WO2024256635A1 - Dpm1 inhibitor for treating cancer - Google Patents

Dpm1 inhibitor for treating cancer Download PDF

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Publication number
WO2024256635A1
WO2024256635A1 PCT/EP2024/066558 EP2024066558W WO2024256635A1 WO 2024256635 A1 WO2024256635 A1 WO 2024256635A1 EP 2024066558 W EP2024066558 W EP 2024066558W WO 2024256635 A1 WO2024256635 A1 WO 2024256635A1
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Prior art keywords
cancer
malignant
carcinoma
cell
dpmi
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French (fr)
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Eric Chevet
Hussein ISSAOUI
Jean-Ehrland RICCI
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Universite de Rennes 1
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Nice Sophia Antipolis UNSA
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Universite de Rennes 1
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Nice Sophia Antipolis UNSA
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    • 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
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/57595—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving intracellular compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91097—Hexosyltransferases (general) (2.4.1)
    • G01N2333/91102—Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention is in the field of oncology. More particularly, the invention relates to methods and composition for treating a cancer.
  • TME tumor microenvironment
  • UPR unfolded protein response
  • BioID biotinylation-based approach
  • DPMI KO prevented tumor growth only in the presence of a functional adaptive immune system (as depleting cytotoxic CD8+ T cells prevented the protective effect).
  • DPMI KO leads to reduced production of key cytokines and cell surface expression of PD-L1 (a major player in the control of adaptive immune response) through ERAD (ER-associated protein degradation).
  • DPMI KO in cancer cells limited M2 -like macrophages polarization overcoming immunosuppression and enhances cytotoxic T cell activity.
  • the inventors’ work reveals how tumoral UPR can limit tumor growth and suggests that DPMI inhibition is a useful strategy for improving cancer immunotherapy.
  • the invention relates to a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Dolichol-phosphate mannosyltransferase (DPMI) inhibitor.
  • DPMI Dolichol-phosphate mannosyltransferase
  • the present invention relates to a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Dolicholphosphate mannosyltransf erase (DPMI) inhibitor.
  • DPMI Dolicholphosphate mannosyltransf erase
  • the present invention relates to a method of improving cancer immunotherapy in a patient suffering from a cancer comprising administering to the patient a therapeutically effective amount of a Dolichol-phosphate mannosyltransferase (DPMI) inhibitor.
  • DPMI Dolichol-phosphate mannosyltransferase
  • the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with or susceptible to be afflicted with cancer.
  • IREla refers to inositol-requiring enzyme 1 (IRE1) and in humans is encoded by the ERN1 gene. It is an endoplasmic reticulum (ER)-resident transmembrane signaling protein and a cellular stress sensor. This protein harbors a cytosolic dual kinase/endoribonuclease activity required for adaptive responses to micro-environmental changes.
  • RNase activity of IRE1 refers to the activity of the endoribonuclease domain of IRE1 which degrades specific RNA (mRNA or microRNA) to avoid their translation or their cellular activity, an activity known as the RIDD (regulated IRE1- dependent decay of RNA), or contributes to the splicing XBP1 (X-box-binding protein 1) mRNA to change the reading frame leading to the production of a novel protein (XBPls), a potent unfolded-protein response transcriptional activator.
  • the IRE1 endonuclease activity is based on recognition of an RNA stem loop structure found twice in substrates HAC1 mRNA in yeast or XBP1 mRNA in metazoans. Cleavage of HAC1 or XBP1 mRNA occurs at both sites resulting in an mRNA fragment whose two ends are ligated in a unique splicing event (Ron et al.; Lee et al.).
  • the spliced HAC1 or XBP1 mRNAs encode transcription factor that activate numerous target genes, including genes involved in the unfolded protein response (UPR).
  • the UPR is a signal transduction cascade that occurs in response to the accumulation of misfolded proteins in the ER.
  • stroma refers to the connective, supportive framework of a biological cell, tissue, or organ.
  • tumor microenvironment refers to the cellular environment in which the tumor exists, including the area immediately surrounding fibroblasts, leukocytes and endothelial cells and the extracellular matrix (ECM).
  • ECM extracellular matrix
  • stromal cells include fibroblasts, leukocytes and vascular cells. Accordingly, cells of a tumor microenvironment comprise malignant cells in association with non-malignant cells that support their growth and survival.
  • the non-malignant cells also called stromal cells, occupy or accumulate in the same cellular space as malignant cells, or the cellular space adjacent or proximal to malignant cells, which modulate tumor cell growth or survival.
  • Non-malignant cells of the tumor microenvironment include fibroblasts, epithelial cells, vascular cells (including blood and lymphatic vascular endothelial cells and pericytes), resident and/or recruited inflammatory and immune (e.g., macrophages, dendritic cells, granulocytes, lymphocytes, etc.). These cells and especially activated fibroblasts actively participate in metastasis development.
  • the “unfolded protein response” is an endoplasmic reticulum stress response characterized by upregulation of UPR-related genes, including protein kinase RNA- like endoplasmic reticulum kinase (PERK), binding immunoglobulin protein (BiP), CCAAT/enhancer-binding protein homologous protein (CHOP), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), endoplasmic reticulum to nucleus signaling 1 (ERN1, which encodes inositol -requiring enzyme 1 (IRE1)), X-box binding protein 1 (XBP1), and XBP1 spliced protein (XBPls).
  • PERK protein kinase RNA- like endoplasmic reticulum kinase
  • BiP binding immunoglobulin protein
  • ATF4 activating transcription factor 4
  • ATF6 activating transcription factor 6
  • Certain food allergens promote food allergy by inducing an epithelial cell UPR, which in turn, causes these cells to express pro-Th2 cytokines, including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP).
  • pro-Th2 cytokines including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP).
  • DPMI knockout (KO) in cancer cells prevents M2 -like macrophages polarization overcoming immunosuppression and enhances cytotoxic T cell activity.
  • macrophage has its general meaning in the art and refers to a type of antigen-presenting cell of the mammalian immune system that have phagocytic activities. These cells are characterized by their distinctive morphology and high levels of surface MHC- class II expression.
  • a macrophage is a monocyte-derived phagocyte which is not a dendritic cell or a cell that derives from tissue macrophages by local proliferation. In the body these cells are tissue specific and refer to e. g. Kupffer cells in the liver, alveolar macrophages in the lung, microglia cells in the brain, osteoclasts in the bone etc.
  • the skilled person is aware how to identify macrophage cells, how to isolate macrophage cells from the body of a human or animal, and how to characterize macrophage cells with respect to their subclass and subpopulation.
  • Macrophages have historically been divided into two phenotypically diverse populations, i.e. a Ml -like -polarized or "classically activated” population, and a macrophage M2 -like -polarized or “alternatively activated” population.
  • Ml -like -polarized or "classically activated” population a macrophage M2 -like -polarized or "alternatively activated” population.
  • Macrophages exhibiting a Ml -like phenotype are pro-inflammatory, and are capable of either direct (pathogen pattern recognition receptors) or indirect (Fc receptors, complement receptors) recognition of pathogens and tumor antigens (i.e. they exhibit anti -tumor activity).
  • Ml-like macrophages produce reactive oxygen species and secrete pro-inflammatory cytokines and chemokines, such as, for example, but without limitation, TNFa, IL-1, IL-6, IL-15, IL-18, IL- 23, and iNOS.
  • Ml-like macrophages also express high levels of MHC, costimulatory molecules, and FCyR.
  • the Ml-like phenotype is triggered by GM-CSF and further stimulated by interferon-y (IFN-y), bacterial lipopolysaccharide (LPS), or tumor necrosis factor a (TNFa), and is mediated by several signal transduction pathways involving signal transducer and activator of transcription (STAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB), and mitogen-activated protein kinases (MAPK).
  • IFN-y interferon-y
  • LPS bacterial lipopolysaccharide
  • TNFa tumor necrosis factor a
  • STAT signal transducer and activator of transcription
  • NF-KB nuclear factor kappa-light-chain-enhancer of activated B cells
  • MAPK mitogen-activated protein kinases
  • macrophages exhibiting a M2 -like phenotype are often characterized as being antiinflammatory and immunosuppressive as they suppress T-cell responses and are involved in the Th2-type immune response.
  • the M2 macrophage phenotype facilitates tissue repair, wound healing, and is profibrotic.
  • M2-like macrophages often undesirably infiltrate and surround tumors, where they provide an immunosuppressive microenvironment that promotes rather than suppresses tumor progression.
  • M2-like macrophages are characterized by high surface expression of I1-4R, FcsR, Dectin-1, CD 136, CD206, and CD209A.
  • M2-like macrophages include IL-4/IL- 13 -stimulated macrophages, IL-10-induced macrophages, and immune complex-triggered macrophages.
  • the term “polarization” refers to the phenotypic features and the functional features of the macrophages.
  • the phenotype can be defined through the surface markers expressed by the macrophages.
  • the functionality can be defined for example based on the nature and the quantity of chemokines and/or cytokines expressed, in particular secreted, by the macrophages. Indeed, the macrophages present different phenotypic and functional features depending of their state, either pro-inflammatory Ml-type macrophage or anti-inflammatory M2 -type macrophage.
  • M2 -type macrophages can be characterized by the expression of surface markers such as CD206, CD 163, PD-L1 and CD200R and then secretion of cytokines such as CCL17, IL-10, TGFb.
  • Ml-type macrophages can be defined by the expression of surface markers such as CD86 and CCR7 and the secretion of cytokines such as IL-6, TNF-a and IL12p40.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, oesophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the patient suffers from a solid cancer selected from the group consisting of skin cancer (e.g. melanoma, nonmelanoma skin cancer), colorectal cancer, adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma), sarcomas such as liposarcoma and soft-tissue sarcoma, brain and central nervous system cancer (e.g.
  • skin cancer e.g. melanoma, nonmelanoma skin cancer
  • colorectal cancer e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer
  • bladder cancer e.g. osteoblastoma, osteosarcoma
  • breast cancer e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ
  • cervical cancer e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ
  • endometrial cancer e.g.
  • esthesioneuroblastoma midline granuloma
  • nasopharyngeal cancer neuroblastoma
  • oral cavity and oropharyngeal cancer ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g.
  • rhabdomyosarcoma e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma
  • salivary gland cancer stomach cancer
  • the patient suffers from a hematological cancer such as leukaemia, lymphoma (such as Hodgkin lymphoma or non-Hodgkin lymphoma) and myeloma.
  • a hematological cancer such as leukaemia, lymphoma (such as Hodgkin lymphoma or non-Hodgkin lymphoma) and myeloma.
  • the patient suffers from a cold tumor.
  • cold tumor refers to a tumor or cancer associated with suppressive immune cells, such as myeloid-derived suppressor cells and/or Tregs.
  • the cold tumor or cancer is ovarian cancer, prostate cancer, lung cancer or pancreatic cancer.
  • the patient suffers from lung cancer (cold tumor).
  • the patient suffers from a hot tumor.
  • the term “hot tumor” refers to a tumor that that contains T cells and expresses neoantigens.
  • the hot tumor is a bladder cancer, head and neck cancer, kidney cancer, liver cancer, colorectal cancer, melanoma, non-small cell lung cancer, or microsatellite instability high cancer.
  • the patient suffers from colorectal cancer.
  • metastasis or “tumour metastasis” is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream or lymphatics, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass.
  • metastatic tumor refers to a tumor that is capable of metastasizing, but has not yet metastasized to tissues or organs elsewhere in the body. In certain embodiments, the term metastatic tumor refers to a tumor that has metastasized to tissues or organs elsewhere in the body.
  • treating refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • improving cancer immunotherapy also known as immunooncology, refers to the improvement of a form of cancer treatment that uses the power of the body’s own immune system to prevent, control, and eliminate cancer.
  • dolichol refers to any of a group of long-chain mostly unsaturated organic compounds that are made up of varying numbers of isoprene units terminating in an a- saturated isoprenoid group, containing an alcohol functional group.
  • DPMI dolichol-phosphate mannosyltransferase subunit 1
  • Dol-P-Man dolichol-phosphate-mannose
  • Dol-P-Man serves as a donor of mannosyl residues in various eukaryotic glycosylation processes, including N-glycosylation of asparagine residues and O-mannosylation of alpha-dystroglycan.
  • the DPMI protein contains 260 amino acids for an estimated molecular weight of 29634 Da. DPMI is having the following UniProt Accession 060762.
  • the terms “inhibit”, “inhibiting”, and “inhibition” refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells.
  • the inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.
  • the inhibitor of DPMI is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.
  • the inhibitor of DPMI is a polypeptide or fragment thereof.
  • polypeptide refers both short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues), and longer peptides (the usual interpretation of "polypeptide", i.e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, oligopeptide, or polypeptide which may be chemically modified by being glycosylated, by being lipidated, or by comprising prosthetic groups).
  • polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art.
  • expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is in particular generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system.
  • Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown.
  • a common, preferred bacterial host is E coli.
  • the polypeptides of the invention and fragments thereof according to the invention can exhibit post-translational modifications, including, but not limited to glycosylations, (e.g., N- linked or O-linked glycosylations), myristylations, palmitylations, acetylations and phosphorylations (e.g., serine/threonine or tyrosine).
  • glycosylations e.g., N- linked or O-linked glycosylations
  • myristylations e.g., palmitylations
  • acetylations and phosphorylations e.g., serine/threonine or tyrosine.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
  • the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
  • adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • the inhibitor of DPMI is a peptidomimetic.
  • peptidomimetic means a peptide-like molecule that has the activity of the peptide upon which it is structurally based.
  • peptidomimetics include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids, and peptoids, and have an activity such as selective homing activity of the peptide upon which the peptidomimetic is derived (see, for example, Goodman and Ro, Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages 803-861).
  • Peptidomimetics may be designed in order to increase peptide stability, bioavailability, solubility, etc.
  • the inhibitor of DPMI is an aptamer.
  • Aptamers refers to a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • the inhibitor of DPMI is a small organic molecule.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • the inhibitor of DPMI is an RNase domain inhibitor.
  • the inhibitor of DPMI is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of metabolites involved in DPMI metabolism.
  • shRNA short hairpin RNA
  • siRNA small interfering RNA
  • antisense oligonucleotide which inhibits the expression of metabolites involved in DPMI metabolism.
  • the inhibitor of DPMI is siRNA.
  • a short hairpin RNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound.
  • RISC RNA-induced silencing complex
  • siRNA Small interfering RNA
  • silencing RNA are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene.
  • RNAi RNA interference
  • the inhibitor of DPMI is an anti-sense oligonucleotides (ASO).
  • ASO anti-sense oligonucleotides
  • Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g.
  • Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno- associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno- associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus
  • the inhibitor of DPMI is an endonuclease.
  • staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer.
  • these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis.
  • the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years.
  • NHEJ errorprone nonhomologous end-joining
  • HDR high-fidelity homology-directed repair
  • the endonuclease is CRISPR-cas.
  • CRISPR-cas has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.
  • the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes.
  • the CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing.
  • the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
  • the inhibitor of DPMI is an antibody.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • the term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical” scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"
  • Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments.
  • the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567.
  • the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409.
  • the antibody is a human antibody.
  • a “human antibody” such as described in US 6,075,181 and 6,150,584.
  • the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388.
  • the inhibitor of DPMI is a monoclonal antibody.
  • Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.
  • the inhibitor of DPMI is an intrabody.
  • intrabody generally refer to an intracellular antibody or antibody fragment.
  • Antibodies in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention.
  • the intrabody is a single domain antibody.
  • the antibody according to the invention is a single domain antibody.
  • single domain antibody sdAb or "VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • the inhibitor of DPMI is an inhibitor of DPMI expression.
  • an “inhibitor of DPMI expression” refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the gene encoding for DPMI.
  • the inhibitor of DPMI expression has a biological effect on one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • the inhibitor of DPMI expression is an antisense oligonucleotide.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of DPMI mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of DPMI proteins, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding DPMI can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
  • the inhibitor of DPMI expression is a shRNA.
  • shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound.
  • RISC RNA-induced silencing complex
  • the inhibitor of DPMI expression is a small inhibitory RNAs (siRNAs).
  • DPMI expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that DPMI expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference or RNAi
  • the siRNA is ALN-PCS02 developed by Alnylam (phase 1 ongoing).
  • inhibitor of DPMI expression is a ribozyme.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of DPMI mRNA sequences are thereby useful within the scope of the present invention.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC.
  • RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable.
  • the suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of DPMI) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • a substance as it exists outside the body (e.g., an inhibitor of DPMI) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • a “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject.
  • a “therapeutically effective amount” to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the inhibitors of DPMI as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as a carboxylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, sulfate, adiluent, encapsulating material or formulation auxiliary of any type.
  • compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the invention refers to a method for treating a subject suffering from a cancer comprising a step of administering said subject with i) a therapeutically effective amount of an inhibitor of DPMI and ii) classical treatment of cancer.
  • the invention also refers to a method for improving cancer immunotherapy in a subject suffering from a cancer comprising a step of administering said subject with i) a therapeutically effective amount of an inhibitor of DPMI and ii) classical treatment of cancer.
  • the invention relates to i) an inhibitor of DPMI and iii) a classical treatment used as a combined preparation for treating a subject suffering from a cancer.
  • the invention relates to i) an inhibitor of DPMI and iii) a classical treatment used as a combined preparation for improving cancer immunotherapy in a subject suffering from a cancer.
  • an inhibitor of DPMI and ii) a classical treatment as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer.
  • a classical treatment as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer.
  • the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication.
  • the combined therapy may be dual therapy or bi-therapy.
  • administration simultaneously refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time.
  • administration separately refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes.
  • administration sequentially refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
  • classical treatment refers to any compound, natural or synthetic, used for the treatment of cancer.
  • the classical treatment refers to radiation therapy, antibody therapy or chemotherapy.
  • an inhibitor of DPMI and ii) immunotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer.
  • an inhibitor of DPMI and ii) immunotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer.
  • the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response.
  • the immunotherapy consists of use of an immune check point inhibitor as described above.
  • an inhibitor of DPMI and ii) chemotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer.
  • an inhibitor of DPMI and ii) chemotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • chemotherapeutic agents include multkinase inhibitors such as sorafenib and sunitinib, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its thiotepa and
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • antihormonal agents that act to regulate or inhibit honnone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • an inhibitor of DPMI and ii) radiation therapy as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer.
  • an inhibitor of DPMI and ii) radiation therapy as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer.
  • radiation therapy or “radiotherapy” have their general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow.
  • One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body.
  • Linear accelerators and betatrons produce x-rays of increasingly greater energy.
  • the use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy.
  • Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay.
  • the radiation therapy is external radiation therapy.
  • external radiation therapy examples include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction
  • an inhibitor of DPMI and ii) immune checkpoint inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer.
  • an inhibitor of DPMI and ii) immune checkpoint inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer.
  • immune checkpoint inhibitor refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins.
  • immune checkpoint protein has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules).
  • Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489).
  • Examples of stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS.
  • inhibitory checkpoint molecules examples include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA.
  • A2AR Adenosine A2A receptor
  • B7-H4 also called VTCN1
  • B and T Lymphocyte Attenuator (BTLA) and also called CD272 has HVEM (Herpesvirus Entry Mediator) as its ligand.
  • HVEM Herpesvirus Entry Mediator
  • Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA.
  • CTLA-4 Cytotoxic T- Lymphocyte- Associated protein 4 and also called CD 152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation.
  • IDO Indoleamine 2,3-dioxygenase
  • TDO tryptophan catabolic enzyme
  • Another important molecule is TDO, tryptophan 2,3-dioxygenase.
  • IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis.
  • KIR Killercell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells.
  • LAG3, Lymphocyte Activation Gene-3 works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells.
  • PD-1 Programmed Death 1 (PD-1) receptor
  • PD-L1 and PD-L2 This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014.
  • An advantage of targeting PD- 1 is that it can restore immune function in the tumor microenvironment.
  • TIM-3 short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines.
  • TIM-3 acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9.
  • VISTA Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti -turn or T-cell response.
  • an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade.
  • the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.
  • the immune checkpoint inhibitor is an antibody.
  • antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
  • the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302.
  • anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).
  • the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897.
  • anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).
  • the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.
  • the immune checkpoint inhibitor inhibits Tim-3 or its ligand.
  • the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and W02013006490.
  • the immune checkpoint inhibitor is a small organic molecule.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
  • small organic molecules interfere with transduction pathway of PD-1 and Tim-3.
  • they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.
  • the small organic molecules interfere with Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor.
  • IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677.
  • IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5- methoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin
  • the IDO inhibitor is selected from 1 -methyl -tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.
  • the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4-fluorophenyl)-N'- hydroxy-4- ⁇ [2-(sulfamoylamino)-ethyl]amino ⁇ -l,2,5-oxadiazole-3 carboximidamide :
  • the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:
  • the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand- 1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015).
  • PD-L1 programmed death ligand- 1
  • VISTA V-domain Ig suppressor of T cell activation
  • the immune checkpoint inhibitor is an aptamer.
  • the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
  • aptamers are DNA aptamers such as described in Prodeus et al 2015.
  • a major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration.
  • aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG).
  • the aptamer is an anti-PD-1 aptamer.
  • the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.
  • the DPMI inhibitor for use according to the invention combined with classical treatment as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • the invention relates to a pharmaceutical composition comprising a DPMI inhibitor for use in the treatment of cancer.
  • the invention relates to a pharmaceutical composition comprising a DPMI inhibitor for use in a method for improving cancer immunotherapy.
  • the pharmaceutical composition according to the invention comprising i) DPMI inhibitor and ii) a classical treatment, as a combined preparation for use in the treatment of cancer.
  • the pharmaceutical composition according to the invention comprising i) DPMI inhibitor and ii) a classical treatment, as a combined preparation for use in a method for improving cancer immunotherapy.
  • DPMI inhibitor as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as a carboxylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, phosphatethacrylate, phosphate, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, sulfate
  • compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intravitreal administration, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Method de screening also relates to a method of screening a drug suitable for the treatment of cancer comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and/or activity of DPMI metabolism.
  • the present invention also relates to a method of screening a drug suitable for improving cancer immunotherapy comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and/or activity of DPMI metabolism.
  • Tests for determining the capacity of a compound to be inhibitor of DPMI are well known to the person skilled in the art. Inhibition of the DPMI may be determined by any techniques well known in the art. For instance, an inhibitor of DPMI can be identified by carrying out the following steps: i) providing a plurality of test substances ii) determining whether the test substances are inhibitors of DPMliii) positively selecting the test substances that are inhibitor of DPMI.
  • the test substances that have been positively selected may be subjected to further selection steps in view of further assaying its properties for the treatment of cancer.
  • the candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties on animal models.
  • the assays may be performed using high throughput screening techniques for identifying test substances for developing drugs (inhibitor of DPMI) that may be useful to the treatment of cancer.
  • High throughput screening techniques may be carried out using multi-well plates (e.g., 96-, 389-, or 1536-well plates), in order to carry out multiple assays using an automated robotic system.
  • multi-well plates e.g., 96-, 389-, or 1536-well plates
  • large libraries of test substances may be assayed in a highly efficient manner. Compounds in the library will be applied one at a time in an automated fashion to the wells of the microtitre dishes. Once the test substances which inhibits the DPMI are identified, they can be positively selected for further characterization.
  • control substance refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression.
  • test compounds capable of activating or inhibiting the activity or expression of DPMI are likely to exhibit similar modulatory capacity in applications in vivo.
  • the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies (e.g. intra-antibodies), aptamers or nucleic acids.
  • the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Invalidation of DPMI affects Tumor Development in a CD8-Dependent in colorectal carcinoma cells (hot tumor).
  • A) CT26 colorectal carcinoma cells (Mock and KO for DPMI) were injected sub-cutaneously in immunocompetent BalbC mice - in which CD8 T cells were depleted (anti-CD8+) or not. Tumor growth was monitored over time. n 10/group.
  • B) average tumor weight in each group. n 9-10 /group.
  • FIG. 2 Invalidation of DPMI affects Tumor Development in a CD8-Dependent in Lung carcinoma cells (cold tumor).
  • A) LLC1 Lung carcinoma cells (Mock and KO for DPMI) were injected sub-cutaneously in immunocompetent C57BL/6 mice - in which CD8 T cells were depleted (anti-CD8+) or not. Tumor growth was monitored over time. n 8-9/group.
  • B) average tumor weight in each group. n 8-9 / group. ** p ⁇ 0,01, ***p ⁇ 0.001.
  • NS non-significant.
  • CT26 cells mock KO or rescued for DPMI expression (DMP1 CT26 KO cells transduced with a control vector or a vector expressing DMP1) were subjected for immunblots for the indicated proteins. P-actin is used as a leading control.
  • FIG. 4 DPMI invalidation overcomes immunotherapy resistance in LLC-1 cold tumor.
  • WT syngeneic BALB/c and C57BL/6 mice were obtained from ENVIGO and housed in our animal facility (C3M-Nice, France). WT syngeneic BALB/c were subcutaneously engrafted with 0.75 / I O 6 CT26 cells while C57BL/6 mice were subcutaneously engrafted with 0.5 x 10 6 LLC1. After subcutaneous engraftment of CT26 and LLC1 cells, mice were inspected every two days for tumor development. Tumor growth was monitored by caliper measurement following the equation (width2 x length)/2.
  • mice were intraperitoneally injected with 100 ug of an anti-CD8-depleting antibody (Bioxcell, clone53-6.7, #BE0004-l) or vehicle (PBS) every second day for seven doses during 2 weeks after tumor cell injection.
  • an anti-CD8-depleting antibody Bioxcell, clone53-6.7, #BE0004-l
  • vehicle PBS
  • mice were intraperitoneally injected with lOmg/Kg of anti-PD-1 blocking antibody (Biolegend, 135248) or anti-CTLA-4 blocking antibody (Bioxcell, Clone 9D9, BX-BE0164) or vehicle (PBS) every three days after the fifth day of tumor cell injection.
  • CT26 cells were obtained from the ATCC (#CRL-2638) and cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (5000 U/mL) (Gibco), and 1% sodium pyruvate (Gibco).
  • LL/2 (LLC1) cells were obtained from the EC ACC (#90020104) and cultured in DMEM (Gibco) supplemented with 10% FBS.
  • the rescue of DPMI expression in CT26 KO cells was generated by the overexpression (OE) of the wild type of form of DPMI to levels equivalent to the endogenous expression using a lentiviral vector coding for mouse IRE la (mDPMl) and eGFP under the control of the EFl A promoter was designed pLV[Exp]-EGFP:T2A:Puro-EFlA>mDpml and purchased from VectorBuilder (VB900002-2967sue). This lentiviral vector was used to generate the control vector that only expresses EGFP. All cell lines were mycoplasma free. All cell lines were incubated at 37°C in a 5% CO2 atmosphere.
  • Nanoblades were produced from transfected producer 293T cells plated at 2.6 x io 6 cells/10 cm plate 24 h before transfection with a calcium phosphate transfection method.
  • Plasmids encoding the GagMLV-CAS9 fusion (3 pg), Gag-POLMLV (3 pg), gRNA expressing plasmid(s) (3 pg), VSV-G (2 pg), the Baboon Endogenous retrovirus Riess glycoprotein (BaEVRless) (2 pg) were cotransfected and supernatants were collected from producer cells after 40 h. For production of serum -free particles, medium was replaced 24 h after transfection by 10 ml of Optimem (Gibco) supplemented with penicillin-streptomycin.
  • Nanoblade-containing medium was clarified by a short centrifugation (500 x g 5 min) and filtered through a 0.45 pm pore-size filter before overnight centrifugation (3000 x g). Pellet was resuspended by gentle agitation in 400 pl of OptiMEM medium.
  • CT26 and LLC1 tumors were dissociated using the Tumor Dissociation Kit for mice (Miltenyi Biotec, #130-096-730) yielding a single-cell suspension. Immune infiltrating cells were characterized using a spectral FACS - Cytek Aurora analysis.
  • LT8 CD45+CD3+CD8+
  • LT4 CD45+CD3+CD4+
  • Tregs CD45+CD3+CD4+CD25+FOXP3+
  • NK CD45+CD3-NK1.1+ or CD49b
  • LB CD45+ B220+ CD19+
  • DC CD45+ CDl lb+ CDl lc+ MHCII+F4/80-CD24+
  • Macrophages Cd45+ CD1 lb+ CD1 lc+ MHCII+ F4/80+ CD24-).
  • the inventors showed that invalidation of DPMI affects Tumor Development in a CD8- Dependent in colorectal carcinoma cells, a hot tumor Figures 1A to ID) and that invalidation of DPMI affects Tumor Development in a CD8-Dependent in Lung carcinoma cells, a cold tumor ( Figures 2A and 2B).
  • the inventors also showed that DPMI invalidation decrease PD- L1 expression and binding to PD1 ( Figures 3A and 3B)
  • the inventors also show that DPMI invalidation overcomes immunotherapy resistance in LLC-1 cold tumor ( Figures 4A and 4B).
  • the inventors thus demonstrated that how tumoral UPR can limit tumor growth and suggests that DPMI inhibition is a useful strategy for improving cancer immunotherapy.

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Abstract

The tumor microenvironment (TME) is characterized by hostile conditions that lead to the induction of the unfolded protein response (UPR) in both cancer cells and immune infiltrating cells. Mechanistically, DPM1 KO leads to reduced production of key cytokines and cell surface expression of PD-L1 (a major player in the control of adaptive immune response) through ERAD (ER-associated protein degradation). Additionally, DPM1 KO in cancer cells limited M2-like macrophages polarization overcoming immunosuppression and enhances cytotoxic T cell activity. Thus, the inventors' work reveals how tumoral UPR can limit tumor growth and suggests that DPM1 inhibition is a useful strategy for improving cancer immunotherapy. The present invention relates to a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Dolichol-phosphate mannosyltransferase (DPM1) inhibitor.

Description

DPMI INHIBITOR FOR TREATING CANCER
FIELD OF THE INVENTION:
The invention is in the field of oncology. More particularly, the invention relates to methods and composition for treating a cancer.
BACKGROUND OF THE INVENTION:
The tumor microenvironment (TME) is characterized by hostile conditions that lead to the induction of the unfolded protein response (UPR) in both cancer cells and immune infiltrating cells. However, how the UPR in cancer cells can modulate the anti-cancer immune response remains to be discovered. To elucidate this effect, the inventors performed a characterization of the IREl-a interactome using a proximity-dependent biotinylation-based approach (BioID). Among the different partners identified, they showed that Dolichol-phosphate mannosyltransferase (DPMI), a key player in proteins N- and O-glycosylation, controlled the immunogenicity of tumoral cells (using colon and lung cancer models). Indeed, they demonstrated in hot and cold tumor models that DPMI KO prevented tumor growth only in the presence of a functional adaptive immune system (as depleting cytotoxic CD8+ T cells prevented the protective effect). Mechanistically, DPMI KO leads to reduced production of key cytokines and cell surface expression of PD-L1 (a major player in the control of adaptive immune response) through ERAD (ER-associated protein degradation). Additionally, DPMI KO in cancer cells limited M2 -like macrophages polarization overcoming immunosuppression and enhances cytotoxic T cell activity. Thus, the inventors’ work reveals how tumoral UPR can limit tumor growth and suggests that DPMI inhibition is a useful strategy for improving cancer immunotherapy.
SUMMARY OF THE INVENTION:
The invention relates to a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Dolichol-phosphate mannosyltransferase (DPMI) inhibitor. In particular, the invention is defined by claims.
DETAILED DESCRIPTION OF THE INVENTION: Method of treatment
The present invention relates to a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Dolicholphosphate mannosyltransf erase (DPMI) inhibitor.
The present invention relates to a method of improving cancer immunotherapy in a patient suffering from a cancer comprising administering to the patient a therapeutically effective amount of a Dolichol-phosphate mannosyltransferase (DPMI) inhibitor.
As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with or susceptible to be afflicted with cancer.
As used herein, the term “IREla” refers to inositol-requiring enzyme 1 (IRE1) and in humans is encoded by the ERN1 gene. It is an endoplasmic reticulum (ER)-resident transmembrane signaling protein and a cellular stress sensor. This protein harbors a cytosolic dual kinase/endoribonuclease activity required for adaptive responses to micro-environmental changes.
As used herein, the term “RNase activity of IRE1” refers to the activity of the endoribonuclease domain of IRE1 which degrades specific RNA (mRNA or microRNA) to avoid their translation or their cellular activity, an activity known as the RIDD (regulated IRE1- dependent decay of RNA), or contributes to the splicing XBP1 (X-box-binding protein 1) mRNA to change the reading frame leading to the production of a novel protein (XBPls), a potent unfolded-protein response transcriptional activator. Typically, the IRE1 endonuclease activity is based on recognition of an RNA stem loop structure found twice in substrates HAC1 mRNA in yeast or XBP1 mRNA in metazoans. Cleavage of HAC1 or XBP1 mRNA occurs at both sites resulting in an mRNA fragment whose two ends are ligated in a unique splicing event (Ron et al.; Lee et al.). The spliced HAC1 or XBP1 mRNAs encode transcription factor that activate numerous target genes, including genes involved in the unfolded protein response (UPR). The UPR is a signal transduction cascade that occurs in response to the accumulation of misfolded proteins in the ER. As used herein, the terms “stroma” or “microenvironment” refer to the connective, supportive framework of a biological cell, tissue, or organ. As used herein, the term “tumor microenvironment” refers to the cellular environment in which the tumor exists, including the area immediately surrounding fibroblasts, leukocytes and endothelial cells and the extracellular matrix (ECM). The term “stromal cells” include fibroblasts, leukocytes and vascular cells. Accordingly, cells of a tumor microenvironment comprise malignant cells in association with non-malignant cells that support their growth and survival. The non-malignant cells, also called stromal cells, occupy or accumulate in the same cellular space as malignant cells, or the cellular space adjacent or proximal to malignant cells, which modulate tumor cell growth or survival. Non-malignant cells of the tumor microenvironment include fibroblasts, epithelial cells, vascular cells (including blood and lymphatic vascular endothelial cells and pericytes), resident and/or recruited inflammatory and immune (e.g., macrophages, dendritic cells, granulocytes, lymphocytes, etc.). These cells and especially activated fibroblasts actively participate in metastasis development.
As used herein, the “unfolded protein response” (UPR) is an endoplasmic reticulum stress response characterized by upregulation of UPR-related genes, including protein kinase RNA- like endoplasmic reticulum kinase (PERK), binding immunoglobulin protein (BiP), CCAAT/enhancer-binding protein homologous protein (CHOP), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), endoplasmic reticulum to nucleus signaling 1 (ERN1, which encodes inositol -requiring enzyme 1 (IRE1)), X-box binding protein 1 (XBP1), and XBP1 spliced protein (XBPls). Certain food allergens promote food allergy by inducing an epithelial cell UPR, which in turn, causes these cells to express pro-Th2 cytokines, including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP).
The inventors show that DPMI knockout (KO) in cancer cells prevents M2 -like macrophages polarization overcoming immunosuppression and enhances cytotoxic T cell activity.
As used herein, the term “macrophage” has its general meaning in the art and refers to a type of antigen-presenting cell of the mammalian immune system that have phagocytic activities. These cells are characterized by their distinctive morphology and high levels of surface MHC- class II expression. A macrophage is a monocyte-derived phagocyte which is not a dendritic cell or a cell that derives from tissue macrophages by local proliferation. In the body these cells are tissue specific and refer to e. g. Kupffer cells in the liver, alveolar macrophages in the lung, microglia cells in the brain, osteoclasts in the bone etc. The skilled person is aware how to identify macrophage cells, how to isolate macrophage cells from the body of a human or animal, and how to characterize macrophage cells with respect to their subclass and subpopulation.
Macrophages have historically been divided into two phenotypically diverse populations, i.e. a Ml -like -polarized or "classically activated" population, and a macrophage M2 -like -polarized or "alternatively activated" population. However, it is well appreciated in the art that a continuum of phenotypes exists between the macrophage Ml- polarized and macrophage M2- polarized populations, and in some cases macrophages assume a phenotype that does not fit well within any of these defined phenotypic groups.
Macrophages exhibiting a Ml -like phenotype are pro-inflammatory, and are capable of either direct (pathogen pattern recognition receptors) or indirect (Fc receptors, complement receptors) recognition of pathogens and tumor antigens (i.e. they exhibit anti -tumor activity). Ml-like macrophages produce reactive oxygen species and secrete pro-inflammatory cytokines and chemokines, such as, for example, but without limitation, TNFa, IL-1, IL-6, IL-15, IL-18, IL- 23, and iNOS. Ml-like macrophages also express high levels of MHC, costimulatory molecules, and FCyR. The Ml-like phenotype is triggered by GM-CSF and further stimulated by interferon-y (IFN-y), bacterial lipopolysaccharide (LPS), or tumor necrosis factor a (TNFa), and is mediated by several signal transduction pathways involving signal transducer and activator of transcription (STAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB), and mitogen-activated protein kinases (MAPK). These events enhance the production of agents such as the reactive oxygen species and nitric oxide (NO) and promote subsequent inflammatory immune responses by increasing antigen presentation capacity and inducing the Thl immunity through the production of cytokines such as IL-12.
In contrast, macrophages exhibiting a M2 -like phenotype are often characterized as being antiinflammatory and immunosuppressive as they suppress T-cell responses and are involved in the Th2-type immune response. The M2 macrophage phenotype facilitates tissue repair, wound healing, and is profibrotic. M2-like macrophages often undesirably infiltrate and surround tumors, where they provide an immunosuppressive microenvironment that promotes rather than suppresses tumor progression. M2-like macrophages are characterized by high surface expression of I1-4R, FcsR, Dectin-1, CD 136, CD206, and CD209A. M2-like macrophages include IL-4/IL- 13 -stimulated macrophages, IL-10-induced macrophages, and immune complex-triggered macrophages.
As used herein, the term “polarization” refers to the phenotypic features and the functional features of the macrophages. The phenotype can be defined through the surface markers expressed by the macrophages. The functionality, can be defined for example based on the nature and the quantity of chemokines and/or cytokines expressed, in particular secreted, by the macrophages. Indeed, the macrophages present different phenotypic and functional features depending of their state, either pro-inflammatory Ml-type macrophage or anti-inflammatory M2 -type macrophage. M2 -type macrophages can be characterized by the expression of surface markers such as CD206, CD 163, PD-L1 and CD200R and then secretion of cytokines such as CCL17, IL-10, TGFb. Ml-type macrophages can be defined by the expression of surface markers such as CD86 and CCR7 and the secretion of cytokines such as IL-6, TNF-a and IL12p40.
As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, oesophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
In a particular embodiment, the patient suffers from a solid cancer selected from the group consisting of skin cancer (e.g. melanoma, nonmelanoma skin cancer), colorectal cancer, adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma), sarcomas such as liposarcoma and soft-tissue sarcoma, brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ), cervical cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinoma), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hepatic adenoma, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma). In a particular embodiment, the patient suffers from a hematological cancer such as leukaemia, lymphoma (such as Hodgkin lymphoma or non-Hodgkin lymphoma) and myeloma.
In some embodiment, the patient suffers from a cold tumor. As used herein, the term “cold tumor” refers to a tumor or cancer associated with suppressive immune cells, such as myeloid-derived suppressor cells and/or Tregs. In some embodiments, the cold tumor or cancer is ovarian cancer, prostate cancer, lung cancer or pancreatic cancer.
In a particular embodiment, the patient suffers from lung cancer (cold tumor).
In some embodiment, the patient suffers from a hot tumor.
As used herein, the term “hot tumor” refers to a tumor that that contains T cells and expresses neoantigens. In some embodiments, the hot tumor is a bladder cancer, head and neck cancer, kidney cancer, liver cancer, colorectal cancer, melanoma, non-small cell lung cancer, or microsatellite instability high cancer.
In a particular embodiment, the patient suffers from colorectal cancer.
As used herein, the term “metastasis” or “tumour metastasis” is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream or lymphatics, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. In certain embodiments, the term metastatic tumor refers to a tumor that is capable of metastasizing, but has not yet metastasized to tissues or organs elsewhere in the body. In certain embodiments, the term metastatic tumor refers to a tumor that has metastasized to tissues or organs elsewhere in the body.
As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
As used herein the terms "improving cancer immunotherapy” also known as immunooncology, refers to the improvement of a form of cancer treatment that uses the power of the body’s own immune system to prevent, control, and eliminate cancer.
As used herein, the terms “dolichol” refers to any of a group of long-chain mostly unsaturated organic compounds that are made up of varying numbers of isoprene units terminating in an a- saturated isoprenoid group, containing an alcohol functional group.
As used herein, the terms “dolichol-phosphate mannosyltransferase subunit 1” (DPMI) refers to an enzyme complex that synthesizes dolichol-phosphate-mannose (Dol-P-Man) from GDP -mannose and dolichol-phosphate. Dol-P-Man serves as a donor of mannosyl residues in various eukaryotic glycosylation processes, including N-glycosylation of asparagine residues and O-mannosylation of alpha-dystroglycan. The DPMI protein contains 260 amino acids for an estimated molecular weight of 29634 Da. DPMI is having the following UniProt Accession 060762.
As used herein, the terms “inhibit”, “inhibiting”, and “inhibition” refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.
In a particular embodiment, the inhibitor of DPMI is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.
In some embodiments, the inhibitor of DPMI is a polypeptide or fragment thereof. As used herein, the term “polypeptide” refers both short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues), and longer peptides (the usual interpretation of "polypeptide", i.e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, oligopeptide, or polypeptide which may be chemically modified by being glycosylated, by being lipidated, or by comprising prosthetic groups).
The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptides for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. In particular, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is in particular generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.The polypeptides of the invention and fragments thereof according to the invention can exhibit post-translational modifications, including, but not limited to glycosylations, (e.g., N- linked or O-linked glycosylations), myristylations, palmitylations, acetylations and phosphorylations (e.g., serine/threonine or tyrosine). In some embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
In some embodiments, the inhibitor of DPMI is a peptidomimetic.
As used herein, the term "peptidomimetic" means a peptide-like molecule that has the activity of the peptide upon which it is structurally based. Such peptidomimetics include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids, and peptoids, and have an activity such as selective homing activity of the peptide upon which the peptidomimetic is derived (see, for example, Goodman and Ro, Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages 803-861). Peptidomimetics may be designed in order to increase peptide stability, bioavailability, solubility, etc.
In a particular embodiment, the inhibitor of DPMI is an aptamer.
As used herein, the term “Aptamers” refers to a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
In a particular embodiment, the inhibitor of DPMI is a small organic molecule.
As used herein, the term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
In a particular embodiment, the inhibitor of DPMI is an RNase domain inhibitor.
In some embodiments, the inhibitor of DPMI is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of metabolites involved in DPMI metabolism.
In a particular embodiment, the inhibitor of DPMI is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene.
In a particular embodiment, the inhibitor of DPMI is an anti-sense oligonucleotides (ASO). Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno- associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.
In some embodiments, the inhibitor of DPMI is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).
In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics.l 13.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.
In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
In some embodiments, the inhibitor of DPMI is an antibody.
As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388.
In a particular embodiment, the inhibitor of DPMI is a monoclonal antibody.
Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.
In a particular, the inhibitor of DPMI is an intrabody. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
In a particular embodiment, the inhibitor of DPMI is an inhibitor of DPMI expression.
An "inhibitor of DPMI expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the gene encoding for DPMI. Typically, the inhibitor of DPMI expression has a biological effect on one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
In some embodiments, the inhibitor of DPMI expression is an antisense oligonucleotide.
Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of DPMI mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of DPMI proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding DPMI can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
In a particular embodiment, the inhibitor of DPMI expression is a shRNA.
As used herein, the term “shRNA” is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound.
In some embodiments, the inhibitor of DPMI expression is a small inhibitory RNAs (siRNAs).
DPMI expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that DPMI expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). In a particular embodiment, the siRNA is ALN-PCS02 developed by Alnylam (phase 1 ongoing).
In some embodiments, inhibitor of DPMI expression is a ribozyme.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of DPMI mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of DPMI) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.
A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
The inhibitors of DPMI as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
Combined preparation:
The invention refers to a method for treating a subject suffering from a cancer comprising a step of administering said subject with i) a therapeutically effective amount of an inhibitor of DPMI and ii) classical treatment of cancer.
The invention also refers to a method for improving cancer immunotherapy in a subject suffering from a cancer comprising a step of administering said subject with i) a therapeutically effective amount of an inhibitor of DPMI and ii) classical treatment of cancer.
Accordingly, the invention relates to i) an inhibitor of DPMI and iii) a classical treatment used as a combined preparation for treating a subject suffering from a cancer.
Accordingly, the invention relates to i) an inhibitor of DPMI and iii) a classical treatment used as a combined preparation for improving cancer immunotherapy in a subject suffering from a cancer.
In a particular embodiment, i) an inhibitor of DPMI and ii) a classical treatment as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer.
In a particular embodiment, i) an inhibitor of DPMI and ii) a classical treatment as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer. As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.
As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
As used herein, the term “classical treatment” refers to any compound, natural or synthetic, used for the treatment of cancer.
In a particular embodiment, the classical treatment refers to radiation therapy, antibody therapy or chemotherapy.
In a particular embodiment, i) an inhibitor of DPMI and ii) immunotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer.
In a particular embodiment, i) an inhibitor of DPMI and ii) immunotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer.
As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response. In a particular embodiment, the immunotherapy consists of use of an immune check point inhibitor as described above.
In a particular embodiment, i) an inhibitor of DPMI and ii) chemotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer. In a particular embodiment, i) an inhibitor of DPMI and ii) chemotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer.
As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include multkinase inhibitors such as sorafenib and sunitinib, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In a particular embodiment, i) an inhibitor of DPMI and ii) radiation therapy as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer.
In a particular embodiment, i) an inhibitor of DPMI and ii) radiation therapy as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer. As used herein, the terms “radiation therapy” or “radiotherapy” have their general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.
In a particular embodiment, i) an inhibitor of DPMI and ii) immune checkpoint inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use for treating a subject suffering from a cancer.
In a particular embodiment, i) an inhibitor of DPMI and ii) immune checkpoint inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use for improving cancer immunotherapy in a subject suffering from a cancer. As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins.
As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T- Lymphocyte- Associated protein 4 and also called CD 152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killercell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD-1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD- 1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti -turn or T-cell response.
In some embodiments, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.
In a particular embodiment, the immune checkpoint inhibitor is an antibody.
Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).
In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS). In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.
In the context of the invention, the immune checkpoint inhibitor inhibits Tim-3 or its ligand.
In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and W02013006490.
In some embodiments, the immune checkpoint inhibitor is a small organic molecule.
The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.
In a particular embodiment, the small organic molecules interfere with Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5- methoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1 -methyl -tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.
In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4-fluorophenyl)-N'- hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-l,2,5-oxadiazole-3 carboximidamide :
Figure imgf000030_0001
In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:
Figure imgf000030_0002
In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand- 1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA-170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.
In some embodiments, the immune checkpoint inhibitor is an aptamer.
Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA. In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.
Pharmaceutical composition
The DPMI inhibitor for use according to the invention combined with classical treatment as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
Accordingly, the invention relates to a pharmaceutical composition comprising a DPMI inhibitor for use in the treatment of cancer.
Accordingly, the invention relates to a pharmaceutical composition comprising a DPMI inhibitor for use in a method for improving cancer immunotherapy.
In a particular embodiment, the pharmaceutical composition according to the invention comprising i) DPMI inhibitor and ii) a classical treatment, as a combined preparation for use in the treatment of cancer.
In a particular embodiment, the pharmaceutical composition according to the invention comprising i) DPMI inhibitor and ii) a classical treatment, as a combined preparation for use in a method for improving cancer immunotherapy.
DPMI inhibitor as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intravitreal administration, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
Method de screening The present invention also relates to a method of screening a drug suitable for the treatment of cancer comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and/or activity of DPMI metabolism.
The present invention also relates to a method of screening a drug suitable for improving cancer immunotherapy comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and/or activity of DPMI metabolism.
Tests for determining the capacity of a compound to be inhibitor of DPMI are well known to the person skilled in the art. Inhibition of the DPMI may be determined by any techniques well known in the art. For instance, an inhibitor of DPMI can be identified by carrying out the following steps: i) providing a plurality of test substances ii) determining whether the test substances are inhibitors of DPMliii) positively selecting the test substances that are inhibitor of DPMI. The test substances that have been positively selected may be subjected to further selection steps in view of further assaying its properties for the treatment of cancer. For example, the candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties on animal models.
The assays may be performed using high throughput screening techniques for identifying test substances for developing drugs (inhibitor of DPMI) that may be useful to the treatment of cancer. High throughput screening techniques may be carried out using multi-well plates (e.g., 96-, 389-, or 1536-well plates), in order to carry out multiple assays using an automated robotic system. Thus, large libraries of test substances may be assayed in a highly efficient manner. Compounds in the library will be applied one at a time in an automated fashion to the wells of the microtitre dishes. Once the test substances which inhibits the DPMI are identified, they can be positively selected for further characterization. Because this assay can readily be performed in a microtitre plate format, the assays described can be performed by an automated robotic system, allowing for testing of large numbers of test samples within a reasonably short time frame. The assays can be used as a screen to assess the activity of a previously untested compound or extract, in which case a single concentration is tested and compared to controls. These assays can also be used to assess the relative potency of a compound by testing a range of concentrations, in a range of 100 pM to 1 pM, for example, and computing the more efficient concentration. As used herein, the term "control substance", "control agent", or "control compound" refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of activating or inhibiting the activity or expression of DPMI, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies (e.g. intra-antibodies), aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1: Invalidation of DPMI affects Tumor Development in a CD8-Dependent in colorectal carcinoma cells (hot tumor). A) CT26 colorectal carcinoma cells (Mock and KO for DPMI) were injected sub-cutaneously in immunocompetent BalbC mice - in which CD8 T cells were depleted (anti-CD8+) or not. Tumor growth was monitored over time. n=10/group. B) average tumor weight in each group. n= 9-10 /group. C) as in A but at day 27 mice were subjected to a second tumor cells injection and tumor growth was monitored over time. The lack of tumor growth indicated that a long-term immune response (memory cells) was engaged by injecting DPMI -KO cells, n=5 mice upon challenge. D) Percentage of CD206+ macrophages (M2 -like, pro-tumoral) from whole tumors presented in A, as quantified by flow cytometry. ** p<0,01, NS: non-significant.
Figure 2: Invalidation of DPMI affects Tumor Development in a CD8-Dependent in Lung carcinoma cells (cold tumor). A) LLC1 Lung carcinoma cells (Mock and KO for DPMI) were injected sub-cutaneously in immunocompetent C57BL/6 mice - in which CD8 T cells were depleted (anti-CD8+) or not. Tumor growth was monitored over time. n=8-9/group. B) average tumor weight in each group. n= 8-9 / group. ** p<0,01, ***p < 0.001. NS: non-significant. Figure 3: DPMI invalidation decrease PD-L1 expression and binding to PD1. A) CT26 cells mock KO or rescued for DPMI expression (DMP1 CT26 KO cells transduced with a control vector or a vector expressing DMP1) were subjected for immunblots for the indicated proteins. P-actin is used as a leading control. B) CT26 cells were treated by IFNy (lOOng/ml). PD-L1 expression on their cell surface was determined by FACS (n=4). PD1 binding to PD-L1 expressed on the cell surface of CT26 cells was decrease by DPMI KO and maintained upon re-expression of DMP1 in KO cells.
Figure 4: DPMI invalidation overcomes immunotherapy resistance in LLC-1 cold tumor. A) LLC-1 cells (Mock and KO) were injected sub-cutaneously C57BL/6 mice - in which CTLA-4 and PD-1 receptors were blocked (using anti-CTLA-4 and anti-PD-1 antibodies) or not. Tumor growth was monitored over time (MOCK, n = 7; DPMI KO, n = 7; MOCK a-PD- 1 a-CTLA-4, n = 7; DPMI KO a-PD-1 a-CTLA-4, n = 6). B) Average tumor weight in each group. *p < 0.05 ; **p < 0.01; ***p < 0.001 ; ****p < 0.0001
EXAMPLE:
Material & Methods
Mice
All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee and the regional ethics committee (approval references PEA-503). All experiments used age-matched five-week-old female littermates. WT syngeneic BALB/c and C57BL/6 mice were obtained from ENVIGO and housed in our animal facility (C3M-Nice, France). WT syngeneic BALB/c were subcutaneously engrafted with 0.75 / I O6 CT26 cells while C57BL/6 mice were subcutaneously engrafted with 0.5 x 106 LLC1. After subcutaneous engraftment of CT26 and LLC1 cells, mice were inspected every two days for tumor development. Tumor growth was monitored by caliper measurement following the equation (width2 x length)/2. Animals were sacrificed when a tumor reached at least 1000 mm3. For antibody -mediated depletion experiments in vivo, mice were intraperitoneally injected with 100 ug of an anti-CD8-depleting antibody (Bioxcell, clone53-6.7, #BE0004-l) or vehicle (PBS) every second day for seven doses during 2 weeks after tumor cell injection. For antibody- mediated blockade experiments in vivo, mice were intraperitoneally injected with lOmg/Kg of anti-PD-1 blocking antibody (Biolegend, 135248) or anti-CTLA-4 blocking antibody (Bioxcell, Clone 9D9, BX-BE0164) or vehicle (PBS) every three days after the fifth day of tumor cell injection.
Cell lines and cell culture conditions
CT26 cells were obtained from the ATCC (#CRL-2638) and cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (5000 U/mL) (Gibco), and 1% sodium pyruvate (Gibco). LL/2 (LLC1) cells were obtained from the EC ACC (#90020104) and cultured in DMEM (Gibco) supplemented with 10% FBS. The rescue of DPMI expression in CT26 KO cells was generated by the overexpression (OE) of the wild type of form of DPMI to levels equivalent to the endogenous expression using a lentiviral vector coding for mouse IRE la (mDPMl) and eGFP under the control of the EFl A promoter was designed pLV[Exp]-EGFP:T2A:Puro-EFlA>mDpml and purchased from VectorBuilder (VB900002-2967sue). This lentiviral vector was used to generate the control vector that only expresses EGFP. All cell lines were mycoplasma free. All cell lines were incubated at 37°C in a 5% CO2 atmosphere.
Generation of CRISPR/Cas9 cells
For the generation of stable CT26 and LLC-1 cells with invalidated DPMI, cells were transfected nanoblades particles with CRISPR-Cas9-expressing knockout plasmid. Nanoblades were produced from transfected producer 293T cells plated at 2.6 x io6 cells/10 cm plate 24 h before transfection with a calcium phosphate transfection method. Plasmids encoding the GagMLV-CAS9 fusion (3 pg), Gag-POLMLV (3 pg), gRNA expressing plasmid(s) (3 pg), VSV-G (2 pg), the Baboon Endogenous retrovirus Riess glycoprotein (BaEVRless) (2 pg) were cotransfected and supernatants were collected from producer cells after 40 h. For production of serum -free particles, medium was replaced 24 h after transfection by 10 ml of Optimem (Gibco) supplemented with penicillin-streptomycin. Nanoblade-containing medium was clarified by a short centrifugation (500 x g 5 min) and filtered through a 0.45 pm pore-size filter before overnight centrifugation (3000 x g). Pellet was resuspended by gentle agitation in 400 pl of OptiMEM medium.
Western Blot
Cells were lysed with RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology) according to the protocol supplied. Protein lyses were quantified using Pierce BCA protein assay kit (Thermo Scientific, #23225). Whole cell lysates (30-50pg) were separated by SDS-PAGE, transferred to PVDF membrane. Membranes were subjected to immunoblot analyses using primary antibodies against PD-L1 (ab213480 Abeam), P-actin (sc-47778, Santa Cruz Biotechnology). Immunoblots were developed using the Amersham ECL Prime Western Blotting Detection Reagent (Cytiva, #RPN2236) and visualized with ImageQuant LAS 4000 (GE Healthcare, Life Science).
Flow cytometry analysis
CT26 and LLC1 tumors were dissociated using the Tumor Dissociation Kit for mice (Miltenyi Biotec, #130-096-730) yielding a single-cell suspension. Immune infiltrating cells were characterized using a spectral FACS - Cytek Aurora analysis. The following markers were used to define the different populations: LT8 (CD45+CD3+CD8+), LT4 (CD45+CD3+CD4+), Tregs (CD45+CD3+CD4+CD25+FOXP3+), NK (CD45+CD3-NK1.1+ or CD49b), LB (CD45+ B220+ CD19+), DC (CD45+ CDl lb+ CDl lc+ MHCII+F4/80-CD24+), and Macrophages (Cd45+ CD1 lb+ CD1 lc+ MHCII+ F4/80+ CD24-).
For PD-L1 and PD-1 FACS staining. Cells were stained on ice for 30 min with Recombinant Mouse PD-1 (CD279)-Fc Chimera (764806, Biolegend) at 10 pg/ml. Following a wash, bound Fc protein was detected with PE-conjugated F(ab’)2 goat anti-human Fc (398004, Biolegend). Samples were analyzed using a spectral FACS - Cytek Aurora.
Results:
The inventors showed that invalidation of DPMI affects Tumor Development in a CD8- Dependent in colorectal carcinoma cells, a hot tumor Figures 1A to ID) and that invalidation of DPMI affects Tumor Development in a CD8-Dependent in Lung carcinoma cells, a cold tumor (Figures 2A and 2B). The inventors also showed that DPMI invalidation decrease PD- L1 expression and binding to PD1 (Figures 3A and 3B) The inventors also show that DPMI invalidation overcomes immunotherapy resistance in LLC-1 cold tumor (Figures 4A and 4B).
The inventors thus demonstrated that how tumoral UPR can limit tumor growth and suggests that DPMI inhibition is a useful strategy for improving cancer immunotherapy.
REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:
1. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Dolichol-phosphate mannosy Itransferase (DPMI) inhibitor.
2. A method of improving cancer immunotherapy in a patient suffering from a cancer comprising administering to the patient a therapeutically effective amount of a Dolicholphosphate mannosyltransferase (DPMI) inhibitor.
3. The method according to 1 or 2 wherein, the inhibitor of DPMI is an antibody.
4. The method according to 1 or 2 wherein, the inhibitor of DPMI is a small molecule.
5. The method according to 1 or 2 wherein, the inhibitor of DPMI is a small inhibitory RNAs (siRNAs) or an antisense oligonucleotide.
6. The method according to 1 or 2 wherein the cancer is a skin cancer, colorectal cancer, adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, sarcomas such as liposarcoma and soft-tissue sarcoma, brain and central nervous system cancer, breast cancer, cervical cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer, neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified nonHodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
7. The method according to 1 or 2 wherein the cancer is a colorectal cancer.
8. The method according to 1 or 2 wherein the cancer is a lung cancer.
9. i) An inhibitor of DPMI and iii) a classical treatment for use as a combined preparation for treating a subject suffering from a cancer.
10. The inhibitor of DPMI and the classical treatment for use according to claim 9 wherein the classical treatment is radiation therapy, immunotherapy or chemotherapy.
11. An inhibitor of DPMI and iii) a classical treatment for use as a combined preparation for improving cancer immunotherapy in a subject suffering from a cancer.
12. The inhibitor of DPMI and the classical treatment for use according to claim 11 wherein the classical treatment is radiation therapy, immunotherapy or chemotherapy.
13. A pharmaceutical composition comprising a DPMI inhibitor for use in the treatment of cancer.
14. A pharmaceutical composition comprising a DPMI inhibitor for use in a method for improving cancer immunotherapy.
15. A method of screening a drug suitable for the treatment of cancer comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and/or activity of DPMI metabolism.
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