Methods of treating cancer by inhibiting ubiquitin conjugating enzyme e2k (UBE2k)

Abstract

To provide methods for the treatment of cancer.SOLUTION: The disclosure provides methods for the treatment of cancer in a subject in need of cancer treatment, comprising administering to the subject an inhibitor of Ubiquitin Conjugating Enzyme E2K (UBE2K), thereby treating cancer in the subject.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 729,348, filed Sep. 10, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The above ASCII copy, created on September 9, 2019, is named 119992_19420_Sequence_Listing.txt and is 3KB in size.

[0003] Technical Field The present invention relates to a method for treating cancer by inhibiting the ubiquitin-conjugating enzyme E2K (UBE2K). [Background technology]

[0004] Cancer is currently one of the leading causes of death in developed countries. A diagnosis of cancer is traditionally accompanied by serious health complications. Cancer can cause disfigurement, chronic or acute pain, lesions, organ failure, or even death. Traditionally, many cancers are treated by surgery, chemotherapy, radiation, or a combination thereof. Chemotherapeutic agents used to treat cancer are known to cause several serious and unpleasant side effects in patients. For example, some chemotherapeutic agents cause neuropathy, nephrotoxicity (e.g., hyperlipidemia, proteinuria, hypoproteinemia, combinations thereof, etc.), stomatitis, mucositis, alopecia, anorexia, esophagitis, amenorrhea, immunosuppression, anemia, high-tone hearing loss, cardiotoxicity, fatigue, neuropathy, bone marrow suppression, or a combination thereof. In many cases, chemotherapy is ineffective or loses effectiveness after a period of effectiveness either during treatment or shortly after the treatment regimen is completed (i.e., the treatment regimen does not result in a cure). Improved methods for the treatment of cancer are still desirable. Summary of the Invention

[0005] In certain aspects, the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a ubiquitin-conjugating enzyme E2K (UBE2K) inhibitor, thereby treating cancer in the subject.

[0006] In certain aspects, the present disclosure relates to a method of reducing the proliferation of cancer cells in a subject in need thereof, comprising administering to the subject a ubiquitin-conjugating enzyme E2K (UBE2K) inhibitor, thereby reducing the proliferation of cancer cells in the subject compared to a subject not administered the UBE2K inhibitor.

[0007] In certain aspects, the present disclosure relates to a method for inducing cancer cell death in a subject in need thereof, comprising administering to the subject a ubiquitin-conjugating enzyme E2K (UBE2K) inhibitor, thereby inducing cancer cell death in the subject. In certain embodiments, the cancer cell death is induced by apoptosis.

[0008] In certain embodiments, the UBE2K inhibitor is a specific inhibitor of UBE2K. In certain embodiments, the UBE2K inhibitor comprises a small molecule. In certain embodiments, the UBE2K inhibitor comprises a nucleic acid inhibitor. In certain embodiments, the nucleic acid inhibitor comprises an antisense nucleic acid molecule. In certain embodiments, the nucleic acid inhibitor comprises a double-stranded nucleic acid molecule. In certain embodiments, the double-stranded nucleic acid molecule comprises a double-stranded RNA selected from the group consisting of siRNA, shRNA, and dicer substrate siRNA (DsiRNA). In certain embodiments, the UBE2K inhibitor comprises an antibody.

[0009] In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the solid tumor is selected from the group consisting of carcinoma, melanoma, sarcoma, and lymphoma. In certain embodiments, the solid tumor is selected from the group consisting of pancreatic cancer, liver cancer, colorectal cancer, and lymphoma. In certain embodiments, the solid tumor is pancreatic cancer or liver cancer. In certain embodiments, the cancer is leukemia.

[0010] In certain embodiments, the UBE2K inhibitor is administered with an additional agent. In certain embodiments, the additional agent is an anti-cancer agent. In certain embodiments, the additional agent is a chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent is selected from the group consisting of gemcitabine, 5-fluorouracil, leucovorin, docetaxel, fludarabine, cytarabine, cyclophosphamide, paclitaxel, docetaxel, busulfan, methotrexate, daunorubicin, doxorubicin, melphalan, cladribine, vincristine, vinblastine, chlorambucil, tamoxifen, taxol, camptothecin, actinomycin-D, mitomycin C, combretastatin, cisplatin, etoposide, verapamil, podophyllotoxin, and 5-fluorouracil. In certain embodiments, the additional agent is an anti-angiogenic agent.

[0011] In certain embodiments, the additional agent is an immunotherapeutic agent. In certain embodiments, the immunotherapeutic agent is an immune checkpoint modulator of an immune checkpoint molecule. In certain embodiments, the immune checkpoint molecule is selected from CD27, CD28, CD40, CD122, OX40, GITR, ICOS, 4-1BB, ADORA2A, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, and VISTA. In certain embodiments, the immune checkpoint molecule is a stimulatory immune checkpoint molecule, and the immune checkpoint modulator is an agonist of the stimulatory immune checkpoint molecule. In certain embodiments, the immune checkpoint molecule is an inhibitory immune checkpoint molecule, and the immune checkpoint modulator is an antagonist of the inhibitory immune checkpoint molecule. In certain embodiments, the immune checkpoint modulator is selected from a small molecule, an inhibitory RNA, an antisense molecule, and an immune checkpoint molecule binding protein. In certain embodiments, the immune checkpoint molecule is PD-1, and the immune checkpoint modulator is a PD-1 inhibitor. In certain embodiments, the PD-1 inhibitor is selected from pembrolizumab, nivolumab, pidilizumab, SHR-1210, MEDI0680R01, BBg-A317, TSR-042, REGN2810, and PF-06801591. In certain embodiments, the immune checkpoint molecule is PD-L1, and the immune checkpoint modulator is a PD-L1 inhibitor. In certain embodiments, the PD-L1 inhibitor is selected from durvalumab, atezolizumab, avelumab, MDX-1105, AMP-224, and LY3300054. In certain embodiments, the immune checkpoint molecule is CTLA-4, and the immune checkpoint modulator is a CTLA-4 inhibitor. In certain embodiments, the CTLA-4 inhibitor is selected from ipilimumab, tremelimumab, JMW-3B3, and AGEN1884. [Brief description of the drawings]

[0012] [Figure 1] 1 shows an overview of the ubiquitin proteasome system. [Diagram 2] 1 shows knockdown of UBE2K transcripts 24 and 96 hours after siRNA transfection in Miapaca2, HepG2, and SKHEP1 cells. [Diagram 3] 1 shows knockdown of UBE2K protein 24 and 96 hours after siRNA transfection in Miapaca2, HepG2, and SKHEP1 cells. [Figure 4] Shows the effect of UBE2K siRNA-mediated knockdown on cell number under basal conditions 96 hours after transfection. [Diagram 5] 1 shows the effect of UBE2K siRNA-mediated knockdown on doxorubicin sensitivity. [Figure 6] 1 shows the effect of UBE2K siRNA-mediated knockdown on related E2 mRNA expression. [Figure 7] Shows the effect of UBE2K siRNA-mediated knockdown on cell number / viability when cultured in medium containing 0.5% or 5% serum. [Figure 8] 1 shows the effect of UBE2K siRNA-mediated knockdown on cell death and cell cycle progression. [Figure 9A] Figures 9A and 9B: Regulation of UBE2K interactors at protein (A) and transcript levels (B) in UBE2K knockdown cells and MiaPaca2 cells transfected with non-targeting siRNA with and without MG132. [Figure 9B] This is a continuation of Figure 9A. [Figure 10] Figure 1 shows cell cycle analysis of synchronous and asynchronous populations of MiaPaca2 cells. Pelleted MiaPaca2 cells were treated with Hoechst stain for 10 minutes. Cell cycle analysis was analyzed by measuring the MFI of Hoechst staining in the FL-3 channel using flow cytometry. [Figure 11] Cell Titer-Fluor cell viability assay of synchronized MiaPaca2 cells with UBE2K knockdown 48, 72, and 96 hours after release from serum deprivation with 20% FBS. [Figure 12] Changes in cyclin concentrations throughout the cell cycle are shown, with a direct correlation between cyclin accumulation and the three major cell cycle checkpoints. [Figure 13] FIG. 1 shows a time course of the levels of key cell cycle regulators in synchronized MiaPaca2 cells. [Figure 14] 1 shows confirmation of Cdc34 and UBE2K knockdown in MiaPaca2 cells 72 hours after transfection. [Figure 15] Figure 2 shows that UBE2K knockdown in MiaPaca2 cells reduced viable cell numbers by approximately 20% 72 hours after transfection. Knockdown of Cdc34 did not affect viable cell numbers. [Figure 16] Figure 1 shows confirmation of UBE2K knockdown in MiaPaca2 cell lines transduced with UBE2K shRNA2 and shRNA3. Increased levels of G2 / M cell cycle regulatory proteins were observed in UBE2K knockdown cells. [Figure 17] 1 shows that Cell Titer-Fluor cell viability assays after 72 hours of plating revealed that the total number of live cells was significantly lower in UBE2K knockdown cells than in non-targeting (NT) siRNA control shRNA cells. [Figure 18] Direct cell counts and individual nuclei counts in MiaPaca2 cells are shown. UBE2K knockdown cells showed significantly fewer cells / well 96 hours after plating when compared to their non-targeting (NT) shRNA control counterparts. [Figure 19] 1 shows the correlation between UBE2K gene expression and survival rate of patients with pancreatic ductal adenocarcinoma. [Figure 20] An example dataset of UBE2K immunohistochemical staining in pancreatic tumor tissue and normal adjacent tissue is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Using discovery platform technology, ubiquitin-conjugating enzyme E2 K (UBE2K) was identified as a central node that is significantly regulated in human cancer cells. Knockdown of UBE2K in cancer cells, such as pancreatic cancer and hepatocellular carcinoma cell lines, reduced cancer cell numbers in vitro, and further studies showed that UBE2K knockdown reduced cancer cell proliferation and induced cancer cell death. Thus, the present invention provides a method of reducing cancer cell proliferation and / or inducing cancer cell death by administering a UBE2K inhibitor. The present invention further provides a method of treating cancer in a subject by administering a UBE2K inhibitor to the subject.

[0014] I. Definition The terms "administer", "administering" or "administering" include any method of delivering a pharmaceutical composition or agent into a subject or to a particular area in or on a subject. In certain embodiments, the agent is delivered orally. In certain embodiments, the agent is administered parenterally. In certain embodiments, the agent is delivered locally, including transmucosally. In certain embodiments, the agent is delivered by inhalation. In certain embodiments of the invention, the agent is administered by parenteral delivery, including intravenous, intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injection. In one embodiment, the compositions provided herein can be administered by direct injection into a tumor. In some embodiments, the agent is delivered by injection or infusion. In certain embodiments, administration is systemic. In certain embodiments, administration is local. In some embodiments, one or more routes of administration may be combined, such as, for example, intravenous and intratumoral, or intravenous and oral, or intravenous and topical, or intravenous and transdermal or transmucosal. Administration of an agent may be performed by the concerted efforts of many people. Administering a drug includes, for example, prescribing a drug to be administered to a subject and / or providing instructions to take a particular drug, directly or otherwise, for self-delivery, e.g., oral delivery, subcutaneous delivery, intravenous delivery through a central line, etc., or for delivery by a trained professional, e.g., intravenous delivery, intramuscular delivery, intratumoral delivery, etc.

[0015] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the above, through at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) variants, multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigenic determinant of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies may be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different well-known subunit structures and three-dimensional configurations. Antibodies may be naked or may be conjugated to other molecules, such as toxins, radioisotopes, etc.

[0016] In some embodiments, the antibody is a non-naturally occurring antibody. In some embodiments, the antibody is purified from natural components. In some embodiments, the antibody is recombinantly produced. In some embodiments, the antibody is produced by a hybridoma.

[0017] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and F. vAntibody fragments include, but are not limited to, Fab fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. The term "antigen-binding fragment" of an antibody includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by certain fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (but are not limited to): (i) Fab fragments, V L , V H , C L , and C H1 (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region (e.g., digestion of an antibody with pepsin produces two fragments, a bivalent antigen-binding F(ab')2 fragment, and a pFc' fragment that does not bind to antigen) and their associated monovalent F(ab') units; (iii) V H and C H1 F consists of domains d (iv) a V fragment of a single arm of an antibody (i.e., a portion of the heavy chain contained in a Fab); L and V H F consists of domains v Fragments and related disulfide-linked F v ;(v)V H These include dAb (domain antibody) or sdAb (single domain antibody) fragments consisting of domains (Ward et al., Nature 341:544-546, 1989), and (vi) isolated complementarity determining regions (CDRs).

[0018] As used herein, the term "CDR" refers to the complementarity determining region in the antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, and they are designated as CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region that can bind to antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs are sometimes referred to as the Kabat CDRs. Chothia and coworkers found that certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level (Chothia et al. (1987) J. MOL. BIOL. 196:901-917, and Chothia et al. (1989) NATURE SYSTEMS AND METHODS OF GENERALIZED GENERATIONS, VOL. 1, pp. 1111-1115, 2003). 342:877-883). These subportions are designated as L1, L2 and L3, or H1, H2 and H3, where "L" and "H" represent the light and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, and have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with the Kabat CDRs are described by Padlan et al. (1995) FASEB J. 9:133-139 and MacCallum et al. (1996) J. MOL. BIOL. 262(5):732-45.Still other CDR boundary definitions may not strictly follow one of the above systems, but still overlap with the Kabat CDRs and may be shortened or extended in light of predictions or experimental results that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs.

[0019] As used herein, a "monoclonal antibody" refers to a homogeneous antibody population that is involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. The term "monoclonal antibody" refers to intact, full-length monoclonal antibodies as well as antibody fragments (Fab, Fab', F(ab'), ... v etc.), single chain (scFv) antibodies, fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies produced in any number of ways, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0020] The term "humanized antibody" as used herein refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding sequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies and antibody fragments are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further improve and optimize the performance of the antibody or antibody fragment. Generally, a humanized antibody or antibody fragment thereof comprises substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of non-human immunoglobulins, and all or a substantial portion of the FR regions being those of human immunoglobulin sequences.A humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin.For further details, see Jones et al. (1986) NATURE 321:522-525; Reichmann et al. (1988) NATURE 332:323-329; and Presta (1992) CURR.OP.STRUCT.BIOL.2:593-596, each of which is incorporated herein by reference in its entirety.

[0021] A "bivalent antibody" refers to an antibody or antigen-binding fragment thereof that contains two antigen-binding sites. The two antigen-binding sites may bind to the same antigen or each may bind to a different antigen, in which case the antibody or antigen-binding fragment is characterized as being "bispecific." A "tetravalent antibody" refers to an antibody or antigen-binding fragment thereof that contains four antigen-binding sites. In certain embodiments, a tetravalent antibody is bispecific. In certain embodiments, a tetravalent antibody is multispecific, i.e., binds to more than two different antigens.

[0022] Fab (Fragment Antigen Binding) antibody fragments consist of heavy chain variable regions (V H ) and heavy chain constant region 1 (C H1 ) portion and a light chain variable (V L ) and light chain constant (C L and a polypeptide comprising a C portion, L and C H1 The moieties are preferably linked together by disulfide bonds between Cys residues.

[0023] The terms "cancer" or "tumor" are well known in the art and refer to the presence in a subject of cells that have characteristics typical of cancer-causing cells, such as, for example, uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, reduced cell death / apoptosis, and certain characteristic morphological features.

[0024] As used herein, "cancer" refers to all types of cancer or neoplasm or malignant tumors found in humans, including, but not limited to, leukemia, lymphoma, melanoma, carcinoma, and sarcoma. As used herein, the terms or language "cancer," "neoplasm," and "tumor" are used interchangeably, either in the singular or plural, and refer to cells that have undergone malignant transformation that becomes pathological to the host organism. Primary cancer cells (i.e., cells obtained from near the site of malignant transformation) can be easily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of cancer cells used herein includes not only primary cancer cells, but also cancer stem cells, and cancer precursor cells, or any cells derived from ancestors of cancer cells. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a hematological tumor (i.e., a non-solid tumor).

[0025] A "solid tumor" is one that is detectable, for example, by procedures such as CAT scan, MR imaging, X-ray, ultrasound, or palpation, based on tumor burden, and / or due to the expression of one or more cancer-specific antigens in a sample that can be obtained from a patient. A tumor need not have measurable dimensions.

[0026] The specific criteria for staging a cancer depend on the particular type of cancer based on tumor size, histological characteristics, tumor markers, and other criteria known to those of skill in the art. In general, cancer stages can be described as follows: Stage 0 – Carcinoma in situ Stage I, Stage II, Stage III - higher numbers indicate more extensive disease: larger tumor size and / or spread of cancer beyond the organ where it first began to nearby lymph nodes and / or tissues or organs adjacent to the primary tumor site Stage IV – Cancer has spread to distant tissues or organs

[0027] RECIST criteria are clinically accepted evaluation criteria used to provide a standard approach to solid tumor measurement, providing definitions for objectively evaluating changes in tumor size for use in clinical trials. Such criteria can also be used to monitor the response of individuals undergoing treatment for solid tumors. RECIST 1.1 criteria are discussed in detail in Eisenhauer et al. (New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1) Eur. J. Cancer. 45: 228-247, 2009), the entire contents of which are incorporated herein by reference. Response criteria for target lesions include: Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have decreased in size to <10 mm in the short axis. Partial Response (PR): At least a 30% reduction in the sum of the diameters of the target lesions, using the baseline sum diameter as the measure. Progression (PD): At least a 20% increase in the sum of the diameters of the target lesions using as the criterion the smallest sum on study (which includes the baseline sum, which is also the smallest on study). In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm. (Note: The appearance of one or more new lesions is also considered progression.) Stable (SD): Using the smallest summed diameter on test as the criterion, there is not enough contraction to be considered PR and not enough increase to be considered PD.

[0028] RECIST 1.1 criteria also take into account non-target lesions, which are defined as lesions that may be measurable but do not need to be measured and should only be assessed qualitatively at the desired time points. Response criteria for non-target lesions include: Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker levels. All lymph nodes must be non-pathological in size (short axis <10 mm). Non-CR / Non-PD: persistence of one or more non-target lesion(s) and / or maintenance of tumor marker levels above normal limits. Progression (PD): Definite progression of existing non-target lesions (emphasis in original). The appearance of one or more new lesions is also considered progression. To achieve "definite progression" based on non-target disease, the overall level of non-target disease must worsen substantially, such that the overall tumor burden has increased sufficiently to merit cessation of treatment, even in the presence of SD or PR in target disease. A slight "increase" in the size of one or more non-target lesions is usually not sufficient to be considered a definite progressive state. Thus, it is extremely rare to designate overall progression based solely on changes in non-target disease in terms of SD or PR in target disease.

[0029] Clinically accepted criteria for response to treatment of acute leukemia are as follows: Complete Response (CR): Patient is free of all symptoms related to leukemia and has an absolute neutrophil count >1.0 × 10 9 / L and platelet count >100 × 10 9 / L, normal bone marrow cells have <5% blasts and no Auer rods.

[0030] Complete response with incomplete blood count recovery (Cri): Like CE, but with residual thrombocytopenia (platelet count <100 × 10 9 / L) or residual neutropenia (absolute neutrophil count <1.0 × 10 9 / L).

[0031] Partial response (PR): >50% reduction in bone marrow blasts to 5-25% abnormal cells in the bone marrow; or, if Auer rods are present, CR with <5% blasts. Treatment failure: treatment failed to achieve CR, Cri, or PR. Relapse.

[0032] Relapse after confirmed CR: Appearance of leukemic blasts in peripheral blood or >5% blasts in the bone marrow not attributable to any other cause (e.g., bone marrow regeneration after consolidation therapy), or appearance of new dysplastic changes.

[0033] "Chemotherapeutic agent" refers to a drug used to treat cancer. Chemotherapeutic agents include, but are not limited to, small molecules, hormones and hormone analogs, and biologics (e.g., antibodies, peptide drugs, nucleic acid drugs). In certain embodiments, chemotherapy does not include hormones and hormone analogs.

[0034] A "chemotherapeutic regimen" is a clinically accepted dosing protocol for the treatment of cancer that includes administering to a subject a specific amount of one or more chemotherapeutic agents on a specific schedule. In certain embodiments, the chemotherapeutic agents may be agents in clinical trials.

[0035] Chemotherapy regimens may include administration of drugs in a predetermined "cycle", which includes intervals of administration and non-administration of one or more drugs for the treatment of cancer. For example, to provide a 4-week cycle, the drug may be administered once or more times a week for 3 consecutive weeks, followed by 1 week without administration of the drug. The cycle may be repeated so that the subject is subjected to 3 weeks of treatment, 1 week without treatment, 3 weeks of treatment, 1 week without treatment, etc., for the desired number of cycles. In certain embodiments, treatment efficacy and laboratory values ​​(e.g., liver enzymes, blood counts, renal function) are evaluated at the end of each cycle or every other cycle.

[0036] As used herein, "immunotherapeutic agent" refers to a pharma- ceutically acceptable compound, composition, or treatment that induces or enhances an immune response. Immunotherapeutic agents include, but are not limited to, immune checkpoint modulators, Toll-like receptor (TLR) agonists, cell-based therapies, cytokines, and cancer vaccines.

[0037] As used herein, an "immune checkpoint" or "immune checkpoint molecule" is a molecule of the immune system that regulates a signal. An immune checkpoint molecule can be a stimulatory checkpoint molecule, i.e., it increases a signal, or it can be an inhibitory checkpoint molecule, i.e., it decreases a signal. As used herein, a "stimulatory checkpoint molecule" is a molecule of the immune system that increases a signal or is co-stimulatory. As used herein, an "inhibitory checkpoint molecule" is a molecule of the immune system that decreases a signal or is co-inhibitory.

[0038] As used herein, an "immune checkpoint modulator" is an agent that can change the activity of an immune checkpoint in a subject. In certain embodiments, the immune checkpoint modulator changes the function of one or more immune checkpoint molecules, including but not limited to CD27, CD28, CD40, CD122, OX40, GITR, ICOS, 4-1BB, ADORA2A, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, and VISTA. The immune checkpoint modulator can be an agonist or antagonist of an immune checkpoint. In some embodiments, the immune checkpoint modulator is an immune checkpoint binding protein (e.g., an antibody, an antibody Fab fragment, a bivalent antibody, an antibody drug conjugate, an scFv, a fusion protein, a bivalent antibody, or a tetravalent antibody). In other embodiments, the immune checkpoint modulator is a small molecule. In certain embodiments, the immune checkpoint modulator is an anti-PD1, anti-PD-L1, or anti-CTLA-4 binding protein, e.g., an antibody or antibody fragment.

[0039] As used herein, the terms "increase" (or "activate") and "decrease" refer to modulation that results in a greater or lesser amount, function or activity of a parameter compared to a baseline. For example, after administration of a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) described herein, a parameter (e.g., tumor size, cancer cell proliferation, or cancer cell death) may increase or decrease in a subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more compared to the amount of the parameter before administration, or compared to a subject or cancer cell that has not been administered a UBE2K inhibitor. In general, the metric is measured at a time when administration has shown the described effect, for example, at least 1 day, 1 week, 1 month, 3 months, 6 months after the treatment regimen has been initiated. Furthermore, the metric may be measured relative to cancer cells or subjects not administered a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K).Similarly, a preclinical parameter (such as cancer cell death or proliferation in vitro and / or reduction of UBE2K activity by a UBE2K inhibitor described herein) may be increased or decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more compared to the amount of the parameter before administration of a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) or compared to a cancer cell or UBE2K enzyme not treated with a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K).

[0040] As used herein, a reduction in expression or activity of UBE2K is understood to include a change in gene and / or protein expression or activity. In one embodiment, expression or activity is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0041] As used herein, a "nucleic acid" UBE2K inhibitor is any nucleic acid-based inhibitor that causes a decrease in the expression and / or activity of UBE2K. In certain embodiments, the nucleic acid inhibitor acts by hybridizing with at least a portion of the RNA transcript from the corresponding gene (UBE2K), resulting in a decrease in the expression of UBE2K. Nucleic acid inhibitors include, for example, single-stranded nucleic acid molecules, such as antisense nucleic acids, and double-stranded nucleic acids, such as siRNAs, shRNAs, dsiRNAs (see, for example, U.S. Patent Publication No. US2007 / 0104688). As used herein, a double-stranded nucleic acid molecule is designed to be double-stranded over at least 12, preferably at least 15 nucleotides. A double-stranded nucleic acid molecule can be a single nucleic acid strand, such as an shRNA, designed to hybridize to itself. It should be understood that the nucleic acid inhibitor can be administered as an isolated nucleic acid. Alternatively, the nucleic acid inhibitor can be administered as an expression construct to produce the inhibitor in a cell. In certain embodiments, the nucleic acid inhibitor includes one or more chemical modifications to improve the activity and / or stability of the nucleic acid inhibitor. Such modifications are well known in the art.The specific modifications used depend, for example, on the type of nucleic acid inhibitor.In certain embodiments, the nucleic acid UBE2K inhibitor is a specific inhibitor of UBE2K, i.e., it does not reduce the expression or activity of other genes or proteins other than UBE2K.

[0042] As used herein, a "small molecule" UBE2K inhibitor is a UBE2K inhibitor molecule having a molecular weight of less than 1000 Da, preferably less than 750 Da, or preferably less than 500 Da. In certain embodiments, a "small molecule" is a synthetic organic compound and does not include a nucleic acid molecule. In certain embodiments, a "small molecule" is a synthetic organic compound and does not include a peptide longer than 3 amino acids. A "small molecule" UBE2K inhibitor is a molecule that specifically binds to UBE2K and at least partially inhibits UBE2K. In certain embodiments, a small molecule UBE2K inhibitor is a specific inhibitor of UBE2K, i.e., does not substantially reduce the expression or activity of other genes or proteins other than UBE2K. For example, in some embodiments, a small molecule UBE2K inhibitor does not reduce the activity of nuclear factor kappa light chain enhancer of activated B cells (NF-κB).

[0043] As used herein, the term "subject" refers to humans and non-human animals, including animal subjects. The term "non-human animals" includes all vertebrates, e.g., mammals, and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a human, and may be referred to as a patient.

[0044] A "therapeutically effective amount" is an amount sufficient to treat a disease in a subject. A therapeutically effective amount can be administered in one or more administrations.

[0045] As used herein, the terms "treat", "treating" or "treatment" preferably refer to actions to obtain a beneficial or desired clinical outcome, such as, but not limited to, alleviation or improvement of one or more signs or symptoms of a disease or condition (e.g., partial or complete regression), reduction in the extent of disease, a state of disease stability (i.e., not worsening, achieving stable disease), improvement or mitigation of pathology, reduction in rate or time of progression, and remission (either partial or total). "Treatment" of cancer can also mean extending survival compared to expected survival in the absence of treatment. Treatment need not be curative. In certain embodiments, treatment includes one or more of a reduction in pain or an increase in quality of life (QOL), as determined by a qualified individual, e.g., the treating physician, using, e.g., an accepted assessment tool for pain and quality of life (QOL). In certain embodiments, a reduction in pain or an increase in quality of life (QOL), as determined by, e.g., the treating physician, using, e.g., an accepted assessment tool for pain and quality of life (QOL), is not considered a "treatment" of cancer.

[0046] The articles "a," "an," and "the" 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, unless clearly indicated otherwise. By way of example, "an element" means one element or more than one element.

[0047] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0048] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0049] The term "such as" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0050] Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless clear from the context, all numerical values ​​provided herein can be modified by the term about.

[0051] Ranges provided herein are to be understood as shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0052] The recitation of a list of chemical group(s) in any definition of a variable herein includes the definition of that variable as any single group or combination of the groups listed. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0053] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein, including, but not limited to, combinations of administration rates, administration times, dosage amounts, treatment methods, monitoring methods, and selection methods.

[0054] II. UBE2K UBE2K is a component of ubiquitin-dependent proteolysis involved in protein degradation. Ubiquitin-dependent proteolysis utilizes ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes to sequentially activate, transfer, and ligate ubiquitin (Ub) to lysine residues of target proteins. During intermediate steps of this cascade, ubiquitin forms a covalent thioester conjugate between its C-terminal carboxylate and the catalytic cysteine ​​of the E2 enzyme (E2~Ub). Transfer to the substrate is then mediated by either a RING E3 ligase that functions as a scaffold protein, or a HECTE3 ligase that forms further covalent thioester conjugates with ubiquitin prior to substrate labeling. Repeated cycles of this process form polyubiquitin chains using one or more of the seven available lysine residues found in ubiquitin (i.e., K6, K11, K27, K29, K33, K48 and / or K63). See Cook et al., 2015, PLoS ONE 10(3):e0120318.

[0055] UBE2K is a protein component of this ubiquitin proteasome system and has been well characterized as an enzyme involved in the ubiquitination of various proteins. It is also known as huntingtin interacting protein 2 (HIP2). One of about 50 E2 enzymes of the ubiquitin system, UBE2K is a 25 kDa protein. Human UBE2K amino acid and nucleic acid sequences are provided herein as SEQ ID NO:1 and SEQ ID NO:2, respectively. UBE2K functions to receive ubiquitin moieties from E1 enzymes and covalently attach them to other protein substrates via thioester bonds, marking them for degradation by the proteasome. UBE2K is also known to interact non-covalently with ubiquitin. UBE2K is known to synthesize polyubiquitin chains linked to Lys 48 and is a member of the class III E2 family. It is unique among other E2s because it possesses a C-terminal ubiquitin-associated (UBA) domain in addition to the conserved UBC domain. The UBA domain of UBE2K has been reported to direct the specificity of polyubiquitin chain binding. It also enhances the processivity in the synthesis of polyubiquitin chains linked to Lys48. Using a yeast two-hybrid screen, UBE2K was found to interact with several RING finger proteins. It was also revealed that the RING finger domain of RNF2 is essential for UBE2K recognition, and RNF2 plays a role as an E3 ligase. Furthermore, UBE2K has the ability to ubiquitinate substrates independently of E3 ligases. In vitro ubiquitination assays showed that mutation of the active site cysteine ​​residues in the UBC domain impaired the ability of UBE2K to form mono- or polyubiquitin chains.

[0056] Although there is extensive literature on the ubiquitin-proteasome system in cancer, few reports exist on the role of specific E2 enzymes. Importantly, UBE2K has been reported to act in concert with two related tumor suppressors to regulate cancer-related phenotypes. UBE2K interacts with BRCA1 and ubiquitinates the G2 / M cell cycle proteins cyclin B1 and cdc25, affecting proliferation, while BRCA1 functions as an E3 ligase in this setting. Furthermore, UBE2K has been shown to ubiquitinate p53 and promote its degradation by using MDM2 as an E3 ligase.

[0057] UBE2K activity can be assayed by SDS-PAGE of ubiquitinated complexes after incubation of UBE2K enzyme with fluorescently labeled ubiquitin. For example, UBE2K can be incubated in a buffer containing fluorescently labeled ubiquitin, followed by addition of magnesium acetate and ATP. The reaction can be stopped by adding 2.5 μl of 10% (w / v) SDS and heating at 75° C. for 6 minutes. The sample is then subjected to SDS-PAGE in the absence of thiols. Methods for assaying UBE2K activity are described, for example, in Cook et al., 2015, PLoS ONE 10(3):e0120318; Middleton et al., Sci. Rep. (5):16793; and Strickson et al., 2013, Biochem Journal 451:427-437, each of which is incorporated herein by reference in its entirety.

[0058] III. UBE2K Inhibitors As used herein, a UBE2K inhibitor refers to a small molecule compound (i.e., a synthetic organic compound), a nucleic acid (e.g., antisense, siRNA, shRNA, dsiRNA, etc.), a protein, or an antibody (e.g., an antibody that specifically binds to the protein or enzyme UBE2K) that partially or completely inhibits the enzyme by reducing the expression and / or activity of the enzyme, e.g., by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 75-fold, or at least 100-fold, etc.; or reduces the expression and / or activity of UBE2K by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. As used herein, a "UBE2K inhibitor" can act by any mechanism, for example, by inhibiting the expression of UBE2K at the RNA or protein level, or by inhibiting the activity of UBE2K, for example, by inhibiting the addition of ubiquitin to UBE2K. In a preferred embodiment, the UBE2K inhibitor is a specific inhibitor of UBE2K, i.e., does not substantially reduce the expression or activity of polypeptides other than UBE2K. In some embodiments, the UBE2K-specific inhibitor preferentially inhibits UBE2K at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 500-fold, or at least 1000-fold compared to one or more different E2 proteins (not UBE2K). In some embodiments, the inhibited UBE2K is human UBE2K. In some embodiments, the UBE2K inhibitor does not reduce the activity of nuclear factor kappa light chain enhancer of activated B cells (NF-κB). In some embodiments, a UBE2K inhibitor does not reduce the expression or activity of other E2 enzymes other than UBE2K.

[0059] UBE2K inhibitors can be identified using methods known in the art.For example, compounds that reduce the expression of UBE2K can be identified by treating cells in vitro with a putative UBE2K inhibitor and measuring its effect on the expression of UBE2K, for example, mRNA or protein expression.The protein level of UBE2K can be measured by suitable techniques known in the art, including ELISA or Western blot.The level of UBE2K nucleic acid (e.g., mRNA) can be measured by suitable techniques known in the art, including polymerase chain reaction (PCR) amplification reaction, reverse transcriptase PCR analysis, quantitative real-time PCR, single-strand conformation polymorphism analysis (SSCP), mismatch cleavage detection, heteroduplex analysis, Northern blot analysis, in situ hybridization, array analysis, deoxyribonucleic acid sequencing, restriction fragment length polymorphism analysis, and combinations or subcombinations thereof.

[0060] UBE2K inhibitors can also be identified by measuring their effect on UBE2K activity using methods known in the art, such as those described above.For example, to determine whether a putative inhibitor prevents the ubiquitination of UBE2K enzyme, a putative UBE2K inhibitor can be incubated with UBE2K enzyme and fluorescently labeled ubiquitin.After incubation, samples are subjected to SDS-PAGE to detect ubiquitin / UBE2K complexes.

[0061] A. Small Molecule UBE2K Inhibitors Small molecule UBE2K inhibitors include (E)3-¥[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082). This compound has been shown to inhibit the addition of ubiquitin to E2 conjugating enzymes such as UBE2K. See Strickson et al., 2013, Biochem Journal 451:427-437, which is incorporated herein by reference in its entirety. BAY11-7082 is described, for example, in US2005 / 0124590 and US2009 / 0082371, each of which is incorporated herein by reference in its entirety. The chemical structure of BAY11-7082 is shown below. [ka]

[0062] In some embodiments, the UBE2K inhibitor does not include BAY 11-7082. In some embodiments, the UBE2K inhibitor does not include a small molecule.

[0063] B. Nucleic Acid UBE2K Inhibitors In some embodiments, the UBE2K inhibitor (e.g., specific inhibitor of UBE2K) is a nucleic acid. Nucleic acid inhibitors are well known in the art. Nucleic acid inhibitors include both single-stranded and double-stranded nucleic acids (i.e., nucleic acids having a complementary region of at least 15 nucleotides in length) that are complementary to a target sequence in a cell. Nucleic acid can be delivered to cells in culture, for example, by adding the nucleic acid to culture medium alone or together with an agent for promoting the uptake of the nucleic acid into the cell. Nucleic acid can be delivered to the cells of a subject, i.e., in vivo, by any administration route. The specific formulation depends on the administration route.

[0064] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising a second nucleotide sequence to form a duplex structure under certain conditions, as would be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing. Other conditions may be applied, such as physiologically suitable conditions that may be encountered within an organism. One of skill in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences, depending on the ultimate use of the hybridized nucleotides.

[0065] When there is base pairing between the nucleotides of the first nucleotide sequence and the nucleotides of the second nucleotide sequence over the entire length of the first and second nucleotide sequences, the sequences can be "fully complementary" to each other. However, when a first sequence is referred to as "substantially complementary" to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than four, three, or two mismatched base pairs upon hybridization while retaining the ability to hybridize under conditions most suitable for their final use. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, as is common in double-stranded nucleic acid therapeutics, such overhangs shall not be considered as mismatches for the purpose of determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides long and another oligonucleotide 23 nucleotides long is still referred to as "fully complementary" for the purposes described herein, even if the longer oligonucleotide comprises a 21 nucleotide sequence that is fully complementary to the shorter oligonucleotide.

[0066] As used herein, "complementary" sequences may also include or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, GU wobble base pairs or Hoogsteen base pairs.

[0067] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to base matches between the sense and antisense strands of a dsRNA, or between an antisense nucleic acid or antisense strand of a dsRNA and a target sequence, as understood from the context in which they are used.

[0068] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding HSP90, particularly HSP90β), including the 5'UTR, open reading frame (ORF), or 3'UTR. For example, a polynucleotide is complementary to at least a portion of a UBE2K mRNA if the sequence is substantially complementary to an uninterrupted portion of an mRNA encoding UBE2K.

[0069] Patents relating to antisense nucleic acid, chemical modification, and therapeutic applications are provided, for example, in U.S. Patent No. 5,898,031, which relates to chemically modified RNA-containing therapeutic compounds, and U.S. Patent No. 6,107,094, which relates to the method of using these compounds as therapeutic agents.U.S. Patent No. 7,432,250, which relates to the method of treating patients by administering single-stranded chemically modified RNA-like compounds; and U.S. Patent No. 7,432,249, which relates to pharmaceutical compositions containing single-stranded chemically modified RNA-like compounds.U.S. Patent No. 7,629,321, which relates to the method of cleaving target mRNA using a single-stranded oligonucleotide having multiple RNA nucleosides and at least one chemical modification.Each of the patents listed in this paragraph is incorporated herein by reference.

[0070] Nucleic acid inhibitors may contain natural (i.e., A, G, U, C, or T) or modified (7-deazaguanosine, inosine, etc.) bases. Furthermore, the bases of the nucleotides may be linked by bonds other than phosphodiester bonds, so long as they do not interfere with hybridization. Thus, inhibitory nucleic acids may be peptide nucleic acids, in which the constituent bases are linked by peptide bonds rather than phosphodiester bonds. Inhibitory nucleic acids may be prepared by converting RNA to cDNA using known methods (see, for example, Ausubel et.al., Current Protocols in Molecular Biology Wiley 1999). Inhibitory nucleic acids may also be cRNA (see, for example, Park et.al., (2004) Biochem. Biophys. Res. Commun. 325(4):1346-52)).

[0071] Nucleic acid inhibitors can be produced by synthesis methods such as phosphoramidite, H-phosphonate, and phosphite trimester methods. Inhibitory nucleic acids can also be produced by PCR methods. Such methods produce cDNA and cRNA sequences complementary to mRNA. The method of synthesis of therapeutic nucleic acids is not a limitation of the present invention.

[0072] Nucleic acid inhibitors typically contain one or more chemical modifications to improve their stability and adjust their pharmacokinetic and pharmacodynamic properties.For example, modifications to nucleotides can include, but are not limited to, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.

[0073] The nucleic acid inhibitor may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may occur on any nucleotide of the sense strand or antisense strand, or both (in the nucleic acid treatment comprising the sense strand), at any position of the strand. For example, the internucleotide bond modification may occur at every nucleotide on the sense strand or antisense strand. Each internucleotide bond modification may occur in an alternating pattern on the sense strand or antisense strand. Or, the sense strand or antisense strand may contain both internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification on the sense strand may be the same or different from that of the antisense strand, and the alternating pattern of the internucleotide bond modification on the sense strand may have a shift compared to the alternating pattern of the internucleotide bond modification on the antisense strand.

[0074] Other modifications include the incorporation of modified bases (or modified nucleosides or nucleotides) that are variations on the standard base, sugar and / or phosphate backbone chemistry occurring in ribonucleic acids (i.e., A, C, G, and U) and deoxyribonucleic acids (i.e., A, C, G, and T). For example, Gm (2'-methoxyguanylic acid), Am (2'-methoxyadenylic acid), Cf (2'-fluorocytidylic acid), Uf (2'-fluorouridylic acid), Ar (riboadenylic acid) fall within this scope. Aptamers may also include cytosine or any cytosine-related base, including 5-methylcytosine, 4-acetylcytosine, 3-methylcytosine, 5-hydroxymethylcytosine, 2-thiocytosine, 5-halocytosine (e.g., 5-fluorocytosine, 5-bromocytosine, 5-chlorocytosine, and 5-iodocytosine), 5-propynylcytosine, 6-azocytosine, 5-trifluoromethylcytosine, N4,N4-ethanocytosine, phenoxazine cytidine, phenothiazine cytidine, carbazole cytidine, or pyridoindole cytidine. The aptamer may further comprise guanine or any guanine-related base including 6-methylguanine, 1-methylguanine, 2,2-dimethylguanine, 2-methylguanine, 7-methylguanine, 2-propylguanine, 6-propylguanine, 8-haloguanine (e.g., 8-fluoroguanine, 8-bromoguanine, 8-chloroguanine, and 8-iodoguanine), 8-aminoguanine, 8-sulfhydrylguanine, 8-thioalkylguanine, 8-hydroxylguanine, 7-methylguanine, 8-azaguanine, 7-deazaguanine, or 3-deazaguanine.The aptamer may further comprise adenine or any adenine related base including 6-methyladenine, N6-isopentenyladenine, N6-methyladenine, 1-methyladenine, 2-methyladenine, 2-methylthio-N6-isopentenyladenine, 8-haloadenine (e.g., 8-fluoroadenine, 8-bromoadenine, 8-chloroadenine, and 8-iodoadenine), 8-aminoadenine, 8-sulfhydryladenine, 8-thioalkyladenine, 8-hydroxyladenine, 7-methyladenine, 2-haloadenine (e.g., 2-fluoroadenine, 2-bromoadenine, 2-chloroadenine, and 2-iodoadenine), 2-aminoadenine, 8-azaadenine, 7-deazaadenine, or 3-deazaadenine. Also, uracil, or 5-halouracil (e.g., 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil), 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, 1-methylpseudouracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, Also included are any of the uracil related bases including uracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, 5-methylaminomethyluracil, 5-propynyluracil, 6-azouracil, or 4-thiouracil.

[0075] Other examples of modified base variants known in the art include, but are not limited to, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-methoxycytidine, 5-carboxymethylaminomethyl-2-thiolysine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpseudouridine, bD-galactosyl eosin, inosine, N6-isopentenyl adenosine, 1-methyl adenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, bD-mannosyl eosin, 5-methoxy Carbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-bD-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-bD-ribofuranosylpurin-6-yl)N-methyl-carbamoyl)threonine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid (v), wybutoxosin (wyb utoxosine), pseudouridine, queosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, N-((9-bD-ribofuranosylpurin-6-yl)carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, and wybutosine, 3-(3-amino-3-carboxypropyl)uridine.

[0076] Also, U.S. Patent Nos. 3,687,808, 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,525,712, 5,525,713, 5,525,714, 5,525,715, 5,525,716, 5,525,717, 5,525,718, 5,525,719, 5,530,302, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,525,712, 5,530,30 ... Also included are modified nucleobases described in Nos. 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,830,653, 5,763,588, 6,005,096, and 5,681,941, each of which is incorporated by reference in its entirety. Examples of modified nucleoside and nucleotide sugar backbone variants known in the art include, but are not limited to, F, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2, CH3, ONO2, NO2, N3, NH2, OCH2CH2OCH3, O(CH2)2ON(CH3)2, OCH2OCH2N(CH3)2, O(C 1-10 alkyl), O(C 2-10 alkenyl), O(C 2-10 alkynyl), S(C 1-10 alkyl), S(C 2-10 alkenyl), S(C2-10 alkynyl), NH(C1-10 alkyl), NH(C 2-10 These include those with 2'-ribosyl substitutions such as 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'OCH2CH2CH2NH2), 2'-O-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-O--CH2-CH=CH2), 2'-amino (2'-NH2), and 2'-fluoro (2'-F). The 2'-substituent may be in the arabino (up) or ribo (down) position.

[0077] Single-stranded nucleic acid therapeutics Antisense nucleic acid therapeutics are single-stranded nucleic acid therapeutics, typically about 16-30 nucleotides in length, that are complementary to a target nucleic acid sequence in a target cell either in culture or in an organism.

[0078] In another embodiment, the agent is a single-stranded antisense RNA molecule. The antisense RNA molecule is complementary to a sequence in the target mRNA. The antisense RNA can stoichiometrically inhibit translation by base pairing with the mRNA and physically interfering with the translation machinery. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The antisense RNA molecule can have about 15-30 nucleotides complementary to the target mRNA. For example, the antisense RNA molecule can have a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides complementary to the target mRNA.

[0079] Patents relating to antisense nucleic acid, chemical modification, and therapeutic applications are provided, for example, in U.S. Patent No. 5,898,031, which relates to chemically modified RNA-containing therapeutic compounds, and U.S. Patent No. 6,107,094, which relates to the method of using these compounds as therapeutic agents.U.S. Patent No. 7,432,250, which relates to the method of treating patients by administering single-stranded chemically modified RNA-like compounds; and U.S. Patent No. 7,432,249, which relates to pharmaceutical compositions containing single-stranded chemically modified RNA-like compounds.U.S. Patent No. 7,629,321, which relates to the method of cleaving target mRNA using a single-stranded oligonucleotide having multiple RNA nucleosides and at least one chemical modification, relates to the entire contents of each of the patents listed in this paragraph, which are incorporated herein by reference.

[0080] Double-stranded nucleic acid therapeutics Nucleic acid inhibitors also include double-stranded nucleic acids. "RNAi agent", "double-stranded RNAi agent", double-stranded RNA (dsRNA) molecule, also referred to as "dsRNA agent", "dsRNA", "siRNA", "iRNA agent", when used interchangeably herein, refers to a complex of ribonucleic acid molecules having a duplex structure that includes antiparallel and substantially complementary nucleic acid strands as defined below. As used herein, RNAi agent can also include dsiRNA (see, for example, US Patent Publication 2007 / 0104688, which is incorporated herein by reference). Generally, the majority of nucleotides in each strand are ribonucleotides, but as described herein, one or both strands can also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. Furthermore, as used herein, "RNAi agent" can include ribonucleotides with chemical modifications, and RNAi agents can include substantial modifications to multiple nucleotides. Such modifications can include all types of modifications disclosed herein or known in the art. When used in siRNA type molecules, such modifications are encompassed by the term "RNAi agent" for the purposes of this specification and claims.

[0081] The two strands forming the duplex structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. If the two strands are part of one larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand forming the duplex structure and the 5' end of the other strand, the connected RNA strands are referred to as "hairpin loops". If the two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3' end of one strand forming the duplex structure and the 5' end of the other strand, the connected structure is referred to as a "linker". The RNA strands may have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides of the shortest strand of the dsRNA minus the overhang present in the duplex. In addition to the duplex structure, the RNAi agent may include one or more nucleotide overhangs. The term "siRNA" is also used herein to refer to the RNAi agent as described above.

[0082] In many embodiments, the duplex region is 15-30 nucleotide pairs in length, hi some embodiments, the duplex region is 17-23 nucleotide pairs in length, 17-25 nucleotide pairs in length, 23-27 nucleotide pairs in length, 19-21 nucleotide pairs in length, or 21-23 nucleotide pairs in length.

[0083] In certain embodiments, each strand has between 15 and 30 nucleotides.

[0084] RNAi agents used in the methods of the present invention include agents having chemical modifications as disclosed, for example, in U.S. Provisional Application No. 61 / 561,710, filed November 18, 2011, International Application No. PCT / US2011 / 051597, filed September 15, 2010, and PCT Publication No. WO2009 / 073809, the entire contents of each of which are incorporated herein by reference. The term "antisense strand" refers to the strand of a double-stranded RNAi agent that includes a region that is substantially complementary to a target sequence (e.g., human TTR mRNA). As used herein, the term "region complementary to a portion of an mRNA encoding transthyretin" refers to a region on the antisense strand that is substantially complementary to a portion of the TTR mRNA sequence. If the region of complementarity is not completely complementary to the target sequence, mismatches are most tolerated in the terminal regions, and if present, are generally within the terminal region(s), e.g., 6, 5, 4, 3, or 2 nucleotides of the 5' and / or 3' termini.

[0085] As used herein, the term "sense strand" refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0086] C. Antibody UBE2K Inhibitors In some embodiments, the inhibitor that reduces the expression or activity of UBE2K is an antibody. The therapeutic method of the present invention may include the use of antibodies, including polyclonal and monoclonal antibodies. The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a population of antibody molecules that contain only one type of antigen binding site that can immunoreact with a specific epitope. In some embodiments, the antibody for use in the methods described herein specifically binds to UBE2K, i.e., does not bind to other polypeptides other than UBE2K. The antibody can be obtained from commercial sources or generated using known methods.

[0087] Polyclonal antibodies can be prepared by immunizing a suitable subject with the protein of the invention as an immunogen. Antibody titers in immunized subjects can be monitored over time by standard techniques, such as by using enzyme-linked immunosorbent assays (ELISAs) using immobilized polypeptides. At an appropriate time after immunization, for example when the titer of specific antibodies is highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies (mAbs) by standard techniques, such as the hybridoma technique first described by Kohler and Milstein (1975) Nature 256:495-497, the human B-cell hybridoma technique (see Kozbor et al., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Cole et al., pp.77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985), or the trioma technique. Techniques for producing hybridomas are well known (see generally, Current Protocols in Immunology, Coligan et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.

[0088] As an alternative to preparing monoclonal antibody-secreting hybridomas, monoclonal antibodies against the proteins of the present invention can be identified and isolated by screening recombinant combinatorial immunoglobulin libraries (e.g., antibody phage display libraries) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01, and Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly suitable for use in generating and screening antibody display libraries can be found in, e.g., U.S. Pat. No. 5,223,409, PCT Publication Nos. WO 92 / 18619; PCT Publication No. WO 91 / 17271; PCT Publication No. WO 92 / 20791; PCT Publication No. WO 92 / 15679; PCT Publication No. WO 93 / 01288; PCT Publication No. WO 92 / 01047; PCT Publication No. WO 92 / 09690; PCT Publication No. WO 90 / 02809; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.

[0089] Recombinant antibodies that specifically bind to the protein of interest (i.e., UBE2K) can also be used in the methods of the present invention. In a preferred embodiment, the recombinant antibody specifically binds to the protein of interest (i.e., UBE2K) or a fragment thereof. Recombinant antibodies include, but are not limited to, chimeric and humanized monoclonal antibodies, single chain antibodies, and multispecific antibodies, which contain both human and non-human portions. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a mouse mAb and a human immunoglobulin constant region. (See, for example, U.S. Pat. No. 4,816,567 to Cabilly et al. and U.S. Pat. No. 4,816,397 to Boss et al., which are incorporated herein by reference in their entirety.) Single chain antibodies have an antigen-binding site and consist of a single polypeptide. They can be produced by techniques known in the art, for example, using the methods described in U.S. Patent No. 4,946,778 to Ladner et al., incorporated herein by reference in its entirety; Bird et al., (1988) Science 242:423-426; Whitlow et al., (1991) Methods in Enzymology 2:1-9; Whitlow et al., (1991) Methods in Enzymology 2:97-105; and Huston et al., (1991) Methods in Enzymology Molecular Design and Modeling: Concepts and Applications 203:46-88. Multispecific antibodies are antibody molecules that have at least two antigen-binding sites that specifically bind different antigens. Such molecules can be produced by techniques known in the art, for example, using the methods described in U.S. Patent No. 4,676,980 to Segal, the disclosure of which is incorporated herein by reference in its entirety; Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Whitlow et al., (1994) Protein Eng. 7:1017-1026, and U.S. Patent No. 6,121,424.

[0090] Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, incorporated herein by reference in its entirety.) Humanized monoclonal antibodies can be made by recombinant DNA techniques known in the art, e.g., PCT Publication No. WO 87 / 02671; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT Publication No. WO 86 / 01533; U.S. Pat. No. 4,816,567; European Patent Application No. 125,023; Better et al. (1988) 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. al.(1987)Proc.Natl.Acad.Sci.USA 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(1985)Science 229:1202-1207;Oi et al.(1986)Bio / Techniques 4:214;USPatent 5,225,539;Jones et al.(1986)Nature 321:552-525;Verhoeyan et al. al. (1988) Science 239:1534; and Beidler It can be produced using the methods described in et al. (1988) J. Immunol. 141:4053-4060.

[0091] More specifically, humanized antibodies can be produced, for example, using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but can express human heavy and light chain genes. The transgenic mice are immunized in the usual manner with a selected antigen, for example, all or a portion of a polypeptide corresponding to a marker of the invention. Monoclonal antibodies against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, such technology can be used to generate therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for generating human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent Nos. 5,625,126, 5,633,425, 5,569,825, 5,661,016, and 5,545,806. Additionally, companies can be engaged to provide human antibodies against a selected antigen using technology similar to that described above.

[0092] Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al., 1994, Bio / technology 12:899-903).

[0093] The antibodies of the invention can be isolated after production (e.g., from the blood or serum of a subject) or synthesis and further purified by well-known techniques. For example, IgG antibodies can be purified using Protein A chromatography. Antibodies specific to the proteins of the invention can be selected (e.g., partially purified) or purified, for example, by affinity chromatography. For example, recombinantly expressed and purified (or partially purified) proteins of the invention are produced as described herein and are covalently or non-covalently linked to a solid support, such as, for example, a chromatography column. The column can then be used to affinity purify antibodies specific to the proteins of the invention from a sample containing antibodies against a number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies. A substantially purified antibody composition, in this context, means that the antibody sample contains at most no more than 30% (by dry weight) of contaminating antibodies against epitopes other than the epitope of the desired protein of the invention, and preferably at most 20%, even more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample are contaminating antibodies. A purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein of the invention.

[0094] Antibodies against the protein (i.e., UBE2K) can be used to isolate the protein by standard techniques such as affinity chromatography or immunoprecipitation. In addition, such antibodies can be used to detect target proteins, such as UBE2K, or fragments thereof (e.g., in cell lysates or cell supernatants) to assess the level and pattern of expression of the target protein. Antibodies can also be used diagnostically to monitor protein levels in tissues or body fluids (e.g., body fluids associated with disease or toxic conditions) as part of a clinical testing procedure, for example, to determine the effectiveness of a given treatment regimen. Detection can be facilitated by using antibody derivatives, including antibodies of the present invention linked to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and an example of a suitable radioactive material is 125 I, 131 I, 35 S or 3 Contains H.

[0095] IV. Cancer Treatment In some embodiments, the present disclosure provides a method for treating cancer in a subject, e.g., a subject in need thereof, by administering to the subject a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K).

[0096] In some embodiments, the disclosure provides a method of reducing the proliferation of cancer cells, comprising contacting the cancer cells with a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K), thereby reducing the proliferation of the cancer cells. In some embodiments, the disclosure provides a method of reducing the proliferation of cancer cells, comprising administering a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) to the cancer cells, thereby reducing the proliferation of the cancer cells, e.g., compared to cancer cells not treated with a UBE2K inhibitor. In some embodiments, the disclosure provides a method of reducing the proliferation of cancer cells in a subject, e.g., a subject in need thereof, comprising administering a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) to the subject, thereby reducing the proliferation of the cancer cells in the subject, e.g., compared to cancer cells in a subject not administered a UBE2K inhibitor.

[0097] In some embodiments, the disclosure provides a method of inducing cancer cell death comprising contacting a cancer cell with a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K), thereby inducing cancer cell death. In some embodiments, the disclosure provides a method of inducing cancer cell death comprising administering a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) to a cancer cell, thereby inducing cancer cell death. In some embodiments, the disclosure provides a method of inducing cancer cell death in a subject, e.g., a subject in need thereof, comprising administering a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) to the subject, thereby inducing cancer cell death.

[0098] In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer comprises leukemia. In certain embodiments, the cancer is treated with a UBE2K inhibitor alone. In certain embodiments, the cancer is treated with a UBE2K inhibitor and an additional agent. In certain embodiments, the additional agent is a chemotherapeutic agent.

[0099] In one embodiment, administration of a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) results in one or more of a reduction in tumor size, weight or volume, an increase in time to progression, inhibition of tumor growth and / or an increase in the survival of a subject with an oncological disorder. In certain embodiments, administration of a UBE2K inhibitor reduces tumor size, weight or volume, increases the time to progression, inhibits tumor growth and / or increases the survival of a subject by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500% compared to a corresponding control subject not administered a UBE2K inhibitor. In some embodiments, administration of a UBE2K inhibitor stabilizes cancer in a subject who had a progressive oncological disorder prior to treatment.

[0100] In the treatment of cancer, the formulation may be a pharma- ceutically acceptable carrier that may be administered in a therapeutically effective amount to a subject, either as monotherapy, in combination with at least one other anti-cancer agent for a given indication, e.g., a chemotherapeutic agent, in combination with radiation therapy following surgical intervention to definitively remove a tumor, in combination with other alternative and / or complementary acceptable treatments for cancer, etc.

[0101] Generally, the formulations and methods of UBE2K inhibitors (e.g., specific inhibitors of UBE2K) described herein can be used to treat any neoplasm. In certain embodiments, the formulations and methods are used to treat solid tumors in subjects. In certain embodiments, the formulations and methods are used to treat non-solid tumors, such as leukemia, in subjects.

[0102] In one embodiment, administration of a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) described herein achieves at least stable disease, reduces tumor size, inhibits tumor growth, and / or extends the survival of a tumor-bearing subject, as compared to a suitable control. Thus, the present invention also relates to a method of treating a tumor in a human or other animal by administering to such a human or animal an effective non-toxic amount of a UBE2K inhibitor. Those skilled in the art will be able to determine by routine experimentation with the guidance provided herein what is an effective non-toxic amount of a UBE2K inhibitor for the purpose of treating cancer. For example, a therapeutically active amount of a UBE2K inhibitor may vary depending on factors such as the stage of the disease (e.g., stage I vs. stage IV), the subject's age, sex, medical complications (e.g., immunosuppressive conditions or diseases, coagulation disorders) and weight, as well as the ability of the UBE2K inhibitor to induce a desired response in the subject. Dosage regimens can be adjusted to provide an optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the treatment situation.

[0103] In certain embodiments of the invention, the method further comprises a treatment regimen comprising any one or combination of surgery, radiation, chemotherapy, e.g., hormonal therapy, antibody therapy, growth factor therapy, cytokines, and anti-angiogenic therapy.

[0104] Cancers for treating with the method of the present invention include all types of cancers or neoplasms or malignant tumors found in mammals, including, but not limited to, leukemia, lymphoma, melanoma, carcinoma and sarcoma. In one embodiment, cancers for treating with the method of the present invention include melanoma, carcinoma and sarcoma. In a preferred embodiment, the UBE2K inhibitor composition is used to treat various types of solid tumors, such as breast cancer, bladder cancer, colon and rectal cancer, endometrial cancer, kidney (renal cell) cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, cervical cancer, liver cancer, stomach cancer, oral cancer, neuroblastoma, testicular cancer, uterine cancer, thyroid cancer, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovarian, sarcoma, stomach, uterine and medulloblastoma, and vulvar cancer. In a particular embodiment, the solid tumor includes breast cancer, including triple negative breast cancer. In certain embodiments, the skin cancer comprises melanoma, squamous cell carcinoma, cutaneous T-cell lymphoma (CTCL). In certain embodiments, the cancer comprises leukemia. In certain embodiments, the leukemia comprises acute leukemia. In certain embodiments, the leukemia comprises chronic leukemia. In certain embodiments, the leukemia comprises acute lymphocytic (or lymphoblastic) leukemia (ALL), acute myeloid (or myeloid or non-lymphocytic) leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML). Further types of leukemia include hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia. However, treatment using UBE2K inhibitor compositions is not limited to these types of cancer.

[0105] In some embodiments, the cancer for treating with UBE2K inhibitor is selected from liver cancer and pancreatic cancer.In some embodiments, the cancer for treating with UBE2K inhibitor is selected from choriocarcinoma, ovarian cancer, leukemia (e.g., T-cell leukemia, T-lymphoblastic leukemia, and chronic myelogenous leukemia), lymphoma (e.g., B-cell lymphoma and anaplastic large cell lymphoma), embryonal carcinoma, osteosarcoma, skin cancer, and colon cancer (e.g., colon adenocarcinoma).

[0106] As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably, either in the singular or plural, to refer to cells that have undergone malignant transformation that becomes pathological to the host organism. Primary cancer cells (i.e., cells obtained from near the site of malignant transformation) can be readily distinguished from noncancerous cells by well-established techniques, particularly histological examination. The definition of cancer cells used herein includes not only primary cancer cells, but any cells derived from a cancer cell ancestor. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that typically manifest as solid tumors, a "clinically detectable" tumor is one that is detectable based on tumor burden, for example, by procedures such as CAT scan, MR imaging, X-ray, ultrasound, or palpation, and / or detectable due to the expression of one or more cancer-specific antigens in a sample that can be obtained from the patient. Leukemia is clinically detectable using one or more of the following: complete blood count, pallor, blood smear, and bone marrow smear. Progressive leukemia in certain subjects may manifest as a solid tumor.

[0107] Examples of non-solid tumors that cannot be detected by imaging or palpation, such as leukemia, can be detected, for example, by neutrophil count, platelet count, and by detection of abnormal cells in the bone marrow, for example, by the presence of otherwise unexplained blasts (e.g., bone marrow regeneration after consolidation therapy), the presence of Auer rods, or the appearance of new dysplastic changes.

[0108] The term "sarcoma" generally refers to a tumor made up of a substance like embryonic connective tissue and generally consists of closely packed cells embedded in a fibrillar or homogeneous substance. Examples of sarcomas that may be treated with the present compositions and optionally additional anti-cancer agents, such as chemotherapeutic agents, include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma, embryonal carcinoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple chromatohemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemic sarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, and / or reticulocytic sarcoma. sarcoma), Rous sarcoma, serous cystic sarcoma, synovial cell sarcoma, and telangiectatic sarcoma.

[0109] The term "melanoma" is taken to mean a tumor arising from the melanocyte system of the skin and other organs.Melanomas that can be treated with the compositions of the present invention and optionally additional anti-cancer agents, such as chemotherapeutic agents, include, but are not limited to, for example, acral lentigo melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0110] The term "carcinoma" refers to a malignant neoplasm composed of epithelial cells that tend to invade surrounding tissues and give rise to metastases. Carcinomas that can be treated with the compositions of the present invention and optionally additional anti-cancer agents, such as chemotherapeutic agents, include, but are not limited to, acinar carcinoma, acinic cell carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adrenal cortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basaloid carcinoma, basal squamous cell carcinoma, bronchiolocarcinoma, bronchogenic carcinoma, encephalomyeloma, cholangiocarcinoma, choriocarcinoma, gelatinous carcinoma, comedocarcinoma, uterine corpus, cribriform carcinoma, armor carcinoma, skin carcinoma, cylindrical carcinoma, cystic ... cancer, columnar cell carcinoma, ductal carcinoma, compact carcinoma, embryonal carcinoma, encephalomatosis, epidermoid carcinoma, epithelial adenoid carcinoma, exophytic carcinoma, ulcerative carcinoma, fibrous carcinoma, gelatinous carcinoma, gelatinous carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, blood-like carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyaline carcinoma, adrenal gland-like carcinoma, childhood embryonal carcinoma, intraepithelial carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, Kronpecker carcinoma, Kurticytsky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma carcinoma), melanoma, soft carcinoma, mucinous carcinoma, mucinous carcinoma, mucosal carcinoma, mucosal carcinoma, mucinous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, atheromatous carcinoma, renal cell carcinoma of the kidney, supplementary cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solenoid carcinoma, globular cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, telangiectatic carcinoma, transitional cell carcinoma, nodular carcinoma, nodular carcinoma, verrucous carcinoma, and choriocarcinoma.

[0111] The term "leukemia" refers to a type of cancer of the blood or bone marrow characterized by an abnormal increase in immature white blood cells called "blasts." Leukemia is a broad term that encompasses a range of diseases. In turn, leukemia is part of a much broader group of diseases that affect the blood, bone marrow, and lymphatic systems, all of which are known as hematological neoplasms. Leukemia is broadly classified into four types: acute lymphocytic (or lymphoblastic) leukemia (ALL), acute myeloid (or myeloid or non-lymphoid) leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML). Further types of leukemia include hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia.

[0112] "Acute leukemia" is characterized by a sudden increase in the number of immature blood cells. When overcrowded with such cells, the bone marrow is unable to produce healthy blood cells. Acute leukemia requires immediate treatment because malignant cells progress and accumulate rapidly, spilling into the bloodstream and spreading to other parts of the body. Acute forms of leukemia are the most common form of leukemia in children.

[0113] "Chronic leukemia" is characterized by an excessive accumulation of relatively mature, but still abnormal, white blood cells. It typically progresses over months or years, with cells being produced at a much faster rate than normal, resulting in many abnormal white blood cells. Acute leukemia needs to be treated immediately, while chronic forms may be monitored for a period of time before treatment to ensure maximum therapeutic benefit.

[0114] Lymphoblastic, or lymphocytic, leukemia is caused by an overgrowth of bone marrow cells that produce lymphocytes (white blood cells), typically B cells.

[0115] Myeloid, or myeloid leukemia, is caused by an overgrowth of bone marrow cells that produce red blood cells, several other types of white blood cells, and platelets.

[0116] Additional cancers that can be treated with the compounds disclosed herein include, for example, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung tumors, primary brain tumors, gastric cancer, colon cancer, malignant pancreatic islet cancer, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, These include esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical carcinoma, prostate cancer, pancreatic cancer, uterine sarcoma, myxoliposarcoma, leiomyosarcoma, chondrosarcoma, osteosarcoma, colon adenocarcinoma of the colon, cervical squamous cell carcinoma, tonsillar squamous cell carcinoma, papillary thyroid carcinoma, adenoid cystic carcinoma, synovial cell sarcoma, malignant fibrous histiocytoma, desmoplastic sarcoma, hepatocellular carcinoma, spindle cell sarcoma, cholangiocarcinoma, and triple-negative breast cancer.

[0117] In some embodiments, the cancer is not a lymphoma. In some embodiments, the cancer is not a B-cell lymphoma. In some embodiments, the cancer is not a lymphoma with a MyD88 mutation.

[0118] V. Combination Therapy The methods of treating cancer provided herein include combination therapy with additional anti-cancer agents or interventions (e.g., radiation, surgery, bone marrow transplantation). In certain embodiments, "combination therapy" includes treatment with a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) to reduce tumor burden and / or improve clinical response. Administering a UBE2K inhibitor with a palliative treatment or a treatment to reduce the side effects of a drug (e.g., to reduce nausea, pain, anxiety, or inflammation, to normalize coagulation) is not considered a combination treatment of cancer.

[0119] In a preferred embodiment, treatment with a UBE2K inhibitor (e.g., a specific inhibitor of UBE2K) is combined with standard treatment for the particular cancer being treated. Standard treatment for a particular cancer type can be determined by those skilled in the art based on, for example, the type and severity of the cancer, the subject's age, weight, sex, and / or medical history, and the success or failure of previous treatments.

[0120] In certain embodiments, treatment of a subject with a solid tumor with a UBE2K inhibitor is combined with one or more of the following therapeutic agents:

[0121] 1. Gemcitabine, preferably 600 mg / m 2 It is administered intravenously in weekly doses starting at 10 mg / kg and the dose is adjusted based on the subject's tolerance to the drug.

[0122] 2. 5-Fluorouracil (5-FU), preferably 100 mg / m 2 350 mg / m2 weekly in combination with leucovorin 2 is administered intravenously at an initial weekly dose of 10 mg / kg / day, with the dose adjusted based on the subject's tolerance to the drug.

[0123] 3. Docetaxel, preferably 20 mg / m 2 is administered intravenously once weekly at a starting dose of 0.01 mg / kg / day, with the dose adjusted based on the subject's tolerance to the drug.

[0124] In certain embodiments, the combination therapy is administered to the subject for 1, 2, 3, 4 or 5 cycles.The subject is evaluated for response criteria at the end of each cycle.The subject is also monitored throughout each cycle for adverse events (e.g., coagulation, anemia, liver and kidney function, etc.) to ensure that the treatment regimen is well tolerated.

[0125] In certain embodiments, the UBE2K inhibitor (e.g., specific inhibitor of UBE2K) is administered in an amount that would be therapeutically effective if delivered alone, i.e., the UBE2K inhibitor is administered and / or acts as a therapeutic agent, rather than as an agent to primarily ameliorate the side effects of other chemotherapy or other cancer treatments. The UBE2K inhibitor and / or pharmaceutical formulation thereof and the other therapeutic agent can act additively, or more preferably synergistically. In one embodiment, the UBE2K inhibitor (e.g., specific inhibitor of UBE2K) and / or formulation thereof is administered simultaneously with administration of an additional anti-cancer agent (e.g., chemotherapeutic agent, anti-angiogenic agent). In another embodiment, the compound and / or pharmaceutical formulation thereof is administered before or after administration of another anti-cancer agent, and both agents are present in the subject at the same time or have therapeutic activity in the subject at the same time. In one embodiment, the UBE2K inhibitor and the additional anti-cancer agent act synergistically. In one embodiment, the UBE2K inhibitor and the additional anti-cancer agent act additively.

[0126] In one embodiment, the therapeutic method of the present invention further comprises the administration of one or more additional therapeutic agents, such as one or more anti-cancer agents, such as anti-angiogenic agents, chemotherapeutic agents, such as small molecule anti-cancer agents, biological anti-cancer agents, including both protein-based therapeutic agents and nucleic acid-based therapeutic agents.For example, in one embodiment, the additional anti-cancer agent for use in the therapeutic method of the present invention is a chemotherapeutic agent.

[0127] Small molecule chemotherapeutic agents generally belong to a variety of classes including, for example: 1. topoisomerase II inhibitors (cytotoxic antibiotics), for example, anthracyclines / anthracenediones, such as doxorubicin, epirubicin, idarubicin, and nemorubicin, anthraquinones, such as mitoxantrone and losoxantrone, and podophyllotoxins, such as etoposide and teniposide; 2. agents that affect microtubule formation (mitotic inhibitors), for example, plant alkaloids (compounds that belong to a family of alkaline, nitrogen-containing molecules derived from plants that are biologically active and cytotoxic), for example, taxanes, for example, paclitaxel and docetaxel, and vinca alkaloids, for example, vinblastine, vincristine, and vinorelbine; 3. Alkylating agents, such as nitrogen mustards, ethylenimine compounds, alkylsulfonates, and other compounds with alkylating action, such as nitrosoureas, dacarbazine, cyclophosphamide, ifosfamide, and melphalan; 4. Antimetabolites (nucleoside inhibitors), such as folates, such as folic acid, fluoropyrimidines, purine or pyrimidine analogues, such as 5-fluorouracil, capecitabine, gemcitabine, methotrexate, and edatrexate; 5. Topoisomerase I inhibitors, such as topotecan, irinotecan, and 9-nitrocamptothecin, camptothecin derivatives, and retinoic acid; 6. Platinum compounds / complexes, such as cisplatin, oxaliplatin, and carboplatin. Exemplary chemotherapeutic agents for use in the methods of the invention include, but are not limited to, amifostine (Ethyol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carnustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), doxorubicin lipo (Doxyl), gemcitabine (Gemzar), daunorubicin, daunorubicin lipo (Daunoxome), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine,Bleomycin, paclitaxel (Taxol), docetaxel (Taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-I1, 10-hydroxy-7-ethyl-camptothecin (SN38), dacarbazine, SI capecitabine, ftorafur, 5'deoxyfluroidine, UFT, eniluracil, deoxycytidine, 5-azacytosine, 5-azadeoxycytosine camptothecin, allopurinol, 2-chloroadenosine, trimetrexate, aminopterin, methylene-10-deazaaminopterin (MDAM), oxaplatin, picoplatin, tetraplatin, satraplatin, platinum-DACH, CI-973, JM-216 and its analogs, epirubicin, etoposide phosphate, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin, TAS 103, vindesine, L-phenylalanine mustard, ifosphamide mefosphamide, perfosfamide, trofosfamide, carmustine, semustine, epothilone A to E, tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, etoposide phosphate, karenitecin, acyclovir, valacyclovir, ganciclovir, amantadine, rimantadine, lamivudine, zidovudine, bevacizumab, trastuzumab, rituximab, 5-fluorouracil, capecitabine, pentostatin, trimetrexate, cladribine, floxuridine , fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, cisplatin, doxorubicin, paclitaxel (taxol), bleomycin, mTor, epidermal growth factor receptor (EGFR) and fibroblast growth factor (FGF),and combinations thereof that would be readily apparent to one of skill in the art based on the appropriate standard of care for a particular tumor or cancer.

[0128] In another embodiment, the additional chemotherapeutic agent for use in the combination therapy of the present invention is a biological agent.A biological agent (also called a biologic) is a product of a biological system, such as an organism, a cell, or a recombinant system.Examples of such biological agents include nucleic acid molecules (e.g., antisense nucleic acid molecules), interferons, interleukins, colony-stimulating factors, antibodies, such as monoclonal antibodies, antiangiogenic agents, and cytokines.Exemplary biological agents are discussed in more detail below and generally belong to various classes, including, for example:1. hormones, hormone analogs, and hormone complexes, such as estrogens and estrogen analogs, progesterone, progesterone analogs and progestins, androgens, adrenocortical steroids, antiestrogens, antiandrogens, antitestosterone, adrenal steroid inhibitors, and antileutinized hormones;2. enzymes, proteins, peptides, polyclonal and / or monoclonal antibodies, such as interleukins, interferons, colony-stimulating factors, etc.

[0129] In one embodiment, the biologic is interferon. Interferon (IFN) is a type of biologic that occurs naturally in the body. Interferons are also produced in laboratories and administered to cancer patients in biotherapy. They have been shown to improve the way that the immune system of cancer patients acts against cancer cells.

[0130] Interferons can act directly on cancer cells to slow their growth or change them into more normally behaving cells. Some interferons can also stimulate natural killer (NK) cells, T cells, and macrophages, types of white blood cells in the bloodstream that help fight cancer cells.

[0131] In one embodiment, the biologic is an interleukin. Interleukins (ILs) are proteins (cytokines and chemokines) that stimulate the growth and activity of many immune cells. They occur naturally in the body, but can also be made in a laboratory. Some interleukins stimulate the growth and activity of immune cells, such as lymphocytes, which act to destroy cancer cells.

[0132] In another embodiment, the biologic is a colony stimulating factor. Colony stimulating factors (CSFs) are proteins given to patients to encourage stem cells in the bone marrow to produce more blood cells. The body constantly needs new white blood cells, red blood cells, and platelets, especially when cancer is present. CSFs are given in conjunction with chemotherapy to help strengthen the immune system. When cancer patients undergo chemotherapy, the bone marrow's ability to produce new blood cells is suppressed, making the patient more susceptible to developing infections. Because parts of the immune system cannot function without blood cells, colony stimulating factors encourage bone marrow stem cells to produce white blood cells, platelets, and red blood cells.

[0133] With adequate cell production, other cancer treatments can continue to allow patients to safely receive higher doses of chemotherapy.

[0134] In another embodiment, the biologic is an antibody. Antibodies, such as monoclonal antibodies, are agents produced in the laboratory that bind to cancer cells.

[0135] Monoclonal antibody agents do not destroy healthy cells. Monoclonal antibodies exert their therapeutic effects through various mechanisms. They can have a direct effect in bringing about apoptosis, or programmed cell death. They can block growth factor receptors, effectively arresting the proliferation of tumor cells. In cells expressing monoclonal antibodies, they can cause the formation of anti-idiotypic antibodies.

[0136] Examples of antibodies that can be used in the combination therapy of the present invention include, but are not limited to, anti-CD20 antibodies, such as cetuximab, tositumomab, rituximab, and ibritumomab. Anti-HER2 antibodies can also be used in combination with UBE2K inhibitors to treat cancer. In one embodiment, the anti-HER2 antibody is trastuzumab (Herceptin). Other examples of antibodies that can be used in combination with UBE2K inhibitors to treat cancer include anti-CD52 antibodies (e.g., alemtuzumab), anti-CD-22 antibodies (e.g., epratuzumab), and anti-CD33 antibodies (e.g., gemtuzumab ozogamicin). Anti-VEGF antibodies can also be used in combination with UBE2K inhibitors to treat cancer. In one embodiment, the anti-VEGF antibody is bevacizumab. In other embodiments, the biologic is an antibody that is an anti-EGFR antibody, such as cetuximab. Another example is the anti-glycoprotein 17-1A antibody edrecolomab. It will be appreciated by those of skill in the art that numerous other anti-tumor antibodies are known in the art and are encompassed by the present invention.

[0137] In another embodiment, the biologic is a cytokine. Cytokine therapy uses proteins (cytokines) to help the subject's immune system recognize and destroy cancerous cells. Cytokines are naturally produced in the body by the immune system, but can be produced in a laboratory. This therapy is used for advanced melanoma and is used with adjuvant therapy (treatment given after or in addition to the main cancer treatment). Cytokine therapy reaches all parts of the body to kill cancer cells and prevent tumors from growing.

[0138] In another embodiment, the biologic is a fusion protein.For example, recombinant human Apo2L / TRAIL (GENETECH) can be used in combination therapy.Apo2 / TRAIL is the first dual pro-apoptotic receptor agonist designed to activate both pro-apoptotic receptors DR4 and DR5, which are involved in the regulation of apoptosis (programmed cell death).

[0139] In one embodiment, the biologic is a therapeutic nucleic acid molecule. Nucleic acid therapeutics are well known in the art. Nucleic acid therapeutics include a therapy of single-stranded and double-stranded nucleic acids (i.e., nucleic acid therapeutics having a complementary region of at least 15 nucleotides in length) that are complementary to target sequences in cells. Therapeutic nucleic acids can target essentially any target nucleic acid sequence in cells. In certain embodiments, nucleic acid therapeutics target the nucleic acid sequence that codes for angiogenesis stimulators, such as VEGF, FGF, or tumor growth stimulators, such as EGFR.

[0140] Antisense nucleic acid therapeutics are single-stranded nucleic acid therapeutics, typically about 16-30 nucleotides in length, that are complementary to a target nucleic acid sequence in a target cell either in culture or in an organism.

[0141] In another embodiment, the agent is a single-stranded antisense RNA molecule. The antisense RNA molecule is complementary to a sequence in the target mRNA. Antisense RNA can stoichiometrically inhibit translation by base pairing with the mRNA and physically interfering with the translation machinery. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The antisense RNA molecule can have about 15-30 nucleotides complementary to the target mRNA. Patents relating to antisense nucleic acids, chemical modifications, and therapeutic applications are provided, for example, in U.S. Pat. No. 5,898,031, which relates to therapeutic compounds containing chemically modified RNA, and U.S. Pat. No. 6,107,094, which relates to methods of using these compounds as therapeutic agents. U.S. Pat. No. 7,432,250, which relates to a method of treating a patient by administering a single-stranded chemically modified RNA-like compound; and U.S. Pat. No. 7,432,249, which relates to a pharmaceutical composition containing a single-stranded chemically modified RNA-like compound. US Patent No. 7,629,321 relates to a method for cleaving a target mRNA using a single-stranded oligonucleotide having multiple RNA nucleosides and at least one chemical modification. The entire contents of each of the patents listed in this paragraph are incorporated herein by reference.

[0142] Nucleic acid therapeutics for use in the methods of the present invention also include double-stranded nucleic acid therapeutics. "RNAi agent", "double-stranded RNAi agent", double-stranded RNA (dsRNA) molecule, also referred to as "dsRNA agent", "dsRNA", "siRNA", "iRNA agent" when used interchangeably herein, refers to a complex of ribonucleic acid molecules having a duplex structure that includes antiparallel and substantially complementary nucleic acid strands as defined below. As used herein, RNAi agent can also include dsiRNA (see, for example, US Patent Publication 2007 / 0104688, which is incorporated herein by reference). Generally, the majority of nucleotides in each strand are ribonucleotides, but as described herein, one or both strands can also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. Furthermore, as used herein, "RNAi agent" can include ribonucleotides with chemical modifications, and RNAi agents can include substantial modifications to multiple nucleotides. Such modifications can include all types of modifications disclosed herein or known in the art. When used in siRNA type molecules, such modifications are included in "RNAi agent" for the purpose of this specification and claims.The RNAi agent used in the method of the present invention includes the agent with chemical modifications as disclosed in, for example, US Provisional Application No. 61 / 561,710 filed on November 18, 2011, International Application No. PCT / US2011 / 051597 filed on September 15, 2010, and PCT Publication WO2009 / 073809, each of which is incorporated herein by reference in its entirety.

[0143] Additional exemplary biologics for use in the methods of the invention include, but are not limited to, gefitinib (Iressa), anastrozole, diethylstilbesterol, estradiol, premarin, raloxifene, progesterone, norethynodrel, ethisterone, dimestisterone, megestrol acetate, medroxyprogesterone acetate, hydroxyprogesterone caproate, norethisterone, methyltestosterone, testosterone, dexamethasone, prednisone, cortisol, solumedrol, tamoxifen, fulvestrant, toremifene, aminoglutethimide, testolactone, droloxifene, anastrozole, bicalutamide, flutamide, nilutamide, goserelin, flutamide, leuprolide, triptorelin, aminoglutethimide, mitotane, goserelin, cetuximab, erlotinib ... Lotinib, Imatinib, Tositumomab, Alemtuzumab, Trastuzumab, Gemtuzumab, Rituximab, Ibritumomab Tiuxetan, Bevacizumab, Denileukin-Diftitox, Daclizumab, Interferon Alpha, Interferon Beta, Anti-4-lBB, Anti-4-lBBL, Anti-CD40, Anti-CD154, Anti-OX40, Anti-OX40L, Anti-CD28, Anti-CD80, Anti-CD86, Anti-CD70 , anti-CD27, anti-HVEM, anti-LIGHT, anti-GITR, anti-GITRL, anti-CTLA-4, soluble OX40L, soluble 4-IBBL, soluble CD154, soluble GITRL, soluble LIGHT, soluble CD70, soluble CD80, soluble CD86, soluble CTLA4-Ig, GVAX®, and combinations thereof that will be readily apparent to one of skill in the art based on the appropriate standard of care for a particular tumor or cancer. Soluble forms of the agent can be made, for example, as a fusion protein, by operably linking the agent, for example, with an Ig-Fc region.

[0144] Immune checkpoint inhibitors In some embodiments, the additional agent is an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an immune checkpoint modulator of an immune checkpoint molecule. Examples include LAG-3 (Triebel et al., 1990, J. Exp. Med. 171: 1393-1405), TIM-3 (Sakuishi et al., 2010, J. Exp. Med. 207: 2187-2194) and VISTA (Wang et al., 2011, J. Exp. Med. 208: 577-592). Examples of costimulatory molecules that improve immune responses include ICOS (Fan et al., 2014, J. Exp. Med. 211:715-725), OX40 (Curti et al., 2013, Cancer Res. 73:7189-7198) and 4-1BB (Melero et al., 1997, Nat. Med. 3:682-685).

[0145] Immune checkpoints can be stimulatory immune checkpoints (i.e., molecules that stimulate an immune response) or inhibitory immune checkpoints (i.e., molecules that inhibit an immune response). In some embodiments, the immune checkpoint modulator is an antagonist of an inhibitory immune checkpoint. In some embodiments, the immune checkpoint modulator is an agonist of a stimulatory immune checkpoint. In some embodiments, the immune checkpoint modulator is an immune checkpoint binding protein (e.g., an antibody, an antibody Fab fragment, a bivalent antibody, an antibody drug conjugate, an scFv, a fusion protein, a bivalent antibody, or a tetravalent antibody). In certain embodiments, the immune checkpoint modulator can bind to or modulate the activity of more than one immune checkpoint. Provided below are examples of stimulatory and inhibitory immune checkpoints, as well as molecules that modulate these immune checkpoints that can be used in the methods of the invention.

[0146] i. Stimulatory immune checkpoint molecules CD27 supports antigen-specific proliferation of naive T cells and is essential for the generation of T cell memory (see, e.g., Hendriks et al. (2000) Nat. Immunol. 171(5):433-40). CD27 is also a memory marker for B cells (see, e.g., Agematsu et al. (2000) Histol. Histopathol. 15(2):573-6). The activity of CD27 is governed by the temporal availability of its ligand, CD70, on lymphocytes and dendritic cells (see, e.g., Borst et al. (2000) Nat. Immunol. 171(5):433-40). (see, e.g., et al. (2005) Curr. Opin. Immunol. 17(3):275-81). Several immune checkpoint modulators specific for CD27 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of CD27. In some embodiments, the immune checkpoint modulator is an agent that binds to CD27 (e.g., an anti-CD27 antibody). In some embodiments, the checkpoint modulator is a CD27 agonist. In some embodiments, the checkpoint modulator is a CD27 antagonist. In some embodiments, the immune checkpoint modulator is a CD27 binding protein (e.g., an antibody). In some embodiments, the immune checkpoint modulator is varlilumab (Celldex Therapeutics).Additional CD27 binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. Pat. Nos. 9,248,183, 9,102,737, 9,169,325, 9,023,999, 8,481,029, U.S. Patent Application Publication Nos. 2016 / 0185870, 2015 / 0337047, 2015 / 0299330, 2014 / 0112942, 2013 / 0336976, 2013 / 0243795, and 2013 / 0183316. , 2012 / 0213771, 2012 / 0093805, 2011 / 0274685, 2010 / 0173324; and PCT Publication Nos. WO2015 / 016718, WO2014 / 140374, WO2013 / 138586, WO2012 / 004367, WO2011 / 130434, WO2010 / 001908, and WO2008 / 051424, each of which is incorporated herein by reference.

[0147] CD28. Cluster of differentiation 28 (CD28) is a protein expressed on T cells that provides a costimulatory signal necessary for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) can provide a strong signal for the production of various interleukins, especially IL-6. Binding to its two ligands CD80 and CD86 expressed on dendritic cells promotes T cell proliferation (see, e.g., Prasad et al. (1994) Proc. Nat'l. Acad. Sci. USA 91(7):2834-8). Several immune checkpoint modulators specific for CD28 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of CD28. In some embodiments, the immune checkpoint modulator is an agent that binds to CD28 (e.g., an anti-CD28 antibody). In some embodiments, the checkpoint modulator is a CD28 agonist. In some embodiments, the checkpoint modulator is a CD28 antagonist. In some embodiments, the immune checkpoint modulator is a CD28 binding protein (e.g., an antibody). In some embodiments, the immune checkpoint modulator is selected from the group consisting of TAB08 (TheraMab LLC), lurizumab (also known as BMS-931699, Bristol-Myers Squibb), and FR104 (OSE immunotherapeutic agent).Additional CD28 binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. Patent Nos. 9,119,840, 8,709,414, 9,085,629, 8,034,585, 7,939,638, 8,389,016, 7,585,960, 8,454,959, 8,168,759, 8,785,604 ...7,939,638, 7,939,638, 7,939,638, 7,939,638, 7,939,638, 7,939,638, 7,939,638, 7,939,638, 7, ,723,482; U.S. Patent Application Publication Nos. 2016 / 0017039, 2015 / 0299321, 2015 / 0150968, 2015 / 0071916, 2015 / 0376278, 2013 / 0078257, 2013 / 0230540, 2013 / 0078236, 2013 / 0109846, 2013 / 0266577, Nos. 2012 / 0201814, 2012 / 0082683, 2012 / 0219553, 2011 / 0189735, 2011 / 0097339, 2010 / 0266605, 2010 / 0168400, 2009 / 0246204, 2008 / 0038273; and PCT Publication Nos. WO2015 / 198147, WO2016 / Nos. WO2010 / 007376, WO2010 / 009391, WO2004 / 004768, WO2002 / 030459, WO2002 / 051871, and WO2002 / 047721, each of which is incorporated herein by reference.

[0148] CD40. Cluster of differentiation 40 (CD40, also known as TNFRSF5) is found on a variety of immune system cells, including antigen-presenting cells. CD40L, also known as CD154, is the ligand for CD40 and mediates activation of CD4 +It is transiently expressed on the surface of T cells. CD40 signaling is known to "license" dendritic cells to mature, thereby leading to T cell activation and differentiation (see, e.g., O'Sullivan et al. (2003) Crit. Rev. Immunol. 23(1):83-107). Several immune checkpoint modulators specific for CD40 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of CD40. In some embodiments, the immune checkpoint modulator is an agent that binds to CD40 (e.g., an anti-CD40 antibody). In some embodiments, the checkpoint modulator is a CD40 agonist. In some embodiments, the checkpoint modulator is a CD40 antagonist. In some embodiments, the immune checkpoint modulator is an agent such as dacetuzumab (Genentech / Seattle Genetics), CP-870,893 (Pfizer), bleselumab (Astellas Pharma), lucatumumab (Novartis), CFZ533 (Novartis; see, e.g., Cordoba et al. (2015) Am. J. Transplant. 15(11):2825-36), RG7876 (Genentech Inc.), FFP104 (PanGenetics, BV), APX005 (Apexigen), BI 655064 (Boehringer Ingelheim), Chi Lob 7 / 4 (Cancer Research UK; see, e.g., Johnson et al. (2015) Clin. Cancer Res. 21(6):1321-8), ADC-1013 (BioInvent International), SEA-CD40 (Seattle Genetics), XmAb 5485 (Xencor), PG120 (PanGenetics BV), teneliximab (Bristol-Myers Squibb;For example, a CD40 binding protein selected from the group consisting of CD40-binding protein A (see, e.g., Thompson et al. (2011) Am. J. Transplant. 11(5):947-57), and AKH3 (Biogen; see, e.g., International Publication No. WO 2016 / 028810). Additional CD40 binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. Patent Nos. 9,234,044, 9,266,956, 9,109,011, 9,090,696, 9,023,360, 9,023,361, 9,221,913, 8,945,564, 8,926,979, 8,828,396, 8,637,032, 8,221,913, and 8,234,044. Nos. 77,810, 8,088,383, 7,820,170, 7,790,166, 7,445,780, 7,361,345, 8,961,991, 8,669,352, 8,957,193, 8,778,345, 8,591,900, 8,551,485, 8,492,531, 8,362,210, and 8,388,971;U.S. Patent Application Publication Nos. 2016 / 0045597, 2016 / 0152713, 2016 / 0075792, 2015 / 0299329, and 2015 / 0057437 2015 / 0315282, 2015 / 0307616, 2014 / 0099317, 2014 / 0179907, 2014 / 0349395, 2014 / 0234344, 2014 / 034883 No. 6, No. 2014 / 0193405, No. 2014 / 0120103, No. 2014 / 0105907, No. 2014 / 0248266, No. 2014 / 0093497, No. 2014 / 0010812, No. 2013 / 00 No. 24956, No. 2013 / 0023047, No. 2013 / 0315900, No. 2012 / 0087927, No. 2012 / 0263732, No. 2012 / 0301488, No. 2011 / 0027276, No. 201 1 / 0104182, 2010 / 0234578, 2009 / 0304687, 2009 / 0181015, 2009 / 0130715, 2009 / 0311254, 2008 / 0199471, No. 2008 / 0085531, No. 2016 / 0152721, No. 2015 / 0110783, No. 2015 / 0086991, No. 2015 / 0086559, No. 2014 / 0341898, No. 2014 / 02056 No. 02, No. 2014 / 0004131, No. 2013 / 0011405, No. 2012 / 0121585, No. 2011 / 0033456, No. 2011 / 0002934, No. 2010 / 0172912, No. 2009 / 0 No. 081242, No. 2009 / 0130095, No. 2008 / 0254026, No. 2008 / 0075727, No. 2009 / 0304706, No. 2009 / 0202531, No. 2009 / 0117111, No. 20 09 / 0041773, 2008 / 0274118, 2008 / 0057070, 2007 / 0098717, 2007 / 0218060, 2007 / 0098718, 2007 / 0110754;and PCT Publication Nos. WO2016 / 069919, WO2016 / 023960, WO2016 / 023875, WO2016 / 028810, WO2015 / 134988, WO2015 / 091853, WO2015 / 091655, WO2014 / 065403, WO2014 / 070934, WO2014 / 065402, WO2014 / 207064, WO2013 / 034904, WO201 2 / 125569, WO2012 / 149356, WO2012 / 111762, WO2012 / 145673, WO2011 / 123489, WO2010 / 123012, WO2010 / 10476 1, WO2009 / 094391, WO2008 / 091954, WO2007 / 129895, WO2006 / 128103, WO2005 / 063289, WO2005 / 063981, WO2 003 / 040170, WO2002 / 011763, WO2000 / 075348, WO2013 / 164789, WO2012 / 075111, WO2012 / 065950, WO2009 / 062 054, WO2007 / 124299, WO2007 / 053661, WO2007 / 053767, WO2005 / 044294, WO2005 / 044304, WO2005 / 044306, W and WO2005 / 044855, WO2005 / 044854, WO2005 / 044305, WO2003 / 045978, WO2003 / 029296, WO2002 / 028481, WO2002 / 028480, WO2002 / 028904, WO2002 / 028905, WO2002 / 088186, and WO2001 / 024823, each of which is incorporated herein by reference.

[0149] CD122 is the beta subunit of the interleukin 2 receptor and is associated with CD8 +It is known to increase the proliferation of effector T cells. See, e.g., Boyman et al. (2012) Nat. Rev. Immunol. 12(3):180-190. Several immune checkpoint modulators specific for CD122 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of CD122. In some embodiments, the immune checkpoint modulator is an agent that binds to CD122 (e.g., an anti-CD122 antibody). In some embodiments, the checkpoint modulator is a CD122 agonist. In some embodiments, the checkpoint modulator is a CD22 agonist. In some embodiments, the immune checkpoint modulator is humanized MiK-Beta-1 (Roche; see, e.g., Morris et al. (2006) Proc Nat'l. Acad. Sci. USA 103(2):401-6, which is incorporated by reference). Additional CD122 binding proteins (eg, antibodies) are known in the art and are disclosed, for example, in US Pat. No. 9,028,830, which is incorporated herein by reference.

[0150] OX40. The OX40 receptor (also known as CD134) promotes the proliferation of effector and memory T cells. OX40 also suppresses the differentiation and activity of regulatory T cells and regulates cytokine production (see, e.g., Croft et al. (2009) Immunol. Rev. 229(1):173-91). Several immune checkpoint modulators specific for OX40 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of OX40. In some embodiments, the immune checkpoint modulator is an agent that binds to OX40 (e.g., an anti-OX40 antibody). In some embodiments, the checkpoint modulator is an OX40 agonist. In some embodiments, the checkpoint modulator is an OX40 antagonist. In some embodiments, the immune checkpoint modulator is an OX40 binding protein (e.g., an antibody) selected from the group consisting of MEDI6469 (AgonOx / Medmmune), pogalizumab (also known as MOXR0916 and RG7888; Genentech, Inc.), taborixizumab (also known as MEDI0562; Medimmune), and GSK3174998 (GlaxoSmithKline).Additional OX-40 binding proteins (e.g., antibodies) are known in the art and are described, e.g., in U.S. Pat. Nos. 9,163,085, 9,040,048, 9,006,396, 8,748,585, 8,614,295, 8,551,477, 8,283,450, 7,550,140; U.S. Pat. Publication No. 2016 / 0068604, Publication No. 2016 / 0031974, Publication No. 2015 / 0315281, Publication No. 2015 / 0132288, Publication No. 2014 / 030827 No. 6, No. 2014 / 0377284, No. 2014 / 0044703, No. 2014 / 0294824, No. 2013 / 0330344, No. 2013 / 0280 275, 2013 / 0243772, 2013 / 0183315, 2012 / 0269825, 2012 / 0244076, 2011 / 0008368, 2011 / 0123552, 2010 / 0254978, 2010 / 0196359, 2006 / 0281072; and PC and WO2014 / 148895, WO2013 / 068563, WO2013 / 038191, WO2013 / 028231, WO2010 / 096418, WO2007 / 062245, and WO2003 / 106498, each of which is incorporated herein by reference.

[0151] GITR. Glucocorticoid-inducible TNFR family-related gene (GITR) is a member of the tumor necrosis factor receptor (TNFR) superfamily that is constitutively or conditionally expressed in Treg, CD4, and CD8 T cells. GITR is rapidly upregulated in effector T cells after TCR ligation and activation. Human GITR ligand (GITRL) is constitutively expressed in APCs in secondary lymphoid organs and some non-lymphoid tissues. Downstream effects of GITR:GITRL interaction induce attenuation of Treg activity and upregulate CD4 +It enhances the activity of T cells, resulting in reversal of Treg-mediated immune suppression and increased immune stimulation. Several immune checkpoint modulators specific for GITR have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that regulates the activity and / or expression of GITR. In some embodiments, the immune checkpoint modulator is an agent that binds to GITR (e.g., an anti-GITR antibody). In some embodiments, the checkpoint modulator is a GITR agonist. In some embodiments, the checkpoint modulator is a GITR antagonist. In some embodiments, the immune checkpoint modulator is a GITR binding protein (e.g., an antibody) selected from the group consisting of TRX518 (Leap Therapeutics), MK-4166 (Merck&Co.), MEDI-1873 (MedImmune), INCAGN1876 (Agenus / Incyte), and FPA154 (Five Prime Therapeutics). Additional GITR binding proteins (e.g., antibodies) are known in the art, see, e.g., U.S. Patent Nos. 9,309,321, 9,255,152, 9,255,151, 9,228,016, 9,028,823, 8,709,424, and 8,388,967; U.S. Patent Application Publication Nos. 2016 / 0145342, 2015 / 0353637, 2015 / 0064204, 2014 / 0348841, 2014 / 0065152, 2014 / 0072566, and 2015 / 0145342. and PCT Publication Nos. WO2014 / 0072565, 2013 / 0183321, 2013 / 0108641, 2012 / 0189639; and PCT Publication Nos. WO2016 / 054638, WO2016 / 057841, WO2016 / 057846, WO2015 / 187835, WO2015 / 184099, WO2015 / 031667, WO2011 / 028683, and WO2004 / 107618, each of which is incorporated herein by reference.

[0152] ICOS.-inducible T cell costimulator (ICOS, also known as CD278) is expressed on activated T cells. Its ligand is ICOSL, which is expressed primarily on B cells and dendritic cells. ICOS is important in T cell effector function. ICOS expression is upregulated upon T cell activation (see, e.g., Fan et al. (2014) J. Exp. Med. 211(4):715-25). Several immune checkpoint modulators specific for ICOS have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of ICOS. In some embodiments, the immune checkpoint modulator is an agent that binds to ICOS (e.g., an anti-ICOS antibody). In some embodiments, the checkpoint modulator is an ICOS agonist. In some embodiments, the checkpoint modulator is an ICOS antagonist. In some embodiments, the immune checkpoint modulator is an ICOS binding protein (e.g., an antibody) selected from the group consisting of MEDI-570 (also known as JMab-136, Medimmune), GSK3359609 (GlaxoSmithKline / INSERM), and JTX-2011 (Jounce Therapeutics). Additional ICOS binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. Pat. Nos. 9,376,493, 7,998,478, 7,465,445, 7,465,444; U.S. Patent Application Publication Nos. 2015 / 0239978, 2012 / 0039874, 2008 / 0199466, 2008 / 0279851; and PCT Publication No. WO2001 / 087981, each of which is incorporated herein by reference.

[0153] 4-1BB. 4-1BB (also known as CD137) is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB (CD137) mediates the expression of primed CD4 + and CD8 + It is a type II transmembrane glycoprotein inducibly expressed in T cells, activated NK cells, DCs, and neutrophils, and acts as a T cell costimulatory molecule upon binding to the 4-1BB ligand (4-1BBL) found on activated macrophages, B cells, and DCs. Ligation of the 4-1BB receptor leads to activation of the NF-κB, c-Jun, and p38 signaling pathways, and inhibits CD8 T cell proliferation, particularly by upregulating the expression of the anti-apoptotic genes BcL-x(L) and Bfl-1. +It has been shown to promote T cell survival. In this way, 4-1BB plays a role in enhancing or even rescuing suboptimal immune responses. Several immune checkpoint modulators specific for 4-1BB have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of 4-1BB. In some embodiments, the immune checkpoint modulator is an agent that binds to 4-1BB (e.g., an anti-4-1BB antibody). In some embodiments, the checkpoint modulator is a 4-1BB agonist. In some embodiments, the checkpoint modulator is a 4-1BB antagonist. In some embodiments, the immune checkpoint modulator is a 4-1BB binding protein, urelumab (also known as BMS-663513; Bristol-Myers Squibb) or utomilumab (Pfizer). In some embodiments, the immune checkpoint modulator is a 4-1BB binding protein (e.g., an antibody).4-1BB binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. Pat. Nos. 9,382,328, 8,716,452, 8,475,790, 8,137,667, 7,829,088, and 7,659,384; U.S. Patent Application Publication Nos. 2016 / 0083474, 2016 / 0152722, 2014 / 0193422, 2014 / 0178368, 2013 / 0149301, 2012 / 0237498, Same No. 2012 / 0141494, No. 2012 / 0076722, No. 2011 / 0177104, No. 2011 / 0189189, No. 2010 / 0183 621, 2009 / 0068192, 2009 / 0041763, 2008 / 0305113, 2008 / 0008716; and PCT Publication Nos. WO2016 / 029073, WO2015 / 188047, WO2015 / 179236, WO2015 / 119923, WO2 012 / 032433, WO2012 / 145183, WO2011 / 031063, WO2010 / 132389, WO2010 / 042433, WO2006 / 126835, WO2005 / 035584, WO2004 / 010947; as well as Martinez-Forero et al. (2013) J. Immunol. 190(12):6694-706, and Dubrot et al. (2010) Cancer Immunol. Immunother. 59(8):1223-33, each of which is incorporated herein by reference.

[0154] ii. Inhibitory immune checkpoint molecules ADORA2A. The adenosine A2A receptor (A2A4) is a member of the G protein-coupled receptor (GPCR) family with seven transmembrane α-helices and is considered an important checkpoint for cancer treatment. The A2A receptor can negatively regulate over-reactive immune cells (see, e.g., Ohta et al. (2001) Nature 414(6866):916-20). Several immune checkpoint modulators specific for ADORA2A have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of ADORA2A. In some embodiments, the immune checkpoint modulator is an agent that binds to ADORA2A (e.g., an anti-ADORA2A antibody). In some embodiments, the immune checkpoint modulator is an ADORA2A binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is an ADORA2A agonist. In some embodiments, the checkpoint modulator is an ADORA2A antagonist. ADORA2A binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. Patent Application Publication No. 2014 / 0322236, which is incorporated herein by reference.

[0155] B7-H3. B7-H3 (also known as CD276) belongs to the B7 superfamily, a group of molecules that costimulate or downregulate T cell responses. B7-H3 potently and consistently downregulates human T cell responses (see, e.g., Leitner et al. (2009) Eur. J. Immunol. 39(7):1754-64). Several immune checkpoint modulators specific for B7-H3 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of B7-H3. In some embodiments, the immune checkpoint modulator is an agent that binds to B7-H3 (e.g., an anti-B7-H3 antibody). In some embodiments, the checkpoint modulator is a B7-H3 agonist. In some embodiments, the checkpoint modulator is a B7-H3 antagonist. In some embodiments, the immune checkpoint modulator is an anti-B7-H3 binding protein selected from the group consisting of DS-5573 (Daiichi Sankyo Co., Ltd.), enoblituumab (MacroGenics, Inc.), and 8H9 (Sloan Kettering Institute for Cancer Research; see, e.g., Ahmed et al. (2015) J. Biol. Chem. 290(50):30018-29). In some embodiments, the immune checkpoint modulator is a B7-H3 binding protein (e.g., an antibody).B7-H3 binding proteins (e.g., antibodies) are known in the art and are described in, e.g., U.S. Patent Nos. 9,371,395, 9,150,656, 9,062,110, 8,802,091, 8,501,471, and 8,414,892; U.S. Patent Application Publication Nos. 2015 / 0352224, 2015 / 0297748, 2015 / 0259434, 2015 / 0274838, 2014 / 032875, 2014 / 0161814, 2013 / 0287798, and 2014 / 0287799. Nos. 2013 / 0078234, 2013 / 0149236, 2012 / 02947960, 2010 / 0143245, 2002 / 0102264; PCT Publication Nos. WO2016 / 106004, WO2016 / 033225, WO2015 / 181267, WO2014 / 057687, WO2012 / 147713, WO2011 / 109400, WO2008 / 116219, WO2003 / 075846, WO2002 / 032375; and Shi et al. (2016) Mol. Med. Rep. 14(1):943-8, each of which is incorporated herein by reference.

[0156] B7-H4. B7-H4 (also known as O8E, OV064, and V-set domain-containing T cell activation inhibitor (VTCN1)) belongs to the B7 superfamily. By arresting the cell cycle, B7-H4 ligation of T cells shows profound inhibitory effects on growth, cytokine secretion, and the development of cytotoxicity. Administration of B7-H4Ig to mice impairs antigen-specific T cell responses, whereas blocking endogenous B7-H4 with specific monoclonal antibodies promotes T cell responses (see, e.g., Sica et al. (2003) Immunity 18(6):849-61). Several immune checkpoint modulators specific for B7-H4 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of B7-H4. In some embodiments, the immune checkpoint modulator is an agent that binds to B7-H4 (e.g., an anti-B7-H4 antibody). In some embodiments, the immune checkpoint modulator is a B7-H4 binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is a B7-H4 agonist. In some embodiments, the checkpoint modulator is a B7-H4 antagonist.B7-H4 binding proteins (e.g., antibodies) are known in the art and are described in, e.g., U.S. Pat. Nos. 9,296,822, 8,609,816, 8,759,490, 8,323,645; U.S. Patent Application Publication Nos. 2016 / 0159910, 2016 / 0017040, 2016 / 0168249, 2015 / 0315275, 2014 / 0134180, 2014 / 0322129, 2014 / 0356364, 2014 / 0328751, 2014 / 0294861, 2014 / 0308259, 2013 / 0058864, 2011 / 0085970, 2009 / 0074660, 2009 / 0208489; as well as PCT Publication Nos. 2016 / 040724, WO2016 / 070001, WO2014 / 159835, WO2014 / 100483, WO2014 / 100439, WO2013 / 067492, WO2013 / 025779, WO2009 / 073533, WO2007 / 067991, and WO2006 / 104677, each of which is incorporated herein by reference.

[0157] B and T lymphocyte attenuator (BTLA), also known as BTLA.CD272, has HVEM (herpes virus entry mediator) as its ligand. Surface expression of BTLA is expressed on human CD8 + Tumor-specific human CD8 T cells are progressively downregulated during differentiation from naive to effector phenotypes. +T cells express high levels of BTLA (see, e.g., Derre et al. (2010) J. Clin. Invest. 120(1):157-67). Several immune checkpoint modulators specific for BTLA have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of BTLA. In some embodiments, the immune checkpoint modulator is an agent that binds to BTLA (e.g., an anti-BTLA antibody). In some embodiments, the immune checkpoint modulator is a BTLA binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is a BTLA agonist. In some embodiments, the checkpoint modulator is a BTLA antagonist. BTLA binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. Pat. Nos. 9,346,882, 8,580,259, 8,563,694, 8,247,537; U.S. Patent Application Publication Nos. 2014 / 0017255, 2012 / 0288500, 2012 / 0183565, 2010 / 0172900, and PCT Publication Nos. WO2011 / 014438, and WO2008 / 076560, each of which is incorporated herein by reference.

[0158] CTLA-4. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is an immunoregulatory CD28-B7 immunoglobulin superfamily member that acts on naive and resting T lymphocytes to promote immune suppression through both B7-dependent and B7-independent pathways (see, e.g., Kim et al. (2016) J. Immunol. Res., Article ID 4683607, 14pp.). CTLA-4 is also referred to as CD152. CTLA-4 regulates the threshold for T-cell activation. See, e.g., Gajewski et al. (2001) J. Immunol. 166(6):3900-7. Several immune checkpoint modulators specific for CTLA-4 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of CTLA-4. In some embodiments, the immune checkpoint modulator is an agent that binds to CTLA-4 (e.g., an anti-CTLA-4 antibody). In some embodiments, the checkpoint modulator is a CTLA-4 agonist. In some embodiments, the checkpoint modulator is a CTLA-4 antagonist. In some embodiments, the immune checkpoint modulator is a CTLA-4 binding protein (e.g., an antibody) selected from the group consisting of ipilimumab (Yervoy; Medarex / Bristol-Myers Squibb), tremelimumab (formerly ticilimumab; Pfizer / AstraZeneca), JMW-3B3 (University of Aberdeen), and AGEN1884 (Agenus).Additional CTLA-4 binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. Patent No. 8,697,845; U.S. Patent Application Publication Nos. 2014 / 0105914, 2013 / 0267688, 2012 / 0107320, 2009 / 0123477; and PCT Publication Nos. WO2014 / 207064, WO2012 / 120125, WO2016 / 015675, WO2010 / 09597, WO2006 / 066568, and WO2001 / 054732, each of which is incorporated herein by reference.

[0159] IDO. Indoleamine 2,3-dioxygenase (IDO) is a tryptophan catabolic enzyme with immunosuppressive properties. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO has been shown to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. Prendergast et al., 2014, Cancer Immunol Immunother. 63(7):721-35, incorporated herein by reference. Several immune checkpoint modulators specific for IDO have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of IDO. In some embodiments, the immune checkpoint modulator is an agent that binds to IDO (e.g., an IDO binding protein such as an anti-IDO antibody). In some embodiments, the checkpoint modulator is an IDO agonist. In some embodiments, the checkpoint modulator is an IDO antagonist. In some embodiments, the immune checkpoint modulator is selected from the group consisting of norharman, rosmarinic acid, COX-2 inhibitors, α-methyl-tryptophan, and epacadostat. In one embodiment, the modulator is epacadostat.

[0160] KIR. Killer immunoglobulin-like receptors (KIR) comprise a diverse repertoire of MHCI-binding molecules that negatively regulate the function of natural killer (NK) cells, protecting them from NK-mediated cytolysis. KIRs are generally expressed on NK cells, but have also been detected on tumor-specific CTLs. Multiple immune checkpoint modulators specific for KIRs have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of KIR. In some embodiments, the immune checkpoint modulator is an agent that binds to KIR (e.g., an anti-KIR antibody). In some embodiments, the immune checkpoint modulator is a KIR-binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is a KIR agonist. In some embodiments, the checkpoint modulator is a KIR antagonist. In some embodiments, the immune checkpoint modulator is lirilumab (also known as BMS-986015; Bristol-Myers Squibb).Additional KIR binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. Patent Nos. 8,981,065, 9,018,366, 9,067,997, 8,709,411, 8,637,258, 8,614,307, 8,551,483, 8,388,970, 8,119, and 8,221,747. ,775; U.S. Patent Application Publication Nos. 2015 / 0344576, 2015 / 0376275, 2016 / 0046712, 2015 / 0191547, 2015 / 0290316, 2015 / 0283234, 2015 / 0197569, 2014 / 0193430, and 2013 / 0143269 Nos. 2013 / 0287770, 2012 / 0208237, 2011 / 0293627, 2009 / 0081240, 2010 / 0189723; and PCT Publication Nos. WO2016 / 069589, WO2015 / 069785, WO2014 / 066532, WO2014 / 055648 No. WO2012 / 160448, No. WO2012 / 071411, No. WO2010 / 065939, No. WO2008 / 084106, No. WO2006 / 072625, No. WO2006 / 072626, and No. WO2006 / 003179, each of which is incorporated herein by reference.

[0161] LAG-3, lymphocyte activation gene 3 (LAG-3, also known as CD223), is a CD4-associated transmembrane protein that competitively binds to MHC II and acts as a co-inhibitory checkpoint for T cell activation (see, e.g., Goldberg and Drake (2011) Curr. Top. Microbiol. Immunol. 344:269-78). Several immune checkpoint modulators specific for LAG-3 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of LAG-3. In some embodiments, the immune checkpoint modulator is an agent that binds to LAG-3 (e.g., an anti-PD-1 antibody). In some embodiments, the checkpoint modulator is a LAG-3 agonist. In some embodiments, the checkpoint modulator is a LAG-3 antagonist. In some embodiments, the immune checkpoint modulator is selected from the group consisting of pembrolizumab (Keytruda; formerly lambrolizumab; Merck & Co., Inc.), nivolumab (Opdivo; Bristol-Myers Squibb), pidilizumab (CT-011, CureTech), SHR-1210 (Incyte / Jiangsu Hengrui Medicine Co., Ltd.), MEDI0680 (also known as AMP-514; Amplimmune Inc. / Medimmune), PDR001 (Novartis), BGB-A317 (BeiGene Ltd.), TSR-042 (also known as ANB011; AnaptysBio / Tesaro, Inc.), REGN2810 (Regeneron), and / or other anti-cancer drugs. The LAG-3 binding protein (e.g., an antibody) is selected from the group consisting of PF-06801591 (Pfizer), PF-06801592 (Pfizer), and PF-06801593 (Pfizer).Additional PD-1 binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. Pat. Nos. 9,181,342, 8,927,697, 7,488,802, 7,029,674; U.S. Patent Application Publication Nos. 2015 / 0152180, 2011 / 0171215, 2011 / 0171220; and PCT Publication Nos. WO2004 / 056875, WO2015 / 036394, WO2010 / 029435, WO2010 / 029434, WO2014 / 194302, each of which is incorporated herein by reference.

[0162] PD-1. Programmed cell death protein 1 (PD-1, also known as CD279 and PDCD1) is an inhibitory receptor that negatively regulates the immune system. In contrast to CTLA-4, which primarily affects naive T cells, PD-1 is more widely expressed on immune cells and regulates the activity of mature T cells in peripheral tissues and the tumor microenvironment. PD-1 inhibits T cell responses by interfering with T cell receptor signaling. PD-1 has two ligands, PD-L1 and PD-L2. Several immune checkpoint modulators specific for PD-1 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of PD-1. In some embodiments, the immune checkpoint modulator is an agent that binds to PD-1 (e.g., an anti-PD-1 antibody). In some embodiments, the checkpoint modulator is a PD-1 agonist. In some embodiments, the checkpoint modulator is a PD-1 antagonist. In some embodiments, the immune checkpoint modulator is selected from the group consisting of pembrolizumab (Keytruda; formerly lambrolizumab; Merck & Co., Inc.), nivolumab (Opdivo; Bristol-Myers Squibb), pidilizumab (CT-011, CureTech), SHR-1210 (Incyte / Jiangsu Hengrui Medicine Co., Ltd.), MEDI0680 (also known as AMP-514; Amplimmune Inc. / Medimmune), PDR001 (Novartis), BGB-A317 (BeiGene Ltd.), TSR-042 (also known as ANB011; AnaptysBio / Tesaro, Inc.), REGN2810 (Regeneron), and / or other anti-cancer drugs. and PF-06801591 (Pfizer).Additional PD-1 binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. Pat. Nos. 9,181,342, 8,927,697, 7,488,802, 7,029,674; U.S. Patent Application Publication Nos. 2015 / 0152180, 2011 / 0171215, 2011 / 0171220; and PCT Publication Nos. WO2004 / 056875, WO2015 / 036394, WO2010 / 029435, WO2010 / 029434, WO2014 / 194302, each of which is incorporated herein by reference.

[0163] PD-L1 / PD-L2. PD ligand 1 (PD-L1, also known as B7-H1) and PD ligand 2 (PD-L2; also known as PDCD1LG2, CD273, and B7-DC) bind to the PD-1 receptor. Both ligands belong to the same B7 family as the B7-1 and B7-2 proteins, which interact with CD28 and CTLA-4. PD-L1 can be expressed on many cell types, including, for example, epithelial cells, endothelial cells, and immune cells. Ligation of PDL-1 reduces the production of IFNγ, TNFα, and IL-2 and stimulates the production of IL10, an anti-inflammatory cytokine associated with reduced T cell reactivity and proliferation, as well as antigen-specific T cell anergy. PDL-2 is expressed primarily on antigen-presenting cells (APCs). Ligation of PDL2 also results in T cell suppression, whereas PDL-1-PD-1 interaction inhibits proliferation via cell cycle arrest in the G1 / G2 phase, engagement by PDL2-PD-1 has been shown to inhibit TCR-mediated signaling by blocking B7:CD28 signals at low antigen concentrations and reduce cytokine production at high antigen concentrations. Multiple immune checkpoint modulators specific for PD-L1 and PD-L2 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of PD-L1. In some embodiments, the immune checkpoint modulator is an agent that binds to PD-L1 (e.g., an anti-PD-L1 antibody). In some embodiments, the checkpoint modulator is a PD-L1 agonist. In some embodiments, the checkpoint modulator is a PD-L1 antagonist. In some embodiments, the immune checkpoint modulator is a PD-L1 binding protein (e.g., an antibody or Fc fusion protein) selected from the group consisting of durvalumab (also known as MEDI-4736; AstraZeneca / Celgene Corp. / Medimmune), atezolizumab (Tecentriq; also known as MPDL3280A and RG7446; Genetech Inc.), avelumab (also known as MSB0010718C; Merck Serono / AstraZeneca); MDX-1105 (Medarex / Bristol-Meyers Squibb), AMP-224 (Amplimmune, GlaxoSmithKline), LY3300054 (Eli Lilly and Co.). Additional PD-L1 binding proteins are known in the art and are disclosed in, for example, U.S. Patent Application Publication Nos. 2016 / 0084839, 2015 / 0355184, 2016 / 0175397, and PCT Publication Nos. WO2014 / 100079, WO2016 / 030350, and WO2013 / 181634, each of which is incorporated herein by reference.

[0164] In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of PD-L2. In some embodiments, the immune checkpoint modulator is an agent that binds to PD-L2 (e.g., an anti-PD-L2 antibody). In some embodiments, the checkpoint modulator is a PD-L2 agonist. In some embodiments, the checkpoint modulator is a PD-L2 antagonist. PD-L2 binding proteins (e.g., antibodies) are known in the art and have been disclosed in, for example, U.S. Patent Nos. 9,255,147, 8,188,238; U.S. Patent Application Publication Nos. 2016 / 0122431, 2013 / 0243752, 2010 / 0278816, 2016 / 0137731, 2015 / 0197571, 2013 / 0291136, 2011 / 0271358; and PCT Publication Nos. WO2014 / 022758, and WO2010 / 036959, each of which is incorporated herein by reference.

[0165] TIM-3. T-cell immunoglobulin mucin 3 (TIM-3, also known as Hepatitis A Virus Cellular Receptor (HAVCR2)) is a type I glycoprotein receptor that binds to the S-type lectin galectin-9 (Gal-9). TIM-3 is a ligand that is widely expressed in lymphocytes, liver, small intestine, thymus, kidney, spleen, lung, muscle, reticulocyte, and brain tissues. Tim-3 was initially identified as being selectively expressed in Th1 and Tc1 cells that secrete IFN-γ (Monney et al. (2002) Nature 415:536-41). Binding of Gal-9 by the TIM-3 receptor triggers downstream signaling and negatively regulates T cell survival and function. Several immune checkpoint modulators specific for TIM-3 have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and / or expression of TIM-3. In some embodiments, the immune checkpoint modulator is an agent that binds to TIM-3 (e.g., an anti-TIM-3 antibody). In some embodiments, the checkpoint modulator is a TIM-3 agonist. In some embodiments, the checkpoint modulator is a TIM-3 antagonist. In some embodiments, the immune checkpoint modulator is an anti-TIM-3 antibody selected from the group consisting of TSR-022 (AnaptysBio / Tesaro, Inc.) and MGB453 (Novartis).Additional TIM-3 binding proteins (e.g., antibodies) are known in the art and are described, e.g., in U.S. Pat. Nos. 9,103,832, 8,552,156, 8,647,623, 8,841,418; U.S. Patent Application Publication Nos. 2016 / 0200815, 2015 / 0284468, 2014 / 0134639, 2014 / 0044728, 2015 / 0200921, and 2015 / 0200722. Nos. 12 / 0189617, 2015 / 0086574, 2013 / 0022623; and PCT Publication Nos. WO2016 / 068802, WO2016 / 068803, WO2016 / 071448, WO2011 / 155607, and WO2013 / 006490, each of which is incorporated herein by reference.

[0166] VISTA, a V-domain Ig suppressor of T-cell activation (VISTA, also known as the platelet receptor Gi24), is an Ig superfamily ligand that negatively regulates T-cell responses. See, e.g., Wang et al., 2011, J. Exp. Med. 208:577-92. VISTA expressed on APCs inhibits the expression of CD4 + and CD8 +It directly inhibits T cell proliferation as well as cytokine production (Wang et al. (2010) J Exp Med. 208(3):577-92). Several immune checkpoint modulators specific for VISTA have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates VISTA activity and / or expression. In some embodiments, the immune checkpoint modulator is an agent that binds to VISTA (e.g., an anti-VISTA antibody). In some embodiments, the checkpoint modulator is a VISTA agonist. In some embodiments, the checkpoint modulator is a VISTA antagonist. In some embodiments, the immune checkpoint modulator is a VISTA binding protein (e.g., an antibody) selected from the group consisting of TSR-022 (AnaptysBio / Tesaro, Inc.) and MGB453 (Novartis). VISTA binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. Patent Application Publication Nos. 2016 / 0096891, 2016 / 0096891; and PCT Publication Nos. WO2014 / 190356, WO2014 / 197849, WO2014 / 190356 and WO2016 / 094837, each of which is incorporated herein by reference.

[0167] In some embodiments, more than one (e.g., 2, 3, 4, 5 or more) immune checkpoint modulators are administered to a subject. When more than one immune checkpoint modulator is administered, the modulators can each target a stimulatory immune checkpoint molecule or each target an inhibitory immune checkpoint molecule. In other embodiments, the immune checkpoint modulators include at least one modulator that targets a stimulatory immune checkpoint molecule and at least one immune checkpoint modulator that targets an inhibitory immune checkpoint molecule. In certain embodiments, the immune checkpoint modulator is a binding protein, e.g., an antibody. The term "binding protein" as used herein refers to a protein or polypeptide that can specifically bind to a target molecule, e.g., an immune checkpoint molecule. In some embodiments, the binding protein is an antibody or an antigen-binding portion thereof, and the target molecule is an immune checkpoint molecule. In some embodiments, the binding protein is a protein or polypeptide that specifically binds to a target molecule (e.g., an immune checkpoint molecule). In some embodiments, the binding protein is a ligand. In some embodiments, the binding protein is a fusion protein. In some embodiments, the binding protein is a receptor. Examples of binding proteins that can be used in the methods of the present invention include, but are not limited to, humanized antibodies, antibody Fab fragments, bivalent antibodies, antibody drug conjugates, scFvs, fusion proteins, bivalent antibodies, and tetravalent antibodies.

[0168] Immune checkpoint modulator antibodies include, but are not limited to, at least four major categories: i) antibodies that block inhibitory pathways directly on T cells or natural killer (NK) cells (e.g., PD-1-targeting antibodies such as nivolumab and pembrolizumab, antibodies targeting TIM-3, and antibodies targeting LAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049, and KIR); ii) antibodies that activate stimulatory pathways directly on T cells or NK cells (e.g., antibodies targeting OX40, GITR, and 4-1BB). These include antibodies that block inhibitory pathways directly on cancer cells, iii) antibodies that block inhibitory pathways on immune cells or that rely on antibody-dependent cellular cytotoxicity to deplete inhibitory populations of immune cells (e.g., CTLA-4-targeting antibodies such as ipilimumab, antibodies targeting VISTA, and antibodies targeting PD-L2, Gr1, and Ly6G), and iv) antibodies that block inhibitory pathways directly on cancer cells or that rely on antibody-dependent cellular cytotoxicity to enhance cytotoxicity against cancer cells (e.g., rituximab, antibodies targeting PD-L1, and antibodies targeting B7-H3, B7-H4, Gal-9, and MUC1). Examples of checkpoint inhibitors include, for example, inhibitors of CTLA-4, such as ipilimumab or tremelimumab; inhibitors of the PD-1 pathway, such as anti-PD-1, anti-PD-L1, or anti-PD-L2 antibodies. Exemplary anti-PD-1 antibodies are described in WO2006 / 121168, WO2008 / 156712, WO2012 / 145493, WO2009 / 014708, and WO2009 / 114335. Exemplary anti-PD-L1 antibodies are described in WO2007 / 005874, WO2010 / 077634, and WO2011 / 066389, and exemplary anti-PD-L2 antibodies are described in WO2004 / 007679.

[0169] In certain embodiments, the immune checkpoint modulator is a fusion protein, e.g., a fusion protein that modulates the activity of the immune checkpoint modulator. In one embodiment, the immune checkpoint modulator is a therapeutic nucleic acid molecule, e.g., a nucleic acid that modulates the expression of an immune checkpoint protein or mRNA. Nucleic acid therapeutics are well known in the art. Nucleic acid therapeutics include both single-stranded and double-stranded nucleic acids (i.e., nucleic acid therapeutics with a complementary region of at least 15 nucleotides) that are complementary to a target sequence in a cell. In certain embodiments, the nucleic acid therapeutic targets the nucleic acid sequence that codes for an immune checkpoint protein.

[0170] It should be noted that more than one, for example, two, three, four, five or more additional anti-cancer agents can be administered in combination with the UBE2K inhibitor (e.g., specific inhibitor of UBE2K) formulation provided herein.For example, in one embodiment, two additional chemotherapeutic agents can be administered in combination with the UBE2K inhibitor.The appropriate dose and administration route of the chemotherapeutic agents provided herein are known in the art. EXAMPLES

[0171] Example 1: Description of the Identification of the Role of UBE2K in Cancer Using Network Biology UBE2K (E2-25K) was identified from the first generation of cancer networks by using the Interrogative Biology™ platform in an in vitro pan-cancer model. The Interrogative Biology™ platform is described, for example, in WO / 2012 / 119129, which is incorporated herein by reference in its entirety. The in vitro pan-cancer model consisted of cultured cancer cells representing various tissues of origin, namely liver (HepG2), pancreas (MIA PaCa2), skin (SKMEL28 melanoma), tongue (SCC-25 squamous cell carcinoma), breast (SkBr-3, MCF7), and prostate (PC-3 and LnCAP), as well as non-tumorigenic cells derived from breast, pancreas, prostate, liver, and skin. The cells were exposed to in vitro conditions designed to simulate hypoxic load, low pH, and low nutrient microenvironments, as follows: Non-tumorigenic and cancer cells were cultured in low (5 mM) or high glucose (22 mM), with or without lactate (12.5 mM), with or without coenzyme Q10 (50 and 100 μM), and in normoxia (~21% oxygen) or hypoxia (2% oxygen). Cell lysates were collected at 24 and 48 h for proteomic analysis, and the results were used as input for a Bayesian network inference model. Candidate oncology targets were prioritized and ranked based on the number of in-degree and out-degree connections (edges) and the frequency / AUC of connections.

[0172] Example 2: Knockdown of UBE2K in cancer cell lines in vitro Rationale for cell line selection Given that UBE2K was identified from pan-cancer in vitro models representing multiple tissues of origin / cell lines, a subset was selected for initial in vitro validity testing. Based on the proteomic data used for network generation, robust changes in UBE2K protein expression were identified in Miapaca2 pancreatic cancer cell line. Furthermore, because UBE2K may act in concert with two related tumor suppressors (p53, BRCA1), additional cell lines were selected that are representative of tumor types in which these tumor suppressors are relevant and included in the pan-cancer in vitro model, namely breast cancer and hepatocellular carcinoma (HCC). SkBr-3 (breast) and HepG2 (HCC) were used for network generation. Taking into account the possibility of capturing canonical and non-canonical functions of UBE2K in these contexts, we also included T47D, MDA-MB231, and BT549 (breast), as well as SKHEP1 and Hep3B (HCC) to provide further diversity with respect to p53 and BRCA1 status. The cell lines used in the initial validation studies are summarized in Table 1. [Table 1]

[0173] Initial Validation Strategy In initial validation studies, UBE2K was transiently knocked down using an siRNA-mediated approach in Miapaca2, HepG2, SKHEP1, Hep3B, MDAMB231, SKBR3, T47D, and BT549 cells. A non-targeting (NT) siRNA sequence was used as a control. Samples for confirming knockdown at transcript and protein levels were harvested 24 and 96 hours post-transfection, and the effect of transient knockdown of UBE2K was assessed by cell viability / cell number using Cell Titer Fluor 96 hours post-transfection. In parallel, the effect of siRNA-mediated UBE2K knockdown on doxorubicin (a drug that causes cell death) sensitivity was assessed in the above cell lines. Cells were exposed to doxorubicin for 72 hours starting 24 hours post-transfection.

[0174] Results of in vitro experiments Using this siRNA-mediated approach, greater than 70% UBE2K knockdown was achieved at both the transcript and protein levels in all cell lines tested 24 hours after transfection (see Figure 2 and Figure 3), providing a rational means to evaluate the effect of UBE2K knockdown in phenotypic assays. Under basal conditions, UBE2K siRNA-mediated knockdown reduced cell numbers in Miapaca2 pancreatic cancer cell line by 50% and in SKHEP1 and HepG2 hepatocellular carcinoma cells by 30% 96 hours after transfection (Figure 4). No effect of UBE2K siRNA was observed in the remaining five cancer cell lines. Furthermore, sensitivity to doxorubicin-induced cell death was unchanged in response to UBE2K siRNA-mediated knockdown in all cell lines tested (Figure 5 and Table 2). Table 3 shows a summary of the results. [Table 2] [Table 3]

[0175] UBE2K knockdown in cells may trigger compensatory regulation of other related E2s. To understand this possibility, we examined the expression of related E2s upon siRNA-mediated knockdown of UBE2K. Related E2s were selected based on either the presence of common E3s for substrate ubiquitination (E2D1, E2D2, E2D3) or their ability to synthesize Lys 48-linked ubiquitin chains (E2N, cdc34, E2D). E2R2 was also included in this study because it is closely related to cdc34. Data from Miapaca2 cells are shown in Figure 6. UBE2K was found to be knocked down 24 and 96 hours after transfection, and the expression of related E2s appears to be largely unchanged by UBE2K knockdown at both time points, suggesting the absence of a compensatory transcriptional response. Similar results were obtained in all other cell lines evaluated (SKHEP1, HepG2, Hep3B, MDAMB231, BT549, SKBR3, and T47D). Thus, siRNA-mediated knockdown of UBE2K does not trigger a compensatory transcriptional response of associated E2s.

[0176] UBE2K is part of the ubiquitin proteasome system and is responsible for the turnover of approximately 80% of proteins in cells. To investigate whether the effect of UBE2K knockdown depends on the proliferation state of the cells, UBE2K knockdown Miapaca2 cells were grown in the presence of 5% serum (medium) or 0.5% serum and cell number was assessed 96 h after transfection. As expected, cell number was increased in 5% serum conditions compared to 0.5% serum, indicating that serum stimulates proliferation. Nevertheless, UBE2K knockdown reduced cell number to a similar extent in both serum conditions (64.1% in 0.5% serum and 64.3% in 5% serum; Figure 8), indicating that the effect of UBE2K knockdown is independent of the proliferation state.

[0177] UBE2K knockdown in Miapaca2 cells resulted in a 50% decrease in cell number. To understand whether this change in cell number was due to increased cell death or decreased cell proliferation, UBE2K siRNA-treated cells and non-targeting siRNA-treated cells were assessed using PI / Annexin V and PI cell cycle analysis, respectively. PI / Annexin V data showed that UBE2K knockdown resulted in 6–8% cell death 96 hours after siRNA transfection. Assessment of cell proliferation in UBE2K knockdown cells using PI cell cycle analysis (48 hours after transfection) revealed that UBE2K siRNA-transfected cells had 20% more cells in G2 / M phase compared to non-targeting siRNA-transfected cells (Figure 9). This increase in the G2 / M cell population corresponded to a decrease in the G1 cell population, suggesting that cells are arrested in G2 / M phase. Serum starvation was used as a positive control for cell cycle changes and, as expected, resulted in G1 arrest. Thus, siRNA-mediated knockdown of UBE2K resulted in a reduction in cancer cell numbers, a robust G2 / M cell cycle arrest, and a slight increase in apoptosis / necrosis.

[0178] To identify possible UBE2K-dependent substrates mechanistically related to the observed phenotypes, a curated list of UBE2K interactors was generated (Source: BioGrid) using available literature for UBE2K yeast two-hybrid screening / co-immunoprecipitation. Substrates were selected based on their interactions with other E2s and their role in cell proliferation / cell death (Table 4). Protein levels of three of the seven UBE2K interactors were found to be increased, two decreased, and two unchanged in response to UBE2K knockdown (Figure 9A and Figure 9B). Expression of these interactors was not observed to change at the transcript level, suggesting that regulation may occur post-translationally. [Table 4]

[0179] Example 3: siRNA-mediated knockdown of UBE2K in synchronized MiaPaca2 pancreatic cancer cells Accumulation of cyclin B1 in cells with siRNA-mediated knockdown of UBE2K coincides with G2 / M arrest, as degradation of cyclin B1 is required to exit M-phase; therefore, its levels remain elevated when arrest in G2 / M occurs. To further understand the mechanistic basis of the effect of UBE2K on cell cycle regulation, in particular, its role in cyclin B1 degradation, we followed the levels of various cell cycle regulatory proteins over time in synchronized MiaPaca2 cells with and without modulated expression levels of UBE2K. The experimental setup was as follows: (A) Synchronization of MiaPaCa2 cells in G0 / G1 phase by serum deprivation followed by transfection with siRNA for 48 h (B) Release of synchronized cells from serum deprivation by adding 2x FBS (20% in DMEM) medium for 4 days (C) Measurement of cell viability and levels of various cell cycle regulators including cyclin D1, E1, A2, B1 and Cdc20.

[0180] Cell cycle analysis comparing asynchronous populations of MiaPaca2 cells (grown in DMEM + 10% FBS) with cells serum starved for 48 h revealed that serum starvation synchronized approximately 80–85% of cells in G1 phase (Figure 10).

[0181] Similar to the observations in asynchronous MiaPaca2 cell populations, a reduction in viable cell numbers under siRNA-mediated UBE2K knockdown conditions was also detected in synchronous MiaPaCa2 populations: specifically, MiaPaCa-2 UBE2K knockdown cells had approximately 40% fewer viable cells than non-targeting (NT) siRNA controls 96 hours after release from synchronization (Figure S11).

[0182] Cyclin family proteins control the progression of cells through the cell cycle by activating cyclin-dependent kinases (Cdks). As cells progress through the different phases of the cell cycle, cyclin levels drop sharply after each checkpoint (G1 / S and G2 / M) as cyclins are degraded by cytoplasmic enzymes. A diagram representing the expression levels of human cyclins through the different phases of the cell cycle is shown in Figure 12. Cyclin B1, which has been shown to interact with UBE2K, is a regulatory protein involved in mitosis. Cyclin B1 accumulates throughout the cell cycle process and is activated during mitosis, but it is important that it is degraded at the end of mitosis for the cell cycle to progress. Cdc20 is a highly conserved activator of the anaphase-promoting complex / cyclosome (APC / C) that promotes cell cycle-regulated ubiquitination and proteolysis of many important cell cycle regulatory targets, including mitotic cyclins (A and B) and securin.

[0183] Immunoblotting was performed with samples obtained from cell synchronization and release to check the levels of various cell cycle regulators. Western blot results confirmed the knockdown of UBE2K by siRNA transfection after 48 hours. Cyclin E1 levels at 0 hours indicated that cells were arrested in G1 phase. Cyclin D1 and E1 levels at 0, 24, and 36 hours demonstrated that cells cycled through G1 and S phases unperturbed by UBE2K knockdown. In contrast, cyclin A2, cyclin B1, and Cdc20 protein levels accumulated to a similar extent, but their levels were slower to return to baseline in UBE2K siRNA-treated cells, suggesting impaired degradation. These observations, together with the reduced number of viable cells after release, indicate that loss of UBE2K in MiaPaca2 cells disrupts the metaphase-to-anaphase transition, leading to G2 / M arrest in cells (Figure 13).

[0184] Example 4: Comparison of the effects of UBE2K knockdown and Cdc34 knockdown on the viability of MiaPaCa2 pancreatic cancer cells UBE2K is known to preferentially catalyze the formation of Lys48-linked polyubiquitin chains on its substrates, leading to proteasomal degradation. Cdc34 (Ube2R1), another Lys-48 chain assembly E2 enzyme in the ubiquitin proteasome system, catalyzes the ubiquitin-mediated degradation of cell cycle G1 regulators and regulates tumor suppressors in multiple cancer types. The above results show that knocking down UBE2K using targeted siRNA in MiaPaca2 cells reduced viable cell numbers as a result of robust G2 / M cell cycle arrest. Because UBE2K is one of approximately 50 E2s found in the ubiquitin system, it is important to assess UBE2K specificity in cells. To determine whether the above phenotypes are specific to the function of UBE2K, the functionally similar E2 enzyme cdc34 was transiently knocked down in MiaPaca2 cells and compared with the effect of UBE2K knockdown. Knockdown was confirmed at the protein level (Figure 14). As expected, UBE2K knockdown reduced cell viability by approximately 20% 72 hours after transfection when compared to NT siRNA-treated MiaPaca2 cells. In contrast, Cdc34 knockdown had minimal effect on MiaPaca2 cell viability (Figure S15), supporting a unique role for UBE2K in this cell type.

[0185] Example 5: Stable sh-RNA-mediated knockdown of UBE2K in MiaPaca2 pancreatic cells For in vivo proof-of-concept studies, MiaPaca2 cell lines with stable UBE2K knockdown were generated. To this end, Miapaca2 cells were transduced with lentiviruses containing shRNAs targeting UBE2K or non-targeting controls. Cell lines were generated as a mixed population of cells ("pool of clones"). Efficient knockdown of UBE2K was verified at the mRNA (data not shown) and protein levels (Figure 16). Furthermore, stable knockdown of UBE2K (shRNA2 and shRNA3) increased the protein levels of cell cycle regulators cyclin A2, cyclin B1, and cdc20 (Figure 16). UBE2K shRNA also induced a slower growth phenotype, as indicated by lower cell or nuclei numbers in UBE2K stable knockdown cells compared to non-targeting shRNA-transduced cells at 72 hours post-inoculation (Figures 17 and 18). Taken together, these data demonstrate that key findings observed using siRNA-mediated knockdown of UBE2K are recapitulated in UBE2K stable knockdown cells.

[0186] Example 6: Expression of UBE2K in human pancreatic tumors Evaluation of UBE2K expression in human pancreatic tumors was performed using two datasets, one from an external publicly available dataset and the other from a commercial tumor tissue array process. Tumor expression of UBE2K (mRNA) and pancreatic ductal adenocarcinoma patient survival data were obtained from the publicly available TCGA database. Results showed a correlation between decreased UBE2K expression and increased patient survival (Figure 19).

[0187] Immunohistochemical analysis of UBE2K expression (protein) was performed using a tissue microarray containing 75 pancreatic tissue samples and matched normal adjacent tissue (Figure 20). Stained tissue was scored by a pathologist and estimated as strong, moderate, or weak staining. Localization of staining was also recorded as membranous, cytoplasmic, or nuclear. Statistical analysis of the staining profile demonstrated that UBE2K staining was significantly stronger in tumor tissue compared to normal adjacent tissue (Table 4, p-value < 2e-16). Furthermore, membrane UBE2K staining was observed only in tumor tissue and not in normal adjacent tissue (Table 5, p-value < 2e-16). [Table 5]

[0188] Example 7: Effect of small molecule inhibitors of UBE2K in human cancer cell cultures Using the methods provided in Example 2 above, the effect of small molecule inhibitors of UBE2K on cell proliferation and cell death of the following human cancer cell lines is determined: choriocarcinoma (JAR), ovarian cancer (PA-1), T-cell leukemia (Jurkat clone E6.1), lymphoma (SR), embryonal carcinoma (NCCIT), DB, B-cell lymphoma (SU-DHL-6), osteosarcoma (MG63), skin cancer (DU4475), T-lymphoblastic leukemia (MOLT-4), chronic myeloid leukemia (K562 and KU812), anaplastic large cell lymphoma (SU-DHL-1), and colorectal adenocarcinoma (SW48). Small molecule inhibitors of UBE2K are expected to reduce cell proliferation and induce cell death in these human cancer cell lines.

[0189] Example 8: Efficacy of small molecule inhibitors of UBE2K in mouse models of human cancer The mouse models of cancer described in Example 7 are evaluated to determine the effect of small molecule UBE2K inhibitors on tumor development in vivo. For each mouse model, human cancer cells (1×10 7) are suspended in MATRIGEL® and injected into immunodeficient mice. Cancers are allowed to develop, on average, for at least 3 weeks before treatment begins. For tumor-forming cancers, treatment is not initiated unless palpable tumors are present. Mice are randomized into two groups as follows: i. Group 1 - no treatment ii. Group 2 - Treatment with small molecule inhibitors of UBE2K

[0190] Mice are observed for survival and secondary symptoms, and tumor growth is monitored by palpation of tumor-forming cancer. At the time of death, tumors are harvested from the mice, measured, weighed, and analyzed for the presence of tumor vasculature. Small molecule inhibitors of UBE2K are expected to increase survival of mice, reduce secondary symptoms, and reduce tumor size.

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Claims

1. 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a ubiquitin-conjugating enzyme E2K (UBE2K) inhibitor, thereby treating cancer in the subject.

2. A method for reducing cancer cell proliferation in a subject in need thereof, comprising administering to the subject a ubiquitin-conjugating enzyme E2K (UBE2K) inhibitor, thereby reducing cancer cell proliferation in the subject compared to a subject not administered the UBE2K inhibitor.

3. A method for inducing cancer cell death in a subject in need thereof, comprising administering to the subject a ubiquitin-conjugating enzyme E2K (UBE2K) inhibitor, thereby inducing cancer cell death in the subject.

4. The method of claim 3 , wherein the death of the cancer cells is induced by apoptosis.

5. The method of any one of claims 1 to 4, wherein the UBE2K inhibitor is a specific inhibitor of UBE2K.

6. The method of any one of claims 1 to 5, wherein the UBE2K inhibitor comprises a small molecule.

7. The method of any one of claims 1 to 5, wherein the UBE2K inhibitor comprises a nucleic acid inhibitor.

8. The method of claim 7 , wherein the nucleic acid inhibitor comprises an antisense nucleic acid molecule.

9. The method of claim 7 , wherein the nucleic acid inhibitor comprises a double-stranded nucleic acid molecule.

10. 10. The method of claim 9, wherein the double-stranded nucleic acid molecule comprises a double-stranded RNA selected from the group consisting of siRNA, shRNA, and Dicer substrate siRNA (DsiRNA).

11. The method of any one of claims 1 to 5, wherein the UBE2K inhibitor comprises an antibody.

12. The method of any one of claims 1 to 11, wherein the cancer comprises a solid tumor.

13. 13. The method of claim 12, wherein the solid tumor is selected from the group consisting of carcinoma, melanoma, sarcoma, and lymphoma.

14. 13. The method of claim 12, wherein the solid tumor is selected from the group consisting of pancreatic cancer, liver cancer, colorectal cancer, and lymphoma.

15. The method of claim 12, wherein the solid tumor is pancreatic cancer or liver cancer.

16. The method of any one of claims 1 to 11, wherein the cancer is leukemia.

17. The method of any one of claims 1 to 16, wherein the UBE2K inhibitor is administered in combination with an additional agent.

18. 20. The method of claim 17, wherein the additional agent is an anti-cancer agent.

19. 20. The method of claim 17, wherein the additional agent is a chemotherapeutic agent.

20. 20. The method of claim 19, wherein the chemotherapeutic agent is selected from the group consisting of gemcitabine, 5-fluorouracil, leucovorin, docetaxel, fludarabine, cytarabine, cyclophosphamide, paclitaxel, docetaxel, busulfan, methotrexate, daunorubicin, doxorubicin, melphalan, cladribine, vincristine, vinblastine, chlorambucil, tamoxifen, taxol, camptothecin, actinomycin-D, mitomycin C, combretastatin, cisplatin, etoposide, verapamil, podophyllotoxin, and 5-fluorouracil.

21. The method of claim 17 , wherein the additional agent is an anti-angiogenic agent.

22. 20. The method of claim 17, wherein the additional agent is an immunotherapeutic agent.

23. 23. The method of claim 22, wherein the immunotherapeutic agent is an immune checkpoint modulator of an immune checkpoint molecule.

24. 24. The method of claim 23, wherein the immune checkpoint molecule is selected from CD27, CD28, CD40, CD122, OX40, GITR, ICOS, 4-1BB, ADORA2A, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, and VISTA.

25. 24. The method of claim 23, wherein the immune checkpoint molecule is a stimulatory immune checkpoint molecule and the immune checkpoint modulator is an agonist of the stimulatory immune checkpoint molecule.

26. 24. The method of claim 23, wherein said immune checkpoint molecule is an inhibitory immune checkpoint molecule and said immune checkpoint modulator is an antagonist of said inhibitory immune checkpoint molecule.

27. 27. The method of any one of claims 23 to 26, wherein the immune checkpoint modulator is selected from a small molecule, an inhibitory RNA, an antisense molecule, and an immune checkpoint molecule binding protein.

28. 24. The method of claim 23, wherein said immune checkpoint molecule is PD-1 and said immune checkpoint modulator is a PD-1 inhibitor.

29. 29. The method of claim 28, wherein the PD-1 inhibitor is selected from pembrolizumab, nivolumab, pidilizumab, SHR-1210, MEDI0680R01, BBg-A317, TSR-042, REGN2810, and PF-06801591.

30. 24. The method of claim 23, wherein said immune checkpoint molecule is PD-L1 and said immune checkpoint modulator is a PD-L1 inhibitor.

31. 31. The method of claim 30, wherein the PD-L1 inhibitor is selected from durvalumab, atezolizumab, avelumab, MDX-1105, AMP-224, and LY3300054.

32. 24. The method of claim 23, wherein said immune checkpoint molecule is CTLA-4 and said immune checkpoint modulator is a CTLA-4 inhibitor.

33. 33. The method of claim 32, wherein the CTLA-4 inhibitor is selected from ipilimumab, tremelimumab, JMW-3B3, and AGEN1884.

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