Immune regulation of the tumor microenvironment

Abstract

The present invention relates to a method for immunomodulation of an immunosuppressive tumor cell microenvironment and prevention of the influence of tumor microbiota through multiple pathways by DNase enzyme therapy.A method for immunomodulation of an immunosuppressive tumor cell microenvironment comprising administering an effective amount of a deoxyribonuclease (DNase) enzyme, alone or in combination with other immunomodulatory agents.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 191,551, filed May 21, 2021, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated by reference in its entirety. The ASCII copy, created on May 19, 2022, is named 252732_000038_SL.txt and is 14,097 bytes in size.

[0003] FIELD OF THEINVENTION The present invention relates to a new method for immune modulation of the immunosuppressive tumor cell microenvironment and prevention of the influence of the tumor microbiota in patients with different cancers using deoxyribonuclease (DNase) enzyme. [Background technology]

[0004] 2. Background of the Invention Patients with malignant tumors have higher levels of circulating cell-free DNA (cfDNA) in plasma and serum compared to healthy individuals (Fleischhacker, 2007). In cancer patients, circulating cfDNA originates from dying non-tumor cells, tumor cells, and neutrophils. Tumors predispose to the release of neutrophil extracellular DNA traps (NETs), which contribute to the establishment of a prothrombotic state, cachexia, and organ failure in cancer patients (Demmers, 2012). Tumor-originating cfDNA may contribute to the development of metastasis and chemoresistance (Garcia-Olmo, 2013). The amount of circulating cfDNA increases with tumor progression (Sawyers, 2008) and reaches maximum levels in patients with advanced and metastatic disease (Butt, 2008). It has been shown that higher amounts of circulating cfDNA are significantly correlated with poor patient survival (Schwarzenbach, 2008).

[0005] The inventors have previously demonstrated that systemic administration of high-dose DNase protein into the circulation of patients can be useful in treating a number of diseases and conditions associated with increased levels of cfDNA in the blood, including cancer (e.g., carcinoma, sarcoma, lymphoma, melanoma; see, e.g., U.S. Patent Nos. 7,612,032; 8,710,012; and 9,248,166), the occurrence of somatic mosaicism (see, e.g., U.S. Patent Application Publication No. 20170056482), and side effects associated with chemotherapy or radiation therapy (see, e.g., U.S. Patent Application Publication No. 20170100463). All of these patents and patent applications are incorporated herein by reference in their entirety. Others have subsequently demonstrated similar effects (Wen, 2013; Cederval, 2015; Tohme, 2016; Patutina, 2011; Li, 2015).

[0006] Immune checkpoint inhibitors (ICIs) are currently approved for a number of cancers, however reported response rates to monotherapy range from 28 to 52% (Darvin et al., 2018). Summary of the Invention [Problem to be solved by the invention]

[0007] Summary of the Invention The present invention addresses a great need in the art for new and more effective treatments for cancer, particularly those cancers that are not effectively treated with immune checkpoint modulator monotherapy. [Means for solving the problem]

[0008] In one aspect, the invention provides a method of immunomodulation of an immunosuppressive tumor cell microenvironment in a subject with cancer, comprising administering to the subject an effective amount of a deoxyribonuclease (DNase) enzyme. In some embodiments, the immunomodulation comprises increased tumor cell killing by cytotoxic CD8 T cells and / or NK cells and / or CAR-T cells within the immunosuppressive tumor cell microenvironment.

[0009] In another aspect, the present invention provides a method for modulating tumor-associated microbiota in a subject having cancer, comprising administering to the subject an effective amount of a deoxyribonuclease (DNase) enzyme.

[0010] In a further aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a deoxyribonuclease (DNase) enzyme and a second immunomodulatory agent.

[0011] In some embodiments of any of the above methods, administration of the DNase enzyme is effective to reduce the number and / or activity of tumor-associated macrophages (TAMs) and / or tumor-infiltrating neutrophils (TINs) in the immunosuppressive tumor cell microenvironment.

[0012] In some embodiments of the above method comprising administering a second immune modulator, the second immune modulator is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is a modulator of an immune checkpoint molecule selected from PD-1, CD28, CTLA-4, CD137, CD40, CD134 (OX-40), ICOS, KIR, LAGS, CD27, TIM-3, BTLA, GITR, TCR, 4-1BB, TIGIT, CD96, CD226, KIR2DL, VISTA, HLLA2, TLIA, DNAM-1, CEACAM1, CD155, IDO, TGF-beta, IL-10, IL-2, IL-15, CSF-1, IL-6, adenosine A2A receptor (A2AR), and their ligands. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically binds CTLA-4, PD-1, OX-40, PD-L1, or PD-L2. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically binds CTLA-4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, TIM-3, VISTA, or SIGLEC7. In some embodiments, the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, atezolizumab, avelumab, durvalumab, cemiplimab, and any combination thereof. In some embodiments, the immune checkpoint inhibitor is pembrolizumab.

[0013] In some embodiments of any of the above methods of the invention, the DNase enzyme is selected from human DNase I, human DNase-1-like 3 (D1L3), human DNase-1-like 2 (D1L2), human DNase-1-like 1 (D1L1), DNase X, DNase gamma, DNase II, DNase II alpha, DNase II beta, and caspase-activated DNase (CAD). In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to human DNase I enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of the DNase 1-like 3 (D1L3) enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of SEQ ID NO:3. In some embodiments, the DNase enzyme comprises the amino acid sequence SEQ ID NO:2.

[0014] In some embodiments of any of the above methods of the invention, the DNase enzyme is administered as a DNase enzyme protein. In some embodiments, the DNase enzyme protein is administered intravenously for at least 2 days. In some embodiments, the DNase enzyme protein is administered intravenously for at least 7 days. In some embodiments, the DNase enzyme protein is administered intravenously for at least 14 days. In some embodiments, the DNase enzyme protein is administered intravenously for at least 16 days. In some embodiments, the DNase enzyme protein is administered intravenously for 1-2 days every 2 or 3 or 4 weeks, the total length of treatment being between 2 weeks and 50 years. In some embodiments, the DNase enzyme protein is administered intravenously for 2-5 days every 2 or 3 or 4 weeks, the total length of treatment being between 2 weeks and 50 years. In some embodiments, the DNase enzyme protein is administered intravenously for 7-14 days every 2 or 3 or 4 weeks, the total length of treatment being between 2 weeks and 50 years. In some embodiments, the DNase enzyme protein is administered at 125-250 μg / kg / day.

[0015] In some embodiments of any of the methods including administration of a second immunomodulator, the DNase enzyme protein is administered 120 hours to 1 hour prior to administration of the second immunomodulator. In some embodiments, the DNase enzyme protein is administered 30 minutes to 2 hours after administration of the second immunomodulator. In some embodiments, the DNase enzyme protein is administered 2 hours to 360 hours after administration of the second immunomodulator.

[0016] In some embodiments of any of the above methods of the invention, the DNase enzyme is encoded by a gene therapy vector. In some embodiments, the gene therapy vector is administered to a subject. In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding the DNase enzyme. In some embodiments, the promoter is selected from a liver-specific promoter, a nervous system-specific promoter, an intestine-specific promoter, a liver-specific / nervous system-specific tandem promoter, and a liver-specific / intestine-specific tandem promoter. In some embodiments, the promoter is specific to a tumor-originating tissue or a metastatic target tissue. In some embodiments, the AAV is selected from serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVLK03, AAVLK06, AAVLK12, AAV-KP1, AAV-F, AAVDJ, AAVhu37, AAVrh64R1, and Anc80.

[0017] In some embodiments of any of the above methods of the present invention, the DNase enzyme is expressed by a cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the cell is administered to a subject. In some embodiments, the CAR-expressing cell or the TCR-expressing cell is administered directly to the site of the tumor. In some embodiments, the CAR-expressing cell or the TCR-expressing cell is single-targeted or multi-targeted. In some embodiments, the CAR comprises an antigen-binding domain capable of specific binding to one or more tumor antigens.

[0018] In some embodiments of any of the above methods of the invention comprising the use of a second immunomodulator, administration of DNase enzyme is effective to reduce the severity of one or more immune-related adverse events associated with the use of the second immunomodulator, hi some embodiments, such immune-related adverse event is cytokine release syndrome (CRS). In some embodiments, the one or more immune-related adverse events are selected from uveitis, Sjogren's syndrome, conjunctivitis, blepharitis, episcleritis, scleritis, retinitis, pneumonia, pleuritis, sarcoid-like granuloma, hepatitis, pancreatitis, autoimmune diabetes, interstitial nephritis, glomerulonephritis, acute kidney injury (AKI), skin rash, pruritus, vitiligo, DRESS, psoriasis, Steven-Johnson syndrome, arthralgia, arthritis, myositis, dermatomyositis, anemia, neutropenia, thrombocytopenia, thrombotic microangiopathy, acquired hemophilia, vasculitis, colitis, enteritis, gastritis, myocarditis, pericarditis, hypophysitis, thyroiditis, adrenalitis, encephalitis, meningitis, polyneuropathy, Guillain-Barré syndrome, and subacute inflammatory neuropathy.

[0019] In some embodiments of any of the above methods of the invention, administration of DNase enzyme results in a change in the content and / or activity of the tumor microbiota in the subject. In some embodiments, the tumor microbiota comprises one or more bacterial taxa selected from the phyla Acidobacteria, Actinobacteria, Bacteroidetes, Chlamydia, Chrysiogenes, Cyanobacteria, Fibrobacteria, Firmicutes, Fusobacteria, Gemmatimonads, Lentisphaera, Proteobacteria, Spirochaetes, Synergistes, Tenericutes, and Verrucomicrobium.

[0020] In some embodiments of any of the above methods of the invention, a DNase enzyme is administered before, with, or after cell therapy. In some embodiments, the cell therapy comprises administering (i) cells comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and / or (ii) NK cells, and / or (iii) CD8 T cells.

[0021] In some embodiments of any of the above methods of the invention, the subject is a human.

[0022] In another aspect, the present invention provides a pharmaceutical composition comprising a deoxyribonuclease (DNase) enzyme, a second immunomodulator, and a pharma- ceutically acceptable carrier or excipient.

[0023] In a related aspect, the invention provides a pharmaceutical dosage form comprising a deoxyribonuclease (DNase) enzyme and a second immunomodulatory agent.

[0024] In a further aspect, the present invention provides a kit comprising a deoxyribonuclease (DNase) enzyme, a second immunomodulatory agent, and optionally instructions for use.

[0025] In some embodiments of any of the above pharmaceutical compositions, dosage forms, and kits, the second immune modulator is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is a modulator of an immune checkpoint molecule selected from PD-1, CD28, CTLA-4, CD137, CD40, CD134 (OX-40), ICOS, KIR, LAGS, CD27, TIM-3, BTLA, GITR, TCR, 4-1BB, TIGIT, CD96, CD226, KIR2DL, VISTA, HLLA2, TLIA, DNAM-1, CEACAM1, CD155, IDO, TGF-beta, IL-10, IL-2, IL-15, CSF-1, IL-6, adenosine A2A receptor (A2AR), and their ligands. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically binds CTLA-4, PD-1, OX-40, PD-L1, or PD-L2. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically binds CTLA-4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, TIM-3, VISTA, or SIGLEC7. In some embodiments, the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, atezolizumab, avelumab, durvalumab, cemiplimab, and any combination thereof. In some embodiments, the immune checkpoint inhibitor is pembrolizumab.

[0026] In some embodiments of any of the above pharmaceutical compositions, dosage forms, and kits, the DNase enzyme is selected from DNase I, human DNase-1-like 3 (D1L3), human DNase-1-like 2 (D1L2), human DNase-1-like 1 (D1L1), DNase X, DNase gamma, DNase II, DNase II alpha, DNase II beta, and caspase-activated DNase (CAD). In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to human DNase I enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of the DNase 1-like 3 (D1L3) enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of SEQ ID NO:3. In some embodiments, the DNase enzyme comprises the amino acid sequence of SEQ ID NO:2.

[0027] In some embodiments of any of the above pharmaceutical compositions, dosage forms, and kits, the DNase enzyme is present in the form of a DNase enzyme protein.

[0028] In some embodiments of any of the pharmaceutical compositions, dosage forms, and kits, the DNase enzyme is present in the form of a gene therapy vector encoding the DNase enzyme. In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding the DNase enzyme. In some embodiments, the promoter is selected from a liver-specific promoter, a nervous system-specific promoter, an intestine-specific promoter, a liver-specific / nervous system-specific tandem promoter, and a liver-specific / intestine-specific tandem promoter. In some embodiments, the promoter is specific to tumor-originating tissue or metastatic target tissue. In some embodiments, the AAV is selected from serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVLK03, AAVLK06, AAVLK12, AAV-KP1, AAV-F, AAVDJ, AAVhu37, AAVrh64R1, and Anc80.

[0029] In some embodiments of any of the above pharmaceutical compositions, dosage forms, and kits, the DNase enzyme is present in the form of a cell that expresses the DNase and also comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the cell expressing the CAR or the cell expressing the TCR is single-targeted or multi-targeted. In some embodiments, the CAR comprises an antigen-binding domain capable of specifically binding to one or more tumor antigens.

[0030] These and other aspects described herein will become apparent to those skilled in the art in the following description, claims and drawings. [Brief description of the drawings]

[0031] [Figure 1]Effect of DNase on (A) TAM and (B) TIN in breast adenocarcinoma (MA), colorectal cancer (CRC), lung cancer (LC), and pancreatic adenocarcinoma (PDAC) models. The amount of TAM and TIM was normalized as a percentage compared to the average TAM and TIM in the PBS group (CD45 in the tumor was set as 100%, control) in each experiment. [Diagram 2] The effect of different treatment regimens of DNase administration on (A) TAM and (B) TIN. The amounts of TAM and TIM were normalized as a percentage compared to the mean TAM and TIM of the PBS group (control, CD45 in the tumor was set as 100%) in each experiment. [Diagram 3] Effect of DNase administration on (A) TAM and (B) TIN at different time periods after tumor implantation. The amount of TAM and TIM was normalized as a percentage compared to the average TAM and TIM of the PBS group (CD45 in the tumor was set as 100%, control) in each experiment. [Figure 4] Effect of DNase administration in combination with checkpoint inhibitors on TAMs at different time periods after tumor implantation. The amount of TAMs was normalized as a percentage compared to the average TAMs in the PBS group (CD45 in the tumor was set as 100%, control) in each experiment. [Diagram 5] Role of DNase in regulating the pro-tumorigenic effects of tumor microbiota. Tumor size was normalized as a percentage compared to the mean size of group 1 (control set as 100%) in each experiment. [Figure 6] DNase prevents inhibition of checkpoint inhibitor activity by the tumor microenvironment. Tumor size was normalized as a percentage compared to the average size of group 1 (control set as 100%) in each experiment. [Figure 7]DNase prevents toxicity associated with checkpoint inhibitor treatment. The graph shows body weight following administration of test substances to C57BL / 6 mice bearing AT3 tumors. Data points represent group mean body weight. Error bars represent standard error of the mean (SEM). Administration of anti-CTLA4 antibody and F. nucleatum arrests weight gain in treated animals. Combination of CTLA4 antibody and F. nucleatum results in intense weight loss. DNase I treatment prevents anti-CTLA4-induced and / or F. nucleatum-induced toxicity and rescues weight gain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description of the Invention The present invention is based on the inventors' hypothesis that targeting neutrophil extracellular traps (NETs), which are extracellular networks of DNA and proteins expelled by neutrophils into the tumor microenvironment, may improve response rates to immune checkpoint therapy. Specifically, the inventors decided to combine treatment with DNase, a NET-removing agent, with immune checkpoint therapy.

[0033] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0034] The singular forms "a," "an," and "the" include plural referents unless the content clearly indicates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0035] The term "about" or "approximately" includes being within a statistically significant range of a value. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably within 10%, more preferably within 5% of a given value or range. The acceptable variation encompassed by the term "about" or "approximately" will depend on the particular system being tested and can be readily discerned by one of ordinary skill in the art.

[0036] The term "immunosuppressive tumor cell microenvironment" refers to non-cancerous resident and infiltrating host cells, secreted factors, and extracellular matrix proteins within tumor cells that collectively prevent tumor cell killing by cytotoxic CD8 T cells, NK cells, and / or CAR-T cells.

[0037] The term "tumor-associated microbiota" refers to microorganisms, their intracellular parts, and their metabolic products that are localized within a tumor.

[0038] The terms "extracellular DNA", "cell-free DNA" and "cfDNA" are used interchangeably to refer to extracellular DNA (e.g., of eukaryotic, viral, archaeal, prokaryotic, intracellular or extracellular parasitic origin), including DNA in extracellular vesicles (e.g., exosomes and microvesicles) found in any body fluid and tissue. cfDNA can be found in blood, lymph, liver, nervous tissue, cerebrospinal fluid (CSF), and / or intestine, including DNA in extracellular vesicles (e.g., exosomes and microvesicles) found in these body fluids and tissues.

[0039] The term "treat" or "treatment" of a condition, disorder or condition encompasses (1) preventing, delaying, or reducing the incidence and / or probability of occurrence of at least one clinical or subclinical symptom of the condition, disorder or condition occurring in a subject who may be afflicted with or predisposed to the condition, disorder or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder or condition; or (2) inhibiting the condition, disorder or condition, i.e., arresting, reducing, or delaying the onset of the disease or its recurrence or at least one clinical or subclinical symptom, or (3) palliating the disease, i.e., causing at least one alleviation of the condition, disorder or condition, or clinical or subclinical symptom. The benefit to the subject being treated is either statistically significant or at least perceptible to the patient or physician.

[0040] The terms "individual," "subject," "animal," "patient," and "mammal" are used interchangeably to refer to mammals, including humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats).

[0041] The term "effective" as applied to a dose or amount refers to the amount of a compound or pharmaceutical composition that is sufficient to produce the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the condition being treated, the individual drug or drugs used, the mode of administration, and the like.

[0042] As used herein, the term "therapeutically effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition that is sufficient to produce a desired activity upon administration to a subject in need thereof. It should be noted that when a combination of active ingredients is administered (e.g., a combination of DNase with another compound), the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered individually.

[0043] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences and is located upstream in the direction of transcription of the transcription start site of the coding sequence and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start site, any other DNA sequences, including but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "tissue-specific" promoter may be preferentially active in a specific type of tissue or cell.

[0044] As used herein, the term "liver-specific expression" refers to predominant or exclusive expression in the liver, ie, expression to a substantially greater extent than in other tissues and organs.

[0045] The term "liver-specific promoter" is used herein to refer to a promoter that is primarily or exclusively active in liver cells (e.g., hepatocytes) and directs / initiats transcription in the liver to a substantially greater extent than other tissues and organs. In this context, the term "primarily" means that at least 50% of the promoter-driven expression, more typically at least 90% of the promoter-driven expression (such as 100% of the promoter expression) occurs in liver cells. The ratio of liver expression to non-liver expression may vary between different liver-specific promoters. In some embodiments, a liver-specific promoter may preferentially direct / initiat transcription in individual liver cell types (e.g., hepatocytes, Kupffer cells, endothelial cells, etc.). Some liver-specific promoters useful in the expression cassette of the present invention include at least one, and typically multiple, hepatic nuclear factor binding sites. Liver-specific promoters useful in the expression cassette of the present invention may be constitutive or inducible promoters. Some non-limiting examples of liver promoters useful in the expression cassettes of the present invention include the albumin promoter (Alb), human alpha-1 antitrypsin (hAAT) promoter, thyroxine-binding globulin (TBG), apolipoprotein E hepatic control region promoter, apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor clade A member 1 (SERPINA1) (hAAT) promoter, cytochrome p450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter P1, murine transthyretin (MTTR) promoter, alpha-fetoprotein (AFP) promoter, lecithin-cholesterol acyltransferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, and mouse prealbumin gene promoter.

[0046] Non-limiting examples of liver-specific promoters include, for example, albumin promoter (Alb), human alpha-1 antitrypsin (hAAT) promoter, thyroxine-binding globulin (TBG) promoter, apolipoprotein E hepatic control region promoter, apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor clade A member 1 (SERPINA1) (hAAT) promoter, cytochrome p450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter P1, murine transthyretin (MTTR) promoter, alpha-fetoprotein (AFP) promoter, thyroid hormone-binding globulin promoter, alcohol dehydrogenase promoter, factor VIII (FVIII) promoter, HBV basic core promoter (BCP) and PreS2 promoter, phosphoenolpyruvate carboxykinase (PEP) promoter, and the like. CK) promoter, liver control region (HCR)-apoCII hybrid promoter, AAT promoter in combination with mouse albumin gene enhancer (Ealb) element, low density lipoprotein promoter, pyruvate kinase promoter, phosphoenolpyruvate carboxykinase promoter, lecithin-cholesterol acyltransferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, transferrin promoter, transthyretin promoter, alpha-fibrinogen and beta-fibrinogen promoters, alpha1-antichymotrypsin promoter, alpha-2HS glycoprotein promoter, heptoglobin promoter, ceruloplasmin promoter, plasminogen promoter, promoters of complement proteins (e.g., Clq, Clr, C2, C3, C4, C5, C6, C8, C9, complement factor I, and factor H), C3 complement activator and [alpha]1 acid glycoprotein promoter. Additional tissue-specific promoters can be found in the Tissue-Specific Promoter Database, TiProD (Nucleic Acids Research, J4:D104-D107 (2006).

[0047] The term "nervous system-specific promoter" is used herein to refer to a promoter that is primarily or exclusively active in nervous system cells and directs / initiat es transcription in the nervous system (e.g., the central nervous system (CNS) and / or the enteric nervous system (ENS), including the brain) to a substantially greater extent than in other tissues and organs. In this context, the term "primarily" means that at least 50% of the promoter-driven expression, more typically at least 90% of the promoter-driven expression (such as 100% of the promoter expression), occurs in cells of the nervous system. The ratio of nervous system to non-neuronal expression may vary among different nervous system-specific promoters. In some embodiments, a nervous system-specific promoter may preferentially direct / initia te transcription in individual CNS and / or ENS cell types (e.g., neurons, glial cells (e.g., oligodendrocytes, astrocytes, ependymal cells, microglia, Schwann cells, satellite cells), enteric neurons, intrinsic afferents, interneurons, motor neurons, etc.). The nervous system specific promoter useful in the expression cassette of the present invention can be a constitutive or inducible promoter.Some non-limiting examples of the nervous system promoter useful in the expression cassette of the present invention include microglia specific promoter (e.g., F4 / 80, CD68, TMEM119, CX3CR1, CMV and Iba1 promoter), myeloid specific promoter (e.g., TTR, CD11b and c-fes promoter), neuron specific promoter (e.g., CMV, NSE, synapsin [SynI, SynII], CamKII, α-CaMKII and VGLUT1 promoter), and other neuronal and glial cell (e.g., oligodendrocyte, astrocyte) type specific promoter (e.g., glial fibrillary acidic protein [GFAP] promoter).

[0048] The term "intestinal specific promoter" is used herein to refer to a promoter that is primarily or exclusively active in intestinal cells and directs / initiatates transcription in the intestine to a substantially greater extent than other tissues and organs. In this context, the term "primarily" means that at least 50% of the promoter-driven expression, more typically at least 90% of the promoter-driven expression (such as 100% of the promoter expression) occurs in intestinal cells. The ratio of intestinal to non-intestinal expression may vary between different intestinal specific promoters. In some embodiments, an intestinal specific promoter may preferentially direct / initiatate transcription in individual intestinal cell types (e.g., enterocytes, goblet cells, enteroendocrine cells). Intestinal specific promoters useful in the expression cassettes of the present invention may be constitutive or inducible promoters. Some non-limiting examples of intestinal promoters useful in the expression cassettes of the present invention include CB / CMV, GFAP, miCMV, CMV+I, tetO-CMV, β-acti-CMV, MUC2, villin, and T3. b Promoters include:

[0049] In some embodiments, for example, when tissue targeting is mediated by a viral capsid protein, the nucleic acid encoding the DNase may be operably linked to a promoter that allows for efficient systemic expression (e.g., a CMV promoter, a chicken beta-actin promoter (CBA), or an EF1a promoter).

[0050] The phrase "pharmaceutical acceptable" used in connection with compositions of the present invention refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to a subject (e.g., a mammal, such as a human). As used herein, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency for use in mammals, more particularly humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.

[0051] As used herein, the terms "viral vector" and "viral construct" refer to a recombinant viral construct that contains one or more heterologous nucleotide sequences (e.g., a nucleotide sequence encoding a DNase enzyme). In some embodiments, the viral vector is replication-defective. In some embodiments, viral structural and non-structural coding sequences are not present in the viral vector and are provided during viral vector production in trans by a vector such as a plasmid or by stably incorporating the sequences into a packaging cell line. Depending on the virus, the viral vector can be packaged in a capsid (e.g., AAV vector) and / or lipid envelope (e.g., lentiviral vector).

[0052] In accordance with the present invention there may be employed conventional pharmacology and molecular biology techniques within the skill of the art, such techniques being explained fully in the literature. See, among others, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (dN Glover ed. 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984); Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. (1985)); Transcription and Translation (BD Hames & SJ Higgins, eds. (1984)); Animal Cell Culture (RI Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel et al. (eds.), See Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0053] Method of the invention In one embodiment, the present invention provides a method of immunomodulation of the tumor microenvironment and / or prevention of tumor microbiota effects in a subject in need thereof comprising administering to the subject a deoxyribonuclease (DNase) enzyme. In another embodiment, the present invention provides a method of reducing the intensity of adverse events, including immune-related adverse events, of immune checkpoint modulator therapy through immunomodulation of the tumor microenvironment and prevention of tumor microbiota effects.

[0054] In one aspect, the present invention provides a method of immunomodulation of an immunosuppressive tumor cell microenvironment in a subject with cancer comprising administering to the subject an effective amount of DNase enzyme.

[0055] In another aspect, the present invention provides a method for modulating tumor-associated microbiota in a subject having cancer, comprising administering to the subject an effective amount of DNase enzyme.

[0056] In a further aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of DNase enzyme and a second immune modulator. In some embodiments, the second immune modulator is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is a modulator of an immune checkpoint molecule selected from PD-1, CD28, CTLA-4, CD137, CD40, CD134 (OX-40), ICOS, KIR, LAGS, CD27, TIM-3, BTLA, GITR, TCR, 4-1BB, TIGIT, CD96, CD226, KIR2DL, VISTA, HLLA2, TLIA, DNAM-1, CEACAM1, CD155, IDO, TGF-beta, IL-10, IL-2, IL-15, CSF-1, IL-6, adenosine A2A receptor (A2AR), and their ligands. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. Non-limiting examples of useful immune checkpoint inhibitors include, for example, antibodies that specifically bind to CTLA-4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, TIM-3, VISTA, or SIGLEC7. In some embodiments, the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, atezolizumab, avelumab, durvalumab, cemiplimab, and any combination thereof. In some embodiments, administration of DNase enzyme is effective in reducing the severity of one or more immune-related adverse events associated with the use of the second immune modulator. In some embodiments, the immune-related adverse event is cytokine release syndrome (CRS).Other non-limiting examples of immune-related adverse events include, for example, uveitis, Sjogren's syndrome, conjunctivitis, blepharitis, episcleritis, scleritis, retinitis, pneumonia, pleuritis, sarcoid-like granuloma, hepatitis, pancreatitis, autoimmune diabetes, interstitial nephritis, glomerulonephritis, acute kidney injury (AKI), skin rash, pruritus, vitiligo, DRESS, psoriasis, Steven-Johnson syndrome, arthralgia, arthritis, myositis, dermatomyositis, anemia, neutropenia, thrombocytopenia, thrombotic microangiopathy, acquired hemophilia, vasculitis, colitis, enteritis, gastritis, myocarditis, pericarditis, hypophysitis, thyroiditis, adrenalitis, encephalitis, meningitis, polyneuritis, Guillain-Barré syndrome, and subacute inflammatory neuritis.

[0057] In some embodiments of any of the methods of the invention, administration of the DNase enzyme results in a change in the content and / or activity of the tumor microbiota in the subject. In some embodiments, the tumor microbiota comprises one or more bacterial taxa selected from the phyla Acidobacteria, Actinobacteria, Bacteroidetes, Chlamydia, Chrysiogenes, Cyanobacteria, Fibrobacteria, Firmicutes, Fusobacteria, Gemmatimonads, Lentisphaera, Proteobacteria, Spirochaetes, Synergistes, Tenericutes, and Verrucomicrobium.

[0058] In one aspect, the present invention provides a method for immune modulation of the tumor microenvironment and prevention of tumor microbiota effects in a subject in need thereof, where the tumor microbiota is represented by bacteria, fungi, and viruses.

[0059] In one aspect, embodiments of the present invention provide methods for reducing the intensity of adverse events, including immune-related adverse events, of immune checkpoint modulator therapy through immune modulation of the tumor microenvironment and prevention of tumor microbiota effects.

[0060] In one aspect, the present invention provides a method for immune modulation of tumor microenvironment and prevention of tumor microbiota effects in a subject in need thereof, wherein the bacteria of the tumor microbiota are from the phylum Acidobacteria, Actinobacteria, Bacteroidetes, Chlamydia, Chrysiogenes, Cyanobacteria, Fibrobacteria, Firmicutes, Fusobacterium, Gemmatimonads, Lentisphaera, Proteobacteria, Spirochaetes, Synergistes, Tenericutes, and / or Verrucomicrobium.

[0061] In one aspect, the present invention provides a method for immune modulation of the tumor microenvironment and prevention of tumor microbiota effects in a subject in need thereof, wherein the bacteria of the tumor microbiota are representatives of the phylum Fusobacterium, the order Enterobacteriale, and / or the order Bacillales.

[0062] In some embodiments of any of the above methods, administration of the DNase enzyme is effective to reduce the number and / or activity of tumor-associated macrophages (TAMs) and / or tumor-infiltrating neutrophils (TINs) in the immunosuppressive tumor cell microenvironment.

[0063] In some embodiments of any of the above methods, the DNase enzyme is selected from DNase I, DNase X, DNase gamma, DNase1L1, DNase1L2, DNase1L3, DNaseII, DNaseII alpha, DNaseII beta, caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), and variants or derivatives thereof.

[0064] In some embodiments of any of the above methods, the DNase enzyme is administered as a DNase enzyme protein. In some embodiments, the DNase enzyme protein is administered parenterally.

[0065] In some embodiments of any of the above methods, the DNase enzyme is DNase I or a mutant or derivative thereof. In some embodiments, the DNase I mutant comprises one or more mutations in the actin binding site. In some embodiments, the one or more mutations in the actin binding site are selected from mutations at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combination thereof. In some embodiments, one of the mutations in the actin binding site is a mutation at Ala-114. In some embodiments, the DNase I mutant comprises one or more mutations that increase DNase activity. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combination thereof. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, N74K and A114F, and any combination thereof. In some embodiments, the DNase I mutant comprises the mutations Q9R, E13R, N74K, and A114F. In some embodiments, the DNase I mutant comprises the mutations Q9R, E13R, N74K, and A114F. The I mutants are H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C, A114C, A114R, H44N:T46S, D53R:Y65A, D53R:E69R, H44A:D The gene includes one or more mutations selected from the group consisting of 53R:Y65A, H44A:Y65A:E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T, and any combination thereof.In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to human DNase I enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of the DNase 1-like 3 (D1L3) enzyme.

[0066] In some embodiments of any of the above methods using DNase enzyme protein, the DNase enzyme protein is injected intravenously for at least 14 days following infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously for at least 16 days following infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously for at least 2 days prior to, with, or following infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously for at least 1 day prior to or following infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously for at least 7 days following infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously for at least 7 days prior to infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously every other day for at least 2 days prior to or following infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously every other day for at least 5 days prior to or following infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously every other day for at least 7 days prior to or following infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously every other day for at least 14 days prior to or following infusion of the immune checkpoint modulator. In some embodiments, the DNase enzyme protein is injected intravenously by an intermittent course for a total of 2-7 days prior to or 2-7 days following infusion of the immune checkpoint modulator.

[0067] Administration of the DNase enzyme protein according to the methods of the present invention can be by any suitable route, including systemic administration and direct administration to the site of disease (e.g., to the primary tumor).Non-limiting examples of useful administration routes include intravenous (IV), subcutaneous (SC), intraperitoneal (IP), oral, and intramuscular.

[0068] In certain embodiments, the DNase enzyme protein is formulated in a pharmaceutical composition together with a pharma- ceutically acceptable carrier or excipient.

[0069] In some embodiments of any of the above methods, the DNase enzyme is encoded by a gene therapy vector. In some embodiments, the gene therapy vector is administered to the subject. In some embodiments, the gene therapy vector is a viral vector. Non-limiting examples of useful viral vectors include, for example, adeno-associated virus (AAV) vectors, adenovirus vectors, retrovirus vectors (e.g., lentivirus vectors), and hepatotropic viral vectors (e.g., Hepatitis B virus (HBV) vectors).

[0070] In some embodiments of any of the above methods using a gene therapy vector, the gene therapy vector is a recombinant adeno-associated virus (rAAV) expression vector comprising: (i) a capsid protein; and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a DNase enzyme. Non-limiting examples of AAV serotypes that can be used to develop AAV expression vectors of the invention include, for example, AAV serotype 1 (AAV1), AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAV-LK03, AAV-LK06, AAV-LK-01-19, AAV-LK12, AAV-KP1, AAVKP2-KP11, AAV-F, AAVrhl64R1, AAVhu37, Anc80, Anc80L65, AAV-DJ, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and chimeras thereof. In some embodiments, the promoter is specific for tumor-originating tissue or metastatic target tissue. In some embodiments, the promoter is selected from a liver-specific promoter, a nervous system-specific promoter, an intestinal specific promoter, a liver-specific / nervous system-specific tandem promoter, and a liver-specific / intestinal specific tandem promoter.

[0071] In some embodiments of any of the above methods, the DNase enzyme is expressed by a cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the cell is administered to a subject. In some embodiments, the CAR-expressing cell or the TCR-expressing cell is administered directly to the site of the tumor. In some embodiments, the CAR-expressing cell or the TCR-expressing cell is single-targeted or multi-targeted. In some embodiments, the CAR comprises an antigen-binding domain capable of specific binding to one or more tumor antigens. In some embodiments of any of the above methods, the CAR-expressing cell or the TCR-expressing cell is further modified to express an immune checkpoint inhibitor molecule. In some embodiments, the CAR-expressing cell is a CAR T cell. In some embodiments, the CAR is a CAR T cell. In some embodiments, the CAR is a CAR T cell. AG, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44V6, CEA, EPCAM, B7H3, KIT, IL-13RA2, IL-11RA, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, FR-1, C-MET, EGFR / CD133, IL13Ra2, HER2, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, reglumin, HPV E6, E7, MAGEA1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, P53, P53 mutant, Prostein, Survivin, Telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, 0Y-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, and mut (ii) a tumor antigen selected from hsp70-2; (iii) an antigen associated with a solid tumor; (iv) mesothelin, EGFRvIII, GD2, CLDN6, Tn Ag, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, reglumin, CD171, PSCA, TARP, MAD-CT-1, Lewis Y, CD24, folate receptor alpha, folate receptor beta, ERBB, MUC1, EGFR, NCAM, PDGFR-beta, MAD-CT-2, Fos-related antigen, SSEA-4, neutrophil elastase, CAIX, HPV E6 E7, ML-IAP, NA17, ALK, androgen receptor polysialic acid, TRP-2, CYP1B1, PLAC1, GloboH, NY-BR-1, sperm protein 17, HMWMAA, beta-human chorionic gonadotropin, AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, and muthsp70-2; (iv) a solid tumor associated antigen present in / on the surface of mesothelioma, lung cancer, pancreatic cancer, esophageal adenocarcinoma, ovarian cancer, breast cancer, colorectal cancer, bladder cancer, or any combination thereof; (v) a tumor antigen associated with a blood cancer; (vi) a tumor antigen present in a disease selected from acute leukemia, including B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), and acute lymphoblastic leukemia (ALL); or one or more chronic leukemias, including chronic myelogenous leukemia (CIVIL) and chronic lymphocytic leukemia (CLL); and (vi) a tumor antigen present in a therapy-resistant cancer.

[0072] DNase enzyme As used herein, the terms "deoxyribonuclease" and "DNase" are used to refer to any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in DNA backbone. A wide variety of deoxyribonucleases are known and can be used in the methods of the present invention. Non-limiting examples of DNases useful in the methods of the present invention include, for example, DNase I (e.g., recombinant human DNase I (rhDNase I) or bovine pancreatic DNase I), analogs of DNase I (e.g., DNase X, DNase gamma, DNase1L1, DNase1L2, DNase 1L3, etc.), DNase II (e.g., DNase IIα, DNase IIβ), caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and their mutants or derivatives. Also encompassed by the present invention are DNase enzymes with extended half-lives (e.g., albumin and / or Fc fusions, or those prevented from binding to actin by modification of the actin-binding site; see, e.g., Gibson et al., (1992) J. Immunol. Methods, 155, 249-256). The actin-binding site of DNase I can be mutated, for example, at the following residues: Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114 of recombinant human DNase I (SEQ ID NO:1). For example, one human DNase I hyperreactive variant contains a mutated Ala-114 residue.Other exemplary mutations include, for example, H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C, A114C, A114R, H44N:T46S, D5 3R:Y65A, D53R:E69R, H44A:D53R:Y65A, H44A:Y65A:E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T (in the sequence of SEQ ID NO:1). Also included are mutations in DNase I that have increased DNase I activity. Non-limiting examples of such mutations are, for example, Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, and A114F of recombinant human DNase I (SEQ ID NO:1). For example, one hyperreactive DNase I mutant contains a combination of Q9R, E13R, N74K, and A114F mutations. DNase I cleaves DNA preferentially at phosphodiester bonds adjacent to pyrimidine nucleotides, resulting in 5'-phosphate-terminated polynucleotides with a free hydroxyl group at the 3' position, generating tetranucleotides on average. DNase I acts on single-stranded DNA, double-stranded DNA, and chromatin.

[0073] In some embodiments, the DNase may be DNase I or a variant or derivative thereof.

[0074] In some embodiments, the DNase I may be a human DNase I or a variant or derivative thereof. In some embodiments, the DNase I may be a non-human DNase I or a variant or derivative thereof, such as, but not limited to, a rodent (e.g., mouse) DNase I or a variant or derivative thereof.

[0075] In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a human DNase I enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to a human DNase I enzyme (SEQ ID NO:1). In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to a human DNase I enzyme variant (SEQ ID NO:2).

[0076] In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 21-305 of the DNase 1-like 3 (D1L3) enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of the human DNase 1-like 3 (D1L3) enzyme (SEQ ID NO:3).

[0077] In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 21-305 of the DNase 1-like 2 (D1L2) enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of the human DNase 1-like 2 (D1L2) enzyme (SEQ ID NO:4).

[0078] In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 21-305 of the DNase 1-like 1 (D1L1) enzyme. In some embodiments, the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of the human DNase 1-like 1 (D1L1) enzyme (SEQ ID NO:5).

[0079] In some embodiments, the DNase is a DNase I mutant comprising one or more mutations in the actin-binding site. In some embodiments, the one or more mutations in the actin-binding site are selected from mutations at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combination thereof. In some embodiments, one of the mutations in the actin-binding site is a mutation at Ala-114.

[0080] In some embodiments, the DNase is a DNase I mutant comprising one or more mutations that increase DNase activity. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combination thereof. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, N74K, and A114F.

[0081] In some embodiments, the DNase is selected from the group consisting of H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C, A114C, A114R, H44N:T46S, D53R:Y65A, D53R:E69R, H DNase I variants comprising one or more mutations selected from the group consisting of 44A:D53R:Y65A, H44A:Y65A:E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T, and any combination thereof. In some embodiments, the DNase I variant is a long-acting DNase. In some embodiments, the DNase I variant is a hyperreactive variant form of DNase. In some embodiments, the DNase I variant comprises the amino acid sequence SEQ ID NO:2.

[0082] In some embodiments, the DNase I mutant comprises the mutations Q9R, E13R, N74K and A114F.

[0083] In some embodiments, the DNase I mutant comprises the mutations Q9R, E13R, N74K and A114F.

[0084] In some embodiments of any of the methods of the invention, the sequence encoding DNase comprises a secretory signal sequence. In some embodiments, when DNase is administered as a recombinant vector, the secretory signal sequence mediates efficient secretion of the enzyme into the circulation of the portal sinusoid of the liver upon administration of the vector to a subject. In some embodiments, the secretory signal sequence is selected from the group consisting of a DNase I secretory signal sequence, an IL2 secretory signal sequence, an albumin secretory signal sequence, a β-glucuronidase secretory signal sequence, an alkaline protease secretory signal sequence, and a fibronectin secretory signal sequence. In some embodiments, the secretory signal sequence comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6). In some embodiments, the secretory signal sequence comprises the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6). In some embodiments, the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6). In some embodiments, the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). In some embodiments, the secretory signal sequence comprises the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). In some embodiments, the secretory signal sequence consists of the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:7).

[0085] In some embodiments, the DNase enzyme is selected from the group consisting of DNase I, DNase X, DNase gamma, DNase1L1, DNase1L2, DNase1L3, DNaseII, DNaseII alpha, DNaseII beta, caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and variants or derivatives thereof. In some embodiments, the enzyme having DNase activity is DNase I or a variant or derivative thereof. In some embodiments, the DNase I is human DNase I or a variant or derivative thereof. In some embodiments, the DNase I variant comprises one or more mutations in the actin binding site. In some embodiments, the one or more mutations in the actin binding site are selected from mutations at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combination thereof. In some embodiments, one of the mutations in the actin binding site is a mutation at Ala-114. In some embodiments, the DNase I mutant comprises one or more mutations that increase DNase activity. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combination thereof. In some embodiments, the one or more mutations that increase DNase activity are selected from the group consisting of Q9R, E13R, N74K and A114F, and any combination thereof. In some embodiments, the DNase I mutant comprises a sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity with the sequence of SEQ ID NO:2. In some embodiments, the DNase I mutant comprises the mutations Q9R, E13R, N74K, and A114F. In some embodiments, the DNase I mutant comprises the sequence of SEQ ID NO:2.In some embodiments, the DNase I mutant consists of the sequence of SEQ ID NO: 2. In some embodiments, the DNase I mutant consists of the sequence of SEQ ID NO: 2. In some embodiments, the DNase I mutant consists of the sequence of SEQ ID NO: 2. In some embodiments, the DNase I mutant consists of the sequence of SEQ ID NO: 2. The enzyme having DNase activity comprises one or more mutations selected from the group consisting of 53R:Y65A, H44A:Y65A:E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T, and any combination thereof. In some embodiments, the enzyme having DNase activity is a fusion protein comprising a DNase enzyme or a fragment thereof linked to (i)(ii) albumin or Fc or a fragment thereof. In some embodiments, the sequence encoding the enzyme having DNase activity comprises a sequence encoding a secretory signal sequence, the secretory signal sequence mediating efficient secretion of the enzyme. In some embodiments, the secretory signal sequence is selected from the group consisting of a DNase I secretory signal sequence, an IL2 secretory signal sequence, an albumin secretory signal sequence, a β-glucuronidase secretory signal sequence, an alkaline protease secretory signal sequence, and a fibronectin secretory signal sequence. In some embodiments, the secretory signal sequence comprises the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). In some embodiments, the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO:7).In some embodiments, the secretory signal sequence comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity with the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO:6), or a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity with the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). In some embodiments, the secretory signal sequence consists of the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO:8). In some embodiments, the secretory signal sequence comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity with the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO:8).

[0086] Administration of the DNase enzyme according to the methods of the present invention may be by any suitable route, including systemic administration and direct administration to the site of disease (e.g., to the primary tumor). Specific non-limiting examples of useful routes of administration include intravenous (IV), subcutaneous (SC), intraperitoneal (IP), oral, and intramuscular.

[0087] The DNase enzyme protein dosage useful in the methods of the present invention depends on the type of additional treatment, the patient's clinical history and response to DNase, and the discretion of the attending physician. Non-limiting examples of useful dosage ranges include 0.005-100 mg / kg / day or 10-200,000 KU / kg / day, preferably 0.05-50 mg / kg / day or 1,000-100,000 Kunitz units (KU) / kg / day, more preferably 1.5-50 mg / kg / day or 3,000-100,000 KU / kg / day, and most preferably 10-50 mg / kg / day or 20,000-100,000 KU / kg / day.

[0088] In some embodiments, the DNase enzyme protein is injected intravenously at 250 μg / kg / day. In some embodiments, the DNase enzyme protein is injected intravenously at 250 μg / kg / day for at least 14 days.

[0089] DNase Compositions and Formulations In certain embodiments, the DNase enzyme protein, a vector encoding the DNase enzyme, or cells expressing the DNase enzyme are formulated in a pharmaceutical composition together with a pharma- ceutically acceptable carrier or excipient, hi certain embodiments, such a composition further comprises a second immunomodulatory agent.

[0090] The formulation used in the method of the present invention can be conventionally presented in unit dosage form and can be prepared by methods known in the art.The amount of active ingredient that can be combined with carrier material to produce a single dosage form will vary depending on the host treated and the individual mode of administration.The amount of active ingredient that can be combined with carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect.

[0091] In general, the formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0092] Pharmaceutical compositions suitable for parenteral administration may contain one or more active ingredients ((i) DNase enzyme protein, a vector encoding the DNase enzyme or cells expressing the DNase enzyme, and, optionally, (ii) another compound [e.g., a second immunomodulator or another anti-cancer compound]) in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that may be constituted into a sterile injectable solution or dispersion immediately prior to use, which may contain antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0093] These compositions may also contain preservatives, wetting agents, emulsifiers and dispersants.Prevention of microbial action may be ensured by including various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol sorbic acid and the like.It may also be desirable to include isotonic agents in the composition, such as sugar, sodium chloride and the like.In addition, prolonged absorption of injectable pharmaceutical forms may be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0094] Injectable depot forms can be made by forming microencapsule matrices of one or more active ingredients in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of active ingredient to polymer and the nature of the particular polymer used, the rate of release of the active ingredient can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot-type injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions that are compatible with body tissues.

[0095] Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, or may be present as a solution or suspension in an aqueous or non-aqueous liquid (e.g., as a mouthwash, as a composition to be swallowed, or as an enema), or as oil-in-water or water-in-oil liquid emulsions, and the like, each containing a predetermined amount of one or more active ingredients.

[0096] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more active ingredients may be mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) saccharides, such as carboxymethylcellulose, alginates, gelatin, polyvinyl chloride, and the like; (3) humectants such as glycerol; (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retarders such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft-filled and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols and the like.

[0097] Suspensions may contain, in addition to the active ingredient or ingredients, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0098] Powders and sprays can contain, in addition to one or more active ingredients, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0099] Cancer and other treatments The methods of the present invention may be used in subjects suffering from a wide range of cancers. Non-limiting examples of relevant cancers include, for example, breast cancer, prostate cancer, multiple myeloma, transitional cell carcinoma, lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer, thyroid cancer, leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia), lymphoma (e.g., B cell lymphoma, T cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma), head and neck cancer, esophageal cancer, gastric cancer, colon cancer, intestinal cancer, colorectal cancer, rectal cancer, pancreatic cancer, liver cancer, bile duct cancer, gallbladder cancer, ovarian cancer, endometrial cancer, vaginal cancer, cervical cancer, bladder cancer, neuroblastoma, sarcoma, osteosarcoma, malignant melanoma, squamous cell carcinoma, primary bone cancer (e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant fibrous histiocytoma, adalimumab, sarcoma ... Bone cancer, including both primary (mantinoma, giant cell tumor, chordoma) and secondary (metastatic) bone cancer, soft tissue sarcoma, basal cell carcinoma, angiosarcoma, angiosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, testicular cancer, uterine cancer, gastrointestinal cancer, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenstrom's macroglobulinemia, papillary adenocarcinoma, cystadenocarcinoma, bronchogenic carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, medullary carcinoma, thymoma, sarcoma, etc.

[0100] In some embodiments, the treatment methods of the present invention, in addition to administering the DNase enzyme and the second immune modulator, may include administering additional anti-cancer agents and / or therapies, including, but not limited to, chemotherapeutic agents, radiation therapy, cell therapy (e.g., cells containing a chimeric antigen receptor (CAR) or a T cell receptor (TCR), NK cells, CD8 T cells, etc.), and any combination thereof.

[0101] In some embodiments of any of the methods of the invention, the subject is a human.

[0102] Working Example The present invention is also described and demonstrated by the following examples. However, the use of these and other examples elsewhere in this specification is merely illustrative and does not limit the scope and meaning of the present invention or any exemplified term. Similarly, the present invention is not limited to any particular preferred embodiment described herein. In fact, many improvements and modifications of the present invention may become apparent to those skilled in the art upon reading this specification, and such modifications may be made without departing from the invention in spirit or scope. Therefore, the present invention is limited only by the terms of the appended claims and the full range of equivalents to which those claims are entitled.

[0103] Example 1. Deoxyribonuclease enzyme reduces the number of tumor-associated macrophages (TAMs) and tumor-infiltrating neutrophils (TINs) in tumor tissue Based on the conditions of the internal environment, circulating monocytes give rise to mature macrophages, and when they are recruited to the tumor microenvironment, they are transformed into tumor-associated macrophages (TAMs). TAMs not only lack the function of phagocytosis of tumor cells, but also help these tumor cells avoid being killed and help them spread to other tissues and organs. Tumor-infiltrating neutrophils (TINs) are engaged in the tumor microenvironment by cytokines and chemokines. TANs stimulate immune suppression, tumor growth, angiogenesis and metastasis by DNA instability or by the release of cytokines and / or chemokines.

[0104] BALB / c mammary adenocarcinoma, MC38 colon carcinoma, Lewis lung carcinoma (LL2), and murine pancreatic adenocarcinoma (Panc2) cancer cell lines were used. 3x10e6 mammary adenocarcinoma, 1x10e6 MC38 cells, 3x10e5 LL2 cells, or 2x10e5 Panc2 cells in 100 μl of RPMI were administered by subcutaneous injection into the mammary fat pad of 6-8 week old syngeneic Balb / c or C57Bl / 6 female mice.

[0105] Six days after tumor implantation, animals were randomized into groups (N=3) and treated daily with IV injections of 75 μg of human recombinant DNase I per mouse, while the control group received the same volume of PBS. To isolate TAMs and TINs, tumors were extracted and treated with 12 U / mL collagenase I (Sigma), 450 U / mL collagenase IV (Sigma), and 50 U / ml DNase I (Kevet). Cell debris and dead cells were removed by density gradient. To purify TAMs, F4 / 80 cells were additionally MACS enriched (with anti-CD11b microbeads) (Sigma).

[0106] The amount of TAM and TIN after treatment with DNase is shown in Figures 1A and 1B, respectively, as a % of the relative control (considered as 100%). The data clearly demonstrate that daily IV injections of DNase I enzyme provide a statistically significant reduction in TAM and TIN.

[0107] Example 2. The effect of different dosing regimens of deoxyribonuclease enzyme on TAM and TIN abundance in tumor tissue 1 × 10e5 Panc2 cells in 100 μl of RPMI were injected subcutaneously into 6-8 week old syngeneic C57Bl / 6 female mice.

[0108] Six days after tumor implantation, animals were randomized into groups (N=3): Group 1: untreated control; Group 2: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 1 mg / kg daily for 7 days; Group 3: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg daily for 14 days; Group 4: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg daily for 7 days; Group 5: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg daily for 2 days; Group 6: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg, 2 days per week for 3 weeks; Group 7: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg for 2 days in weeks 1 and 3 of 3 weeks.

[0109] To isolate TAMs and TINs, tumors were extracted and treated with 12 U / mL collagenase I, 450 U / mL collagenase IV, and 50 U / mL DNase I. Cell debris and dead cells were removed by density gradient. To purify TAMs, F4 / 80 cells were additionally MACS enriched (with anti-CD11b microbeads).

[0110] The amounts of TAM and TIN are shown in Figures 2A and 2B, respectively.

[0111] Surprisingly, all of the employed regimens of DNase I administration provided a statistically significant (p<0.05) reduction in TAM and TIN.

[0112] Example 3. The effect of different times of deoxyribonuclease enzyme therapy on the abundance of TAM and TIN in tumor tissue 3×10e6 mammary adenocarcinoma cells in 100 μl of RPMI were injected subcutaneously into 6-8 week old syngeneic Balb / c female mice.

[0113] Animals were randomly assigned to groups (N=3): Group 1: untreated control; Group 2: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 2 after tumor implantation for a total of 21 days; Group 3: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 6 after tumor implantation for a total of 21 days; Group 4: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 10 after tumor implantation for a total of 21 days; Group 5: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 14 after tumor implantation for a total of 21 days; Group 6: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 18 after tumor implantation for a total of 21 days;

[0114] To isolate TAMs and TINs, tumors were extracted and treated with 12 U / mL collagenase I, 450 U / mL collagenase IV, and 50 U / mL DNase I. Cell debris and dead cells were removed by density gradient. To purify TAMs, F4 / 80 cells were additionally MACS enriched (with anti-CD11b microbeads).

[0115] The amounts of TAM and TIN are shown in Figures 3A and 3B, respectively.

[0116] Unexpectedly, administration of DNase I provided a statistically significant (p<0.05) reduction in TAMs and TINs, even at late stages of tumor development.

[0117] Example 4. The effect of combining deoxyribonuclease enzyme therapy with immune checkpoint inhibitors on TAM and TIN abundance in tumor tissue 1x10e6 CT26 or MC38 in 100 μl of RPMI were injected into 6-8 week old female mice.

[0118] Treatment was initiated on days 3 or 10 after tumor implantation.

[0119] Animals were randomly assigned to groups (N=3): Group 1: untreated control; Group 2: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 3 after tumor implantation; Group 3: IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 10 after tumor implantation for a total of 21 days; Group 4: IV injection of anti-mouse CTLA-4 antibody (BEO164, InVivoMAb, 200 μg / dose) every 3 days starting on day 3 after tumor implantation for a total of 21 days; Group 5: IV injection of anti-mouse CTLA-4 antibody (BEO164, InVivoMAb, 200 μg / dose) every 3 days starting on day 10 after tumor implantation for a total of 21 days; Group 6: IV injection of anti-mouse CTLA-4 antibody (BEO164, InVivoMAb, 200 μg / dose) every 3 days starting on day 3 after tumor implantation + IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 3 after tumor implantation for a total of 21 days; Group 7: IV injection of anti-mouse PD1 antibody (BEO146, InVivoMAb, 200 μg / dose) every 3 days starting on day 10 after tumor implantation + IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 10 after tumor implantation for a total of 21 days; Group 8: IV injection of anti-mouse PD1 antibody (BEO146, InVivoMAb, 200 μg / dose) every 3 days starting on day 3 after tumor implantation for a total of 21 days; Group 9: IV injection of anti-mouse PD1 antibody (BEO146, InVivoMAb, 200 μg / dose) every 3 days starting on day 10 after tumor implantation for a total of 21 days; Group 10: IV injection of anti-mouse PD1 antibody (BEO146, InVivoMAb, 200 μg / dose) every 3 days starting on day 3 after tumor implantation + IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 3 after tumor implantation for a total of 21 days; Group 11: IV injection of anti-mouse PD1 antibody (BEO146, InVivoMAb, 200 μg / dose) every 3 days starting on day 10 after tumor implantation + IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 10 after tumor implantation for a total of 21 days; Group 12: IV injection of anti-mouse OX-40 antibody (BE0031, InVivoMAb, 200 μg / dose) every 3 days starting on day 3 after tumor implantation for a total of 21 days; Group 13: IV injection of anti-mouse OX-40 antibody (BE0031, InVivoMAb, 200 μg / dose) every 3 days starting on day 3 after tumor implantation + IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 3 after tumor implantation for a total of 21 days; Group 14: IV injection of anti-mouse OX-40 antibody (BE0031, InVivoMAb, 200 μg / dose) every 3 days starting on day 10 after tumor implantation for a total of 21 days; Group 15: IV injection of anti-mouse OX-40 antibody (BE0031, InVivoMAb, 200 μg / dose) every 3 days starting on day 10 after tumor implantation + IV injection of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg every 3 days starting on day 10 after tumor implantation for a total of 21 days.

[0120] TAMs were extracted from tumors as previously described. The amounts of TAMs and TINs are shown in FIG. 4.

[0121] When used alone, checkpoint inhibitors statistically significantly reduced TAM abundance at tumor sites at early stages of tumor progression, but failed to do so when they were used at more advanced stages of disease (when used in animals 10 days after tumor implantation). Unexpectedly, when combined with DNase I, immune checkpoint inhibitors continued to significantly (p<0.05) reduce TAMs.

[0122] Example 5. Effect of different forms of DNase enzyme on tumor microbiota Fusobacterium nucleatum (F. nucleatum) is an oral anaerobe that has recently been found to be abundant in human colorectal cancer (CRC), breast cancer, and several other cancers associated with poor treatment outcomes and a tumor immunosuppressive environment. Inoculation with F. nucleatum leads to tumor colonization, inhibits the accumulation of tumor-infiltrating T cells, and promotes tumor growth and metastatic progression. See, e.g., Kostic et al., Cell host & microbe, 2013, 14(2):207-15; Van der Merwe et al., Immunology Letters, 2021,232:60-66.

[0123] F. nucleatum (ATCC 25586) was cultured anaerobically at 37°C and 5% CO2 on Columbia agar supplemented with 5% sheep red blood cells. The breast cancer cell line AT3 (ATCC) was injected into the mammary fat pad of 6-8 week old C57BL / 6 mice. Animals were randomly assigned to groups (N=3). The study ended on day 24. Group 1: untreated control; Group 2: injected with 7.5 × 10e7 F. nucleatum ATCC23726; Group 3: injected with 7.5×10e7 F. nucleatum ATCC23726 and animals were treated with 10 mg / kg metronidazole on days 2, 3, 4, 6 and 7 after tumor F. nucleatum implantation. Group 4: injected with 7.5×10e7 F. nucleatum ATCC23726 and treated with a 2mg / kg dose of human recombinant DNase I enzyme (Kevelt AS; SEQ ID NO:1) given every 3 days starting on day 1 after bacterial injection; Group 5: injected with 7.5×10e7 F. nucleatum ATCC23726 and treated with a 2mg / kg dose of mutant DNase I enzyme (Kevelt AS) (SEQ ID NO:2) given every 3 days starting on day 1 after bacterial injection; Group 6: injected with 7.5 × 10e7 F. nucleatum ATCC23726 and treated with DNase I-like 3 (Human Microbiology Institute, NY, USA) (SEQ ID NO:3) at a dose of 2 mg / kg given every 3 days starting on day 1 after bacterial injection; Group 7: injected with 7.5×10e7 F. nucleatum ATCC23726 and treated with DNase I-like 3 (SEQ ID NO:3) at a dose of 2mg / kg given on days 1 and 4 after bacterial injection; Group 8: injected with 7.5 × 10e7 F. nucleatum ATCC23726 and treated with DNase I-like 2 (Human Microbiology Institute, NY, USA) (SEQ ID NO:4) at a dose of 2 mg / kg given every 3 days starting on day 1 after bacterial injection; Group 9: injected with 7.5x10e7 F. nucleatum ATCC23726 and treated with DNase I-like 1 (Kevelt AS) (SEQ ID NO:5) at a dose of 2mg / kg given every 3 days starting on day 1 after bacterial injection.

[0124] Tumor volumes were measured with calipers and estimated using the ellipsoid formula. The data are shown in Figure 5. The data clearly show that IV injection of different forms of human recombinant DNase I enzyme provided a statistically significant (p<0.05) prevention of F. nucleatum-induced tumor growth and prevented the pro-tumorigenic effect of F. nucleatum.

[0125] At the end of the study, the presence of F. nucleatum in the tumor tissue was confirmed by inoculating tumor homogenates into nutrient medium and incubating for 48 hours at 37° C. under anaerobic conditions. Additional PCR analysis of both tumor tissue and colonies that were allowed to grow was performed. The data are shown in Table 1. [Table 1]

[0126] The data clearly show that DNase did not alter the amount of F. nucleatum in the tumors and has no antibacterial activity in these conditions. Unexpectedly, DNase I significantly inhibited the protumorigenic effect of F. nucleatum. In this study, the regimen of DNase I administration had little effect on the results and the various types of DNase worked equally well.

[0127] Example 6. Influence of the tumor microbiota on the antitumor activity of immune checkpoint inhibitors F. nucleatum ATCC 25586 was cultured anaerobically at 37°C and 5% CO2 on Columbia agar supplemented with 5% sheep red blood cells. The breast cancer cell line AT3 was injected into the mammary fat pad of 6-8 week old C57BL / 6 mice weighing 18-22 g. Animals were randomly assigned to groups (N=5). The study was terminated on day 21. Group 1: untreated control; Group 2: injected with 7.5 × 10e7 F. nucleatum ATCC23726; Group 3: injected with 7.5×10e7 F. nucleatum ATCC23726 and animals were treated with 10 mg / kg metronidazole (Sigma) on days 2, 3, 4, 5, 6, and 7 after tumor F. nucleatum implantation. Group 4: injected with 7.5×10e7 F. nucleatum ATCC23726 and treated with a 2mg / kg dose of human recombinant DNase I (Kevelt AS; SEQ ID NO:1) given every 3 days starting on day 1 after bacterial injection; Group 5: injected with anti-mouse CTLA-4 antibody (BEO164, InVivoMAb, 200 μg / dose) every 2 days; Group 6: injected with 7.5 × 10e7 F. nucleatum ATCC23726 and treated with anti-mouse CTLA-4 antibody (BEO164, InVivoMAb, 200 μg / dose) given every 2 days starting from bacterial injection; Group 7: treated with human recombinant DNase I (Kevelt AS; SEQ ID NO:1) at a dose of 2 mg / kg given every 3 days starting on day 1 after bacterial injection and treated with anti-mouse CTLA-4 antibody (BEO164, InVivoMAb, 200 μg / dose) given every 2 days starting on day 1 after bacterial injection; Group 8: injected with 7.5x10e7 F. nucleatum ATCC23726 and treated with human recombinant DNase I (Kevelt AS; SEQ ID NO:1) at a dose of 2mg / kg given every 3 days starting on day 1 after bacterial injection, and treated with anti-mouse CTLA-4 antibody (BEO164, InVivoMAb, 200μg / dose) given every 2 days starting on day 1 after bacterial injection.

[0128] Tumor volumes were measured with calipers and estimated using the ellipsoid formula. The data are shown in Figure 6.

[0129] At the end of the study, the presence of F. nucleatum in the tumor tissue was confirmed by inoculating tumor homogenates into nutrient medium and incubating at 37° C. for 48 hours under anaerobic conditions. Additional PCR analysis of both tumor tissue and colonies that were allowed to grow was performed. The data are shown in Table 2. [Table 2]

[0130] The data demonstrate that F. nucleatum significantly inhibited the anti-cancer activity of the immune checkpoint inhibitor anti-CTLA-4 antibody. Unexpectedly, combined treatment with checkpoint inhibitors and DNase I enzyme reduced the pro-tumorigenic and anti-cancer immune activity of F. nucleatum (p<0.05) but did not affect the viability of this microorganism.

[0131] Another unexpected finding from this study was that F. nucleatum increased the toxic effects of anti-CTLA-4 antibody checkpoint inhibitor treatment, and that combined treatment with this checkpoint inhibitor and DNase I reduced F. nucleatum- and CTLA-4 antibody-induced toxicity. Specifically, as shown in Figure 7, administration of anti-CTLA4 antibody (3 mg / kg) or F. nucleatum (7.5 x 10e7) individually arrested weight growth in treated animals, the combination of anti-CTLA4 antibody and F. nucleatum led to intensive weight loss, and DNase I treatment (at 2 mg / kg) prevented anti-CTLA4 antibody- and / or F. nucleatum-induced toxicity and led to weight gain.

[0132] array SEQ ID NO:1- Mature wild type (WT) human DNase I (without secretory signal sequence; Genbank Accession No. 4AWN_A): LKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAI VPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO:2 - mature human DNAse I variant (without secretory signal sequence); mutated residues compared to SEQ ID NO:1 are shown in bold and underlined: LKIAAFNIRTFGRTKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAI VPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO:3 - human DNase-1-like 3 MSRELAPLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEI KDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAE NFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS SEQ ID NO:4 - human DNase-1-like 2 MGGPRALLAALWALEAAGTAALRIGAFNIQSFGDSKVSDPACGSIIAKILAGYDLALVQEVRDPDLSAVSALMEQINSVSEHEYSFVSSQPLGRDQYKEMYLFVYRKDAVSVVDTYLYPDPEDVFSREPFVVKFSAPGTGERAPPLPSRRALTPPPLPAAAQNLVLIPLHAAPHQAVAEIDALYDVYLDVIDKWGTDDMLFLGDFNADCSYVRAQDWAAIRLRSSEVFKWLIPDSADTTVGNSDCAYDRIVACGARLRRSLKPQSATVHDFQEEFGLDQTQALAISDHFPVEVTLKFHR SEQ ID NO:5 - Human DNase-1-like 1 MHYPTALLFLILANGAQAFRICAFNAQRLTLAKVAREQVMDTLVRILARCDIMVLQEVVDSSGSAIPLLLRELNRFDGSGPYSTLSSPQLGRSTYMETYVYFYRSHKTQVLSSYVYNDEDDVFAREPFVAQFSLPSNVLPSLVLVPLHTTPKAVEKELNALYDVFLEVSQHWQSKDVILLGDFNADCASLTKKRLDKLELRTEPGFHWVIADGEDTTVRASTHCTYDRVVLHGERCRSLLHTAAAFDFPTSFQLTEEEALNISDHYPVEVELKLSQAHSVQPLSLTVLLLLSLLSPQLCPAA

[0133] Reference materials: Song, L. et al., “NLRP3 Inflammasome in Neurological Diseases, from Functions to Therapies” Front. Cell. Neurosci., 2017, Vol. 11, No. 63. Fleischhacker M. et al., “Circulating nucleic acids(CNAs) and cancer: a survey” Biochim biophys Acta, 2007, 1775(1): 181-232. Demers, M. at al., “Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis” PNAS, 2012, 109(32):13076-13081. Garcia-Olmo D.C. and Garcia-Olmo, D. “Biological role of cell-free nucleic acids in cancer: the theory of genometastasis” Crit Rev Oncolog., 2013, 18:153-161. Sawyers 2008 C.L., “The cancer biomarker problem” Nature, 2008, 452(7187):548-552. Butt A.N. et al. Overview of circulating nucleic acids in plasma / serum. Ann. N.Y. Acad. Sci., 2008, 1137:236-242. Schwarzenbach H. et al., “Detection and monitoring of cell-free DNA in blood of patients with colorectal cancer” Ann. N.Y. Acad. Sci., 2008, 1137:190-196. Zenaro E, et al., Neutrophils promote Alzheimer’s disease-like pathology and cognitive decline via LFA-1 integrin. Nat Rev Nephrol., 2015, Author manuscript; available in PMC 2017 Jul 14. Fushi, W. et al., “Extracellular DNA in Pancreatic Cancer Promotes Cell Invasion and Metastasis” Cancer Res., 2013, 73:4256-4266. Tohme, S. et al., “Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress” Cancer Res., 2016, 76(6): 1367-1380. Patutina, O. et al., Inhibition of metastasis development by daily administration of ultralow doses of RNase A and DNase I. Biochimie., 2011, 93(4): 689-96. Dan Li, DNase I Treatment Reduces GVHD in Mice. Biology of Blood and Marrow Transplantation, Volume 21, Issue 2, Page S339. Darvin, P. et al., “Immune checkpoint inhibitors: recent progress and potential biomarkers” Experimental & molecular medicine, 2018, 50(12):1-1.

[0134] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0135] All patents, patent applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present in this specification.

Claims

1. A method for immunomodulation of an immunosuppressive tumor cell microenvironment in a subject with cancer comprising administering to the subject an effective amount of a deoxyribonuclease (DNase) enzyme.

2. 2. The method of claim 1, wherein the immunomodulation comprises increased tumor cell killing by cytotoxic CD8 T cells and / or NK cells and / or CAR-T cells within an immunosuppressive tumor cell microenvironment.

3. A method for modulating tumor-associated microbiota in a subject with cancer, comprising administering to the subject an effective amount of a deoxyribonuclease (DNase) enzyme.

4. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a deoxyribonuclease (DNase) enzyme and a second immunomodulatory agent.

5. 5. The method of any one of claims 1 to 4, wherein administration of the DNase enzyme is effective to reduce the number and / or activity of tumor-associated macrophages (TAM) and / or tumor-infiltrating neutrophils (TIN) in an immunosuppressive tumor cell microenvironment.

6. 5. The method of claim 4, wherein the second immunomodulator is an immune checkpoint modulator.

7. 7. The method of claim 6, wherein the immune checkpoint modulator is a modulator of an immune checkpoint molecule selected from PD-1, CD28, CTLA-4, CD137, CD40, CD134 (OX-40), ICOS, KIR, LAGS, CD27, TIM-3, BTLA, GITR, TCR, 4-1BB, TIGIT, CD96, CD226, KIR2DL, VISTA, HLLA2, TLIA, DNAM-1, CEACAM1, CD155, IDO, TGF-beta, IL-10, IL-2, IL-15, CSF-1, IL-6, adenosine A2A receptor (A2AR), and ligands thereof.

8. 7. The method of claim 6, wherein the immune checkpoint modulator is an immune checkpoint inhibitor.

9. The method of claim 8, wherein the immune checkpoint inhibitor is an antibody that specifically binds CTLA-4, PD-1, OX-40, PD-L1, or PD-L2.

10. 9. The method of claim 8, wherein the immune checkpoint inhibitor is an antibody that specifically binds to CTLA-4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, TIM-3, VISTA, or SIGLEC7.

11. 9. The method of claim 8, wherein the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, atezolizumab, avelumab, durvalumab, cemiplimab, and any combination thereof.

12. 12. The method of claim 11, wherein the immune checkpoint inhibitor is pembrolizumab.

13. 13. The method of any one of claims 1 to 4 and 6 to 12, wherein the DNase enzyme is selected from human DNase I, human DNase-1-like 3 (D1L3), human DNase-1-like 2 (D1L2), human DNase-1-like 1 (D1L1), DNase X, DNase gamma, DNase II, DNase II alpha, DNase II beta, and caspase-activated DNase (CAD).

14. 13. The method of any one of claims 1 to 4 and 6 to 12, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity with human DNase I enzyme.

15. 15. The method of claim 14, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

16. 13. The method of any one of claims 1-4 and 6-12, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of a DNase 1-like 3 (D1L3) enzyme.

17. 17. The method of claim 16, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity with amino acids 21 to 305 of SEQ ID NO:

3.

18. 13. The method of any one of claims 1 to 4 and 6 to 12, wherein the DNase enzyme comprises the amino acid sequence SEQ ID NO:

2.

19. The method of any one of claims 1 to 4 and 6 to 12, wherein the DNase enzyme is administered as a DNase enzyme protein.

20. 20. The method of claim 19, wherein the DNase enzyme protein is administered intravenously for at least two days.

21. 21. The method of claim 20, wherein the DNase enzyme protein is administered intravenously for at least 7 days.

22. 22. The method of claim 21, wherein the DNase enzyme protein is administered intravenously for at least 14 days.

23. 23. The method of claim 22, wherein the DNase enzyme protein is administered intravenously for at least 16 days.

24. 20. The method of claim 19, wherein said DNase enzyme protein is administered intravenously for 1-2 days every 2 or 3 or 4 weeks, with a total length of treatment ranging from 2 weeks to 50 years.

25. 20. The method of claim 19, wherein the DNase enzyme protein is administered intravenously for 2-5 days every 2 or 3 or 4 weeks, with a total length of treatment ranging from 2 weeks to 50 years.

26. 20. The method of claim 19, wherein the DNase enzyme protein is administered intravenously for 7 to 14 days every 2 or 3 or 4 weeks, with a total length of treatment ranging from 2 weeks to 50 years.

27. 20. The method of claim 19, comprising administering the DNase enzyme protein to the subject 120 hours to 1 hour prior to administering the second immunomodulator.

28. 20. The method of claim 19, comprising administering the DNase enzyme protein to the subject 30 minutes to 2 hours after administering the second immunomodulator.

29. 20. The method of claim 19, comprising administering the DNase enzyme protein to the subject between 2 hours and 360 hours after administering the second immunomodulator.

30. 20. The method of claim 19, wherein the DNase enzyme protein is administered at 125-250 μg / kg / day.

31. The method of any one of claims 1 to 4 and 6 to 12, wherein the DNase enzyme is encoded by a gene therapy vector.

32. 32. The method of claim 31 , wherein the gene therapy vector is administered to the subject.

33. 32. The method of claim 31, wherein the gene therapy vector is a recombinant adeno-associated virus (rAAV) expression vector comprising a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding (i) a capsid protein and (ii) the DNase enzyme.

34. 34. The method of claim 33, wherein the promoter is selected from a liver-specific promoter, a nervous system-specific promoter, an intestinal-specific promoter, a liver-specific / nervous system-specific tandem promoter, and a liver-specific / intestinal-specific tandem promoter.

35. 34. The method of claim 33, wherein the promoter is specific to a tumor-initiating tissue or a metastatic target tissue.

36. 34. The method of claim 33, wherein the AAV is selected from serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAVLK03, AAVLK06, AAVLK12, AAV-KP1, AAV-F, AAVDJ, AAVhu37, AAVrh64R1, and Anc80.

37. 13. The method of any one of claims 1 to 4 and 6 to 12, wherein the DNase enzyme is expressed by a cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the cell is administered to the subject.

38. 38. The method of claim 37, wherein the CAR-expressing cells or the TCR-expressing cells are administered directly to the site of the tumor.

39. 38. The method of claim 37, wherein the CAR-expressing cell or the TCR-expressing cell is single-targeted or multi-targeted.

40. 38. The method of claim 37, wherein the CAR comprises an antigen-binding domain capable of specifically binding to one or more tumor antigens.

41. 13. The method of any one of claims 4 and 6-12, wherein administration of the DNase enzyme is effective to reduce the severity of one or more immune-related adverse events associated with use of the second immunomodulatory agent.

42. 42. The method of claim 41, wherein the immune-related adverse event is cytokine release syndrome (CRS).

43. 42. The method of claim 41, wherein the one or more immune-related adverse events are selected from uveitis, Sjogren's syndrome, conjunctivitis, blepharitis, episcleritis, scleritis, retinitis, pneumonia, pleuritis, sarcoid granuloma, hepatitis, pancreatitis, autoimmune diabetes, interstitial nephritis, glomerulonephritis, acute kidney injury (AKI), skin rash, pruritus, vitiligo, DRESS, psoriasis, Steven-Johnson syndrome, arthralgia, arthritis, myositis, dermatomyositis, anemia, neutropenia, thrombocytopenia, thrombotic microangiopathy, acquired hemophilia, vasculitis, colitis, enterocolitis, gastritis, myocarditis, pericarditis, hypophysitis, thyroiditis, adrenal inflammation, encephalitis, meningitis, polyneuropathy, Guillain-Barré syndrome, and subacute inflammatory neuropathy.

44. The method of any one of claims 1 to 4 and 6 to 12, wherein administration of the DNase enzyme results in a change in the content and / or activity of the tumor microbiota in the subject.

45. 45. The method of claim 44, wherein the tumor microbiota comprises one or more bacterial taxa selected from the phyla Acidobacteria, Actinobacteria, Bacteroidetes, Chlamydiae, Chrysiogenes, Cyanobacteria, Fibrobacteria, Firmicutes, Fusobacteria, Gemmatimonads, Lentisphaera, Proteobacteria, Spirochaetes, Synergistes, Tenericutes, and Verrucomicrobium.

46. 13. The method of any one of claims 1-4 and 6-12, wherein the DNase enzyme is administered prior to, together with, or after cell therapy.

47. 47. The method of claim 46, wherein the cell therapy comprises administering (i) cells comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and / or (ii) NK cells, and / or (iii) CD8 T cells.

48. The method of any one of claims 1 to 4 and 6 to 12, wherein the subject is a human.

49. A pharmaceutical composition comprising a deoxyribonuclease (DNase) enzyme, a second immunomodulator, and a pharmaceutically acceptable carrier or excipient.

50. 50. The pharmaceutical composition of claim 49, wherein the second immunomodulator is an immune checkpoint modulator.

51. 51. The pharmaceutical composition of claim 50, wherein the immune checkpoint modulator is a modulator of an immune checkpoint molecule selected from PD-1, CD28, CTLA-4, CD137, CD40, CD134 (OX-40), ICOS, KIR, LAGS, CD27, TIM-3, BTLA, GITR, TCR, 4-1BB, TIGIT, CD96, CD226, KIR2DL, VISTA, HLLA2, TLIA, DNAM-1, CEACAM1, CD155, IDO, TGF-beta, IL-10, IL-2, IL-15, CSF-1, IL-6, adenosine A2A receptor (A2AR), and ligands thereof.

52. 51. The pharmaceutical composition of claim 50, wherein the immune checkpoint modulator is an immune checkpoint inhibitor.

53. 53. The pharmaceutical composition of claim 52, wherein the immune checkpoint inhibitor is an antibody that specifically binds to CTLA-4, PD-1, OX-40, PD-L1, or PD-L2.

54. 53. The pharmaceutical composition of claim 52, wherein the immune checkpoint inhibitor is an antibody that specifically binds to CTLA-4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, TIM-3, VISTA, or SIGLEC7.

55. 53. The pharmaceutical composition of claim 52, wherein the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, atezolizumab, avelumab, durvalumab, cemiplimab, and any combination thereof.

56. 56. The pharmaceutical composition of claim 55, wherein the immune checkpoint inhibitor is pembrolizumab.

57. 57. The pharmaceutical composition of any one of claims 49 to 56, wherein the DNase enzyme is selected from human DNase I, human DNase-1-like 3 (D1L3), human DNase-1-like 2 (D1L2), human DNase-1-like 1 (D1L1), DNase X, DNase gamma, DNase II, DNase II alpha, DNase II beta, and caspase-activated DNase (CAD).

58. 57. The pharmaceutical composition of any one of claims 49 to 56, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity with human DNase I enzyme.

59. 59. The pharmaceutical composition of claim 58, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

60. 57. The pharmaceutical composition of any one of claims 49-56, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of a DNase 1-like 3 (D1L3) enzyme.

61. 61. The pharmaceutical composition of claim 60, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of SEQ ID NO:

3.

62. 57. The pharmaceutical composition of any one of claims 49 to 56, wherein the DNase enzyme comprises the amino acid sequence SEQ ID NO:

2.

63. 57. The pharmaceutical composition of any one of claims 49 to 56, wherein the DNase enzyme is present in the composition in the form of a DNase enzyme protein.

64. 57. The pharmaceutical composition of any one of claims 49 to 56, wherein the DNase enzyme is present in the composition in the form of a gene therapy vector encoding the DNase enzyme.

65. 65. The pharmaceutical composition of claim 64, wherein the gene therapy vector is a recombinant adeno-associated virus (rAAV) expression vector comprising a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding (i) a capsid protein and (ii) the DNase enzyme.

66. 66. The pharmaceutical composition of claim 65, wherein the promoter is selected from a liver-specific promoter, a nervous system-specific promoter, an intestinal-specific promoter, a liver-specific / nervous system-specific tandem promoter, and a liver-specific / intestinal-specific tandem promoter.

67. 66. The pharmaceutical composition of claim 65, wherein the promoter is specific to tumor-initiating tissue or metastasis target tissue.

68. 66. The pharmaceutical composition of claim 65, wherein the AAV is selected from serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAVLK03, AAVLK06, AAVLK12, AAV-KP1, AAV-F, AAVDJ, AAVhu37, AAVrh64R1, and Anc80.

69. 57. The pharmaceutical composition of any one of claims 49 to 56, wherein the DNase enzyme is present in the composition in the form of cells that express the DNase and also comprise a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

70. 70. The pharmaceutical composition of claim 69, wherein the CAR-expressing cell or the TCR-expressing cell is single-targeted or multi-targeted.

71. 70. The pharmaceutical composition of claim 69, wherein the CAR comprises an antigen-binding domain capable of specifically binding to one or more tumor antigens.

72. A pharmaceutical dosage form comprising a deoxyribonuclease (DNase) enzyme and a second immunomodulator.

73. 73. The pharmaceutical dosage form of claim 72, wherein the second immunomodulatory agent is an immune checkpoint modulator.

74. 74. The pharmaceutical dosage form of claim 73, wherein the immune checkpoint modulator is a modulator of an immune checkpoint molecule selected from PD-1, CD28, CTLA-4, CD137, CD40, CD134 (OX-40), ICOS, KIR, LAGS, CD27, TIM-3, BTLA, GITR, TCR, 4-1BB, TIGIT, CD96, CD226, KIR2DL, VISTA, HLLA2, TLIA, DNAM-1, CEACAM1, CD155, IDO, TGF-beta, IL-10, IL-2, IL-15, CSF-1, IL-6, adenosine A2A receptor (A2AR), and ligands thereof.

75. 74. The pharmaceutical dosage form of claim 73, wherein the immune checkpoint modulator is an immune checkpoint inhibitor.

76. 76. The pharmaceutical dosage form of claim 75, wherein the immune checkpoint inhibitor is an antibody that specifically binds CTLA-4, PD-1, OX-40, PD-L1, or PD-L2.

77. 76. The pharmaceutical dosage form of claim 75, wherein the immune checkpoint inhibitor is an antibody that specifically binds to CTLA-4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, TIM-3, VISTA, or SIGLEC7.

78. 76. The pharmaceutical dosage form of claim 75, wherein the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, atezolizumab, avelumab, durvalumab, cemiplimab, and any combination thereof.

79. 79. The pharmaceutical dosage form of claim 78, wherein the immune checkpoint inhibitor is pembrolizumab.

80. 80. The pharmaceutical dosage form of any one of claims 72 to 79, wherein the DNase enzyme is selected from human DNase I, human DNase-1-like 3 (D1L3), human DNase-1-like 2 (D1L2), human DNase-1-like 1 (D1L1), DNase X, DNase gamma, DNase II, DNase II alpha, DNase II beta, and caspase-activated DNase (CAD).

81. 80. The pharmaceutical dosage form of any one of claims 72 to 79, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity with the human DNase I enzyme.

82. 82. The pharmaceutical dosage form of claim 81, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

83. 80. The pharmaceutical dosage form of any one of claims 72 to 79, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21 to 305 of a DNase 1-like 3 (D1L3) enzyme.

84. 84. The pharmaceutical dosage form of claim 83, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity with amino acids 21 to 305 of SEQ ID NO:

3.

85. 80. The pharmaceutical dosage form of any one of claims 72 to 79, wherein the DNase enzyme comprises the amino acid sequence SEQ ID NO:

2.

86. 80. The pharmaceutical dosage form of any one of claims 72 to 79, wherein the DNase enzyme is present in the dosage form in the form of a DNase enzyme protein.

87. 80. The pharmaceutical dosage form of any one of claims 72 to 79, wherein the DNase enzyme is present in the dosage form in the form of a gene therapy vector encoding the DNase enzyme.

88. 88. The pharmaceutical dosage form of claim 87, wherein the gene therapy vector is a recombinant adeno-associated virus (rAAV) expression vector comprising a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding (i) a capsid protein and (ii) the DNase enzyme.

89. 89. The pharmaceutical dosage form of claim 88, wherein the promoter is selected from a liver-specific promoter, a nervous system-specific promoter, an intestinal-specific promoter, a liver-specific / nervous system-specific tandem promoter, and a liver-specific / intestinal-specific tandem promoter.

90. 89. The pharmaceutical dosage form of claim 88, wherein the promoter is specific to tumor-initiating tissue or metastatic target tissue.

91. 89. The pharmaceutical dosage form of claim 88, wherein the AAV is selected from serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAVLK03, AAVLK06, AAVLK12, AAV-KP1, AAV-F, AAVDJ, AAVhu37, AAVrh64R1, and Anc80.

92. 80. The pharmaceutical dosage form of any one of claims 72 to 79, wherein the DNase enzyme is present in the dosage form in the form of cells that express the DNase and also comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

93. 93. The pharmaceutical dosage form of claim 92, wherein the CAR-expressing cell or the TCR-expressing cell is single-targeted or multi-targeted.

94. 93. The pharmaceutical dosage form of claim 92, wherein the CAR comprises an antigen-binding domain capable of specifically binding to one or more tumor antigens.

95. A kit comprising a deoxyribonuclease (DNase) enzyme, a second immunomodulator, and optionally instructions for use.

96. 96. The kit of claim 95, wherein the second immune modulator is an immune checkpoint modulator.

97. 97. The kit of claim 96, wherein the immune checkpoint modulator is a modulator of an immune checkpoint molecule selected from PD-1, CD28, CTLA-4, CD137, CD40, CD134 (OX-40), ICOS, KIR, LAGS, CD27, TIM-3, BTLA, GITR, TCR, 4-1BB, TIGIT, CD96, CD226, KIR2DL, VISTA, HLLA2, TLIA, DNAM-1, CEACAM1, CD155, IDO, TGF-beta, IL-10, IL-2, IL-15, CSF-1, IL-6, adenosine A2A receptor (A2AR), and ligands thereof.

98. 97. The kit of claim 96, wherein the immune checkpoint modulator is an immune checkpoint inhibitor.

99. The kit of claim 98, wherein the immune checkpoint inhibitor is an antibody that specifically binds CTLA-4, PD-1, OX-40, PD-L1, or PD-L2.

100. 99. The kit of claim 98, wherein the immune checkpoint inhibitor is an antibody that specifically binds to CTLA-4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, TIM-3, VISTA, or SIGLEC7.

101. 99. The kit of claim 98, wherein the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, atezolizumab, avelumab, durvalumab, cemiplimab, and any combination thereof.

102. 102. The kit of claim 101, wherein the immune checkpoint inhibitor is pembrolizumab.

103. 103. The kit of any one of claims 95 to 102, wherein the DNase enzyme is selected from human DNase I, human DNase-1-like 3 (D1L3), human DNase-1-like 2 (D1L2), human DNase-1-like 1 (D1L1), DNase X, DNase gamma, DNase II, DNase II alpha, DNase II beta, and caspase-activated DNase (CAD).

104. 103. The kit of any one of claims 95 to 102, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity with human DNase I enzyme.

105. 105. The kit of claim 104, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

106. 103. The kit of any one of claims 95-102, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity to amino acids 21-305 of a DNase 1-like 3 (D1L3) enzyme.

107. 107. The kit of claim 106, wherein the DNase enzyme comprises an amino acid sequence having at least 90% sequence identity with amino acids 21 to 305 of SEQ ID NO:

3.

108. 103. The kit of any one of claims 95 to 102, wherein the DNase enzyme comprises the amino acid sequence SEQ ID NO:

2.

109. 103. The kit of any one of claims 95 to 102, wherein the DNase enzyme is present in the kit in the form of a DNase enzyme protein.

110. 103. The kit of any one of claims 95 to 102, wherein the DNase enzyme is present in the kit in the form of a gene therapy vector encoding the DNase enzyme.

111. The kit of claim 110, wherein the gene therapy vector is a recombinant adeno-associated virus (rAAV) expression vector comprising a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding (i) a capsid protein and (ii) the DNase enzyme.

112. 112. The kit of claim 111, wherein the promoter is selected from a liver-specific promoter, a nervous system-specific promoter, an intestinal-specific promoter, a liver-specific / nervous system-specific tandem promoter, and a liver-specific / intestinal-specific tandem promoter.

113. 112. The kit of claim 111, wherein the promoter is specific to a tumor-initiating tissue or a metastasis target tissue.

114. 112. The kit of claim 111, wherein the AAV is selected from serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAVLK03, AAVLK06, AAVLK12, AAV-KP1, AAV-F, AAVDJ, AAVhu37, AAVrh64R1, and Anc80.

115. 103. The kit of any one of claims 95 to 102, wherein the DNase enzyme is present in the kit in the form of cells that express the DNase and also comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

116. The kit of claim 115, wherein the CAR-expressing cell or the TCR-expressing cell is single-targeted or multi-targeted.

117. 116. The kit of claim 115, wherein the CAR comprises an antigen-binding domain capable of specifically binding to one or more tumor antigens.

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