor size of untreated GR1 -depeted mice at the end of each experiment.

[0145] FIG.1D is a graph of tumor size over time for NOX2 knock-out mice, inoculated with EL-4 cells, and treated with HDC, or untreated (control). The tumor size is normalized against the mean tumor size of untreated NOX2-KO mice at the end of each experiment.

[0146] FIG. 2A is a graph depicting accumulation of intratumoral and splenic MDSCs in EL-4-bearing mice. Content of MDSCs was examined in control mice (n=31 for intratumoral MDSCs, n=l9 for splenic MDSCs) and in HDC-treated mice (n=33 for intratumoral MDSCs, n=2l for splenic MDSCs). [0147] FIG. 2B is a graph depicting counts of splenocytes in tumor-free (naive) and control or HDC-treated 4T1 -bearing mice.

[0148] FIG. 2C is a graph depicting a correlation between intratumoral MDSCs and tumor size in control (black) and HDC-treated (grey) EL-4-bearing mice.

[0149] FIG. 2D is a graph depicting a correlation between intratumoral MDSCs and tumor size in control (black) and HDC-treated (grey) 4T1 -bearing mice.

[0150] FIG. 2E is a graph depicting mean D peptide-induced ROS production from leukocytes recovered from tumors of control (solid line, n=l8) and HDC-treated (HDC, dotted line, n=l7) EL-4-bearing mice.

[0151] FIG. 2F is a graph depicting ROS formation (area under the curve) in response to D-peptide by single cell suspensions from tumors, spleens or splenocyte-derived GR1+ cells isolated from control (tumor n=l8, spleen n=20, GR1+ n=9) or HDC-treated (tumor n=l7, spleen n=l9, GR1+ n=l l) EL-4-bearing mice. ROS formation was normalized against the mean ROS formation of tumor-bearing control mice.

[0152] FIG. 2G is a graph depicting mean D peptide- induced ROS production from leukocytes recovered from tumors of control (solid line, n=l5) and HDC-treated (HDC, dotted line, h=14) 4T1 -bearing mice.

[0153] FIG. 2H is a graph depicting ROS formation (area under the curve) in response to D-peptide stimulation by single cell suspensions from tumors or spleens isolated from control (tumor n=l 5, spleen n=l 5) or HDC-treated (tumor n=l5, spleen h=15) 4T1- bearing mice. ROS formation was normalized against the mean ROS formation of tumor bearing control mice.

[0154] FIG. 21 is a graph depicting ROS formation in response to D-peptide from GR1+ (solid line, n=3) and GR1- (dotted line, n=3) cells isolated from tumors of control EL- 4-bearing mice.

[0155] FIG. 2J is a graph depicting ROS formation in response to D-peptide from GR1+ (solid line, n=3) and GR1- (dotted line, n=3) cells isolated from spleens of control EL- 4-bearing mice.

[0156] FIG. 2K is a graph depicting proliferation of OT-l CD8+ T cells and shows representative histograms of CellTraceViolet-stained SIINFEKL-stimulated OT-l CD8+ splenocytes in the absence of GR1+ cells (SIINFEKL, No GR1+) or in the presence of GR1 + cells isolated from spleens of control or HDC-treated EL-4-bearing mice.

[0157] FIG. 2L is a graph depicting the percentage of proliferating CD8+ T cells in the absence of stimuli (n=3), in response to a control peptide (gplOO, n=3) or in response to an OT-l specific peptide (n=3). Where indicated, GR1+ cells isolated from control (n=5) or HDC-treated (n=6) EL-4 bearing mice of GR1 + cells isolated from naive tumor free mice (n=2) were present in the co-culture.

[0158] FIG. 3A is a graph depicting ROS production from cultured monocytes (control, dotted line) and MDSC-like cells (IL-6+GM-CSF, solid line) in response to stimulation with fMLF.

[0159] FIG. 3B is a graph depicting expression of HLA-DR on monocytes after five days of culture in absence of stimuli (Control) and in presence of IL-6 and GM-CSF (n=7).

[0160] FIG. 3C is a graph depicting expression of HLA-DR on monocytes cultured for five days with IL-6 and GM-CSF in the absence or presence of 100mM HDC (n=7).

[0161] FIG. 3D is a graph depicting expression of H2R and gp9lphox on M- MDSCs for a representative patient with acute myeloid leukemia (AML).

[0162] FIG. 3E is a graph depicting the frequency of M-MDSCs before (cycle 1, day 1; C1D1) and after the first treatment cycle (cycle 1, day 21; C1D21) and at the beginning (cycle 3, day 1 ; C3D1) and end (cycle 3, day 21 ; C3D21) of the third treatment cycle. The patients were AML patients in complete remission, that received ten 21 day-cycles of HDC/IL- 2 for relapse prevention.

[0163] FIG. 3F is a graph depicting the absolute counts of M-MDSCs before (C1D1) and after the first treatment cycle (C1D21) and at the beginning (C3D1) and end (C3D21) of the third HDC/IL-2 treatment cycle.

[0164] FIG. 3G is a graph depicting the leukemia-free survival (LFS) of AML patients showing a below median reduction (solid line) or above median reduction (dotted line) of M-MDSC counts during the first HDC/IL-2 treatment cycle (n=48).

[0165] FIG. 3H is a graph depicting the LFS of AML patients showing a below median reduction (solid line) or above median reduction (dotted line) of M-MDSCs from the start of cycle 1 to the end of cycle 3 (n=36). [0166] FIG. 4A is a series of graphs depicting expression of PD-L1 (solid line) in EL-4 and MC-38 cells. The dashed line shows fluorescence of unstained cells.

[0167] FIG. 4B is a graph depicting growth of EL-4 tumors in control (solid line), a-PD-l/a-PD-Ll -treated (dotted line), or HDC/a-PD-l/a-PD-Ll -treated (dashed line) mice.

[0168] FIG. 4C is a graph depicting growth of MC-38 tumors in control (solid line), a-PD-l/a-PD-Ll -treated (dotted line), or HDC/a-PD-l/a-PD-Ll -treated (dashed line) mice.

[0169] FIG. 5A is a graph depicting tumor growth in wild-type mice inoculated with MC-38 cells and treated with HDC, or untreated (control).

[0170] FIG. 5B is a graph depicting in vitro proliferation of EL-4 cells treated with HDC, or untreated.

[0171] FIG. 5C is a graph depicting in vitro effects of HDC on cell cycling of EL- 4 cells at G0/G1 phase.

[0172] FIG. 5D is a graph depicting in vitro effects of HDC on cell cycling of EL- 4 cells at S phase.

[0173] FIG. 5E is a graph depicting in vitro effects of HDC on cell cycling of EL- 4 cells at G2/M phase.

[0174] FIG. 5F is a graph depicting in vitro proliferation of MC-38 cells treated with HDC, or untreated.

[0175] FIG. 5G is a graph depicting effect of previous in vitro exposure of EL-4 cells to HDC on tumor growth in vivo.

[0176] FIG. 6A is a graph depicting percentage GR1+ cells out of live CD45+ cells in mice inoculated with EL-4 cells and treated or untreated with HDC, and depleted or undepleted of GR1+ cells.

[0177] FIG. 6B is a graph depicting tumor size in mice inoculated with EL-4 cells and treated with HDC or not (Ctrl), and depleted or not depleted of GR1+ cells.

[0178] FIG. 7A is a graph depicting percentage of MDSCs out of viable CD45+ cells in tumor or spleen of mice inoculated with 4T1 cells, and treated or not with HDC.

[0179] FIG. 7B is a graph depicting percentage of G- and M-MDSCs among viable CD45+ cells in tumor or spleen of mice inoculated with EL-4 cells, and treated or not with HDC. [0180] FIG. 7C is a graph depicting percentage of G- and M-MDSCs among viable CD45+ cells in tumor or spleen of mice inoculated with 4T1 cells, and treated or not with HDC.

[0181] FIG. 8A is a graph depicting percentage of CD8+ T cells among lymphocytes in tumor or spleen of mice inoculated with EL4 cells, and treated or not with HDC.

[0182] FIG. 8B is a graph depicting percentage of CD8+ T cells among lymphocytes in tumor or spleen of mice inoculated with 4T1 cells, and treated or untreated with HDC.

[0183] FIG. 8C is a graph depicting a correlation between intratumoral CD8+ T cells and intratumoral MDSCs in mice inoculated with EL-4 cells.

[0184] FIG. 8D is a graph depicting a correlation between intratumoral CD8+ T cells and intratumoral MDSCs in mice inoculated with 4T1 cells.

[0185] FIG. 8E is a graph depicting percentage of CD8+ T cells with an effector phenotype among all CD8+ T cells in tumor or spleen of mice inoculated with EL-4 cells, and treated or not with HDC.

[0186] FIG. 8F is a graph depicting percentage CD8+ T cells with an effector phenotype among all CD8+ T cells in tumor or spleen of mice inoculated with 4T1 cells, and treated or not with HDC.

[0187] FIG. 8G is a graph depicting percentage of CD4+ T cells among lymphocytes in tumor or spleen of mice inoculated with EL-4 cells, and treated or not with HDC.

[0188] FIG. 8H is a graph depicting percentage of NK cells among lymphocytes in tumor or spleen of mice inoculated with EL-4 cells, and treated or not with HDC.

[0189] FIG. 81 is a graph depicting percentage of B cells among lymphocytes in tumor or spleen of mice inoculated with EL-4 cells, and treated or not with HDC.

[0190] FIG. 9 depicts a gating strategy to obtain M-MDSCs from peripheral blood mononuclear cells (PBMCs).

[0191] FIG. 10A is a graph depicting percentage of MDSCs among live CD45+ cells in mice inoculated with EL-4 cells and treated or not with HDC in combination with a- PD-l/a-PD-Ll treatment (PD). [0192] FIG. 10B is a graph depicting percentage of CD8+ T cells among lymphocytes in mice inoculated with EL-4 cells and treated or not with HDC in combination with a-PD-l/a-PD-Ll treatment (PD).

[0193] FIG. 10C is a graph depicting percentage of CD8+ T cells with an effector phenotype among all CD8+ T cells, in mice inoculated with EL-4 cells and treated or not with HDC in combination with a-PD-l/a-PD-Ll treatment (PD).

[0194] FIG. 10D is a graph depicting percentage of CD4+ T cells among lymphocytes in mice inoculated with EL-4 cells and treated or not with HDC in combination with a-PD-l/a-PD-Ll treatment (PD).

[0195] FIG. 10E is a graph depicting percentage of NK cells among lymphocytes in mice inoculated with EL-4 cells and treated or not with HDC in combination with a-PD- l/a-PD-Ll treatment (PD).

[0196] FIG. 11A is a graph depicting tumor size in mice inoculated with MC-38 cells and treated or not with HDC in combination with a-PD-l/a-PD-Ll treatment (PD-l+PD- Ll).

[0197] FIG. 11B is a graph depicting percentage of CD8+ T cells among lymphocytes in mice inoculated with MC-38 cells and treated or not with HDC in combination with a-PD-l/a-PD-Ll treatment (PD).

[0198] FIG. 11C is a graph depicting percentage of CD8+ T cells with an effector phenotype among all CD8+ T cells, in mice inoculated with MC-38 cells and treated or untreated with HDC in combination with a-PD-l/a-PD-Ll treatment (PD).

[0199] FIG. 11D is a graph depicting percentage of CD4+ T cells among lymphocytes in mice inoculated with MC-38 cells and treated or not with HDC in combination with a-PD-l/a-PD-Ll treatment (PD).

[0200] FIG. 11E is a graph depicting percentage of NK cells among lymphocytes in mice inoculated with MC-38 cells and treated or not with HDC in combination with a-PD- l/a-PD-Ll treatment (PD).

DETAILED DESCRIPTION

[0201] Some embodiments of the methods and compositions provided herein relate to the amelioration or treatment of a cancer in a subject, such as a colon cancer or a breast cancer. In some, embodiments, a cancer in a subject can be ameliorated or treated by reducing the activity of NADPH oxidase 2 (NOX2) in a cell of the subject. In some embodiments, a cancer in a subject can be ameliorated or treated by reducing the activity of NOX2 in a cell of the subject in combination with reducing the activity of programmed cell death receptor 1 (PD- 1) in a cell of the subject, and/or reducing the activity of programmed cell death receptor ligand 1 (PD-L1) in a cell of the subject.

[0202] Myeloid-derived suppressor cells (MDSCs) are immature monocytes and granulocytes that impede immune-mediated clearance of malignant cells by multiple mechanisms, including the formation of immunosuppressive reactive oxygen species (ROS) via the myeloid cell NOX2. Histamine dihydrochloride (HDC), a NOX2 inhibitor, exerts anti cancer efficacy in several experimental tumor models but the detailed mechanisms are insufficiently understood. To determine effects of HDC on the MDSC compartment, three murine cancer models which accumulate MDSC were studied. These models include an EL-4 lymphoma model, an MC-38 colorectal carcinoma model, and a 4T1 mammary carcinoma model.

[0203] In some embodiments described herein, treatment with HDC delayed in vivo tumor growth in models for lymphoma (EL-4 cells), breast cancer (4T1 cells), and colon cancer (MC38 cells). In some embodiments described herein, in vivo treatment of a colon tumor with a NOX2 inhibitor (NOX2), in combination with a PD-l inhibitor (anti-PD-l antibody) and a PD-L1 inhibitor (anti-PD-Ll antibody) provided a surprising 100% clearance in the subject.

[0204] In some embodiments, treatment with HDC reduce the ROS formation by intratumoral MDSCs. HDC treatment of EL-4 bearing mice reduced the accumulation of intratumoral MDSCs and reduced MDSC-induced suppression of T cells ex vivo. In some embodiments, the use of GR1 -depleted and Nox2 knock out mice supported the hypothesis that the anti-tumor efficacy of HDC required presence of NOX2+ GR1+ cells in vivo. In addition, treatment with HDC enhanced the anti-tumor efficacy of programmed cell death receptor 1 (PD-l) and PD-l ligand checkpoint blockade in EL-4- and MC-38-bearing mice.

[0205] In some embodiments described herein, immunomodulatory effects of a HDC-containing regimen on MDSCs were further analyzed in a phase IV trial (ClinicalTrials.gov; NCT01347996) where patients with acute myeloid leukemia received HDC in conjunction with low-dose IL-2 (HDC/IL-2) for relapse prevention. Peripheral CDl4⁺HLA-DR ^/low MDSCs (M-MDSCs) were reduced during cycles of HDC/IL-2 therapy and a pronounced reduction of M-MDSCs during HDC/IL-2 treatment heralded favorable clinical outcome. In some embodiments, anti-tumor properties of HDC may comprise the targeting of MDSCs.

[0206] Immature myeloid cells (IMCs) accumulate in peripheral organs and in the tumor microenvironment in human and experimental cancer. IMCs normally differentiate into mature myeloid cells such as macrophages, dendritic cells and granulocytes upon migration from the bone marrow (BM) to the periphery. This differentiation is frequently defective in cancer with ensuing expansion of IMCs, presumably as the result of the formation of differentiation-inhibitory factors by malignant cells. IMCs may be further activated to acquire immunosuppressive properties by factors produced by activated T cells and tumor stroma cells. These immature immunosuppressive cells are denoted MDSCs.

[0207] ROS are short-lived compounds that arise from electron transfer across biological membranes to form superoxide anion (O² ) from molecular oxygen. ROS comprise oxygen radicals such as O² and hydroxyl radicals (.OH) along with non-radicals, including hydrogen peroxide. ROS, formed by NOX2, are pivotal mediators in the defense against microorganisms. When released into the extracellular space, ROS may also trigger dysfunction and apoptosis in neighboring cells, including lymphocytes. This mechanism of immunosuppression is exploited by MDSCs, which show increased ROS production by virtue of up-regulated NOX2 activity. In the absence of functional NOX2, MDSCs are less prone to suppress T cells and instead differentiate into macrophages and dendritic cells.

[0208] Human and murine MDSCs occur in granulocytic (G-MDSCs) and monocytic forms (M-MDSCs). Phenotypically, human G-MDSCs share the surface markers of neutrophils but differ in buoyant density. Human M-MDSCs are phenotypically distinguished from monocytes by their expression density of HLA-DR, where monocytes are CD 14⁺HLA-DR^high whereas M-MDSCs are CDl4⁺HLA-DR^/low. Human M-MDSCs as well as G-MDSCs reportedly produce NOX2-derived ROS and suppress T cell functions in a ROS- dependent manner. Murine MDSCs express GR1 and CDl lb, and the murine G-MDSC and M-MDSC subsets are distinguished by their expression of the GR1 epitopes Ly6G and Ly6C. Hence, G-MDSCs are CD1 lb⁺Ly6G⁺Ly6C^low whereas M-MDSCs are CD 1 1 tTLy6G Ly6C^hiah. In mice, the capacity to suppress T cells via ROS production is largely confined to the G- MDSC subset whereas murine M-MDSCs rely on nitric oxide synthase for their immunosuppressive properties.

[0209] The presence of MDSCs is assumed to facilitate the growth and spread of tumors and may also dampen the efficacy of cancer immunotherapies. Several approaches to target MDSCs have been proposed, including blocking the recruitment of MDSCs to the tumor microenvironment, eliminating MDSCs, targeting their immunosuppressive features, or facilitating their maturation. Histamine is a pleiotropic biogenic amine stored in mast cells and basophilic leukocytes. Administration of HDC, a histamine salt that dissociates into histamine in solution, promotes the development of monocyte-derived dendritic cells in vitro and in vivo, and these pro-differentiating properties were mediated by inhibition of NOX2. In addition, mice that lack the histamine-forming histidine decarboxylase, with ensuing histamine deficiency in tissues, are highly susceptible to chemically induced cancer. These histamine- deficient mice were reported to accumulate MDSCs to a higher extent than their wild-type counterparts during the progression of solid tumors.

[0210] Beyond its purported role in myelopoiesis, HDC inhibits ROS production by myeloid cells in a NOX2-dependent manner and thus reduces the immunosuppressive features of various NOX2+ myeloid cells. HDC is approved in Europe, in conjunction with low-dose IL-2, for relapse prevention in patients with acute myeloid leukemia (AML) who have achieved complete remission after chemotherapy. While not wishing to be bound by any one theory, the anti-leukemic action of the HDC component may include HDC targeting NOX2-derived immunosuppressive ROS to protect anti-tumor lymphocytes from ROS- induced inactivation.

[0211] Some embodiments provided herein include effects of HDC on MDSCs in three murine tumor models that include pronounced MDSC accumulation. It has been discovered that the systemic administration of HDC, by targeting NOX2, rendered intratumoral MDSCs less immunosuppressive and delayed the growth of murine EL-4 lymphoma and 4T1 breast cancer and, also, that these properties of HDC translated into improved anti-tumor efficacy of antibodies against PD- 1 and PD-L 1 in EL-4- and MC-38-bearing mice. In addition, the administration of HDC/IL-2 to AML patients in complete remission was associated with reduced counts of M-MDSCs in blood, which predicted reduced risk of leukemic relapse. Embodiments provided herein are consistent with anti-tumor effects of HDC target MDSCs.

Methods of treatment

[0212] Some embodiments of the methods and compositions provided herein include preventing, treating or ameliorating a subject having a cancer, such as a colon cancer, or a breast cancer. As used herein,“subject” can include a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. As used herein,“treat,” “treatment,” or“treating,” can include administering a pharmaceutical composition to a subject for therapeutic purposes, and can include reducing the symptoms or consequences of a disorder, such as preventing the occurrence of a colon or breast tumor, reducing the number of tumor cells of a colon or breast tumor or inhibiting the growth of tumor cells of a colon or breast tumor; and can include curing a disorder, such as eliminating the symptoms of a disorder, such as the elimination of colon or breast tumor cells in a subject. As used herein, “ameliorate”, or“ameliorating” can include a therapeutic effect which relieves, to some extent, one or more of the symptoms of a disorder. As used herein, "prevent," "preventing" and "prevention" can include inhibiting the occurrence of a disorder, and can include preventing a an action that occurs before a subject begins to suffer from the regrowth of the cancer and/or which inhibits or reduces the severity of the cancer. As used herein, an“effective amount” can include an amount, such as a dose, of a therapeutic compound sufficient to treat a disorder. As used herein, reducing the activity of NOX2 can include reducing the activity of NADPH oxidase 2, and/or reducing the activity of a NADPH oxidase holoenzyme which includes the NOX2 protein. In some embodiments, a cell is a hematopoietic cell. Hematopoietic cells include myeloid cells and lymphoid cells. In some embodiments, the cell is a myeloid cell. Examples of myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets. In some embodiments, the cell is a CDl lb+ myeloid cell. In some embodiments, the cell is a lymphoid cell. Examples of lymphoid cells include T cells, B cells, and NK cells.

[0213] Some embodiments include reducing the activity of NOX2 by contacting a cell with an agent that reduces the activity of NOX2 in the cell, such as a NOX2 inhibitor or an isolated nucleic acid which reduces the level of expression of a nucleic acid encoding NOX2 in the cell or the level of expression of NOX2 protein in the cell. Some embodiments include reducing the activity of NOX2 by contacting a cell with a NOX2 inhibitor. In some embodiments, an effective amount of a NOX2 inhibitor can be administered to a subject in need thereof. Examples of NOX2 inhibitors include histamine dihydrochloride (HDC) (CEPLENE), GSK2795039, apocynm, GKT136901, GKT137831, ML171, VAS2870, VAS3947, celastrol, ebselen, perhexiline, grindelic acid, NOX2ds-tat, NOXAlds, fulvene-5, ACD 084, and shionogi. Altenhofer, S. et al,“Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement”, Antioxid Redox Signal. 2015 23: 406- 427; Hirano, K. et al,“Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor”, Antioxid Redox Signal. 2015 23: 358-374, which are each incorporated by reference in its entirety. More examples of NOX2 inhibitors include histamine, N-methyl- histamine, 4-methyl-histamine, histamine phosphate, and histamine diphosphate. In some embodiments, the NOX2 inhibitor is HDC.

[0214] In some embodiments, a NOX2 inhibitor can include RAC1 inhibitors and RAC2 inhibitor, such as NSC23766, CAS 1177865-17-6, and CAS 1090893-12-1. RAC1 and RAC2 can each be associated with NOX2 holoenzyme, and inhibition of RAC 1 or RAC 2 can inhibit NOX2. See e.g., Veluthakal R., etal, (2016)“NSC23766, a Known Inhibitor of Tiaml- Racl Signaling Module, Prevents the Onset of Type 1 Diabetes in the NOD Mouse Model” Cell Physiol Biochem 39:760-767; and Cifuentes-Pagano, E., et al, (2014)“The Quest for Selective Nox Inhibitors and Therapeutics: Challenges, Triumphs and Pitfalls” Antioxid Redox Signal. 20: 2741-2754, which are each incorporated by reference in its entirety. More examples of RAC1 inhibitors are disclosed in Arnst, J.L. et al, (2017)“Discovery and characterization of small molecule Racl inhibitors”, Oncotarget. 8: 34586-34600.

[0215] Some embodiments include reducing the activity of PD-l by contacting a cell with an agent that reduces the activity of PD-l in the cell, such as a PD-l inhibitor or an isolated nucleic acid which reduces the level of expression of a nucleic acid encoding PD-l in the cell or the level of expression of PD-l protein in the cell. Some embodiments include reducing the activity of PD-l by contacting a cell with a PD-l inhibitor. In some embodiments, an effective amount of a PD-l inhibitor can be administered to a subject in need thereof. Examples of PD-l inhibitors include pembrolizumab, nivolumab, pidilizumab, cemiplimab, AMP-224, AMP-514, and PDR001. In some embodiments, the PD-l inhibitor is an anti -PD- 1 antibody or antigen binding fragment thereof, such as pembrolizumab, nivolumab, pidilizumab, or cemiplimab.

[0216] Some embodiments include reducing the activity of PD-L1 by contacting a cell with an agent that reduces the activity of PD-L1 in the cell, such as a PD-L1 inhibitor or an isolated nucleic acid which reduces the level of expression of a nucleic acid encoding PD- Ll in the cell or the level of expression of PD-l protein in the cell. Some embodiments include reducing the activity of PD-L1 by contacting a cell with a PD-L1 inhibitor. In some embodiments, an effective amount of a PD-L1 inhibitor can be administered to a subject in need thereof. Examples of PD-L1 inhibitors include atezolizumab, avelumab, durvalumab, BMS-936559, and CK-301. In some embodiments, the PD-L1 inhibitor is an anti-PD-Ll antibody or an antigen binding fragment thereof, such as atezolizumab, avelumab, and durvalumab.

Reducing expression levels of a protein or nucleic acid

[0217] Some embodiments of the methods and compositions provided herein include reducing the activity of NOX2, PD-l and/or PD-L1 in a cell by reducing the expression level of a nucleic acid encoding NOX2, PD-l and/or PD-L1, or the expression level of a NOX2 protein, PD-l protein and/or PD-L1 protein in the cell. Some embodiments include reducing the expression level of a nucleic acid encoding NOX2, PD-l and/or PD-L1, or the expression level of a NOX2 protein, PD-l protein and/or PD-L1 protein in a cell using either RNA interference, RNA antisense technologies or a CRISPR based system, such as a CRISPR/C<xs9 system.

[0218] Some embodiments include reducing the expression level of a nucleic acid encoding NOX2, PD-l and/or PD-L1, or the expression level of a NOX2 protein, PD-l protein and/or PD-L1 protein in a cell using a CRISPR based system, such as a CRISPR/Ca.v9 system. In some embodiments, a CRISPR (clustered regularly interspaced short palindromic repeats) system can be used to modify a cell to reduce the expression level of a nucleic acid encoding NOX2, PD-l and/or PD-L1, or the expression level of a NOX2 protein, PD-l protein and/or PD-L1 protein in the cell. For example, a cell can be modified such that a target gene, such as NOX2 gene, PD-l gene, or PD-L1 gene can be functionally knocked-out. In some embodiments, a cell can be obtained from a subject. In some embodiments, the cell can be modified by a CRISPR system ex vivo. In some embodiments, the modified cell can be delivered to a subject. Examples of CRISPR systems useful with the methods and compositions provided herein are disclosed in U.S. Pat. App. Pub. 20180201951, U.S. Pat. App. Pub. 20180177893, and U.S. Pat. App. Pub. 20180105834 which are each incorporated by reference in its entirety.

[0219] A CRISPR system includes a microbial nuclease system involved in defense against invading phages and plasmids that provides a form of acquired immunity. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures. Cas9 forms a complex with the 3' end of the sgRNA, and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5' end of the sgRNA sequence and a predefined 20 bp DNA sequence, known as the protospacer. This complex is directed to homologous loci of pathogen DNA via regions encoded within the crRNA, i.e., the protospacers, and protospacer-adjacent motifs (PAMs) within the pathogen genome. The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). By simply exchanging the 20 bp recognition sequence of the expressed sgRNA, the Cas9 nuclease can be directed to new genomic targets. CRISPR spacers are used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.

[0220] Three classes of CRISPR systems (Types I, II and III effector systems) are known. The Type II effector system carries out targeted DNA double-strand break in four sequential steps, using a single effector enzyme, Cas9, to cleave dsDNA. Compared to the Type I and Type III effector systems, which require multiple distinct effectors acting as a complex, the Type II effector system may function in alternative contexts such as eukaryotic cells. The Type II effector system consists of a long pre-crRNA, which is transcribed from the spacer-containing CRISPR locus, the Cas9 protein, and a tracrRNA, which is involved in pre- crRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, thus initiating dsRNA cleavage by endogenous RNase III. This cleavage is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9, forming a Cas9:crRNA-tracrRNA complex.

[0221] The Cas9:crRNA-tracrRNA complex unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a "protospacer" sequence in the target DNA and the remaining spacer sequence in the crRNA. Cas9 mediates cleavage of target DNA if a correct protospacer-adjacent motif (PAM) is also present at the 3' end of the protospacer. For protospacer targeting, the sequence must be immediately followed by the protospacer-adjacent motif (PAM), a short sequence recognized by the Cas9 nuclease that is required for DNA cleavage. Different Type II systems have differing PAM requirements. The Streptococcus pyogenes CRISPR system may have the PAM sequence for this Cas9 (SpCas9) as 5' -NRG-3', where R is either A or G, and characterized the specificity of this system in human cells. A unique capability of the CRISPR/Cas9 system is the straightforward ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs. For example, the S. pyogenes Type II system naturally prefers to use an "NGG" sequence, where "N" can be any nucleotide, but also accepts other PAM sequences, such as "NAG" in engineered systems (Hsu et ah, Nature Biotechnology (2013) doi: l0. l038/nbt.2647). Similarly, the Cas9 derived from Neisseria meningitidis (NmCas9) normally has a native PAM of NNNNGATT, but has activity across a variety of PAMs, including a highly degenerate NNNNGNNN PAM (Esvelt et al. Nature Methods (2013) doi: 10.1038/nmeth.2681 ).

[0222] An engineered form of the Type II effector system of Streptococcus pyogenes was shown to function in human cells for genome engineering. In this system, the Cas9 protein was directed to genomic target sites by a synthetically reconstituted "guide RNA" ("gRNA", also used interchangeably herein as a chimeric single guide RNA ("sgRNA")), which is a crRNA-tracrRNA fusion that obviates the need for RNase III and crRNA processing in general. Provided herein are CRISPR/Cas9-based engineered systems for use in genome editing. The CRISPR/Cas9-based engineered systems may be designed to target any gene, such as a gene encoding NOX2. The CRISPR/Cas9-based systems may include a Cas9 protein or Cas9 fusion protein and at least one gRNA. The Cas9 fusion protein may, for example, include a domain than has a different activity that what is endogenous to Cas9, such as a transactivation domain.

[0223] The CRISPR/Cas9-based system may include a Cas9 protein or a Cas9 fusion protein. Cas9 protein is an endonuclease that cleaves nucleic acid and is encoded by the CRISPR loci and is involved in the Type II CRISPR system. The Cas9 protein may be from any bacterial or archaea species, such as Streptococcus pyogenes. The Cas9 protein may be mutated so that the nuclease activity is inactivated. An inactivated Cas9 protein from Streptococcus pyogenes (iCas9, also referred to as "dCas9") with no endonuclease activity has been recently targeted to genes in bacteria, yeast, and human cells by gRNAs to silence gene expression through steric hindrance. As used herein, "iCas9" and "dCas9" can include a Cas9 protein that has the amino acid substitutions D10A and H840A and has its nuclease activity inactivated.

[0224] The CRISPR/Cas9-based system may include a fusion protein. The fusion protein may comprise two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein and the second polypeptide domain has nuclease activity that is different from the nuclease activity of the Cas9 protein. The fusion protein may include a Cas9 protein or a mutated Cas9 protein, as described above, fused to a second polypeptide domain that has nuclease activity. A nuclease, or a protein having nuclease activity, is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Nucleases are usually further divided into endonucleases and exonucleases, although some of the enzymes may fall in both categories. Well known nucleases are deoxyribonuclease and ribonuclease.

[0225] In some embodiments, a gRNA provides the targeting of the CRISPR/Cas9- based system. The gRNA is a fusion of two noncoding RNAs: a crRNA and a tracrRNA. The gRNA may target any desired DNA sequence, such as a DNA sequence encoding a NOX2 protein, by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system. This duplex, which may include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, acts as a guide for the Cas9 to cleave the target nucleic acid. The "target region", "target sequence" or "protospacer" as used interchangeably herein refers to the region of the target gene to which the CRISPR/Cas9-based system targets. The CRISPR/Cas9-based system may include at least one gRNA, wherein the gRNAs target different DNA sequences. The target DNA sequences may be overlapping. The target sequence or protospacer is followed by a PAM sequence at the 3' end of the protospacer. Different Type II systems have differing PAM requirements. For example, the Streptococcus pyogenes Type II system uses an "NGG" sequence, where "N" can be any nucleotide.

[0226] The gRNA may target any nucleic acid sequence such as an endogenous gene, such as a NOX2 gene, a PD-l gene, or a PD-L1 gene. The CRISPR/Cas9-based system may use gRNA of varying sequences and lengths. The gRNA may comprise a complementary polynucleotide sequence of the target DNA sequence followed by a PAM sequence. The gRNA may comprise a "G" at the 5' end of the complementary polynucleotide sequence. The gRNA may comprise at least a 10 base pair, at least a l l base pair, at least a 12 base pair, at least a 13 base pair, at least a 14 base pair, at least a 15 base pair, at least a 16 base pair, at least a 17 base pair, at least a 18 base pair, at least a 19 base pair, at least a 20 base pair, at least a 21 base pair, at least a 22 base pair, at least a 23 base pair, at least a 24 base pair, at least a 25 base pair, at least a 30 base pair, or at least a 35 base pair complementary polynucleotide sequence of the target DNA sequence followed by a PAM sequence. The PAM sequence may be "NGG", where "N" can be any nucleotide. The gRNA may target at least one of the promoter region, the enhancer region or the transcribed region of the target gene.

[0227] In some embodiments, a target gene can include the NOX2 gene also known as the CYBB gene which encodes a NOX2 protein, also known as cytochrome b-245 beta chain protein. In some embodiments, a target gene can encode a polypeptide that binds to or is associated with the NOX2 protein in vivo. Examples of such target genes include the CYBA gene which encodes a p22phox protein, the NCF1 gene which encodes neutrophil cytosolic factor 1 protein, the NCF2 gene which encodes a neutrophil cytosolic factor 2 protein, the NCF4 gene which encodes a neutrophil cytosolic factor 4 protein, the RAC1 gene which encodes a Racl protein, and the RAC2 gene which encodes a Rac2 protein.

[0228] In some embodiments, a target gene can include the PD-l gene. In some embodiments, a target gene can include the PD-L1 gene. Accession numbers for example human genomic DNA sequences that contain certain target genes and are useful to generate targeted nucleic acids for use in a CRISPR system to reduce activity of a NOX2 protein, PD- 1 and/or PD-L1 in a cell are listed in TABLE 1.

TABLE 1

[0229] Adeno-associated virus (AAV) vectors may be used to deliver CRISPRs to the cell using various construct configurations. For example, AAV may deliver Cas9 and gRNA expression cassettes on separate vectors. Alternatively, if the small Cas9 proteins, derived from species such as Staphylococcus aureus or Neisseria meningitidis, are used then both the Cas9 and up to two gRNA expression cassettes may be combined in a single AAV vector within the 4.7 kb packaging limit.

[0230] In some embodiments, the delivery of the CRISPR/Cas9-based system may be the transfection or electroporation of the CRISPR/Cas9-based system as a nucleic acid molecule that is expressed in the cell and delivered to the surface of the cell. The nucleic acid molecules may be electroporated using BioRad Gene Pulser Xcell or Amaxa Nucleofector lib devices. Several different buffers may be used, including BioRad electroporation solution, Sigma phosphate-buffered saline product #D8537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (N. V.). Transfections may include a transfection reagent, such as Lipofectamine 2000. Upon delivery of the CRISPR/Cas9 system to the tissue, and thereupon the vector into the cells of the mammal, the transfected cells will express the CRISPR/Cas9- based system and/or a site-specific nuclease. In some embodiments, a modified AAV vector can be capable of delivering and expressing the site-specific nuclease in the cell of a subject. For example, the modified AAV vector may be an AAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy 23:635-646). The modified AAV vector may be based on one or more of several capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. The modified AAV vector may be based on AAV2 pseudotype with alternative muscle-tropic AAV capsids, such as AAV2/1, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2.5 and AAV/SASTG vectors that efficiently transduce skeletal muscle or cardiac muscle by systemic and local delivery (Seto et al. Current Gene Therapy (2012) 12: 139-151). In some embodiments, a cell can be modified ex vivo, and the modified cell can be delivered to a subject. In some embodiments, modified cells may be injected or implanted into a subject, used exogenously, or developed into tissue engineered constructs.

[0231] Some embodiments include reducing the expression level of a nucleic acid encoding NOX2, PD-l and/or PD-L1, or the expression level of a NOX2 protein, PD-l protein and/or PD-Llprotein in a cell by RNA interference and/or antisense technologies. RNA interference is an efficient process whereby double-stranded RNA (dsRNA), also referred to as siRNAs (small interfering RNAs) or ds siRNAs (double-stranded small interfering RNAs), induces the sequence-specific degradation of targeted mRNA in animal or plant cells (Hutvagner, G. et al. (2002) Curr. Opin. Genet. Dev. 12:225-232); Sharp, P. A. (2001) Genes Dev. 15:485-490). RNA interference can be triggered by various molecules, including 21- nucleotide duplexes of siRNA (Chiu, Y.-L. et al. (2002) Mol. Cell. 10:549-561. Clackson, T. et al. (1991) Nature 352:624-628.; Elbashir, S. M. et al. (2001) Nature 411 :494-498), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which can be expressed in vivo using DNA templates with RNA polymerase III promoters (Zheng, B. J. (2004) Antivir. Ther. 9:365-374; Paddison, P. J. et al. (2002) Genes Dev. 16:948-958; Lee, N. S. et al. (2002) Nature Biotechnol. 20:500-505; Paul, C. P. et al. (2002) Nature Biotechnol. 20:505-508; Tuschl, T. (2002) Nature Biotechnol. 20:446-448; Yu, J.-Y. et al. (2002) Proc. Natl. Acad. Sci. USA 99(9):6047-6052; McManus, M. T. et al. (2002) RNA 8:842-850; Sui, G. et al. (2002) Proc. Natl. Acad. Sci. USA 99(6):55l5-5520, each of which are incorporated herein by reference in their entirety).

[0232] In some embodiments, reducing the expression level of a nucleic acid encoding NOX2, PD-l and/or PD-L1 or the expression level of NOX2, PD-l protein and/or PD-L1 protein in a cell can include contacting the cell with an isolated nucleic acid selected from the group consisting of a guide RNA (gRNA), small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme. In some embodiments, the isolated nucleic acid comprises a sequence encoding NOX2 or a fragment thereof, a sequence encoding antisense NOX2 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding NOX2 or a fragment thereof. In some embodiments, the isolated nucleic acid comprises a sequence encoding PD-l or a fragment thereof, a sequence encoding antisense PD-l or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-l or a fragment thereof. In some embodiments, the isolated nucleic acid comprises a sequence encoding PD-L1 or a fragment thereof, a sequence encoding antisense PD-L1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-L1 or a fragment thereof.

[0233] A fragment of a polynucleotide sequence can include any nucleotide fragment having, for example, at least about 5 successive nucleotides, at least about 12 successive nucleotides, at least about 15 successive nucleotides, at least about 18 successive nucleotides, or at least about 20 successive nucleotides of the sequence from which it is derived. An upper limit for a fragment can include, for example, the total number of nucleotides in a full-length sequence encoding a particular polypeptide. A fragment of a polypeptide sequence can include any polypeptide fragment having, for example, at least about 5 successive residues, at least about 12 successive residues, at least about 15 successive residues, at least about 18 successive residues, or at least about 20 successive residues of the sequence from which it is derived. An upper limit for a fragment can include, for example, the total number of residues in a full-length sequence of a particular polypeptide.

[0234] Some embodiments include reducing the expression level of a nucleic acid encoding NOX2, PD-l, and/or PD-L1, or the expression level of a NOX2 protein, PD-l protein, and/or PD-L1 protein in a cell by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage within a range of any two of the foregoing percentages.

[0235] As used herein,“antisense polynucleotide” can include a nucleic acid that binds to a target nucleic acid, such as a RNA or DNA. An antisense polynucleotide can upregulate or downregulate expression and/or function of a target nucleic acid. An antisense polynucleotide can include any exogenous nucleic acid useful in therapeutic and/or diagnostic methods. Antisense polynucleotides can include antisense RNA or DNA molecules, micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

[0236] As used herein,“short hairpin RNA” (“shRNA”), also known as“small hairpin RNAs”, refers to an RNA (or RNA analog) including a first portion and a second portion, having sufficient complementarity to anneal or hybridize to form a duplex or double- stranded stem portion. The two portions need not be fully or perfectly complementary. The first and second“stem” portions are connected by a portion having a sequence that has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as a "loop" portion in the shRNA molecule. shRNA molecules are processed to generate siRNAs. shRNAs can also include one or more bulges, such as extra nucleotides that create a small nucleotide“loop” in a portion of the stem, for example a one-, two- or three-nucleotide loop. The stem portions can be the same length, or one portion can include an overhang of, for example, 1-5 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. Such Us are notably encoded by thymidines (Ts) in the shRNA-encoding DNA which signal the termination of transcription.

[0237] In some embodiments, a shRNA can include a portion of the duplex stem is a nucleic acid sequence that is complementary (e.g., perfectly complementary or substantially complementary, e.g., anti-sense) to a NOX2 target sequence, a PD-l target sequence, and/or a PD-L1 target sequence. In some embodiments, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA, such as a NOX2, PD-l or PD-L1 mRNA sequence, to mediate degradation or cleavage of said target RNA via RNA interference (RNAi). Alternatively, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA (e.g., such as a NOX2, PD-l or PD-L1 mRNA sequence) to inhibit translation of said target RNA via RNA interference (RNAi). Thus, engineered RNA precursors include a duplex stem with two portions and a loop connecting the two stem portions. The antisense portion can be on the 5' or 3' end of the stem. The stem portions of a shRNA are preferably about 15 to about 50 nucleotides in length. Preferably the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In some embodiments, the length of the stem portions should be

21 nucleotides or greater. When used in mammalian cells, the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses like the interferon pathway.

[0238] As used herein, the term“small interfering RNA” (“siRNA”), also referred to in the art as“short interfering RNAs”, refers to an RNA or RNA analog comprising between about 10-50 nucleotides or nucleotide analogs which is capable of directing or mediating RNA interference. Preferably, an siRNA comprises between about 15-30 nucleotides or nucleotide analogs, between about 16-25 nucleotides or nucleotide analogs, between about 18-23 nucleotides or nucleotide analogs, or between about 19-22 nucleotides or nucleotide analogs, such as 19, 20, 21 or 22 nucleotides or nucleotide analogs. The term“short” siRNA can refer to a siRNA comprising about 21 nucleotides or nucleotide analogs, for example, 19, 20, 21 or

22 nucleotides. The term“long” siRNA can refer to a siRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, such as enzymatic processing to a short siRNA.

[0239] As used herein,“microRNA” (“miRNA”), also referred to in the art as “small temporal RNAs” (“stRNAs”), can refer to a small (10-50 nucleotide) RNA or nucleotide analogs which can be genetically encoded, such as by viral, mammalian, or plant genomes, or synthetically produced and is capable of directing or mediating RNA silencing. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein- coding or non-coding. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products. The mature miRNA is incorporated into an RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA.

[0240] In some embodiments, an siRNA is a duplex consisting of a sense strand and complementary antisense strand, the antisense strand having sufficient complementary to a NOX2 sequence, a PD-l sequence, or a PD-L1 sequence, to mediate RNAi. In some embodiments, an miRNA is optionally a duplex consisting of a 3' strand and complementary 5' strand, the 5' strand having sufficient complementary to a NOX2 sequence to mediate RNAi. In some embodiments, the siRNA or miRNA molecule has a length from about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). In some embodiments, the siRNA or miRNA molecule has a length from about 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is sufficiently complementary to a target region. In some embodiments, the strands are aligned such that there are at least 1, 2, or 3 bases at the end of the strands which do not align (i.e., for which no complementary bases occur in the opposing strand) such that an overhang of 1 , 2 or 3 residues occurs at one or both ends of the duplex when strands are annealed. In some embodiments, the siRNA molecule has a length from about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). In some embodiments, the siRNA or miRNA molecule has a length from about 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially complementary to a target sequence, and the other strand is identical or substantially identical to the first strand. siRNAs or miRNAs can be designed by using any method known in the art. The siRNAs or miRNAs provided herein can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from, e.g., shRNA. The dsRNA molecules can be designed using any method known in the art.

[0241] In some embodiments, miRNAs can regulate gene expression at the post transcriptional or translational level. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotides precursor RNA stem-loop, probably by Dicer, an RNase Ill-type enzyme, or a homolog thereof. By substituting the stem sequences of the miRNA precursor with miRNA sequence complementary to the target mRNA, a vector construct that expresses the novel miRNA can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells ( See e.g., Zheng, B. J. (2004) Antivir. Ther. 9:365-374). When expressed by DNA vectors containing polymerase PI promoters, micro- RNA designed hairpins can silence gene expression, such as NOX2 expression.

[0242] An example method for designing dsRNA molecules is provided in the pSUPER RNAi SYSTEM™ (OligoEngine, Seattle, WA). The system provides inducible expression of a siRNA in a transfected cell. To effect silencing of a specific gene, a pSUPERIOR vector is used in concert with a pair of custom oligonucleotides that include a unique l9-nt sequence derived from the mRNA transcript of the gene targeted for suppression (the“N- 19 target sequence”). The N-19 target sequence corresponds to the sense strand of the pSUPER-generated siRNA, which in turn corresponds to a l9-nt sequence within the mRNA. In the mechanism of RNAi, the antisense strand of the siRNA duplex hybridizes to this region of the mRNA to mediate cleavage of the molecule. These forward and reverse oligonucleotides are annealed and cloned into the vector so that the desired siRNA duplex can be generated. The sequence of the forward oligonucleotide includes the unique N- 19 target in both sense and antisense orientation, separated by a 9-nt spacer sequence. The resulting transcript of the recombinant vector is predicted to fold back on itself to form a 19-base pair stem-loop structure. The stem-loop precursor transcript is quickly cleaved in the cell to produce a functional siRNA (T.R. Brummelkamp, et al , Science 296, 550 (2002)). More example methods are provided in Taxman D.J. et al. (2006) BMC Biotechnol. 6:7; and McIntyre G. J. et al. (2006) BMC Biotechnol. 6: 1, each of which is incorporated by reference in its entirety.

[0243] As used herein,“ribozyme” can include a catalytic RNA molecule that cleaves RNA in a sequence specific manner. Ribozymes that cleave themselves are known as cis- acting ribozymes, while ribozymes that cleave other RNA molecules are known as trans acting ribozymes. The term "cv.s-acting ribozyme sequence" as used herein refers to the sequence of an RNA molecule that has the ability to cleave the RNA molecule containing the cv.s-acting ribozyme sequence. A cv.s-acting ribozyme sequence can contain any sequence provided it has the ability to cleave the RNA molecule containing the cv.s-acting ribozyme sequence. For example, a cv.s-acting ribozyme sequence can have a sequence from a hammerhead, axhead, or hairpin ribozyme. In addition, a cv.s-acting ribozyme sequence can have a sequence from a hammerhead, axhead, or hairpin ribozyme that is modified to have either slow cleavage activity or enhanced cleavage activity. For example, nucleotide substitutions can be made to modify cleavage activity (Doudna and Cech, Nature, 418:222- 228 (2002)). Examples of ribozyme sequences that can be used with the methods and compositions described herein include those described in U.S. Patent No. 6,271,359, and U.S. Patent No. 5,824,519, incorporated by reference in their entireties. One example method for preparing a ribozyme is to synthesize chemically an oligodeoxyribonucleotide with a ribozyme catalytic domain (approximately 20 nucleotides) flanked by sequences that hybridize to the target mRNA. The oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers. The amplified product is cloned into a eukaryotic expression vector. A ribozyme can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ohkawa et ah, Nucleic Acids Symp. Ser., 27: 15-6 (1992); Taira et ah, Nucleic Acids Res., 19: 5125-30 (1991); Ventura et ah, Nucleic Acids Res., 21, 3249-55 (1993).

[0244] In some embodiments, an isolated nucleic acid can include an antisense nucleic acid sequence selected such that it is complementary to the entirety of NOX2 or to a portion of NOX2. In some embodiments, an isolated nucleic acid can include an antisense nucleic acid sequence selected such that it is complementary to the entirety of PD-l or to a portion of PD-l . In some embodiments, an isolated nucleic acid can include an antisense nucleic acid sequence selected such that it is complementary to the entirety of PD-L1 or to a portion of PD-L 1. In some embodiments, a portion can refer to at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, and at least about 80%, at least about 85%, at least about 90%, at least about 95%, or any portion within a range of any two of the foregoing percentages. In some embodiments, a portion can refer up to 100%. Example mRNA sequences of human NOX2 (SEQ ID NO:0l), human PD-l (SEQ ID NO:02), and human PD-L1 (SEQ ID NO:03) are shown in TABLE 2.

TABLE 2

[0245] In some embodiments, an antisense oligonucleotide can have a length of at least about 5 nucleotides, at least about 7 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucleotides. An antisense nucleic acid of disclosed herein can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation, namely, RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest. The antisense nucleic acid molecules can be administered to a subject, such as systemically or locally by direct injection at a tissue site, or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding NOX2, PD-l and/or PD-L1 to thereby inhibit its expression. Alternatively, antisense nucleic acid molecules can be modified to target particular cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to particular cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter can be used.

[0246] In some embodiments, antisense oligonucleotide include a-anomeric nucleic acid molecules. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier, C. et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide, or a chimeric RNA-DNA analogue (Inoue, H. et al. (1987) Nucleic Acids Res. 15:6131-6148; Inoue, H. et al. (l987a) FEBS Lett. 215:327-330).

[0247] In some embodiments, an isolated nucleic acid can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability, and/or half-life. The conjugation can be accomplished by methods known in the art, such as the methods of Lambert, G. et al. (2001) Drug Deliv. Rev. 47(1): 99-112 (describes nucleic acids loaded to polyalky lcyanoacrylate (PACA) nanoparticles); Fattal et al. (1998) J. Control Release 53(1-3): 137-43 (describes nucleic acids bound to nanoparticles); Schwab et al. (1994) Ann. Oncol. 5 Suppl. 4:55-58 (describes nucleic acids linked to intercalating agents, hydrophobic groups, poly cations or PACA nanoparticles); and Godard, G. etal. (1995) Eur. J. Biochem. 232(2):404-l0 (describes nucleic acids linked to nanoparticles). Because RNAi is believed to progress via at least one single stranded RNA intermediate, the skilled artisan will appreciate that ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also be designed as described herein and utilized according to the claimed methodologies.

[0248] Some embodiments reducing the expression level of a nucleic acid encoding NOX2, PD-l, and/or PD-L1, or the expression level of a NOX2 protein, PD-l protein, and/or PD-L1 protein in a cell can include delivering an isolated nucleic acid, such as an siRNA to a cell by methods known in the art, including cationic liposome transfection and electroporation. In some embodiments, an siRNA can show short term persistence of a silencing effect which may be beneficial in certain embodiments. To obtain longer term suppression of expression for targeted genes, such as NOX2, PD-l or PD-L1, and to facilitate delivery under certain circumstances, one or more siRNA duplexes, such as a ds-siRNA, can be expressed within cells from recombinant DNA constructs. Such methods for expressing siRNA duplexes within cells from recombinant DNA constructs to allow longer-term target gene suppression in cells are known in the art, including mammalian Pol III promoter systems (e.g., Hl or U6/snRNA promoter systems (Tuschl, T. (2002) Nature Biotechnol. 20:446-448) capable of expressing functional double-stranded siRNAs; (Lee, N. S. et al. (2002) Nature Biotechnol. 20:500-505; Miyagishi, M. and Taira, K. (2002) Nature Biotechnol. 20:497-500; Paul, C. P. et al. (2002) Nature Biotechnol. 20:505-508; Yu, J.-Y. etal. (2002) Proc. Natl. Acad. Sci. USA 99(9): 6047- 6052; Sui, G. et al. (2002) Proc. Natl. Acad. Sci. USA 99(6): 5515-5520). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence. The siRNA is complementary to the sequence of the target gene in 5'-3' and 3'-5' orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by an Hl or U6 snRNA promoter can be expressed in cells, and can inhibit target gene expression. Constructs containing siRNA sequence(s) under the control of a T7 promoter also make functional siRNAs when co-transfected into the cells with a vector expressing T7 RNA polymerase (Jacque J.-M. et al. (2002) Nature 418:435-438). A single construct may contain multiple sequences coding for siRNAs, such as multiple regions of the NOX2 gene, PD-l gene, or PD-L1 gene, such as a nucleic acid encoding the NOX2 mRNA, PD-l mRNA, and/or PD-L1 mRNA. and can be driven, for example, by separate Pol III promoter sites.

[0249] Some embodiments reducing the expression level of a nucleic acid encoding NOX2, PD-l, and/or PD-L1 or the expression level of a NOX2 protein, PD-l protein, and/or PD-L1 protein in a cell can include viral-mediated delivery of certain isolated nucleic acids to a cell. In some such embodiments, specific silencing of targeted genes through expression of certain nucleic acids, such as an siRNA by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al. (2002) Nature Biotechnol. 20(10): 1006-10). Injection of recombinant adenovirus vectors into transgenic mice expressing the target genes of the siRNA results in in vivo reduction of target gene expression. In adult mice, efficient delivery of siRNA can be accomplished by the "high-pressure" delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA containing solution into animal via the tail vein (Lewis, D. L. (2002) Nature Genetics 32: 107-108). Nanoparticles, liposomes and other cationic lipid molecules can also be used to deliver siRNA into animals. A gel-based agarose/liposome/siRNA formulation is also available (Jiamg, M. et al. (2004) Oligonucleotides l4(4):239-48).

Combination therapies

[0250] Some embodiments of the methods and compositions provided herein include contacting a cell and/or administering to a subject an agent which reduces the activity of NOX2 in a cell, such as a NOX2 inhibitor or an isolated nucleic acid which reduces the activity of NOX2 in a cell, in combination with an additional therapeutic agent. As used herein, administering in combination can include administering two or more agents to a subject, such as a NOX2 inhibitor or isolated nucleic acid, and an additional therapeutic agent, such that the two or more agents may be found in the subject’s bloodstream at the same time, regardless of when or how they are actually administered. In some embodiments, the agents are administered simultaneously. In some such embodiments, administration in combination is accomplished by combining the agents in a single dosage form. When combining the agents in a single dosage form, they may be physically mixed, such as by co-dissolution or dry mixing, or may form an adduct or be covalently linked such that they split into the two or more active ingredients upon administration to the subject. In some embodiments, the agents are administered sequentially. In some embodiments, the agents are administered through the same route, such as orally. In some embodiments, the agents are administered through different routes, such as one being administered orally and another being administered i.v.

[0251] In some embodiments, an additional therapeutic agent can include an agent which reduces the activity of PD-l in a cell, such as a PD-l inhibitor, or an isolated nucleic acid which reduces the activity of PD-l in a cell. Examples of PD-l inhibitors include pembrolizumab, nivolumab, pidilizumab, cemiplimab, AMP-224, AMP-514, and PDROOl . In some embodiments, the PD-l inhibitor is an anti-PD-l antibody or antigen binding fragment thereof, such as pembrolizumab, nivolumab, pidilizumab, or cemiplimab. In some embodiments, an additional therapeutic agent can include an agent to reduce the activity of PD-L1 in a cell, such as a PD-L1 inhibitor, or an isolated nucleic acid which reduces the activity of PD-L1 in a cell. Examples of PD-L1 inhibitors include atezolizumab, avelumab, durvalumab, BMS-936559, and CK-301. In some embodiments, the PD-L1 inhibitor is an anti- PD-L1 antibody or antigen binding fragment thereof, such as atezolizumab, avelumab, and durvalumab. In some embodiments, an agent which reduces the activity of NOX2 in a cell can be administered to a subject in combination with both an agent which reduces the activity of PD-l in a cell, and an agent which reduces the activity of PD-L1 in a cell.

[0252] In some embodiments, administration to a subject of an agent which reduces the activity of NOX2 in a cell, such as a NOX2 inhibitor or an isolated nucleic acid which reduces the activity of NOX2 in a cell is sufficient to achieve a reduction in a volume of a tumor of the cancer of a subject. In some embodiments, the reduction in a volume of a tumor can be by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or a percentage within a range of any two of the foregoing percentages.

[0253] In some embodiments, administration to a subject an agent which reduces the activity of NOX2 in a cell, such as a NOX2 inhibitor or an isolated nucleic acid which reduces the activity of NOX2 in a cell, in combination with an additional therapeutic agent, such as an agent which reduces the activity of PD-l in a cell and/or an agent which reduces the activity of PD-L1 in a cell, is sufficient to achieve a reduction in a volume of a tumor of the cancer of a subject. In some embodiments, the reduction in a volume of a tumor can be by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or a percentage within a range of any two of the foregoing percentages.

[0254] In some embodiments, administration to a subject an agent which reduces the activity of NOX2 in a cell, such as a NOX2 inhibitor or an isolated nucleic acid which reduces the activity of NOX2 in a cell, in combination with an additional therapeutic agent, such as an agent which reduces the activity of PD-l in a cell and/or an agent which reduces the activity of PD-L1 in a cell, is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells. In some embodiments, the increase in a fraction of intratumoral lymphocytes which are CD8+ T cells can be by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200%, or a percentage increase within a range of any two of the foregoing percentages.

[0255] In some embodiments, administration to a subject an agent which reduces the activity of NOX2 in a cell, such as a NOX2 inhibitor or an isolated nucleic acid which reduces the activity of NOX2 in a cell, in combination with an additional therapeutic agent, such as an agent which reduces the activity of PD-l in a cell and/or an agent which reduces the activity of PD-L1 in a cell, is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cell with an effector phenotype. In some embodiments, the increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cells with an effector phenotype can be by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200%, or a percentage increase within a range of any two of the foregoing percentages.

[0256] In some embodiments, administration to a subject an agent which reduces the activity of NOX2 in a cell, such as a NOX2 inhibitor or an isolated nucleic acid which reduces the activity of NOX2 in a cell, in combination with an additional therapeutic agent, such as an agent which reduces the activity of PD-l in a cell and/or an agent which reduces the activity of PD-L1 in a cell, is sufficient to achieve a increase in a fraction of intratumoral lymphocytes which are natural killer (NK) cells. In some embodiments, the increase in a fraction of intratumoral lymphocytes which are NK cells can be by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200%, 600%, or a percentage increase within a range of any two of the foregoing percentages.

Pharmaceutical compositions and formulations

[0257] Some embodiments of the methods and compositions provided herein include pharmaceutical compositions, and administration of such compositions. In some embodiments, a pharmaceutical composition can include an agent which reduces the activity of NOX2 in a cell, such as a NOX2 inhibitor or an isolated nucleic acid which reduces the activity of NOX2 in a cell; an agent which reduces the activity of PD-l in a cell, such as a PD- 1 inhibitor, or an isolated nucleic acid which reduces the activity of PD-l in a cell; and/or an agent to reduce the activity of PD-L1 in a cell, such as a PD-L1 inhibitor, or an isolated nucleic acid which reduces the activity of PD-L1 in a cell. In some embodiments, a pharmaceutical composition can include an agent, such as a NOX2 inhibitor, a PD-l inhibitor, and/or a PD-L1 inhibitor, or an isolated nucleic acid which can reduce the activity of NOX2, PD-l or PD-L1 in a cell, and a pharmaceutically acceptable excipient. As used herein, a“pharmaceutically acceptable” can include a carrier, diluent or excipient that does not abrogate the biological activity and properties of a NOX2 inhibitor, a PD-l inhibitor, and/or a PD-L1 inhibitor, or an isolated nucleic acid which can reduce the activity of NOX2, PD-l or PD-L1 in a cell. Standard pharmaceutical formulation techniques can be used, such as those disclosed in Remington's The Science and Practice of Pharmacy, 2lst Ed., Lippincott Williams & Wilkins (2005), incorporated by reference in its entirety.

[0258] In some embodiments, a pharmaceutical composition can be administered to a subject by any of the accepted modes of administration for agents that serve similar utilities including, but not limited to, orally, subcutaneously, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. [0259] In some embodiments, a pharmaceutical composition comprising a NOX2 inhibitor, a PD-l inhibitor, and/or a PD-L1 inhibitor, or an isolated nucleic acid which can reduce the activity of NOX2, PD-l or PD-L1 in a cell, can be administered at a therapeutically effective dosage, such as a dosage sufficient to provide treatment for a disorder. The amount of active compound administered will, of course, be dependent on the subject and disease state being treated, the severity of the disorder, the manner and schedule of administration and the judgment of the prescribing physician. The actual dose of the active compounds, such as NOX2 inhibitors, PD-l inhibitors, and/or PD-L1 inhibitors, or an isolated nucleic acid which can reduce the activity of NOX2, PD-l or PD-L1 in a cell, can depend on the specific compound, and on the condition to be treated; the selection of the appropriate dose is well within the knowledge of the skilled artisan.

[0260] In some embodiments, the pharmaceutical composition is administered subcutaneously. Solutions of an active compound, such as a NOX2 inhibitor, a PD-l inhibitor, and/or a PD-L1 inhibitor, as a free acid or a pharmaceutically-acceptable salt may be administered in water with or without a surfactant such as hydroxypropyl cellulose. Dispersions are also contemplated such as those utilizing glycerol, liquid polyethylene glycols and mixtures thereof and oils. Antimicrobial compounds may also be added to the preparations. Injectable preparations may include sterile aqueous solutions or dispersions and powders which may be diluted or suspended in a sterile environment prior to use. Carriers such as solvents dispersion media containing, e.g., water, ethanol polyols, vegetable oils and the like, may also be added. Coatings such as lecithin and surfactants may be utilized to maintain the proper fluidity of the composition. Isotonic agents such as sugars or sodium chloride may also be added as well as products intended for the delay of absorption of the active compounds such as aluminum monostearate and gelatin. Sterile injectable solutions are prepared as is known in the art and filtered prior to storage and/or administration. Sterile powders may be vacuum dried freeze dried from a solution or suspension containing them. In some embodiments, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intra-muscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In some embodiments, the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration. In some embodiments, the pharmaceutical compositions are administered intra- arterially and are thus formulated in a form suitable for intra-arterial administration. In some embodiments, the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra-muscular administration.

[0261] Proper formulation is dependent upon the route of administration selected. For injection, the agents of the compounds may be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0262] For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers known in the art. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained using a solid excipient in admixture with the active ingredient (agent), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include: fillers such as sugars, comprising lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0263] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agents.

[0264] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0265] For administration intranasally or by inhalation, the compounds, such as NOX2 inhibitors, PD-l inhibitors, and/or PD-L1 inhibitors, for use according to the present disclosure may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, such as carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0266] In some embodiments, agents such as NOX2 inhibitors, PD-l inhibitors, and/or PD-L1 inhibitors, or an isolated nucleic acid which can reduce the activity of NOX2, PD-l or PD-L1 in a cell, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit- dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0267] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds, such as NOX2 inhibitors, PD-l inhibitors, and/or PD-L1 inhibitors, or an isolated nucleic acid which can reduce the activity of NOX2, PD-l or PD-L1 in a cell, in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0268] In addition to the formulations described herein, agents, such as NOX2 inhibitors, PD-l inhibitors, and/or PD-L1 inhibitors, an isolated nucleic acid which can reduce the activity of NOX2, PD-l or PD-L1 in a cell, may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation, such as subcutaneously or intramuscularly, or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. A pharmaceutical carrier for hydrophobic compounds is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the non-polar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD: 5 W) contains VPD diluted 1 : 1 with a 5% dextrose in water solution. This co solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. The proportions of a co-solvent system may be suitably varied without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity non-polar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.

[0269] In some embodiments, delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity due to the toxic nature of DMSO. Additionally, the compounds, such as NOX2 inhibitors, may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained- release materials have been established and are known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

[0270] The pharmaceutically acceptable formulations can contain a compound, or a salt or solvate thereof, in an amount of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg. Additionally, the pharmaceutically acceptable formulations may contain a compound such as NOX2 inhibitor, a PD-l inhibitor, and/or a PD-L1 inhibitor, or a salt or solvate thereof, in an amount from about 0.5 w/w % to about 95 w/w %, or from about 1 w/w % to about 95 w/w %, or from about 1 w/w % to about 75 w/w %, or from about 5 w/w % to about 75 w/w %, or from about 10 w/w % to about 75 w/w %, or from about 10 w/w % to about 50 w/w %.

Kits

[0271] Some embodiments of the methods and compositions provided herein include kits comprising an agent for reducing activity of NOX2 in a cell such as a NOX2 inhibitor and/or an isolated nucleic acid which can reduce the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell. In some embodiments, the NOX2 inhibitor can include histamine dihydrochloride (HDC), histamine, N-methyl- histamine, 4-methyl-histamine, histamine phosphate, histamine diphosphate, GSK2795039, apocynm, GKT136901, GKT137831, ML171, VAS2870, VAS3947, celastrol, ebselen, perhexiline, grindelic acid, NOX2ds-tat, NOXAlds, fulvene-5, ACD 084, NSC23766, CAS 1177865-17-6, and CAS 1090893-12-1, and shionogi. In some embodiments, the NOX2 inhibitor is HDC.

[0272] Some embodiments also include an agent for reducing activity of PD-l in a cell such as a PD-l inhibitor and/or an isolated nucleic acid which can reduce the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell. In some embodiments the PD-l inhibitor can include pembrolizumab, nivolumab, pidilizumab, cemiplimab, AMP-224, AMP-514, and PDR001. In some embodiments, the PD-l inhibitor is an anti -PD-l antibody or antigen binding fragment thereof, such as pembrolizumab, nivolumab, pidilizumab, or cemiplimab.

[0273] Some embodiments also include an agent for reducing activity of PD-L1 in a cell such as a PD-L1 inhibitor and/or an isolated nucleic acid which can reduce the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell. In some embodiments the PD-L1 inhibitor can include atezolizumab, avelumab, durvalumab, BMS-936559, and CK-301. In some embodiments, the PD-L1 inhibitor is an anti-PD-Ll antibody or antigen binding fragment thereof, such as atezolizumab, avelumab, and durvalumab.

[0274] In some embodiments, an isolated nucleic acid which can reduce the expression level of a nucleic acid encoding NOX2, PD-l, or PD-L1, or the expression level of a NOX2 protein, a PD-l protein, or a PD-L1 protein, in a cell can include a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme. In some embodiments, the isolated nucleic acid comprises a sequence encoding NOX2, PD-l, or PD-L1, or a fragment thereof; a sequence encoding antisense NOX2, PD-l, or PD-L1, or a fragment thereof; or an antisense nucleic acid complementary to a sequence encoding NOX2, PD-l, or PD-Llor a fragment thereof.

[0275] In some embodiments, a kit can include reagents to generate the modified cell. In some such embodiments, a kit can include reagents useful for use with a CRISPR system. In some embodiments, reagents can include a modified AAV vector and a nucleotide sequence encoding a site-specific nuclease. The site-specific nuclease may include a ZFN, a TALEN, or CRISPR/Cas9-based system that specifically binds and cleaves a modified target gene, such as a modified NOX2 gene, a modified PD-l gene, or a modified PD-L1 gene. The site-specific nuclease may be included in the kit to specifically bind and target a particular region in the endogenous target gene, such as a NOX2 target gene, a PD-l target gene, or a PD-L1 target gene. The kit may further include donor DNA, a gRNA, or a transgene. In some embodiments, a kit can include a Cas9 protein or Cas9 fusion protein, a nucleotide sequence encoding a Cas9 protein or Cas9 fusion protein, and/or at least one gRNA. The CRISPR/Cas9- based system may be included in the kit to specifically bind and target a particular target region upstream, within or downstream of the coding region of the target gene, such as a NOX2 gene, a PD-l target gene, or a PD-L1 target gene. For example, a CRISPR/Cas9-based system may be specific for a promoter region of a target gene or a CRISPR/Cas9-based system may be specific for the coding region. EXAMPLES

Example 1— HDC reduces tumor progression by targeting NOX2+ MDSCs

[0276] HDC reduced the growth of EL-4 lymphoma and 4T1 mammary carcinoma in mice. Mice were either untreated or treated with HDC thrice weekly starting one day before tumor cell inoculation. Tumor growth was measured thrice weekly. Tumor size was normalized against the mean tumor size of control mice at the end of each experiment. Results were analyzed using two-way ANOVA

[0277] Systemic administration of HDC significantly reduced the in vivo growth of EL-4 lymphomas, 4T1 mammary carcinoma, and MC-38 colon tumor (FIG. 1A, FIG. 1B, FIG. 5A). To elucidate the role of MDSCs for the anti-tumor effect of HDC, mice inoculated with EL-4 lymphoma cells were depleted of GR1+ cells using the GR1 -neutralizing antibody RB6- 8C5. As determined by FACS analysis at the end of the experiment, intratumoral GRl+CDl lb+ MDSCs were reduced by approximately 75% following GR1 antibody treatment (FIG. 6A). In GR1 -depleted animals, treatment with HDC did not affect EL-4 lymphoma growth (FIG. 1C) but significantly reduced lymphoma growth in simultaneously performed experiments in non-GRl -depleted animals (p=0.03 at day 10, Students' t test, FIG. 6B). Treatment with GR1 -neutralizing antibodies per se did not significantly impact on EL-4 lymphoma growth (FIG. 6B).

[0278] The effect of HDC treatment on EL-4 lymphoma growth was also evaluated in NOX2 knock-out (Nox2-KO) mice, where MDSCs accumulate but do not generate NOX2- derived ROS. Systemic treatment of mice with HDC did not alter lymphoma growth in the Nox2-KO mice (FIG. 1D). HDC did not affect the proliferation or cell cycling of EL-4 or 4T1 cells, but reduced the proliferation of MC-38 cells after two days in culture (FIG.s 5B-5F). In vivo growth of EL-4 cells was not affected by previous in vitro exposure to HDC (FIG. 5G).

Example 2— Effects of HDC on myeloid and lymphoid populations in tumor-bearing mice

[0279] HDC reduced the immunosuppressive properties of MDSCs in mice carrying EL-4 and 4T1 tumors. EL-4-bearing mice were sacrificed after 2 weeks and 4T1- bearing mice after 3 weeks of tumor growth when the mean tumor size of untreated mice reached approximately 1.5 cm². The accumulation of intratumoral and splenic MDSCs in EL- 4-bearing mice was measured, and the content of MDSCs was examined in control mice (n=31 for intratumoral MDSCs, n=l9 for splenic MDSCs) and in HDC-treated mice (n=33 for intratumoral MDSCs, n=2l for splenic MDSCs). Counts of splenocytes in tumor-free (naive) and control or HDC-treated 4T1 -bearing mice were determined. A correlation between intratumoral MDSCs and tumor size in control and HDC-treated EL-4-innoculated mice and 4Tl-innoculated was measured. Mean D peptide-induced ROS production from leukocytes recovered from tumors of control (n=l8) and HDC-treated (n=l7) EL-4-bearing mice was determined. ROS formation (area under the curve) in response to D-peptide by single cell suspensions from tumors, spleens or splenocyte-derived GR1+ cells isolated from control (tumor n=l8, spleen n=20, GRl + n=9) or HDC-treated (tumor n=l7, spleen n=l 9, GRl+ n=l l) EL-4-bearing mice was measured. Mean D peptide-induced ROS production from leukocytes recovered from tumors of control (n=l 5) and HDC-treated (n=l4) 4Tl-bearing mice was measured. ROS formation (area under the curve) in response to D-peptide stimulation by single cell suspensions from tumors or spleens isolated from control (tumor n=l 5, spleen n=l5) or HDC-treated (tumor n=l5, spleen h=15) 4T1 -bearing mice was measured. ROS formation was normalized against the mean ROS formation of tumor-bearing control mice in each experiment. ROS formation in response to D-peptide from GR1+ (solid line, n=3) and GR1- (dotted line, n=3) cells isolated from tumors and spleens of control EL-4-bearing mice was measured. Proliferation of OT-l CD8+ T cells was determined after three days of culture. Statistical differences between two groups were evaluated using Student’s t test. One-way ANOVA followed by Holm-Sidak’s multiple comparisons test was used for comparison between several groups and linear regression was utilized to analyze correlations. * p<0.05, ***p<0.00l .

[0280] EL-4 and 4T1 growth was associated with a pronounced increase of MDSCs in tumors and spleens (FIG. 2A and FIG. 7A). Treatment of mice with HDC significantly reduced the accumulation of MDSCs within EL-4 lymphomas, but not in spleen (FIG. 2A). Mice inoculated with 4T1 cells acquired enlarged spleens where approximately 52 % ± 2.5 (n=30) of splenocytes were MDSCs. Treatment of mice with HDC reduced the number of splenocytes but did not alter the content of MDSCs in tumors or spleens in this model (FIG. 2B and FIG. 7A). The vast majority of MDSCs in tumor-bearing mice were G-MDSCs. HDC did not affect the distribution of MDSC subtypes in EL-4-bearing mice (FIG. 7B) but significantly reduced the accumulation of splenic and tumor-infiltrating M-MDSCs in 4T1- bearing mice (FIG. 7C).

[0281] No significant increase in the number of tumor-infiltrating or splenic CD8+ T cells in HDC-treated EL-4 or 4T1 -bearing mice was observed (FIG. 8A and FIG. 8B). However, a negative correlation was noted between the percentage of intratumoral MDSCs and tumor-infiltrating CD8+ T cells in both models (FIG. 8C and FIG. 8D). Treatment of mice with HDC significantly enhanced the proportion of CD62L- cells (comprising CD44+ effector memory and CD44- effector populations) among CD8+ T cells in EL-4-bearing mice with a similar trend in the 4T1 model (FIG. 8E and FIG. 8F). Treatment of EL-4-bearing mice with HDC did not significantly alter the percentage of intratumoral CD4+ T cells, NK cells or B cells but slightly reduced the percentage of CD4+ T cells and NK cells in spleens (FIG.s 8G- 81).

Example 3— HDC reduces NOX2-dependent immunosuppression in MDSCs

[0282] A correlation was assessed between tumor size with MDSC content in the EL-4 model and the number of splenocytes in the 4T1 model to determine whether the reduction in MDSCs and splenocytes following HDC treatment was secondary to the reduced tumor size. A positive correlation was noted between the size of tumors and the percentage of intratumoral MDSCs in EL-4-bearing control mice and with splenocytes in 4T1 -bearing control mice (FIG. 2C and FIG. 2D). No such correlations were observed in HDC-treated EL- 4- or 4T1 -bearing mice suggesting that MDSCs accumulating in tumors following HDC treatment might be less immunosuppressive.

[0283] In vivo administration of HDC reduced the capacity of leukocytes isolated from EL-4 and 4T1 tumors as well as from splenocytes of EL-4-bearing mice to generate NOX2-derived ROS, with a similar trend observed for isolated GR1+ cells (FIG.s 2E-2H). Leukocytes recovered from tumors and spleens of EL-4-bearing control mice were separated into GR1+ and GR1- fractions and analyzed for ROS-forming capacity, which confirmed that ROS production was confined to the GR1+ MDSC population (FIG. 21 and FIG. 2J). GR1+ cells recovered from EL-4-bearing control mice strongly suppressed T cell proliferation and were significantly more suppressive than MDSCs recovered from HDC-treated mice (FIG. 2K and FIG. 2L). MDSCs may also exert immunosuppression via additional mechanisms, including nitric oxide synthase-derived NO production. No significant difference in nitric oxide synthase expression between MDSCs isolated from HDC-treated and control EL-4 bearing mice was observed (p=0.24, n=4, Students' t test).

Example 4— HDC reduces the in vitro generation of human MDSC-like cells

[0284] HDC targeted human MDSCs in vitro and in vivo. Human monocytes were cultured in the absence of stimuli or in the presence of IL-6 and GM-CSF for five days to induce MDSC-like cells. ROS production from cultured monocytes and MDSC-like cells (IL- 6+GM-CSF, solid line) in response to stimulation with fMLF was measured. Expression of HLA-DR on monocytes after five days of culture in absence of stimuli (Control) and in presence of IL-6 and GM-CSF (n=7) was measured. Expression of HLA-DR on monocytes cultured for five days with IL-6 and GM-CSF in the absence or presence of 100 mM HDC (n=7) was measured. AML patients in complete remission received HDC/IL-2 immunotherapy in three-week cycles. Expression of H2R and gp9lphox on M-MDSCs was measured. The frequency and the absolute counts of M-MDSCs before (cycle 1, day 1 ; C1D1) and after the first treatment cycle (cycle 1, day 21 ; C1D21) and at the beginning (cycle 3, day 1; C3D1) and end (cycle 3, day 21 ; C3D21) of the third treatment cycle were measured. Results were analyzed by Student's paired t test or by the log rank test. * p<0.05, ** pO.Ol, *** pO.OOl .

[0285] HDC facilitates the maturation of human and murine myeloid cells. The effects of HDC on the cytokine-induced generation of human MDSCs in vitro were determined. IL-6 and GM-CSF induced an MDSC-like phenotype in monocytes characterized by an enhanced production of NOX2-derived ROS in response to A'-formyl-Met-Leu-Phe (FIG. 3 A) and a reduced expression of HLA-DR in all donors (n=l2) albeit to a variable degree (10-70 % reduction in MFI of HLA-DR). Donors showing a robust cytokine-induced generation of MDSCs, as determined by a >50 % reduction in monocytic HLA-DR expression (7 out of 12 donors, FIG. 3B), incubation with HDC significantly reduced the cytokine-induced down-regulation of HLA-DR (FIG. 3C).

Example 5— Effect of HDC-based immunotherapy on human monocytic MDSCs

[0286] The content of MDSCs in blood samples from patients with AML who had been treated with HDC in conjunction with low dose IL-2 were analyzed, to determine effects

-el - of treatment with HDC on human MDSCs in vivo. PBMCs from patient blood samples were analyzed for content of M-MDSCs before and after treatment cycle one and three (i.e. cycle 1 day 1 and day 21 (C1D1 and C1D21)) and cycle 3 day 1 and day 21 (C3D1 and C3D21)). The gating strategy from a representative sample is shown in FIG. 9. M-MDSCs were found to consistently express high levels of gp9lphox, the catalytic subunit of NOX2, and H2R (FIG. 3D). The frequency and absolute counts of M-MDSCs in blood was significantly reduced during treatment with HDC/IL-2 (FIG. 3E and FIG. 3F). When patients were dichotomized based on above or below median reduction in total number of M-MDSCs within cycle one or between the onset of therapy (C1D1) and the end of cycle three (C3D21), it was observed that a strong reduction in M-MDSC counts significantly predicted leukemia free survival (FIG. 3G and FIG. 3H).

Example 6— HDC enhances the anti -tumor efficacy of q-PD-l and q-PD-Ll antibodies

[0287] HDC improved the anti-tumor efficacy of a-PD-l/a-PD-Ll immunotherapy. PD-L1 expression was measured in EL-4 and MC-38 cells. Growth of EL-4 and MC-38 tumors in control, or a-PD-l/a-PD-Ll-treated, or HDC/a-PD-l/a-PD-Ll -treated mice was measured. In experiments using EL-4 cells, tumor size was normalized against the mean tumor size of control mice at the end of each of four experiments, and results were analyzed using two-way ANOVA. In the MC-38 model, the difference in slope between HDC/a-PD-l/a-PD-Ll and a-PD-l/a-PD-Ll treatment was analyzed by linear mixed models.

[0288] The impact of HDC on the efficiency of CD8+ T cell-enhancing immunotherapy was investigated. EL-4 cells expressed high levels of PD-L1 (FIG. 4A). Also, 77 % ± 5.5 (mean ± SEM; n=l 1) of intratumoral M-MDSCs and 76 % ± 2.3 (mean ± SEM; n=l 1) of intratumoral G-MDSCs expressed PD-L1, and 77 % ± 2.8 (mean ± SEM; n=20) of tumor- infiltrating CD8+ T cells expressed PD-l in this model. Treatment of mice with HDC in vivo did not alter the expression of PD-L1 on MDSCs or PD-l on CD8+ T cells (data not shown). Treatment of EL-4-bearing mice with a-PD-l/a-PD-Ll antibodies tended to reduce tumor growth rate. The combination of HDC and a-PD-l/a-PD-Ll was superior to monotherapy with either HDC or a-PD-l/a-PD-Ll in reducing EL-4 tumor growth (FIG. 4B). Analysis of infiltrating immune populations in EL-4 lymphomas showed that a-PD-l/a-PD- Ll treatment did not affect MDSC, T or NK cell accumulation, while it slightly increased the fraction of CD8+ T cells displaying an effector phenotype (FIG.s 10A-E). The combined regimen of HDC/a-PD-l/a-PD-Ll was also assessed in the 4T1 model. 4T1 tumor growth was unaffected by a-PD-l/a-PD-Ll treatment. In this model, the combination of HDC/a-PD-l/a- PD-Ll was not superior to HDC alone in reducing tumor growth (data not shown).

[0289] Murine colorectal MC-38 cells expressed high levels of PD-L1 (FIG. 4A), and 70 % ± 5.5 (mean ± SEM; n=l 1) of G-MDSCs and 59% ± 3.2 (mean ± SEM; n=l 1) of M- MDSCs were also PD-Ll±. The expression of PD-l was modest in MC-38 tumor- infiltrating CD8± T cells (data not shown). Surprisingly, MC-38 tumor growth was nevertheless strongly reduced by treatment with a-PD-l/a-PD-Ll ; in these mice, tumors expanded during the first week after tumor cell inoculation and then regressed. Treatment with HDC further improved the anti-tumor efficacy of a-PD-l/a-PD-Ll (FIG. 4C). At days 10 and 13, tumor reduction in the HDC/a-PD-l/a-PD-Ll group was superior to treatment with a-PD-l/a-PD-Ll (p=0.0l and 0.04, respectively). At the end of the experiment 50 % of mice treated with a-PD-l/a-PD-Ll monotherapy were tumor-free whereas complete tumor clearance was noted in 100 % of mice receiving HDC/a-PD-l/a-PD-Ll . To enable analysis of MC-38 infiltrating immune populations following immunotherapy, mice were inoculated with a higher number of tumor cells to reduce the likelihood of complete tumor eradication at the experimental endpoint. The added benefit of HDC to a-PD-l/a-PD-Ll therapy was demonstrated also following inoculation of a higher number of MC-38 tumor cells (FIG. 11 A). In these experiments, treatment of MC-38 tumor-bearing mice with a-PD-l/a-PD-Ll or HDC/a-PD-l/a-PD-Ll tended to increase the fraction of intratumoral CD8± T cells and significantly increased the fraction of CD8± T cell with an effector phenotype (FIG. 11B and FIG. 11C). The percentage of intratumoral CD4± T cells was not altered, while a reduction in tumor infiltrating NK cells was noted (FIG. 11D and FIG. 11E).

[0290] Depletion of MDSCs in EL-4-bearing mice by 5-fluorouracil or therapeutic peptides can reduce tumor growth rate and promote CD8± T cell immunity likely by reducing immunosuppression. Some embodiments described herein include examining anti-tumor properties of HDC, an inhibitor of NOX2-derived ROS, by targeting MDSCs. Some anti-tumor properties of HDC rely on the presence of NOX2± GRl± cells because HDC had no significant anti-tumor activity in mice genetically deficient in NOX2 and in mice where MDSCs were depleted by GR1 -neutralizing antibodies. These findings support the anti-tumor efficacy of HDC in experimental cancer. Treatment of mice with HDC reduced the accumulation of intratumoral MDSCs and the number of splenocytes in two experimental tumor models and the use of a HDC-based regimen reduced MDSC counts in blood of AML patients in complete remission.

[0291] HDC did not affect the in vitro proliferation of EL-4 lymphoma and 4T1 mammary carcinoma cells. Additionally, no correlation was observed between tumor growth on the one hand and intratumoral MDSC or splenomegaly on the other in HDC-treated mice, which is consistent with a finding that the reduction of MDSC was not secondary to the reduced tumor size. Without wishing to be bound by any one theory, the reduction of MDSCs may be explained by pro-differentiating properties of HDC resulting in increased numbers of intratumoral dendritic cells. Endogenous histamine may also have a role in appropriate maturation of myeloid cells. Furthermore, MDSCs isolated from Nox2-KO mice more readily differentiate into dendritic cells and macrophages. The finding that NOX2 inhibition, via HDC, limits cytokine-induced generation of human MDSCs in vitro is consistent with ROS preventing the differentiation of myeloid cells, and HDC overcoming the insufficient differentiation. The administration of all-trans retinoic acid to tumor-bearing mice was shown to reduce MDSC counts in several experimental tumor models and to promote the maturation of MDSCs into dendritic cells and macrophages. The pro- differentiating properties of all-trans retinoic acid were secondary to neutralization of elevated ROS levels in MDSCs. The findings reported herein are consistent with targeting NOX2-derived ROS for appropriate myeloid cell maturation.

[0292] The number of cells with MDSC-like phenotype was low in AML patients who had achieved complete remission after receiving chemotherapy. Counts of M-MDSCs in blood were further reduced during the first cycle of HDC/IL-2, and a strong reduction heralded a favorable course of disease. The results reported herein support a finding that treatment with HDC may affect the human MDSC compartment, and MDSCs may constitute a targetable population for the efficiency of immunotherapy in AML.

[0293] In addition to limiting the accumulation of MDSCs in tumor-bearing mice, treatment with HDC reduced the formation of NOX2-derived ROS ex vivo. Consistent with this finding, MDSCs isolated from HDC-treated mice showed a reduced capacity to suppress CD8+ T cell proliferation. This implied that HDC targets a significant effector function in MDSC-mediated immunosuppression. Notably, treatment of mice with HDC did not improve tumor infiltration of CD8+ T cells, despite a positive correlation between accumulation of intratumoral MDSCs and tumor-infiltrating CD8+ T cells. Instead, it was observed that treatment with HDC was accompanied by the accumulation of intratumoral effector CD8+ T cells in EL-4-bearing mice. This observation supports a finding that HDC may promote effector functions of tumor-infiltrating CD8+ T cells.

[0294] The effect of HDC on the anti -tumor efficacy of checkpoint inhibition was investigated. HDC enhanced the efficacy of a-PD-l/a-PD-Ll in reducing EL-4 and MC-38 tumor growth. In the MC-38 model, but not in the EL-4 model, a-PD-l/a-PD-Ll treatment triggered an influx of CD8+ effector T cells to tumors. In patients, an optimal anti-tumor efficacy of a-PD-l therapy is generally believed to depend on pre-existing tumor-infiltrating CD8+ T cells. This suggests that combining HDC and a-PD-l/a-PD-Ll therapy with agents that enhance T cell infiltration, such as chemotherapy or a-VEGF antibodies, might further improve anti-tumor efficacy.

[0295] Results provided herein support a finding that in vivo treatment with HDC reduces the accumulation and immunosuppressive features of MDSCs and improves the anti tumor efficacy of checkpoint blockade in murine EL-4 lymphoma and MC-38 colon carcinoma.

Methods

Culture of EL-4. 4T1 and MC-38 cells

[0296] The EL-4 lymphoma and the 4T1 mammary cancer cell lines were maintained in RPMI 1640 (VWR, Stockholm, Sweden) and the MC-38 colon carcinoma cells in DMEM without sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA). Medium was supplemented with 10 % fetal calf serum (FCS), 100 pg/ml penicillin, 100 pg/ml streptomycin and 2 mM L-glutamine (EL-4 and 4T1 cells) at 37°C and 5 % C02. Adherent 4T1 and MC-38 cells were detached by trypsinization before expansion. Cells were cultured in vitro for one to two weeks prior to inoculation into mice.

Tumor cell proliferation assay [0297] EL-4 and MC-38 cells were stained with CellTraceViolet Proliferation Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cells were cultured in the presence or absence of 100 mM HDC (Sigma- Aldrich) for one to four days following assessment of proliferation using a four-laser BD LSRFortessa (405, 488, 532, and 640nm from BD Biosciences, San Diego, CA, USA) and analyzed using FACSDiva software (version 6 or later; BD Biosciences). The 4T1 cells were cultured for five days in the presence or absence of 100 mM HDC. At 30 minutes or eight hours prior to collection of cells, BrDU at a final concentration of 10 mM was added to the medium. The cells were then fixed, permeabilized, incubated with DNase A and analyzed on a BD LSRFortessa for BrdU incorporation using the BD Pharmingen BrdU Flow Kit (BD Biosciences).

EL-4 4T1 and MC-38 models

[0298] Six to eight week old female C57BL/6J and BALB/c mice were obtained from Charles River (Charles River Laboratories Sulzfeld, Germany). B6. l29S6-Cybbtmldin (Nox2-knock out (KO)) mice were originally obtained from Jackson Laboratory (Bar Harbor, ME, USA) and bred in-house. C57BL/6J mice and Nox2-KO mice were injected subcutaneously (s.c.) with 1.75 - 3 x 10⁵ EL-4 cells or 5 - 10 x 10⁵ MC-38 cells. BALB/c mice were injected s.c. with 4 x 10⁵ 4T1 cells. Mice were treated by intraperitoneal (i.p.) injections of HDC at 1,500 pg/mouse (EL-4- and MC-38-bearing mice) or 1,000 pg/mouse (4Tl-bearing mice) three times per week starting one day before tumor inoculation, or with i.p. injections of a mixture of antibodies against PD-l (a-PD-l ; 100-240 pg/mouse; RMP1-14; Nordic Biosite, Stockholm, Sweden) and PD-L1 (a-PD-Ll; 100-240 pg/mouse; 10F.9G2; Nordic Biosite) three, six and ten days after tumor inoculation, or with a combination of HDC and a-PD-l/a- PD-L1 antibodies. Effects of HDC on EL-4 tumor growth are consistent with a previous study (Martner A, el al., (2015) J Immunol 194:5014-5021). In some experiments, EL-4 cells were treated with 100 mM HDC in vitro for three to five days prior to tumor inoculation. Mice inoculated with in vitro HDC-treated cells did not receive further in vivo treatment. GR1+ cells depletion in EL-4-bearing mice was achieved by i.p. injections of GR1 -neutralizing antibodies (250 pg, RB6-8C5, BioXcell, West Lebanon, USA) every other day starting once tumors became palpable. The size of tumors was estimated three times per week as the length x width and normalized against the mean tumor size of untreated control mice or untreated GR1 depleted mice at the termination of each experiment. Mice were sacrificed and tumors and spleens harvested two to three weeks after tumor cell inoculation when the size of the largest tumors had reached a diameter of 1-1.5 cm.

Processing of spleens tumors and BM

[0299] Single cell suspensions of tumors were prepared by enzymatic digestion using a Tumor Dissociation Kit (Miltenyi Biotec, Lund, Sweden) along with mechanical dissociation utilizing a gentleMACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions. BM cells were isolated from femur and tibia of tumor-free naive mice. BM cells were rinsed, and spleens were extracted through a 70 pm strainer and depleted of erythrocytes by Red Blood Cell Lysis buffer (Sigma- Aldrich).

Flow cytometry analysis of murine samples

[0300] Single cell suspensions from tumors and spleens were incubated for five minutes with Fc-block (BD Biosciences) and then stained with either a myeloid panel of antibodies comprising CD45-BV786 (Clone 30-F11, BD Biosciences), GR1-PE (Clone RB6- 8C5, BD Biosciences), CDl lb-BV7l l (Clone MI/70, BD Biosciences), Ly6C-PerCpCy5.5 (Clone AL-21, BD Biosciences), Ly6G-FITC (Clone IA8, BD Biosciences) and DAPI (Invitrogen) or a lymphoid panel of antibodies comprising CD45-AlexaFlur700 (Clone 30- Fl l, BD Biosciences), CD3-PE (Clone 145-201, eBioscience), NKp46-PE-Cy7 (Clone 29A1.4, eBioscience), CD4-APC (Miltenyi Biotec), CD8-FITC (Miltenyi Biotec), CD44- BV711 (Clone IM7, BD Biosciences), CD62L-BV786 (Clone MEL- 14, BD Biosciences), PD- 1-BV605 (Clone J43, BD Biosciences) and DAPI (Invitrogen). Cells were acquired on a BD LSRFortessa and analyzed using FACSDiva.

T cell suppression assay

[0301] GR1 + cells were isolated from spleens of EL-4-bearing mice and from BM of tumor-free naive mice. Single cell suspensions were stained with a Ly6G/C-biotin antibody (clone RB6-8C5, BD Biosciences) followed by incubation with streptavi din-conjugated magnetic beads and positively selected by use of a MACS magnet (Miltenyi Biotech) according to the manufacturer’s instructions. The purity was consistently >80%. The purified GR1+ cells expressed CD1 lb (98 % ± 0.48, (mean ± standard error of the mean (SEM)), n=6). Splenocytes from OT-l mice (Rag2/OT-l, Taconic, USA) were stained with CellTraceViolet Proliferation Kit (Invitrogen) according to the manufacturer’s instructions. CellTraceViolet+ OT-l splenocytes were cultured at a 1 : 1 ratio with GR1+ cells from EL-4-bearing or naive mice in the presence of 10 pg/ml the OT-l T cell specific peptide SIINFEKL (Sigma- Aldrich) or the control peptide gpl 00 IMDQVPFSV (AnaSpec, Fremont, USA). The cells were cultured for three days in RPM1 1640 supplemented with 10 % FCS, 100 pg/ml penicillin, 100 pg/ml streptomycin and 2 mM L-glutamine at 37°C and 5 % C02 and thereafter stained with FITC- anti-CD8 (Miltenyi Biotec) before measuring T cell proliferation by flow cytometry. Results were analyzed with FlowJo Version 10.1 (TreeStar, Ashland, USA).

Generation of human monocyte-derived MDSCs

[0302] PBMCs were prepared from healthy blood donor buffy coats by Ficoll- Paque (Lymphoprep, Nycomed, Oslo, Norway) density centrifugation. Monocytes were isolated by adherence and cultured in Iscoves’ medium supplemented with 10 % human AB serum, 2 mM L-glutamine, 100 pg/ml penicillin, 100 pg/ml streptomycin, 1 ng/ml interleukin 6 (hIL-6, Sigma- Aldrich) and 10 ng/ml granulocyte macrophage colony-stimulating factor (hGM-CSF, Peprotech, Rocky Hill, USA) in the presence or absence of 100 pM HDC. In control experiments, adherent monocytes were cultured in the absence of cytokines. One half of the medium was replaced and HDC was again added after two days of culture. Cells were examined for expression of HLA-DR (antibody: HLA-DR-APC-Cy7, Clone C243, BD Biosciences) by flow cytometry after five days of culture.

Detection of ROS by chemiluminescence

[0303] Superoxide anion production in response to the hexapeptide Typ-Lys-Tyr- Met-Val-d-Met (D-peptide, R&D Systems, Minneapolis, MN, USA) or N-formyl-Met-Leu- Phe (fMLF, Sigma-Aldrich) by murine cells from tumors and spleens or by human cytokine- induced MDSCs was measured by isoluminol chemiluminescence (CL) as described in Dahlgren C, Karlsson A (1999) J Immunol Methods 232 (l-2):3-l4. doi: l0. l0l6/s0022- 1759(99)00146-5. Results are presented as curves displaying continuous ROS formation or as the area under the curve normalized to the mean area under the curve for cells from tumor bearing control mice.

MDSCs in a clinical trial of HDC/IL-2

[0304] In a phase IV trial (Re:Mission; ClinicalTrials.gov; NCT01347996), 84 patients with AML in first complete remission received ten consecutive 2l-day cycles of HDC and interleukin-2 (HDC/IL-2) for eighteen months or until relapse or death. The trial is described in detail in Rydstrom A, et al, (2017) J Leukoc Biol 102. doi: l0. l l 89/jlb.5VMAl l l6-455R. Blood was collected before and after the first and third HDC/IL-2 treatment cycle. PBMCs were isolated and cryopreserved at local sites and shipped on dry ice to the central laboratory at the Sahlgrenska Cancer Center, University of Gothenburg, Sweden.

[0305] PBMCs were stained with a panel of antibodies against myeloid cells to determine the content of MDSCs in blood as described Rydstrom A, et al, (2017). The panel included the following antibodies from BD Biosciences: CD3-PerCpCy5.5 (clone HIT3A), CDl9-PerCPCy5.5 (SJ25C1), CD l6-Bnlliant Violet 605 (3G8), HLA-DR-APCH7 (G46-6), CDl4-PECy7 (McpP9), and CD56-PerCp eflour 710 from CMSSB, Thermo Fischer Scientific, USA. Stained samples were acquired on a BD FACSAria. PBMCs were also stained to determine the expression level of H2R and gp9lphox (the catalytic subunit of NOX2) on MDSCs using the following stains and antibodies: FIVE/DEAD fixable yellow stain (Fife Technologies, Grand Island, NY, USA), CD33-PECy7 (P67.6), CD16-APC-H7 (3G8), HFA- DR-Qdot605 (G46-6) (all from BD Biosciences), CDl4-Qdot655 (TiiK4, Fife Technologies), anti-histamine H2 receptor (polyclonal rabbit IgG, MBF International, Woburn, MA, USA), goat anti-rabbit-PerCpCy5.5 and gp9lphox-FITC (7D5, MBF International). Samples were analyzed on a four-laser BD FSRFortessa flow cytometer and data analysis were performed by using FlowJo software, version 7.6.5 or later (TreeStar, AShlandm OR).

Statistics

[0306] Statistical analyses were performed using GraphPad Prism software (version 6.0 or later). Paired and unpaired t-tests were utilized for comparisons between two groups and one and two-way ANOVA followed by Holm-Sidak’s test was used for comparisons between > two groups. In experiments evaluating multiple treatments in MC-38 tumor-bearing mice were some tumors were completely eradicated by immunotherapy the linear mixed effects model was employed to compare the slope of tumor growth curves starting day 6 and finishing at the experimental endpoint, or at the first size=0 measurement. For survival analysis, the logrank (Mantel-Cox) test was utilized to compare patients showing a strong or a low/no reduction of MDSCs (dichotomized by the median reduction) during treatment with HDC/IL-2.

[0307] The term“comprising” as used herein is synonymous with“including,” “containing,” or“characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0308] The articles“a” and“an” are used herein to refer to one or to more than one (for example, at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

[0309] By“about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0310] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

[0311] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS:

1. A method of treating or ameliorating a cancer in a subject, wherein the cancer is a breast cancer or a colon cancer, the method comprising reducing the activity of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject.

2. The method of claim 1 , further comprising reducing the activity of programmed cell death protein 1 (PD-l) or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject.

3. The method of claim 1 , further comprising reducing the activity of programmed cell death protein ligand 1 (PD-L1) or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

4. The method of claim 1, wherein reducing the activity of NOX2 comprises administering an effective amount of a NOX2 inhibitor to the subject.

5. The method of claim 4, wherein the NOX2 inhibitor is selected from the group consisting of histamine dihydrochloride (HDC), histamine, N-methyl-histamine, 4-methyl- histamine, histamine phosphate, histamine diphosphate, GSK2795039, apocynin, GKT136901, GKT137831, ML171, VAS2870, VAS3947, celastrol, ebselen, perhexihne, grmdelic acid, NOX2ds-tat, NOXAlds, fulvene-5, ACD 084, NSC23766, CAS 1177865-17- 6, and CAS 1090893-12-1, and shionogi.

6. The method of 1, wherein reducing the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell comprises contacting the cell with an isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

7. The method of claim 6, wherein the isolated nucleic acid comprises a sequence encoding NOX2 or a fragment thereof, a sequence encoding antisense NOX2 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding NOX2 or a fragment thereof.

8. The method of claim 6, wherein the isolated nucleic acid comprises a gRNA comprising a sequence complementary to the sequence of a target gene selected from the group consisting of NOX2, CYBA, NCF1, NCF2, NCF4, RAC1, and RAC2.

9. The method of claim 2, wherein reducing the activity of PD-l comprises administering an effective amount of a PD-l inhibitor and an effective amount of a NOX2 inhibitor to the subject.

10. The method of claim 9, wherein the PD-l inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, AMP-224, AMP-514, and PDR001.

11. The method of claim 9, wherein the PD-l inhibitor is an anti -PD-l antibody or antigen binding fragment thereof selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, and cemiplimab.

12. The method of claim 2, wherein reducing the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell comprises contacting the cell with an isolated nucleic acid selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

13. The method of claim 12, wherein the isolated nucleic acid comprises a sequence encoding PD-l or a fragment thereof, a sequence encoding antisense PD-l or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-l or a fragment thereof.

14. The method of claims 3, wherein reducing the activity of PD-L1 comprises administering an effective amount of a PD-L1 inhibitor to the subject.

15. The method of claim 14, wherein the PD-L1 inhibitor is selected from an anti- PD-L1 antibody, an antigen binding fragment, atezolizumab, avelumab, durvalumab, BMS- 936559, and CK-301.

16. The method of claim 3, wherein reducing the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell comprises contacting the cell with an isolated nucleic acid selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

17. The method of claim 16, wherein the isolated nucleic acid comprises a sequence encoding PD-L1 or a fragment thereof, a sequence encoding antisense PD-L1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-L1 or a fragment thereof.

18. The method of claim 3, wherein an agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered concurrently with an agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, and/or an agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

19. The method of claim 3, wherein an agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered sequentially with an agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, and/or an agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

20. The method of claims 1, wherein the cell is a hematopoietic cell, a myeloid cell, a myeloid-derived suppressor cell (MDSC), an intratumoral MDSC, a peripheral CDl4⁺HLA- DR /low MDSC, a GRl⁺ MDSC, a monocytic MDSC, and a granulocytic MDSC.

21. The method of claim 1, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 10% to about 95%.

22. The method of claim 1, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 50% to about 200%.

23. The method of claim 1, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cell with an effector phenotype by at least about 5% to about 20%.

24. The method of claims 1, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are natural killer (NK) cells by at least about 50% to about 600%.

25. Use of a first agent to treat or ameliorate a cancer in a subject, wherein the cancer is a breast cancer or a colon cancer, wherein the first agent reduces the activity of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject.

26. The use of the first agent of claim 25 in combination with a second agent which reduces the activity of programmed cell death protein 1 (PD-l) or the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject.

27. The use of the first agent of claim 26 in combination with a third agent which reduces the activity of programmed cell death protein ligand 1 (PD-L1) or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

28. The use of any one of claims 25, wherein the first agent comprises a NOX2 inhibitor.

29. The use of claim 28, wherein the NOX2 inhibitor is selected from the group consisting of histamine dihydrochloride (HDC), histamine, N-methyl-histamine, 4-methyl- histamine, histamine phosphate, histamine diphosphate, GSK2795039, apocynin, GKT136901, GKT137831, ML171, VAS2870, VAS3947, celastrol, ebselen, perhexilme, grmdelic acid, NOX2ds-tat, NOXAlds, fulvene-5, ACD 084, NSC23766, CAS 1177865-17- 6, and CAS 1090893-12-1, and shionogi.

30. The use of claim 25, wherein the first agent comprises an isolated nucleic acid which reduces the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject, wherein the isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

31. The use of claim 30, wherein the isolated nucleic acid comprises a sequence encoding NOX2 or a fragment thereof, a sequence encoding antisense NOX2 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding NOX2 or a fragment thereof.

32. The use of claim 30, wherein the isolated nucleic acid comprises a gRNA comprising a sequence complementary to the sequence of a target gene selected from the group consisting of NOX2, CYBA, NCF1, NCF2, NCF4, RAC1, and RAC2.

33. The use of claim 26, wherein the second agent comprises a PD-l inhibitor.

34. The use of claim 33, wherein the PD-l inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, AMP-224, AMP-514, and PDR001.

35. The use of claim 33, wherein the PD-l inhibitor is an anti -PD-l antibody or antigen binding fragment thereof selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, and cemiplimab.

36. The use of claim 26, wherein the second agent comprises an isolated nucleic acid which reduces the expression level of a nucleic acid encoding PD-l or the expression level of PD-l protein in a cell of the subject, wherein the isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

37. The use of claim 36, wherein the isolated nucleic acid comprises a sequence encoding PD-l or a fragment thereof, a sequence encoding antisense PD-l or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-l or a fragment thereof.

38. The use of claim 27, wherein the third agent comprises a PD-L1 inhibitor.

39. The use of claim 38, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS-936559, and CK-301

40. The use of claim 38, wherein the PD-L1 inhibitor is an anti-PD-Ll antibody or antigen binding fragment thereof selected from the group consisting of atezolizumab, avelumab, and durvalumab.

41. The use of claim 27, wherein the third agent comprises an isolated nucleic acid which reduces the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject, wherein the isolated nucleic acid is selected from the group consisting of a guide RNA (gRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.

42. The use of claim 41, wherein the isolated nucleic acid comprises a sequence encoding PD-L1 or a fragment thereof, a sequence encoding antisense PD-L1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding PD-L1 or a fragment thereof.

43. The use of claim 27, wherein the first agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered concurrently with the second agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD- 1 protein in a cell of the subject, and/or the third agent to reduce the activity of PD-L 1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

44. The use of claim 27, wherein the first agent to reduce the activity of NOX2 or the expression level of a nucleic acid encoding NOX2 or the expression level of NOX2 protein in a cell of the subject is administered sequentially with the second agent to reduce the activity of PD-l or the expression level of a nucleic acid encoding PD-l or the expression level of PD- 1 protein in a cell of the subject, and/or the third agent to reduce the activity of PD-L1 or the expression level of a nucleic acid encoding PD-L1 or the expression level of PD-L1 protein in a cell of the subject.

45. The use of claim 25, wherein the cell is a hematopoietic cell, a myeloid cell, or a myeloid-derived suppressor cell (MDSC), an intratumoral MDSC, a peripheral CD 14 ILA- DR /low MDSC, a GRl⁺ MDSC, a monocytic MDSC, a granulocytic MDSC.

46. The use of claims 25, wherein the treatment is sufficient to achieve a reduction in a volume of a tumor of the cancer by at least about 10% to about 100%.

47. The use of claim 25, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are CD8+ T cells by at least about 50% to about 200%.

48. The use of claim 25, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral CD8+ T cells which are CD8+ T cells with an effector phenotype by at least about 5% to about 20%.

49. The use of of claims 25, wherein the treatment is sufficient to achieve an increase in a fraction of intratumoral lymphocytes which are natural killer (NK) cells by at least about 50% to about 600%.