WO2012052995A2 - Traitement et prévention du carcinome hépatocellulaire au moyen de modulateurs des récepteurs de chimiokines - Google Patents

Traitement et prévention du carcinome hépatocellulaire au moyen de modulateurs des récepteurs de chimiokines Download PDF

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WO2012052995A2
WO2012052995A2 PCT/IL2011/000816 IL2011000816W WO2012052995A2 WO 2012052995 A2 WO2012052995 A2 WO 2012052995A2 IL 2011000816 W IL2011000816 W IL 2011000816W WO 2012052995 A2 WO2012052995 A2 WO 2012052995A2
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rantes
ccr5
subject
derivative
polypeptide
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PCT/IL2011/000816
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WO2012052995A3 (fr
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Amnon Peled
Eithan Galun
Neta Barashi
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Hadasit Medical Research Services And Development Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES

Definitions

  • the present invention is directed to use of CCR5 modulators in the treatment, prevention and inhibition of hepatocellular carcinoma.
  • Hepatocellular carcinoma is a highly aggressive carcinoma of the liver which has been reported to occur world-wide in increasing numbers. Intra-hepatic metastatic recurrences via the portal vein are the main cause of death in HCC patients who have undergone partial hepatectomy or liver transplantation (Korn, World J. Gastroenterol., 7:777-8, 2001). However, the molecular events and main players that promote HCC continue to be a challenge for the treatment and prevention of this disease.
  • CCR5 and CCR1 are members of the chemokine receptor subclass of the G- Coupled- Protein Receptor superfamily. These chemokine receptors were identified as functional receptors for several inflammatory CC-chemokines, including Macrophage Inflammatory Protein (MIP)- la, ⁇ - ⁇ ⁇ , and RANTES. CCR5 and CCR1 are expressed on peripheral blood leukocytes, including macrophages, NK cells and T-cells. The inventors of the present invention have recently showed that CCR5 but not CCR1 regulates the trafficking of T, NK, NKT cells and macrophages into the liver under normal conditions.
  • MIP Macrophage Inflammatory Protein
  • RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted), also known as CCL5, is an 8kDa protein, shown to interact with CCR3 in addition to CCR1 and
  • This chemokine was found to be produced by various cells in the body including platelets, macrophages, eosinophils, and fibroblasts, as well as endothelial, epithelial, and endometrial cells, and to have chemotactic activity towards T cells, dendritic cells, eosinophils, NK cells, mast cells, and basophils.
  • RANTES has been suggested to exert a beneficial role in bringing immune cells to areas of infection and injury.
  • RANTES was also suggested to mediate detrimental effects by recruiting immune cells that enhance untoward inflammatory processes.
  • beneficial as well as harmful effects for this chemokine during the development of different neoplastic conditions.
  • RANTES and its receptors Various agonists and antagonists of RANTES and its receptors have been disclosed. Agonists or antagonists of the CCR5 receptor and use thereof for the treatment and/or prophylaxis of certain disease states, other than cancer, liver cancer or hepatocellular carcinoma are disclosed in US Patent Application, Publication No. 2004/0038982.
  • US Pat. No. 6,168,784 discloses N-terminally modified RANTES derivatives, useful for the treatment of asthma, allergic rhinitis, atopic dermatitis, atheroma/atherosclerosis, rheumatoid arthritis and HIV infection. The derivatives are optionally modified by the grafting of polyethylene glycol (PEG) or PEG-based chains.
  • PEG polyethylene glycol
  • 7,666,400 discloses methods and compositions for forming PEGylated complexes of defined stoichiometry and structure.
  • the publication provides a long list of potential target agents that may be modified, including chemokines such as RANTES.
  • chemokines such as RANTES.
  • liver cancer a malignant neoplasm originating from liver cancer.
  • CCR5 antagonists are particularly known for their contribution in the treatment of HIV.
  • US Patent No. 6,440,946 discloses a compound comprising at least two compounds having anti-HIV activity and having no affinity for cell surface proteins; said compounds bound together with at least one linkage, wherein said compounds include a CCR5 antagonist.
  • US Patent No. 7,247,631 discloses a method of treating patients co-infected with HIV-1 and Hepatitis C Virus which comprises administering a combination of several compounds, including a therapeutically effective amount of small molecules which are CCR5 antagonist.
  • Chemokine Receptor (CCR5) HDGNR10 is also suggested by US Pat. Appl. Pub. Nos. 2003/0125380 and 2009/0226416. Among the extensive lists of conditions recited by these publications as potentially treatable by such agonists or antagonists is cancer, inter alia liver cancer.
  • US Pat. Appl. Pub. No. 2002/0077339 discloses heterocyclic compounds that bind to chemokine receptors, including CXCR4 and CCR5, and demonstrates protective effects against infection of target cells by a human immunodeficiency virus (HIV). The disclosure further suggests the use of these compounds for the treatment of various diseases, including cancer.
  • US Patent Application, Publication No. 2010/011 1898 discloses a method of treating a fibrotic or fibroproliferative disorder in patient, comprising administering to a patient a combination of one or more fibrocyte suppressors and one or more profibrotic factor antagonists, inter alia, CCR5 antagonists, or anti-fibrotic agents.
  • the specification states that liver fibrosis is a precursor to the complications associated with liver cirrhosis, such as hepatocellular carcinoma.
  • CCR5 and CCL5 are disclosed to play different roles during HCV infection.
  • homozygosis to a CCR5 mutation is disclosed to adversely affect HCV infection
  • HCV patients are disclosed to have upregulated chemokine receptors (including CCR5) in liver portal tracts and liver-derived chemokines, and down-regulated expression in peripheral blood T cells. It is disclosed that the effects of CCR5 blocking on HCV progression are different to assess and may either ameliorate or exacerbate the disease.
  • Nahon et al. (World J Gastroenterol. 2008 Feb 7;14(5):713-9) examined the influence of polymorphism in various chemokine and chemokine receptor genes in HCC patients. The reported findings suggest lack of association of CCR5 polymorphism with death and HCC occurrence in cirrhotic HCV-infected patients.
  • the publication discloses that the various chemokines might be involved both in beneficial inflammatory processes that lead to viral clearance and in perpetuation of chronic inflammation that results in liver damage.
  • Liu et al. (Am J Gastroenterol. 2004 Jun;99(6): l 1 1 1-21 ; Clin Immunol. 2005 Feb; 1 14(2): 174-82) examine the expression of chemokine receptors including CCR5 on various lymphocyte populations, and report enhanced CCR5 expression on HCC tumor lymphocytes compared to peripheral blood lymphocytes.
  • Yoong et al. (Hepatology. 1999 Jul;30(l):100-l l) discloses expression of CCR5 among other chemokine receptors on tumor infiltrating lymphocytes in human HCC. The use of such tumor infiltrating lymphocytes is suggested for the development of immunotherapy for HCC.
  • CCRl- and CCR5 -deficient mice display substantially reduced hepatic fibrosis and macrophage infiltration in two mouse models for fibrosis; bile duct ligation (BDL) and CC14 treatment induced fibrosis (Seki, J Clin Invest., 2009, 1 19(7):1858-70).
  • BDL bile duct ligation
  • CC14 CC14 treatment induced fibrosis
  • RANTES functions as an adjuvant to boost anti-tumor immunity by diverse protocols such as co-immunization of recombinant RANTES protein with tumor-associated antigen, vaccination with RANTES-expressing tumor cells, or viral vector delivery of RANTES cDNA into growing tumor.
  • RANTES may also promote tumor cell survival, proliferation and invasion.
  • co- vaccination with chemokine (mRANTES) and tumor-specific (hgplOO) genes in a specific time sequence is more effective at suppressing tumor growth and metastasis than hgplOO alone.
  • US Pat. Appl. Pub. No. 2002/0034494 discloses a method of treating disease states comprising administering to an individual in need thereof an amount of chemokine sufficient to increase the migration of immature dendritic cells to the site of antigen delivery, inter alia RANTES, preferably in combination with a disease-associated antigen.
  • chemokine sufficient to increase the migration of immature dendritic cells to the site of antigen delivery, inter alia RANTES, preferably in combination with a disease-associated antigen.
  • liver cancer is liver cancer.
  • PCT Pub. No. WO 2001/051077 provides a method for treating a disease associated with increased IL-12 production in a subject, comprising administering to the subject a CCR5 antagonist in an amount effective in reducing the disease-associated effect of IL-12, and a method for increasing IL-12 production in a subject, comprising administering and effective amount of a CCR5 agonist to the subject. It is disclosed that increased IL-12 production may be desirable when a subject has an infectious disease, an atopic/allergic condition or cancer. Hepatoma is disclosed as a specific example for cancer.
  • US Pat. Appl. Pub. No. 2010/0203027 discloses a negative-strand RNA viral vector that carries a cytokine gene, optionally encoding a chemokine such as RANTES.
  • the vector may be intended for treatment of cancer.
  • the publication discloses that an anti-tumor effect can be produced when a heparin-binding cytokine such as granulocyte macrophage colony stimulating factor (GM-CSF) and a chemokine such as TARC or RANTES are expressed in vivo using a viral vector based on a negative-strand RNA virus.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • TARC or RANTES chemokine
  • Kirovski et al. (2010) report on elevated hepatic as well as circulating RANTES levels in response to hepatic steatosis.
  • Katzenellenbogen et al. (2007) show, using genetic profiling of tumorous and non-tumorous liver tissue, elevation of RANTES in Mdr2 -knockout mice, which serve as a model for ⁇ -catenin-negative subgroup of human HCCs characterized by low nuclear cyclin Dl levels in tumor cells and by down-regulation of multiple tumor suppressor genes.
  • the invention is directed to the use of CCR5 inhibitors for treating, preventing and suppressing the onset and progression of hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • the invention further relates to therapeutic and prophylactic modalities for elevating the level of the CCR5 and CCRl ligand RANTES, specifically in the blood of a subject having or predisposed to HCC.
  • certain embodiments of the invention encompass the use of RANTES conjugates and derivatives enabling prolonged blood retention and reduced liver clearance.
  • the present invention is based, in part, on the surprising discovery that, in a HCC mouse model induced by a knockout mutation in the Mdr2 gene (Mdr2 'A mice), down-regulation of CCR5 expression and enhancement of RANTES plasma levels beyond a specific threshold are associated with significantly lower tumor incidence and development. In comparison, down- regulated CCRl expression resulted in earlier tumor onset and no further elevation in RANTES blood plasma levels.
  • the invention provides safe and effective means for treating HCC or inhibiting or delaying the onset of a HCC tumor using CCR5 modulators.
  • novel treatment regimes are provided for elevating blood plasma levels of RANTES or derivatives thereof.
  • a method for treating hepatocellular carcinoma (HCC) or for inhibiting or delaying the onset of a HCC tumor formation comprising administering to a subject in need thereof an isolated RANTES polypeptide or derivative thereof, so as to enhance the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • the method is used for treating HCC.
  • the method is used for inhibiting or delaying the onset of a HCC tumor formation.
  • said subject is afflicted with HCC.
  • the subject in need thereof is susceptible to HCC.
  • the subject in need thereof is a patient with chronic liver inflammation.
  • Mdr2 "/_ is a model for inflammation-associated HCC, specifically Mdr2 'A may serve as a model for a ⁇ -catenin-negative subgroup of human HCCs characterized by low nuclear cyclin Dl levels in tumor cells and by down-regulation of multiple tumor suppressor genes.
  • the HCC is characterized by lack of ⁇ -catenin expression in the tumor cells.
  • the HCC is characterized by nuclear cyclin Dl levels in tumor cells that are lower than nuclear cyclin Dl levels in non-tumor cells.
  • the HCC is characterized by down-regulation of at least one tumor suppressor gene selected from the group consisting of Cadherin-1 (Cdhl), Deleted in lymphocytic leukemia 2 (Dleu2), Pleomorphic adenoma gene-like 1 (Plagll), and Retinoblastoma-like 2 (Rbl2).
  • said subject has progressive steatosis.
  • the method comprises:
  • RANTES polypeptide or derivative thereof so as to enhance the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • the method comprises administering intravenously to said subject a therapeutically effective amount of an isolated RANTES polypeptide or a derivative thereof. In another embodiment, the method comprises administering to said subject intravenously a therapeutically effective amount of an isolated RANTES polypeptide (e.g. a mammalian RANTES or particularly a human RANTES). In another embodiment, the method comprises administering to said subject intravenously a therapeutically effective amount of an isolated RANTES derivative.
  • the RANTES derivative is a RANTES agonist.
  • the RANTES derivative is a RANTES partial agonist.
  • the RANTES derivative is substantially devoid of chemotactic activity. In yet another particular embodiment, the RANTES derivative down- regulates CCR5 expression.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient or diluent.
  • the composition is formulated in the form of a sustained release formulation.
  • the composition is not administered in the form of a vaccine further comprising an additional antigen.
  • the composition is not administered by a therapeutic regimen adapted for vaccination.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is at least 4 fold higher than mean blood plasma levels of native RANTES in healthy individuals. In another embodiment the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is 4-10 fold higher than mean blood plasma levels of native RANTES in healthy individuals. In another embodiment the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is 4-6 fold higher than mean blood plasma levels of native RANTES in healthy individuals. In another embodiment the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is about 5 fold higher than mean blood plasma levels of native RANTES in healthy individuals.
  • said isolated RANTES polypeptide or a derivative thereof is administered to said subject by continuous intravenous infusion. In another embodiment said isolated RANTES polypeptide or a derivative thereof is administered to said subject at a dosing regime comprising periodical intravenous injections.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject in a form enabling a significantly longer plasma half-life of said RANTES polypeptide or derivative compared to native RANTES polypeptide.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject in the form of a complex or conjugate having a significantly longer plasma half-life compared to native RANTES polypeptide.
  • the RANTES derivative is characterized by a significantly longer plasma half-life compared to native RANTES polypeptide.
  • the plasma half-life of the RANTES form, complex or conjugate may be at least 5 times longer or at least 10 times longer compared to native RANTES polypeptide.
  • said RANTES polypeptide or derivative may be conjugated to (covalently) or complexed with (non-covalently) a compound selected from the group consisting of: polyethylene glycol, a copolymer of ethylene glycol, a polypropylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-l ,3-dioxolane , a poly-l,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycos
  • said RANTES polypeptide or derivative is conjugated to or complexed with polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • said RANTES polypeptide or derivative is conjugated to or complexed with an immunoglobulin Fc domain or portion thereof.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject in a form enabling a significantly lower liver uptake compared to native RANTES polypeptide.
  • the method further comprises administering to said subject at least one inhibitor of a chemokine receptor selected from the group consisting of CCRl and CCR5.
  • the inhibitor may be e.g. a double-stranded RNA, a compound antagonizing the binding of the chemokine receptor to its ligand, neutralizing antibody to CCR5, neutralizing antibody to CCRl , a ligand corresponding to a neutralizing antibody to CCR5, a ligand corresponding to a neutralizing antibody to CCRl , an isolated peptide derived from the sequences of CCR5 or analogs thereof capable of inhibiting CCR5, an isolated peptide derived from the sequences of CCRl or analogs thereof capable of inhibiting CCRl, antisense nucleic acids, antagonist micro RNA and an enzymatic RNA molecule.
  • the at least one inhibitor of a chemokine receptors is a CCR5 antagonist.
  • the inhibitor may be administered to said subject so as to inhibit, antagonize or down-regulate the expression of CCR5 in the liver, tumor cells or lymphocytes of said subject, wherein each possibility represents a separate embodiment of the invention.
  • the present invention provides a method for treating hepatocellular carcinoma, comprising administering to a subject in need thereof at least one inhibitor of CCR5.
  • inhibitor and “antagonist” as used herein are interchangeable and refer to a substance that interferes with the expression of a gene, specifically, the gene encoding CCRl or CCR5, or interferes with the activity of the gene product, specifically, interferes with the activity of CCR5 and/or CCRl .
  • Interference with gene expression includes, but is not limited to, inhibition of translation or induction of gene transcript cleavage.
  • the inhibitor is selected from the group consisting of: double-stranded RNA (e.g. an RNA interfering oligonucleotide), a compound antagonizing the binding of the chemokine receptor to its ligand, a compound antagonizing the binding of RANTES (the natural ligand to CCR5) to CCR5, neutralizing antibody to CCR5, a ligand corresponding to a neutralizing antibody to CCR5, an isolated peptide derived from the sequences of CCR5 or analogs thereof capable of inhibiting CCR5, antisense nucleic acids, antagonist microR A and enzymatic RNA molecule.
  • double-stranded RNA e.g. an RNA interfering oligonucleotide
  • RANTES the natural ligand to CCR5
  • neutralizing antibody to CCR5 a ligand corresponding to a neutralizing antibody to CCR5
  • the double-stranded RNA is a small interfering RNA
  • the enzymatic RNA molecule is a ribozyme.
  • the method further comprises administering the at least one inhibitor of a chemokine receptor in combination with at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is selected from the group consisting of: a cytokine, an anti-tumor agent and antiviral agents.
  • the at least one additional therapeutic agent is an inhibitor of CCR1.
  • the cytokine is RANTES or a derivative thereof.
  • the subject in need thereof is susceptible to HCC. According to yet another embodiment, the subject in need thereof is a patient with chronic liver inflammation.
  • the HCC is characterized by lack of ⁇ -catenin expression in the tumor cells. In another embodiment the HCC is characterized by nuclear cyclin Dl levels in tumor cells that are significantly lower than nuclear cyclin Dl levels in non-tumor cells.
  • the present invention provides a method for ameliorating
  • the present invention provides use of at least one inhibitor of a chemokine receptor selected from the group consisting of CCR1 and CCR5 for the manufacture of a medicament for treating hepatocellular carcinoma.
  • the present invention provides use of CCR5 inhibitor for the manufacture of a medicament for ameliorating the onset of hepatocellular carcinoma.
  • Figure 1A presents the increase in the CCR5/1 ligand RANTES in Mdr2 knockout mice (Mdr2 " " , empty column) compared to wild-type mice (WT, full column).
  • Figures IB - ID exhibit the increase in the levels of ALP, ALT or AST liver enzymes, respectively, in various mouse strains.
  • WT full squares
  • Mdr2 v" empty squares
  • Figure 2 shows the increase of P AN-CK staining for bile ducts in liver sections of Mdr2 " '- mice, MDR2 "/" CCR5 'A mice and MDR2 "A CCR1 _/” mice.
  • Figure 3A presents the increase of F4/80 + cells in liver sections of Mdr2 " " mice (white), MDR2 " “ CCR5 “ “ mice (vertical lines) and MDR-T ⁇ CCRl “7” mice (horizontal lines) compared to WT mice (gray).
  • Figure 3B exhibits the inability of MAC-1 + cells derived from CCR5 _ " mice to migrate in response to RANTES. WT - black, CCR5 _ " - white.
  • Figure 3C shows the inability of F4/80 + CFSE + cells to migrate in vivo to the liver of MDR2 KO. From the left: migration of WT cells into liver of WT mice ("WT into WT”); migration of CCR5 "7” cells into liver of WT mice (“CCR5 into WT”); migration of WT cells into liver of MDR2 "7”” mice (“WT into Mdr2 KO”); migration of CCR5 "A cells into liver of MDR2 "7'” mice (“CCR5 into Mdr2 KO”).
  • Figure 3D presents F4/80 staining for macrophages in 3 months old WT, MDR2 _ " ("Mdr2 KO”) and MDR2 "/" CCR5 “ " (“Mdr2CCR5 DKO”) mice.
  • Figure 3E presents PAN-CK immunohistochemical staining (left), immunohistochemical detection of BrdU positive cells (center), and assessment of extracellular collagen deposits by SIRIUS RED chemical staining reaction (right) in WT (top), MDR2 " ' (Mdr2 KO, middle) and MDR2 "/" CCR5 “/' (Mdr2CCR5 DKO, bottom) mice.
  • Figure 4A presents the staining for ECM collagen deposit that was significantly higher in MDR2 ";” CCR1 “A and Mdr2 “A mice compared to MDR2- /” CCR5 “ “ mice.
  • Figure 4B presents expression of TGF- ⁇ in liver extracts as detennined by real time PCR in WT (black), MDR2 " ' “ (MDR2 KO, gray), and MD ⁇ CCRS " - (Mdr2CCR5 DKO, vertical lines) mice.
  • Figure 4C shows immunoblotting for the HSC activation marker a-SMA on liver tissues from 3 months old WT, Mdr2 "/_ (Mdr2 KO) and MD ⁇ CCRS “7" (Mdr2CCR5 DKO) mice.
  • Figure 5A presents representative MRI images of WT, Mdr2 "/_ , Mdr2 “/” CCR5 ' “ and Mdr2 “/” CCR1 '/” at 16 months of age and the number of mice with visible tumors assessed by MRI.
  • Figure 5B shows representative images of harvested liver and tumors in the livers of WT, Mdr2 'A , Mdr2- /" CCR5 " ' and Mdr2 "/_ CCR1 "7" mice.
  • Figure 5C shows liver/body mass in wild-type (WT, black) Mdr2 _/" mice (Mdr2 KO, gray), MDR2 "/" CCR5 “/” mice (Mdr2CCR5 DKO, vertical lines) and MDR2 " ' CCR1 "/” mice (Mdr2CCRl DKO, white).
  • Figure 5D shows the incidence of tumors in Mdr2 " _ mice (Mdr2 KO, gray), MDR2 "/_ 5 CCR5 “7” mice (Mdr2CCR5 DKO, vertical lines) and MDR2 "/' CCR1 " " mice (Mdr2CCRl DKO, white).
  • Figure 5E presents the incidence of tumors in Mdr2 "/_ mice, (Mdr2 KO) MDR2 " ⁇ CCR5 " ⁇ (Mdr2CCR5 KO) and MDR2 " _ CCR1 " _ mice (Mdr2CCRl KO).
  • Figure 5F presents tumor volume in Mdr2 "7" mice (Mdr2 KO, gray), MDR2 " “ CCR5 “ “ ) (Mdr2CCR5 DKO, vertical lines) and MDR2 " " CCR1 "/” mice (Mdr2CCRl DKO, white).
  • FIG. 6A shows RANTES levels in livers of wild-type (WT, black) Mdr2 "/_ mice (Mdr2, gray), MD ⁇ CCRS “ " mice (Mdr2CCR5, vertical lines) and MDR2 " " CCR1 "/” mice (Mdr2CCRl , white).
  • FIG. 6B shows RANTES levels in sera of wild-type (WT, black) Mdr2 "A mice (Mdr2, ; gray), MDR2 ' CCR5 " " mice (Mdr2CCR5, vertical lines) and MDR2 "A CCR 1 "A mice (Mdr2CCRl , white).
  • the invention is directed to the use of CCR5 inhibitors for treating, preventing and suppressing the onset and progression of hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • the invention further relates to therapeutic and prophylactic modalities for elevating the level of the CCR5 and CCR1 ligand RANTES, specifically in the blood of a subject having (or predisposed to) HCC.
  • embodiments of the invention encompass the use of formulations and derivatives of RANTES enabling prolonged blood retention and reduced liver clearance of the chemokine.
  • the present disclosure is based, in part, on studies performed using a HCC mouse model induced by a knockout mutation in the Mdr2 gene (Mdr2 "A mice), which optionally carry a second knockout mutation in the CCR5 or CCR1 chemokine receptor genes (Mdr2 " " CCR5 “ “ mice and Mdr2 “ “ CCR1 “/” mice, respectively).
  • the present invention is based in part on the unexpected discovery that in Mdr2 "/" CCR5 “/” mice, the incidence and development of tumors is significantly lower and tumor progression is inhibited compared to Mdr2 " _ mice, although liver damage associated with Mdr2 deficiency is not inhibited and resembles the level in Mdr2 "A mice.
  • Chemokines represent a large family of chemoattractant cytokines that regulate leukocyte trafficking by mediating the adhesion of leukocytes to endothelial cells, the initiation of transendothelial migration and tissue invasion. Chemokines are small (5-20 kDa) proteins that
  • chemokines 5 are rich in basic amino acids and contain conserved cysteine motifs forming essential disulfide bonds between the first and third, and the second and fourth cysteines.
  • the number and spacing of the first cysteines in the amino acid sequence are used to classify chemokines into four subfamilies: CXC (or a), CC (or ⁇ ), CX 3 C, and C chemokines. If the first two cysteines are adjacent to each other, they are classified in the CC family consisting of CCL1- CCL28.
  • chemokines lymphotactin/XCLl and fractalkine/CX 3 CLl
  • chemokines represent the members of the C and CX 3 C subfamilies. Functionally, the chemokines are grouped into two subfamilies: inflammatory and homeostatic chemokines. Inflammatory chemokines mainly control the recruitment of leukocytes to the sites of inflammation and tissue injury, while the homeostatic chemokines mediate primarily the navigation of leukocytes to secondary lymphoid and hematopoietic organs.
  • Chemokines mediate their biologic effects by binding to specific cell-surface receptors.
  • the chemokine receptors which have also been classified into four subfamilies, are G-protein- coupled seven transmembrane spanning molecules.
  • Chemokine receptors as well as chemokines 3 share 25%-80% of the amino acid sequence identity. Despite variable levels of sequence homology, they adopt a characteristic fold.
  • Chemokines have been implicated in pathological conditions such as autoimmunity, inflammatory diseases, HIV pathogenesis, and cancer. It is now widely accepted that interactions of cancer cells with components of their tumor microenvironment are bi-directional
  • chemokines that are inducible in normal tissues as well as functional chemokine receptors.
  • Extensive networks of chemokines and chemokine receptors play multifunctional roles in malignant processes, providing directional signals for cancer cell migration and metastasis, regulating angiogenesis, controlling leukocyte infiltration into tumors
  • HCC in humans is an example of inflammation induced cancer mostly following cirrhosis that may be caused by chronic viral hepatitis, metabolic liver diseases or alcohol abuse. HCC is the third leading cause of cancer mortality worldwide, causing 662,000 deaths per year. Chemokines and Chemokine receptors are main contributors for the initiation and maintenance of inflammation, but nowhere in the art has it been disclosed or suggested that chemokine receptors, particularly CCR5, have a significant role in tumorigenesis of the liver, and most specifically, in the manifestation of HCC.
  • the present invention is directed to methods for treating, inhibiting the onset of, or preventing hepatocellular carcinoma, using antagonists of the chemokine receptors, specifically, CCR1 and CCR5.
  • the invention is partially based on the discovery that in a mouse HCC model where the gene CCR5 was knocked out, liver state was generally improved and liver destruction was reduced, with a restricted injury scar that localized only in the periductal area, and did not invade the liver parenchyma. This observation led to conclude that CCR5 is a pivotal player in the tumorigenesis process.
  • the methods according to embodiments of the present invention for treatment, prevention and onset prevention of HCC are particularly advantageous since they can be readily executed using CCRl and CCR5 antagonists that are already available in the market.
  • Another significant advantage of the methods of the invention is based on the known fact that the population of patients susceptible to HCC is at least partially defined. Accordingly, the population to which the method of the invention for the prevention of HCC or at least for I ameliorating the onset of this disease is particularly directed, is known and much defined.
  • the methods of the invention comprise administering to a subject in need thereof a therapeutically effective amount of at least one inhibitor of CCR5 and/or CCRl .
  • the inhibitor may be selected from the group consisting of: double- stranded RNA (e.g. a CCR5 or CCRl -specific siRNA or other RNA interfering oligonucleotides), a compound inhibiting or antagonizing the binding of the chemokine receptor (e.g.
  • CCR5 or CCRl to its ligand, a compound antagonizing the binding of RANTES (the natural ligand to CCR5) to CCR5, neutralizing antibody to CCR5, a neutralizing ' antibody to CCRl, a ligand corresponding to a neutralizing antibody to CCR5 (e.g. a peptide epitope derived from the corresponding chemokine, e.g.
  • RANTES which peptide epitope binds the CCR5 epitope recognized by the neutralizing antibody
  • a ligand corresponding to a neutralizing antibody to CCRl an isolated peptide derived from the sequences of CCR5 or analogs thereof capable of inhibiting CCR5
  • an isolated peptide derived from the sequences of CCRl or analogs thereof capable of inhibiting CCRl antisense nucleic acids, antagonist microRNA and enzymatic RNA molecule, as described herein.
  • Each possibility represents a separate embodiment of the invention.
  • inhibitor and “antagonist” as used herein are interchangeable and refer to a substance that interferes with the expression of a gene, specifically, the gene encoding CCRl or CCR5, or interferes with the activity of the gene product, specifically, interferes with the activity of CCR5 and/or CCRl .
  • the inhibitor interferes with CCR5 expression. In another embodiment the inhibitor interferes with CCRl expression.
  • Interference with gene expression includes, but is not limited to, inhibition of translation or induction of gene transcript cleavage.
  • expression-inhibiting oligonucleotides may conveniently be used.
  • compounds that interact with chemokine receptors and induce their internalization and/or enhance their intracellular sequestering have been described and may be used in embodiments of the invention.
  • the inhibitor interferes with CCR5 activity. In another embodiment, the inhibitor interferes with CCRl activity.
  • Interference with gene activity includes specific inhibition or reduction of at least one biological activity mediated by the gene, e.g. at least one activity mediated by CCR5 or CCRl upon binding of their respective chemokine ligands. Specific inhibition of activity means that other cellular activities not mediated by or associated with the chemokine receptor are not substantially inhibited.
  • the inhibitors induce a direct inhibiting activity, i.e. by binding to the chemokine receptor or its chemokine ligand. Activities mediated by chemokine receptors may include chemokine mediated signaling, chemotaxis and Ca ++ influx.
  • N' derived RANTES analogs produced by total synthesis such as aminooxypentane (AOP)-RANTES activate early signaling events following binding to CCR5, including triggering of tyrosine phosphorylation and activation of the Janus activated kinase (JAK)/STAT pathway, but do not induce sustained cell polarization and chemotaxis.
  • compounds such as N-nonanoyl (NNY)-RANTES, l-Thia-Pro2,l-a-cyclohexyl-Gly3-NNY- RANTES (PSC-RANTES), induce internalization and inhibit the re-expression of CCR5 (Mosier et al. Journal of Virology, May 1999, p. 3544-3550, Vol. 73, No. 5).
  • the present invention is directed in some embodiments to the use of CCRl and CCR5 antagonists including the various CCRl and CCR5 inhibitors and antagonist that are known in the art.
  • the inhibitors may include peptides, polypeptides, antibodies, nucleic acids and small molecule inhibitors, as described hereinbelow. Each possibility represents a separate embodiment of the invention.
  • compounds inhibiting or antagonizing the binding of the chemokine receptor e.g. CCR5 or CCRl
  • the chemokine receptor typically include small or low molecular weight compounds including but not limited to piperazine derivatives, anilide derivatives, benzanilides, benzazepine derivatives, pyrrolidine compounds and other substituted heterocyclic compounds known in the art as capable of antagonizing the binding of the chemokine receptor to its chemokine ligand.
  • US Patent No. 7,384,944 discloses piperazine derivatives useful as selective CCR5 antagonists useful in the treatment of Human Immunodeficiency Virus (HIV).
  • US Patent No. 6,689,783 discloses aryl oxime-piperazines useful as CCR5 antagonists.
  • Other examples of CCR5 antagonists include a compound that antagonizes binding of RANTES as a
  • non-peptide molecules may be synthesized or isolated by methods well known in the art, for example by organic chemistry methods as described in the above-mentioned publications.
  • Additional antagonist suitable for treating or preventing HCC according to the principles of the present invention further include immunogenic molecules, such as, antibodies to CCR5, CCR1 and their respective ligands.
  • antibody refers to an antibody, preferably a monoclonal antibody, or fragments thereof, including, but not limited to, a full length antibody having a human immunoglobulin constant region, a monoclonal IgG, a single chain antibody, a humanized monoclonal antibody, an F(ab') 2 fragment, an F(ab) fragment, an Fv fragment, a labeled antibody, an immobilized antibody and an antibody conjugated with a heterologous compound.
  • the antibody is a monoclonal antibody.
  • the antibody is a polyclonal antibody.
  • the antibody is a humanized antibody.
  • Antibodies may be generated via any one of several known methods, which may employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries, or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique. Besides the conventional method of raising antibodies in vivo, antibodies can be generated in vitro using phage display technology, by methods well known in the art (e.g. Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1).
  • An antigen e.g. CCR5 or immunogenic complex (e.g. a CCR5 epitope conjugated to a protein carrier such as BSA) can be injected into suitable mammalian subjects such as mice,
  • Suitable protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule designed to boost production of antibodies in the serum.
  • the titers of the immune serum can readily be measured using immunoassay procedures which are well known in the art.
  • the antisera obtained can be used directly (e.g. as diluted sera or as purified polyclonal antibodies), or monoclonal antibodies may be obtained, as described
  • a monoclonal antibody is a substantially homogeneous population of antibodies to a specific antigen.
  • mAbs may be obtained by methods known to those skilled in the art. See, for example US patent 4,376,1 10; Ausubel et al ("Current Protocols in Molecular Biology," Volumes I-III, John Wiley & Sons, Baltimore, Maryland, 1994).
  • a hybridoma producing a mAb 5 may be cultivated in vitro or in vivo. High titers of mAbs can be obtained in in vivo production where cells from the individual hybridomas are injected intraperitoneally into pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs.
  • MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • Antibody fragments may be obtained using methods well known in the art. (See, for example, Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary (CHO) cell culture or other protein expression
  • antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • (Fab') 2 antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Ample guidance for practicing such methods is provided in the literature of the art (for example, refer to: U.S.
  • An Fv is composed of paired heavy chain variable and light chain variable domains. This association may be noncovalent. Alternatively, as described hereinabove, the variable domains may be linked to generate a single-chain Fv by an intermolecular disulfide bond, or alternately such chains may be cross-linked by chemicals such as glutaraldehyde.
  • the Fv is a single-chain Fv.
  • Single-chain Fvs are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains.
  • Ample guidance for producing single-chain Fvs is provided 5 in the literature of the art. Improved bivalent miniantibodies, with identical avidity as whole antibodies, may be produced by high cell density fermentation of Escherichia coli. (U.S. Pat. No. 4,946,778).
  • human antibody includes antibodies having variable and constant regions corresponding substantially to human germline immunoglobulin sequences known in the art.
  • Human antibodies used in embodiments of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, CDR3.
  • the human antibody can have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline
  • Chimeric antibodies are molecules, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Antibodies which have variable region framework residues substantially from human antibody (termed an acceptor antibody) and complementarity determining regions substantially from a mouse antibody (termed a donor antibody) are also referred to as humanized antibodies.
  • Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (e.g.
  • CDR grafting may be performed to alter certain properties of the antibody molecule including affinity or specificity.
  • a non-limiting example of CDR grafting is disclosed in US patent 5,225,539. Further methods for producing chimeric antibodies are described, for example, in US patent 4,81 ,567. These references are hereby incorporated by reference.
  • humanized antibodies are preferably used.
  • Humanized forms of non-human (e.g., murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having (preferably minimal) portions derived from non-human antibodies.
  • Humanized antibodies include antibodies in which the CDRs of a human antibody (recipient antibody) are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired functionality.
  • donor antibody such as mouse, rat, or rabbit
  • the Fv framework residues of the human antibody are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the framework regions correspond to those of a relevant human consensus sequence.
  • Humanized antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as imported residues, which are typically taken from an imported variable domain. Humanization can be performed as is known in the art (see, for example: U.S. Pat. No. 4,816,567), by substituting human CDRs with corresponding rodent CDRs. Accordingly, humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies may be typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various additional techniques known in the art, including phage-display libraries or other well known methods (e.g. U.S. Pat. No. 5,545,807).
  • antibodies After antibodies have been obtained, they may be tested for activity, for example via enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the antibodies of the present invention are anti-CCR5 or anti- CCR1 antibodies, i.e. Abs that specifically bind to CCR5 or CCR1, respectively.
  • the terms "specific binding” or “specifically binds” refers to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the association constant K A is higher than 10 6 M "1 . If necessary, nonspecific binding can be reduced without substantially affecting specific binding by varying the binding conditions.
  • binding conditions such as concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g., serum albumin, milk casein), etc.
  • concentration of a blocking agent e.g., serum albumin, milk casein
  • concentration of a blocking agent e.g., serum albumin, milk casein
  • concentration of a blocking agent e.g., serum albumin, milk casein
  • detection of the capacity of an antibody to specifically bind an antigen e.g. CCR5 or CCR1 or their respective chemokine ligands, may be performed by quantifying specific antigen-antibody complex formation (e.g. by ELISA).
  • the present invention is directed to CCR5 or CCR1 -neutralizing antibodies.
  • a "neutralizing antibody” as used herein refers to a molecule having an antigen binding site to a target molecule, e.g. a chemokine, which is capable of reducing or inhibiting (blocking) activity or signaling mediated by the chemokine and/or the respective chemokine receptor. This activity or signaling is conveniently determined by in vivo or in vitro assays, as per the specification.
  • CCR5 -neutralizing antibodies of the invention are anti-CCR5 antibodies that inhibit CCR5 activity.
  • the phrase “inhibit” or “antagonize” activity of a chemokine receptor e.g.
  • CCR5 and CCR1 and its cognates refers to a reduction, inhibition, or otherwise diminution of at least one activity mediated by the chemokine receptor due to the activity of a chemokine receptor antagonist (e.g. binding an anti-CCR5 antibody), wherein the reduction is relative to the activity of the chemokine receptor in the absence of the same antagonist.
  • the activity can be measured using any technique known in the art, including, for example, as described in the Examples. Inhibition or antagonism does not necessarily indicate a total elimination of the receptor's biological activity.
  • a reduction in activity may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • a "ligand corresponding to a neutralizing antibody” means a sequence derived (obtained) from the chemokine ligand of the chemokine receptor to which the neutralizing antibody is directed.
  • This sequence (polypeptide or peptide) is capable of specifically binding the epitope recognized by the neutralizing antibody, thus mimicking the activity of the antibody in inhibiting receptor-chemokine binding.
  • the sequence may also contain derivatizations and substitutions (e.g. conservative substitutions) compared to the native sequence of the chemokine, as long as the binding requirements and hence the functional characteristics are maintained.
  • Suitable inhibitory molecules further include isolated peptides derived from the sequences of CCR5, CCR1, their respective ligands (e.g. RANTES or other chemokines capable of binding and inducing a biological activity mediated by these receptors), or analogs capable of inhibiting CCR5 or CCR1 dependent activities.
  • CCR5 CCR5
  • CCR1 their respective ligands
  • analogs capable of inhibiting CCR5 or CCR1 dependent activities.
  • Such peptides are readily chosen or designed by the skilled artisan according to the published sequences of the corresponding chemokine receptors.
  • Candidate sequences may be screened for the desired inhibitory activity using cell culture assays (e.g. inhibition of RANTES- induced chemotaxis using the Transwell system) or animal models known in the art.
  • the resulting peptide inhibitors may be synthesized by well known chemical or recombinant methods, e.g. as detailed hereinbelow.
  • amino acid sequence of human CCR5 may be represented by SEQ ID NO: 2, as follows:
  • amino acid sequence of human CCR1 may be represented by SEQ ID NO: 3, as follows:
  • Suitable compounds for inhibiting gene expression include, but are not limited to, double-stranded RNA (such as short- or small-interfering RNA or "siRNA”), antisense nucleic acids, antagonist microRNAs, such as, antagomiRs, and enzymatic RNA molecules, such as ribozymes.
  • siRNA double-stranded RNA
  • antisense nucleic acids such as short- or small-interfering RNA or "siRNA”
  • antagonist microRNAs such as, antagomiRs
  • enzymatic RNA molecules such as ribozymes.
  • Each possibility represents a separate embodiment of the invention.
  • Each of these compounds can be targeted to a given gene and interfere with the expression of (e.g., inhibit translation of, induce cleavage or destruction of) the target gene product. This ability to silence a gene has broad potential for treating human diseases, and many researchers and commercial entities are currently investing considerable resources in developing therapies based on this technology.
  • expression-inhibiting oligonucleotides Such DNA and RNA-based compounds are further referred to collectively as "expression-inhibiting oligonucleotides".
  • This term as used herein denotes an oligonucleic acid capable of specifically reducing the expression of the gene products, i.e. the level of mRNA encoding the protein and/or the level of the protein, below the level that is observed in the absence of the oligonucleic acid.
  • expression inhibition may be determined by measuring the activity of the protein, e.g., in the case of chemokine receptors, by measuring an activity mediated by the chemokine receptor upon contacting its respective chemokine ligand.
  • gene expression is down-regulated by at least 25%, preferably at least 50%, at least 70%, 80% or at least 90%. In certain other embodiments, partial down-regulation is preferred.
  • expression-inhibiting (down-regulating or silencing) oligonucleic acids are antisense molecules, RNA interfering molecules (RNAi), and enzymatic nucleic acid molecules, as detailed herein.
  • the length of the molecule may vary between short oligonucleic acids of about 20 nucleic acid residues or base pairs to longer polynucleic acids as long as thousands of nucleic acid residues, as detailed further below.
  • the expression-inhibiting oligonucleic acid comprises at least one nucleic acid sequence substantially complementary to one region of the target gene or transcript thereof.
  • “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence. A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule, which can form hydrogen bonds (e.g., Watson- Crick base pairing) with a second nucleic acid sequence. “Fully complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • substantially complementary refers to a molecule in which about 80% of the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In some embodiments substantially complementary refers to 85%, 90%, 95% of the contiguous residues of a nucleic acid sequence hydrogen bonding with the same number of contiguous residues in a second nucleic acid sequence.
  • Double stranded RNA induced gene silencing can occur on at least three different levels: (i) transcription inactivation, which refers to RNA guided DNA or histone methylation; (ii) siRNA induced mRNA degradation; and (iii) mRNA induced transcriptional attenuation. Efficient sequence specific gene silencing through the use of siRNA is possible if particular siRNAs are selected by rational design.
  • RNAi RNA induced silencing
  • Gene expression according to the present invention can be inhibited by inducing RNA interference of the gene with an isolated double-stranded RNA molecule which has at least 90%, at least 95%, at least 98%, at least 99%, or 100%, sequence homology with at least a portion of the gene.
  • the dsRNA molecule is a short small interfering RNA or siRNA.
  • siRNA useful in the present methods comprise short double-stranded RNA from about 10 nucleotides to about 40 nucleotides in length.
  • the siRNA comprises a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base- pairing interactions (hereinafter "base-paired").
  • the sense strand comprises a nucleic acid sequence that is substantially identical to a nucleic acid sequence contained within the target gene.
  • the RNA interference molecule can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule.
  • the RNAi molecule can be a single-stranded hairpin polynucleotide having self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule or it can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active molecule capable of mediating RNAi.
  • RNAi is a generic term used throughout the specification to include small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species that can be cleaved in vivo to form siRNAs.
  • RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving rise to transcripts which form dsRNAs or hairpin RNAs in cells, and/or transcripts that can produce siRNAs in vivo.
  • the siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex.
  • the siRNAs are around 19-30 nucleotides in length, and even more preferably 21-23 nucleotides in length, e.g., corresponding in length to the fragments generated by nuclease "dicing" of longer double-stranded RNAs.
  • one or both strands of the siRNA of the invention can also comprise a 3' overhang.
  • a "3' overhang” refers to at least one unpaired nucleotide extending from the 3 '-end of an RNA strand.
  • the siRNA of the invention comprises at least one 3' overhang of from 1 to about 6 nucleotides in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length.
  • each strand of the siRNA of the invention can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid (“UU").
  • TT dithymidylic acid
  • UU diuridylic acid
  • RNAi molecules suitable for use with the present invention can be effected as follows.
  • the nucleic acid sequence target e.g. CCR5
  • Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites.
  • potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites that exhibit significant homology to other coding sequences are filtered out.
  • Qualifying target sequences are selected as template for siRNA synthesis.
  • Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %.
  • Several target sites are preferably selected along the length of the target gene for evaluation.
  • a negative control is preferably used in conjunction.
  • Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome.
  • a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • An encoded siRNA agent of the present invention are of at least 10, at least 15, at least 17 or at least 19 bases specifically hybridizable with a CCR5 or CCR1 mRNA.
  • a nucleic acid sequence specifically hybridizable with a chemokine receptor mRNA has a preference for hybridizing (in cells, under physiological conditions) with a chemokine receptor mRNA as opposed to a non-related RNA molecule (e.g. GAPDH).
  • said sequence has at least a 5-fold preference for hybridizing with a chemokine receptor mRNA as opposed to a non-related RNA molecule.
  • a siRNA specifically hybridizable with a chemokine receptor mRNA has sufficient complementarity to an RNA product of a chemokine receptor gene for the siRNA molecule to direct cleavage of said RNA via RNA interference.
  • RNAi molecules and methods for producing RNAi oligonucleotides can be found for example in US Patent Application Publication Nos. 20050100907, 20020137210 and the like.
  • Antagomirs or "antagonist microRNA”, as used herein, refer to engineered oligonucleotides (sometimes together with chemical modifications) that are used to antagonize gene functions, based on complementation and hybridization.
  • an antisense nucleic acid refers to a nucleic acid molecule that binds to target RNA by means of RNA-RNA, RNA-DNA or RNA-peptide nucleic acid interactions, which alters the activity of the target RNA.
  • Antisense nucleic acids suitable for use in the present methods are single-stranded nucleic acids (e.g. RNA, DNA, RNA-DNA chimeras, peptide nucleic acid (PNA)) that generally comprise a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a gene.
  • the antisense nucleic acid can comprise a nucleic acid sequence that is 50-100% complementary, 75-100% complementary, or 95-100% complementary to a contiguous nucleic acid sequence in the gene product.
  • Antisense oligonucleotides are nucleic acids that are complementary (or antisense) to the coding strand (sense strand) of the mRNA encoding a particular protein (also known as the negative strand). Although antisense oligonucleic acids are typically R A based, they can also be DNA based. Additionally, antisense oligonucleotides are often modified to increase their stability. These modifications are known in the art and include, but are not limited to modifying the backbone of the oligonucleotide, modifying the sugar moieties, or modifying the base. Also inclusive in these modifications are various DNA-RNA hybrids or constructs commonly referred to as "gapped" oligonucleotides.
  • the binding of these antisense molecules to the mRNA is believed to induce stretches of double stranded RNA that trigger degradation of the messages by endogenous RNAses.
  • ribosomes which are in the process of making the protein from the RNA, are blocked from progressing as they cannot move along the regions of double stranded RNA that are formed.
  • the oligonucleotides are specifically designed to bind near the promoter of the message, and under these circumstances, the antisense oligonucleotides may additionally interfere with translation of the message.
  • antisense oligonucleotides Regardless of the specific mechanism by which antisense oligonucleotides function, their administration to a cell or tissue allows the degradation of the mRNA encoding a specific protein or prevention of its translation. Accordingly, antisense molecules decrease the expression and/or activity of a particular protein.
  • an antisense oligonucleic acid that specifically binds to and mediates the degradation of a particular protein, it is important that the sequence recognized by the oligonucleic acid is unique or substantially unique to that particular protein. For example, sequences that are frequently repeated across protein may not be an ideal choice for the design of an oligonucleic acid that specifically recognizes and degrades a particular message.
  • One skilled in the art can design an oligonucleic acid, and compare the sequence of that oligonucleic acid to nucleic acid sequences that are deposited in publicly available databases to confirm that the sequence is specific or substantially specific for a particular protein.
  • the antisense molecule may be selected from the group consisting of : a) a DNA antisense molecule; b) a RNA antisense molecule; c) a triplex forming molecule (see below); and d) analogs of a) or b) or c), wherein the oligonucleic acid comprises a sequence substantially complementary to at least a part of a CCR5 or CCR1 transcript.
  • the antisense molecule is an RNA antisense molecule.
  • An encoded antisense molecule according to the invention may be as short as about 20 nucleotides in length, however longer sequences are preferred.
  • the antisense molecule is at least 25, preferably at least 30, and more preferably at least 100 nucleotides in length.
  • longer sequences of about 1000 nucleotides or more are preferred (e.g. a molecule substantially complementary to a full-length CCR5 or CCR1 transcript).
  • An antisense molecule is typically about 100-2500 bases in length.
  • an antisense molecule of the invention comprises at least one nucleic acid sequence substantially complementary to at least one target sequence of a CCR5 or CCR1 transcript.
  • said encoded antisense molecule comprises at least one sequence that is fully complementary to a target sequence of about 20 to about 30 nucleotides of a CCR5 or CCR1 transcript. More preferably, said encoded antisense molecule comprises at least two sequences that are fully complementary to a target sequence of about 20 to about 30 nucleotides of the transcript.
  • Another particular antisense molecules includes oligonucleic acids that bind to double- stranded or duplex nucleic acids (e.g., in a folded region of the target RNA or in the target gene), forming a triple helix-containing, or "triplex" nucleic acid.
  • Triple helix formation results in inhibition of gene expression by, for example, preventing transcription of the gene, thereby reducing or eliminating its activity in a cell. Without intending to be bound by any particular mechanism, it is believed that triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules to occur.
  • Triplex oligo- and polynucleotides are constructed using the base-pairing rules of triple helix formation (see, e.g., Rigas et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:9591) and the target gene mRNA and/or gene sequence.
  • the triplex-forming oligonucleotides of the invention comprise a specific sequence of from about 10 to at least about 25 nucleotides or longer complementary to a specific sequence in the target RNA or gene (i.e., large enough to form a stable triple helix, but small enough, depending on the mode of delivery, to administer in vivo, if desired).
  • oligonucleotides are designed to bind specifically to the regulatory regions of the target gene (e.g., the 5'-flanking sequence, promoters, and enhancers) or to the transcription initiation site (e.g., between -10 and +10 from the transcription initiation site or translation start site, e.g., at a methionine residue).
  • the regulatory regions of the target gene e.g., the 5'-flanking sequence, promoters, and enhancers
  • the transcription initiation site e.g., between -10 and +10 from the transcription initiation site or translation start site, e.g., at a methionine residue.
  • an "enzymatic nucleic acid” refers to a nucleic acid comprising a substrate binding region that has complementarity to a contiguous nucleic acid sequence of a gene, and which is able to specifically cleave the gene.
  • the enzymatic nucleic acid substrate binding region can be, for example, 50-100% complementary, 75-100% complementary, or 95-100% complementary to a contiguous nucleic acid sequence in a gene.
  • the enzymatic nucleic acids can also comprise modifications at the base, sugar, and/or phosphate groups.
  • An exemplary enzymatic nucleic acid for use in the present methods is a ribozyme.
  • enzymatic nucleic acid is used interchangeably with for example, ribozymes, catalytic RNA, enzymatic R A, catalytic DNA, aptazyme or aptamer-binding ribozyme, catalytic oligonucleotide, nucleozyme, DNAzyme, RNAenzyme.
  • ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers, and specific somatic mutations in genetic disorders
  • Ribozymes and ribozyme analogs are described, for example, in US Patent Nos. 5,436,330; 5,545,729 and 5,631 ,1 15.
  • ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy particular mRNAs
  • other ribozymes include hammerhead ribozymes.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • the construction and production of hammerhead ribozymes is well known in the art, see for example WO 2004/041197.
  • Ribozymes also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one which occurs naturally in 25 Tetrahymena thermophila (known as the IVS, or L- 19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (see e.g. published International patent application No. WO 88/04300).
  • Cech-type ribozymes such as the one which occurs naturally in 25 Tetrahymena thermophila (known as the IVS, or L- 19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (see e.g. published International patent application No. WO 88/04300).
  • the Cech-type ribozymes have an eight base pair active site that hybridizes to a target RNA sequence "hereafter cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes that target eight base- pair active site sequences. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • DNAzyme molecule Another agent capable of silencing a target gene is a DNAzyme molecule, which is capable of specifically cleaving an mRNA transcript or a DNA sequence of a target gene.
  • DNAzymes are single-stranded polynucleotides that are capable of cleaving both single- and double-stranded target sequences. Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single- and double-stranded target cleavage sites are disclosed in US Patent No. 6,326, 174.
  • the 10-23 DNA enzyme comprises a loop structure that connects two arms.
  • the two arms provide specificity by recognizing the particular target nucleic acid sequence while the loop structure: provides catalytic function under physiological conditions.
  • the unique or substantially sequence is a G/C rich of approximately 18 to 22 nucleotides. High G/C content helps insure a stronger interaction between the DNA enzyme and the target sequence.
  • the specific antisense recognition sequence that will target the enzyme to the message is divided so that it comprises the two arms of the DNA enzyme, and the DNA enzyme loop is placed between the two specific arms.
  • Nucleic acid inhibitors such as those described above may be readily designed according to the published nucleic acid sequences of these genes and coding sequences. Such sequences may be derived e.g. from the above-identified amino acid sequences and accession numbers. Enterz Genebank gene identification numbers of human CC 1 and CCR5 are 1230 and 1234, respectively.
  • RANTES refers to a cytokine (which may be mammalian) capable of binding to CCR5, CCR3 and/or CCR1 receptors, having an amino acid sequence of a naturally occurring mammalian RANTES polypeptide (full length or mature form), e.g., an amino acid sequence shown as SEQ ID NO: 1 , as follows: MKVSAAALAVILIATALCAPASASPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPE
  • KKWVREYINSLEMS SEQ ID NO: 1 ; genebank no. AF043341, human RANTES precursor.
  • the RANTES polypeptide may bind to CCR5, CCR3 and CCR1 receptors of mammalian origin, e.g., human CCR5.
  • the use of RANTES derivatives is contemplated.
  • the derivatives may be a fragment or homolog of a naturally occurring RANTES capable of binding to CCR5, CCR3 and/or CCR1 receptors that exhibits at least one of the following features: (1) an amino acid sequence substantially identical to, e.g., at least 85%, 90%, 95%o, 96%, 97%, 98%, 99% or 100% identical to, an amino acid sequence shown as SEQ ID NO: 1 or a fragment thereof (e.g.
  • the derivative is a chemical derivatives of RANTES.
  • RANTES polypeptides and derivatives according to the invention induce a biological activity, typically a CCR5 mediated biological activity, upon receptor binding.
  • the RANTES derivative is a RANTES agonist.
  • the RANTES derivative is a RANTES partial agonist (a derivative capable of inducing partial response mediated by a RANTES receptor but does not exert the full spectrum of biological activities associated with the chemokine).
  • a RANTES derivative may be substantially devoid of chemotactic activity or induce down-regulation of CCR5 expression.
  • derivatives may retain 80%, 90%, 95% or 100% of a level of a biological activity mediated by binding of RANTES to CCR5.
  • RANTES polypeptides and derivatives may be synthesized e.g. by chemical synthesis or recombinant methods known in the art, e.g. as detailed above.
  • RANTES polypeptides may be isolated from samples of a biological source, by methods known in the art.
  • RANTES derivatives may comprise peptide homologs essentially based on the amino acid sequence of a RANTES polypeptide but having one or more amino acid residues deleted, substituted or added.
  • amino acid residues are substituted, such conservative replacements which are envisaged are those which do not significantly alter the structure or biological activity of the peptide.
  • basic amino acids will be replaced with other basic amino acids, acidic ones with acidic ones and neutral ones with neutral ones.
  • homologs comprising non-conservative amino acid substitutions are further envisaged, as long as said homologs essentially retain the biological activities of the RANTES polypeptide, as detailed herein.
  • RANTES polypeptides may be N' truncated, C truncated or both (e.g. by 1 -5 amino acids).
  • the present invention encompasses RANTES derivatives containing non-natural amino acid derivatives or non-protein side chains.
  • the RANTES polypeptides and derivatives may be used having a terminal carboxy acid, as a carboxy amide, as a reduced terminal alcohol or as any pharmaceutically acceptable salt, e.g., as metal salt, including sodium, potassium, lithium or calcium salt, or as a salt with an organic base, or as a salt with a mineral acid, including sulfuric acid, hydrochloric acid or phosphoric acid, or with an organic acid e.g., acetic acid or maleic acid.
  • any pharmaceutically acceptable salt of the polypeptides and peptides utilized in methods of the invention may be used, as long as the biological activities of the polypeptide are maintained.
  • Chemical derivatives may have one or more residues chemically derivatized by reaction of side chains or functional groups.
  • the RANTES polypeptide or derivative may be administered in the form of a complex or conjugate having a significantly longer plasma half-life compared to native RANTES polypeptide.
  • the polypeptides and derivatives may be conjugated with a half-life elongating compound.
  • Exemplary half-life extending moieties that can be used, in accordance with the present invention, include an immunoglobulin Fc domain, or a portion thereof, or a biologically suitable polymer or copolymer, for example, a polyalkylene glycol compound, such as a polyethylene glycol or a polypropylene glycol.
  • a polyalkylene glycol compound such as a polyethylene glycol or a polypropylene glycol.
  • Other appropriate polyalkylene glycol compounds include, but are not limited to, charged or neutral polymers of the following types: dextran, polylysine, colominic acids or other carbohydrate based polymers, polymers of amino acids, and biotin derivatives.
  • half-life extending moiety examples include a copolymer of ethylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-l ,3-dioxolane, a poly-l ,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid (e.g., polylysine), a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an
  • a C3 ⁇ 4 domain of Fc an albumin (e.g., human serum albumin (HSA)); see, e.g., U.S. Pat. No. 6,926,898 and US 2005/0054051 ; U.S. Pat. No. 6,887,470), a transthyretin (TTR; see, e.g., US 2003/0195154 Al ; 2003/0191056 Al), or a thyroxine-binding globulin (TBG).
  • HSA human serum albumin
  • said RANTES polypeptide or derivative may be conjugated to a compound selected from the group consisting of: polyethylene glycol, a copolymer of ethylene glycol, a polypropylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-l,3-dioxolane , a poly-l ,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n- vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid,
  • said RANTES polypeptide or derivative is conjugated to or complexed with polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • polyethylene glycol or “PEG” is meant a polyalkylene glycol compound or a derivative thereof, with or without coupling agents or derivatization with coupling or activating moieties (e.g., with aldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate, azirdine, oxirane, orthopyridyl disulphide, vinylsulfone, iodoacetamide or a maleimide moiety).
  • useful PEG includes substantially linear, straight chain PEG, branched PEG, or dendritic PEG. (See, e.g., U.S. Pat. No.
  • PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art.
  • Pegylation of RANTES polypeptides may be carried out by any of the pegylation reactions known in the art, as described for example in EP 0154316; EP 0401384 and U.S. Pat. No. 4,179,337.
  • Methods for preparing pegylated RANTES polypeptides will generally comprise the steps of (a) reacting the polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby RANTES polypeptide becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s).
  • polyethylene glycol such as a reactive ester or aldehyde derivative of PEG
  • the optimal reaction conditions for the acylation reactions will be determined based on known parameters and the desired result. For example, the larger the ratio of PEG: protein, the greater the percentage of poly-pegylated product .
  • the RANTES polypeptide derivative will have a single PEG moiety at the amino terminus. See U.S. Pat. No. 5,234,784.
  • n is 20 to 2300.
  • the combined or total molecular mass of PEG used in a PEG-conjugated peptide of the present invention is from about 3,000 Da or 5,000 Da, to about 50,000 Da or 60,000 Da (total n is from 70 to 1 ,400), or from about 10,000 Da to about 40,000 Da (total n is about 230 to about 910).
  • combined mass for PEG is from about 20,000 Da to about 30,000 Da (total n is about 450 to about 680).
  • the number of repeating units "n" in the PEG is approximated for the molecular mass described in Daltons. It is preferred that the combined molecular mass of PEG on an activated linker is suitable for pharmaceutical use. Thus, in typical embodiments the combined molecular mass of the PEG molecule should not exceed about 100,000 Da.
  • Activated PEG such as PEG-aldehydes or PEG-aldehyde hydrates
  • PEG-aldehydes or PEG-aldehyde hydrates can be chemically synthesized by known means or obtained from commercial sources, e.g., Shearwater Polymers, (Huntsville, Ala.) or Enzon, Inc. (Piscataway, N.J).
  • pegylation may reduce liver uptake and enhance blood plasma circulation.
  • said RANTES polypeptide or derivative is conjugated to or complexed with an immunoglobulin Fc domain or portion thereof
  • the polypeptides may be fused at either the N-terminus or the C-terminus to one or more domains of an Fc region of human IgG.
  • the RANTES polypeptide may be fused at either the N-terminus or C-terminus using methods known to the skilled artisan (e.g. recombinant technology).
  • the resulting fusion protein may be purified by use of a Protein A or Protein G affinity column.
  • Peptides and proteins fused to an Fc region have been found to exhibit a substantially greater half-life in vivo than the unfused counterpart.
  • the Fc region may be a naturally occurring Fc region, or may be altered to improve certain qualities, such as therapeutic qualities, circulation time, decrease aggregation problems, etc.
  • Fc domain encompasses native Fc and Fc variant molecules and sequences, and includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
  • native Fc refers to molecule or sequence comprising the sequence of a non- antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form.
  • the original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgGl or IgG2 are preferred.
  • Native Fc's are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgGA2).
  • class e.g., IgG, IgA, IgE
  • subclass e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgGA2
  • a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG.
  • Fc variant refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn.
  • FcRn a binding site for the salvage receptor
  • Several published patent documents describe exemplary Fc variants, as well as interaction with the salvage receptor. See International Applications WO 97/34 631 ; U.S. Pat. No. 6,096,891,; and WO 04/1 10 472.
  • the term “Fc variant” includes a molecule or sequence that is humanized from a non-human native Fc.
  • a native Fc comprises sites that can be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention.
  • Fc variant includes a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Polysaccharide polymers are another type of water-soluble polymer that can be used for protein modification.
  • Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by a. 1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kDa to about 70 kDa.
  • Dextran is a suitable water-soluble polymer for use in the present invention as a half-life extending moiety by itself or in combination with another half-life extending moiety (e.g., Fc). See, for example, WO 96/1 1953 and WO 96/05309.
  • dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0315456.
  • Dextran of about 1 kDa to about 20 kDa is preferred when dextran is used as a half-life extending moiety in accordance with the present invention.
  • polypeptides and peptides of the invention may be isolated or synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g. Boc or f-Moc chemistry) and solution phase synthesis methods.
  • solid phase e.g. Boc or f-Moc chemistry
  • solution phase synthesis methods e.g. Boc or f-Moc chemistry
  • the peptides can be synthesized by a solid phase peptide synthesis method of Merrifield (1963, J Am Chem Soc 85, 2149).
  • a peptide of the present invention can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, 1984) or by any other method known in the art for peptide synthesis.
  • polypeptides and peptides may be produced by recombinant technology.
  • Recombinant methods for designing, expressing and purifying proteins and peptides are known in the art (see, e.g. Sambrook et al., 1989, 1992, 2001 , Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York).
  • Nucleic acid molecules may include DNA, RNA, or derivatives of either DNA or RNA.
  • An isolated nucleic acid sequence encoding a polypeptide or peptide can be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof.
  • a nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional peptide.
  • a polynucleotide or oligonucleotide sequence can be deduced from the genetic code of a protein, however, the degeneracy of the code must be taken into account, as well as the allowance of exceptions to classical base pairing in the third position of the codon, as given by the so-called "Wobble rules". Polynucleotides that include more or less nucleotides can result in the same or equivalent proteins.
  • selected host cells e.g. of a microorganism such as E. coli or yeast, are transformed with a hybrid viral or plasmid DNA vector including a specific DNA sequence coding for the polypeptide or polypeptide analog and the polypeptide is synthesized in the host upon transcription and translation of the DNA sequence.
  • sequences may be derived directly from the corresponding sequence of the receptor or chemokine (such that they may be identical to a portion of a sequence of the chemokine or chemokine receptor) or may contain certain derivatizations and substitutions.
  • the use of salts and functional derivatives of these sequences are contemplated, as long as they retain the respective biologic functions, as detailed herein. Accordingly the present invention encompasses peptide homologs containing non-natural amino acid derivatives or non-protein side chains.
  • the peptide homologs of the invention may be used having a terminal carboxy acid, as a carboxy amide, as a reduced terminal alcohol or as any pharmaceutically acceptable salt, e.g., as metal salt, including sodium, potassium, lithium or calcium salt, or as a salt with an organic base, or as a salt with a mineral acid, including sulfuric acid, hydrochloric acid or phosphoric acid, or with an organic acid e.g., acetic acid or maleic acid.
  • any pharmaceutically acceptable salt of the peptide of the invention may be used, as long as the biological activities of the peptide are maintained.
  • the present invention encompasses derivatives containing non-natural amino acid derivatives or non-protein side chains.
  • the polypeptides and derivatives may be used having a terminal carboxy acid, as a carboxy amide, as a reduced terminal alcohol or as any pharmaceutically acceptable salt, e.g., as metal salt, including sodium, potassium, lithium or calcium salt, or as a salt with an organic base, or as a salt with a mineral acid, including sulfuric acid, hydrochloric acid or phosphoric acid, or with an organic acid e.g., acetic acid or maleic acid.
  • any pharmaceutically acceptable salt of the polypeptides and peptides utilized in methods of the invention may be used, as long as the biological activities of the polypeptide are maintained.
  • amino acid residues described herein are preferred to be in the "L” isomeric form.
  • residues in the "D” isomeric form can be substituted for any L-amino acid residue, as long as the peptide substantially retains the desired functional property.
  • Chemical derivatives may have one or more residues chemically derivatized by reaction of side chains or functional groups.
  • derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p- toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine.
  • chemical derivatives those peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline may be substituted for proline; 5 -hydroxyl ysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine may be substituted for lysine.
  • a derivative can differ from the natural sequence of the polypeptides or peptides of the invention by chemical modifications including, but are not limited to, terminal- 5 NH 2 acylation, acetylation, or thioglycolic acid amidation, and by terminal-carboxlyamidation, e.g., with ammonia, methylamine, and the like.
  • the present invention refers to a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient an active agent according to the invention (e.g. a 0 RANTES polypeptide or derivative thereof and/or a CCR5 or CCRl inhibitor), for use in therapy.
  • an active agent according to the invention e.g. a 0 RANTES polypeptide or derivative thereof and/or a CCR5 or CCRl inhibitor
  • the methods of the invention employ administration of an isolated RANTES polypeptide or derivative thereof to said subject in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient or diluent.
  • the methods of the invention employ administration of at least one inhibitor of a chemokine receptor selected from the group consisting of CCRl and CCR5 is administered to said subject in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient or diluent.
  • compositions may be in any pharmaceutical form suitable for administration to a d patient, including but not limited to solutions, suspensions, lyophilized powders for reconstitution with a suitable vehicle or dilution prior to usage, capsules, tablets, sustained- release formulations and the like.
  • the compositions may comprise a therapeutically effective amount of an agent of the present invention, preferably in purified form, and a pharmaceutical excipient.
  • pharmaceutical excipient includes solvents, dispersion media, 5 coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents etc. and combinations thereof, which are compatible with pharmaceutical administration.
  • compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
  • the composition consists essentially of a RANTES polypeptide or derivative thereof and/or a CCR5 or CCRl inhibitor and one or more pharmaceutical excipients.
  • the composition consists of a RANTES polypeptide or derivative thereof and/or a CCR5 or CCRl inhibitor and one or more pharmaceutical excipients.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. Examples of suitable excipients and modes for formulating the compositions are described in the latest edition of "Remington's Pharmaceutical Sciences" by E. W. Martin.
  • compositions according to the invention are typically liquid formulations suitable for injection or infusion.
  • administration of a pharmaceutical composition include oral ingestion, inhalation, intravenous and continues infusion, intraperitoneal, intramuscular, intracavity, subcutaneous, cutaneous, or transdermal administration.
  • the compositions are suitable for intralesional (e.g. intratumoral) administration.
  • the compositions are suitable for intravenous administration.
  • saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • Solutions or suspensions used for intravenous administration typically include a carrier such as physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.), ethanol, or polyol.
  • a carrier such as physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.), ethanol, or polyol.
  • the composition must be sterile and fluid for easy syringability. Proper fluidity can often be obtained using lecithin or surfactants.
  • the composition must also be stable under the conditions of manufacture and storage. Prevention of microorganisms can be achieved with antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.
  • isotonic agents sucrose
  • polyalcohols mannitol and sorbitol
  • sodium chloride may be included in the composition.
  • Prolonged absorption of the composition can be accomplished by adding an agent which delays absorption, e.g., aluminum monostearate and gelatin.
  • the composition may also include a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by 5 infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions include an inert diluent or edible carrier.
  • the composition can be 0 enclosed in gelatin or compressed into tablets.
  • the active agent can be incorporated with excipients and placed in tablets, troches, or capsules.
  • Pharmaceutically compatible binding agents or adjuvant materials can be included in the composition.
  • the tablets, troches, and capsules may optionally contain a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a 5 disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; or a sweetening agent or a flavoring agent.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a 5 disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate
  • a glidant such as colloidal silicon dioxide
  • sweetening agent or a flavoring agent may optionally contain a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose,
  • composition may also be administered by a transmucosal or transdermal route.
  • antibodies that comprise an Fc portion may be capable of crossing mucous membranes
  • Transmucosal administration can be accomplished through the use of lozenges, nasal sprays, inhalers, or suppositories.
  • Transdermal administration can also be accomplished through the use of a composition containing ointments, salves, gels, or creams known in the art.
  • penetrants appropriate to the barrier to be permeated are used.
  • administration by inhalation is accomplished through the use of a composition containing ointments, salves, gels, or creams known in the art.
  • the antibodies are delivered in an aerosol spray from a pressured container or dispenser, which contains a propellant (e.g., liquid or gas) or a nebulizer.
  • a propellant e.g., liquid or gas
  • a nebulizer e.g., nebulizer
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Solutions or suspensions used for intradermal or subcutaneous application typically include at least one of the following components: a sterile diluent such as water, saline solution, SO fixed oils, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and tonicity agents such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases.
  • Such preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials.
  • polypeptide active agents e.g. antibodies and RANTES polypeptides
  • carriers to protect the polypeptide against rapid elimination from the body.
  • Biodegradable polymers e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid
  • Methods for the preparation of such formulations are known by those skilled in the art.
  • Liposomal suspensions can be used as pharmaceutically acceptable carriers too.
  • the liposomes can be prepared according to established methods known in the art (U.S. Pat. No. 4,522,81 1).
  • liposomes containing PEG moieties or glycolipids may advantageously be used to enhance blood plasma retention and/or to reduce liver uptake.
  • larger liposomes e.g. 300 nm or more
  • smaller liposomes e.g. 40 nm or less
  • liposomes of 40-300nm are used, which may have enhanced blood plasma retention.
  • the liposomes may further contain polymers such as PEG (see, for example, Litzinger et al. , Biochim Biophys Acta. 1994 Feb 23;1190(l):99-107).
  • US 2011160642 discloses pegylated liposomal formulations having reduced accumulation in the liver and spleen. A range of liposomes formulated to evade uptake by the reticuloendothelial system and circulate for longer are described in U.S. Pat. No. 6,284,267.
  • antibodies of the present invention may be administered with various effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins.
  • Continuous as well as intermittent intravenous administration can also be achieved using an implantable or external pump (e.g., INFUSAID pump).
  • INFUSAID pump e.g., INFUSAID pump
  • the use of such pumps and adjustment of dosing protocols to the required parameters are well within the abilities of the skilled artisan.
  • composition is formulated in the form of a sustained release formulation.
  • polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention.
  • examples of polymers used in sustained release formulations include, but are not limited to, poly(2 -hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • Sustained-release compositions also include liposomally entrapped polypeptides. Liposomes containing polypeptides useful in the methods of the invention are prepared by methods known per se, e.g. U.S. Pat. Nos. 4,485,045 and 4,544,545.
  • the composition is formulated in the form of a transdermal patch.
  • sustained release formulations enable a prolonged elevation of RANTES plasma levels.
  • nucleic acid active agents e.g. expression-inhibiting oligonucleotides
  • excipients include, without limitation, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • a therapeutic composition further comprises a pharmaceutically acceptable carrier.
  • a “carrier” refers to any substance suitable as a vehicle for delivering a nucleic acid molecule of the present invention to a suitable in vivo or in vitro site.
  • carriers can act as a pharmaceutically acceptable excipient of a therapeutic composition containing a nucleic acid molecule of the present invention.
  • Preferred carriers are capable of maintaining a nucleic acid molecule of the present invention in a form that, upon arrival of the nucleic acid molecule to a cell, the nucleic acid molecule is capable of entering the cell and being expressed by the cell.
  • Carriers of the present invention include: (1) excipients or formularies that transport, but do not specifically target a nucleic acid molecule to a cell (referred to herein as non-targeting carriers); and (2) excipients or formularies that deliver a nucleic acid molecule to a specific site in a subject or a specific cell (i.e., targeting carriers).
  • non-targeting carriers include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum- containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols.
  • Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity.
  • Suitable auxiliary substances for use with nucleic acid agents include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer.
  • Auxiliary substances can also include preservatives, such as thimerosal, m- and o-cresol, formalin and benzol alcohol.
  • Preferred auxiliary substances for aerosol delivery include surfactant substances non-toxic to a subject, for example, esters or partial esters of fatty acids containing from about six to about twenty-two carbon atoms.
  • esters include, caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric, and oleic acids.
  • Other carriers can include metal particles (e.g., gold particles) for use with, for example, a biolistic gun through the skin.
  • Therapeutic compositions of the present invention can be sterilized by conventional methods.
  • Targeting carriers are herein referred to as "delivery vehicles”.
  • Delivery vehicles of the present invention are capable of delivering a therapeutic composition of the present invention to a target site in a subject.
  • a "target site” refers to a site in a subject to which one desires to deliver a therapeutic composition.
  • Examples of delivery vehicles include, but are not limited to, artificial and natural lipid-containing delivery vehicles. Natural lipid-containing delivery vehicles include cells and cellular membranes. Artificial lipid-containing delivery vehicles include liposomes and micelles.
  • a delivery vehicle of the present invention can be modified to target to a particular site in a subject, thereby targeting and making use of a nucleic acid molecule of the present invention at that site.
  • Suitable modifications include manipulating the chemical formula of the lipid portion of the delivery vehicle and/or introducing into the vehicle a compound capable of specifically targeting a delivery vehicle to a preferred site, for example, a preferred cell type.
  • Specifically targeting refers to causing a delivery vehicle to bind to a particular cell by the interaction of the compound in the vehicle to a molecule on the surface of the cell.
  • Suitable targeting compounds include ligands capable of selectively (i.e., specifically) binding another molecule at a particular site. Examples of such ligands include antibodies, antigens, receptors and receptor ligands.
  • an antibody specific for an antigen found on the surface of a target cell can be introduced to the outer surface of a liposome delivery vehicle so as to target the delivery vehicle to the target cell.
  • Manipulating the chemical formula of the lipid portion of the delivery vehicle can modulate the extracellular or intracellular targeting of the delivery vehicle.
  • a chemical can be added to the lipid formula of a liposome that alters the charge of the lipid bilayer of the liposome so that the liposome fuses with particular cells having particular charge characteristics.
  • a preferred delivery vehicle of the present invention is a liposome.
  • a liposome is capable of remaining stable in a subject for a sufficient amount of time to deliver a nucleic acid molecule of the present invention to a preferred site in the subject.
  • a liposome of the present invention is preferably stable in the subject into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour and even more preferably for at least about 24 hours. W
  • Suitable liposomes for use with nucleic acid agents include any liposome.
  • Preferred liposomes for use with nucleic acid agents include those liposomes standardly used_ in, for example, gene delivery methods known to those of skill in the art.
  • more preferred liposomes comprise liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • compositions may also be included in a container, pack, or dispenser and optionally instructions for administration.
  • the kit may contain instructions for administering the composition to a subject afflicted with HCC, as detailed herein.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. All formulations for administration should be in dosages suitable for the chosen route of administration. More specifically, a "therapeutically effective" dose means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure and Examples provided herein.
  • Unit dosage form refers to physically discrete units suited for the patient.
  • Each unit dosage contains a predetermined quantity of an active agent calculated to produce a therapeutic effect in association with the carrier.
  • the unit dosage depends on the characteristics of the agent and the particular therapeutic effect to be achieved.
  • Toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition), LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) and the maximal tolerated dose for a subject compound.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5o/ED 50 .
  • Antibodies that exhibit large therapeutic indices may be less toxic and/or more therapeutically effective.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • _dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.
  • a pharmaceutical composition may be provided that comprises an isolated RANTES polypeptide or derivative thereof in a form enabling a significantly longer plasma half-life of said RANTES polypeptide or derivative compared to native RANTES polypeptide.
  • the isolated RANTES polypeptide or derivative thereof may be in the form of a complex or conjugate having a significantly longer plasma half- life compared to native RANTES polypeptide, e.g. conjugated to polyethylene glycol (PEG) or an immunoglobulin Fc domain or a portion thereof.
  • the pharmaceutical composition may comprise liposomes, PEG and/or glycolipids, as detailed herein.
  • the pharmaceutical composition may be used for the treatment, prevention or amelioration of the onset of HCC.
  • a method for treating hepatocellular carcinoma (HCC) or for inhibiting or delaying the onset of a HCC tumor formation comprising administering to a subject in need thereof an isolated RANTES polypeptide or derivative thereof, so as to enhance the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • HCC hepatocellular carcinoma
  • said subject is afflicted with HCC.
  • the subject in need thereof is susceptible to HCC.
  • the subject in need thereof is a patient with chronic liver inflammation.
  • said subject has progressive steatosis (accumulation of fat in the interstitial tissue of an organ).
  • a method for treating HCC comprising administering to a subject in need thereof an isolated RANTES polypeptide or derivative thereof, so as to enhance the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • a method for inhibiting or delaying the onset of a HCC tumor formation comprising administering to a subject in need thereof an isolated
  • RANTES polypeptide or derivative thereof so as to enhance the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • a method for preventing HCC comprising administering to a subject in need thereof an isolated RANTES polypeptide or derivative thereof, so as to enhance the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • a method for reducing the incidence of HCC tumors comprising administering to a subject in need thereof an isolated RANTES polypeptide or derivative thereof, so as to enhance the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • a method for reducing HCC tumor volume comprising administering to a subject in need thereof an isolated RANTES polypeptide or derivative thereof, so as to enhance the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • a method for ameliorating the onset of HCC comprising administering to a subject in need thereof an isolated RANTES polypeptide or derivative thereof, so as to enhance the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • the HCC may be characterized by lack of ⁇ -catenin expression in the tumor cells (e.g. in at least a portion of the cells of the HCC tumor). In the methods of the invention, the HCC may be characterized by reduced ⁇ -catenin expression in the tumor cells compared to non-tumor cells. In another embodiment, the HCC is characterized by nuclear cyclin Dl levels in tumor cells that are lower than nuclear cyclin Dl levels in non-tumor cells. In another embodiment, the HCC is characterized by lack of cyclin Dl expression in tumor cells. In another embodiment the HCC is characterized by down-regulation (e.g.
  • the HCC is characterized by lack of expression of at least one tumor suppressor gene selected from the group consisting of Cdhl, Dleu2, Plagll, and Rbl2, wherein each possibility represents a separate embodiment of the invention.
  • the HCC is characterized by lack of ⁇ -catenin expression in the tumor cells and nuclear cyclin Dl levels in tumor cells that are lower than nuclear cyclin Dl levels in non-tumor cells.
  • the HCC is characterized by lack of ⁇ -catenin expression in the tumor cells and down-regulation of at least one tumor suppressor gene selected from the group consisting of Cdhl, Dleu2, Plagll, and Rbl2, wherein each possibility represents a separate embodiment of the invention.
  • the HCC is characterized by lack of ⁇ -catenin expression in the tumor cells, nuclear cyclin Dl levels in tumor cells that are lower than nuclear cyclin Dl levels in non-tumor cells and down-regulation of at least one tumor suppressor gene selected from the group consisting of Cdhl , Dleu2, Plagll, and Rbl2, wherein each possibility represents a separate embodiment of the invention.
  • the method comprises:
  • the method may further comprise determining expression of at least one tumor suppressor gene selected from the group consisting of Cdhl , Dleu2, Plagll , and Rbl2, wherein down-regulation (e.g. compared to non-tumor cells) of the at least one tumor suppressor gene indicates that said subject is amenable for treatment.
  • VDLACTPTDV RDVDI (SEQ ID NO: 5, human cyclin Dl, accession no. AAH00076);
  • ADAPNTPAWE AVYTILNDDG GQFVVTTNPV NNDGILKTAK GLDFEAKQQY ILHVAVTNVV PFEVSLTTST
  • VINIIDADLP PNTSPFTAEL THGASANWTI
  • QYNDPTQESI ILKPK ALEV GDYKINLKLM DNQNKDQVTT
  • human Del2 also known as leukemia-associated protein 2, accession no. 043262;
  • Determining the expression (including determining nuclear levels) of gene products may be performed by methods well known in the art, including, but not limited to, enzyme-linked immunosorbent assay (ELISA), reverse-transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry, fluorescence activated cell sorting (FACS), Western blot and other commonly used assays for measuring mRNA transcripts or their polypeptide products.
  • ELISA enzyme-linked immunosorbent assay
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • FACS fluorescence activated cell sorting
  • Western blot and other commonly used assays for measuring mRNA transcripts or their polypeptide products.
  • RT-PCR methods use PCR amplification of RNA molecules which may be relatively rare.
  • RNA molecules are purified from cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as oligo-dT, random hexamers, or gene-specific primers.
  • a reverse transcriptase enzyme such as an MMLV-RT
  • primers such as oligo-dT, random hexamers, or gene-specific primers.
  • a PCR amplification reaction is carried out in a PCR machine.
  • Those of ordinary skill in the art are capable of selecting the length and sequence of the gene-specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles, and the like) that are suitable for detecting specific RNA molecules.
  • a semiquantitative RT-PCR reaction can be employed, by adjusting the number of PCR cycles and comparing the amplification product to known controls.
  • ELISA involves fixation of a sample containing a protein substrate (e.g., fixed cells or a proteinaceous solution) to a surface such as a well of a microtiter plate.
  • a substrate-specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody.
  • Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced.
  • a substrate standard is generally employed to improve quantitative accuracy.
  • Western blot involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nitrocellulose, nylon, or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody-binding reagents.
  • Antibody-binding reagents may be, for example, protein A or secondary antibodies.
  • Antibody-binding reagents may be radiolabeled or enzyme-linked, as described hereinabove. Detection may be by autoradiography, colorimetric reaction, or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane indicative of the protein's migration distance in the acrylamide gel during electrophoresis, resulting from the size and other characteristics of the protein.
  • FACS involves detection of a substrate in situ in cells bound by substrate-specific, fluorescently labeled antibodies.
  • the substrate-specific antibodies are linked to fluorophores. Detection is by means of a cell-sorting machine, which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
  • negative control samples or values corresponding to healthy (non-tumor) cells or tissue and optionally positive control samples or values corresponding to HCC tumor cells.
  • negative control sample may contain non- tumor liver cells e.g. obtained from healthy individuals.
  • samples, as well as samples isolated by e.g. tissue biopsy from the candidate subject may readily be obtained by commonly used methods.
  • reduced expression of the aforementioned markers in tumor cells of said subject compared to a negative control sample either statistically significant or to an extent recognized significant by a skilled artisan (e.g. physician) indicates that said subject is amenable for treatment by the methods of the invention.
  • reduced expression may be by 20%, 40%, 60% 80%, 90% or 100% compared to the negative control.
  • the method comprises administering intravenously to said subject a therapeutically effective amount of an isolated RANTES polypeptide or a derivative thereof.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient or diluent.
  • the composition is not administered in the form of a vaccine further comprising an additional antigen.
  • the composition is not administered by a therapeutic regimen adapted for vaccination.
  • Vaccines and vaccination protocols are well known in the art. Vaccines typically contain one or more antigens to which a desired immune response is to be elicited (e.g. attenuated cancer cells or tumor associated antigens), and often contain additional adjuvants, used to boost or enhance the immune response (e.g. Alum. Or mineral oil).
  • the level of RANTES may be enhanced specifically in blood plasma of said subject.
  • the level of RANTES may be enhanced preferentially or 5 exclusively in blood plasma, for example plasma RANTES levels may be enhanced to a significant extent more than its relative enhancement in other cells or organs (such as the liver).
  • enhancement in the level of RANTES engulfed by surveillance macrophages and directed to degradation in the liver or spleen does not represent enhancement of the biologically active chemokine.
  • Specific enhancement of RANTES may also
  • the methods refer to enhancement of at least 4 fold.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is 4-10 fold higher than mean blood plasma levels of native RANTES in healthy individuals.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is 4-10 fold higher than mean blood plasma levels of native RANTES in healthy individuals.
  • RANTES or derivative thereof is administered to said subject so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is 4-6 fold higher than mean blood plasma levels of native RANTES in healthy individuals.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is about 5 fold higher than mean
  • Steady state plasma concentration refers to plasma drug concentration at steady- state during a constant rate intravenous infusion.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve an average plasma concentration of RANTES or derivative thereof that is at least 4 fold higher than mean blood plasma levels of native RANTES in healthy individuals. In another embodiment the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve an average plasma concentration of RANTES or derivative thereof that is 4-10 fold higher than mean blood plasma levels of native RANTES in healthy individuals. In another embodiment the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve an average plasma concentration of RANTES or derivative thereof that is 4-6 fold higher than mean blood plasma levels of native RANTES in healthy individuals. In another embodiment the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve an average plasma concentration of RANTES or derivative thereof that is about 5 fold higher than mean blood plasma levels of native RANTES in healthy individuals.
  • Average plasma concentration refers to average plasma concentration of drug during a dosage interval at steady-state (multiple dosing).
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a plasma concentration of RANTES or derivative thereof that is at least 4 fold higher than mean blood plasma levels of native RANTES in healthy individuals for at least 6 hours (e.g. for 6-12 hours or for 6-8 hours). In another embodiment the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a plasma concentration of RANTES or derivative thereof that is 4-10 fold higher than mean blood plasma levels of native RANTES in healthy individuals for at least 6 hours. In another embodiment the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a plasma concentration of RANTES or derivative thereof that is 4-6 fold higher than mean blood plasma levels of native RANTES in healthy individuals for at least 6 hours. In another embodiment the isolated RANTES polypeptide or derivative thereof is administered to said subject so as to achieve a plasma concentration of RANTES or derivative thereof that is about 5 fold higher than mean blood plasma levels of native RANTES in healthy individuals for at least 6 hours.
  • the isolated RANTES polypeptide or derivative thereof may be administered to said subject so as to achieve a steady state or average plasma concentration of l -4ng/ml, e.g. 1.5 or 2 ng/ml.
  • doses of 1-1 ,000 ⁇ g kg, typically 1- 10 ⁇ g kg, 1-4 ⁇ g kg, or 5-10 ⁇ g kg of a RANTES polypeptide may be administered to a human subject, e.g. at daily or twice-daily intravenous administrations.
  • a daily dose of e.g. 100-500 ⁇ g may be administered intravenously to a human subject afflicted with HCC.
  • 100-300 ⁇ g/kg of the RANTES polypeptide may be administered.
  • said isolated RANTES polypeptide or a derivative thereof is administered to said subject by continuous intravenous infusion.
  • said isolated RANTES polypeptide or a derivative thereof is administered to said subject at a dosing regime comprising periodical intravenous injections (e.g. once, twice, three or four times a day, or once every two days).
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject in a form enabling a significantly longer plasma half-life of said RANTES polypeptide or derivative compared to native RANTES polypeptide.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject in the form of a complex or conjugate having a significantly longer plasma half-life compared to native RANTES polypeptide.
  • the RANTES derivative is characterized by a significantly longer plasma half-life compared to native RANTES polypeptide.
  • the plasma half-life of the RANTES form, complex or conjugate may be at least 5 times longer or at least 10 times longer compared to native RANTES polypeptide.
  • the isolated RANTES polypeptide or derivative thereof is administered to said subject in a form enabling a significantly lower liver uptake compared to native RANTES polypeptide
  • the obtained enhancement in RANTES levels is not sufficient to increase the migration of immature dendritic cells to the tumor site. In other embodiments, the methods of the invention do not substantially increase the migration of immature dendritic cells to the tumor site.
  • the method further comprises administering to said subject at least one inhibitor of a chemokine receptor selected from the group consisting of CCR1 and CCR5.
  • the inhibitor may be administered to said subject so as to inhibit, antagonize or down-regulate the expression of CCR5 in the liver, tumor cells or lymphocytes of said subject, wherein each possibility represents a separate embodiment of the invention.
  • the present invention provides a method for treating hepatocellular carcinoma, comprising administering to a subject in need thereof at least one inhibitor of CCR5.
  • the method further comprises administering the at least one inhibitor of CCR5 in combination with at least one additional therapeutic agent.
  • the at least one additional therapeutic agent may be selected from the group consisting of: a cytokine (e.g. RANTES or interferon alpha), an anti- tumor agent (e.g. a chemotherapy such as taxol and cisplatin or an immunotherapy such as an antibody directed to a tumor associated antigen) and antiviral agents (e.g. an anti-HCV agent, such as weekly injections of interferon alfa-2b combined with daily oral administration of ribavirin which is standard care for patients infected with hepatitis C, or protease inhibitors such as Telaprevir).
  • a cytokine e.g. RANTES or interferon alpha
  • an anti- tumor agent e.g. a chemotherapy such as taxol and cisplatin or an immunotherapy such as an antibody directed to a tumor associated antigen
  • antiviral agents e.g. an anti-HCV agent, such as weekly injections of interferon alfa-2b combined with daily oral administration of
  • the cytokine is RANTES or a derivative thereof.
  • the subject in need thereof is susceptible to HCC. According to yet another embodiment, the subject in need thereof is a patient with chronic liver inflammation.
  • the HCC is characterized by lack of ⁇ -catenin expression in the tumor cells. In another embodiment the HCC is characterized by nuclear cyclin Dl levels in tumor cells that are significantly lower than nuclear cyclin Dl levels in non-tumor cells.
  • the present invention provides a method for ameliorating (e.g. inhibiting or delaying) the onset of hepatocellular carcinoma, comprising administering to a subject in need thereof at least one inhibitor of CCR5.
  • the present invention provides at least one inhibitor of a chemokine receptor selected from the group consisting of CCR1 and CCR5 for treating hepatocellular carcinoma.
  • the present invention provides a CCR5 inhibitor for ameliorating the onset of hepatocellular carcinoma.
  • an isolated RANTES polypeptide or derivative thereof formulated so as to enhance the level of RANTES or derivative thereof specifically in blood plasma, for treating hepatocellular carcinoma (HCC) or for inhibiting or delaying the onset of a HCC tumor formation.
  • HCC hepatocellular carcinoma
  • treating HCC refers to taking steps to obtain beneficial or desired results, including but not limited to, alleviation or amelioration of one or more symptoms of HCC, diminishment of extent of disease, tumor shrinkage or disappearance, delay or slowing of disease progression and/or onset, amelioration, palliation or stabilization of the disease state, partial or complete remission, prolonged survival and other beneficial results known in the art.
  • beneficial or desired results including but not limited to, alleviation or amelioration of one or more symptoms of HCC, diminishment of extent of disease, tumor shrinkage or disappearance, delay or slowing of disease progression and/or onset, amelioration, palliation or stabilization of the disease state, partial or complete remission, prolonged survival and other beneficial results known in the art.
  • beneficial or desired results including but not limited to, alleviation or amelioration of one or more symptoms of HCC, diminishment of extent of disease, tumor shrinkage or disappearance, delay or slowing of disease progression and/or onset, amelioration, palliation or stabilization of the disease state, partial or
  • treating also refers to administering a therapy in amount, manner, and/or mode effective to improve a condition, symptom, or parameter associated with a disorder or to prevent progression of a disorder, to either a statistically significant degree or to a degree detectable to one skilled in the art.
  • An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject.
  • treating HCC may refer in some embodiments to administering a RANTES polypeptide or derivative thereof and/or a CCR5 inhibitor in an amount, manner, and/or mode effective to reduce tumor volume or in other embodiments to reduce the number of tumors in a subject afflicted with HCC, to either a statistically significant degree or to a degree detectable to one skilled in the art.
  • inhibiting or “reducing” refer to either statistically significant inhibition or reduction, or to inhibition or reduction to a significant extent as determined by a skilled artisan, e.g. the treating physician. It should be understood, that inhibition or reduction does not necessarily indicate a total elimination of the measured function or biological activity. A reduction in activity may be for example about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • chemokine receptor selected from the group consisting of CCR1 and CCR5
  • use of at least one inhibitor of a chemokine receptor selected from the group consisting of CCR1 and CCR5 is further contemplated.
  • the inhibitor is selected from the group consisting of: double-stranded RNA, a compound antagonizing the binding of the chemokine receptor to its ligand, neutralizing antibody to CCR5, neutralizing antibody to CCR1, a ligand corresponding to a neutralizing antibody to CCR5, a ligand corresponding to a neutralizing antibody to CCR1 , an isolated peptide derived from the sequences of CCR5 or analogs thereof capable of inhibiting CCR5, an isolated peptide derived from the sequences of CCR1 or analogs thereof capable of inhibiting CCR1, antisense nucleic acids, antagonist microRNA and enzymatic RNA molecule.
  • the double-stranded RNA is siRNA.
  • the enzymatic RNA molecule is a ribozyme.
  • the at least one inhibitor is provided in a composition comprising at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is selected from the group consisting of: a cytokine, an anti-tumor agent and antiviral agents.
  • the at least one inhibitor of a chemokine receptors is a CCR5 antagonist.
  • the invention relates to the use of a CCR5 inhibitor in the manufacture of a medicament for ameliorating the onset of hepatocellular carcinoma.
  • the inhibitor is selected from the group consisting of: double- stranded RNA, a compound antagonizing the binding of the chemokine receptor to its ligand, neutralizing antibody to CCR5, a ligand corresponding to a neutralizing antibody to CCR5, an isolated peptide derived from the sequences of CCR5 or analogs thereof capable of inhibiting CCR5, antisense nucleic acids, antagonist microRNA and enzymatic RNA molecule.
  • the double-stranded RNA is siRNA.
  • the enzymatic RNA molecule is a ribozyme.
  • the at least one inhibitor is provided in a composition comprising at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is selected from the group consisting of: a cytokine, an anti-tumor agent and antiviral agents.
  • the invention relates to the use of an isolated RANTES polypeptide or derivative thereof for the preparation of a medicament for treating hepatocellular carcinoma (HCC) or for inhibiting or delaying the onset of a HCC tumor formation, wherein the medicament enhances the level of RANTES or derivative thereof specifically in blood plasma of said subject.
  • HCC hepatocellular carcinoma
  • the HCC is characterized by lack of ⁇ -catenin expression in the tumor cells and/or by nuclear cyclin Dl levels in tumor cells that are significantly lower than nuclear cyclin Dl levels in non-tumor cells.
  • expression of ⁇ -catenin and cyclin Dl in HCC tumor cells of said subject is determined, wherein lack of ⁇ -catenin expression and significantly lower nuclear cyclin Dl levels in tumor cells compared to non-tumor cells indicates that said subject is amenable for treatment with said medicament.
  • said medicament is formulated for intravenous administration.
  • the RANTES polypeptide is human RANTES.
  • said medicament is formulated in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient or diluent.
  • said medicament is formulated so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is at least 4 fold higher than mean blood plasma levels of native RANTES in healthy individuals. In another embodiment said medicament is formulated so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is 4-10 fold higher than mean blood plasma levels of native RANTES in healthy individuals. In another embodiment said medicament is formulated so as to achieve a steady state plasma concentration of RANTES or derivative thereof that is about 5 fold higher than mean blood plasma levels of native RANTES in healthy individuals. In another embodiment said medicament is formulated so as to achieve an average plasma concentration of RANTES or derivative thereof that is 4-10 fold higher than mean blood plasma levels of native RANTES in healthy individuals.
  • said medicament is formulated for administration by continuous intravenous infusion or for administration at a dosing regime comprising periodical intravenous injections.
  • said medicament is formulated in a form enabling a significantly longer plasma half-life of said RANTES polypeptide or derivative compared to native RANTES polypeptide.
  • said medicament is formulated in the form of a complex or conjugate having a significantly longer plasma half-life compared to native RANTES polypeptide.
  • said RANTES polypeptide or derivative is conjugated to or complexed with polyethylene glycol (PEG) and/or an immunoglobulin Fc domain or portion thereof
  • said medicament further comprises at least one inhibitor of a chemokine receptor selected from the group consisting of CCR1 and CCR5.
  • the inhibitor is selected from the group consisting of: double-stranded RNA, a compound antagonizing the binding of the chemokine receptor to its ligand, neutralizing antibody to CCR5, neutralizing antibody to CCR1 , a ligand corresponding to a neutralizing antibody to CCR5, a ligand corresponding to a neutralizing antibody to CCR1, an isolated peptide derived from the sequences of CCR5 or analogs thereof capable of inhibiting CCR5, an isolated peptide derived from the sequences of CCR1 or analogs thereof capable of inhibiting CCR1, antisense nucleic acids, antagonist microR A and enzymatic R A molecule.
  • the at least one inhibitor of a chemokine receptors is a CCR5 antagonist.
  • KO and "-/-” are used interchangeably throughout the specification and drawings to indicate a knockout mutation in both alleles of a gene, e.g. Mdr2.
  • DKO indicates double knock-out, i.e. knockout mutations as described above in two genes (e.g. Mdr2 and CCR5).
  • WT indicates wild-type (non-mutated) cells or animals.
  • mice Animal experiments were performed according to a protocol approved by The Animal Care Committee of the Hebrew University. All animals were kept on a 12h light/dark cycle in a pathogen-free animal facility with free access to food and water. Wildtype C57B1/6J and CCR5- deficient mice were purchased from Jackson Laboratories (Bar Harbor, ME). CCR1 - deficient mice were acquired from the Taconic Farms (Germantown, NY). FVB.129P2- Abcb4tmlBor (Mdr2-KO) mice - Jackson Laboratory, Bar Harbor, ME. Mice were crossed into the C57B1/6 genetic background for at least nine generations.
  • Double mutant Mdr2 "/" CCR1 "/” and Mdr2 "/” CCR5 '/” mice were generated by crossing Mdr2 'A with either CCR5 or CCR1 deficient mice and their progeny were identified by PCR analysis.
  • mice were sacrificed by a lethal dose of isoflurane anesthesia and livers were excised and weighed. All mice were injected i.p with BrdU (Sigma-Aldrich, Rehovot, Israel) lmg/mouse in ⁇ per lg body weight 3 and 24h before they were sacrificed. Liver specimens were either fixed in 4% buffered formalin or snap-frozen in liquid nitrogen for further analysis.
  • ALT Alanine aminotransferase
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase
  • MRI Magnetic resonance Imaging
  • Mice were anaesthetized (30 mg/kg pentobarbital, i.p) and placed supine with the liver located at the centre of the coil. Eight mice from each group (i.e.
  • Blots were incubated overnight at 4°C in a blocking buffer containing 5% skim milk and then incubated with either anti-SMA (Dako, Carpintera, USA) or beta-actin (Sigma-Aldrich, Rehovot, Israel) mouse monoclonal antibody diluted 1/2000, for 2 h at room temperature, and subsequently, with peroxidase-conjugated goat anti-mouse IgG (Dako, Carpintera, USA) for lh at room temperature.
  • anti-SMA Novo, Carpintera, USA
  • beta-actin Sigma-Aldrich, Rehovot, Israel
  • RNA Extraction and real-time PCR Total RNA was extracted from livers of 1 and 3 months old mice (WT, Mdr2 _/” , Mdr2 "/” CCR5 “/” and Mdr2 “/” CCR1 “/” ) using TRIzol reagent (Invitrogen Life Technologies) according to manufacture's recommended protocol.
  • cDNA was obtained by reverse transcription of 1 mg of total RNA in a final reaction volume of 25 ⁇ containing lx M-MLV RT buffer, 2.5 ⁇ / ⁇ random hexamers, 0.5 mmol/1 of each dNTP, 3 mmol/1 MgC12, 0.4 U/ ⁇ RNase inhibitor, and 100 U/ ⁇ M-MLV RT (Promega, Madison, WI).
  • Quantitative real-time PCR assays containing the primers and probe mix for TGF- ⁇ and RANTES were purchased from Applied Biosystems (Foster City, CA) and utilized according to the manufacturer's instructions.
  • PCR reactions were carried out in a final reaction volume of 20 ⁇ containing 100 ng cDNA template, ⁇ ⁇ TaqMan Universal Master Mix (Applied Biosystems), and ⁇ gene and probe mix. All reactions were run in triplicates and the housekeeping gene GAPDH (Applied Biosystems) was amplified in a parallel reaction for normalization.
  • BM cells were isolated from the tibia and femur of either WT, CCR5 7" or CCRl 7" mice filtered through a cell strainer FACS tube (Falcon, BD Biosciences, San Jose, CA, USA), washed twice in sterile PBS and diluted to 5* 10 7 cells/ml. Purified BM cells were labeled with 0.5 ⁇ / ⁇ CFSE for 15 min. washed in PBS and treated with FCS to neutralize CFSE activity. Labeled BM cells were injected into the tail vein of three months old MDR2 " or WT mice, 2* 10 6 cells per mouse in a total volume of 200 ⁇ L ⁇ .
  • mice were sacrificed and the liver was harvested. Liver tissue was homogenized and immune cells were collected oh a Ficoll gradient (Histopaque- 1077-1, Sigma- Aldrich, Rehovot, Israel). Recruitment of total CFSE positive cells and CFSE-F4/80 (Alexa Fluor 647 Anti-Mouse F4/80, Ebioscience, San Diego, CA) double positive cells to the liver was assessed by Facscalibur (Becton Dickinson Immunocytometry Systems).
  • liver tissue was cut into 5 mm sections, deparaffinized with xylene and hydrated through graded ethanols. Endogenous peroxidase was blocked by incubation for 5 min in 3% H 2 0 2 .
  • BrdU M0744, DAKO
  • Nitrotyrosine AB7048, Abeam
  • the Mdr2 -knockout mouse strain serves as a model for studying inflammation-associated HCC. This strain of mice spontaneously develops chronic cholestatic hepatitis and fibrosis that is eventually followed by HCC in 100% of animals over 12 months of age (Galun E. et al., Nature, 2004, 431 (7007):461-6).
  • Mdr2-knockout mice Genetic profiling of Mdr2-knockout mice (Mdr2 "A mice) revealed a marked elevation of the chemokine RANTES which is the ligand of the chemokine receptors CCRl and CCR5 (Katzenellenbogen et al., Mol Cancer Res., 2007, 5(11):1 159-70) in the liver of FVB.129 Mdr2 " " mice compared to control WT.
  • RANTES is expressed 30 fold higher in the liver of the generated C57B1 Mdr2-knockout mice used in the experiments described herein, compared to control WT (Fig 1 A).
  • Mdr2 KO mice that represent a model for chronically induced HCC serve as a good tool for the understanding of the mechanisms that govern the turnover from inflammation to tumorigenesis.
  • the inventors of the present invention generated novel strains of MDR2 'A , MDR2 _/" CCR5 V” and MDR2 "/" CCR1 " " double knockout (DKO) mice on the C57B1 background.
  • the mouse Mdr2 gene encodes a P-glycoprotein that is present on the bile canalicular membrane of hepatocytes. Mice deficient of this canalicular phospholipid flippase (Mdr2 "A mice) are unable to secrete phospholipids into bile resulting in regurgitation/secretion of bile into the portal tracts leading to hepatic and bile cell death and liver injury.
  • ALT, AST and ALP were detected in the serum of 1-6 months old Mdr2 "A mice compared to WT controls.
  • the elevated ALT, AST and ALP levels were also found in MDR2 ⁇ /" CCR5 "/” and MDR2- /' CCR1 "/ - mice. This indicates an onset of liver damage in all Mdr2 knockout strains, namely Mdr2 _/” , MDR2 "/” CCR5 “/' and MDR2 " " CCR1 " " mice (Fig. 1B-D).
  • Liver damage was also characterized by evaluating bile duct proliferation and inflammation that represent the most harmful damages in the assaulted liver of the Mdr2 KO mouse model. Proliferation (ductular reaction) is thought to have a key role in the initiation and progression of liver damage and cirrhosis progression, and therefore acts as an indicative marker in the prognosis of liver state.
  • liver sections from Mdr2 "A and MDR2 "/” CCR /” mice exhibit an intense inflammatory process with massive infiltration of immune cells
  • Mdr2 "/_ CCR5 " ' ' mice reside with only a mild inflammatory process that is located only in the periductal area not invading the parenchyma of the liver.
  • Knocking-out CCR5 resulted in complete augmentation of macrophage accumulation in the liver, with only a minor stained area (F4-80 stained area - wt -3.5%, Mdr2 " - 17.4% and Mdr2 "/" CCR5 "/" - 2.7%).
  • macrophages accumulate throughout the entire liver parenchyma they predominantly locate to specific sites of bile duct proliferation and their accumulation is in direct proportion to the high proliferative index of bile ducts.
  • Mdr2CCR5 DKO mice reduced inflammation correlated with reduced proliferation and fibrosis, as demonstrated by PAN-CK immunohistochemical staining for cholangiocytes (Fig. 3E, left panels). Mice were injected with BrdU and proliferation was assessed by immunohistochemical detection of BrdU positive cells (Fig. 3E, middle panels).
  • Example 6 In-vitro trans ell migration assay The recruitment of macrophages to the liver was next examined. An in-vitro transwell migration assay was performed in order to evaluate the effect of CCR5 depletion on MAC1 + monocytes mobility in response to RANTES. The results indicate that in the absence of CCR5 (cells obtained from CCR5 _/" mice) the migration of these cells in response to RANTES was completely abrogated (Fig. 3B).
  • Example 7 In-vivo mobility assays The role of CCR5 in the trafficking of F4/80 + macrophages to the liver of Mdr2 v" mice was studied. CFSC stained immune cells were adoptively transferred from the bone marrow of WT, CCRl "7" KO or CCR5 _/" KO mice into healthy C57/B1 WT or Mdr2 "7" mice.
  • Fig. 3D demonstrates enhanced infiltration of F4/80 + cells to the liver of Mdr2 _/" mice compared to WT mice, which was abrogated in MDR2 "/" CCR5 '/" mice.
  • periductal inflammation is accompanied by an intense proliferative activity of cholangiocyte and is not controlled by liver damage itself. Rather, without being bound to a specific mechanism, it is postulated that periductal inflammation is dominated by the inflammatory cells.
  • Example 8 Fibrogenesis profile in Mdr2CCR5 DKO mice
  • Hepatic stellate cells are the main liver cells in the fibrogenesis process, producing most of the extracellular collagen deposits when activated.
  • damaged cells and infiltrating immune cells secrete a vast number of cytokines in the assaulted area, these in turn can induce activation of hsc predominantly by TGF- ⁇ , and thereby arouse the initiation of fibrosis in the damaged tissue.
  • ⁇ -SMA a marker for activation of fibrogenic stellate cells in the liver
  • mice were scanned by MRI monthly from the age of 9 months to assess tumor development and hepatomegaly (Fig. 5A). Age matched WT mice were used as controls. Arrows indicate tumor foci. The results are summarized in Table 1., Table 1 - number of mice with visible tumors assessed by MRI
  • mice tumors were first detectable by MRI at the age of 13 months, where about 15% of the mice presented with tumors. At 16 months, 75% of Mdr2 " mice were presented with tumors detectable by MRI. Surprisingly, in Mdr2 "/" CCR5 “ “ mice, no tumors could be detected by MRI at 13 months, and by the age of 16 months only 33.33% of the mice presented with tumors. In comparison, in Mdr2 "/" CCR1 " " mice, at the age of 13 months 68.75% of the mice were already diagnosed with MRI detectable tumors, and at 16 months tumor incidence was 88.88%.
  • liver to body weight ratio was increased from about 0.06 in wild-type mice to 0.11 in MDR "7" mice.
  • Mdr2 "/" CCR5 " * mice average liver/body weight ratio was only about 0.07.
  • the ratio was about 0.1, resembling that of MDR 7' mice.
  • the Mdr2 "/” CCRr " mice which have shown an earlier tumor onset than the Mdr2 ⁇ ' ⁇ mice, exhibited small tumors with a 5 fold decrease in tumor volume compared with the Mdr2 _/" strain (Fig 5F).
  • the tumors in the Mdr2 "/_ CCR1 mice did not progress into massive and aggressive HCC tumors.
  • Example 10 - RANTES levels in liver and blood plasma
  • RANTES mRNA expression was found to be 30 fold higher in the liver of C57B1 Mdr2 7" mice used in the experiments described herein, compared to control WT.
  • ELISA testing shows elevated levels of RANTES in protein lysates of liver cells obtained from Mdr2 ";” , Mdr2 "/" CCR5 “/” and Mdr2 ⁇ /' CCRr /" mouse strains compared to WT mice.
  • enhancement of RANTES plasma levels beyond a specific threshold is associated with significantly lower tumor incidence and development.
  • RANTES (1 ⁇ g per mouse) was administered intravenously to WT mice. Subsequently, blood samples were collected and RANTES blood plasma concentrations were determined after 1, 3, 6 and 24 hours of administration, using ELISA. The results, depicted in Fig. 7, demonstrate that administration of a single RANTES dose at the specified amount resulted in RANTES blood plasma levels of 1000 pg/ml after one hour of administration, i.e. 10 fold over native RANTES concentrations in wt mice. An average RANTES level of about 3-4 fold over the baseline level was maintained 3 hours and 6 hours following administration, which declined 24 hours after administration.

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Abstract

L'invention concerne des moyens pour augmenter spécifiquement le niveau de RANTES, le ligand des récepteurs CCR5 et CCR1, dans le sang de sujets atteints de carcinome hépatocellulaire (CHC) ou prédisposés au développement de ce cancer. Des schémas posologiques, des formulations, des dérivés et des conjugués avantageux de RANTES favorisant une rétention prolongée dans le sang peuvent être particulièrement utiles pour traiter, prévenir ou inhiber l'apparition et l'évolution de sous-populations du CHC. Par ailleurs, l'invention concerne l'utilisation d'inhibiteurs des CCR5 et/ou CCR1.
PCT/IL2011/000816 2010-10-21 2011-10-11 Traitement et prévention du carcinome hépatocellulaire au moyen de modulateurs des récepteurs de chimiokines WO2012052995A2 (fr)

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GONZALEZ ET AL.: 'A hammerhead ribozyme targeted to the human chemokine receptor CCR5.' BIOCHEM BIOPHYS RES COMMUN. vol. 251, no. 2, 20 October 1998, pages 592 - 6 *
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