WO2018112032A1 - Methods and compositions for targeting tumor-infiltrating tregs using inhibitors of ccr8 and tnfrsf8 - Google Patents

Abstract

The present disclosure relates to methods and compositions related to the diagnosis, treatment or prevention of cancer using an agent that interacts with tumor infiltrating T regulatory cells (TITRs). Examples are provided with anti CD30 antibodies and anti CCR8 antibodies. Also disclosed are methods for identifying TITRs in a subject (e.g., identifying TITRs in a tumor present in a subject).

Description

METHODS AND COMPOSITIONS FOR TARGETING TUMOR-INFILTRATING TREGS USING

INHIBITORS OF CCR8 AND TNFRSF8

Related Applications:

This application claims the benefit of priority to U.S. Provisional Patent Application serial number 62/433347, filed December 13, 2016, which is herein incorporated by reference in their entireties.

Background

Regulatory T cells characterized by expression of transcription factor FOXP3 (Treg) are critical to the maintenance of immunologic homeostasis, the enforcement of tolerance to self, and the prevention of runaway immune responses. Tregs regulate the activation and differentiation of conventional CD4⁺ T cells, as well as many other cells of the innate and adaptive immune systems, through a variety of mechanisms. A subset of Tregs known as tumor infiltrating Tregs (TITRs) are known to infiltrate cancerous tumors and are believed to play a role in the suppression of a host's immune response against the infiltrated tumors. TITRs are therefore an attractive therapeutic target for the treatment of cancer.

Summary

Provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor (e.g., a tumor type disclosed herein) present in a subject (e.g., a subject with cancer) by administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene. In some aspects, provided herein, are methods of inducing an anti-tumor immune response (e.g., long lasting responses, such as a response that is at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 30 days, at least 3 months, at least 6 months or at least one year) by decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject, the method comprising administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene. Also provided herein are methods of inducing an anti-tumor immune response (e.g., long lasting responses, such as a response that is at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 30 days, at least 3 months, at least 6 months or at least one year) by decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject by conjointly administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene and an immune checkpoint inhibitor. An anti-tumor response, as used herein, may be, for example, any decrease in tumor growth rate. In some embodiments, an anti-tumor response is characterized by any decrease of tumor growth after administration of a composition or combination therapy described herein. In some aspects, provided herein are methods of treating a tumor in a subject, the method comprising administering to the subject an agent that inhibits the activity or expression of a product of either the CCR8 or TNFRSF8 gene (e.g., a protein or mRNA product). In other aspects, provided herein are methods of increasing the amount of T effector cells in a tumor in a subject by administering to the subject an agent, or a pharmaceutical composition comprising the agent, that inhibits the activity or expression of a product of either the CCR8 or

TNFRSF8 gene. The agent may be an antibody, peptide, small molecule, or an interfering nucleic acid. In some embodiments, the methods describe administering a second agent (e.g., a chemotherapeutic agent, immune checkpoint inhibitor, or a tumor vaccine).

In some aspects, the methods provided herein relate to methods of targeting an agent to TITRs in a subject (e.g., a subject with cancer) by administering to the subject the agent (e.g., a drug, such as a toxin disclosed herein) that is conjugated to a polypeptide or protein that binds to the protein product of either the CCR8 or TNFRSF8 gene. In some

embodiments, the polypeptide or protein is an antibody specific for a protein product of either the CCR8 or TNFRSF8 gene. In some embodiments, the protein product is CD30.

In some aspects, provided herein are methods of determining whether an agent is an anti-cancer therapeutic agent comprising determining whether the test agent inhibits the expression or activity of a protein product of either the CCR8 or TNFRSF8 gene, wherein the test agent is determined to be an anti-cancer therapeutic agent if the test agent inhibits the expression or activity of a protein product of either the CCR8 or TNFRSF8 gene. The test agent may, for example, be a member of a library of test agents. The agent may be an antibody, peptide, small molecule, a protein drug conjugate, or an interfering nucleic acid.

In some aspects, provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of the CCR8 or TNFRSF8 gene. In other aspects, provided herein are methods of treating a tumor in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of the CCR8 or TNFRSF8 gene. In some aspects, provided herein are methods of increasing the amount of T effector cells in a tumor in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of the CCR8 or TNFRSF8 gene. The cytotoxicity may be antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). The agent may be an antibody, for example, peptide, small molecule, a protein drug conjugate, or an interfering nucleic acid.

In some aspects, disclosed herein are methods of identifying TITRs in a subject by expression level of the CCR8 or TNFRSF8 gene. In some embodiments, provided herein are methods of targeting and killing TITRs by first measuring expression level of the CCR8 or TNFRSF8 gene, and, if the expression level is above a determined threshold, targeting and killing the TITR.

Brief Description of the Drawings

Figure 1, has three Parts, 1-3, and shows a flow chart describing the generation of three independent and cross-confirming datasets. Part 1 shows the purification and profiling of Treg cells infiltrating three different transplantable tumors in immunocompetent mice. Part 2 shows the purification of TITR cells from patients with colorectal tumors, and comparison of their gene expression profiles with those of Treg cells purified from normal human colon (many from the same donors). Part 3 shows the mining of large datasets from TCGA for genes whose expression correlated with that of the Treg-defining factor FOXP3. Ultimately, these three datasets were combined to identify genes specifically overexpressed in TITRs.

Figure 2 has three Parts, A-C, and shows identification of TITR signature. Part A shows fold change (FC) x FC plots depicting the FC in expression of genes in tumor versus spleen for one tumor type versus another tumor type (i, ii) MC38 x B16, (iii) B16 x BP, (iv) MC38 x BP. An additive, filtered gene-set (genes with FC in expression > 4 in TITRs versus splenic Tregs, for each of the three transplantable tumor models) is highlighted in red on Part Aii-iv. Part B shows a heatmap depicting FC values for each gene in the TITR gene-set in tumor/spleen or tissue/spleen are depicted in the heatmap (color scale is blue to red; the darker the red, the greater the FC value). Part C shows tumor-preferential genes

(highlighted) were revealed by plotting the filtered TITR gene-set on a FC x FC plot where the x-axis is FC in expression of gene X in tissue Tregs compared to splenic Tregs (average FC across colon, pancreas, adipose tissue (fat) and muscle) and the y-axis is the FC in expression of gene X in TITRs vs splenic Tregs (average FC across MC38, B 16 and BP). Figure 3 has four Parts, A-D, and shows the conservation and derivation of TITR signature across species and individual human colon cancer patients. Part A shows the transcriptomic profile of human colon tumor Tregs versus normal colonic mucosa Tregs. The plot shows FC and p values for the expression of each gene in tumor/standard Tregs.

Annotated are genes known to be involved in Treg activity and/or co-stimulation. Part B shows the comparison of batches 1 and 2 of the human tumor versus normal colonic Tregs. Genes up two-fold or greater in tumor versus standard Tregs in both human data sets are highlighted in red. Part C shows the FC values for these highlighted genes for individual patients are presented in the heatmap. Known targets and T FRSF8 are annotated. Part D shows the mouse TITR signature is highlighted in purple on a human tumor/standard Treg data set.

Figure 4 has four Parts, A-D, and shows the correlation to FOXP3 in TCGA. Part A shows the correlation of genes to FOXP3 in raw data (x-axis) cf. data with immunocyte infiltrate regressed out (y-axis). Part B shows a plot of how genes correlate with FOXP3 in two different cancer types (colon and breast) from TCGA (immunocyte infiltrate regressed). Genes that highly correlate with FOXP3 are circled in green. Part C shows a heatmap with FOXP3 correlation coefficients for each of the four tumor types analyzed from TCGA: breast (BRCA), colon (COAD), lung (LUSC) and pancreas (PAAD). Part D shows classic Treg signature highlighted in pink on breast and colon datasets.

Figure 5 has two Parts, A-B, and shows the combinatorial data integration. Part A shows the overall score for human (x-axis) and mouse (y-axis) tumor Tregs. Highlighted are genes at the top of the ranking in both species (red), with high scores in the mouse and still in top 10% of differential transcripts in human (blue), or highest in the human ranking, but not in the mouse (green). Part B shows a combination of overall score for over-expression in human tumor Tregs (x-axis) with average correlation score derived from the whole-tumor TCGA datasets (y-axis). CCR8 and TNFRSF8 are highlighted in red.

Figure 6 shows the examination of how the combined TITR signature looks in 4 different cancers (colon, breast, pancreas and lung) from TCGA. Red or white clusters represent genes whose expression correlates with expression of the other genes in a given cluster in these TCGA data. Known, validated targets and FOXP3, IL2RB are annotated in grey.

Figure 7 has five Parts, A-E, and shows a summary of the data from single agent monoclonal antibody (mAb) treatments. MC38 tumor-bearing mice received anti-CD30, CCR8, IL1RL1, IL21R, CXCR3 or CXCR6 mAb treatment on days 7, 10 and 13 or 10, 13 and 16 following tumor cell injection. The delta in tumor volume from the time treatment began until experiment end (Day 21) is the metric plotted. Excellent responses to treatment were observed following treatment with anti-CD30 or anti-CCR8 mAbs, with 63% or 47% mice responding to anti-CD30 or anti-CCR8 mAb treatment, respectively (panel A). These results demonstrate that a modest response was observed when anti-ILlRLl (a.k.a. ST2) mAb was given (panel B) and no change in tumor growth was observed when mice received mAbs targeting IL21R or CXCR6 (panel C, D). Unexpectedly, significant worsening of the tumor occurred following administration of anti-CXCR3 mAb (panel E), suggesting that not all TITR targets function similarly.

Figure 8 shows that combination therapy improves the anti-tumor response versus single agent therapy. The tumor growth plots track tumor volume over time for mAb or control treated mice. mAbs were either given alone, as for CD30, CCR8, PD-1 or in combination, as for CD30+PD-1, CCR8+PD-1. These results demonstrate that combination mAb therapy outperforms single agent mAb therapy such that it causes more mice to exhibit resolution (zero tumor volume) or improvement (slower tumor growth) of their tumor burden, suggesting that anti-CCR8 or anti-CD30 mAbs are useful complements of checkpoint blockade therapy.

Figure 9 demonstrates that 'cured' mice (those whose MC38 tumors resolved completely following mAb treatment on days 10, 13 and 16 after original tumor cell injection) are protected from re-challenge with the same tumor type. A zero tumor volume measurement was recorded for at least 30 days prior to re-challenge with MC38 cells. Each line on the plot represents an individual mouse and the key indicates the treatment administered initially after original tumor inoculation.

Figure 10 has two Parts, A-B, shows data on the effects of single agent mAb treatments. Part A shows that there is no immediate clearance of TITR with either anti- CD30 or anti-CCR8 mAb treatment at 40 hrs after one treatment. Part B shows a heatmap of gene expression of the TITR-enriched geneset in Table 2 in TITRs from anti-CD30-, anti- CCR8- or control-treated mice at 40 hrs after one treatment. The color code reflects the range (low to high = blue to red) of fold change in expression value between TITR and splenic

Treg cells. There is a bias in the TITR signature in all anti-CD30 treated mice and one of the anti-CCR8 treated mice, such that these cells express less of the transcripts typically overexpressed in TITRs. These data suggest that blockade of these TITR-specific targets did not kill the TITRs, but modified their phenotype away from the tumor-specific pattern.

Detailed Description

In some aspects, provided herein are methods and compositions related to the treatment or prevention of cancer (e.g., by targeting a tumor in a subject with cancer) by administering to the subject an agent disclosed herein. In certain aspects, the invention relates to targeting tumors in a subject, and/or decreasing the number of tumor infiltrating T regulatory cells (TITRs) (e.g., TITRs present within a tumor) in a subject by administering to the subject an agent disclosed herein. In some aspects, the methods include decreasing the number or activity of TITR cells in a tumor present in a subject and/or increasing the amount of T effector cells in a tumor in a subject by administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene. Also provided herein are methods of determining whether a test agent is an anti-cancer agent (e.g., an agent that decreases the number and/or activity of TITRs). In some embodiments, methods disclosed herein relate to methods of determining whether a cell is a TITR cell. In some aspects, provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of the CCR8 or TNFRSF8 gene. In other aspects, provided herein are methods of treating a tumor in a subject by administering to the subject an agent that that induces cytotoxicity in cells that express a product of the CCR8 or TNFRSF8 gene. In some aspects, provided herein are methods of identifying a TITR in a subject by determining the expression level of the CCR8 or TNFRSF8 gene. In some aspects, provided herein are methods of identifying a TITR in a subject by determining the expression level of a CCR8 or TNFRSF8 gene product in the

TITR. In some embodiments, provided herein are methods of targeting and killing the TITR by first measuring the expression level of CCR8 or TNFRSF8 in the TITR, and, if the expression level is above a determined threshold, targeting and killing the TITR. TNFRSF8 is a gene which encodes for a cell membrane protein of the tumor necrosis factor receptor family and tumor marker. CCR8 is a gene encodes a member of the beta chemokine receptor family, which is predicted to be a seven transmembrane protein similar to G protein-coupled receptors. Chemokines and their receptors are important for the migration of various cell types into inflammatory sites. The present disclosure relates, at least in part, to the discovery of new targets for tumor immunotherapy, namely genes (i.e., CCR8 or TNFRSF8 ) specifically over-expressed in TITR versus Tregs from secondary lymphoid organs or normal tissues. This Treg- specific gene signature is conserved across species (human and mouse), is consistently expressed in tumor Tregs across individual colon cancer patients, and is present in at least four different types of tumors in The Cancer Genome Atlas. Novel immunotherapies can be developed based on these targets (i.e., CCR8 or TNFRSF8), aiming to reduce the function and/or number of Tregs in tumors, and hence alleviate their suppressive effects and unleash an efficient anti-tumor immune response. This goal may be achieved by blocking the function of these molecules in TITR through, for example, infusion of antibodies, peptides, interfering nucleic acids, or small molecule inhibitors disclosed herein, hence inhibiting TITR homeostasis or function; by using these molecules, for example, as targets of lytic antibodies, via complement- or ADCC- mediated toxicity, to preferentially deplete TITRs; and/or, for example, by genetic means (e.g., RNAi) to perturb the expression of these genes in TITRs. These agents may be delivered alone, or coupled to other molecules that enhance the specificity for TITRs over other Tregs or immunocytes. The agents and methods described herein may be combined with other known agents and methods. Definitions

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "agenf is used herein to denote a chemical compound, a small molecule, a mixture of chemical compounds and/or a biological macromolecule (such as a nucleic acid, an antibody, an antibody fragment, a protein or a peptide). The activity of such agents may render them suitable as a "therapeutic agent" which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject. An agent may be cytotoxic to a cell (e.g., TITR). An agent disclosed herein may target cells for cytotoxicity via CDC or ADCC-mediated cytotoxicity, or as a drug-antibody conjugate where the drug is cytotoxic.

Unless otherwise specified here within, the terms "antibody" and "antibodies" broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody

derivatives may comprise a protein or chemical moiety conjugated to an antibody.

The term "antibody" as used herein also includes an "antigen-binding portion" of an antibody (or simply "antibody portion"). The term "antigen-binding portion", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Examples of binding fragments encompassed within the term "antigen- binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989)

Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequences of specific scFv can be linked to human

immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-

6448; Poljak e/ a/. (1994) Structure 2: 1121-1123).

An antibody for use in the instant invention may be a bispecific antibody. A bispecific antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by a hybrid hybridoma or a trioma are disclosed in U.S.

Patent 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83 : 1453, and Staerz and

Bevan (1986) Immunol. Today 7:241). Bispecific antibodies are also described in U.S.

Patent 5,959,084. Fragments of bispecific antibodies are described in U.S. Patent 5,798,229.

Bispecific agents can also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling both antibodies. They can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and

Fv sequences.

Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31 : 1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and

immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

Antibodies may also be humanized which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term "humanized antibody", as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

As used herein, the term "cancer" is limited to solid tumors. The term "cancer" further encompasses primary and metastatic cancers.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies that specifically bind to the same epitope, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.

The terms "polynucleotide" , and "nucleic acid' are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or

ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non- limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA

(mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present,

modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation with a labeling component. The term "recombinant" polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.

The terms "prevent, " "preventing, " "prevention, " and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.

The term "small molecule^'" is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.

As used herein, the term "subject' means a human or non-human animal selected for treatment or therapy.

The phrases "therapeutically-effective amount" and "effective amount' as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment.

"Treating" a disease in a subject or "treating" a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.

Tumor Infiltrating Tregs (TITRs)

Tregs are often found at elevated frequencies in blood and tumors of human cancer patients and, for many cancers, a high density of Tregs correlates with poor prognosis. The involvement of Tregs in tumors has been demonstrated in animal models where their depletion via administration of anti-CD25 antibody or transfer of cells depleted of CD25⁺ Tregs, mostly eliminated different types of tumors. Treg depletion increased the number of CD4⁺ and/or CD8⁺ effector T cells (Teff) in the tumor, which exhibited robust tumor-specific killing activity. Removal of Treg-mediated suppression of the anti-tumor immune response can therefore result in tumor irradication.

In some aspects, provided herein are methods and compositions that relate, at least in part, to the targeting of a tumor, decreasing the number or activity of TITRs in a tumor, e.g., by inhibiting the expression of at least one product of CCR8 or TNFRSF8.

In some aspects, disclosed herein are methods of decreasing the number or activity of TITRs in a subject (e.g., TITRs in a tumor present in a subject), the method comprising administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene. In some aspects, provided herein are methods related to treating a tumor in a subject by administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene. Disclosed herein are methods related to increasing the amount of T effector cells in a tumor in a subject by administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene. In some aspects, provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject by administering to the subject an agent that induces cytotoxicity (e.g., ADCC or CDC cytotoxicity, or a cytotoxic drug-protein conjugate) in cells that express a product of the CCR8 or TNFRSF8 gene. In other aspects, provided herein are methods of treating a tumor in a subject by administering to the subject an agent that induces cytotoxicity in cells that express a product of the CCR8 or TNFRSF8 gene.

Modulators of Tumor Infiltrating Tregs (TITRs)

Provided herein are methods related to treating or preventing a tumor (e.g., a tumor present in a subject with cancer) by decreasing the number or activity of TITRs (i.e., TITRs in the tumor) by inhibiting the expression and/or activity of any one of the CCR8 or

TNFRSF8 gene. An agent disclosed herein may be an antibody, a small molecule, a peptide, or an interfering nucleic acid. An agent may reduce the number of TITRs (e.g., TITRs in a tumor) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%>, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. An agent disclosed herein may reduce the activity or expression of a product of either the CCR8 or TNFRSF8 gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. An agent disclosed herein may reduce the mRNA of a product of either the CCR8 or TNFRSF8 gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

In some aspects, provided herein are methods related to decreasing the number of TITRs in a tumor. TITRs in the tumor may be identified using any technique known in the art, including detecting the expression of a product of CCR8 or TNFRSF8 by cells in the tumor, wherein expression of a product (e.g., an mRNA product or a protein product) of CCR8 or TNFRSF8 by a cell in the tumor indicates that there is a TITR in the tumor. In some aspects, provided herein are methods of identifying TITRs in a tumor by expression level of CCR8 or TNFRSF8 or a gene product of CCR8 or TNFRSF8. In some embodiments, the TITR is identified as a TITR if the TITR has an expression level of CCR8 or TNFRSF8 or a gene product of CCR8 or TNFRSF8 that is above a certain threshold. In some embodiments, provided herein are methods of targeting and killing a TITR by first measuring the expression level of CCR8 or TNFRSF8 or gene product of CCR8 or TNFRSF8 and, if the expression level is above a determined threshold, killing the TITR. A gene product (e.g., an mRNA product) may be detected by nucleic acid amplification, a nucleic acid probe, or through sequencing. A protein product may be detected by using an antibody specific for the protein product, through immunocytochemistry (IHC), or by flow cytometry (e.g., FACS).

Also provided herein are methods for determining whether an agent is an anti-cancer therapeutic agent by determining whether the test agent inhibits the expression or activity of a protein product of CCR8 or TNFRSF8, such as CCR8 or TNFRSF8, wherein the test agent is an anti-cancer therapeutic agent if the test agent inhibits the expression or activity of a protein product of CCR8 or TNFRSF8. In some embodiments, the test agent is a member of a library of test agents. The test agent may be any agent disclosed herein, including an interfering nucleic acid, a peptide, a small molecule, an antibody, or a peptide-drug conjugate. A test agent disclosed herein may inhibit the expression or activity of a protein product of either the CCR8 or TNFRSF8 gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. A gene product (e.g., an mRNA product) may be detected by nucleic acid

amplification, a nucleic acid probe, or through sequencing. A protein product may be detected, for example, by using an antibody specific for the protein product, through immunocytochemistry (IHC), or by flow cytometry (e.g., FACS). Also provided herein are methods of treating a subject with a tumor, comprising administering an agent described herein that inhibits the expression or activity of a product of CCR8 or TNFRSF8.

Antibody Agents

Provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor (e.g., a tumor type disclosed herein) present in a subject (e.g., a subject with cancer) by administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene. In some aspects, provided herein are methods of treating a tumor in a subject, the method comprising administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of CCR8 or TNFRSF8. In other aspects, provided herein are methods of increasing the amount of T effector cells in a tumor in a subject by administering to the subject an antibody (e.g., a monoclonal antibody), or a pharmaceutical composition comprising the agent, that inhibits the activity or expression of a product of CCR8 or TNFRSF8. In some embodiments, the agent described herein is an antibody specific for a protein product of CCR8 or TNFRSF8. In some embodiments, the antibody induces cytotoxicity in cells that express a product of CCR8 or TNFRSF8. An antibody disclosed herein may inhibit expression or activity of a product of either the CCR8 or TNFRSF8 gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

An antibody disclosed herein may inhibit the binding of a protein product of either the CCR8 or TNFRSF8 gene to another protein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. An antibody provided herein may have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% specificity for a product of either the CCR8 or TNFRSF8 gene.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human. The terms "monoclonal antibodies" and "monoclonal antibody composition", as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term "polyclonal antibodies" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it

immunoreacts.

In certain embodiments, the methods and compositions provided herein relate to antibodies and antigen binding fragments thereof that bind specifically to a product of CCR8 or TNFRSF8. In some embodiments, the antibodies inhibit the function of the protein, such as inhibiting the activity of the protein, or interfering with protein-protein interactions. Such antibodies can be polyclonal or monoclonal and can be, for example, murine, chimeric, humanized or fully human. In some embodiments, the agent may be a recombinant antibodies specific for a product of CCR8 or TNFRSF8, such as chimeric or humanized monoclonal antibodies, can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in US Pat No. 4,816,567; US Pat. No. 5,565,332; Better et al. (\9%%) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.

80: 1553-1559); Morrison, S. L. (\9 5) Science 229: 1202-1207; Oi et al. (1986)

Biotechniques 4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141 :4053- 4060.

Human monoclonal antibodies specific for a product of CCR8 or TNFRSF8 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. For example, "HuMAb mice" which contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856 859).

Accordingly, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human ¾GK monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of

Experimental Pharmacology 113 :49 101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13 : 65 93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y Acad. Sci 764:536 546). The preparation of HuMAb mice is described in Taylor, L. et al. (1992)

Nucleic Acids Research 20:6287 6295; Chen, J. et al. (1993) International Immunology 5: 647 656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci USA 90:3720 3724; Choi et al. (1993) Nature Genetics 4: 117 123; Chen, J. et al. (1993) EMBO J. 12: 821 830; Tuaillon et al. (1994) J. Immunol. 152:2912 2920; Lonberg et al, (1994) Nature 368(6474): 856 859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113 :49 101; Taylor, L. et al. (1994) International Immunology 6: 579 591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13 : 65 93; Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536 546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845 851. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625, 126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and 5,545,807.

In some embodiments, the antibody is a CD30 antibody (e.g., an anti-CD30 monoclonal antibody). In some embodiments, the antibody is HRS-3. In some embodiments, the antibody is HRS-3.6. In some embodiments, the antibody is Ber-H2. For more information on CD-30 antibodies, please see Reusch U. et al., MABS. 2014; 6(3):727-738, or Falini B, Flenghi L, Fedeli L, et al. In vivo targeting of Hodgkin and Reed- Sternberg cells of Hodgkin's disease with monoclonal antibody Ber-H2 (CD30): immunohistological evidence. Br J Haematol. 1992;82: 38-45, which are hereby incorporated in their entirety. Polypeptide Agents

In some embodiments, the agent provided herein is a polypeptide agent (e.g., a polypeptide that binds to a protein expressed by the CCR8 or TNFRSF8 gene. In some embodiments, the polypeptide induces cytotoxicity in cells that express a product of the CCR8 or TNFRSF8 gene. A polypeptide agent disclosed herein may inhibit the expression or activity of a product of the CCR8 or TNFRSF8 gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. A polypeptide agent disclosed herein may inhibit the binding of a product of either the CCR8 or TNFRSF8 gene to another protein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%), at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, the agent may be a chimeric or fusion polypeptide. A fusion or chimeric polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety.

The polypeptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding a polypeptide(s). Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N. Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif ; Merrifield, J. (1969) J. Am. Chem. Soc. 91 :501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11 :255; Kaiser et al. (1989) Science 243 : 187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference. Interfering Nucleic Acid Agents

In certain embodiments, interfering nucleic acid molecules that selectively target and inhibit the activity or expression of a product (e.g., an mRNA product) of a gene listed in either the CCR8 or TNFRSF8 gene are provided herein and/or used in methods described herein. In some embodiments, the interfering nucleic acid induces cytotoxicity in cells that express a product of the CCR8 or TNFRSF8 gene. An agent may inhibit the expression or activity of a product (e.g., an mRNA product) of a gene in either the CCR8 or TNFRSF8 gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. An agent disclosed herein may comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) complementarity to a product (e.g., an mRNA product) of the CCR8 or TNFRSF8 gene.

In some embodiments, the inhibiting nucleic acid is a siRNA, a shRNA, a PNA, or a miRNA molecule. Interfering nucleic acids generally include a sequence of cyclic subunits, each bearing a base-pairing moiety, linked by intersubunit linkages that allow the base- pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence. Interfering RNA molecules include, but are not limited to, antisense molecules, siRNA molecules, single-stranded siRNA molecules, miRNA molecules and shRNA molecules.

Typically at least 17, 18, 19, 20, 21, 22 or 23 nucleotides of the complement of the target mRNA sequence are sufficient to mediate inhibition of a target transcript. Perfect complementarity is not necessary. In some embodiments, the interfering nucleic acid molecule is double-stranded RNA. The double-stranded RNA molecule may have a 2 nucleotide 3' overhang. In some embodiments, the two RNA strands are connected via a hairpin structure, forming a shRNA molecule. shRNA molecules can contain hairpins derived from microRNA molecules. For example, an RNAi vector can be constructed by cloning the interfering RNA sequence into a pCAG-miR30 construct containing the hairpin from the miR30 miRNA. RNA interference molecules may include DNA residues, as well as RNA residues.

Interfering nucleic acid molecules provided herein can contain RNA bases, non-RNA bases or a mixture of RNA bases and non-RNA bases. For example, interfering nucleic acid molecules provided herein can be primarily composed of RNA bases but also contain DNA bases or non-naturally occurring nucleotides.

The interfering nucleic acids can employ a variety of oligonucleotide chemistries. Examples of oligonucleotide chemistries include, without limitation, peptide nucleic acid (PNA), linked nucleic acid (LNA), phosphorothioate, 2'0-Me-modified oligonucleotides, and morpholino chemistries, including combinations of any of the foregoing. In general,

PNA and LNA chemistries can utilize shorter targeting sequences because of their relatively high target binding strength relative to 2'0-Me oligonucleotides. Phosphorothioate and 2'0- Me-modified chemistries are often combined to generate 2'0-Me-modified oligonucleotides having a phosphorothioate backbone. See, e.g., PCT Publication Nos. WO/2013/112053 and WO/2009/008725, incorporated by reference in their entireties.

Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone is structurally homomorphous with a deoxyribose backbone, consisting of N-(2-aminoethyl) glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligonucleotides obeying Watson- Crick base-pairing rules, and mimic DNA in terms of base pair recognition (Egholm, Buchardt et al. 1993). The backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well-suited for antisense applications (see structure below). The backbone is uncharged, resulting in PNA/DNA or PNA/RNA duplexes that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases.

Despite a radical structural change to the natural structure, PNAs are capable of sequence-specific binding in a helix form to DNA or RNA. Characteristics of PNAs include a high binding affinity to complementary DNA or RNA, a destabilizing effect caused by single-base mismatch, resistance to nucleases and proteases, hybridization with DNA or RNA independent of salt concentration and triplex formation with homopurine DNA.

PANAGENE.TM. has developed its proprietary Bts PNA monomers (Bts; benzothiazole-2- sulfonyl group) and proprietary oligomerization process. The PNA oligomerization using Bts PNA monomers is composed of repetitive cycles of deprotection, coupling and capping. PNAs can be produced synthetically using any technique known in the art. See, e.g., U.S. Pat. Nos. 6,969,766, 7,211,668, 7,022,851, 7, 125,994, 7, 145,006 and 7,179,896. See also U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 for the preparation of PNAs. Further teaching of PNA compounds can be found in Nielsen et al., Science, 254: 1497-1500, 1991. Each of the foregoing is incorporated by reference in its entirety.

Interfering nucleic acids may also contain "locked nucleic acid" subunits (LNAs). "LNAs" are a member of a class of modifications called bridged nucleic acid (BNA). BNA is characterized by a covalent linkage that locks the conformation of the ribose ring in a C30- endo (northern) sugar pucker. For LNA, the bridge is composed of a methylene between the 2'-0 and the 4'-C positions. LNA enhances backbone preorganization and base stacking to increase hybridization and thermal stability.

The structures of LNAs can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998) 54:3607, and Accounts of Chem. Research (1999) 32:301); Obika, et al., Tetrahedron Letters (1997) 38:8735; (1998) 39:5401, and Bioorganic Medicinal Chemistry (2008) 16:9230. Compounds provided herein may incorporate one or more LNAs; in some cases, the compounds may be entirely composed of LNAs. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligonucleotides are described, for example, in U.S. Pat. Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461, each of which is incorporated by reference in its entirety. Typical intersubunit linkers include phosphodiester and phosphorothioate moieties; alternatively, non-phosphorous containing linkers may be employed. One embodiment is an LNA containing compound where each LNA subunit is separated by a DNA subunit. Certain compounds are composed of alternating LNA and DNA subunits where the intersubunit linker is phosphorothioate.

"Phosphorothioates" (or S-oligos) are a variant of normal DNA in which one of the nonbridging oxygens is replaced by a sulfur. The sulfurization of the internucleotide bond reduces the action of endo-and exonucleases including 5' to 3' and 3' to 5' DNA POL 1 exonuclease, nucleases SI and PI, RNases, serum nucleases and snake venom

phosphodiesterase. Phosphorothioates are made by two principal routes: by the action of a solution of elemental sulfur in carbon disulfide on a hydrogen phosphonate, or by the method of sulfurizing phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1, 2- bensodithiol-3-one 1, 1-dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990). The latter methods avoid the problem of elemental sulfur' s insolubility in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also yield higher purity phosphorothioates.

"2'0-Me oligonucleotides" molecules carry a methyl group at the 2' -OH residue of the ribose molecule. 2'-0-Me-RNAs show the same (or similar) behavior as DNA, but are protected against nuclease degradation. 2'-0-Me-RNAs can also be combined with phosphothioate oligonucleotides (PTOs) for further stabilization. 2'0-Me oligonucleotides (phosphodiester or phosphothioate) can be synthesized according to routine techniques in the art (see, e.g., Yoo et al., Nucleic Acids Res. 32:2008-16, 2004).

The interfering nucleic acids described herein may be contacted with a cell or administered to an organism (e.g., a human). Alternatively, constructs and/or vectors encoding the interfering RNA molecules may be contacted with or introduced into a cell or organism. In certain embodiments, a viral, retroviral or lentiviral vector is used. In some embodiments, the vector has a tropism for cardiac tissue. In some embodiments the vector is an adeno-associated virus.

In some embodiments, the interfering nucleic acid molecule is a siRNA molecule.

Such siRNA molecules should include a region of sufficient homology to the target region, and be of sufficient length in terms of nucleotides, such that the siRNA molecule down- regulate target RNA. The term "ribonucleotide" or "nucleotide" can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions. It is not necessary that there be perfect complementarity between the siRNA molecule and the target, but the correspondence must be sufficient to enable the siRNA molecule to direct sequence-specific silencing, such as by RNAi cleavage of the target RNA. In some embodiments, the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-strand character of the molecule.

In addition, an siRNA molecule may be modified or include nucleoside surrogates. Single stranded regions of an siRNA molecule may be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates.

Modification to stabilize one or more 3'- or 5'-terminus of an siRNA molecule, e.g., against exonucleases, or to favor the antisense siRNA agent to enter into RISC are also useful.

Modifications can include C3 (or C6, C7, CI 2) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, CI 2, abasic, tri ethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.

A "small hairpin RNA" or "short hairpin RNA" or "shRNA" includes a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNAs provided herein may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).

Non-limiting examples of shRNAs include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions. In some embodiments, the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

Additional embodiments related to the shRNAs, as well as methods of designing and synthesizing such shRNAs, are described in U.S. patent application publication number 2011/0071208, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

In some embodiments, provided herein are micro RNAs (miRNAs). miRNAs represent a large group of small RNAs produced naturally in organisms, some of which regulate the expression of target genes. miRNAs are formed from an approximately 70 nucleotide single-stranded hairpin precursor transcript by Dicer. miRNAs are not translated into proteins, but instead bind to specific messenger RNAs, thereby blocking translation. In some instances, miRNAs base-pair imprecisely with their targets to inhibit translation.

In certain embodiments, antisense oligonucleotides may be 100% complementary to the target sequence, or may include mismatches, e.g., to improve selective targeting of allele containing the disease-associated mutation, as long as a heteroduplex formed between the oligonucleotide and target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo. Hence, certain oligonucleotides may have about or at least about 70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence.

Oligonucleotide backbones that are less susceptible to cleavage by nucleases are discussed herein. Mismatches, if present, are typically less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.

Interfering nucleic acid molecules can be prepared, for example, by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, or other methods known in the art. See Hannon, GJ, 2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002, The rest is silence. RNA 7: 1509-1521; Hutvagner G et al., RNAi: Nature abhors a double- strand. Curr. Opin. Genetics & Development 12: 225-232; Brummelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553; Lee NS, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J. (2002). Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnol. 20:500-505; Miyagishi M, and Taira K. (2002). U6-promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnol. 20:497-500; Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, and Conklin DS. (2002). Short hairpin RNAs (shRNAs) induce sequence- specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul CP, Good PD, Winer I, and Engelke DR. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnol. 20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester WC, and Shi Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99(6):5515-5520; Yu J-Y, DeRuiter SL, and Turner DL. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99(9):6047-6052.

In the present methods, an interfering nucleic acid molecule or an interfering nucleic acid encoding polynucleotide can be administered to the subject, for example, as naked nucleic acid, in combination with a delivery reagent, and/or as a nucleic acid comprising sequences that express an interfering nucleic acid molecule. In some embodiment, the interfering nucleic acid is administered directly to a tumor in a subject. In some embodiments, the nucleic acid comprising sequences that express the interfering nucleic acid molecules are delivered within vectors, e.g. plasmid, viral and bacterial vectors. Any nucleic acid delivery method known in the art can be used in the methods described herein. Suitable delivery reagents include, but are not limited to, e.g., the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes. The use of atelocollagen as a delivery vehicle for nucleic acid molecules is described in Minakuchi et al. Nucleic Acids Res., 32(13):el09 (2004); Hanai et al. Ann NY Acad Sci., 1082:9-17 (2006); and Kawata et al. Mol Cancer Ther., 7(9):2904-12 (2008); each of which is incorporated herein in their entirety. Exemplary interfering nucleic acid delivery systems are provided in U.S. Patent Nos. 8,283,461, 8,313,772, 8,501,930. 8,426,554, 8,268,798 and 8,324,366, each of which is hereby incorporated by reference in its entirety.

In some embodiments of the methods described herein, liposomes are used to deliver an inhibitory oligonucleotide to a subject. Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are herein incorporated by reference.

The liposomes for use in the present methods can also be modified so as to avoid clearance by the mononuclear macrophage system ("MMS") and reticuloendothelial system ("RES"). Such modified liposomes have opsonization-inhibition moieties on the surface or incorporated into the liposome structure.

Small Molecule Agents

Certain embodiments of the methods and compositions disclosed herein relate to the use of small molecule agents e.g., small molecule agents that inhibit the expression or activity of a product of the CCR8 or TNFRSF8 gene, for decreasing the number or activity of TITRs (e.g., TITRs in a tumor), in a subject, or increasing the number or activity of T effector cells in a tumor in a subject. In some embodiments, the small molecule induces cytotoxicity in cells that express a product of the CCR8 or TNFRSF8 gene. Such agents include those known in the art and those identified using the screening assays described herein. A small molecule provided herein may have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% specificity for a product of the CCR8 or TNFRSF8 gene.

In some embodiments, assays used to identify agents (e.g., anti-cancer agents) in the methods described herein include obtaining a population of cells and a small molecule agent, wherein the cells are incubated with a small molecule agent and the number or activity of TITRs in the population of cells is subsequently measured. Agents identified via such assays, may be useful, for example, for decreasing the number or activity of TITRs (e.g., TITRs in a tumor), in a subject, or increasing the number or activity of T effector cells in a tumor in a subject.

Agents useful in the methods disclosed herein may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Agents may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al, 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261 : 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.

33 :2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33 :2061; and in Gallop et al.

(1994) J. Med. Chem. 37: 1233.

Libraries of agents may be presented in solution (e.g., Houghten, 1992, Biotechniques 13 :412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, USP 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89: 1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra.).

Agents useful in the methods disclosed herein may be identified, for example, using assays for screening candidate or test agents e.g., agents that decrease the activity or expression of a product of the CCR8 or TNFRSF8 gene or that decrease the number or activity of TITRs in a sample or population of cells. Combination Methods

Provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor (e.g., a tumor type disclosed herein) present in a subject (e.g., a subject with cancer) by conjointly administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene and an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitor disclosed herein). In some aspects, provided herein are methods of treating a tumor in a subject, the method comprising conjointly administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of CCR8 or TNFRSF8 and an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitor disclosed herein). In other aspects, provided herein are methods of increasing the amount of T effector cells in a tumor in a subject by conjointly administering to the subject an antibody (e.g., a monoclonal antibody), or a pharmaceutical composition comprising the agent, that inhibits the activity or expression of a product of CCR8 or TNFRSF8 and an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitor disclosed herein). In some embodiments, the immune checkpoint inhibitor inhibits an immune checkpoint protein. Immune checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response. Examples of immune checkpoint proteins are CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. Provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor (e.g., a tumor type disclosed herein) present in a subject (e.g., a subject with cancer) by conjointly administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene and an oncolytic virus (e.g., any one of the oncolytic viruses disclosed herein). In some aspects, provided herein are methods of treating a tumor in a subject, the method comprising conjointly administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of CCR8 or TNFRSF8 and an oncolytic virus (e.g., any one of the oncolytic viruses disclosed herein). In other aspects, provided herein are methods of increasing the amount of T effector cells in a tumor in a subject by conjointly administering to the subject an antibody (e.g., a monoclonal antibody), or a pharmaceutical composition comprising the agent, that inhibits the activity or expression of a product of CCR8 or TNFRSF8 and an oncolytic virus (e.g., any one of the oncolytic viruses disclosed herein). Oncolytic virotherapy is an emerging treatment modality which uses replication competent viruses to destroy cancers. Oncolytic viruses can kill infected cancer cells in many different ways, ranging from direct virus-mediated cytotoxicity through a variety of cytotoxic immune effector mechanisms. Conventional concepts of cell death (apoptosis, necrosis or autophagy) are generally inadequate to fully describe the complex cell killing scenarios encountered in virotherapy. Specific targeting of cancer cells is obviously the sine qua non for oncolytic virotherapy and can be achieved in several ways. Some viruses such as HI autonomously replicating parvoviruses, reovirus, Newcastle Disease Virus, Mumps virus, Moloney leukemia virus have a natural preference for cancer cells, whereas such as measles, adenovirus, Vesicular Stomatitis Virus, vaccinia and Herpes Simplex Virus can be adapted or engineered to make them cancer-specific. Surface markers such as EGF receptor, Her2-neu, Folate receptor, Prostate Specific Membrane Antigen and CD20, and nuclear transcription factors PSA, hTERT, COX-2, osteocalcin expressed selectively by tumor cells can be targeted by using them as receptors for virus entry or as essential cofactors for viral gene expression.

Provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor (e.g., a tumor type disclosed herein) present in a subject (e.g., a subject with cancer) by conjointly administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene and a kinase inhibitor (e.g., any one of the kinase inhibitors disclosed herein). In some aspects, provided herein are methods of treating a tumor in a subject, the method comprising conjointly administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of CCR8 or TNFRSF8 and a kinase inhibitor (e.g., any one of the kinase inhibitors disclosed herein). In other aspects, provided herein are methods of increasing the amount of T effector cells in a tumor in a subject by conjointly administering to the subject an antibody (e.g., a monoclonal antibody), or a pharmaceutical composition comprising the agent, that inhibits the activity or expression of a product of CCR8 or TNFRSF8 and a kinase inhibitor (e.g., any one of the kinase inhibitors disclosed herein). Small molecule kinase inhibitors include afatinib, alectinib, apatinib, ASP-3026, axitinib, bafetinib, baricitinib, binimetinib, bosutinib, brigatinib, cabozantinib, canertinib, cediranib, CEP-37440, ceritinib, cobimetinib, copanlisib, crenolanib, crizotinib, CYT387, dabrafenib, damnacanthal, dasatinib, doramapimod, enterctinib, erlotinib, everolimus, filgotinib, foretinib, fostamatinib, gefitinib, grandinin, ibrutinib, icotinib, idelalisib, imatinib, IPI-145, JSI-124, lapatinib, lenvatinib, lestaurtinib, linifanib, masitinib, motesanib, mubritinib, neratinib, nilotinib, nintedanib, pacritinib, palbociclib, pazopanib, pegaptanib, perifosine, PF-06463922, ponatinib, PX-866, quizartinib, radotinib, regorafenib, ruxolitinib, selumetinib, sirolimus, sorafenib, staurosporine, sunitinib, SU6656, temsirolimus, TG101348, tivozanib, toceranib, tofacitinib, trametinib, TSR-011, vandetanib, vemurafenib, and X-396. Large molecule kinase inhibitors include aflibercept, bevacizumab, catumaxomab, panitumumab, ranibizumab, and trastuzumab.

In some embodiments, the kinase inhibitor is a tyrosine kinase inhibitor, such as afatinib, alectinib, apatinib, axitinib, bafetinib, baricitinib, binimetinib, bosutinib, brigatinib, cabozantinib, canertinib, cediranib, CEP-37440, ceritinib, cobimetinib, crenolanib, crizotinib, CYT387, damnacanthal, dasatinib, doramapimod, entrectinib, erlotinib, filgotinib, foretinib, fostamatinib, grandinin, gefitinib, ibrutinib, icotinib, imatinib, JSI-124, lapatinib, lestaurtinib, lenvatinib, linifanib, masitinib, motesanib, mubritinib, neratinib, nilotinib, nintedanib, pacritinib, pazopanib, pegaptanib, PF-06463922, ponatinib, quizartinib, radotinib, regorafenib, ruxolitinib, selumetinib, semaxanib, sorafenib, staurosporine, sunitinib, SU6656, TG101348, tivozanib, toceranib, tofacitinib, trametinib, TSR-011, vandetanib, vatalanib, vemurafenib, or X-396. In certain embodiments, the kinase inhibitor is a receptor tyrosine kinase inhibitor.

In some embodiments, the kinase inhibitor is a multi -targeted kinase inhibitor, such as a multi -targeted receptor tyrosine kinase inhibitor. In certain embodiments, the kinase inhibitor is a VEGF receptor kinase inhibitor, PDGF receptor kinase inhibitor, and/or inflammasome inhibitor. In certain embodiments, the kinase inhibitor is apatinib, axitinib, cabozantinib, cediranib, crenolanib, foretinib, lenvatinib, linifanib, masitinib, motesanib, nintedanib, pazopanib, pegaptanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, or vatalanib. In some preferred embodiments, the kinase inhibitor is sorafenib.

Provided herein are methods of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor (e.g., a tumor type disclosed herein) present in a subject (e.g., a subject with cancer) by conjointly administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene and adoptive cell transfer (e.g., cancer-specific chimeric antigen receptor (CAR) T-cells). In some aspects, provided herein are methods of treating a tumor in a subject, the method comprising conjointly administering to the subject an antibody (e.g., a monoclonal antibody) that inhibits the activity or expression of a product of CCR8 or TNFRSF8 and adoptive cell transfer (e.g., cancer-specific chimeric antigen receptor (CAR) T-cells). In other aspects, provided herein are methods of increasing the amount of T effector cells in a tumor in a subject by conjointly administering to the subject an antibody (e.g., a monoclonal antibody), or a pharmaceutical composition comprising the agent, that inhibits the activity or expression of a product of CCR8 or TNFRSF8 and adoptive cell transfer (e.g., cancer-specific chimeric antigen receptor (CAR) T-cells). CAR T-cell therapy is a type of cellular immunotherapy which targets a patient's own immune cells to use directly against their cancer cells. A patient's blood is collected through a process called apheresis. T-cells are separated from the collected blood. After the T-cells are separated, they are then engineered and multiplied to locate and attack cancer cells. The engineered t-cells are then infused back into the same patient.

In certain embodiments, agents of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents). For example, the different therapeutic agents can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. In certain embodiments, the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic agents.

Pharmaceutical Compositions

In certain embodiments, provided herein is a composition, e.g., a pharmaceutical composition, containing at least one agent (e.g., a test agent, such as an antibody, an interfering nucleic acid, a peptide, or a small molecule) described herein together with a pharmaceutically acceptable carrier. In one embodiment, the composition includes a combination of multiple (e.g., two or more) agents described herein.

In some embodiments, the pharmaceutical composition is delivered locally or systemically. In some embodiments, the pharmaceutical composition may be administered to a tumor present in the subject. In some embodiments, the agent or pharmaceutical composition is administered with a second cancer therapeutic agent. In some embodiments, the second cancer therapeutic agent is a chemotherapeutic agent. In some embodiments, the pharmaceutical composition further comprises a second agent for treatment of cancer. In some embodiments, the second agent is a tumor vaccine. In some embodiments, the second agent is an agent that decreases the activity or expression of a gene or gene product known to be upregulated in TITRs. In certain embodiments, the second therapeutic agent is a chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; emylerumines and memylamelamines including alfretamine, triemylenemelamine, triethylenephosphoramide,

triethylenethiophosphoramide, and trimemylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (articularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin phili); dynemicin, including dynemicin A;

bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores),

aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carrninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (Adramycin™) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as demopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replinisher such as frolinic acid; aceglatone; aldophosphamide glycoside;

aminolevulinic acid; eniluracil; amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- tricUorotriemylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiopeta; taxoids, e.g., paclitaxel (Taxol™, Bristol Meyers Squibb Oncology, Princeton, N.J.) and docetaxel (Taxoteret™, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine

(Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide;

mitroxantrone; vancristine; vinorelbine (Navelbine™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in the definition of "chemotherapeutic agent" are anti -hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston™); inhibitors of the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprohde, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In some embodiments, the second cancer therapeutic agent is an immune checkpoint inhibitor, e.g., inhibitors of immune checkpoint proteins such as CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TEVI-3, TEVI-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.

As described in detail below, the pharmaceutical compositions and/or agents disclosed herein may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous, intrathecal, intracerebral or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation. Methods of preparing pharmaceutical formulations or compositions include the step of bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Pharmaceutical compositions suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, dimethyl sulfoxide (DMSO), 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.

Therapeutic Methods

In some aspects, provided herein are methods of treating a cancer by administering to a subject (e.g., to a tumor present in a subject) an agent and/or a pharmaceutical composition described herein.

In some embodiments, the methods described herein may be used to treat a solid tumor. Cancers that may be treated by methods and compositions provided herein include, but are not limited to, cancer cells from the bladder, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp;

adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant;

branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell

adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma;

cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;

infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary paget's disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; and malignant roblastoma;

Sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma;

glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma;

malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma;

myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal

rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma;

malignant struma ovarii; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioendothelioma; kaposi's sarcoma; malignant hemangiopericytoma;

lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma;

ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma;

ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal;

cerebellar sarcoma; ganglion euroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; and immunoproliferative small intestinal disease.. In some embodiments, the subject has cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a blood born tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a Rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymus tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms tumor. Actual dosage levels of the active ingredients in the pharmaceutical compositions or agents to be administered may be varied so as to obtain an amount of the active ingredient (e.g., an agent described herein) which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could prescribe and/or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

Exemplification:

Example 1: Methods

In order to identify which genes were differentially expressed in TITRs compared with standard Tregs from control tissues, the objective and multi -pronged approach described in Figure 1 was followed, which provided three independent and cross-confirming datasets. First, Treg cells infiltrating three transplantable tumors in immunocompetent mice were purified and profiled. Second, TITR cells from patients with colorectal tumors were purified, and compared their gene expression profiles with those of Treg cells purified from normal human colon (many from the same donors). Third, large datasets from The Cancer Genome Atlas (TCGA) were mined for genes whose expression correlated with that of the Treg-defining factor FOXP3. Each of these datasets was analyzed alone, and their intersection then defined high-quality predictions, conserved across species and tumor- types, of genes specifically overexpressed in tumor- infiltrating Tregs.

Example 2: Transcripts specific to mouse tumor Tregs.

Three mouse models of cancer were used, namely the transplantable MC38 colon adenocarcinoma (MC38), B16-F10 melanoma (B16), and BRAF.PTEN melanoma (BP). In each case, tumors were inoculated subcutaneously into immunocompetent Foxp3-GFP/C57BL6 reporter mice, in which Treg cells are uniquely identified and readily sorted on the basis of the fluorescent GFP reporter. After establishment and growth of these tumors (21 days), immunocytes were isolated from the tumors and spleens from these animals, stained with fluorochrome-conjugated antibodies, and 1,000 Tregs

(Foxp3(GFP)+CD4+CD8-TCRb+CD45+ cells) were highly purified (double sorting by flow cytometry) from each tissue. RNA from these cells was then used for gene expression profiling by whole-transcriptome shotgun sequencing (RNAseq, SmatseqV2 procedure, 5- 10x106 mappable reads/sample). Replicate data were generated for each sample type. They were filtered for quality, mapped to the mouse genome (TopHat), normalized

(Cuffnorm), and filtered to avoid low-expression transcripts and resulting noise.

Example 3: Genes Differentially Expressed In Tumor Versus Control Tregs,

In order to identify genes that were differentially expressed in tumor versus control Tregs, the fold change (FC) in expression of each gene in tumor versus splenic Tregs was calculated. As demonstrated in Figure 2, part A, a sizeable set of genes was overexpressed in tumor versus splenic Tregs. Moreover, the vast majority of genes overexpressed in TITRs from one mouse model were similarly upregulated in the other murine tumor models (e.g. MC38 and B 16 comparison shown in Figure 2, Part Ai). The analyses also identified genes that were differentially downregulated in TITRs versus splenic Tregs. Given that such genes were under-expressed in TITRs (hence, expressed at very low levels), they offered less desirable targets and so exploration of these was paused here. Moving forward with the analysis of the over-expressed genes, genes were selected with a FC in expression > 4 and a p value of < 10-3 in TITRs versus splenic Tregs, in either of the three transplantable tumor models. A group of differential transcripts were typical of tumor infiltrating myeloid cells (identified using myeloid cell signatures from the ImmGen database) and were discounted as residual contamination. Overexpression of this filtered gene-set was strongly conserved across the three tumor types that were examined (filtered

gene-set highlighted in red in Figures 2, part Aii-iv). Interestingly, the gene-selection pathway led us to notable transcripts already known for their role in Treg function and/or costimulation (C£>27¥, HAVCR2, TNFRSF9 (aka PDLl, TFM3 and 4- IBB respectively), LAG3, ICOS and TIGIT; annotated on Figure 2, part Aiv). The presence of such genes in the TITR gene set lends credence to their lesser known counterparts, in the same gene set, providing effective targets for Treg modulation. There is growing awareness that Tregs have an extra-immunologic role in several tissues, regulating the harmful side effects of inflammation and promoting effective tissue repair. Examples of such "tissue-Tregs" include populations in the visceral adipose tissue or injured muscle (reviewed in (Panduro et al., 2016)). In line with these wide-ranging roles, Treg cells can adopt distinct subphenotypes, which differ by their preferential expression of chemokine receptors, effector molecules, and cofactors that collaborate with FoxP3 to drive these functional nuances (Feuerer et al., 2009b; Campbell and Koch, 2011). Given the goal to identify molecules specific of TITR, as opposed to Treg cells in other locations, and because some of the transcripts overexpressed in TITRs were reminiscent of tissue Tregs, the transcriptomes were compared of these two Treg populations. The TITR gene-set was selected as described above, and analyzed for how these genes behaved in different tissue Treg subsets, namely those from colon, pancreas, adipose tissue and muscle. Although 2-3 clusters of genes were similarly upregulated in TITRs and Tregs from colon, fat, and muscle, there were large and distinct sets of genes which exhibited increased upregulation in TITRs versus tissue Tregs (Figure 2, Part B; FC values for tumor/spleen or tissue/spleen are depicted in the heatmap). Tumor- preferential overexpression was also revealed by plotting the filtered TITR gene-set on a FC x FC plot where the x-axis is the FC in expression in tissue Tregs compared to splenic Tregs (average FC across colon, pancreas, adipose tissue and muscle) and the y-axis is the FC in expression in TITRs versus splenic Tregs (average FC across MC38, B16 and BP) (Figure 2, Part C). Some transcripts (area A) are preferentially overexpressed in tissue Tregs e.g. 1110 and Areg (the latter known to be upregulated in muscle Tregs (Burzyn et al., 2013)), others (on the diagonal; area B) are similarly upregulated in both tumor and tissue Tregs e.g. Lilrb4 and Ccr2, and others still (area C) are clearly preferentially overexpressed in TITRs e.g. Ill2rb2 and Tnfrsf9.

Example 4: Transcripts Speci fic to Human Colorectal Tumor Tregs.

Tregs were purified from freshly harvested human colorectal tumors or normal colon tissue and then cryopreserved. These were profiled by RNAseq. After dissociation, immunocytes from both tissues were stained with antibodies, and 1,000 Tregs were purified by flow cytometry (CD25+CD127-CD4+CD8-CD3+CD45+, double-sorting). RNAseq was performed on these purified Treg samples, and the data were processed, normalized and filtered as above, (the profiles were generated in two different batches). Next, to detect genes that were differentially expressed in tumor versus normal tissue Tregs, the FC was calculated in expression of each gene in tumor Tregs versus normal colon Tregs, and the p. value associated with this difference. As illustrated in Figure 3, Part A there was a significant bias in the transcriptome of these tumor tregs, with 38 transcripts induced relative to normal colon Tregs (at a threshold fold change of 2, and t.test p.value<10-2). As for the tumor Treg data in the mouse models, these over-expressed human transcripts included several genes previously recognized to have activity in Treg cells and/or costimulatory function (ENTPD1 (encodes CD39), DUSP4, TNFRSF4, TNFRSF9, TNFRSF18 (aka OX40, 4- IBB, GITR, respectively) and FOXP3 itself). Conversely, this same analysis identified genes that were downregulated in tumor verus control Tregs, perhaps surprisingly including genes known to be involved in Treg function, namely IL10, CCR7 and CXCR5 (highlighted in Fig. 3A; (Murai et al., 2009; Schneider et al., 2007; Chung et al., 2011)). Importantly, these differences in gene expression were very reproducible between individuals, a pattern which was not necessarily expected given the heterogeneity of human genetics and the different stages of tumors represented in the sample. The ratios of expression in tumor vs colon Tregs were comparable in samples from batch 1 and batch2 (Figure 3, Part B). The heatmap representation of Figure 3, Part C, which focuses on the top 151 genes from this comparison, shows that essentially all of these genes were up-regulated in all of the eleven tumors. Thus, the tumor Treg signature is a general one, at least among the colorectal tumors tested. Finally, it was asked whether the genes over-expressed in TITRs in the mouse were also differentially represented in human tumor Tregs. Indeed, there was significant overlap of the tumor Treg signature between the mouse and human data sets. In the example volcano plot in Figure 3D, genes that are upregulated 4-fold or greater in tumor versus splenic Tregs in the mouse were highlighted on human data. They were clearly biased towards over-expression in the human data as well (77/90 mouse genes were up and 13/90 down, chisq p.value <10-10). These data clearly demonstrate conservation of the tumor Treg signature across species.

Example 5: Genes Correlated Specifically with FOXP3 in the TCGA Tumor Datasets

FOXP3 is the key transcription factor that defines Treg cells, conditioning a substantial portion of their transcriptional identity (Josefowicz et al., 2012). Thus, genes that are specifically expressed in TITR, relative to other tumor-infiltrating immunocytes, should exhibit a tight correlation to FOXP3 transcripts across sets of gene expression profiles generated from whole tumors. To this end, TCGA, a publically available database of gene expression in 33 types of cancer from more than 11,000 patients was analyzed. Four different types of cancer were selected, representing different frequencies of mutational load, and hence likelihood of anti -tumor immune response (Schumacher and Schreiber, 2015) (namely colon, breast, pancreas and lung, with 285, 1093, 178 and 501 tumor samples, respectively). First attempts at correlation to FOXP3 in these datasets brought forth a large number of genes, many of which were typical of non-Treg cells (i.e. immunoglobulin transcripts from B cells, or Teff cell transcripts like THEMIS). This observation indicated that the proportion of Treg cells in the tumor samples was generally parallel to the overall degree of immune infiltration. To account for, and mathematically remove, this confounder, prototypical signature gene-sets corresponding to the major types of

immunocytes that can be found in a tumor (namely granulocytes, dendritic cells,

macrophages, CD4+ Teff cells and B lymphocytes) were manually curated and determined an "infiltration index" that accounts for the degree of immune infiltration by each

inflammatory cell type in each of the TCGA tumor samples. A linear model was fitted between gene expression and these infiltration indices, and used the residuals from this fit as the component of gene expression that could not be accounted for by overall

degree of immunocyte infiltration. The correlation was recomputed to FOXP3 in this dataset. In keeping with the hypothesis, and as illustrated in Fig. 4A, the correlation to FOXP3 dropped dramatically for most of the genes that correlated strongly in the raw datasets, except for a small minority of genes whose correlation with FOXP3 resisted the normalization process. Fittingly, these included some well-known Treg-specific genes such as those encoding costimulatory molecules CTLA4, CD80 and ICOS, but also chemokine and chemokine receptors like CCR8 and CCL22. Interestingly, positive correlation to FOXP3 was similar in different types of tumors (Figure 4, Part B, C), supporting the notion that these are not spurious correlations. This sharing also indicated that the tumor-Treg specific transcriptome is largely shared between human tumor types. In addition, these FOXP3- correlated transcripts were not simply a phenocopy of the classic Treg signature (Ferraro et al., 2014). Only a subset of the Treg-up signature was positively correlated with FOXP3, as illustrated for the breast and colon tumor datasets in Figure 4, Part D. Similarly, only a fraction of transcripts correlated to FOXP3 actually belong to the classic Treg signature.

Example 6: Data integration

Next, combinatorial approaches were taken to integrate these three inputs and select the set of transcripts most specific of TITR across different tumors. Since the value types were different, the rankings of orthologous genes were combined in the two species, rather than the expression metrics: ranked, for each gene and in each of the eleven samples of colon tumor Tregs, the induction of expression relative to expression in batch-matched Tregs from normal colon tissue. These ranks were then summed across all samples, which yielded an overall rank of Treg genes preferentially induced in human colorectal cancer Tregs. For the mouse tumor datasets, the fold change in expression was ranked of each gene in TITR relative to the splenic Tregs controls, and summed these ranks across the three datasets (MC38, B16 and BP infiltrating Tregs), to yield an overall rank of Treg genes

preferentially induced in the mouse tumor models. From the correlation to FOXP3 in the TCGA tumor datasets, given the high concordance in correlation to FOXP3 in the four

TCGA tumor types analyzed (breast, colon, pancreas and lung), the correlation coefficients was averaged to yield a combined correlation to FOXP3 across these tumors. Figure 5, Part A plots the overall score for human and mouse tumor Tregs (x and y axes,

respectively). Overall, and consistent with the trans-species conservation of tumor-specific gene induction described above, there was a strong correlation between these rankings (r=0.32, p<10-60). A small group of transcripts was on top of the ranking for tumor- specificity in both species (red box in Figure 5, A), extending to another large gene-set that scored highly in the mouse and still in the top 10% of differential transcripts in human tumors (blue box). Interestingly, a smaller group of genes led the rankings for induced expression in human tumor Tregs but not in mice (green box), suggesting that at least some of the differences are species- specific (or might be tied to "natural" vs transplanted tumors). Figure 5, Part B combines the same overall score for over-expression in human tumor Tregs with the average correlation score derived from the TCGA datasets. Many, of the Treg transcripts with the highest rank for over-expression in human TITRs also showed significant association to FOXP3 in the TCGA datasets (e.g. CCR8, TNFRSF9, IL21R), although this did not apply all (e.g. DUSP4, IRAK2 OR CCNG2). Also correlated with FOXP3 were other genes known to be valid immunotherapy targets in tumors (e.g. CTLA4 or TIGIT). From these three inputs, a list was generated of genes differentially expressed in TITR. This list combines (i) transcripts that ranked in the top 3% for differential expression in human colorectal TITRs; (ii) transcripts that ranked in the top 3% for differential expression in mouse TITRs and ranked in the top 10% for differential expression in human colorectal TITRs; (iii) transcripts that ranked in the top 2% for correlation with FOXP3 in TCGA tumor data (average of all 4 tumor-types) and ranked in the top 10% for differential expression in human colorectal TITRs. This selection yielded a total of 184 transcripts, of which 127 were removed from consideration as potential targets because they were found to have significant expression in heart or nervous system tissue in public gene expression databases, and hence to carry a higher likelihood of adverse events if used as therapeutic targets.

Example 7: Curative e ffect ofmAbs targeting TITRs on tumor progression.

Monoclonal antibodies specific for TITR targets were administered to tumor-bearing mice to pre-clinically test the effect of TITR target modulation. The transplantable MC38 colon adenocarcinoma (MC38) was used. Tumors were inoculated subcutaneously into immunocompetent C57BL/6 mice and allowed to grow for 7 days before treatment groups were assigned according to tumor volume. Mice received 200 ug single agent mAb i.p. on day 7 or day 10 and this dose was repeated twice at 72 hour intervals, for a total of three doses. Control mice received either 200 ug of appropriate isotype control or an irrelevant mAb, or were left untreated. Tumor volumes were measured at regular intervals from Day 7 until Day 21. The delta in tumor volume from time of first treatment until the end of the experiment (Day 21) was used as a readout. Table 1 provides a summary of the mAb treatments. The table denotes the number of mice per treatment group whose MC38 tumors resolved (zero tumor volume measurement, no palpable tumor remained) or exhibited improvement (slower or delayed growth), no change (no difference) or worsening (increased tumor growth), following mAb treatment compared to controls. As can be seen in Figure 7, excellent responses to treatment were observed following treatment with anti-CD30 or anti- CCR8 mAbs, with 63% or 47% mice responding to anti-CD30 or anti-CCR8 mAb treatment, respectively (panel A). These results demonstrate that a modest response was observed when anti-ILlRLl (ST2) mAb was given (panel B) and no change in tumor growth was observed when mice received mAbs targeting IL21R or CXCR6 (panel C). Unexpectedly, significant worsening of the tumor occurred following administration of anti-CXCR3 mAb (panel C). These proof-of-concept results indicate that some of the identified TITR transcripts in Examples 1 to 6 can indeed serve as valuable targets for antibody-based cancer immunotherapy, and in particular that antibodies for T FRSF8 (CD30) and CCR8 can be valuable therapeutic agents to treat established tumors.

Example 8: Combination mAb therapy improves the anti-tumor response compared to single agent mAb therapy.

To further explore the success of single agent mAb treatments targeting CD30 or

CCR8, we next tested these treatments in combination with a known, established

immunotherapy, namely anti-PD-1 mAb treatment. The transplantable MC38 colon adenocarcinoma (MC38) was used here. Tumors were inoculated subcutaneously into immunocompetent C57BL/6 mice and allowed to grow for 7 days before treatment groups were assigned according to tumor volume. Mice received 200 ug single agent mAb (as for

CD30, CCR8, PD-1) or a combination of two mAbs (as for CD30+PD-1, CCR8+PD-1) i.p. on days 10, 13 and 16 after tumor cell injection. Control mice received either 200 ug of appropriate isotype control or an irrelevant mAb, or were left untreated. Tumor volumes were measured at regular intervals from Day 7 until Day 21. The tumor growth plots track tumor volume over time. As shown in Figure 8, combination mAb therapy outperforms single agent mAb therapy such that it causes more mice to exhibit resolution (zero tumor volume) or improvement (slower tumor growth) of their tumor burden, suggesting that anti- CCR8 or anti-CD30 mAbs are useful complements of checkpoint blockade therapy.

Example 9: 'Cured' mice are protected from re-challenge with the same tumor type.

As for example 7, 1 x 10⁶ MC38 cells were injected on the flank of

immunocompetent C57BL/6 mice and allowed to grow for seven days before treatment groups were assigned according to tumor volume. Mice received 200 ug single agent mAb (CD30, CCR8, PD-1) or a combination of mAbs (CD30+PD-1, CCR8+PD-1) i.p. on days 10, 13 and 16 after tumor cell injection. Tumor volumes were measured at regular intervals from Day 7 until Day 21. Responders were monitored until Day 60 (Day 50 after first mAb treatment), at which time 1 x 10⁶ MC38 cells were injected s.c. on the opposite flank from the original tumor site in mice whose tumors had completely resolved (0 measurement recorded for at least 30 days prior to re-challenge). Tumors grew in all mice re-challenged, but tumor growth peaked at Day 4 after re-challenge and all tumors quickly began to resolve. These studies demonstrate that 'cured' mice (those whose original MC38 tumors resolved completely following mAb treatment) are protected from re-challenge with the same tumor type (Figure 9), and that anti-CD30 and anti-CCR8, alone or in combinations, can induce long-lasting anti-tumor immune responses. Example 10: Effects of mAb treatment on TITRs and the tumor microenvironment.

The transplantable MC38 colon adenocarcinoma (MC38) was used here. Tumors were inoculated subcutaneously into immunocompetent C57BL/6 or Foxp3-GFP/C57BL6 reporter mice, the latter in which Treg cells are uniquely identified and readily sorted on the basis of the fluorescent GFP reporter. Tumors were allowed to grow for seven days before treatment groups were assigned according to tumor volume. Mice received 1 x 200 ug single agent mAb (CD30, CCR8, PD-1) i.p. on day 10 after tumor cell injection. Control mice received either 200 ug of appropriate isotype control or were left untreated. At 40 hrs after mAb treatment, immunocytes were isolated from the tumors and spleens from these animals, stained with fluorochrome-conjugated antibodies and T cell populations, namely Tregs (CD4+CD25+CD8-TCRb+CD45+ cells), were quantified by flow cytometry.. As shown in Figure 10, panel A, there is no immediate clearance of the TITR population at this early time point after one mAb treatment (numbers represent Tregs as % total CD4+CD8- TcRb+CD45+ T cell population in the tumor). Also at 40 hrs after mAb treatment, 1,000 Tregs (Foxp3(GFP)+CD4+CD8-TCRb+CD45+ cells) were highly purified (double sorting by flow cytometry) from tumors and spleens from Foxp3-GFP/C57BL/6 mice. RNA from these highly purified cells was then used for gene expression profiling by whole- transcriptome shotgun sequencing (RNAseq, SmartseqV2 procedure, 5-10xlO^A6 mappable reads/sample). Replicate data were generated for each sample type. They were filtered for quality, mapped to the mouse genome (TopHat), normalized (Cuffnorm), and filtered to avoid low-expression transcripts and resulting noise. As shown in Figure 10, panel B, there is a bias in the TITR signature in all anti-CD30 treated mice and one of the anti-CCR8- treated mice, such that these cells express less of the transcripts typically overexpressed in TITRs. The heatmap depicts gene expression of the TITR-enriched geneset of Table 2 in TITRs from anti-CD30-, anti-CCR8- or control -treated mice. The color code reflects the range (low to high = blue to red) of fold change in expression value between TITR and splenic Treg cells. This observation suggests that blockade of these TITR-specific targets did not kill the TITRs, but modified their phenotype away from the tumor-specific pattern.

Incorporation by Reference

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web and/or the National Center for Biotechnology Information (NCBI) on the World Wide Web.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject, the method comprising administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene.

2. A method of inducing an anti-tumor immune response by decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject, the method comprising administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene.

3. The method of claim 2, wherein the anti -tumor response is long-lasting.

4. A method of treating a tumor in a subject, the method comprising administering to the subject an agent that inhibits the activity or expression of a product of a the CCR8 or TNFRSF8 gene.

5. A method of increasing the amount of T effector cells in a tumor in a subject, the method comprising administering to the subject an agent that inhibits the activity or expression of a product of a either the CCR8 or TNFRSF8 gene.

6. The method of any one of claims 1 to 5, wherein the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a nueroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a Rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymus tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms tumor.

7. The method of any one of claims 1 to 6, wherein the tumor is a colorectal tumor.

8. The method of any one of claims 1 to 6, wherein the tumor is an adenocarcinoma.

9. The method of any one of claims 1 to 6, wherein the tumor is a breast cancer tumor, a pancreatic cancer tumor, or a lung cancer tumor.

10. The method of any one of claims 1 to 9, wherein the tumor is a primary tumor.

11. The method of any one of claims 1 to 9, wherein the tumor is a metastatic tumor.

12. The method of any one of the preceding claims, wherein the agent is an antibody specific for a protein product of the either the CCR8 or TNFRSF8 gene.

13. The method of claim 12, wherein the antibody is a polyclonal antibody.

14. The method of claim 12, wherein the antibody is a monoclonal antibody.

15. The method of claim 12, wherein the antibody is a chimeric antibody.

16. The method of claim 12, wherein the antibody is a humanized antibody.

17. The method of claim 12, wherein the antibody is an antibody fragment.

18. The method of claim 12, wherein the antibody is bispecific antibody.

19. The method of any one of claims 1 to 11, wherein the agent is a peptide that specifically binds to a protein product of either the CCR8 or TNFRSF8 gene.

20. The method of any one of claims 1 to 11, wherein the agent is a small molecule that inhibits the activity of a protein product of either the CCR8 or TNFRSF8 gene.

21. The method of any one of claims 1 tol 1, wherein the agent is an interfering nucleic acid specific for an mRNA product of either the CCR8 or TNFRSF8 gene.

22. The method of claim 21, wherein the interfering nucleic acid is a siRNA.

23. The method of claim 21, wherein the interfering nucleic acid is a shRNA.

24. The method of claim 21, wherein the interfering nucleic acid is a miRNA.

25. The method of claim 21, wherein the interfering nucleic acid is a peptide nucleic acid.

26. The method of any one of the previous claims, wherein the agent is administered to the subject systemically.

27. The method any one of claims 1 to 25, wherein the agent is administered intravenously.

28. The method any one of claims 1 to 25, wherein the agent is administered subcutaneously.

29. The method any one of claims 1 to 25, wherein the agent is administered intramuscularly.

30. The method any one of claims 1 to 25, wherein the agent is administered orally,

31. The method any one of claims 1 to 25, wherein the agent is administered locally,

32. The method any one of claims 1 to 25, wherein the agent is administered locally to the tumor in the subject.

33. The method of any one of the previous claims, wherein the agent is administered to the subject in a pharmaceutically acceptable formulation.

34. The method of any one of claim 1 to 33, wherein the method further comprises administering a second agent.

35. The method of claim 34, wherein the second agent is a chemotherapeutic agent.

36. The method of claim 34, wherein the second agent is an immune checkpoint inhibitor, a kinase inhibitor, a CAR-T cell, or a oncolytic virus..

37. The method of claim 36, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

38. The method of any one of claims 1 to 37, wherein the second agent is a tumor vaccine.

39. The method of any one of the preceding claims, wherein the subject is a human.

40. The method of any preceding claim, wherein the subject has cancer.

41. The method of claim 40, wherein the cancer is bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.

42. A method of targeting an agent to TITRs in a subject, the method comprising administering to the subject the agent is conjugated to a polypeptide or protein that binds to the protein product of either the CCR8 or TNFRSF8 gene.

43. The method of claim 42, wherein the polypeptide or protein is an antibody specific for a protein product of either the CCR8 or TNFRSF8 gene.

44. The method of claim 43, wherein the antibody is a polyclonal antibody.

45. The method of claim 43, wherein the antibody is a monoclonal antibody.

46. The method of claim 43, wherein the antibody is a chimeric antibody.

47. The method of claim 43, wherein the antibody is a humanized antibody.

48. The method of claim 43, wherein the antibody is an antibody fragment.

49. The method of claim 43, wherein the antibody is bispecific antibody.

50. The method of any one of claims 42 to 48, wherein the agent is a drug.

51. The method of claim 50, wherein the drug is a toxin.

52. The method of claims 51, wherein the toxin is MMAE, DM-1, a maytansinoid, a doxorubicin derivative, an auristatin, a calcheamicin, CC-1065, aduocarmycin or a anthracycline.

53. The method of any one of claims 42 to 52, wherein the agent is administered to the subject systemically.

54. The method of any one of claims 42 to 52, wherein the agent is administered intravenously.

55. The method of any one of claims 42 to 52, wherein the agent is administered subcutaneously.

56. The method of any one of claims 42 to 52, wherein the agent is administered intramuscularly.

57. The method of any one of claims 42 to 52, wherein the agent is administered orally.

58. The method any one of claims 42 to 52, wherein the agent is administered locally.

59. The method claim 58, wherein the agent is administered locally to the tumor in the subject.

60. The method of any one of claims 42 to 59, wherein the agent is administered to the subject in a pharmaceutically acceptable formulation.

61. The method of any one of claims 42 to 60, wherein the subject is a mammal.

62. The method of claim 61, wherein the subject is a human.

63. The method of any one of the preceding claims, wherein the subject has cancer.

64. The method of claim 63, wherein the cancer is bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.

65. A method of determining whether an agent is an anti-cancer therapeutic agent comprising determining whether the test agent inhibits the expression or activity of a protein product of either the CCR8 or TNFRSF8 gene, wherein the test agent is determined to be an anti-cancer therapeutic agent if the test agent inhibits the expression or activity of a protein product of either the CCR8 or TNFRSF8 gene.

66. The method of claim 65, wherein the test agent is a member of a library of test agents.

67. The method of claim 65 or 66, wherein the test agent is a interfering nucleic acid.

68. The method of claim 65 or 66, wherein the test agent is a peptide.

69. The method of claim 65 or 66, wherein the test agent is a small molecule.

70. The method of claim 65 or 66, wherein the test agent is antibody.

71. The method of claim 65 or 66, wherein the test agent is a protein drug conjugate.

72. The method of any one of claims 65 to 71, wherein the test agent inhibits the activity or expression of a protein product of either the CCR8 or TNFRSF8 gene by at least 50%.

73. The method of any one of claims 65 to 71, wherein the test agent inhibits the activity or expression of a protein product of either the CCR8 or TNFRSF8 gene by at least 60%.

74. The method of any one of claims 65 to 71, wherein the test agent inhibits the activity or expression of a protein product of either the CCR8 or TNFRSF8 gene by at least 70%.

75. The method of any one of claims 65 to 71, wherein the test agent inhibits the activity or expression of a protein product of either the CCR8 or TNFRSF8 gene by at least 80%.

76. The method of any one of claims 65 to 71, wherein the test agent inhibits the activity or expression of a protein product of either the CCR8 or TNFRSF8 gene by at least 90%.

77. The method of any one of claims 65 to 71, wherein the test agent inhibits the activity or expression of a protein product of either the CCR8 or TNFRSF8 gene by at least 100%.

78. The method of any one of claims 65 to 77, further comprising treating a subject by administering an agent that inhibits the expression or activity of a product of either the CCR8 or TNFRSF8 gene.

79. A method of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject, the method comprising conjointly administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene and an immune checkpoint inhibitor.

80. A method of inducing an anti-tumor immune response by decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject, the method comprising conjointly administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene and an immune checkpoint inhibitor.

81. The method of claim 80, wherein the anti -tumor response is long-lasting.

82. A method of treating a tumor in a subject, the method comprising conjointly administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene and an immune checkpoint inhibitor.

83. A method of increasing the amount of T effector cells in a tumor in a subj ect, the method comprising conjointly administering to the subject an agent that inhibits the activity or expression of a product of the CCR8 or TNFRSF8 gene and an immune checkpoint inhibitor.

84. The method of any one of claims 79 to 83, wherein the agent is an antibody specific for a protein product of the CCR8 or TNFRSF8 gene.

85. The method of any one of claims 79 to 83, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

86. A method of decreasing the number or activity of tumor infiltrating T regulatory cells (TITR) in a tumor present in a subject, the method comprising administering to the subject an agent that induces cytotoxicity in cells that express a product of a either the CCR8 or TNFRSF8 gene.

87. A method of treating a tumor in a subject, the method comprising administering to the subject an agent that that induces cytotoxicity in cells that express a product of either the CCR8 or TNFRSF8 gene.

88. A method of increasing the amount of T effector cells in a tumor in a subject, the method comprising administering to the subject an agent that that induces cytotoxicity in cells that express a product of either the CCR8 or TNFRSF8 gene.

89. The method of claims 86 to 88, wherein the cytotoxicity is antibody-dependent cell- mediated cytotoxicity (ADCC).

90. The method of claims 86 to 88, wherein the cytotoxicity is complement- dependent cytotoxicity (CDC).

91. The method of any one of claims 86 to 90, wherein the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a nueroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a Rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymus tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms tumor.

92. The method of any one of claims 86 to 91, wherein the tumor is a colorectal tumor.

93. The method of any one of claims 86 to 91, wherein the tumor is an adenocarcinoma.

94. The method of any one of claims 86 to 91, wherein the tumor is a breast cancer tumor, a pancreatic cancer tumor, or a lung cancer tumor.

95. The method of any one of claims 86 to 94, wherein the tumor is a primary tumor.

96. The method of any one of claims 86 to 94, wherein the tumor is a metastatic tumor.

97. The method of any one of the claims 86 to 96, wherein the agent is an antibody.

98. The method of claim 97, wherein the antibody is a polyclonal antibody.

99. The method of claim 97, wherein the antibody is a monoclonal antibody.

100. The method of claim 97, wherein the antibody is a chimeric antibody.

101. The method of claim 97, wherein the antibody is a humanized antibody.

102. The method of claim 97, wherein the antibody is an antibody fragment.

103. The method of claim 97, wherein the antibody is bispecific antibody.

104. The method of any one of claims 86 to 96, wherein the agent is a peptide.

105. The method of any one of claims 86 to 96, wherein the agent is a small molecule.

106. The method of any one of claims 86 to 96, wherein the agent is an interfering nucleic acid.

107. The method of claim 106, wherein the interfering nucleic acid is a siRNA.

108. The method of claim 106, wherein the interfering nucleic acid is a shRNA.

109. The method of claim 106, wherein the interfering nucleic acid is a miRNA.

110. The method of claim 106, wherein the interfering nucleic acid is a peptide nucleic acid.

111. The method of any one of claims 86 to 110, wherein the agent is administered to the subject systemically.

112. The method any one of claims 86 to 110, wherein the agent is administered intravenously.

113. The method any one of claims 86 to 110, wherein the agent is administered subcutaneously.

114. The method any one of claims 86 to 110, wherein the agent is administered intramuscularly.

115. The method any one of claims 86 to 110, wherein the agent is administered orally.

116. The method any one of claims 86 to 110, wherein the agent is administered locally.

117. The method any one of claims 86 to 110, wherein the agent is administered locally to the tumor in the subject.

118. The method of any one of claims 86 to 117, wherein the agent is administered to the subject in a pharmaceutically acceptable formulation.

119. The method of any one of claim 86 to 117, wherein the method further comprises administering a second agent.

120. The method of claim 119, wherein the second agent is a chemotherapeutic agent.

121. The method of claim 119, wherein the second agent is an immune checkpoint inhibitor, a kinase inhibitor, an oncolytic virus, or a CAR-T cell.

122. The method of any one of claims 86 to 121, wherein the second agent is a tumor vaccine.

123. The method of any one of claims 86 to 122, wherein the subject is a human.

124. The method of any one of claims 86 to 123, wherein the subject has cancer.

125. The method of claim 124, wherein the cancer is bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.