HK40069964B - Suppression of myeloid derived suppressor cells and immune checkpoint blockade - Google Patents

Description

Suppression of suppressor cells derived from bone marrow and blockade of immune checkpoints

This application is a divisional application. The original application was filed on July 13, 2015, with application number 2015800490412 and invention title "Inhibition of Suppressive Cells Derived from Bone Marrow and Blockade of Immune Checkpoints".

This invention was made with funding from the U.S. government. The U.S. government retains certain rights in this invention under the terms of grant numbers CA062924 and CA043460 from the National Institutes of Health.

Technical Field

This invention relates to the field of cancer treatment. Specifically, it relates to combination therapies for treating refractory tumors.

Background Technology

The mammalian immune system is precisely regulated to effectively combat attacks from invading foreign invaders such as bacteria and viruses with minimal bystander casualties. This requires functionally redundant regulatory mechanisms to ensure safety (1-3). Cancer exhibits the ability to hijack these mechanisms to avoid immune destruction. Several regulatory mechanisms employed by cancer have been identified. These include regulatory T cells (Tregs), circulating MDSCs, resident tumor-associated macrophages and neutrophils, checkpoints that inhibit receptors on T cells, and immunosuppressive cytokines (4-8). Recently, checkpoints protected by PD-1 and CTLA-4 receptors have been extensively studied due to the availability of antibodies that can inhibit their function. Current clinical trials using anti-CTLA-4, anti-PD-1, and anti-Pd-L1 monoclonal antibodies (mAbs) have shown significant therapeutic responses (9-12), highlighting the potential therapeutic usefulness of disrupting immune checkpoints. However, targeted responses have been observed in only a minority of treated patients and tumor types, and the reasons why some tumors respond while others do not remain a mystery. CT26 and 4T1 are among the most prevalent syngeneic tumor models used to evaluate novel therapeutic approaches. CT26 is derived from undifferentiated colorectal cancer induced in BALB/c mice by repeated intrarectal instillation of N-nitroso-N-methylurethane and has shown adequate immunogenicity (13, 14), whereas 4T1 is derived from spontaneous mammary tumors in BALB/c mice (15). 4T1 exhibits poor immunogenicity and high metastatic potential, characteristics shared with advanced human cancers (16). Despite their widespread use in cancer research, very little genetic characterization is available for either of them.

There is a persistent need in this field to address the problem of treating refractory cancers, enabling remissions to be sustained for longer periods and to be more widely available to the treated population.

Summary of the Invention

According to one aspect of the invention, a method for treating tumor-bearing mammals is provided. At least one first agent that inhibits bone marrow-derived suppressor cells (MDSCs) is administered to the mammal. At least one second agent that blocks immune checkpoints is administered to the mammal. The tumor may or may not be non-small cell lung cancer (NSLC).

According to another aspect of the invention, a kit is provided. The kit comprises a single encapsulated package containing at least one first agent that inhibits bone marrow-derived suppressor cells (MDSCs). It further contains at least two second agents that block at least two immune checkpoints.

In another aspect of the invention, a composition is provided. The composition comprises at least one first agent that inhibits bone marrow-derived suppressor cells (MDSCs) and at least two second agents that block at least two immune checkpoints.

According to another aspect of the invention, a method for treating tumor-bearing mammals is provided. The mammal is given at least one first agent that inhibits bone marrow-derived suppressor cells (MDSCs). The mammal is given at least two second agents that block at least two immune checkpoints.

In another aspect of the invention, a method for treating mammals with bacterial or viral infections is provided. The method involves administering at least one agent that inhibits myeloid-derived suppressor cells (MDSCs). The agent is selected from the group consisting of histone deacetylase inhibitors, DNA methyltransferase inhibitors, p110α subunits of phosphonoinositol 3-kinase (PI3K) inhibitors, and combinations thereof.

Those skilled in the art will understand upon reading this specification that these and other embodiments provide therapeutic formulations and methods for treating refractory tumors.

Attached Figure Description

Figures 1A to 1G. Therapeutic response in tumor-bearing mice. BALB/c mice carrying different tumors were treated with various therapeutic modalities as indicated. IgG, IgG control; P, anti-PD-1 antibody; C, anti-CTLA-4 antibody; AZA, 5-azacytidine; ENT, entinostat. Tumor volume (Figures 1A, 1C, and 1E) and animal survival (Figures 1B, 1D, and 1F) were recorded. (Figures 1A and 1B) BALB/c mice with moderately sized CT26 tumors. (Figures 1C and 1D) BALB/c mice with large CT26 tumors. (Figures 1E and 1F) BALB/c mice with metastatic 4T1 tumors. (Figure 1G) 4T1 tumor-bearing mice treated as indicated and euthanized 6 weeks after tumor implantation. Primary tumors from each mouse were measured and metastatic lesions (metabolic equivalents) in different organs were statistically analyzed. Means and standard deviations are shown. It also indicates the number of animals (n) used in each experimental group and the p-value. *P<0.05, **P<0.01, ***P<0.001, ns, not significant.

Figures 2A through 2H. Immune cell responses following immune checkpoint blockade and epigenetic regulation. BALB/c mice carrying metastatic 4T1 tumors were treated in a therapeutically indicated manner, followed by FACS and immunohistofluorescence analysis to assess tumor-infiltrating and circulating immune cells. Mean and standard deviation are shown, where p-values are indicated. (Figure 2A) FACS results for tumor-infiltrating CD8 ⁺ T cells. (Figure 2B) Representative immunohistofluorescence staining of tumor-infiltrating CD8 ⁺ T cells. Scale bar, 50 μm. (Figure 2C) FACS results for tumor-infiltrating CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs. (Figure 2D) Representative FACS data showing the percentage of FoxP3 and CD25 double-positive cells in CD45 ⁺ CD3 ⁺ CD4 ⁺ gated tumor-infiltrating cells. (Figure 2E) FACS results for circulating G-MDSCs. (F) shows representative FACS data for the percentage of Ly6G ⁺ Ly6C ^lo cells in CD45 ⁺ CD11b ⁺ F4/80 ^- MHC-II ^- gated circulating cells. (Fig. 2G) FACS results for tumor-infiltrating G-MDSCs. (Fig. 2H) Representative immunohistofluorescence staining of tumor-infiltrating Ly6G ⁺ cells. Scale bar, 50 μm.

Figures 3A to 3C. Ly6G ⁺ cells derived from bone marrow induce resistance to immune checkpoint blockade. (Figure 3A) BALB/c mice carrying 4T1 tumors are treated with various antibodies or antibody combinations as indicated, and tumor volume is recorded over time. αLy6G, anti-Ly6G antibody; αCD25, anti-CD25 antibody. (Figure 3B) FACS results for circulating G-MDSCs after treatment with different antibodies or antibody combinations. (Figure 3C) 4T1 tumor-bearing mice are treated with anti-PD-1/anti-CTLA-4 antibodies plus an epigenetic modulator containing or not containing adoptive MDSCs isolated from 4T1 tumor-bearing animals via affinity purification. Tumor volume is recorded after treatment. Mean and standard deviation are shown, where p-values are indicated.

Figures 4A to 4D. Direct effects of epigenetic regulators on cultured cells. (Figures 4A and 4B) 4T1 cells, purified CD8 ⁺ T cells, or G-MDSCs were treated with different concentrations of entenoxetine (Figure 4A) or AZA (Figure 4B). Cell viability was assessed using a metabolism-based colorimetric assay. (Figure 4C) IFN-γ content in regulated culture media co-cultured with different ratios of G-MDSCs and CD8 ⁺ T cells was analyzed. (Figure 4D) IFN-γ content in regulated culture media co-cultured with a 1:1 ratio of G-MDSCs to CD8 ⁺ T cells was collected after 24 hours of continuous treatment with entenoxetine and analyzed. Mean and standard deviation of data from at least three replicate wells are shown. p-values are indicated.

Figure 5. Body weight measurements. BALB/c mice carrying 4T1 tumors were treated with the indicated therapeutic method. Their body weight was measured and recorded regularly after treatment. The mean versus standard deviation data are shown.

Figures 6A to 6B. Higher G-MDSC levels induced by 4T1 tumors. (Figure 6A) Peripheral blood was collected at the indicated time points after 4T1 tumor implantation, and G-MDSC levels were analyzed by FACS. (Figure 6B) Peripheral blood, spleen, and tumor were collected from healthy mice or tumor-bearing mice on day 18 after 4T1 tumor implantation, and G-MDSC levels were analyzed by FACS. Data are shown as mean versus standard deviation.

Figure 7. Gene expression involved in MHC-I presentation. RNA was isolated from in vitro cultured 4T1 and CT26 tumor cells after treatment with epigenetic regulators. The expression of different genes involved in MHC-I presentation was assessed by RT-PCR. β-actin was used as an internal reference. Figure 7A, untreated; Figure 7B, treated with AZA; Figure 7C, treated with entenoxate; Figure 7D, treated with AZA/entenoxate.

Figures 8A through 8C. PI3K inhibitors eradicate 4T1 tumors by consuming G-MDSCs when combined with immune checkpoint blockade. (Figure 8A) G-MDSCs, CD8 ⁺ T cells, and 4T1 tumor cells were treated in vitro with various concentrations of J32. Cell viability was assessed using a metabolism-based colorimetric assay. The mean and standard deviation of data from three replicate wells are shown. (Figure 8B) BALB/c mice carrying 4T1 tumors were treated with the indicated therapeutic modality. FACS analysis was performed to quantify circulating G-MDSCs. (Figure 8C) BALB/c mice carrying 4T1 tumors were treated with J32, anti-PD-1/anti-CTLA-4 antibody, or a combination thereof, and tumor volume was recorded. The mean and standard deviation are shown, where p-values are indicated.

Figure 9. Meier-Kaplan curves for combination therapy. From lowest to highest curve: Clostridium novilis alone -NT; anti-PD-1/anti-CTLA-4 alone; anti-PD-1/anti-CTLA-4 + Clostridium novilis -NT; anti-PD-1/anti-CTLA-4 + ENT/AZA; anti-PD-1/anti-CTLA-4 + ENT/AZA + Clostridium novilis -NT.

Detailed Implementation

The inventors have developed a therapeutic method involving agents that act on host cells such as MDSCs in the immune system to attenuate and/or inhibit them. Such agents can be epigenetic modulators such as histone deacetylase inhibitors or DNA methyltransferase inhibitors. When used in conjunction with immune checkpoint blockade, epigenetic modulators kill MDSCs at concentrations much lower than those required to kill tumor cells in vitro. At the doses used, epigenetic modulators have at best only a small effect on tumor cells in vivo; reducing MDSCs with antibodies against them has antitumor effects similar to those observed with epigenetic modulators. In adoptive transfer experiments, the therapeutic effect of epigenetic modulation was eliminated from MDSCs purified from untreated tumor-bearing mice. Inhibition of MDSCs with a completely different class of agents (PIK3 inhibitors) has effects similar to those of epigenetic modulators.

Tumors that can be treated according to the methods and/or kits and/or compositions of the present invention are both solid tumors and hematologic malignancies. Exemplary tumors include adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain/CNS tumors, pediatric brain/CNS tumors, breast cancer, male breast cancer, cancer in adolescents, cancer in children, cancer in young adults, unknown primary cancer, Kassman disease, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing family tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, adult leukemia-acute lymphoblastic (ALL), leukemia-acute myeloid (AML), leukemia-chronic lymphoblastic (CLL), leukemia-chronic myeloid (CML), and leukemia-chronic myeloid monocytic (CM) ML), childhood leukemia, liver cancer, lung cancer - non-small cell lung cancer, lung cancer - small cell lung cancer, lung tumors, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

Types of bacterial infections treatable by the method according to the present invention include Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella ovis, Brucella swine, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis and Enterococcus faecium, Escherichia coli (usually), and enterotoxic Escherichia coli (E. coli). TEC), enteropathogenic Escherichia coli, Escherichia coli O157:H7, Tula Francisella, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia spp., Salmonella typhi, Salmonella typhimurium, Shigella spp., and Staphylococcus aureus.

The types of viral infections treatable according to the present invention include both chronic and acute infections. Exemplary infections include respiratory viruses such as adenovirus, avian influenza, influenza A virus, influenza B virus, measles, parainfluenza virus, respiratory syncytial virus (RSV), rhinovirus, SARS-CoV, gastrointestinal viruses such as Coxsackievirus, enterovirus, poliovirus, rotavirus, hepatitis viruses such as hepatitis B virus, hepatitis C virus, bovine diarrhea virus (alternative), and herpesviruses such as herpes simplex virus 1, herpes simplex virus 2, human cytomegalovirus, and varicella-zoster virus. Retroviruses such as human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), simian immunodeficiency virus (SIV), sima-human immunodeficiency virus (SHIV), and viral selective agents/emerging viral pathogens such as avian influenza, dengue virus, hantavirus, febrile hemorrhagic virus, lymphovirus, smallpox virus substitutes, cowpox, monkeypox, rabbitpox, cowpox virus, Venezuelan equine encephalomyelitis virus (VEE), West Nile virus, and yellow fever virus.

Antibodies that can be used for targeted immune checkpoints or MSDCs can be of any isotype. They can be humanized or chimeric or derived from other mammals or animals. They can bind to other parts such as toxins. They can be monoclonal or polyclonal. They can be single-chain antibodies, fragments of antibodies, or portions of antibodies.

The administration modalities for therapeutic agents may be those used in this art. Examples include oral, topical, inhalation, and injection. Sites of administration include epidermal or topical, nasal, intra-articular, intra-articular, intracardiac, intramuscular, intradermal, intralesional, intraosseous, intraperitoneal, intrathecal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, percutaneous, transmucosal, avitreal, subcutaneous, percutaneous, and transmucosal.

Treatable mammals include any of the mammals susceptible to tumors, blood cancers, bacterial infections, or viral infections. These include: the South American porcupine (formerly known as Przewalski's hooves), alpaca, anteater, giant anteater, armadillo, southern (formerly known as La Plata) tribanded armadillo, striped mongoose, sloth bear, bear, Andean or striped-eyed bear, beaver, American beaver, bison, American bison (SCBI Front Roval only), lynx-caracal, cat, fishing cat, guinea pig, rock guinea pig, cheetah-African cheetah, clouded leopard-clouded leopard, South American raccoon-white-nosed raccoon, colobus monkey, black and white or East African black and white colobus monkey, cow, Hereford cattle-temperate yellow cattle, cow, Holstein dairy cattle-temperate Holstein dairy cattle, deer, Burmese brow deer (Slope deer) Eld's deer (Cervus eldi thamin) (SCBI Front Royal only), deer, tufted crested deer (SCBI Front Royal only), stag rodent (Cervus eldi thamin), donkey, miniature African wild ass, elephant, Asian elephant, elephant shrew, short-eared elephant shrew, ferret, black-footed stoat, antelope (Oryza satyr), gibbon, white-cheeked gibbon, goat, Nigerian dwarf goat, goat, Nubian black goat, goat, St. Clementi-St. Clementi black goat, gorilla, Weston lowland gorilla, pig, Osborne wild boar, horse, Przewalski's horse or Przewalski's horse, hyrax (African hyrax) Lemurs, Ring-tailed Lemurs, Red-fronted Brown Lemurs (a subspecies of brown lemurs) - Brown Lemurs, Lemurs, Red-collared Lemurs, Lions, Barbary Lions, Macaques, Sulawesi Black-crested Macaques (formerly known as Sulawesi), Lion-tailed Macaques, (White-headed Marmosets or Truncatula Marmosets) - Marmosets, Meerkats - Civets, Moles, Damaralan Rat, Moles, Naked Mole-Rat, Monkeys, Black Howlers - Black Howlers (Alouatta caraya), Meerkats, Pygmy Meerkats, Central Asian Wild Asses, Persian Wild Asses (SCBI Front Roval only), Orangutans, Bornean Apes, Orangutans, Sumatran-Bornean Apes, Antelopes, Slanted Antelopes, Water Otters, Asian small-clawed otters, otters, North American river otters, pandas, giant pandas, red pandas, wild boars, peccaries, porcupines, Brazilian tree porcupines, prairie dogs, groundhogs, rabbits, silver fox-rabbit, lemurs, Guyana (grey-headed) white-faced lemurs, seals, grey seals, swathenogias, sloths, two-toed sloths, squirrels, Prevost squirrels, tamarisks, golden lion-marionettes, tamarisks, golden lion-faced marmosets, Madagascar hedgehogs, large Madagascar hedgehogs, small Madagascar hedgehogs, tigers, Sumatran tigers, marmosets, dark gibbons, tree shrews, northern tree shrews, wolves, maned wolves and zebras, Grevy's zebras, pets, draft animals, and wild animals can be treated like humans.

Kits are typically packaged or contained in separate or unseparated containers holding multiple components. Components may be separate or mixed. They may be pharmaceuticals and/or delivery devices and/or instructions. They may include mixing containers or devices.

Clostridium novipae spores can also be used in combination with another agent described herein. Adding Clostridium spores achieves arbitrary or even high levels of survival in tumor-bearing individuals, even if it is less effective alone than other treatments. The amount of spores administered can be, for example, ^10⁵ to ^10⁹ , ^10⁶ to ^10⁸ , or 1 × ^10⁷ to ^10⁸ /mouse. In one embodiment, the amount of spores is 5.0 × ^10⁷ /mouse. Balb/c mice average approximately 20-30g. The amount of spores can be adjusted proportionally according to the recipient's weight.

Although the precise mechanism of action of the spores is not yet known, it is well known that Clostridium novilis-NT infection can induce a CD8+ T cell-mediated adaptive immune response against Clostridium novilis-NT-infected tumors. Furthermore, it is well known that CD8 ⁺ T cells from Clostridium novilis-NT-cured mice can confer adoptive resistance in a tumor-specific manner. Therefore, CD8 ⁺ T cells are involved in beneficial effects. See Agrawal et al., Proceedings of the National Academy of Sciences of the United States of America (Proc Natl AcadSci U S A.), Oct 19, 2004; 101(42): 15172-15177. One hypothesis is that stable inflammatory responses induced by bacterial infection generally promote adaptive immune responses (against both bacteria and tumor cells).

While the applicant does not wish to be bound by any theory regarding the described mechanism of action, it is reasonable to assume that, in order to generate an effective antitumor immune response, two crucial components should be in the appropriate place: (a) if a strong tumor antigen is absent, a strong tumor antigen or a stable inflammatory response to enhance the adaptive antitumor immune response (e.g., provided by Clostridium novie NT infection); and (b) cytotoxic T cells not inactivated by immune checkpoints (e.g., provided by anti-PD-1/anti-CTLA/4 antibody therapy) or MDSCs (e.g., provided by entenostatin/5-azacitidine).

Current clinical studies suggest that epigenetic regulation plays a major therapeutic role in a small subset of patients with non-small cell lung cancer (NSCLC) (36). Other studies have shown that 5-azacitidine upregulates genes and pathways related to both innate and adaptive immunity, as well as immune evasion genes in the NSCLC lineage (35). These crucial studies, along with current clinical trials involving immune checkpoint blockade, have led to the initiation of a clinical trial in NSCLC patients combining a PD-1 antibody, 5-azacitidine, and entinostat (http://clinicaltrials.gov/ct2/show/NCT01928576?term=entinostat+pd-1&rank=1). It is significant to determine the importance of both changes in gene expression in tumor cells and changes in the number and function of MDSCs in this trial. Our observations raise several questions. For example, what is the underlying mechanism of selective inhibition of MDSCs by epigenetic and PI3K inhibitors? Can other methods targeting immunosuppressive cells (e.g., myelosuppressive agents) synergize with immune checkpoint blockade to completely eradicate solid tumors and their metastases? Does initiating immune checkpoint blockade with epigenetic inhibitors work as effectively as the two-component administrations performed in the current study? Experiments addressing these questions could lead to more effective therapies utilizing anti-interference capabilities.

The foregoing disclosure generally describes the invention. All references disclosed herein are expressly incorporated by way of reference. A more thorough understanding can be obtained by referring to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1

Materials and methods

Reagents. Heklon RPMI 1640 and McCoy's 5A containing L-glutamic acid were purchased from Invitrogen Life Technologies. Heklon fetal bovine serum (FBS) was purchased from Thermo Scientific. Collagenase from Clostridium histolyticum, type IV, was purchased from Sigma-Aldrich. The following antibodies and reagents were used in animal experiments: mCD152 (mCTLA-4) monoclonal antibody (9H10, BioXCell), mPD-1 monoclonal antibody (RMP1-14, BioXCell), mCD25 monoclonal antibody (PC61.5.3, BioXCell), mLy6G monoclonal antibody (RB6-8C5, BioXCell), polyclonal Hampster IgG (BioXCell), entenitol (BPS Bioscience), and 5-azacytidine (Invivogen).

Cell lines. 4T1 (CRL-2539, mouse mammary tumor cells) and CT26 (CRL-2638, mouse colorectal adenocarcinoma) were purchased from ATCC. Both tumor cell lines were grown in McCoy's 5A supplemented with 10% fetal bovine serum at 37°C and 5% CO2.

Preparation of Illumina Genomic DNA Library. The genomic DNA library was prepared according to Illumina's (Illumina) recommended method with the following modifications. 2 μg to 3 μg of genomic DNA was diluted with TE buffer to a final volume of 100 μl and sheared to an average size of 200 bp in a Covaris sonicator. The DNA was then purified using a Nucleospin kit (Macherey-Nagel) and eluted with 50 μl of elution buffer. Unless otherwise noted, all reagents used in the following steps were from New England Biolabs (NEB). 45 μl of purified DNA was then mixed with 40 μl of ddH2O, 10 μl of end-repair buffer, and 5 μl of end-repair enzyme buffer. The mixture was incubated at 20 °C for 30 min, purified using a Qiagen PCR purification kit (Qiagen), and eluted with 42 μl of elution buffer (EB) heated to 70 °C. The end-repair reaction was then performed using 42 μl of end-repaired DNA, 5 μl of 10×dA tailing reaction buffer, and 5 μl of Clenno fragment (3' to 5' beyond) for A-tailing, and incubated at 37°C for 30 min, followed by purification using the MinElute PCR Purification Kit (Kaiger). The purified DNA was eluted with 27 μl of EB at 65°C. Conjugation was performed using 25 μl of A-tailed DNA, 10 μl of PE conjugate (Illumina), 10 μl of 5X conjugation buffer, and 5 μl of rapid T4 conjugase. The conjugation mixture was incubated at 20°C for 15 min. Purification was then performed by mixing 50 μl of the conjugation mixture with 200 μl of NT buffer from the NucleoSpin Extraction II Kit (Clontech) and loading it onto a NucleoSpin column. The column was centrifuged at 14,000g for 1 min in a benchtop centrifuge, washed once with 600 μl of wash buffer (NT3 from Clontech), and centrifuged again for 2 min until completely anhydrous. DNA was eluted in 50 μl of elution buffer included in the kit. The purified conjugating DNA was amplified under the following conditions. Approximately 10 reactions were set up, consisting of the following: 32.5 μl H2O, 2.5 μl (DMSO), 10 μl 5X Phusion HF buffer, 1.0 μl of a dNTP mixture containing 10 mM of each dNTP, 0.5 μl Illumina PE primer #1, 0.5 μl Illumina PE primer #2, 0.5 μl Hotstart Phusion polymerase, and 5 μl conjugating DNA. The PCR program used was: 98°C for 1 minute; 10 to 16 cycles of 98°C for 20 seconds, 65°C for 30 seconds, and 72°C for 30 seconds; and 72°C for 5 minutes. The reaction was then pooled and purified using a 1:2 mixture of PCR product from the NucleoSpin Extract II kit and NT buffer, as per the kit's included purification protocol. Library DNA was eluted with 70°C elution buffer, and DNA concentration was assessed using nanodrop absorbance at 260 nm. The samples were then subjected to SureSelect exome separation.

Exome capture. Mouse exomes were captured according to the method from the Agilent SureSelect Paired Exome Kit (Agilent), with the following modifications. (1) A hybrid mixture was prepared containing 25 μl of SureSelect Hyb#1, 1 μl of SureSelect Hyb#2, 10 μl of SureSelect Hyb#3, and 13 μl of SureSelect Hyb#4. (2) 3.4 μl (0.5 μg) of the above PE-library DNA, 2.5 μl of SureSelect blocking #1, 2.5 μl of SureSelect blocking #2 and 0.6 μl of blocking #3; add to one well of a 384-well diamond PCR plate (catalog number AB-1111, Thermal Science), seal with two layers of microamplifier transparent adhesive film (catalog number 4306311; ABI), and place in a GeneAmp 9700 (Life Sciences) system at 95°C for 5 min, then maintain at 65°C (with a heat-sealed lid). (3) 25 μl of hybrid buffer from step (1) is heated at 65°C for at least 5 min in another sealed plate with a heat-sealed lid. (4) Mix 5 μl of SureSelect Oligo Capture Library, 1 μl of nuclease-free water, and 1 μl of diluted RNase blocker (A 1:1 RNase blocker and nuclease-free water mixture) and heat at 65°C for 2 min in another sealed 384-well plate. (5) While maintaining the reaction at 65°C, rapidly add 13 μl of hybridization buffer from step (3) to 7 μl of the SureSelect capture library mixture from step (4), followed by the entire contents (9 μl) of the library from step (2). Blow the mixture up and down 10 times. (6) Seal the 384-well plate tightly and incubate the hybridization mixture at 65°C with a heated lid for 24 hours. Following hybridization, five steps were performed to restore and amplify the captured DNA library: (1) 50 μl of Dynal MyOne streptococcal C1 magnetic beads (CAT#650.02, Invitrogen Dynal) were placed in a 1.5 ml microcentrifuge tube and vigorously resuspended on a vortex mixer. The beads were washed three times with 200 μl of SureSelect binding buffer, vortexed for five seconds, and then placed in a Dynal magnetic separator tube after removing the supernatant. After the third wash, the beads were resuspended in 200 μl of SureSelect binding buffer. (2) To bind the captured DNA, the entire hybrid mixture (29 μl) was transferred directly from the thermal cycler to the bead solution and then inverted four times to mix; the hybrid mixture/bead solution was then incubated in an Eppendorf thermal mixer at 850 rpm at room temperature for 30 min. (3) To wash the beads, the supernatant was removed from the beads after applying the Dynal magnetic separator, and the beads were resuspended in 500 μl of SureSelect wash buffer #1 by mixing on a vortex mixer for 4 seconds and incubating at room temperature for 15 min. After magnetic separation, wash buffer #1 was then removed from the beads. After incubating at 65°C for 10 min, the beads were washed three more times, each time with 500 μl of preheated SureSelect wash buffer #2. After the final wash, SureSelect wash buffer #2 was completely removed. (4) To elute the captured DNA, the beads were suspended in 50 μl of SureSelect elution buffer, vortexed, and incubated at room temperature for 10 min. After magnetic separation, the supernatant was removed, collected in a new 1.5 ml microcentrifuge tube, and mixed with 50 μl of SureSelect neutralization buffer. The mixture was purified using a Qiager MinElute column and eluted in 17 μl of 65°C buffer EB to obtain a 15 μl captured DNA library. (5) The captured DNA library was amplified as follows: approximately 15 PCR reactions, each containing 9.5 μl _H₂O , 3 μl 5x Phusion HF buffer, 0.3 μl 10 mM dNTPs, 0.75 μl DMSO, 0.15 μl Illumina PE primer #1, 0.15 μl Illumina PE primer #2, 0.15 μl Hotstart Phusion polymerase, and 1 μl of the captured exome library. The PCR program used was: 14 cycles of 98°C for 30 min; 98°C for 10 s, 65°C for 30 s, 72°C for 30 s; and 72°C for 5 min. To purify the PCR product, 225 μl of the PCR mixture (from the 15 PCR reactions) was mixed with 450 μl of NT buffer from the NucleoSpin Extract II kit and purified as described above. The final library DNA was eluted with 30 μl of 65°C elution buffer and the DNA concentration was estimated by OD ₂₆₀ measurement.

Preparation of cDNA for sequencing analysis. mRNA was prepared from 5 μg to 10 μg of total RNA using Dynal oligodeoxythymidine magnetic beads according to the manufacturer's method (BioTechnology) with two rounds of poly-A selection, followed by a second elution with 13 μl of elution buffer. Double-stranded (ds) cDNA was prepared using a top-labeled ds-cDNA kit (Invitrogen) with the following modifications: 12 μl of isolated mRNA was added to 2 μl of 50 ng/μL random hexamer and incubated at 70 °C for 10 min, then placed on ice. A mixture of 4.4 μl of 5X first-strand buffer, 2.2 μl of 0.1 M DTT, and 1.1 μl of 10 mM dNTPs was added to the tube and incubated at 45 °C for 2 min. 1.5 μl of SSII enzyme was added, and the entire mixture was incubated for another 1 h, then placed on ice. Second-strand cDNA was prepared by adding 90 μl of _ddH₂O , 30 μl of 5X second-strand buffer, 3 μl of 10 mM dNTPs, 4 μl of DNA PolI, 1 μl of RNase H, and 1 μl of E. coli DNA conjugase to the first-strand reaction. The mixture was then incubated at 16°C for 2 hours, followed by the addition of 2 μl of T4 conjugase and incubation for another 5 minutes. The resulting cDNA was then purified using a Qiager PCR purification kit according to the manufacturer's instructions, eluting twice with 50 μl of 70°C elution buffer each time. 100 μl of cDNA was used to construct an Illumina library according to the genomic DNA library method using cDNA in place of genomic DNA.

Somatic mutation identification. The library was sequenced on an Illumina GAIIx or HiSeq genome analyzer. Sequencing reads were analyzed using CASAVA (Illumina) and aligned to the mouse genome mm9. Mismatched bases were identified as mutations only if (i) the mismatched bases consisted of at least two reads in the forward direction and two in the reverse direction, or if (ii) the number of different markers containing the specific mismatched bases was at least 30% of the total number of different markers.

The identification of the hypothetical H2-(d) epitope expression was determined by cross-referencing RNASeq data with control RNASeq data. Eight amino acids upstream and downstream of the mutated residues were isolated, corresponding to amino acid changes associated with positive mutations, and then used for epitope identification. These seventeen amino acid sequences were processed using the netMHC epitope identification algorithm (netMHC v3.4) for potential nine-amino acid binding agents up to H2-K(d), H2-L(d), and H2-d(d). A cutoff value of 500 nM affinity or higher was used, corresponding to moderate to high affinity binding agents. Gene expression was confirmed if the normalized count (normalized length/million readings) of the RNASeq data was greater than 0.5.

Animal models. Animal studies were approved and regulated by the Johns Hopkins University Institutional Animal Care and Use Committee. Large female BALB/c mice (Harlan Laboratories) at weeks 6–8 were used for all animal experiments. 5 × ^10⁶ 4T1 tumor cells or 5 × ^10⁶ CT26 tumor cells were subcutaneously inoculated into the appropriate side of each mouse. Tumors were allowed to grow for 11 days prior to randomization and treatment. Mice carrying CT26 tumors were administered 10 mg/kg of anti-PD-1 and/or anti-CTLA-4 antibodies intraperitoneally on days 11, 13, 15, 17, 20, 23, and 26 post-tumor implantation. Mice carrying 4T1 tumors were administered 10 mg/kg of anti-PD-1 and/or anti-CTLA-4 antibodies intraperitoneally on days 11, 13, 15, and 17 post-tumor implantation. Mice carrying 4T1 tumors were administered a single dose of 10 mg/kg anti-CD25 antibody or 10 mg/kg anti-Ly6G antibody intraperitoneally on day 12 post-tumor implantation for cell consumption studies. All antibodies were diluted to appropriate concentrations in 100 μl of sterile PB at pH 7.4 (Invitrogen Life Technologies). Entenolol and 5-azacytidine were administered on day 12 post-tumor implantation at doses of 20 mg/kg and 0.8 mg/kg, respectively. Mice carrying 4T1 tumors received intraperitoneal injections on days 12, 14, 16, and 18. J32 was administered intraperitoneally at 22 mg/kg on days 12, 14, 16, and 18 post-tumor implantation of 4T1 tumors. Tumors were measured 30 days from the start of treatment at the specified intervals in the Results section. Tumor volume was calculated as length × ^width² × 0.5.

Metastasis analysis. Mice carrying 4T1 tumors were euthanized according to the IACUC guidelines on day 46 post-tumor implantation. Lungs, livers, and spleens were harvested and fixed in 10% neutral buffered formalin (Sigma-Aldrich), and metastases were counted from at least three mice in each group.

Flow cytometry. The following antibodies and reagents were used for flow cytometry: CD16/32 (BD Biosciences), CD3e Alexa fluorescent agent 488 (14-C11; BD Biosciences), CD4 Brilliant Violet 421 (GK1.5, BD Biosciences), CD8a PerCP-Cy5.5 (53-6.7, BD Biosciences), CD25 PE (PC61, BD Biosciences), Foxp3 Alexa fluorescent agent 647 (MF23, BD Biosciences), CD11b Alexa fluorescent agent 700 (M1/70, BD Biosciences), Ia/Ie Alexa fluorescent agent 488 (M5/114.15.2, BD Biosciences), Ly-6C PerCP-Cy5.5 (Al-21, BD Biosciences), CD11c PE (HL3, BD Biosciences), F4/80APC (BM8, Biolegend), Ly-6G Pacific Blue (1A8, Biolegend), CD45 Pacific Orange (30-F11, Ingenium Biotech), and staining for viable/dead fixed near-IR dead cells (Ingenium Biotech). Flow cytometry was performed using LSR II (BD Biosciences) and data were analyzed using FACS Diva software (BD Biosciences). To assess circulating G-MDSC cell counts, blood samples were collected from mice 7 days after initiation of anti-PD-1/anti-CTLA-4 antibody treatment with or without 5-azine/entinolide. 150 μl of blood was collected from the right or left superficial vein in _K2 EDTA BD microsampling (BD Biosciences). RBCs from anticoagulated blood samples were immediately lysed for 3 minutes using 2 ml of 1X BD FACS (BD Biosciences) and the samples were washed twice in ice-cold BD FACS buffer (BD Biosciences). After 5 minutes of incubation with live/dead fixable near-IR dead cells and washing twice with ice-cold BD FACS buffer, samples were stained with appropriate antibodies. For analysis, we used previously established phenotypic markers of CD45 ⁺ CD11b ⁺ Ly6G ⁺ Ly6C ^lo F4/80MHCII cells and total CD45-positive cells as common characteristics. To equalize intratumoral CD8 ⁺ and regulatory T cell population levels, lymphocytes were first purified from mouse tumor samples after 7 days of treatment with anti-PD-1/anti-CTLA-4 antibodies, with or without 5-AZA/entinolide. In short, primary tumor tissue was collected, weighed, and minced to a fine fragment. 1 mg/ml collagenase IV (Sigma-Aldrich) in HBSS (Ingenie Life Sciences) was added to each sample at a ratio of 1 ml/200 mg tumor tissue. Samples were incubated at 37°C for 30 minutes on a vertical separator. The resulting tissue homogenate was 0.4 μm, filtered, washed three times in ice ^- cold BD FACS buffer (BD Biosciences), and 5 × ^10⁶ cells/sample were used for antibody labeling. ^CD8⁺ T cell levels were assessed using a previously established phenotypic marker ^{of CD45⁺CD3⁺CD8⁺} , ^with total ^{CD45⁺CD3⁺} cells used ^as a common characteristic. Treg cell ^levels were assessed using a previously established ^phenotypic marker ^{of CD45⁺CD3⁺CD4⁺CD25⁺FoxP3⁺ ,} with total ^{CD45⁺CD3⁺CD4⁺ cells} used as a common characteristic.

Cell isolation. MDSCs from animals carrying 4T1 tumors were isolated from the spleen using a bone marrow-derived suppressor cell isolation kit, mice (Miltenyi Biotec), and a BD FACS Aria III cell sorter (BD Biosciences). CD8 ⁺ cells were isolated from the spleen of animals carrying 4T1 tumors treated with anti-PD-1 and anti-CTLA-4 antibodies using a CD8a+ T cell isolation kit II, mice (Miltenyi Biotec), and a BD FACS Aria III cell sorter (BD Biosciences). MDSCs, Tregs, and CD8 ⁺ T cells were isolated and cultured according to the manufacturer's and published protocols (38 ^, 39). As determined by flow cytometry, the purity of the G-MDSC (CD11b ⁺ Ly6G ⁺ Ly6C ^lo F4/80 ^- MHC-II ^- ) and CD8 ⁺ (CD3 ⁺ CD8 ⁺ ) groups is greater than 95%, and for these groups, as determined by trypan blue staining, the viability is greater than 95%.

In vitro viability assay. MDSCs and CD8 ⁺ T cells were plated in 96-well plates at 2 × ^10⁶ cells/mL in RPMI 1640 medium supplemented with 10% FBS. CD8 ⁺ T cell culture medium was supplemented with 2000 U/mL of recombinant interleukin-2 (Yingjie Life Technology). 4T1 cells in McCoy 5A supplemented with 10% FBS were plated in 96-well plates and cultured until they reached >70% confluence. Cells were cultured for 24 hours at 37°C and 5% _CO₂ using entenoxate, 5-azacytidine, or J32 at concentrations ranging from 0 μM to 50 μM. The viable cell percentage was determined by incubating cells at 37°C for 3 hours with 10% (v/v) cell proliferation reagent WST-1 (Roche Applied Science) and measuring the OD ₄₅₀ absorbance of the resulting formaldehyde product.

IFN-γ assay. MDSCs and CD8 ⁺ T cells isolated from animals carrying 4T1 tumors were cultured at MDSC/CD8 ⁺ T cell ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 in the presence of 2000 U/mL recombinant IL-2 (Ingenie Life Sciences) and CD3/CD28 antibody-coated beads (Miltenyi). MDSCs and CD8 ⁺ T cells were cultured at a 1:1 ratio with 0 μM entenoxate at a concentration range of 0 μM. After 24 hours of incubation at 37°C, cell-free supernatant was collected, and IFN-γ levels were determined using the Mouse IFN-γ DuoSet Elisa Development Kit (R&D Systems) according to the manufacturer's instructions.

MDSC Consumption and Adoptive Transfer. To consume MDSCs in vivo, mice bearing 4T1 tumors were treated intraperitoneally with a single-particle mLy6G monoclonal antibody at 10 mg/kg 11 days after tumor implantation. For adoptive transfer of MDSCs, spleens were collected from mice bearing 4T1 tumors, and MDSCs were purified using a bone marrow-derived suppressor cell isolation kit (Mice, Technion Biotechnology). After column purification using two sequences, cells were washed twice in ice-cold 1×PBS (Ingenie Biotech) and the cell concentration was adjusted to 1× ^10⁸ cells/mL. Cell viability was greater than 95% as determined by trypan blue staining, and cell purity was greater than 90% as determined by flow cytometry. Immediately after isolation, 100 μl of MDSCs at 1× ^10⁸ cells/mL were administered via tail vein injection on days 11, 13, and 15 post-4T1 tumor implantation.

Immunofluorescence. Mice were euthanized according to JHU IUCAC guidelines, and primary tumors from 4T1 tumor-bearing mice were excised using a sterile, disposable surgical scalpel (Bard-Parker). Excised tissues were placed in a tissue-Tek cryo-10-month (Andwin Scientific) alkali mold and stored at -80°C until use. Frozen tissues were sectioned using a Leica CM3050 S cryostat (Leica Biosystems) and fixed for 10 minutes in 1X PBS (Ingenie Life Sciences) with 4% paraformaldehyde (Alfa Aesar) and 0.3% Triton X-100 (Sigma-Aldrich). Sections were washed three times in 1X PBS with 0.05% Tween-20 (Sigma-Aldrich), followed by three 5-minute washes in 1X PBS with 0.05% Tween-20. Tissue was blocked for 30 minutes in 1X PBS using 3% BSA (Sigma-Aldrich) and 0.05% Tween-20, followed by a further 30 minutes of blocking with 10% common goat serum (Yingjie Life Technology). The blocked tissue was incubated overnight at 4°C in anti-CD8 (YTS 169.4, Abcam) or in anti-Ly6G (RB6-8C5, Abcam) at concentrations of 1:50, 1:100, and 1:200. After overnight incubation, the tissue was stained with primary antibodies, washed three times with 0.05% Tween-20 in 1×PBS, and incubated for 1 hour at 20°C with 1:500 goat anti-rat AF488 (Yingjie Life Technology) or goat anti-rat AF594 (Yingjie Life Technology) secondary antibodies. Sections were washed five times in 1×PBS with 0.05% Tween-20. Before placing the coverslip, one drop of Gold/DAPI (Ingenium Life Sciences) was added to the tissue sample, and the sections were stored in the dark at 4°C. For imaging, a Nikon C1 Laser Scanning Confocal System was used, which included an ECLIPSE TE2000-E microscope and an EZ-LIMO for the Nikon C1 confocal v.2.30.

Reverse transcription PCR (RT-PCR). 5 × ^10⁶ CT26 or 4T1 cells were resuspended in 0.75 ml Trizol LS reagent (Ingentech) and 0.25 ml chloroform (Sigma-Aldrich). The sample was vortexed for 15 seconds and incubated at 20°C for 10 minutes. After centrifugation at 12000 g for 15 minutes at 4°C, the supernatant was collected and 0.5 ml of 100% isopropanol (Sigma-Aldrich) was added. The sample was then centrifuged at 12000 g for 10 minutes at 4°C and incubated at 20°C after 10 minutes. The resulting globules were air-dried for less than 10 minutes and resuspended in RNase-free water (Ingentech) to a final concentration of 500 ng/μl. PCR was performed using a single-step RT-PCR system with platinum Taq DNA polymerase (Ingentech) and top-labeled III. For H-2D(d), β2m, and TAP1, the annealing temperature was set at 55°C, and for β-actin, it was set at 60°C. Samples were analyzed on 1% agarose gel. The following primers were used for RT-PCR: H-2D(d) forward 5'-agggcaatgagcagagtttc-3' (SEQ ID NO:1), H-2D(d) reverse 5'-CCACGTTTTCAGGTCTTCGT-3' (SEQ ID NO:2), β2m forward 5'-ATTCACCCCCACTGAGACTG-3' (SEQ ID NO:3), β2m reverse 5'-GCTATTTCTTTCTGCGTGCAT-3' (SEQ ID NO:4), TAP1 forward 5'-GAGACATGCTGTGTCGGATG-3' (SEQ ID NO:5), TAP1 reverse 5'-TGGTGAGAATGGACATGAGC-3' (SEQ ID NO:6), β-actin forward 5'-TTCTTTGCAGCTCCTTCGTTGCCG-3' (SEQ ID NO:7), β-actin reverse 5'-TGGATGGCTACGTACATGGCTGGG-3' (SEQ ID NO:7). ID NO:8).

Statistical analysis was performed using Prism 5.0 (GraphPad Software, Inc.). Primary tumor growth curves were first analyzed using two-way ANOVA, with each group compared to two tailed Wilcoxon rank-sum tests. Kaplan-Meier survival curves were analyzed using log-rank tests. Statistical significance for metastatic lesions, flow cytometry analysis, and in vitro assays was assessed using two tailed Wilcoxon rank-sum tests.

Example 2

Genetic analysis. First, we sequenced the exomes (24,306 genes) of both CT26 and 4T1 cells. The resulting octaves and 3.5 gigabases were mapped to the genomes of CT26 and 4T1, respectively. 83.5% (CT26) and 72.3% (4T1) of the bases in the target regions were covered by at least 10 unique reads in the tumor DNA. Exome sequencing revealed 683 and 47 somatic mutations in CT26 and 4T1, respectively.

~10% of the mutant amino acid-induced epitopes created by somatic mutations in human colorectal and breast cancers have been shown, and their predictions are pending identification via the patient's MHC-I allele (17). To determine whether they are true mouse colorectal (CT26) and breast (4T1) tumors, we used an established algorithm to map somatic mutant epitopes to BALB/c MHC-I. Thus, predictions are only meaningful when the mutant gene is expressed, and we used RNA-seq to determine the transcriptomes of both cell lines. Of the 683 mutants detected in CT26 present in the expressed gene, 28 mutant epitopes were predicted to bind with at least moderate affinity to the H2-(d)MHC-I allele found in BALB/c mice (Table 1). 4T1 cells held 27 mutants in the expressed gene, of which only one was predicted to bind to the H2-(d)MHC-I allele. These data are consistent with the suggestion that CT26 is more immunogenic than 4T1 because its morphogenetic form has more mutant epitopes. They are also consistent with observations that human tumors associated with environmental mutagens (such as UV light and cigarette smoke) have more mutants than other tumors (18).

Table 1. Mutant MHC-I epitopes predicted by genomic and transcriptomic analysis

Example 3

The effect of immune checkpoint blockade. We then tested the effect of immune checkpoint blocking antibodies on tumors derived from these cells in mice. BALB/c mice of moderate size (~400 ^mm³ ) carrying subcutaneous CT26 tumors were used for preliminary experiments. Although repeated treatment with anti-CTLA-4 or anti-PD-1 antibodies, as a single agent, delayed tumor growth, no tumor eradication was observed (Fig. 1A and Fig. 1B). Combination therapy with both antibodies produced tumor eradication in most mice. Conversely, tumors larger than 600 ^mm³ did not respond to combined anti-PD-1/anti-CTLA4 treatment (Fig. 1C), with only 4 out of 11 showing long-term survival (Fig. 1D).

Subsequently, BALB/c mice (~400 ^mm³ ) with well-established 4T1 tumors were evaluated; these tumors had spontaneously metastasized to the lungs and other organs. The 4T1 tumor model was highly resistant to most treatments, including immunotherapy (16). Animals typically died from metastatic disease, even when the primary tumor was surgically removed (19). A smaller number of primary tumors showed a durable response to antibody therapy. Similar to mice with large numbers of CT26 tumors, only 3 out of 10 animals showed complete regression of their primary tumors when treated with anti-PD-1 and anti-CTLA-4 antibodies, and only these were long-term survivors (Figs. 1E and 1F).

Example 4

Epigenetic regulation. We hypothesized that incurable tumors in animals could be caused by downregulating the expression of MHC-I-related genes through epigenetic silencing in tumor cells. In fact, this hypothesis forms the basis for a therapy incorporating epigenetic regulation (20) using either DNA methyltransferase or histone deacetylase (HDAC) inhibitors. To assess this possibility, we treated animals carrying large CT26 tumors (>600 mm³) as described above with anti-PD-1/anti-CTLA-4 antibodies, ⁵ -azacytidine (AZA, a DNA methyltransferase inhibitor), and entenoxetine (ENT, a class I HDAC inhibitor). The tumors responded remarkably well to this treatment, with primary tumor eradication in 10 out of 11 mice and 100% survival 60 days after tumor implantation (Fig. 1D). Similarly, mice with 4T1 tumors (~400 ^mm³ ) treated with anti-PD-1/anti-CTLA-4 plus azirconazole/ENT showed complete regression of all primary tumors after week 3 of treatment and 80% survival for 100 days after tumor implantation (Figs. 1E and 1F). Temporary self-limiting toxicities, as indicated by changes in body weight, were observed when entenostatin was used. However, the addition of anti-PD-1/anti-CTLA-4 antibodies did not add toxicity.

Concurrently with the experiment, we treated 4T1 tumor-bearing mice as described above, but sacrificed them 6 weeks after tumor implantation. We then examined their primary tumors and metastases in the lungs and other organs. Primary tumors were eradicated in all mice treated with anti-PD-1/anti-CTLA-4 antibody plus AZA/entenostatin, and no cancer metastases were observed in any of them (Figure 1G and Table 2). In contrast, all five mice treated with anti-PD-1/anti-CTLA-4 still had substantial primary tumors and an average of 11 lung cancer metastases. We also treated tumor-bearing mice with anti-PD-1/anti-CTLA-4 antibody plus entenostatin or AZA. No tumors or metastases were found in any mice treated with anti-PD-1/anti-CTLA-4 antibody plus entenostatin, indicating that when combined with PD-1/CTLA-4 dual blockade, class I HDAC inhibitors alone (in the absence of DNA methylation inhibitors) are sufficient to eradicate both primary tumors and metastases (Figure 1G and Table 2). In mice treated with anti-PD-1/anti-CTLA-4 antibodies plus azaza, primary tumors were not eradicated, but no metastases were observed. Without PD-1/CTLA-4 inhibition, entenoxate, azazaza, alone or in combination, failed to eradicate primary tumors or metastases (Figure 1G and Table 2). When no PD-1/CTLA-4 inhibition was applied, metastatic lesions were observed in multiple organs besides those in the lungs.

Table 2. Primary and metastatic lesions of 4T1 tumors

Example 5

Mechanistic Study. As mentioned above, we expected that epigenetic regulators would increase the expression of MHC-I-related genes, thereby making cancer cells more susceptible to T cell killing. To test this expectation, we analyzed the expression of genes involved in MHC-I expression via reverse transcription polymerase chain reaction (RT-PCR) in CT26 and 4T1 cells treated with AZA, entinolide, or a combination of both. The expression of MHC-I, β-2 microglobulin (B2M), and the transporter associated with the antigen treatment 1 (TAP1) gene was detected in two tumor cell lines in the absence of treatment. However, exposure to epigenetic regulators did not significantly increase expression (A-D in Figure 7).

We then determined whether the epigenetic modulators affected T cell accumulation within the tumor. As assessed by flow cytometry, tumor-infiltrating CD8+ T cells increased approximately fourfold after PD-1/CTLA-4 inhibition (Figs. 2A and 2B). Addition of AZA and entenostatin did not further increase tumor-infiltrating CD8 ⁺ T cells. However, the inclusion of AZA and entenostatin in the treatment regimen produced a significant reduction in tumor-infiltrating FoxP3+ Tregs compared to untreated tumors or tumors treated with anti-PD-1/CTLA-4 antibodies (Figs. 2C and 2D).

We then analyzed MDSCs by flow cytometry, as these immature cells derived from bone marrow are often found in higher concentrations in tumor-bearing hosts and possess potent immunosuppressive activity (21, 22). We found that 4T1 tumor-bearing mice had a 5- to 7-fold increase in circulating granulocyte MDSCs (G-MDSCs, defined as CD11b ⁺ Ly6G ⁺ Ly6C ^lo MHC- ^II- ) compared to tumor-free animals (Fig. 2E, Fig. 6A, and Fig. 6B). Abundant G-MDSCs were also observed in the spleen and tumor (Fig. 6B). Adding entenostatin or AZA to PD-1/CTLA-4 inhibitors resulted in a striking reduction in the number of circulating G-MDSCs produced, bringing them down to levels similar to those observed in non-tumor-bearing mice (Fig. 2E and Fig. 2F). Interestingly, epigenetic regulators alone or AZA plus anti-PD-1/anti-CTLA-4 antibodies failed to reduce the amount of said G-MDSCs. When combined with immune checkpoint blockade, epigenetic modulators also substantially reduce the number of tumor-infiltrating G-MDSCs (Figure 2G and Figure 2H).

These data are consistent with the hypothesis that immune checkpoint blockade induces an increase in cytotoxic effector T cells (Teffs), but that T cells (Teffs) are not fully functional unless immunosuppressive cells are reduced by treatment with epigenetic modulators. To further test this hypothesis, we used neutralizing antibodies CD25 or Ly6G to deplete Tregs or G-MDSCs (23-25) in mice carrying 4T1 tumors, respectively. We found that anti-Ly6G was as effective as an epigenetic modulator when used in combination with anti-PD-1/anti-CTLA-4 antibodies (Fig. 3A). Flow cytometry showed a substantial reduction in G-MDSC levels after anti-Ly6G treatment (Fig. 3B). In contrast, anti-CD25 treatment, when combined with immune checkpoint blockade, showed only a minor improvement in efficacy (Fig. 3A). However, it should be noted that anti-CD25 treatment can also affect the activation of Teffs, which transiently express CD25. As expected, anti-CD25 and anti-Ly6G are ineffective in the absence of immune checkpoint blockade (Figure 3A).

To directly assess the ability of tumor-induced G-MDSCs to interfere with immune checkpoint blockade effects, we isolated them from 4T1 tumor-bearing mice via affinity purification. We then injected the purified G-MDSCs into 4T1 tumor-bearing mice treated with anti-PD-1/anti-CTLA-4 antibodies plus AZA/entenoxate. Adoptive transfer of G-MDSCs significantly reduced the response to combination therapy (Figure 3C). Based on these results, we conclude that epigenetic regulatory effects are more likely to deplete G-MDSCs than direct depletion of Tregs.

To investigate whether epigenetic regulation directly affects G-MDSCs, we purified these cells from 4T1 tumor-bearing mice as described above and treated them in vitro with entenoxate or AZA. Following treatment with entenoxate in a dose-dependent manner, G-MDSCs showed a significant reduction in viability (Fig. 4A). Conversely, AZA had no effect at comparable concentrations (Fig. 4B). We also treated 4T1 tumor cells with the same concentrations of entenoxate or AZA and found them unresponsive (Fig. 4A and Fig. 4B). Importantly, entenoxate had a modest effect only on CD8 ⁺ T cells (Fig. 4A), creating a large therapeutic window in which G-MDSCs could be consumed while avoiding CD8 ⁺ T cells. Finally, we co-cultured CD8 ⁺ T cells with G-MDSCs and analyzed the concentration of interferon-γ (IFN-γ) in the culture medium via enzyme-linked immunosorbent assay (ELISA) after T cell activation using CD3 and CD28 antibodies. G-MDSCs inhibit IFN-γ secretion (Fig. 4C), however, the inhibition is reversed in a dose-dependent manner when entenostatin is included in the culture medium (Fig. 4D). These data support the concept that G-MDSCs directly inhibit CD8 ⁺ T cell function and that entenostatin alleviates inhibition by directly inhibiting G-MDSCs.

To further confirm this conclusion, and to provide additional therapeutic approaches to achieve the same goal, we searched for other therapeutic agents that can inhibit G-MDSC function. Phosphatidylinositol 3-kinase (PI3K) is known to play a crucial role in hematopoietic cell biology and can activate Gr1 ⁺ /CD11b ⁺ bone marrow cells (26). We previously developed different arrays of PI3K inhibitors and tested one (J32) with high cellular efficacy (27-29). J32 was shown to be cytotoxic to G-MDSCs at nanomolar concentrations (14.3 nM _EC50 ) and much less toxic to CD8 ⁺ T cells (94.6 nM EC50) (Fig. 8A). Treatment of 4T1 tumor-bearing mice with a relatively low dose of J32 (22 mg/kg) in combination with anti-PD-1/anti-CTLA-4 antibodies resulted in a significant reduction in circulating G-MDSCs (Fig. _8B ) and eradication of 4T1 tumors in 80% of the animals (Fig. 8C). J32 alone has no significant effect on the growth of 4T1 tumors.

Example 5

4T1 tumor cells were subcutaneously injected into BALB/c mice. On days 10, 12, 14, and 16 post-tumor cell injection, anti-PD-1 (10 mg/kg) and anti-CTLA-4 (10 mg/kg) antibodies were administered intraperitoneally to mice in groups 2, 3, 4, and 5. On days 11, 13, 15, and 17, entenostatin (ENT, 20 mg/kg) and 5-azacytidine (AZA, 0.8 mg/kg) were administered intraperitoneally to mice in groups 4 and 5. On days 13 and 15, Clostridium novitidis-NT spores (50 million/mouse) were directly subcutaneously injected into the 4T1 tumors in mice in groups 1, 3, and 5. Mice survival was then closely monitored until day 100 post-tumor cell injection. Survival curves are shown in Figure 9. For pathological evaluation, dead mice are dissected, and they always have extensive lung metastases.

The efficacy of checkpoint blockade (anti-PD-1/anti-CTLA-4) plus epigenetic repression (ENT/AZA) in this experiment (50% cure rate) is shown to be somewhat lower than the cure rate (80% cure rate) shown in previous examples. However, the efficacy of anti-PD-1/anti-CTLA-4 alone was lower in this experiment (10% cure rate here compared to 30% in previous examples). The efficacy boost observed in combination therapy has not diminished. The variation in efficacy may be due to the quality of antibodies from different manufacturing batches.

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Claims (19)

1. The use of a first agent that inhibits bone marrow-derived suppressor cells (MDSCs) and a second agent that blocks one or more immune checkpoints in the preparation of a medicament for treating breast cancer and colorectal cancer in tumor-bearing mammals, wherein said treatment comprises: Administer the first agent; and The second agent is administered, wherein the first agent comprises entenoxetine and the second agent comprises a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody. 2. The application according to claim 1, wherein the first agent comprises a combination of entinolide and 5-azacytidine. 3. The application according to claim 1, wherein the treatment further comprises the step of administering spores of Clostridium novi-NT. 4. The application according to claim 1, wherein the antibody is a monoclonal antibody. 5. The application according to claim 1, wherein the tumor is colorectal cancer. 6. The application according to claim 1, wherein the tumor is breast cancer. 7. The application according to claim 1, wherein the tumor is a metastatic colorectal cancer. 8. The application according to claim 1, wherein the tumor is a metastatic breast cancer. 9. The application according to claim 1, wherein the first agent is administered at a dose insufficient to inhibit tumor cell growth alone. 10. A kit for treating breast cancer and colorectal cancer in tumor-bearing mammals, comprising in a single package: The first-line drug to inhibit myeloid-derived suppressor cells (MDSCs); and A second agent that blocks one or more immune checkpoints. The first drug agent includes entinolide, and the second drug agent includes a combination of anti-PD-1 antibody and anti-CTLA-4 antibody. 11. The kit according to claim 10, wherein the antibody is a monoclonal antibody. 12. The kit of claim 10, wherein the first agent comprises a combination of entinolide and 5-azacytidine. 13. The kit according to claim 10, further comprising spores of Clostridium noviperi-NT. 14. The kit of claim 10, comprising one or more unit doses, wherein the first agent is in an amount insufficient to inhibit tumor cell growth alone. 15. A composition for treating breast cancer and colorectal cancer in tumor-bearing mammals, comprising: The first-line drug to inhibit myeloid-derived suppressor cells (MDSCs); and A second agent that blocks one or more immune checkpoints. The first drug agent includes entinolide, and the second drug agent includes a combination of anti-PD-1 antibody and anti-CTLA-4 antibody. 16. The composition of claim 15, wherein the antibody is a monoclonal antibody. 17. The composition of claim 15, wherein the first agent comprises a combination of entinolide and 5-azacytidine. 18. The composition of claim 15, further comprising spores of Clostridium novi-NT. 19. The composition of claim 15, wherein the first agent is in a unit dose, and the unit dose is insufficient to inhibit tumor cell growth on its own.