Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
• • 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
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity

Definitions

• the present invention relates to methods and pharmaceutical compositions for the treatment of osteoclast diseases in a subject in need thereof.
• Osteoclasts are large multinucleated highly specialised cells capable of breaking down both the inorganic (hydroxyapatite) and organic (collagen and multiple-non-collagenous proteins) matrix of bone, dentine and mineralised cartilage and are unique in their ability to resorb mineralised bone matrix (Helfrich et al., 2003). They form as a result of the fusion of mononuclear precursors derived from the monocyte/macrophage lineage, and this differentiation is controlled by interactions between osteoblasts and/or stromal cells and preosteoclasts (Crockett et al., 2011).
• monocytes/macrophages are at the origin of pathological bone erosion in RA due to their excessive differentiation into OCs, which are the only cells specialized in bone resorption (Thurlings et al., 2009). Their differentiation is mediated by two major cytokines, M-CSF and RANKL (receptor activator of nuclear factor kB ligand). M-CSF binds to its receptor c-FMS, (cellular-feline McDonough strain sarcoma virus oncogene homologue, or CSF-1 receptor, or CD115), which in turn induces the expression of RANK on monocytes (Davignon et al., 2013).
• M-CSF binds to its receptor c-FMS, (cellular-feline McDonough strain sarcoma virus oncogene homologue, or CSF-1 receptor, or CD115), which in turn induces the expression of RANK on monocytes (Davignon et al., 2013).
• Inflammatory diseases such as rheumatoid arthritis, spondylitis ankylosing, psoriatic disease are characterized by bone erosion mediated by OCs.
• Osteoporosis is due to imbalance between bone resorption and bone deposition. For example, menopause is increasing the risk of osteoporosis due accelerated bone resorption.
• Bone resorption in cancer metastases is also due to OCs hyperactivity.
• osteoclast diseases which are the result of overactivity of OCs and deregulation of osteoclastogenesis and OC differentiation
• PDB Paget's disease of bone
• JPD Juvenile Paget's disease
• ESH expansile skeletal hyperphosphatasia
• FEO familiar expansile osteolysis
• the clinical phenotype of these diseases is one of uncontrolled bone remodelling, with presence of many, often enlarged, osteoclasts accompanied by areas of active bone formation (Helfrich et al., 2005). Accordingly, there is a need to develop new approaches and drugs that will be suitable for effective and efficient treatment of osteoclast diseases. In this way, it has been suggested that characterization of new therapeutic compounds in osteoclast diseases may be highly desirable.
• the present invention relates to methods and pharmaceutical compositions for the treatment of osteoclast diseases in a subject in need thereof.
• the inventors also demonstrated that HCMV infection inhibits cell proliferation and differentiation during osteoclastogenesis.
• the inventors also demonstrated that HCMV-mediated osteoclastogenesis inhibition is mediated by QKI5 upregulation during infection and that experimental induction of QKI5 expression inhibits osteoclastogenesis by inducing CSF-1R mRNA degradation and RANK inhibition.
• RA Rheumatoid Arthritis
• the present invention relates to a QKI5 polypeptide and/or an agent for QKI5 polypeptide expression for use in the treatment of osteoclast disease in a subject in need thereof.
• a subject denotes a mammal.
• a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with osteoclast disease.
• a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with bone erosion diseases, osteoporosis, rheumatoid arthritis, chronic inflammatory rheumatism, bone cancer or bone cancer metastasis.
• osteoclast disease has its general meaning in the art and includes, but is not limited to, bone erosion diseases and bone cancer.
• osteoclast disease that may be treated by methods and compositions of the present invention include, but are not limited to, bone erosion diseases, osteoporosis, arthritis, rheumatoid arthritis, chronic inflammatory arthritis, rheumatism, chronic inflammatory rheumatism, bone cancer, osteosarcoma, bone cancer metastasis, Paget' s disease of bone (PDB), Juvenile Paget' s disease, periodontal disease, Familial Expanile Osteolysis (FEO), Expansile Skeletal Hyperphosphatasia (ESH) and Camurati Engelmann Disease (CED).
• FEO Familial Expanile Osteolysis
• ESH Expansile Skeletal Hyperphosphatasia
• CED Camurati Engelmann Disease
• the subject suffers from bone erosion diseases, osteoporosis, rheumatoid arthritis, chronic inflammatory rheumatism, bone cancer, osteosarcoma or bone cancer metastasis.
• treatment refers to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects 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., disease manifestation, etc.]).
• QKI5 has its general meaning in the art and refers to quaking (QK) 1-5 protein.
• QKI5 refers to the RNA-binding protein belonging to the evolutionarily conserved signal transduction and activator of RNA (STAR) family implicated in embryogenesis and central nervous system development.
• the QKI (quaking, Qk) locus encodes a diverse set of proteins created by alternative splicing, and the three QKI proteins QKI-5, QKI- 6, and QKI-7 different by their carboxyl tails (Wu et al, 1999; Fu et al, 2012).
• the term “QKI5" also refers to QKI5 polypeptide deposited in the National Center for Biotechnology Information, U.S. National Library of Medicine under the NCBI accession number: NP 001288014.1 (SEQ ID NO: 1).
• the term “QKI5" also refers to QKI5 polypeptide encoded by the nucleic acid sequence deposited in the NCBI under the NCBI accession number NM 001301085.1 (SEQ ID NO: 2).
• the QKI5 polypeptide of the invention is an isolated, synthetic or recombinant QKI5 polypeptide.
• said QKI5 polypeptide comprises a sequence as set forth by SEQ ID NO: l.
• polypeptide of the present invention comprises or consists of an amino acid sequence having at least 70% of identity with SEQ ID NO: 1.
• a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99, or 100%) of identity with the second amino acid sequence.
• Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990).
• the polypeptide of the invention is a functional conservative variant of the polypeptide according to the invention.
• a “function-conservative variant” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Accordingly, a “function-conservative variant” also includes a polypeptide which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent polypeptide to which it is compared. Functional properties of the polypeptide of the invention could typically be assessed in any functional assay as described in the EXAMPLE.
• a further aspect of the present invention relates to a fusion protein comprising the polypeptide according to the invention that is fused to at least one heterologous polypeptide.
• fusion protein refers to the polypeptide according to the invention that is fused directly or via a spacer to at least one heterologous polypeptide.
• the fusion protein comprises the polypeptide according to the invention that is fused either directly or via a spacer at its C-terminal end to the N-terminal end of the heterologous polypeptide, or at its N-terminal end to the C-terminal end of the heterologous polypeptide.
• the term "directly” means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the polypeptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide.
• the last amino acid of the C-terminal end of said polypeptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said polypeptide is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous polypeptide.
• spacer refers to a sequence of at least one amino acid that links the polypeptide of the invention to the heterologous polypeptide. Such a spacer may be useful to prevent steric hindrances.
• the heterologous polypeptide is a cell-penetrating peptide, a
• TAT Transactivator of Transcription
• cell-penetrating peptides are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).
• the heterologous polypeptide is an osteoclast targeting agent.
• Osteoclast targeting agent include but are not limited to ligands or antibodies directed against the integrin ⁇ 3, calcitonin receptors, the vitronectin receptor (VNR), vacuolar ATPases (V- ATPases), the transcription factors PU.l, c-Fos, and NFKB I and 2, the growth factors M-CSF (macrophage colony stimulating factor) and RANKL (TNFRSF11), the receptor RANK ((receptor activator of NFKB), member 11 A of the tumour necrosis factor (TNF) receptor superfamily), the cathepsin K (CTSK), the tartrate-resistant acid phosphatase (TrAP), ClC-7 chloride channel, or antibodies or agents interacting with osteoclast membrane-bound and intracellular targets.
• VNR vitronectin receptor
• V- ATPases vacuolar ATPases
• PU.l vitronectin receptor
• V-ATPases vacuolar ATPases
• the heterologous polypeptide is an osteoclast disease therapeutic polypeptide.
• osteoclast disease therapeutic polypeptide refers to any polypeptide that has anti-osteoclast disease activities such as described below.
• polypeptides are known in the art such as described in Sun et al, 2008; Araujo et al, 2009; Tang et al., 2010; Broadhead et al, 2011; Kim and Moon, 2013.
• polypeptides or fusion proteins of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides or fusion proteins, by standard techniques for production of amino acid sequences. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the polypeptides or fusion proteins of the invention can be synthesized by recombinant DNA techniques as is now well- known in the art.
• these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (polypeptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.
• Polypeptides or fusion proteins of the invention can be used in an isolated (e.g., purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).
• a vector such as a membrane or lipid vesicle (e.g. a liposome).
• polypeptides or fusion proteins according to the invention may be modified in order to improve their therapeutic efficacy and their stability using well-known techniques.
• 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.
• a strategy for improving drug stability is the utilization of water-soluble polymers.
• Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
• water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
• Pegylation is a well-established and validated approach for the modification of a range of polypeptides (Chapman, 2002).
• the benefits include among others: (a) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and/or through evasion of cellular clearance mechanisms; (b) reduced antigenicity and immunogenicity of the molecule to which PEG is attached; (c) improved pharmacokinetics; (d) enhanced proteolytic resistance of the conjugated protein (Cunningham- Rundles et.al, 1992); and (e) improved thermal and mechanical stability of the PEGylated polypeptide.
• the polypeptides of the invention may be covalently linked with one or more polyethylene glycol (PEG) group(s).
• PEG polyethylene glycol
• One skilled in the art can select a suitable molecular mass for PEG, based on how the pegylated polypeptide will be used therapeutically by considering different factors including desired dosage, circulation time, resistance to proteolysis, immunogenicity, etc.
• the PEG of the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CFb ("methoxy PEG").
• a PEG can consist of one or more PEG side-chains which are linked together. PEGs with more than one PEG chain are called branched PEGs. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide.
• One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et al., 1995).
• the hydroxyl end groups of the polymer molecule must be provided in activated form, i. e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate (SPA), succinimidyl carboxymethylate (SCM),benzotriazole carbonate (BTC), N- hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)).
• Suitable activated polymer molecules are commercially available, e. g.
• the polymer molecules can be activated by conventional methods known in the art, e. g. as disclosed in WO 90/13540.
• activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference).
• activated PEG polymers include the following linear PEGs : NHS-PEG (e.g.
• SPA-PEG SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM- PEG
• NOR-PEG BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS.
• the conjugation of the polypeptides or fusion proteins and the activated polymer molecules is conducted by use of any conventional method.
• Conventional methods are known to the skilled artisan.
• the skilled person will be aware that the activation method and/or conjugation chemistry to be used depends on the attachment group(s) of the polypeptides as well as the functional groups of the PEG molecule (e.g., being amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate).
• polypeptides are conjugated with PEGs at amino acid D and E (for COOH), T, Y and S (for OH), K (for NH 2 ), C (for SH if at least one cysteine is conserved) or/and Q and N (for the amide function).
• additional sites for PEGylation can be introduced by site-directed mutagenesis by introducing one or more lysine residues. For instance, one or more arginine residues may be mutated to a lysine residue.
• additional PEGylation sites are chemically introduced by modifying amino acids on polypeptides of the invention.
• PEGs are conjugated to the polypeptides or fusion proteins through a linker.
• Suitable linkers are well known to the skilled person.
• a preferred example is cyanuric chloride ((Abuchowski et al, 1977); US 4,179, 337).
• the pegylated polypeptides provided by the invention have a serum half- life in vivo at least 50%, 75%, 100%, 150% or 200% greater than that of an unmodified polypeptide.
• the agent for QKI5 polypeptide expression of the invention is selected from the group consisting of an isolated, synthetic or recombinant nucleic acid encoding for QKI5 polypeptide, a nucleic acid sequence encoding for the fusion protein, a nucleic acid encoding a fragment of a QKI5 polypeptide, a cell expressing QKI5 polypeptide, and agent inducing QKI5 gene and polypeptide expression and their combinations.
• said nucleic acid encoding for QKI5 polypeptide comprises a sequence as set forth by SEQ ID NO: 2.
• the nucleic acid encoding for QKI5 polypeptide for example comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 2, preferably at least 85% identical to sequence SEQ ID NO: 2, more preferably at least 90% identical to sequence SEQ ID NO: 2, still more preferably at least 95%, at least 96%>, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 2.
• a sequence "encoding" an expression product such as a RNA, polypeptide, protein, or enzyme
• a sequence "encoding" an expression product is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme.
• a coding sequence for a protein may include a start codon (usually ATG) and a stop codon.
• nucleic acid sequences can be obtained by conventional methods well known to those skilled in the art.
• said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.
• a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule encoding for a polypeptide or a fusion protein of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted.
• suitable elements for controlling transcription in particular promoter, enhancer and, optionally, terminator
• recombinant vectors may, for example, be cloning vectors, or expression vectors.
• vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
• a DNA or RNA sequence e.g. a foreign gene
• Any expression vector for animal cell can be used.
• suitable vectors include pAGE107 (Miyaji et al, 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al,
• Plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.
• viral vectors include adenoviral, lentiviral, retroviral, herpes virus and AAV vectors.
• recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
• virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.
• Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.
• promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami and Itoh, 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana et al, 1987), promoter (Mason et al,
• a further aspect of the invention relates to a host cell comprising a nucleic acid molecule encoding for a polypeptide or a fusion protein according to the invention or a vector according to the invention.
• a subject of the present invention is a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule or vector according to the invention.
• transformation means the introduction of a "foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence.
• a host cell that receives and expresses introduced DNA or RNA has been "transformed”.
• prokaryotic cells in particular E. coli cells, will be chosen.
• prokaryotic cells have the advantages to produce protein in large amounts. If a eukaryotic context is needed, yeasts (e.g. saccharomyces strains) may be particularly suitable since they allow production of large amounts of proteins. Otherwise, typical eukaryotic cell lines such as CHO, BHK-21 , COS-7, C127, PER.C6, YB2/0, HEK293, mononuclear macrophage/monocyte-lineage hematopoietic precursors, Haematopoietic stem cells, Mononuclear precursor cells, osteoblast or inactive osteoclast could be used, for their ability to process to the right post-translational modifications of the fusion protein of the invention.
• yeasts e.g. saccharomyces strains
• typical eukaryotic cell lines such as CHO, BHK-21 , COS-7, C127, PER.C6, YB2/0, HEK293, mononuclear macrophage/monocyte-lineage hematop
• the construction of expression vectors in accordance with the invention, and the transformation of the host cells can be carried out using conventional molecular biology techniques.
• the polypeptide or the fusion protein of the invention can, for example, be obtained by culturing genetically transformed cells in accordance with the invention and recovering the polypeptide or the fusion protein expressed by said cell, from the culture. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractional precipitation, in particular ammonium sulfate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins may be used for producing the proteins in accordance with the invention.
• a further aspect of the invention relates to a method for producing a polypeptide or a fusion protein of the invention comprising the step consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said polypeptide or fusion protein; and (ii) recovering the expressed polypeptide or fusion protein.
• the agent for QKI5 polypeptide expression of the invention is an agent inducing QKI5 gene and polypeptide expression selected from the group consisting of, but not limited to, Human Cytomegalovirus (HCMV), VHL/E HCMV strain, and TB40/E HCMV strain.
• HCMV Human Cytomegalovirus
• VHL/E HCMV strain VHL/E HCMV strain
• TB40/E HCMV strain TB40/E HCMV strain.
• the present invention relates to the QKI5 polypeptide and/orthe agent for QKI5 polypeptide expression according to the invention in combination with one or more anti-osteoclast compound for use in the treatment of osteoclast disease in a subject in need thereof.
• anti-osteoclast compound has its general meaning in the art and refers to compounds and therapeutic active agent used in anti-osteoclast therapy such as Dendrimer; Calcium; vitamin D; antioxidant; Bisphosphonates such as ibandronate, aminobisphosphonates (e.g., risedronate, alendronate and zoledronate) and non-aminobisphosphonates (e.g., clodronate, tiludronate and etidronate); Calcitonin; Teriparatide; 1-34 parathyroid hormone (PTH 1-34); Raloxifene (selective estrogen receptor modulator (SERM)); Strontium ranelate; Disease-modifying antirheumatic drugs (DMARDs) such as adalimumab, etanercept, infliximab, agents that inhibit TNF-a; Nonsteroidal anti-inflammatory drugs; selective cyclooxygenase-2 inhibitors; agent targeting the RANK/RANKL/OP
• anti-osteoclast compound also refers to osteoclast differentiation inhibitors such as autophagy-activating compounds such as mammalian target of rapamycin (mTOR)- independent autophagy inducers including spermidine and its derivatives, and promethazine and its derivatives, e.g., trifluoperazine, and mTOR-dependent autophagy inducers, such as rapamycin and everolimus; Ceramide glucosyltransferase (CGT) inhibitors and glucosidase inhibitors such as miglustat (iminosugar N-butyl-deoxynojirimycin, also referred to as NB- DNJ); calcitonin (CT) peptide and bisphosphonate-linked calcitonin; Indenone derivatives such as 6-(2-morpholinoethoxy)-3-phenyl-2-(pyridin-3-yl)-lH-inden-l-one hydrochloride salt; Cystatin C-mim
• the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent.
• said additional active compounds may be contained in the same composition or administrated separately.
• the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression according to the invention as described above are administered to the subject in a therapeutically effective amount.
• a "therapeutically effective amount" of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention as above described is meant a sufficient amount of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression for treating osteoclast disease at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression 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 QKI5 polypeptide and/or the agent for QKI5 polypeptide expression 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 QKI5 polypeptide and/or the agent for QKI5 polypeptide expression employed; the duration of the treatment; drugs used in combination or coincidental with the specific QKI5 polypeptide and/or the agent for QKI5 polypeptide expression 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 QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention 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 QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention, preferably from 1 mg to about 100 mg of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention.
• 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 QKI5 polypeptide and/or the agent for QKI5 polypeptide expression according to the invention may be used in a concentration between 0.01 ⁇ and 20 ⁇ , particularly, the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 ⁇ .
• the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention is administered to the subject in the form of a pharmaceutical composition.
• the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
• pharmaceutically acceptable excipients such as antibiotics, anti-soluble polymers, anti-soluble polymers, anti-soluble polymers, anti-soluble polymers, anti-release matrices, anti-release matrices, anti-release matrices, anti-release matrices, anti-release matrices, anti-release matrices, anti-release matrices, anti-release matrices, anti-release matrices, anti-release matrices, anti-release matrices, anti-release matrices, antigenitrate, antigen, antigen, antigen, antigen, anti
• the active principle in the pharmaceutical 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.
• Solutions comprising QKI5 polypeptide and/or the agent for QKI5 polypeptide expression 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 QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention 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 compounds 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 agent of the present inventions 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 typical methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention plus any additional desired ingredient from a previously sterile- filtered solution thereof.
• the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
• solutions 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.
• 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.
• the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of osteoclast disease in a subject in need thereof.
• the present invention relates to a method for treating osteoclast disease in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the invention.
• the invention also provides kits comprising the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the invention. Kits containing the QKI5 polypeptide and/orthe agent for QKI5 polypeptide expression of the invention find use in therapeutic methods.
• FIGURES are a diagrammatic representation of FIGURES.
• HCMV infection inhibits CSF-IR expression in a replication-dependent manner.
• Purified monocytes were incubated with recombinant M-CSF and RANKL (both at 50 ng/mL) and after 24 hours, infected or not (Ni) with infectious (VHL/E) or UV-inactivated HCMV (VHL/E UV).
• VHL/E infectious or not
• VHL/E UV UV-inactivated HCMV
• FIG. 2 HCMV infection inhibits RANK expression in a replicative dependent manner.
• Purified monocytes were incubated with recombinant M-CSF and RANKL (both at 50 ng/mL) and after 24 hours, infected or not (Ni) with infectious (VHL/E) or UV-inactivated HCMV (VHL/E UV).
• VHL/E infectious or not
• VHL/E UV UV-inactivated HCMV
• Scale bar represents 50 ⁇ .
• HCMV infection inhibits monocytes proliferation and differentiation during osteoclastogenesis in a replication-dependent manner.
• Purified monocytes were incubated with recombinant M-CSF and RANKL (both at 50 ng/mL) and after 24 hours, infected or not (Ni) with UV-inactivated HCMV or not (VHL/E or VHL/E UV).
• A Cell proliferation was determined using CellTiter 96® Aq ue ous One Solution Cell Proliferation Assay at days 0, 2, 6, 8 and 10 after infection.
• FIG. 4 HCMV inhibitis osteoclastogenesis through a downregulation of CSF-IR mediated by QKI5 upregulation.
• Purified monocytes were incubated with recombinant M- CSF and RANKL (both at 50 ng/mL) and after 24 hours, infected (VHL/E) or not (Ni) with HCMV.
• Cells were harvested at TO, 2, 4, 8, 16 and 24 hours after infection to assess CSF-1R, QKI5, RANK and UL122 mRNA expression by qPCR. Scale bar represents 50 ⁇ .
• FIG. 5 Over-expression of QKI5 decrease CSF-1R and RANK expression and inhibitis osteoclastogenesis.
• HCMV seropositivity protects against bone erosion during rheumatoid arthritis, study of ESPOIR cohort.
• FIG. 7 Control of arthritis by lentivirus expressing qki5.
• Mice were injected intraperitoneally with K/BxN serum to induce arthritis. Arthritic score was evaluated on the rear left paws.
• Table 1 Demographic and disease characteristics of patients with RA at inclusion.
• RA rheumatoid arthritis
• ACPA+ anti- citrullinated peptide antibody presence
• RF+ rheumatoid factor presence
• CMV+ HCMV seropositivity
• DAS28 disease Activity Score on 28 joints
• n number of patients
• Table 2 Demographic and disease characteristics of patients with RA at 1 year after inclusion. Based on the report of the hands and feet radiographs, the documented disease stage was determined in all patients according to Sharp Score. Sharp score was separated in two different observations: Erosion score and Joint space narrowing score.
• RA rheumatoid arthritis
• ACPA+ anti-citrullinated peptide antibody presence
• n number of patients
• MRC5 fibroblasts, HCT116 and Hek-293T cells were propagated in DMEM medium (InVitrogen) containing 10% fetal calf serum and 5% penicillin/streptomycin.
• Monocytes were purified from HCMV seropositive and negative donors buffy coat, obtained at the EFS (Etablatorium Francais du Sang, France), layered on Pancoll gradient (PanBiotech, Aidenbach, Germany), and purified by adherence 30 min in cell cultures plates and PBS washes to discard lymphocytes. Purified monocytes were cultures for osteoclast differentiation.
• VHL/E HCMV strain (gift from Christian Sinzger, Germany) was used in this study.
• Virus stocks were generated in MRC5 or HUVEC cells, collecting particles when cytopathic effects were >90%. Supematants were clarified of cell debris by centrifugation at l,500xg for 10 min, ultracentrifuged at 100,000xg for 30 min at 4°C, pellets were resuspended and virus stocks were stored at -80°C until use.
• Virus titers were determined by plaque assay on MRC5 cells using standard methods (MEM2X diluted two fold with solution of 1.6% agarose). UV irradiation of HCMV was performed with a Spectroline irradiator (EF-140/F) for 20 min. Monocytes were infected the day after purification and start of culture at an m.o.i. of 3.
• Recombinant Human M-CSF was purchased from Biolegend (San Diego, CA, USA) and Recombinant Human RANKL from Peprotech (Rocky Hill, NJ, USA).
• Anti- Cytomegalovirus IE1 and IE2 (ab53495) and anti-Histone H3 (abl791) antibodies were purchased from Abeam (Cambridge, UK), anti-RANK monoclonal antibody (MA5-16153) from ThermoFisher Scientific (Waltham, MA, USA), anti-QKI5 polyclonal antibody (AB9904) from Millipore (Temecula, CA, USA), anti-CSF-lR antibody (sc-692) from Santa Cruz (Dallas, TX, USA) and anti-FOXPl monoclonal antibody (3210-S) from EPITOMICS (Burlingame, CA, USA).
• Purified monocytes were incubated with M-CSF and RANKL (both at 50 ng/ml) and cultured for 12 days at 37°C, 5% C02 in RPMI supplemented with 10% FCS (Invitrogen). Culture medium was replaced with fresh one every 4 days with M-CSF and RANKL (both at 50 ng/ml). Osteoclast formation was evaluated by Leukocyte Acid Phosphatase TRAP Kit (Sigma, Saint-Louis, MO, USA) staining and manual counting of the number of multinucleated TRAP+ cells.
• Lentiviral vectors pseudotyped with the vesicular stomatitis G protein were generated by transfection into 293T cells of a packaging construct, pCMVR8.74 (Addgene #22036), a plasmid producing the vesicular stomatitis G protein envelope (pMD2.G, Addgene #12259); and the vector itself.
• pRRL empty backbone pRRL. empty, Addgene #12252
• pRRL.QKI PGK promoter controlling QKI gene
• pLKO vectors coding an shQKI5 were constructed by replacing shKAPl in previously described experiments.
• Viral titers infectious particles were determined by transductions of HCT1 16 cells with serial dilutions of the vector preparation in a 12-well plate. 72 h later, cells were analyzed for eGFP expression by FACS and the infectious particles titer was determined (IP.ml-1).
• the number of viable cells in proliferation was determined using CellTiter 96®
• Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Cells were cultured in 96 wells plate at a concentration a 104 cells per wells in presence of M-CSF and RANKL (both at 50ng/ml) and infected or not with HCMV (m.o.i. of 3). Cell proliferation was measured at day 3, day 6, day 8 and day 10, by adding 20 ⁇ of reagent per wells and incubated 2 hours at 37°C. Absorbance was recorded at 490 nm using a 96-well plate reader.
• Cells lysates were subjected to SDS/PAGE on 4-12% polyacrylamide gels (ThermoFisher scientific, Walham, MA, USA). After transferring on 0.22 ⁇ nitrocellulose membrane, proteins were revealed using specific antibodies and anti-rabbit or anti-mouse HRP- linked polyclonal antibodies (Cell Signaling, Danvers, MA, USA).
• CSF-IR expression is down-regulated during HCMV infection. It has already been shown that infection of macrophages with HCMV clinical strain TB40/E down regulates cell surface expression of CSF-IR (CD115, MCSF-R) (1). Consequently, we asked if HCMV infection could also interfere with CSF-IR cell-surface expression during osteoclastogenesis by infecting monocytes in osteoclast differentiation (stimulated with recombinant M-CSF and RANKL, both at 50 ng/mL) with an HCMV clinical strain (VHL/E) at an m.o.i. of 3.
• Figure 1 shows that HCMV infection lead to a rapid inhibition of CSF-IR mRNA transcription (Fig.1) and protein expression (data not shown) implicating a strong decrease of cell surface expression (data not shown). This inhibition was also observed by fluorescence microscopy in VHL/E infected cells in comparison with non- infected ones (data not shown). Interestingly, viral gene expression was required for this inhibition as UV-inactivated virus showed no effect on CSF- IR expression.
• HCMV infection inhibits cell proliferation and differentiation during osteoclastogenesis. Since M-CSF and RANKL signalization are essential to osteoclastogenesis, we then examined the effect of HCMV infection on osteoclast differentiation. As expected, HCMV infection impaired cell proliferation and in a viral gene expression-dependent mechanism (Fig.3 A). After 12 days of differentiation, osteoclasts were labelled using a TRAP assay and counted. We clearly observed that HCMV infection completely blocked OCs differentiation in a replicative dependent manner (Fig. 3B) whereas in non-infected control wells, large multinucleated osteoclastic cells had clearly developed (data not shown). New experiments using with TRAP staining are ongoing. These results demonstrated for the first time that HCMV infection of monocytes interfere with osteoclast differentiation.
• HCMV-mediated osteoclastogenesis inhibition is mediated by QKI5 upregulation during infection.
• RA Rheumatoid Arthritis
• HCMV HCMV reactivated during osteoclastogenesis?
• HCMV could reactivate from latently infected monocytes or hematopoietic stem cells when they differentiate to macrophages 6 and this phenomenon could occur during articular inflammation, inducing infection of osteoclastic progenitors and inhibiting their differentiation.
• the other possibility is that differentiation of monocytes into osteoclast could also reactivate HCMV from latency and directly induce a viral release during erosion and better target this articular erosion.
• Frascaroli G., et al. Human cytomegalovirus paralyzes macrophage motility through down-regulation of chemokine receptors, reorganization of the cytoskeleton, and release of macrophage migration inhibitory factor. J Immunol 182, 477-488 (2009).
• RNA-binding protein QKI5 is a direct target of C/EBPalpha and delays macrophage differentiation. Mol Biol Cell 23, 1628-1635 (2012).

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Physical Education & Sports Medicine (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Immunology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Epidemiology (AREA)
• Rheumatology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Virology (AREA)
• Gastroenterology & Hepatology (AREA)
• Microbiology (AREA)
• Zoology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Toxicology (AREA)
• Genetics & Genomics (AREA)
• Mycology (AREA)
• Molecular Biology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)

Abstract

Applications Claiming Priority (2)

Publications (1)

Family

ID=59772566

Family Applications (1)

Country Status (1)

Citations (10)

• 2018
  • 2018-08-24 WO PCT/EP2018/072876 patent/WO2019038420A1/fr active Application Filing

Patent Citations (10)

Non-Patent Citations (21)

Similar Documents

Legal Events