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WO2025072709A2 - Suppression pharmacologique de la liaison otud4 et cd73, méthodes de traitement du cancer, agents anticancéreux et compositions thérapeutiques - Google Patents

Suppression pharmacologique de la liaison otud4 et cd73, méthodes de traitement du cancer, agents anticancéreux et compositions thérapeutiques Download PDF

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Publication number
WO2025072709A2
WO2025072709A2 PCT/US2024/048907 US2024048907W WO2025072709A2 WO 2025072709 A2 WO2025072709 A2 WO 2025072709A2 US 2024048907 W US2024048907 W US 2024048907W WO 2025072709 A2 WO2025072709 A2 WO 2025072709A2
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WIPO (PCT)
Prior art keywords
cancer
otud4
0tud4
cells
alkyl
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WO2025072709A3 (fr
Inventor
Yong Wan
Yueming Zhu
Ivet Bahar
Anupam BANERJEE
Jiyoung Lee
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Emory University
Research Foundation of the State University of New York
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Emory University
Research Foundation of the State University of New York
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86

Definitions

  • 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.
  • TNBCs Triple negative breast cancers
  • PD-L1 inhibitors demonstrate promising clinical benefits in certain subsets of patients with TNBC.
  • compositions 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.
  • 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.
  • the therapeutic agent is a small molecule, antibody, or other specific binding agent.
  • 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.
  • 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.
  • 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.
  • the agent pharmaceutical agent specifically binds an amino acid residue within positions 275 to 311 in CD73 VPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLN (SEQ ID NO: 1)
  • the agent pharmaceutical agent specifically binds an amino acid residue within position 380 to 410 in OTUD4, RLQHPSGVRQHAFSSHSSGSQSQKFSSEHKN (SEQ ID NO: 2).
  • this disclosure relates to pharmaceutical composition
  • a pharmaceutically acceptable excipient comprising a pharmaceutically acceptable excipient and a compound that suppress OTUD4 and CD73 binding interactions, derivatives, prodrugs, esters, or salts thereof.
  • 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.
  • 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.
  • the subject is diagnosed with breast cancer, triple negative breast cancer, lung cancer, colon cancer, or other cancer.
  • 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.
  • Figures 1A-1F show data used in the identification of OTUD4 as a key driver for CD73 accumulation in immune-suppressive TNBC breast tumors.
  • 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.
  • Figure IB shows data for adenosine levels that were determined in MDA-MB231, MDA- MB468, 4T1, and EO771 breast cancer cells with OTUD4 overexpression.
  • Figure 1C shows data for adenosine levels that were determined in MDA-MB231, MDA- MB468, 4T1, and EO771 breast cancer cells with ShOTUD4 overexpression.
  • 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).
  • 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.
  • 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.
  • FIG. 2B shows data where MDA-MB231 breast cancer cells were treated with various concentrations of ST80.
  • FIG. 2C shows data where MDA-MB231 breast cancer cells were treated with Z22.
  • 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.
  • Figure 2E shows data on the percentage of GzmB+ in CD8+ T cells.
  • 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.
  • 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 mm 3 , 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.
  • Figure 3B shows data on tumor size over time for 4Tl-hPD-Ll.
  • Figure 3C shows data on tumor size over time for 4Tl-hPDLl-OTUD4.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the therapeutic agent is a small molecule, antibody, or other specific binding agent.
  • a “subject” refers any animal, preferably a human patient, livestock, or domestic pet.
  • 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.
  • 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.
  • co-administration 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the agent pharmaceutical agent specifically binds an amino acid residue within positions 275 to 311 in CD73 VPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLN (SEQ ID NO: 1).
  • the agent pharmaceutical agent specifically binds an amino acid residue within position 380 to 410 in OTUD4.
  • RLQHPSGVRQHAFSSHSSGSQSQKFSSEHKN SEQ ID NO: 2.
  • 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.
  • cancer is breast cancer.
  • cancer is triple negative breast cancer.
  • cancer is lung cancer.
  • cancer is ovarian cancer or colon cancer.
  • 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.
  • 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.
  • 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, ave
  • 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,
  • 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.
  • 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).
  • identifying the suppression of 0TUD4 and CD73 binding interactions can be accomplished using in vitro immunofluorescence staining and proximity ligation assays as reported herein.
  • 0TUD4 and/or CD73 or fragments are conjugated to a label e.g., a fluorophore and the other contains an alternative fluorophore or quencher.
  • a label e.g., a fluorophore
  • 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.
  • 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.
  • 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.
  • labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
  • 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).
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as 18 F, 35 S or 131 I) 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.
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • this disclosure relates to fusion protein containing 0TUD4 and/or CD73 or fragments thereof as disclosed
  • 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.
  • 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.
  • 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.
  • a specific binding agent such as an ligand, receptor, enzyme, antibody or binding region/fragment thereof
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • variable region refers 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.”
  • CDRs binding complementarity- determining regions
  • the CDRs can be determined through epitope studies and sequence alignment comparisons of the constant and framework regions for the specific animal.
  • CDRs can be defined through epitope studies and sequence alignment comparisons of the constant and framework regions for the specific animal.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the hinge region may be associated with either the Fab or Fc fragment.
  • 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.
  • 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.
  • 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.
  • CDRs complementarity-determining regions
  • 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.
  • humanization e.g., framework region humanization
  • 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.
  • 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 are commonly derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca, and guanaco.
  • this disclosure relates to compounds and other specific binding agent such as antibodies that suppress binding interactions of 0TUD4 with CD73.
  • 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.
  • 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.
  • the compound has the following formula I:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , or R 15 are 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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10
  • R 17 is 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 R 17 is optionally substituted with one or more, the same or different, R 18 ; and
  • R 1 is a halogen
  • R 1 is a fluoro
  • R 8 is methyl
  • R 11 is hydrogen
  • the compound has the following Formula II:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , or R 12 are 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 R 1 , R 2 , R 3 , R 4 , R ⁇ R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , or R 12 are optionally substituted with
  • R 17 is 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 R 17 is optionally substituted with one or more, the same or different, R 18 ; and
  • 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.
  • “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, 2 H, or tritium 3 H.
  • enriched halogen isotopes are contemplated, e.g., 18 F, 19 F, or 76 Br.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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) n X, wherein X is typically an H, alkyl, alkoxy, halogenated alkoxy, or alkanoyl, and n is typically 1-50, 1-100, or 1-1,000.
  • halogen and “halo” refer to fluorine, chlorine, bromine, and iodine.
  • halogenated alkyl refers to an alkyl partially or entirely substituted with halogens, e.g., CF3.
  • 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.
  • salts refer to derivatives of the disclosed compounds where the parent compound is modified making acid or base salts thereof.
  • 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.
  • 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.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like
  • organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, mal
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition is in the form of a pill, tablet, capsule, gel, gel capsule, powder, granule, or cream.
  • the pharmaceutical composition is in the form of a pH buffered saline solution optionally comprising a saccharide or polysaccharide.
  • 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
  • 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.
  • 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.
  • compositions 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.
  • the isomer may be present in a composition with enantiomeric excess or diastereomeric excess of greater than 60%.
  • the R isomer may be present in enantiomeric excess or diastereomeric excess of greater than 70%.
  • the isomer may be present in enantiomeric excess or diastereomeric excess greater than 80%.
  • the isomer may be present in enantiomeric excess, diastereomeric excess, of greater than 90%.
  • the isomer may be present in enantiomeric excess diastereomeric excess of greater than 95%.
  • the pharmaceutical composition is in the form of a tablet, pill, capsule, powders, granules, gel, gel capsule, or cream.
  • 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
  • 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.
  • 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.
  • CAP cellulose acetate phthalate
  • PVAP polyvinyl acetate phthalate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • 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.
  • inert diluents commonly used in the art, such as water or other solvents, solubilizing agents
  • 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.
  • the pharmaceutical compositions are in a form for inhalation.
  • the pharmaceutical composition comprises a compound disclosed herein and a propellant.
  • an aerosolizing propellant is compressed air, ethanol, nitrogen, carbon dioxide, nitrous oxide, hydrofluoroalkanes (HFAs), or combinations thereof.
  • the disclosure contemplates a pressurized or unpressurized container comprising a compound herein.
  • the container is a manual pump spray, inhaler, meter-dosed inhaler, dry powder inhaler, nebulizer, vibrating mesh nebulizer, jet nebulizer, or ultrasonic wave nebulizer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compounds disclosed herein and a pharmaceutical carrier are admixed to provide a pharmaceutical composition.
  • 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.
  • the container is preferably a syringe for administering the reconstituted pharmaceutical composition after contact with the diluent.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition is provided in dry in order to prevent degradation and enable storage stability.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • TNBC immune-suppressive triple negative breast cancer
  • 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.
  • adenosine receptors such as AIR, A2AR, A2BR, and A3R
  • 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.
  • 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.
  • 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.
  • TNBC tissue microarray
  • Tandem Affinity Purification 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.
  • TRIM21 was identified as the ubiquitin E3 ligase that suppresses CD73 expression levels through ubiquitin- proteasomal degradation.
  • 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.
  • 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.
  • 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.
  • TRIM21 acts as an E3 ligase responsible for the ubiquitin proteasomal degradation of CD73.
  • 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.
  • 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.
  • 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.
  • CD73 V300P/I301Q interaction mutant notably accelerated CD73 turnover.
  • data showed that cells harboring CD73 V300P/I301Q mutation exhibited substantially lower adenosine levels compared with CD73WT cells.
  • 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.
  • Stabilization of CD73 by OTUD4 in immune-cold tumors is orchestrated in response to TGF- P signaling.
  • 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.
  • the IFN-y signaling pathway may be counteracted by the TGF-P pathway in immunosuppressive tumor microenvironments.
  • TNBC cells with high CD73 expression exhibited increased protein expression related to the TGF- P signaling pathway.
  • GSEA analysis and TIMER2.0 analysis indicated a significant correlation between high OTUD4 expression and increased TGF-P signaling pathway activity.
  • 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.
  • 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.
  • 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.
  • 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.
  • OTUD4-OE modified for OTUD4 overexpression
  • OTUD4-KD OTUD4 knock down
  • 4T1 and EO771 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.
  • 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.
  • Elevated levels of CD73 are often associated with an increase in immunosuppressive cell populations such as Tregs, MDSCs, and tumor-associated macrophages (TAMs).
  • TAMs tumor-associated macrophages
  • 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.
  • 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.
  • 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.
  • Pharmacological blockade of interaction of OTUD4 and CD73 promotes tumor immunogenicity and inhibits tumor progression in immune-cold breast cancer.
  • 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.

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Abstract

L'invention concerne des agents pharmaceutiques qui inhibent ou suppriment des interactions de liaison OTUD4 et CD73 ; et sont ainsi utiles dans le contexte de la gestion de maladies et d'états associés à des interactions de liaison OTUD4 et CD73, telles que des traitements anticancéreux. Dans certains modes de réalisation, la présente divulgation concerne une méthode de traitement du cancer comprenant l'administration d'une quantité efficace d'un agent pharmaceutique qui supprime les interactions de liaison OTUD4 et CD73. Dans certains modes de réalisation, l'agent thérapeutique est une petite molécule, un anticorps ou un autre agent de liaison spécifique.
PCT/US2024/048907 2023-09-27 2024-09-27 Suppression pharmacologique de la liaison otud4 et cd73, méthodes de traitement du cancer, agents anticancéreux et compositions thérapeutiques Pending WO2025072709A2 (fr)

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