Pharmacological suppression of OTUD4 and CD73 binding, methods of treating cancer, anticancer agents, and therapeutic compositions

Abstract

Disclosed herein are pharmaceutical agents that inhibit or suppress OTUD4 and CD73 binding interactions; thus, useful in the context of managing diseases and conditions associated with OTUD4 and CD73 binding interactions, such as cancer treatments. In certain embodiments, this disclosure relates to method of treating cancer comprising administering an effective amount of a pharmaceutical agent that suppress OTUD4 and CD73 binding interactions. In certain embodiments, the therapeutic agent is a small molecule, antibody, or other specific binding agent.

Description

[0001] PHARMACOLOGICAL SUPPRESSION OF OTUD4 AND CD73 BINDING, METHODS

[0002] OF TREATING CANCER, ANTICANCER AGENTS, AND THERAPEUTIC

[0003] COMPOSITIONS

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 585,613 filed September 27, 2023. The entirety of this application is hereby incorporated by reference for all purposes.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with government support under CA258857 and GM139297 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0008] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

[0009] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 24012PCT.xml. The XML file is 2,690 bytes, was created on September 27, 2024, and is being submitted electronically via the USPTO patent electronic filing system.

[0010] BACKGROUND

[0011] Triple negative breast cancers (TNBCs) are an aggressive mammary carcinoma subtype and have few effective targeted therapies. PD-L1 inhibitors demonstrate promising clinical benefits in certain subsets of patients with TNBC. However, the intricacies of the tumor microenvironment, with its inherent immunosuppressive properties and varied immunogenicity profiles, limit the broad efficacy of such therapies. Thus, there is a need to identify improvements.

[0012] Zhao et al. report noncanonical regulation of alkylation damage resistance by the OTUD4 deubiquitinase. The EMBO Journal, 2015, 34(12): 1687-1703.

[0013] Zhao Y, et al. report OTUD4 is a phospho-activated K63 deubiquitinase that regulates MyD88-dependent signaling. Mol Cell, 2018, 69(3): 505-516. Allard et al. report targeting the CD73-adenosine axis in immuno-oncology. Immunol Lett, 2019, 205:31-39.

[0014] Jaynes et al. report OTUD4 enhances TGFP signalling through regulation of the TGF receptor complex. Sci Rep, 2020, 10(1): 15725.

[0015] Du et al. report the function and regulation of OTU deubiquitinases. Front Med, 2020,14(5):542-563.

[0016] Fu et al. report proteolytic regulation of CD73 by TRIM21 orchestrates tumor immunogenicity. Sci Adv, 2023, 9(l):eadd6626.

[0017] References cited herein are not an admission of prior art.

[0018] SUMMARY

[0019] Disclosed herein are pharmaceutical agents that inhibit or suppress OTUD4 and CD73 binding interactions; thus, useful in the context of managing diseases and conditions associated with OTUD4 and CD73 binding interactions, such as cancer. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering to a subject in need thereof an effective amount of a pharmaceutical agent that suppress OTUD4 and CD73 binding interactions. In certain embodiments, the therapeutic agent is a small molecule, antibody, or other specific binding agent.

[0020] In certain embodiments, the compounds are derivatives of 2-(5,7-diphenyl-4,5,6,7- tetrahydro-[l,2,4]triazolo[l,5-a]pyrimidin-2-yl)-3a,4,7,7a-tetrahydro-lH-isoindole-l,3(2H)- dione, prodrugs, esters, or salts thereof. In certain embodiments, the compound is (3aR,7aS)-2- ((5S,7R)-7-(4-fhiorophenyl)-5-(p-tolyl)-4,5,6,7-tetrahydro-[l,2,4]triazolo[l,5-a]pyrimidin-2-yl)- 3a,4,7,7a-tetrahydro-lH-isoindole-l,3(2H)-dione (ST80 / ZINC9345994), prodrugs, esters, or salts thereof. In certain embodiments, the compound is 2,2'-(pyrimidine-2,4-diylbis(piperidine-l,3- diyl))bis(lH-benzo[d]imidazole) (Z22 / ZINC 1336656227), derivative, prodrug, ester, or salt thereof.

[0021] In certain embodiments, the agent pharmaceutical agent that suppress OTUD4 and CD73 binding interactions is a small molecule compound, peptide, or antibody that specifically binds OTUD4 and CD73 bindings cites. In certain embodiments, the agent pharmaceutical agent specifically binds an amino acid residue within positions 275 to 311 in CD73 VPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLN (SEQ ID NO: 1) In certain embodiments, the agent pharmaceutical agent specifically binds an amino acid residue within position 380 to 410 in OTUD4, RLQHPSGVRQHAFSSHSSGSQSQKFSSEHKN (SEQ ID NO: 2).

[0022] In certain embodiments, this disclosure relates to pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound that suppress OTUD4 and CD73 binding interactions, derivatives, prodrugs, esters, or salts thereof.

[0023] In certain embodiments, this disclosure relates to methods of treating or preventing diseases and conditions associated with OTUD4 and CD73 interactions, such as cancer by administering an effective amount of a compound or pharmaceutical agent as disclosed herein to a subject in need thereof.

[0024] In certain embodiments, this disclosure relates to methods of treating or preventing any cancer, breast, lung, colon, or ovarian cancer comprising administering an effective amount of a compound or pharmaceutical agent as disclosed herein to a subject in need thereof. In certain embodiments, the subject is diagnosed with breast cancer, triple negative breast cancer, lung cancer, colon cancer, or other cancer.

[0025] In certain embodiments, this disclosure relates to the production of a medicament for use in the management of diseases and conditions associated with OTUD4 and CD73 binding interactions, such as cancer.

[0026] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0027] Figures 1A-1F show data used in the identification of OTUD4 as a key driver for CD73 accumulation in immune-suppressive TNBC breast tumors.

[0028] Figure 1A shows the MFI of membrane-expressed CD73 that was determined by flow cytometry in both MDA-MB231 and MDA-MB231 -OTUD4 breast cancer cells.

[0029] Figure IB shows data for adenosine levels that were determined in MDA-MB231, MDA- MB468, 4T1, and EO771 breast cancer cells with OTUD4 overexpression.

[0030] Figure 1C shows data for adenosine levels that were determined in MDA-MB231, MDA- MB468, 4T1, and EO771 breast cancer cells with ShOTUD4 overexpression.

[0031] Figure ID shows data for adenosine levels in MDA-MB468-CD73WT, MDA-MB468- CD73WT and OTUD4, MDA-MB468-CD73 and WT-ShOTUD4 cells. Figure IE shows data for MDA-MB468 and MDA-MB468-ShOTUD4 cells that were cocultured with human PBMCs with or without APCP treatment, and CD8+ IFN-y+ T cell populations measured and quantified using flow cytometry (ShCon, control shRNA).

[0032] Figure IF shows data for MDA-MB468-OTUD4-OE cells that were cocultured with human PBMCs with or without APCP treatment, and CD8+ IFN-y+ T cell population was measured and quantified using flow cytometry.

[0033] Figure 2A shows data where MDA-MB231 breast cancer cells were cocultured with human PBMCs at different Effector (E) to Target (T) ratios (2: 1 or 4: 1) and treated with ST80 and Z22 (0.5 pM). Cell proliferation rates were determined.

[0034] Figure 2B shows data where MDA-MB231 breast cancer cells were treated with various concentrations of ST80.

[0035] Figure 2C shows data where MDA-MB231 breast cancer cells were treated with Z22.

[0036] Figure 2D shows data where MDA-MB231 breast cancer cells were cocultured with human PBMCs and treated with 0.5 pM ST80 or Z22. Percentage of IFN-y+ in CD8+ T cells were measured and quantified using flow cytometry.

[0037] Figure 2E shows data on the percentage of GzmB+ in CD8+ T cells.

[0038] Figure 2F shows data where HCC827 lung cancer cells, SKOV3 ovarian cancer cells and HCT116 colon cancer cells were cocultured with human PBMCs at different Effector (E) to target (T) ratio (3: 1) and treated with ST80 (0.5pM), cell proliferation rate was measured by % confluence.

[0039] Figure 3A shows a schematic diagram of experiments, where basal mouse PD-L1 was replaced with a human counterpart, and 4Tl-hPD-Ll or 4Tl-hPDLl-OTUD4 cells (4T1 cells are stage IV human breast cancer cells) were orthotopically injected into the right fourth mammary fat pad and allow to grow to around 100 mm3, followed by injection of ST80 (5 mg / kg, i.p.) 2 times / week, and PD-L1 antibody durvalumab was administered (5 mg / kg, i.p.) 3 times. PBS and IgG were used in control groups.

[0040] Figure 3B shows data on tumor size over time for 4Tl-hPD-Ll.

[0041] Figure 3C shows data on tumor size over time for 4Tl-hPDLl-OTUD4.

[0042] Figure 3D shows data on tumor sized over time for EO771 comparing the effects of ST80 treatment with anti-CD73 antibody treatment in C57BL / 6 mice. DETAILED DISCUSSION

[0043] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. An “embodiment” refers to an example, and the claims are not necessarily limited to such example. The scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0045] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0046] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0047] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0048] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0049] As used herein, the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In certain embodiments, about means within a standard deviation using measurements generally acceptable in the art. In certain embodiments, about means a range extending to + / - a percentage of the specified value and includes the specified value. In certain embodiments, the term “about” can include a 5 % or 10 % difference.

[0050] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0051] "Consisting essentially of' or "consists of' or the like, have the meaning ascribed to them in U.S. Patent law in that when applied to methods and compositions encompassed by the present disclosure refers to the idea of excluding certain prior art element(s) as an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

[0052] The term “comprising” in reference to a peptide having an amino acid sequence refers a peptide that may contain additional N-terminal (amine end) or C-terminal (carboxylic acid end) amino acids, i.e., the term is intended to include the amino acid sequence within a larger peptide. The term “consisting of’ in reference to a peptide having an amino acid sequence refers to a peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids expressly specified in the claim. In certain embodiments, the disclosure contemplates that the “N-terminus of a peptide consists of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids specified in the claim however the C-terminus may be connected to additional amino acids, e.g., as part of a larger peptide. Similarly, the disclosure contemplates that the “C-terminus of a peptide consists of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim however the N-terminus may be connected to additional amino acids, e.g., as part of a larger peptide. Method of use

[0053] In certain embodiments, this disclosure relates to methods of treating or preventing cancer comprising administering to a subject in need thereof an effective amount of a pharmaceutical agent that suppress 0TUD4 and CD73 binding interactions. In certain embodiments, the therapeutic agent is a small molecule, antibody, or other specific binding agent.

[0054] A "subject" refers any animal, preferably a human patient, livestock, or domestic pet.

[0055] The term "effective amount" refers to that amount of a compound or pharmaceutical agent described herein that is sufficient to effect the intended application including, but not limited to, disease treatment as illustrated below. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose will vary depending on, for example, the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0056] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g. patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression.

[0057] The terms "co-administration" or "administered in combination with," and their grammatical equivalents, as used herein, encompass administration of two or more agents to a subject so that both agents and / or their metabolites are present in the subject at the same time. Coadministration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0058] In certain embodiments, the small molecule compound is 2-(5,7-diphenyl-4,5,6,7- tetrahydro-[l,2,4]triazolo[l,5-a]pyrimidin-2-yl)-3a,4,7,7a-tetrahydro-lH-isoindole-l,3(2H)- dione, derivatives, prodrugs, esters, or salts thereof. In certain embodiments, the compound is (3aR,7aS)-2-((5S,7R)-7-(4-fluorophenyl)-5-(p-tolyl)-4,5,6,7-tetrahydro-[l,2,4]triazolo[l,5- a]pyrimidin-2-yl)-3a,4,7,7a-tetrahydro-lH-isoindole-l,3(2H)-dione (ST80 / ZINC9345994), prodrugs, esters, or salts thereof. In certain embodiments, the compound is 2,2'-(pyrimidine-2,4- diylbis(piperidine-l,3-diyl))bis(lH-benzo[d]imidazole) (Z22 / ZINC 1336656227), derivative, prodrug, ester, or salt thereof.

[0059] In certain embodiments, the agent pharmaceutical agent that suppress 0TUD4 and CD73 binding interactions is a small molecule compound, peptide, or antibody that specifically binds 0TUD4 and CD73 bindings cites. In certain embodiments, the agent pharmaceutical agent specifically binds an amino acid residue within positions 275 to 311 in CD73 VPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLN (SEQ ID NO: 1). In certain embodiments, the agent pharmaceutical agent specifically binds an amino acid residue within position 380 to 410 in OTUD4. RLQHPSGVRQHAFSSHSSGSQSQKFSSEHKN (SEQ ID NO: 2).

[0060] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a compound or pharmaceutical agent disclosed herein to a subject in need thereof. In certain embodiments, cancer is breast cancer. In certain embodiments, cancer is triple negative breast cancer. In certain embodiments, cancer is lung cancer. In certain embodiments, cancer is ovarian cancer or colon cancer.

[0061] "Cancer" refers any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or others.

[0062] The cancer to be treated in the context of the present disclosure may be any type of cancer or tumor. These tumors or cancer include, and are not limited to, tumors of the hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies, tumors that affect the blood, bone marrow, lymph, and lymphatic system. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells; the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias, and myeloma are from the lymphoid line, while acute and chronic myelogenous leukemia, myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

[0063] Also contemplated are malignancies located in the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax and genito-urinary apparatus and, more particularly, childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular cancer, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant tumors, anal cancer, astrocytoma, cancer of the biliary tract, cancer of the bladder, bone cancer, brain stem glioma, brain tumors, breast cancer, cancer of the renal pelvis and ureter, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cancer of the cervix, childhood (primary) hepatocellular cancer, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumors, childhood Hodgkin's disease, childhood Hodgkin's lymphoma, childhood visual pathway and hypothalamic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood supratentorial primitive neuroectodermal and pineal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, cancer of the colon, cutaneous T-cell lymphoma, endocrine pancreatic islet cells carcinoma, endometrial cancer, ependymoma, epithelial cancer, cancer of the esophagus, Ewing's sarcoma and related tumors, cancer of the exocrine pancreas, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic biliary tract cancer, cancer of the eye, breast cancer in women, Gaucher's disease, cancer of the gallbladder, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, tricoleukemia, head and neck cancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, Kaposi's sarcoma, cancer of kidney, cancer of the larynx, cancer of the lip and mouth, cancer of the liver, cancer of the lung, lymphoproliferative disorders, macroglobulinemia, breast cancer in men, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, occult primary metastatic squamous neck cancer, primary metastatic squamous neck cancer, metastatic squamous neck cancer, multiple myeloma, multiple myeloma / plasmatic cell neoplasia, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, paranasal sinus and nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, nonmelanoma skin cancer, non-small cell lung cancer, metastatic squamous neck cancer with occult primary, buccopharyngeal cancer, malignant fibrous histiocytoma, malignant fibrous osteosarcoma / histiocytoma of the bone, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancer of the penis, phaeochromocytoma, hypophysis tumor, neoplasia of plasmatic cells / multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell cancer, cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, cancer of the salivary glands, sarcoidosis, sarcomas, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, pineal and supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional renal pelvis and ureter cancer, trophoblastic tumors, cell cancer of the renal pelvis and ureter, cancer of the urethra, cancer of the uterus, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, cancer of the vulva, Waldenstrom's macroglobulinemia, Wilms' tumor and any other hyperproliferative disease, as well as neoplasia, located in the system of a previously mentioned organ.

[0064] In certain embodiments, methods of treatment include administering a pharmaceutical agent that suppress 0TUD4 and CD73 binding interactions is a small molecule compound, peptide, or antibody that specifically binds 0TUD4 and CD73 bindings cites in combination with another anticancer agent. In certain embodiments, the anticancer agent is a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is anti-PD-1, anti-PD-Ll, or anti-CTLA4 antibody or combinations thereof. In certain embodiments, the antibody is durvalumab, atezolizumab, avelumab, ipilimumab, tremelimumab, nivolumab, pembrolizumab, cemiplimab, or combinations thereof. In certain embodiments, anticancer agent is an anti-CD73 antibody. In certain embodiments, the anticancer agent anticancer agent atezolizumab or avelumab

[0065] In certain embodiments, agents disclosed herein may be administered in combination with another pharmaceutical agent or anticancer agent. A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent,” or the like, refer to molecules that are recognized to aid in the treatment of a cancer. Contemplated examples include the following molecules or derivatives such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa- 2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5 -fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MVAC).

[0066] In certain embodiments, the chemotherapy agent is an anti-PD-1, anti-PD-Ll anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e g., nivolumab, pembrolizumab, cemiplimab) and anti-PD-Ll (e g., atezolizumab, avelumab, durvalumab).

[0067] In certain embodiments, the method of administration is in a subject with a lymphodepleted environment due to prior or concurrent administration of lymphodepl eting agents. In certain embodiments, lymphodepleting agents (e.g., cyclophosphamide and fludarabine).

[0068] In certain embodiments, the method of administration is in combination with a therapeutic antibody such as, antibodies selected from the group consisting of abagovomab, abciximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, afutuzumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, andecaliximab, anetumab, anifrolumab, anrukinzumab, apolizumab, aprutumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atlizumab, atorolimumab, avelumab, azintuxizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belantamab, belimumab, bemarituzumab, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bersanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, birtamimab, bivatuzumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab, briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, camidanlumab, camrelizumab, canakinumab, cantuzumab, caplacizumab, capromab, carlumab, carotuximab, catumaxomab, cedelizumab, cemiplimab, cergutuzumab, certolizumab, cetrelimab, cetuximab, cibisatamab, citatuzumab, cixutumumab, clazakizumab, clenoliximab, clivatuzumab, codrituzumab, cofetuzumab, coltuximab, conatumumab, concizumab, cosfroviximab, crenezumab, crizanlizumab, crotedumab, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab, daratumumab, dectrekumab, demcizumab, denintuzumab, denosumab, depatuxizumab, derlotuximab, detumomab, dezamizumab, dinutuximab, diridavumab, domagrozumab, dorlimomab, drozitumab, duligotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab, enavatuzumab, enfortumab, enlimomab, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faricimab, farletuzumab, fasinumab, felvizumab fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frunevetmab, fulranumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gemtuzumab, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab, golimumab, gomiliximab, gosuranemab, guselkumab, ianalumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, ifabotuzumab, igovomab, iladatuzumab, imalumab, imaprelimab, imciromab, imgatuzumab, inclacumab, indatuximab, indusatumab, inebilizumab, inflectra, infliximab, intetumumab, inolimomab, inotuzumab, ipilimumab, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacnotuzumab, ladiratuzumab, lampalizumab, lanadelumab, landogrozumab, laprituximab, larcaviximab, lebrikizumab, lemalesomab, lendalizumab, lenvervimab, lenzilumab, lerdelimumab, leronlimab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifastuzumab, ligelizumab, loncastuximab, losatuxizumab, lilotomab, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab, lucatumumab, lulizumab, lumiliximab, lumretuzumab, lupartumab, lutikizumab, mapatumumab, margetuximab, marstacimab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirikizumab, mirvetuximab, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab, nacolomab, namilumab, naptumomab, naratuximab, narnatumab, natalizumab, navicixizumab, navivumab, naxitamab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, oleclumab, olendalizumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab, oregovomab, orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab, pintumomab, placulumab, plozalizumab, pogalizumab, polatuzumab, ponezumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab, raxibacumab, ravagalimab, ravulizumab, refanezumab, regavirumab, remtolumab, reslizumab, rilotumumab, rinucumab, risankizumab, rituximab, rivabazumab, robatumumab, roledumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab, rovelizumab, rozanolixizumab, ruplizumab, sacituzumab, samalizumab, samrotamab, sapelizumab, sarilumab, satralizumab, satumomab, secukinumab, selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirtratumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab, tadocizumab, talacotuzumab, talizumab, tamtuvetmab, tanezumab, taplitumomab, tarextumab, tavolimab, tefibazumab, telimomab, telisotuzumab, tenatumomab, teneliximab, teplizumab, tepoditamab, teprotumumab, tesidolumab, tetulomab, tezepelumab, tibulizumab, tildrakizumab, tigatuzumab, timigutuzumab, timolumab, tiragotumab, tislelizumab, tisotumab, tocilizumab, tomuzotuximab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, tregalizumab, tremelimumab, trevogrumab, tucotuzumab, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vanalimab, vandortuzumab, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, volociximab, vonlerolizumab, vopratelimab, vorsetuzumab, votumumab, vunakizumab, xentuzumab, zalutumumab, zanolimumab, zenocutuzumab, ziralimumab, zolbetuximab, and zolimomab.

[0069] In certain embodiments, this disclosure relates to methods of screening a test compound for the ability to suppress OTUD4 and CD73 binding interactions comprising contacting OTUD4 and CD73 or fragments thereof as disclosed herein optionally conjugated to a label and a test compound and detecting whether the test compound suppresses 0TUD4 and CD73 binding interactions. Detecting whether the test compound suppresses 0TUD4 and CD73 binding interactions can be accomplished in multiple ways known in art.

[0070] In certain embodiments, whether the test compound suppresses 0TUD4 and CD73 binding or non-binding interactions is recorded on a non-transitory computer readable medium, e.g. in the form of a detection value or non-detection value, measured or quantitative value.

[0071] Binding assays typically include quantitative / qualitative detection of binding partners as well as the resulting binding complexes. For this purpose, either the target biomolecule, ligands or both may be labelled. Numerous labelling / detection techniques may be used in binding assays including radioligand assays, affinity chromatography, surface plasmon resonance, isothermal titration calorimetry and light-based techniques using absorbance, fluorescence, or luminescence readouts. Commercially available kits utilize typically utilize light detection modes such as absorbance, fluorescence, luminescence, FRET (Fluorescence Resonance Energy Transfer), Fluorescence polarization (FP), TRF (time-resolved fluorescence), TR-FRET (time-resolved fluorescence energy transfer). Absorbance ELISA binding assays typically utilize antibody pairs specific for the binding partners, e.g., 0TUD4 and CD73 biomolecule. While one antibody is used to bind and antigen, e.g., 0TUD4 or CD73 to a surface, e.g., microtiter plate surface, the other antibody binds the other binding partner. The binding complex is typically detected by a colorimetric enzymatic reaction coupled to the second antibody, while any unbound binding partners in solution are optionally removed by washing step(s). In certain embodiments, identifying the suppression of 0TUD4 and CD73 binding interactions can be accomplished using in vitro immunofluorescence staining and proximity ligation assays as reported herein.

[0072] In certain embodiments, it is contemplated that 0TUD4 and / or CD73 or fragments are conjugated to a label e.g., a fluorophore and the other contains an alternative fluorophore or quencher. The fluorophores and / or quencher are attached within 0TUD4 and CD73 sequences so that so that they are within close proximity when bound together and fluorescence intensity is quenched or is heightened. In certain embodiments, when the test compound interruptus binding of 0TUD4 and CD73 to each other, the fluorophore physically separates from the quencher, allowing a fluorescence or other label signal to be emitted, e.g., upon excitation, the signal intensity changes e.g., is heightened or diminished. Thus, detection of a change in the fluorescent signal provides an indication that the test compound suppresses or interrupts or weakens the ability 0TUD4 and CD73 to bind together, and when the signal is generated. In certain embodiments, it is contemplated that a specific binding agent, e.g., a first labeled antibody binds 0TUD4 and a second labeled antibody CD73 wherein the antibodies bind outside the domains where 0TUD4 and CD73 bind to each other.

[0073] A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a "label receptor" refers to incorporation of a heterologous polypeptide in the receptor. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moi eties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as18F,35S or131I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0074] In certain embodiments, this disclosure relates to fusion protein containing 0TUD4 and / or CD73 or fragments thereof as disclosed In certain embodiments, this disclosure relates to nucleic acids e.g., DNA, RNA, or mRNA encoding 0TUD4 and / or CD73, or fragments or fusions thereof as disclosed herein. In certain embodiments, this disclosure relates to recombinant vector comprising a nucleic acid encoding 0TUD4 and / or CD73 or fragments thereof as disclosed herein in operable combination with heterologous promoter. In certain embodiments, this disclosure relates to cells (somatic cells) comprising nucleic acids or recombinant vectors encoding a 0TUD4 and / or CD73 or fragments thereof as disclosed herein. The term "specific binding agent" refers to a molecule, such as a proteinaceous molecule, that binds a target molecule with a greater affinity than other random molecules or proteins. Examples of specific binding agents include an antibody that bind an epitope of an antigen or a receptor which binds a ligand. In certain embodiments, "Specifically binds" refers to the ability of a specific binding agent (such as an ligand, receptor, enzyme, antibody or binding region / fragment thereof) to recognize and bind a target molecule or polypeptide, such that its affinity (as determined by, e.g., affinity ELISA or other assays) is at least 10 times as great, but optionally 50 times as great, 100, 250 or 500 times as great, or even at least 1000 times as great as the affinity of the same for any other or other random molecule or polypeptide.

[0075] In certain contexts, an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule, i.e., an antigen to the antibody. The immune system of the animal will create an antibody to specifically bind the antigen, and thereby targeting the antigen for degradation or elimination, or any cell or organism attached to the antigen. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody. Thus, the term “antibody” is intended to include natural antibodies, monoclonal antibody, or non-naturally produced synthetic antibodies, such as specific binding single chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications. The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

[0076] From a structural standpoint, an antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. The heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence. Similarly, the two light chains typically have the same amino acid sequence. Each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. The variable segments are often referred to as the antigen binding domains. The antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope." Epitopes usually consist of surface groupings of molecules, for example, amino acids or carbohydrates. The terms “variable region,” "antigen binding domain," and "antigen binding region" refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigenbinding domain that typically interact with the epitope are also commonly alternatively referred to as the "complementarity-determining regions, or CDRs."

[0077] With regard to variable chain immunoglobulins, the location of binding complementarity- determining regions (CDRs) sometimes varies depending on the specific sequence context and animal. The CDRs can be determined through epitope studies and sequence alignment comparisons of the constant and framework regions for the specific animal. As is well-known in the art that there are multiple conventions to define and describe the CDRs of a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability) and the Chothia definition (which is based on the location of the structural loop regions).

[0078] In general, identifying CDRs can be accomplished utilized the following rules using Kabat or Chothia antibody sequence criteria. Note that there are examples where these constant features do not occur; however, the Cys residues are the most common conserved feature.

[0079] For CDR-L1, the start residue is approximately 24 to 30 after the first amino acid and typically after a Cys. The residue after is typically a Trp such as Trp-Tyr-Gln, Trp-Leu-Gln, Trp- Phe-Gln, or Trp-Tyr-Leu. Length is typically 10 to 17 residues. CDR-L2 starts about 16 residues after the end of LI . The residues before are typically Ile-Tyr, Val-Tyr, Ile-Lys, Ile-Phe, with a length of about 4-7 residues. CDR-L3 starts at about 33 residues after end of L2 before a Cys residue with a length of about 7 to 11 residues typically ending before a Phe-Gly.

[0080] For CDR-H1, the start residue is approximately 26 to 30 after the first amino acid and typically 4 amino acids after a Cys and typically ends with Trp, e.g., Trp-Val, but also, Trp-Ile, Trp-Ala. The length is typically about 6 to 12 residues. CDR-H2 typically starts at about 4-15 residues after the end of CDR-H1. Residues before the start are typically Trp-Ile-Gly but can be a number of variations, and residues after typical ends with Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala- Thr / Ser / Ile / Ala. The length can vary from about 8 to 20 amino acids; CDR-H3 is typically about 30-33 residues after the end of CDR-H2, and often identified 3 amino acids after a Cys, such as in the example Cys-Ala-Arg. The end is sometimes identified before residues such as Trp-Gly. The length can vary widely, e.g., 4-25 or more depending on the animal.

[0081] A "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such that the entire molecule is not naturally occurring. Examples of chimeric antibodies include those having a variable region derived from a non-human antibody and a human immunoglobulin constant region. The term is also intended to include antibodies having a variable region derived from one human antibody grafted to an immunoglobulin constant region of a predetermined sequences or the constant region from another human for which there are allotypic differences residing in the constant regions of any naturally occurring antibody having the variable regions, e.g., CDRs 1, 2, and 3 of the light and heavy chain. Human heavy chain genes exhibit structural polymorphism (allotypes) that are inherited as a haplotype. The serologically defined allotypes differ within and between population groups. See Jefferis et al. mAb, 1 (2009), pp. 332-338. In certain embodiments, the antibody, antigen binding fragment, the light chain, or the heavy chain comprises a non-naturally occurring chimeric amino acid sequence such that there is at least one mutation that is not present in naturally occurring antibodies comprising one or all of the six CDRs.

[0082] The term "antibody fragment" refers to an antibody which comprises less than a complete, intact antibody. Complete antibodies comprise two functionally independent parts or fragments: an antigen binding fragment known as "Fab," and a carboxy terminal crystallizable fragment known as the "Fc" fragment. The Fab fragment includes the first constant domain from both the heavy and light chain (CHI and CL1) together with the variable regions from both the heavy and light chains that bind the specific antigen. Each of the heavy and light chain variable regions includes three complementarity determining regions (CDRs) and framework amino acid residues which separate the individual CDRs. The Fc region comprises the second and third heavy chain constant regions (CH2 and CH3) and is involved in effector functions such as complement activation and attack by phagocytic cells. In some antibodies, the Fc and Fab regions are separated by an antibody "hinge region," and depending on how the full-length antibody is proteolytically cleaved, the hinge region may be associated with either the Fab or Fc fragment. For example, cleavage of an antibody with the protease papain results in the hinge region being associated with the resulting Fc fragment, while cleavage with the protease pepsin provides a fragment wherein the hinge is associated with both Fab fragments simultaneously. Because the two Fab fragments are covalently linked following pepsin cleavage, the resulting fragment is termed the F(ab')2 fragment.

[0083] The term, "humanized" refers to an antibody containing one or more amino acid mutations so that immunogenicity upon administration in human patients, e.g., due to "pre-existing antibodies", is reduced, made highly unlikely, or nonexistent. Anaphylaxis is a severe allergic reaction to an allergen, e.g., polypeptide. Non-human proteins contain amino acid residues that may be immunogenic when targeted by preexisting antibodies circulating in a human patient. Thus, it is desirable to mutate residues within a therapeutic antibody so that the peptide sequences are similar to peptide sequences that commonly occurs in human antibodies / proteins, provided that the desirable therapeutic properties are retained, thereby reducing the risk of undesirable allergic reactions. In antibodies, this is typically accomplished by transfer of complementarity-determining regions (CDRs) of a non-human antibody to a human framework sequence, yielding a human like antibody with reduced immunogenicity. Another method entails comparing sequences, preferably framework sequences, and identifying amino acid substitutions providing "humanized" sequences frequently found within human antibody sequence repertoire. These humanized sequences reduce the risk of undesirable immune reactions providing an antibody that is substantially non- immunogenic in humans and retain the affinity and activity of the original polypeptide. In one example humanization, e.g., framework region humanization, one screens the available human consensus sequences for existing known sequences that are most similar to the original sequence. Using computer databases to compare human consensus frameworks for humanizing antibodies is well-known.

[0084] "Immunoglobulin single variable domains" are antibodies whose complementarity determining regions (CDRs) are part of a single domain polypeptide such as antibodies naturally devoid of light chains, single domain antibodies may be derived from conventional 4-chain antibodies. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule are commonly derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca, and guanaco. Pharmaceutical agents for suppression of OTUD4 and CD73 interactions

[0085] In certain embodiments, this disclosure relates to compounds and other specific binding agent such as antibodies that suppress binding interactions of 0TUD4 with CD73.

[0086] In certain embodiments, the compounds are derivatives of 2-(5,7-diphenyl-4,5,6,7- tetrahydro-[l,2,4]triazolo[l,5-a]pyrimidin-2-yl)-3a,4,7,7a-tetrahydro-lH-isoindole-l,3(2H)- dione, prodrugs, esters, or salts thereof. In certain embodiments, the compound is (3aR,7aS)-2- ((5S,7R)-7-(4-fluorophenyl)-5-(p-tolyl)-4,5,6,7-tetrahydro-[l,2,4]triazolo[l,5-a]pyrimidin-2-yl)- 3a,4,7,7a-tetrahydro-lH-isoindole-l,3(2H)-dione (ST80 / ZINC9345994), prodrugs, esters, or salts thereof.

[0087] In certain embodiments, the compound has the following formula I:

[0088] Formula I or salt thereof wherein

[0089] R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, or R15are each, the same or different, hydrogen, alkyl, halogenated alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, glycol, alkylthio, alkylamino, (alkyl)2amino, alkyl sulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, or R13are optionally substituted with one or more, the same or different, R16; R16is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R16is optionally substituted with one or more, the same or different, R17;

[0090] R17is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R17is optionally substituted with one or more, the same or different, R18; and

[0091] R18is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N- ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methyl sulfinyl, ethyl sulfinyl, mesyl, ethyl sulfonyl, methoxy carbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0092] In certain embodiments, R1is a halogen.

[0093] In certain embodiments, R1is a fluoro.

[0094] In certain embodiments, R8is alkyl.

[0095] In certain embodiments, R8is methyl.

[0096] In certain embodiments, R11is hydrogen.

[0097] In certain embodiments, the compound has the following Formula II:

[0098]

[0099] Formula II or salt thereof wherein

[0100] R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, or R12are each, the same or different, hydrogen, alkyl, halogenated alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, glycol, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R1, R2, R3, R4, R\ R6, R7, R8, R9, R10, R11, or R12are optionally substituted with one or more, the same or different, R16; R16is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkyl sulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R16is optionally substituted with one or more, the same or different, R17;

[0101] R17is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R17is optionally substituted with one or more, the same or different, R18; and

[0102] R18is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, di methyl ami no, diethylamino, N-methyl-N- ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methyl sulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0103] For any element disclosed herein expressly, by chemical formula, by inference, or by a single letter designation, the element is contemplated to include that element in its natural abundance or versions accessible in enriched forms in excess of natural abundance using standard synthetic techniques. For example, “H” or “hydrogen” refers to the hydrogen element or versions accessible as enriched in excess of natural abundance for common isotopes thereof, e.g., deuterium,2H, or tritium3H. Similarly, enriched halogen isotopes are contemplated, e.g.,18F,19F, or76Br.

[0104] As used herein, “alkyl” means a noncyclic straight chain or branched, unsaturated or saturated hydrocarbon such as those containing from 1 to 10 carbon atoms, typically 1 to 6 carbon atoms. Within any embodiments, herein alkyl may refer to an alkyl with 1 to 6 carbons (Ci-ealkyl). Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an “alkenyl” or “alkynyl,” respectively). Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1 -pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2- butenyl, 2,3- dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3- methyl-1- butynyl, and the like.

[0105] Non-aromatic mono or polycyclic alkyls are referred to herein as "carbocycles" or "carbocyclyl" groups. Representative saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated carbocycles include cyclopentenyl and cyclohexenyl, and the like. "Heterocarbocycles" or heterocarbocyclyl" groups are carbocycles which contain from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur which may be saturated or unsaturated (but not aromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Heterocarbocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0106] "Aryl" means an aromatic carbocyclic monocyclic or polycyclic ring such as phenyl or naphthyl. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic.

[0107] As used herein, "heteroaryl" refers an aromatic heterocarbocycle having 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, and containing at least 1 carbon atom, including both mono- and polycyclic ring systems. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that the use of the term "heteroaryl" includes N-alkylated derivatives such as a 1-methylimidazol- 5-yl substituent.

[0108] As used herein, "heterocycle" or "heterocyclyl" refers to mono- and polycyclic ring systems having 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, and containing at least 1 carbon atom. The mono- and polycyclic ring systems may be aromatic, non-aromatic or mixtures of aromatic and non-aromatic rings. Heterocycle includes heterocarbocycles, heteroaryls, and the like.

[0109] "Alkylthio" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through a sulfur bridge. An example of an alkylthio is methylthio, (i.e., -S-CH3).

[0110] "Alkoxy" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, s-butoxy, t-butoxy. " Alkylamino" refers an alkyl group as defined above attached through an amino bridge. An example of an alkylamino is methylamino, (i.e., -NH-CH3).

[0111] "Alkanoyl" refers to an alkyl as defined above attached through a carbonyl bride (i.e., -(C=O)alkyl).

[0112] “Alkoxycarbonyl” refers to an alkyl as defined above attached through a carboxy bridge (i.e., -(C=O)OalkyL

[0113] “Alkylcarbamoyl” refers to an alkyl as defined above attached through a carbonyl bridge (i.e., -(C=O)NHalkyl).

[0114] “Alkanoyl” refers to an alkyl as defined above attached through a carbonyl bridge (i.e., -(C=O)alkyl).

[0115] "Alkylsulfonyl" refers to an alkyl as defined above attached through a sulfonyl bridge (i.e., -S(=O)2alkyl) such as mesyl and the like, and "Arylsulfonyl" refers to an aryl attached through a sulfonyl bridge (i.e., -S(=O)2aryl).

[0116] "Alkylsulfonamide" refers to an alkyl as defined above attached through a sulfamoyl bridge (i.e., -S(=O)2NHalkyl), and an "Aryl sulfonamide" refers to an alkyl attached through a sulfamoyl bridge (i.e., (i.e., - S(=O)2NHaryl).

[0117] "Alkylsulfinyl" refers to an alkyl as defined attached through a sulfinyl bridge (i.e. -S(=O)alkyl).

[0118] “Glycol” refers to an alkyl group substitute with terminal oxygen atoms, and the term is intended to include repeating glycol units, e.g., polyethylene glycol -O(CH2CH2O)nX, wherein X is typically an H, alkyl, alkoxy, halogenated alkoxy, or alkanoyl, and n is typically 1-50, 1-100, or 1-1,000.

[0119] The terms “halogen” and “halo” refer to fluorine, chlorine, bromine, and iodine.

[0120] The term “halogenated alkyl” refers to an alkyl partially or entirely substituted with halogens, e.g., CF3.

[0121] The term "substituted" refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are "substituents." The molecule may be multiply substituted. In the case of an oxo substituent ("=O"), two hydrogen atoms are replaced. Example substituents within this context may include halogen, hydroxy, alkyl, alkoxy, halogenated alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarb ocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaNRb, -NRaC(=O)ORb, - NRaSChRb, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -ORa, -SRa, -SORa, - S(=O)2Ra, -OS(=O)2Ra and -S(=O)2ORa. Ra and Rb in this context may be the same or different and independently hydrogen, halogen hydroxyl, alkyl, alkoxy, halogenated alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl.

[0122] The term "optionally substituted," as used herein, means that substitution is optional and therefore it is possible for the designated atom to be un substituted.

[0123] As used herein, "salts" refer to derivatives of the disclosed compounds where the parent compound is modified making acid or base salts thereof. Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkylamines, or dialkylamines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. In certain embodiment the salts are conventional nontoxic pharmaceutically acceptable salts including the quaternary ammonium salts of the parent compound formed, and non-toxic inorganic or organic acids. Contemplated salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

[0124] The term “prodrug” refers to an agent that is converted into a biologically active form in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like.

[0125] As used herein, the term “small molecule” refers to any variety of covalently bound molecules with a molecular weight of less than 1000 or 1500. Typically, the majority of atoms include carbon, hydrogen, oxygen, nitrogen, and to a lesser extent sulfur and / or a halogen. Examples include steroids, short peptides, mono or polycyclic aromatic or non-aromatic, heterocyclic compounds.

[0126] As used herein, the term “derivative” refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue. The derivative may be structurally similar because it is lacking one or more atoms, substituted, a salt, in different hydration / oxidation states, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing a hydrogen with a deuterium, oxygen atom with a sulfur atom or replacing an amino group with a hydroxyl group. The derivative may be a prodrug. Derivatives may be prepared by any variety of synthetic methods or appropriate adaptations presented in synthetic or organic chemistry textbooks, such as those provide in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference.

[0127] Pharmaceutical compositions

[0128] Disclosed herein are pharmaceutical agents that suppress OTUD4 and CD73 binding interactions; thus, useful in the context of managing diseases and conditions associated with OTUD4 and CD73 binding interactions, such as cancer.

[0129] In certain embodiments, the pharmaceutical composition is in the form of a pill, tablet, capsule, gel, gel capsule, powder, granule, or cream. In certain embodiments, the pharmaceutical composition is in the form of a pH buffered saline solution optionally comprising a saccharide or polysaccharide. In certain embodiments, the excipient is selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N- pyrrolidone, crospovidone, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, corn starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

[0130] Pharmaceutical compositions typically comprise an effective amount of compounds and a suitable pharmaceutical acceptable excipient or carrier. The preparations can be prepared in a manner known per se, which usually involves mixing the compounds according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. Pharmaceutically acceptable salts, solvates, and hydrates of the compounds listed are also useful in the method of the disclosure and in pharmaceutical compositions of the disclosure.

[0131] In certain embodiments, a composition comprising a pharmaceutical agent disclosed herein in enantiomeric excess, diastereomeric excess, or racemic mixture or salt thereof as reported herein of the present disclosure can be administered to a subject either alone or as a part of a pharmaceutical composition.

[0132] In certain embodiments, the isomer may be present in a composition with enantiomeric excess or diastereomeric excess of greater than 60%. In certain embodiments, the R isomer may be present in enantiomeric excess or diastereomeric excess of greater than 70%. In certain embodiments, the isomer may be present in enantiomeric excess or diastereomeric excess greater than 80%. In certain embodiments, the isomer may be present in enantiomeric excess, diastereomeric excess, of greater than 90%. In certain embodiments, the isomer may be present in enantiomeric excess diastereomeric excess of greater than 95%.

[0133] In certain embodiments, the pharmaceutical composition is in the form of a tablet, pill, capsule, powders, granules, gel, gel capsule, or cream. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or: (a) fdlers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia gum, (c) humectants, as for example, glycerol (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. Solid dosage forms can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. In certain embodiments, pharmaceutical composition is in solid form surrounded by an enteric coating. In certain embodiments, the enteric coating comprises methyl acrylate-methacrylic acid copolymers, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, or combinations thereof.

[0134] In certain embodiments, this disclosure contemplates an intravenous formulation with pH buffering agents and tonicity in a range representing physiological values (pH 7 to 8) or for bolus administration, e g., containing normal saline or dextrose optionally containing pH buffering agents. In certain embodiments, the pharmaceutical composition is in the form of a sterilized pH buffered aqueous salt solution or a saline phosphate buffer between a pH of 6 to 8, optionally comprising a saccharide or polysaccharide.

[0135] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable (such as olive oil, sesame oil) and injectable organic esters such as ethyl oleate.

[0136] These compositions may also contain preserving, emulsifying, and dispensing agents. Prevention of the action of microorganisms may be controlled by addition of any of various antibacterial and antifungal agents, example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0137] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, com germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.

[0138] The pharmaceutical compositions of the present disclosure can be administered to subjects either topically to the skin, orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), intraci stemally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments, or drops), or as a buccal or nasal spray.

[0139] In certain embodiments, the pharmaceutical compositions are in a form for inhalation. In certain embodiments, the pharmaceutical composition comprises a compound disclosed herein and a propellant. In certain embodiments, an aerosolizing propellant is compressed air, ethanol, nitrogen, carbon dioxide, nitrous oxide, hydrofluoroalkanes (HFAs), or combinations thereof.

[0140] In certain embodiments, the disclosure contemplates a pressurized or unpressurized container comprising a compound herein. In certain embodiments, the container is a manual pump spray, inhaler, meter-dosed inhaler, dry powder inhaler, nebulizer, vibrating mesh nebulizer, jet nebulizer, or ultrasonic wave nebulizer.

[0141] Pharmaceutical compositions typically comprise an effective amount of a composition or preparation that can be prepared in a manner known per se, which usually involves mixing the at least one compound according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. It is well known that ester prodrugs are readily degraded in the body to release the corresponding alcohol. See e.g., Imai, Drug Metab Pharmacokinet, 2006, 21(3): 173- 85, entitled “Human carboxylesterase isozymes: catalytic properties and rational drug design.

[0142] The pharmaceutical preparations of the disclosure are preferably in a unit dosage form, and can be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which can be properly labeled); optionally with one or more leaflets containing product information and / or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure e g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

[0143] In certain embodiments, production processes are contemplated which two components, compounds disclosed herein and a pharmaceutical carrier, are provided already in a combined dry form ready to be reconstituted together. In other embodiments, it is contemplated that compounds disclosed herein and a pharmaceutical carrier are admixed to provide a pharmaceutical composition.

[0144] Providing a pharmaceutic composition is possible in a one-step process, simply by adding a suitable pharmaceutically acceptable diluent to the composition in a container. In certain embodiments, the container is preferably a syringe for administering the reconstituted pharmaceutical composition after contact with the diluent. In certain embodiments, the coated compounds can be filled into a syringe, and the syringe can then be closed with the stopper. A diluent is used in an amount to achieve the desired end-concentration. The pharmaceutical composition may contain other useful component, such as ions, buffers, excipients, stabilizers, etc.

[0145] A "dry" pharmaceutical composition typically has only a residual content of moisture, which may approximately correspond to the moisture content of comparable commercial products, for example, has about 12% moisture as a dry product. Usually, the dry pharmaceutical composition according to the present invention has a residual moisture content preferably below 10% moisture, more preferred below 5% moisture, especially below 1% moisture. The pharmaceutical composition can also have lower moisture content, e g. 0.1% or even below. In certain embodiments, the pharmaceutical composition is provided in dry in order to prevent degradation and enable storage stability.

[0146] A container can be any container suitable for housing (and storing) pharmaceutically compositions such as syringes, vials, tubes, etc. The pharmaceutical composition may then preferably be applied via specific needles of the syringe or via suitable catheters. Atypical diluent comprises water for injection, and NaCl (preferably 50 to 150 mM, especially 110 mM), CaC12 (preferably 10 to 80 mM, especially 40 mM), sodium acetate (preferably 0 to 50 mM, especially 20 mM) and mannitol (preferably up to 10% w / w, especially 2% w / w). Preferably, the diluent can also include a buffer or buffer system so as to buffer the pH of the reconstituted dry composition, preferably at a pH of 6.2 to 7.9, especially at pH of 6.9 to 7.1. In certain embodiments, the diluent is provided in a separate container. This can preferably be a syringe. The diluent in the syringe can then easily be applied to the container for reconstitution of the dry compositions. If the container is also a syringe, both syringes can be finished together in a pack. It is therefore preferred to provide the dry compositions in a syringe, which is finished with a diluent syringe with a pharmaceutically acceptable diluent for reconstituting, said dry and stable composition.

[0147] In certain embodiments, this disclosure contemplates a kit comprising a pharmaceutical composition disclosed herein and a container with a suitable diluent. Further components of the kit may be instructions for use, administration means, such as syringes, catheters, brushes, etc. (if the compositions are not already provided in the administration means) or other components necessary for use in medical (surgical) practice, such as substitute needles or catheters, extra vials or further wound cover means. In certain embodiments, the kit comprises a syringe housing the dry and stable hemostatic composition and a syringe containing the diluent (or provided to take up the diluent from another diluent container).

[0148] Pharmacological suppression of the OTUD4 / CD73 proteolytic axis revives antitumor immunity against immune-suppressive breast cancers

[0149] Despite widespread utilization of immunotherapy, treating immune-cold tumors remains a challenge. Multiomic analyses and experimental validation identified the OTUD4 / CD73 proteolytic axis as a promising target in treating immune-suppressive triple negative breast cancer (TNBC). Although it is not intended that embodiments of this disclosure be limited by any particular mechanism, it is contemplated that deubiquitylation of CD73 by 0TUD4 counteracted its ubiquitylation by TRIM21, resulting in CD73 stabilization inhibiting tumor immune responses. TGF-P signaling is involved in the OTUD4 / CD73 proteolytic axis within tumor cells. Spatial transcriptomics profiling uncovered spatially resolved features of interacting malignant and immune cells pertaining to expression levels of 0TUD4 and CD73. In addition, the compound ST80 specifically disrupted proteolytic interaction between CD73 and 0TUD4, leading to reinvigoration of cytotoxic CD8+ T cell activities. In preclinical models of TNBC, ST80 treatment sensitized refractor tumors to anti-PD-Ll therapy. The experiments indicate that targeting the immunosuppressive OTUD4 / CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80. Poor clinical response by immune-suppressive tumors is a challenge in use of immune checkpoint inhibitors. Triple negative breast cancers (TNBCs) are the most aggressive mammary carcinoma subtype and have few effective targeted therapies. The intricacies of the tumor microenvironment limit certain therapies. Based on multiomic analyses for improving anticancer immune responses against TNBCs, targeting CD73 and 0TUD4 was identified as a promising avenue.

[0150] CD73, also known as Ecto-5 '-nucleotidase, is an enzyme expressed in a range of tissues and cell types. It is contemplated that CD73 acts together with CD39 in the conversion of extracellular ATP to provide immunosuppressive adenosine. This adenosinergic cascade leads to the suppression of cytotoxic T cells and the creation of an immune-suppressive tumor microenvironment. CD73 is implicated in advancing tumor progression by facilitating tumor angiogenesis and interacting with cancer-associated fibroblasts through adenosine receptors such as AIR, A2AR, A2BR, and A3R through multiple types of immune cells such as Tregs (Foxp3+) T cells, effector T cells, natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs), and macrophages, and B cells.

[0151] It is contemplated that potential posttranslational modifications (PTMs) of CD73 may have significance in regulating other immune checkpoint proteins, such as PD-L1 and B7-H4. 0TUD4 (OTU domain-containing protein 4) acts as a deubiquitinase that counteracts E3 ligase-mediated protein ubiquitylation, stabilizing the targeted protein. The impact of 0TUD4 in regulating tumor immune response and tumor progression remains largely unknown.

[0152] Experiments reported herein indicate that the OTUD4 / CD73 proteolytic axis is a specific target in in identifying agents for treating immune-suppressive TNBCs. While E3 ligase TRIM21 catalyzes the ubiquitylation of CD73 for degradation, cleaving off the ubiquitin-conjugated chain from CD73 by 0TUD4 results in the stabilization of CD73 that dampens CD8+ T cell function. The molecular axis OTUD4 / CD73 / adenosine is dictated by TGF-P signaling, whereas the TRIM21 / CD73 axis is regulated in response to IFN-y signaling. Clinical experiments indicate that the OTUD41o / CD731o signature in a subset of human breast malignancies was associated with a favorable immune profile. The compound ST80, was identified to resume CD73 proteolysis and restore capacity to elicit CD8+ interferon- " / -producing T cell responses through the blockade of the interaction between 0TUD4 and CD73. These finding indicate a strategy for targeting the immunosuppressive proteolytic axis OTUD4 / CD73 in treating immune-suppressive TNBCs and other cancers.

[0153] Elevated expression of CD73 is dramatically associated with an unfavorable tumor immune response and prognosis in immune-suppressive TNBCs

[0154] To search for potential therapeutic targets for immune-cold breast cancer, a bioinformatic analysis of proteomic data was performed concerning over 300 immune-relevant proteins from 59 breast cancer patients in The Cancer Genome Atlas (TCGA). Elevated expression of immunerelevant proteins, including VTCN1, CD274 and CD73, were detected in the basal TNBC subtype of breast cancer compared with other subtypes. Subsequent hierarchical cluster analysis revealed 2 distinct subgroups of patients with TNBC, each characterized by a unique immune-related protein expression profile. Further differential protein expression analysis spotlighted the significant upregulation of CD73 in one of these subgroups. Given the status of PD-L1 as a recognized biomarker for the “immune hot” classification of patients with TNBC, the expression levels of both PD-L1 and CD73 were examined across the 2 TNBC cohorts. Interestingly, while the average PD-L1 expression remained consistent across both cohorts except for the change in the width of the distribution, CD73 was markedly upregulated in one subgroup (TNBC), suggesting the presence of a TNBC subgroup with a potential immune-suppressive profile.

[0155] Additionally, the data indicated that the mRNA expression of CD73 and several mRNAs involved in the adenosinergic pathway were significantly upregulated in immune-noninflamed TNBC samples but downregulated in immune-inflamed and restrained samples from patients with TNBC. In contrast, the mRNAs downregulated in immune-noninflamed TNBCs but upregulated in immune-inflamed TNBCs were mostly associated with IFN-y signaling pathway, which facilitated tumor cell recognition and elimination by recruiting cytotoxic T lymphocytes to tumor cells. To confirm the observations, a tissue microarray (TMA) comprising 110 distinct breast samples was examined using IHC. Elevation in CD73 protein levels was detected in the majority of TNBC tissue specimens. Systematic analysis of both protein expression and mRNA levels of CD73 across a wide spectrum of breast cancer cell lines were performed, encompassing a total of 45 different lines. A similar expression pattern was measured in both CD73 protein and mRNA levels, where CD73 was significantly elevated in TNBC cells in comparison with other breast cancer cell lines. To investigate the potential association between heightened CD73 expression and reduced tumor immunogenicity, a Gene Ontology Biological Process (GOBP) analysis using the TCGA breast cancer database was conducted. Results indicated a strong correlation between increased CD73 expression and biological processes related to tumor immune response, such as adaptive immune response. Additionally, the CIBERSORT analysis verified the correlation between elevated CD73 expression in breast tumors and a reduced CD8+ T cell infiltration. To validate the observations, the association between CD73 expression and CD8+ T cell infiltration was examined. A marked reduction in CD8+ T cell staining in TNBC specimens with elevated CD73 expression was observed. Moreover, Spearman correlation analysis indicated a subset of TNBC tumors with diminished CD8+ T cell infiltration, particularly in cases with high CD73 expression. Likewise, a rise in CD73 expression was associated with the downregulation of immune response, T cell activation, and proliferation, using the GOBP enrichment score and immune suppressive gene correlation analysis. To further determine the prognostic significance of CD73 expression in patients with TNBC, 1,080 TNBC samples from TCGA database were examined. There was a positive correlation between high CD73 expression and increased hazard risk and adverse outcomes in overall survival (OS), as indicated by univariate Cox regression models. A marked improvement in patient survival was observed among those with a high CD8+ T cell population and low CD73 expression. These findings demonstrated a significant correlation between aberrant increase in CD73 and poor prognosis of immune-suppressive TNBCs.

[0156] Identification of OTUD4, a deubiquitinase, as a key driver for CD73 accumulation in immune-suppressive TNBCs

[0157] To explore the underlying mechanism that governs the abnormal accumulation of CD73 in immune-suppressive TNBC breast tumors, Tandem Affinity Purification (TAP) coupled with mass spectrometry analyses was conducted to identify the CD73 interactome. Flag / HA-tagged hCD73 protein was ectopically expressed in MDA-MB-468 breast cancer cells and subsequently purified using an affinity capture approach, with cells transfected with the 3 x Flag vector serving as the control. This approach led to the identification of OTUD4 as a putative critical driver for upregulating CD73 in TNBC cells. In addition, TRIM21 was identified as the ubiquitin E3 ligase that suppresses CD73 expression levels through ubiquitin- proteasomal degradation. To validate the biochemical consequences as well as physiological relevance of interaction between OTUD4 and CD73, a series of immunoprecipitation assays were performed in the MDA-MB468 cells. Endogenous CD73 coimmunoprecipitated with 0TUD4 was observed, and CD73 was detected on 0TUD4 immunocomplexes. To further validate the generality of the OTUD4 / CD73 interaction, a series of immunoprecipitation assays across diverse cancer cell lines, including the non-small cell lung cancer cell HCC827, human ovarian cancer cell SKOV3, and human colorectal cancer cell HCT116, consistently showed endogenous interaction between CD73 and 0TUD4. To examine the cellular compartmentalization of the interaction between CD73 and 0TUD4, cell lysate fractionation was conducted followed by Western blotting. As shown in Figure 2D, it was observed that both 0TUD4 and TRIM21 principally existed in the cytosol. The intracellular interaction site between 0TUD4 and CD73 was identified using an in vitro immunofluorescence staining and proximity ligation assay. Although CD73 was observed in both the membrane and cytosol and 0TUD4 was observed in both the nucleus and cytosol, the interaction between CD73 and 0TUD4, in the cytosol as determined by proximity ligation assay suggested a potential link between OTUD4-guided cytosolic deubiquitylation of CD73 and its subsequent displacement onto the cell membrane. To assess whether 0TUD4 could directly affect CD73 turnover via its deubiquitinase activity, MDA-MB468 and MDA-MB231 human breast cancer cell lines were engineered with stable 0TUD4 expression or knockdown (KD). Elevated 0TUD4 resulted in increased CD73 protein levels. Experiments were performed to assess whether the deubiquitylation of CD73 protein guided by 0TUD4, leading to its stabilization, could result in enhanced membrane presentation. The results indicated that the fluorescence intensity of membrane-bound CD73 in OTUD4-overexpressing (OE) MDA-MB231 (Figure 1A) and MDA-MB468 cells was significantly higher than that in the control cells. In addition, immunofluorescence staining in MDA-MB468-OTUD4 further confirmed that enhanced 0TUD4 expression leads to increased membrane-bound CD73 fluorescence intensity. In contrast, the KD of 0TUD4 using shRNA in MDA-MB23 1 and MDA-MB468 cells led to a downregulation of both total and membrane-bound CD73 protein expression. To determine if 0TUD4 exerted a direct effect on CD73 turnover dynamics, a pulse-chase assay in MDA-MB468 cells stably overexpressing 0TUD4 (0E-0TUD4) was performed. The results indicated that overexpression of 0TUD4 significantly slowed down CD73 protein turnover. To examine the role of 0TUD4 on CD73 degradation through catalyzing CD73 deubiquitylation, a CD73 ubiquitylation assay using MDA-MB468 cells with a 0TUD4 stable KD was performed. The results indicated that endogenous CD73 underwent ubiquitylation, and the knockdown of 0TUD4 markedly increased the ubiquitin-conjugated CD73.

[0158] To ascertain whether the physiological relevance for the accumulation of CD73 is regulated by 0TUD4, adenosine production in both human and mouse TNBC breast cancer cells (MDA- MB231, MDA-MB468, 4T1, and EO771) was evaluated. The data indicated that 0TUD4 overexpression in breast cancer cells significantly augmented adenosine production compared with the control, whereas 0TUD4 KD resulted in a reduction of adenosine production. Similar results were also observed even in the case of CD73 -overexpressed MDA-MB486 and MDA-MB231 cells. In addition, experiments were performed to determine whether modulation of 0TUD4 could influence other enzymes within the adenosinergic pathway, including CD39 and adenosine deaminase (ADA). Results demonstrated that neither AMP production nor ADA activity was significantly altered by the levels of 0TUD4 expression in MDA-MB468 cells. Further, the effect of CD73 deubiquitylation by 0TUD4 in cancer cells on T cell function was investigated in vitro using a coculture experiment. T cell proliferation activity and IFN-y production were elevated when 0TUD4 was knocked down in tumor cells, whereas 0TUD4 overexpression in tumor cells led to a decrease in T cell proliferation activity and IFN-y production. These results indicate that deubiquitinase 0TUD4 sustains CD73 protein accumulation with concomitant extracellular adenosine production, thereby limiting T cell function in immune-suppressive TNBCs.

[0159] Mapping and structural modeling of the interaction between CD73 and OTUD4 by integrated use of experimental data and docking simulations followed by experimental validation

[0160] To elucidate the in-depth mechanism by which CD73 is regulated by 0TUD4, the interaction domains that facilitate the recognition and binding between 0TUD4 and CD73 were identified. To this end, Flag / HA-tagged CD73 fragments and V5-tagged 0TUD4 fragment mutants were constructed and co-transfected into HEK293T cells, followed by co-immunoprecipitation experiments. A series of V5-tagged 0TUD4 truncation mutants with Flag / HA-tagged CD73 were co-transfected. Pull-down assays of individual V5-tagged 0TUD4 mutants were conducted, followed by immunoblotting with anti -Flag antibodies to detect Flag / HA-CD73. Amino acids stretching from 336 to 550 on OTUD4 mediated the interaction with CD73. Additional experiments indicated that amino acids stretching from 380 to 500 on OTUD4 mediated the interactions between 0TUD4 and CD73. A series of 0TUD4 mutants were engineered, specifically deleting the amino acid segments 380-410, 410-440, and 440-470. the OTUD4 mutant without the R380-N410 segment lost its ability to bind by coimmunoprecipitation with CD73.

[0161] A similar strategy was utilized to identify the molecular motifs on CD73 that are responsible for the interaction with OTUD4. The amino acid residues from 275 to 311 on CD73 were pinpointed as the region mediating the binding between CD73 and OTUD4. TRIM21 acts as an E3 ligase responsible for the ubiquitin proteasomal degradation of CD73. To evaluate the interplay of TRIM21 -mediated ubiquitylation and how OTUD4-guided deubiquitylation regulates CD73 proteolysis, structural modeling of the interaction between CD73, TRIM21, and OTUD4 was conducted by integrated use of experimental data and docking simulations followed by experimental validation.

[0162] Experiments revealed that OTUD4 residues R380-N410 engaged with CD73 residues V275-D311. Predominant interfacial residue pairs are believed to include H304(CD73)- S397(OTUD4), the salt bridge E293(CD73)-K403(OTUD4), and the hydrophobic contacts of CD73 V300 and 1301 with OTUD4 F404. Given the significance of hydrophobic contacts at the CD73-OTUD4 interface, CD73 V300 and 1301 were evaluated. A V300P and I301Q double mutation was calculated to elevate the energy by 1 kcal / mol, while reducing CD73 by 5.2 kcal / mol. Experiments were performed to further confirm if amino acids V300 and 1301 on CD73 directly mediate its binding to OTUD4. The results revealed a marked decrease in the interaction between mutant CD73 and OTUD4 when the amino acids V300 and 1301 are mutated in both MDA-MB231 and MDA-MB468 cells. Additionally, a rise in the level of ubiquitin conjugates was observed in the CD73 V300P / I301Q double mutant, thereby indicating that V3OO / I3O1 on CD73 mediates the interaction and subsequently influences CD73 stability through regulation of deubiquitylation. In addition, in MDA-MB468 cells, while the turnover of ectopically expressed CD73 displayed a half-life of approximately 4 hours, the CD73 V300P / I301Q interaction mutant notably accelerated CD73 turnover. To further demonstrate that the CD73 V300P / I301Q mutant also influences CD8+ T cell proliferation via CD73 -dependent enzymatic adenosine production, data showed that cells harboring CD73 V300P / I301Q mutation exhibited substantially lower adenosine levels compared with CD73WT cells.

[0163] MDA-MB468-CD73WT cells had a decrease in IFN-y production and proliferation capacity of CD8+ T cell population when compared to MDA-MB468 vehicle control cells, and the disruption of the CD73-OTUD4 interaction by mutating the V3OO / I3O1 sites restored the IFN-y production and proliferation capacity of CD8+ T cell population comparable to those of the control vector. These data indicate that the specific interaction between CD73 and 0TUD4, dictated through the V300 and 1301 residues on CD73, effect CD73 protein stability and subsequent adenosinergic effects, particularly on modulation of T cell activity.

[0164] Stabilization of CD73 by OTUD4 in immune-cold tumors is orchestrated in response to TGF- P signaling.

[0165] TRIM21 -mediated ubiquitylation and subsequent degradation of CD73 by a feed-forward mechanism involve IFN-y. Analysis using TCGA database corroborated this by indicating a positive correlation between high TRIM21 expression and increased gene signatures related to the IFN-y pathway in patients with TNBC. Experiments were performed to determine whether IFN-y affects the deubiquitylation of CD73 orchestrated by OTUD4. There was no change in OTUD4 protein levels in MDA-MB468-ShTRIM21 cells with IFN-y treatment, suggesting a small contribution of IFN-y to affect OTUD4 activity in tumor cells. The IFN-y signaling pathway may be counteracted by the TGF-P pathway in immunosuppressive tumor microenvironments. Consistent with the importance of TGF-P signaling pathway for sustaining CD73 expression, TNBC cells with high CD73 expression exhibited increased protein expression related to the TGF- P signaling pathway. Further, GSEA analysis and TIMER2.0 analysis indicated a significant correlation between high OTUD4 expression and increased TGF-P signaling pathway activity. These findings indicate that, in a subset of immunosuppressed breast tumors, upregulated TGF-P signaling may co-opt OTUD4 to hyperactively deubiquitylate CD73, thereby enhancing its stability and membrane abundance, immune evasion, and maintaining a protumor microenvironment.

[0166] Experiments were performed, wherein MDA-MB468 and MDA-MB231 breast cancer cells were treated with 3 ng / ml of TGF-p. Distinct from IFN-y, TGF-P treatment increased the protein levels of OTUD4 together with CD73. A significant reduction in the ubiquitylation of CD73 following TGF-P treatment was observed. Additionally, an increase of cytosolic OTUD4 was noted following TGF-P exposure in both TGF-P-treated MDA-MB468 and MDA-MB231 breast cancer cells. An increase in extracellular adenosine production was measured in both MDA- MB468 and MDA-MB231 breast cancer cells treated with TGF-p. A pulse-chase assay was done in TGF-P treated MDA-MB468 cells. TGF-P treatment significantly slowed down CD73 protein turnover. To further elucidate the influence of TGF-P on the OTUD4 / CD73 axis, MDA-MB468 and MDA-MB231 cells we treated with stable KD of 0TUD4 with 3 ng / mL TGF-p. Depletion of 0TUD4 resulted in unchanged CD73 levels after TGF- P treatment, indicating the importance of 0TUD4 in maintaining CD73 stability. The TGF-P- induced increase in adenosine production was abolished completely when 0TUD4 was knocked down. Additionally, knocking down of TGFBR or loss of TGF-P by LY2109761 led to reduced protein levels of 0TUD4 as well as CD73. However, the interaction between 0TUD4 and CD73, was observed, although its proportion correlated with the decreased level of 0TUD4 and CD73 proteins. Collectively, these results indicate that TGF-P may enhance 0TUD4 levels, contributing to the stabilization of CD 73.

[0167] To confirm the role of amino acids V300 and 1301 in facilitating the TGF-P-mediated deubiquitylation of CD73 by OTUD4, experiments indicate that TGF-P treatment reduced the ubiquitylation level, which in turn stabilized the CD73 protein in MDA-MB-468-CD73 cells. In contrast, the TGF-P-induced upregulation of CD73 in MDA-MB468 cells was abrogated completely when the interaction sites between CD73 and OTUD4 were disrupted. Additionally, the degradation rate of CD73 protein remained unchanged in MDA-MB-468-CD73V300P / I301Q cells, regardless of TGF-P treatment, as demonstrated by a pulse-chase assay. In line with these results, an increase in adenosine production was noted only in the CD73WT cells but not in the CD73V300P / I301Q mutant cells. These findings indicate the importance of a TGF-P-mediated OTUD4 / CD73 proteolytic axis for the modulation of tumor immunogenicity.

[0168] Development of pharmacological inhibitors that blocks OTUD4 / CD73 interaction in restoring tumor immune responses in immune-suppressive TNBCs.

[0169] To evaluate the therapeutic consequence of blocking the OTUD4 / CD73 complex formation as a method restoring tumor immune response in immune-suppressive TNBCs, a methodology to identify small molecules that could block the interaction between CD73 and OTUD4 was implemented. Preliminary in silico evaluations were done to identify compounds that disrupt CD73 residues V275-D311 that bind to OTUD4. This approach led to promising small molecules which could be procured from chemical libraries. Notably, these small molecules are believed to target distinct sites both adjoining the interfacial segment V275-D311. To confirm whether the compounds identified could bind CD73 to block its interaction with 0TUD4 and therefore affect CD73 protein levels, MDA-MB231 and MDA-MB468 breast cancer cells were treated with 0.5 pM of the compounds for 24 hours and then assessed for CD73 protein levels. Two compounds, ZINC000009345994 (ST80) and ZINC001336656227 (Z22), were identified as the most potent inhibitors in suppressing CD73 protein abundance by blocking the OTUD4 / CD73 interaction. Additionally, both ST80 and Z22 increased the turnover of the CD73 protein compared with the control in MDA-MB231 cells with 0.5 pM of both ST80 and Z22.

[0170] To confirm whether the inhibitory effects of ST80 and Z22 were attributed specifically to targeting the interaction site of OTUD4 and CD73, we first treated mutant MDA-MB231- CD73V300P / I301Q and MDA-MB468-CD73V300P / I30f Q cells were treated with compounds. ST80 and Z22 treatment (0.5 pM) decreased CD73 protein levels with increased ubiquitylation levels in MDA-MB231-CD73WT cells but not in MDA-MB23 f-CD73V300P / I301Q cells. These results together suggest that both ST80 and Z22 are potential pharmacological inhibitors that specifically block OTUD4 / CD73 interaction to destabilize CD73 protein.

[0171] Although it is not intended that embodiments of this disclosure be limited by any particular mechanism, it is predicted by molecular modeling that ST80 binds to a hydrophobic area defined by residues 171, P254, Y289, P307, 1308, andL309, and Z22 interacts directly with certain residues within the V275-D3 f 1 binding region, as well as with residues P264, 1266, R273, and K274 of CD73.

[0172] To further understand the inhibitory effects of ST80 and Z22 on the regulation of CD73 stability, a series of experiments were performed to determine the optimal dose and treatment duration for ST80 and Z22 to disrupt the OTUD4 / CD73 interaction. MDA-MB231 and MDA- MB468 breast cancer cells were treated with ST80 and Z22 at concentrations ranging from 10 nM to 1 pM for 24 hours. ST80 reduced CD73 protein levels starting from 50 nM while Z22 reduced CD73 protein levels starting from 500 nM in MDA-MB231 and MDA-MB468 breast cancer cells. MDA-MB23 1 and MDA-MB468 cells were treated with various concentrations of ST80 and Z22 in conjunction with a 3 ng / mL administration of TGF-p. ST80 was able to counteract the TGF-P- induced increase in CD73 protein levels starting at a concentration of 500 nM while Z22 required a 1 pM dose to yield comparable inhibitory effects. Further, the inhibitory effects of ST80 were evident as early as 4 hours after treatment and persisted for up to 48 hours in MDA-MB468 cells and MDA-MB231 cells. In contrast, Z22 exerted its effects starting from 12 hours after treatment but only lasted 24 hours.

[0173] To determine the subsequent effects of ST80 and Z22 in cancer cell proliferation and tumor immune response, MDA-MB231 and MDA-MB468 cells were treated with 0.5 pM of ST80 and Z22 for 24 hours, followed by a clonogenic survival assay. Figure 2A highlighted that compounds ST80 and Z22 markedly enhanced the growth inhibition of cancer cells induced by PBMC, even at a low E:T ratio of 2:1. To further determine the impact of ST80 and Z22 on tumor immune responses, it was demonstrated that ST80 reduced adenosine production starting at concentrations as low as 100 nM in MDA-MB231 and MDA-MB468 cells. In contrast, the effects of Z22 become evident at either 100 nM or 500 nM concentrations, depending on the cell line. Further, upon coculturing MDA-MB231 cells with PBMCs, both compounds resulted in an increase in expression of IFN-y (Figure 2D), GzmB (Figure 2E), Ki67, and TNF-a in the CD8+ T cell population.

[0174] To determine if ST80 could exert a broad therapeutic effect in a spectrum of cancers, experiments indicated that ST80 treatment led to a pronounced reduction in CD73 protein levels at a concentration of 500 nM in HCC827 lung cancer cells. Moreover, ST80 treatment appeared to enhance the cytotoxic function of T cells, leading to increased cytotoxic effects of PBMC in multiple cancer types, including HCC827 lung cancer cells, SKOV3 ovarian cancer cells, and HCT116 colon cancer cells (Fig. 2F). These findings indicate that ST80 and Z22 specifically disrupt the OTUD4 / CD73 protein interaction, thereby reducing CD73 protein stability and ultimately boosting tumor immune responses.

[0175] Modulation of CD73 proteolysis by OTUD4 alters tumor growth and capacity to elicit antitumor CD8+ T cell responses

[0176] To evaluate the role of OTUD4 in tumor growth in vivo, murine cell lines were created with modified for OTUD4 overexpression (OTUD4-OE). OTUD4-OE and OTUD4 knock down (OTUD4-KD) versions were developed in both 4T1 and EO771 cells. The effect on tumor growth was then assessed using a syngeneic mouse model. Engineered 4T1 control and 4T1 OTUD4-OE breast cancer cells were subcutaneously injected into the mammary fat pad of female BALB / C mice. A significant increase in both the tumor growth rate and tumor weight was observed 21 days postinjection of the tumor cells. Furthermore, OTUD4 overexpression resulted in an increase in membrane-bound expression levels of CD73 on tumor cells from tumor-bearing mice. The data support a tumor promoting role of the OTUD4 / CD73 proteolytic axis in vivo.

[0177] Elevated levels of CD73 are often associated with an increase in immunosuppressive cell populations such as Tregs, MDSCs, and tumor-associated macrophages (TAMs). To analyze the effect of 0TUD4 in modulating tumor immunity, the tumor immune infdtrates were examined using a high-dimensional spectral flow cytometry panel that incorporated hallmark markers for major immune populations. A dimensionality reduction tool was employed to compare 4T1 control and 4T1 0TUD4-0E tumors. Live intact single cells gated from CD45+ tumor infdtrates could be grouped into distinct subsets, including CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), proliferating CD8+ T cells (CD3+CD8+Ki67+), CD8+ tissue resident memory T cells (CD69+CD103+, TRM), exhausted CD8+ T cells (PD-1+TIM3+, TEX), stem-like progenitors of exhausted CD8+ T cells (CD69+LylO8+, TPEX), Treg (CD3+CD4+ Foxp3+CD25+), non-Treg CD4+ T cells (CD3+CD4+ Foxp3-), B cells (CD 19+ CD3-), dendritic cells (MHC- II+CDl lc+CDllb+, DC), TAMs (Grl- F4 / 80+CDllb+, TAM), polymorphonuclear MDSCs (Ly6G+ Ly6CloCDl lb+, PMN-MDSC), monocytic MDSC (Ly6Glo Ly6C+CDllb+, M-MDSC), NK (CD3- NKp46+), and NKT (CD3+NKp46+). Frequencies of proliferating CD8+ T cells, NK, and NKT among CD45+ tumor infdtrates in 4T1 0TUD4-0E tumor-bearing mice were significantly decreased compared with those in the control mice. By contrast, the frequency of immunosuppressive TAM and PMN-MDSC was increased. Although there was no statistical difference in the frequency of CD8+ TRM, TEX, or TpEX between 4T1 control and 0TUD4-0E tumors, terminal effector-like TEX were more abundant in 0TUD4-0E tumors, as indicated by increased expression levels of CX3CR1, KLRG1, and CD101 with decreased levels of Ki67. 0TUD4-0E in tumor cells enhanced a tumor-promoting M2-like phenotype in TAMs, usually defined by the expression of CD 163. Further, there was a significant reduction in IFN-y and TNF- a secretion by infiltrating CD8+ and CD4+ T cells in 0TUD4-0E tumors.

[0178] Conversely, 0TUD4-KD in either 4T1 or EO771 murine breast cancer cell lines hindered tumor development compared with the control group. Additionally, there was increased tumor infiltration of CD8+ T cells expressing higher levels of IFN-y and Ki67 in EO771-OTUD4-KD tumor-bearing mice compared with those in the control mice. To evaluate the importance of the physical interaction between 0TUD4 and CD73 for tumor growth, both 0TUD4 and CD73WT (0TUD4-0E + CD73WT) or 0TUD4 and CD73V300P / I301Q with mutated interaction sites (0TUD4-0E + CD73V300P / I301 Q) were overexpressed in EO771 cells. While there was no significant difference in tumor growth between mice with EO77I-OTUD4-CD73V300P / I301Q and EO771 vehicle control, tumor growth was accelerated in EO771-OTUD4-CD73WT tumorbearing mice. The results underscore the importance of OTUD4-mediated deubiquitylation of CD73 for tumor growth.

[0179] Pharmacological blockade of interaction of OTUD4 and CD73 promotes tumor immunogenicity and inhibits tumor progression in immune-cold breast cancer.

[0180] To determine the therapeutic relevance of the inhibitor ST80, the antitumor effect of ST80 was examined by single treatment or in combination with anti-hPD-Ll drug durvalumab, using a TNBC 4T1 model where the endogenous mouse PD-L1 was knocked out and replaced with the human counterpart. Combining ST80 with durvalumab led to enhanced suppression in tumor growth and prolonged survival without evident mouse body weight changes. Figure 3A and 3B. Further, similar therapeutic benefits of combination treatment were observed in mice bearing 4T1- hPDLl-OTUD4-OE tumors (Figure 3C), where durvalumab monotherapy failed to suppress tumor growth compared with IgG control. These findings indicate a therapeutic value of pharmacologically targeting the proteolytic interaction of 0TUD4 and CD73 to overcome the resistance of immune checkpoint inhibitors (e.g., anti-PD-Ll antibodies), especially in high CD73- expressing, immunologically suppressive TNBC. The antitumor efficacy of ST80 was compared with the anti-CD73 monoclonal antibody and it was found that both ST80 and anti-CD73 antibodies significantly suppressed tumor progression (Fig. 3D). Further, depletion of CD8+ T cells completely abrogated the efficacy of ST80, indicating a role of CD8+ T cells in ST80- mediated antitumor effect. To investigate whether ST80 treatment affected immunosuppressive cells like Tregs, TAMs, and MDSCs, immune profiling analysis was performed on ST80-treated E0771 tumor infiltrates, which showed that ST80 treatment caused a significant reduction of infiltration of immunosuppressive Tregs, TAMs, and PMN-MDSCs, accompanying increased infiltration of M-MDSCs. Further, ST80 treatment enhanced tumor infiltration of IFN-y+ CD8+ T cells and decreased their expression of PD-1. These results were consistent with those from the immune characterization of 0TUD4-KD and 0TUD4-0E tumor infiltrates. In addition, ST80 treatment decreased surface expression levels of CD73 and PD-L1 on tumor cells from tumor- bearing mice. The data together support pharmacological targeting of the proteolytic interaction of 0TUD4 and CD73 to mitigate tumor immune evasion and revive antitumor T cell immunity.

[0181] Experiments indicates that an unfavorable immune profde of tumors with CD73 high expression correlating with high 0TUD4 expression in an immune-suppressive subset of TNBCs. Stabilization of CD73 catalyzed by 0TUD4 suppresses cytotoxic CD8+ T cell function, leading to tumor immune evasion. It is believed that there is a role of TGF-P signaling in orchestrating the OTUD4 / CD73 proteolytic axis that promotes tumor progression. A pharmacologic inhibitor, ST80 specifically inhibits the physical interaction of 0TUD4 and CD73 to promote ubiquitylation of CD73, thereby causing CD73 degradation and revival of CD8+ T cell function. Notably, treatment with ST80 sensitized TNBC tumors to anti-PD-Ll therapy even in the case of tumors with high expression of OTUD4 and CD73. This study introduces a therapeutic paradigm by specific targeting of a proteolytic interaction for improving tumor immunogenicity and circumventing resistance to anti-PD-Ll therapy for immunotherapy nonresponsive TNBC.

Claims

CLAIMSWhat is claimed is:1 . A pharmaceutical agent that suppress 0TUD4 and CD73 binding interactions.

2. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the pharmaceutical agent that suppress 0TUD4 and CD73 binding interactions of claim 1 which is a compound having the following formula I:Formula I or salt thereof whereinR1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, or R15are each, the same or different, hydrogen, alkyl, halogenated alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, glycol, alkylthio, alkylamino, (alkyl)2amino, alkyl sulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R1, R2, R3, R4, R3, R6, R7, R8, R9, R10, R11, R12, R13, R14, or R15are optionally substituted with one or more, the same or different, R16;R16is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R16is optionally substituted with one or more, the same or different, R17;R17is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R17is optionally substituted with one or more, the same or different, R18; andR18is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N- ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethyl sulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

3. The pharmaceutical composition of claim 2, wherein R1is a halogen.

4. The pharmaceutical composition of claim 2, wherein R1is a fluoro.

5. The pharmaceutical composition of claim 2, wherein R8is alkyl.

6. The pharmaceutical composition of claim 2, wherein R8is methyl.

7. The pharmaceutical composition of claim 2, wherein R11is hydrogen.

8. The pharmaceutical composition of claim 2, wherein the compound is (3aR,7aS)-2- ((5S,7R)-7-(4-fluorophenyl)-5-(p-tolyl)-4,5,6,7-tetrahydro-[l,2,4]triazolo[l,5-a]pyrimidin-2-yl)- 3a,4,7,7a-tetrahydro-lH-isoindole-l,3(2H)-dione or salt thereof (ST80).

9. The pharmaceutical composition of claim 2, wherein the compound is2,2'-(pyrimidine-2,4- diylbis(piperidine-l,3-diyl))bis(lH-benzo[d]imidazole) (Z22), derivative, prodrug, ester, or salt thereof.

10. The pharmaceutical composition of claim 2 in the form of a pill, tablet, capsule, gel, gel capsule, powder, granule, or cream.

11. The pharmaceutical compositions of claim 2 in the form of a pH buffered saline solution optionally comprising a saccharide or polysaccharide.

12. The pharmaceutical compositions of claim 2 wherein the excipient is selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N-pyrrolidone, crospovidone, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, corn starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

13. A method of treating cancer comprising administering an effective amount of a compound as in any of claims 1-9 to a subject in need thereof.

14. The method of claim 13, wherein the cancer is breast cancer or triple negative breast cancer.

15. The method of claim 13, wherein the cancer is ovarian cancer, lung cancer or colon cancer.

16. The method of claim 13, wherein the compound is administered in combination with another anticancer agent.

17. The method of claim 16, wherein the anticancer agent is a checkpoint inhibitor.

18. The method of claim 17, wherein the checkpoint inhibitor is anti-PD-1, anti-PD-Ll, or anti - CTLA4 antibody or combinations thereof.

19. The method of claim 18, wherein the antibody is durvalumab, atezolizumab, avelumab, ipilimumab, tremelimumab, nivolumab, pembrolizumab, cemiplimab, or combinations thereof.

20. The method of claim 16, wherein the anticancer agent is an anti-CD73 antibody.

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