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WO2008146272A2 - Compositions and methods for treating cxcr6/cxcl16 associated diseases - Google Patents

Compositions and methods for treating cxcr6/cxcl16 associated diseases Download PDF

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Publication number
WO2008146272A2
WO2008146272A2 PCT/IL2008/000663 IL2008000663W WO2008146272A2 WO 2008146272 A2 WO2008146272 A2 WO 2008146272A2 IL 2008000663 W IL2008000663 W IL 2008000663W WO 2008146272 A2 WO2008146272 A2 WO 2008146272A2
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Prior art keywords
cxcr6
acid sequence
amino acid
cxcl16
diseases
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WO2008146272A3 (en
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Nathan Karin
Gizi Wildbaum
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Rappaport Family Institute for Research in the Medical Sciences
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Rappaport Family Institute for Research in the Medical Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel molecules and, more particularly, to methods of using same for treating CXCR6/CXCL16 associated diseases, such as autoimmune inflammatory diseases.
  • Chemokines are small (-8-14 kDa), structurally cytokine-like, secreted proteins that regulate cell trafficking. They are produced and secreted by a wide variety of cell types in response to early inflammatory mediators, such as IL- l ⁇ or TNF- ⁇ , and in response to bacterial or viral infection. Chemokines function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils and other effector cells from the blood to sites of infection or damage. They can be released by many different cell types (e.g. macrophages) and can mediate a range of pro-inflammatory effects on leukocytes, such as triggering of chemotaxis, degranulation, synthesis of lipid mediators, and integrin activation.
  • Chemokines can be subdivided into four classes, the C-C, C-X-C, C and C— X3-C chemokines, depending on the location of the first two cysteines in their protein sequence.
  • the interaction of these soluble proteins with their specific receptors which belong to the superfamily of seven-transmembrane domain G-protein-coupled receptors (GPCRs), mediate their biological effects resulting in, among other responses, rapid increase in intracellular calcium concentration, changes in cell shape, increased expression of cellular adhesion molecules, degranulation and promotion of cell migration.
  • GPCRs seven-transmembrane domain G-protein-coupled receptors
  • chemokines have been indicated as important mediators in multiple sclerosis (MS), allergic responses, asthma, atherosclerosis, glomerulonephritis, pancreatitis, restenosis, rheumatoid arthritis (RA), diabetic nephropathy, pulmonary fibrosis, transplant rejection and in cancer.
  • MS multiple sclerosis
  • RA rheumatoid arthritis
  • CXC Chemokine Receptor 6 (CXCR6, NM 006564), which former nomenclature included STRL33, BONZO, and TYMSTR, is expressed on T-cells, including T helper, T-cytotoxic and natural killer T (NKT) cells [Aust et al., Eur J Endocrinol (2005) 152(4):635-43; Matloubian et al., Nat Immunol (2000) 1(4):298-
  • CXCR6 binds specifically to its chemokine ligand CXCL 16 [Wilbanks et al., J Immunol (2001) 166(8):5145-54].
  • CXCL16 is produced by several cells, including dendritic cells [Matloubian et al. supra] and macrophages [van der Voort et al., Arthritis Rheum (2005) 52(5):1381-91], following induction by the inflammatory cytokines IFN- ⁇ and TNF- ⁇ [Abel et al., J Immunol (2004) 172(10):6362-72].
  • CXCL 16 is an unusually large chemokine (254 amino acids) which is composed of a CXC chemokine domain, a mucin-like stalk, a transmembrane domain and a cytoplasmic tail containing a potential tyrosine phosphorylation site.
  • AU of these are unusual features for a chemokine which allow CXCLl 6 to be a soluble chemokine or to be expressed as a cell surface bound molecule.
  • CXCL 16 functions as an adhesion molecule by binding to its receptor CXCR6 expressed on activated T-cells and bone marrow plasma cells [Nakayama et al., J Immunol (2003) 170:1136-1 140; Shimaoka et al., J Leukoc Biol (2004) 75:267-274].
  • CXCL 16 also exists as a soluble molecule, inducing chemotaxis of CXCR6 expressing lymphocytes, including T helper, T-cytotoxic and NKT cells [Matloubian et al. supra; Wilbanks et al. supra; Nakayama et al. supra].
  • CXCR6 Bonzo chemokine receptor- specific monoclonal antibodies (mAb) and ligands.
  • CXCR6 monoclonal antibodies compete for receptor binding thereby blocking natural responses by interfering with ligand-receptor interactions.
  • the contemplated uses of these antibodies include treating chronic inflammatory diseases, such as multiple sclerosis.
  • the inventors have disclosed a CXCR6 ligand [e.g., Spleen Extracted Chemokine (SExCkine)] which could potentially be administered to recruit killer T cells (expressing CXCR6) to solid tumors or sites of infection.
  • SExCkine Spleen Extracted Chemokine
  • This invention has the disadvantages of using mAb: 1) they may induce anti idiotypic responses in the host; 2) they compete for receptor binding yet they do not neutralize the ligands which may still transmit activating signals. Furthermore, the use of CXCR6 ligand does not block CXCR6 activity but rather enhances it.
  • CXCR6 protein containing an N-terminal Hemagglutinin (HA) epitope
  • CXCR6 protein functional variants such as those having deletions (N-terminal, C-terminal or internal deletions) which retain their binding activity, a signaling activity and/or ability to stimulate a cellular response, yet such proteins have not been disclosed, probably since the ligand binding site of CXCR6 has not been discovered.
  • PCT Publication No. WO04019046 discloses a human CXCR6 fusion protein and CXCR6 specific monoclonal antibodies. The use of such antibodies was contemplated for diagnostics and therapeutics of several diseases including hematological diseases, cardiovascular diseases, disorders of the peripheral and central nervous system, genitourinary diseases, cancer and respiratory diseases. This invention has the disadvantages of using mAb as described hereinabove.
  • the fusion protein disclosed in this application is a full-length CXCR6 protein.
  • WO04019046 contemplates the possibility of creating a CXCR6 soluble fusion protein, it in fact teaches away from soluble chemokine receptors by specifically mentioning that transmembrane (TM) domains of such receptors are crucial for ligand binding.
  • TM transmembrane
  • the ligand binding sites of some G-protein coupled receptors (GPCRs), possibly including CXCR6, are believed to comprise hydrophilic sockets formed by several GPCR transmembrane domains (including TM3, TM5, TM6, TM6 and TM7).
  • PCT Publication No. WO03066830 discloses monoclonal antibodies against membrane proteins including CXCR6.
  • This invention enables generation of high affinity monoclonal antibodies against peptide fragments of chemokine receptors by using fusion proteins as an immunogen that contains a peptide fragment derived from the membrane protein. Administering these antibodies to a host may offer a wide variety of therapeutic and diagnostic applications, including treatment of cancer and other diseases associated with abnormal chemokine activities.
  • This invention has the disadvantages of using mAb as described hereinabove. Extracellular fragments of the CXCR6 receptor are contemplated for antibody production but the N terminal domain is not mentioned, again, probably due to the fact that it was not recognized essential for ligand binding.
  • PCT Publication No. WO05049799 discloses chimeric chemokine receptors.
  • This invention teaches chimeric chemokine receptors comprising N terminal and TM region of a first chemokine receptor, such as CCR3, and the intracellular C terminus of a second chemokine receptor, such as CCR2.
  • the first or second chemokine receptors may comprise CXCR6 domains.
  • PCT Publication No. WO05049799 teaches away soluble receptors by specifically mentioning that GPCRs retain all of their known ligand binding regions within the extracellular regions and TM domains 2 through 7.
  • U.S. Publication No. 20060257359 discloses means of modulating phenotypes of macrophage related cells for the treatment of diseases, such as multiple sclerosis. Modulating the cellular phenotype is accomplished by introducing to macrophage related cells effectors, such as a protein, an antibody or a RNA molecule (e.g., a short interfering RNA), thereby altering gene expression and cell phenotype (e.g., secretion of cytokines or cell migration).
  • effectors such as a protein, an antibody or a RNA molecule (e.g., a short interfering RNA), thereby altering gene expression and cell phenotype (e.g., secretion of cytokines or cell migration).
  • CXCR6 and CXCR6 ligand thus in fact teaching away from down-regulating this pathway for treating MS.
  • an isolated soluble polypeptide comprising a heterologous amino acid sequence conjugated to a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCL 16, and wherein the molecule is non-immunogenic in a human subject.
  • an isolated soluble polypeptide comprising an amino acid sequence which comprises an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCLl 6.
  • an isolated soluble polypeptide comprising at least two CXCR6 amino acid sequences each being capable of binding CXCL 16.
  • an isolated soluble polypeptide comprising a tag attached to a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCL 16.
  • an isolated soluble polypeptide comprising an amino acid sequence as set forth in SEQ ID NO. 4 or 8.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding any of the soluble polypeptides.
  • a pharmaceutical composition comprising the soluble polypeptide and a pharmaceutically acceptable carrier.
  • a method of treating a CXCR6/CXCL16 associated disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the molecule, thereby treating the CXCR6/CXCL16 associated disease in the subject.
  • soluble polypeptide for the manufacture of a medicament identified for treating a CXCR6/CXCL16 associated disease.
  • a method of isolating CXCR6 ligand from a biological sample comprising: (a) contacting the biological sample with the soluble polypeptide such that the CXCR6 ligand and the soluble polypeptide form a complex; and (b) isolating the complex to thereby isolate the CXCR6 ligand from the biological sample.
  • the isolated soluble polypeptide is attached to a non-proteinaceous moiety.
  • the soluble polypeptide is non-immunogenic in a human subject.
  • the heterologous amino acid sequence comprises an immunoglobulin amino acid sequence.
  • the CXCR6 amino acid sequence is as set forth in SEQ ID NO: 4. According to still further features in the described preferred embodiments the
  • CXCR6 amino acid sequence is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 3 or 7.
  • CXCR6/CXCL16 associated disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, hematological diseases, cardiovascular diseases, disorders of the peripheral and central nervous system, genitourinary diseases, respiratory diseases and cancer.
  • the tag is an epitope tag.
  • the soluble polypeptide is attached to a solid support.
  • the non-proteinaceous moiety is selected from the group consisting of polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA).
  • PEG polyethylene glycol
  • PVP Polyvinyl pyrrolidone
  • SMA poly(styrene comaleic anhydride)
  • DIVEMA divinyl ether and maleic anhydride copolymer
  • a method of diagnosing Multiple Sclerosis in a subject comprising, detecting expression and/or activity of CXCR6 in cells of the subject, wherein expression and/or activity of the CXCR6 in the cells of the subject above a predetermined threshold is indicative of Multiple Sclerosis in the subject.
  • detecting expression of the CXCR6 is effected by detecting CXCR6 protein.
  • detecting expression of the CXCR6 protein is effected via an assay selected from the group consisting of immunohistochemistry, ELISA, RIA, Western blot analysis, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.
  • detecting expression of the CXCR6 is effected by detecting CXCR6 mRNA.
  • detecting expression of the CXCR6 mRNA is effected via an assay selected from the group consisting of PCR, RT-PCR, chip hybridization, RNase protection, in-situ hybridization, primer extension, Northern blot and dot blot analysis.
  • composition comprising as an active ingredient an anti-Multiple
  • Sclerosis drug and an agent capable of reducing activity and/or expression of CXCR6/CXCL16 and/or of an effector thereof and a pharmaceutically acceptable carrier.
  • the agent is selected from the group consisting of: (i) an oligonucleotide directed to an endogenous nucleic acid sequence expressing the CXCR6/CXCL16 or the effector thereof; (ii) a chemical inhibitor directed to the CXCR6 or the effector thereof; (iii) a neutralizing antibody directed at CXCR6/CXCL16 or the effector thereof; and (iv) a non-functional derivative of the CXCR6 or the effector thereof.
  • the non-functional derivative of the CXCR6/CXCL16 comprises any of the soluble polypeptides.
  • the anti-Multiple Sclerosis drug is selected from the group consisting of Interferon Beta Ia, Interferon Beta Ib, Glatiramer Acetate, Mitoxantrone, MethylPrednisolone,
  • Prednisone Prednisolone
  • Dexamethasone Adreno-corticotrophic Hormone (ACTH) and Corticotropin.
  • ACTH Adreno-corticotrophic Hormone
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing novel molecules and methods of using same for treating CXCR6/CXCL16 associated diseases, such as autoimmune inflammatory diseases.
  • FIG. 1 is a photograph depicting the presence of CXCR6 mRNA in spinal cords of EAE induced mice.
  • cDNA from the spinal cord of EAE induced mouse (day 18, middle lane) or from a na ⁇ ve mouse (left lane) was amplified by PCR using specific primers for the entire murine CXCR6 gene coding region.
  • a water sample was amplified as a negative control (right lane).
  • GADPH was used as a positive control (bottom panel).
  • FIGs. 2A-C are FACS analysis results depicting CXCR6 expressing T helper cells in spinal cords of EAE induced mice at the peak of disease.
  • Figure 2A shows spleen cells of an EAE induced mouse;
  • Figure 2B shows cervical lymph node (LN) cells of an EAE induced mouse;
  • Figure 2C shows spinal cord (SC) cells of an EAE induced mouse. Results were read and analyzed by FACS.
  • FIGs. 3A-B are schematic illustrations depicting the generation of the expression construct encoding the murine CXCRo-IgG peptide of the present invention (SEQ ID NO: 17).
  • Figure 3 A shows insertion of the CXCR6 cDNA sequence encoding the extracellular N-terminal region of murine CXCR6 (SEQ ID NO: 13) to pSecTag vector along with a sequence encoding a part of murine IgGl heavy chain (SEQ ID NO: 5);
  • Figure 3B shows the resulting chimeric protein of CXCR6 and IgGl.
  • FIGs. 4A-B are images depicting purification of the soluble receptor CXCR6-
  • FIG. 4A shows Western blots analysis with mouse anti-myc antibody; and Figure 4B shows Coomassie staining.
  • lane no. 1 is of a size marker (kDa)
  • lane no. 2 is of medium in which DG44-CXCR6IgG transfected cells were grown
  • lanes no. 3-7 are of five sequential elution fractions
  • lane no. 8 is of the flow through medium.
  • the soluble receptor CXCRo-IgG is a 42Kd polypeptide.
  • FIG. 5 is a bar graph depicting the binding specificity of the murine CXCR6- IgG fusion protein to various commercially available murine chemokines. Specific binding was determined by direct ELISA. Where indicated, the soluble receptor CCR2-IgG was added instead of CXCRo-IgG. Results are shown as O.D. reading at 450 nm. Bars are average of triplicates. The data shown represent one of three experiments completed. Error bars: ⁇ standard deviation.
  • FIG. 6 is a graph depicting the ability of CXCRo-IgG to suppress ongoing
  • mice Experimental Autoimmune Encephalomyelitis (EAE) in mice.
  • EAE Experimental Autoimmune Encephalomyelitis
  • MOGp35-55 on day 0
  • Pertussis toxin on days 0 and 2.
  • the mice were treated with repeated intravenous administrations (every other day) of 300 ⁇ g/mouse of either a CXCRo-IgG (indicated by diamonds) or isotype matched IgG (indicated by X).
  • the data are representative of three independent experiments.
  • FIGS. 7A-F are images depicting histochemistry of spinal cords of naive mice and EAE induced mice left untreated or treated with the composition of the present invention.
  • Figures 7A-B show the lumbar spinal cords of a na ⁇ ve mouse (xlOO and x400 magnifications, respectively);
  • Figures 7C-D show the lumbar spinal cords of an EAE induced mouse that was treated with control isotype matched (IgGl) antibody (xlOO and x400 magnifications, respectively);
  • Figures 7E-F show an EAE induced mouse that was treated with the soluble receptor CXCRo-IgG (xlOO and x400 magnifications, respectively).
  • Spinal cords were removed 22 days after MOG immunization.
  • FIGs. 8A-D are bar graphs depicting ex- vivo cytokine secretion by EAE derived splenocytes treated by CXCRo-IgG.
  • CXCRo-IgG three groups of mice (three mice in each group) were subjected to active induction of EAE by MOGp35- 55. Mice were treated with the soluble receptor CXCR6, an isotype matched IgG (mlgG) or PBS (control).
  • mlgG isotype matched IgG
  • PBS control
  • the splenocytes were harvested and restimulated for 72 hours with 50 ⁇ g/ml MOGp35-55 (solid bars) or cell medium (striped bars).
  • Figure 8A is a graph showing IL-2 secretion
  • Figure 8B is a graph showing IFN- ⁇ secretion
  • Figure 8C is a graph showing TNF- ⁇ secretion
  • Figure 8D is a graph showing IL- 12 secretion. Bars indicate average of triplicate wells. Error bars represent ⁇ SD.
  • FIGs. 9A-B are histograms depicting the macrophage levels in CXCR6 treated mice.
  • two groups of C57/B mice three mice in each group were subjected to active induction of EAE by MOGp35-55.
  • Figure 9A shows mice that were not treated (control); and
  • Figure 9B shows mice that were treated with the soluble receptor CXCR6.
  • the splenocytes were harvested and stained anti-CD 1 Ib-FITC and analyzed by FACS. The percentage of CDl Ib positive cells is indicated above the peak of each appropriate histogram.
  • FIGs. 10A-D are FACS analysis results depicting a decrease in IFN ⁇ producing cells in vitro.
  • Splenocytes from C57/B mice were harvested, washed and incubated with MOGp35-55 alone ( Figures 10A-B) or MOGp35-55 and CXCRoIgG ( Figures 1 OC-D).
  • Cells were stimulated with 30 nM PMA and 1 ⁇ M ionomycin for 5 hours and stained with anti CD4-PERCP and anti CXCR6-PE. After permeabilization, the cells were stained intracellularly with anti-IL-4-APC and anti IFN ⁇ -FITC.
  • Figures 1OA and 1OC show analysis of total lymphocytes; and Figures 1OB and 1OD show analysis of CXCR6+ gated lymphocytes.
  • the present invention is of compositions of down-regulating signaling through CXCLl 6 and uses of same for treating a myriad of medical conditions such as Multiple Sclerosis.
  • MS Multiple sclerosis
  • CNS central nervous system
  • MS and its animal model, experimental autoimmune encephalomyelitis (EAE) are believed to result from autoimmune mediated activated immune cells, such as T- and B-lymphocytes as well as macrophages and microglia, and is considered to be an inflammatory neurodegenerative disease.
  • EAE experimental autoimmune encephalomyelitis
  • MS is characterized by perivenous infiltration of lymphocytes and macrophages into the CNS parenchyma, resulting in demyelinative lesions termed plaques. These plaques, which are the hallmark of MS, are associated with oligodendrocytes death, axonal damage and neuronal loss.
  • the etiology of MS has not yet been fully elucidated and it is attributed to both genetic and environmental causes, yet factors which regulate leukocyte entry into the CNS may play a role in MS development as well as in lesion pathogenesis. While reducing the present invention to practice, the present inventors have discovered that the CXCR6/CXCL16 axis is directly involved in MS pathogenesis and therefore, the present inventors envision therapeutics of MS by suppressing signaling through the CXCR6/CXCL16 pathway.
  • CXCR6 N-ter, amino acid coordinates 1-32 of GenBank Accession No. NP_006555; SEQ ID NO: 4
  • This finding is crucial for the generation of new therapeutic tools for treating diseases which involve the CXCR6/CXCL16 axis such as MS.
  • CXCR6 is selectively expressed by T helper (Th) cells in spinal cords of E AE- induced mice (Examples 1-2 of the Examples section which follows), suggesting that CXCR6 may be involved in MS pathogenesis.
  • Example 3 the present inventors have constructed soluble CXCR6 fusion polypeptides and expressed them in mammalian cell systems (see Example 3 of the Examples section which follows). Functionality of the fusion was shown by binding of CXCRo-IgG to CXCL 16 as was demonstrated in Example 4.
  • the fusion polypeptide was proven therapeutic for the treatment of MS as was manifested by suppression of ongoing encephalomyelitis (EAE) in vivo (see Examples 5 and 6) and ex vivo by suppression of pro-inflammatory cytokine production by splenocytes (see Example 7).
  • EAE ongoing encephalomyelitis
  • Example 7 the present results illustrate that CXCRo-IgG treatment leads to a decrease in IFN-gamma producing cells and does not cause depletion in Macrophages (see Examples 8-9).
  • a method of treating MS in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent capable of reducing activity and/or expression of CXCR6/CXCL16 or of an effector thereof, thereby treating MS in the subject.
  • treating refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of Multiple Sclerosis.
  • Multiple Sclerosis refers to the inflammatory, demyelinating disease of the central nervous system (CNS) which is typically characterized by various symptoms of neurological dysfunction.
  • Multiple sclerosis refers to any type of multiple sclerosis (e.g., stage, severity) as outlined infra.
  • Relapsing-remitting describes the initial course of 85 % to 90 % of individuals with MS. This subtype is characterized by unpredictable attacks (relapses) followed by periods of months to years of relative quiet (remission) with no new signs of disease activity. Deficits suffered during the attacks may either resolve or may be permanent. When deficits always resolve between attacks, this is referred to as "benign" MS. Secondary progressive - Secondary progressive describes around 80 % of those with initial relapsing-remitting MS, who then begin to have neurological decline between their acute attacks without any definite periods of remission. This decline may include new neurological symptoms, worsening cognitive function, or other deficits. Secondary progressive is the most common type of MS and causes the greatest amount of disability.
  • Primary progressive - Primary progressive describes the approximately 10 % of individuals who never have remission after their initial MS symptoms. Decline occurs continuously without clear attacks. The primary progressive subtype tends to affect people who are older at disease onset.
  • Progressive relapsing describes those individuals who, from the onset of their MS, have a steady neurological decline but also suffer superimposed attacks; and is the least common of all subtypes.
  • a subject in need thereof refers to a mammal, preferably a human subject who has been diagnosed with probable or definite multiple sclerosis, e.g., a subject who experienced one neurological attack affecting the CNS and accompanied by demyelinating lesions on brain magnetic resonance imaging (MRJ).
  • MRJ brain magnetic resonance imaging
  • the neurological attack can involve acute or sub-acute neurological symptomatology (attack) manifested by various clinical presentations like unilateral loss of vision, vertigo, ataxia, incoordination, gait difficulties, sensory impairment characterized by paresthesia, dysesthesia, sensory loss, urinary disturbances until incontinence, diplopia, dysarthria, various degrees of motor weakness until paralysis, cognitive decline either as a monosymptomatic or in combination.
  • the symptoms usually remain for several days to few weeks, and then partially or completely resolve.
  • the diagnosis MS can also include laboratory tests involving evaluation of IgG synthesis and oligoclonal bands (immunoglobulins found in 85-95 % of subjects diagnosed with definite MS) in the cerebrospinal fluid (CSF, obtained by e.g., lumbar puncture) which provide evidence of chronic inflammation of the central nervous system.
  • CSF cerebrospinal fluid
  • oligoclonal bands can help make a definite diagnosis of MS.
  • the method is effected by administering to the subject a therapeutically effective amount of an agent capable of reducing activity and/or expression of CXCR6/CXCL16 or of an effector thereof, thereby treating MS in the subject.
  • CXCR6 refers to a CXCR6 gene product (i.e., protein or mRNA) such as set forth in GenBank Accession Nos. NM 006564 or NP 006555.
  • CXCR6 activity refers to cell signaling activity (e.g., G protein signaling, NF-kappa B signaling), chemokine binding activity (CXCL 16), viral replication and/or co-receptor for SIM and HIV, cell adhesion, cell proliferation or chemotaxis.
  • cell signaling activity e.g., G protein signaling, NF-kappa B signaling
  • CXCL 16 chemokine binding activity
  • CXCR6 expression refers to expression of the chemokine receptor CXCR6 either at the protein level or at the mRNA level. Typically, CXCR6 is expressed on T cells, such as without limitation, T helper, T-cytotoxic and NKT cells, as well as on bone marrow plasma cells.
  • CXCL 16 refers to CXCL 16 gene product (i.e., protein or mRNA) such as set forth in GenBank Accession Nos. NM 022059 or NP_071342.
  • CXCL 16 expression refers to expression of CXCL 16 chemokine either at the protein or mRNA level.
  • CXCL 16 is expressed as a soluble chemokine or as a cell surface bound molecule, such as for example without limitation, on monocytes and dendritic cells.
  • an effector of CXCR6/CXCL16 refers to a molecule which mediates signaling via CXCR6.
  • a CXCR6/CXCL16 effector can be, for example, heterotrimeric G proteins (e.g., Gi proteins, GenBank Accession Nos. NM 002069 and NP 002060), focal adhesion signaling components (e.g., PB-K [Chandrasekar et al., J Biol Chem. (2004) 279(5):3188-96]), down stream signaling components (e.g., kinases such as PDK-I GenBank Accession Nos. NP 002601 and NM_002610, and Akt GenBank Accession Nos. NM 001014431, NMJ)Ol 014432, or NM 005163) and NF-kappa B signaling pathway components (e.g., NF-kappa B
  • a number of agents can be used in accordance with this aspect of the present invention to reduce/down-regulate the activity or expression of CXCR6/CXCL16 or an effector thereof. Assays for qualifying such agents will be apparent to those of skill in the art. Thus, any in vitro or in vivo activity assay (e.g., signaling, reduction in EAE score) can be used such as described in the Examples section which follows. Thus, for example the agent can be a non-functional derivative of CXCR6 or an effector thereof.
  • non-functional derivative of CXCR6 refers to a CXCR6 amino acid sequence which competes with native CXCR6 (e.g., on ligand or effector molecule binding) but is designed so as to block signaling from same.
  • a non-functional derivative is also referred to in the art of signaling as a "dominant negative molecule”.
  • a non-functional derivative of CXCR6 is the N-terminal domain (amino acid coordinates 1-32 of GenBank Accession No. NP 006555, SEQ ID NOs: 3 and 4) of CXCR6 being capable of binding a ligand (e.g., CXCL 16) without transmitting a signal.
  • a ligand e.g., CXCL 16
  • the present inventors have uncovered that the N-terminal domain of CXCR6 is sufficient to bind CXCL 16 (see Example 4).
  • the present invention provides for an isolated soluble polypeptide comprising a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCL 16, and wherein the molecule is non-immunogenic in a human subject (for maximizing therapeutic efficacy).
  • non-immunogenic refers a substance which is substantially incapable of producing an immune response in a subject administered therewith.
  • non-immunogenic in a human means that upon contacting the molecule of this aspect of the present invention with the appropriate tissue of a human, no state of sensitivity or resistance to the molecule is demonstrable upon the second administration of the molecule after an appropriate latent period (e.g., 8 to 14 days).
  • Such molecules are preferably devoid of other CXCR6 extracellular domains which are not necessary for ligand binding. Such domains are listed infra.
  • soluble N-terminal sequence of CXCR6 may be used per se, it may be modified to improve its availability and pharmacokinetics. Such modifications are provided below.
  • soluble refers to the ability of the molecules of the present invention to dissolve in a physiological aqueous solution (pH about 7, e.g., solubility level in aqueous media of >100 ⁇ g/ml) without substantial aggregation.
  • soluble CXCR6 are preferably devoid of any hydrophobic transmembrane domains (listed in Table 1 above).
  • CXCR6 amino acid sequence refers to a peptide portion of a mammalian (e.g., human) chemokine C-X-C receptor 6 protein having binding affinity for CXCR6 ligands (e.g., CXCL 16). Binding affinity refers to a minimal K D value of at least 10 "6 M., 10 "7 M, lO "8 M, 10 "9 M, 10 '10 M. Methods of assaying ligands for qualified affinity are well known in the art and include Scatchard plotting.
  • CXCR6 amino acid sequence may be included in the molecules of the present invention, but inclusion of at least two CXCR6 amino acid sequences (e.g., of similar affinity), each being capable of binding CXCR6 ligand (preferably with high affinity) may be preferred. Due to increased avidity, these polypeptides may be used as potent inhibitors of CXCR6 ligand activity and lower dosages may be administered.
  • An example of a CXCR6 amino acid sequence which can be used in accordance with the teachings of the present invention is set forth in SEQ ID NO: 4 or 8.
  • Such CXCR6 amino acid sequences may be encoded by a nucleic acid sequences as set forth in SEQ ID NO. 3 or 7. Due to the soluble nature of the molecules of the present invention, it would be preferred that the CXCR6 amino acid sequence is no longer than 50 amino acids in length.
  • the CXCR6 amino acid sequence of the present invention comprises the N terminus (N-ter) part of the receptor. As shown in Example 4 of the Examples section which follows, binding of CXCR6 to its ligand CXCL 16 is mediated by the N-ter domain of CXCR6.
  • polypeptide encompasses native peptides (either degradation products, synthetically synthesized peptides, or recombinant peptides), peptidomimetics (typically, synthetically synthesized peptides), and the peptide analogues peptoids and semipeptoids, and may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Ramsden, C. A., ed. (1992), Quantitative Drug Design, Chapter 17.2, F. Choplin Pergamon Press, which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinbelow.
  • Natural aromatic amino acids, Tip, Tyr, and Phe may be substituted for synthetic non-natural acids such as, for instance, tetrahydroisoquinoline-3-carboxylic acid (TIC), naphthylelanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe, and o-methyl-Tyr.
  • the polypeptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g., fatty acids, complex carbohydrates, etc.).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other less common amino acids, including but not limited to 2- aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine, and ornithine.
  • amino acid includes both D- and L-amino acids.
  • Tables 2 and 3 below list naturally occurring amino acids (Table 2) and non- conventional or modified amino acids (Table 3) which can be used with the present invention.
  • peptide mimetics e.g., which comprise a CXCR6 amino acid sequence with various natural and/or synthetic alterations but which still display dominant negative activity
  • generation of peptide mimetics can be effected using various approaches, including, for example, display techniques.
  • the present invention contemplates a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 5, at least 7, at least 11, at least 15, at least 20, at least 25 consecutive amino acids derived from polypeptide sequences of the N-ter domain of CXCR6 (e.g., SEQ ID NO: 4).
  • display vehicles such as phages, viruses or bacteria
  • Peptide mimetics can also be uncovered using computational biology.
  • the soluble polypeptides of the present invention preferably includes a heterologous amino acid sequence
  • heterologous amino acid sequence refers to a non- immunogenic amino acid sequence which does not form a part of the CXCR6 amino acid sequence. This sequence preferably confers solubility to the molecule of this embodiment of the present invention, preferably increasing the half-life of the chimeric molecule in the serum.
  • heterologous amino acid sequence is generally localized at the amino- or carboxyl- terminus of the CXCR6 peptide of the present invention.
  • the at least one heterologous amino acid sequence can be conjugated to the CXCR6 amino acid sequence of the present invention.
  • the at least one CXCR6 amino acid sequence may be embedded between two heterologous sequences, such as described Hoogenboom (1991) MoI. Immunol. 28: 1027-1037.
  • the heterologous amino acid sequence may be attached to the CXCR6 amino acid sequence by any of peptide or non-peptide bond. Attachment of the CXCR6 amino acid sequence to the heterologous amino acid sequence may be effected by direct covalent bonding (peptide bond or a substituted peptide bond) or indirect binding such as by the use of a linker having functional groups.
  • R" is independently hydrogen, alkyl, cycloalkyl or aryl).
  • heterologous amino acid sequence which may be used in accordance with this aspect of the present invention is an immunoglobulin amino acid sequence, such as the hinge and Fc regions of an immunoglobulin heavy domain (see U.S. Pat. No. 6,777,196).
  • the immunoglobulin moiety in the chimeras of this aspect of the present invention may be obtained from IgGl, IgG2, IgG3 or IgG4 subtypes,
  • IgA, IgE, IgD or IgM as further discussed hereinbelow.
  • Immunoadhesins Chimeras constructed from a receptor sequence linked to an appropriate immunoglobulin constant domain sequence (immunoadhesins) are known in the art. Immunoadhesins reported in the literature include fusions of the T cell receptor
  • L-selectin (homing receptor) [(Watson et al., J. Cell. Biol., 110:2221-2229 (1990); Watson et al., Nature, 349: 164-167 (1991)]
  • CD44 [Aruffo et al., Cell, 61: 1303-1313 (1990)]
  • CD28 and B7 (Linsley et al., J. Exp. Med., 173: 721-
  • the chimeric molecule will retain at least functionally active hinge and CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions can also be generated to the C-terminus of the
  • Fc portion of a constant domain or immediately N-terminal to the CHl of the heavy chain or the corresponding region of the light chain.
  • the exact site at which fusion (conjugation) between the heterologous sequence and the CXCR6 amino acid sequence is not critical. Particular sites are well known in the art and may be selected in order to optimize the biological activity, secretion or binding characteristics of the chimeric molecules of this aspect of the present invention (see Example 3 of the Example section which follows).
  • the CXCR6 amino acid sequence of the present invention may be conjugated to the CXCR6 amino acid sequence of the present invention, it is preferable to fuse shorter sequences. For example, a sequence beginning in the hinge region just upstream of the papain cleavage site, which defines IgG Fc chemically; residue 216, taking the first residue of heavy chain constant region to be 114, or analogous sites of other immunoglobulins, is used in the fusion.
  • the CXCR6 amino acid sequence is fused to the hinge region and CH2 and CH3, or to the CHl, hinge, CH2 and CH3 domains of an IgGl, IgG2, or IgG3 heavy chain (see U.S. Pat. No. 6,777,196).
  • the precise site at which the fusion is made is not critical, and the optimal site can be determined by routine experimentation.
  • the immunoglobulin sequences used in the construction of the chimeric molecules of this aspect of the present invention may be from an IgG immunoglobulin heavy chain constant domain.
  • the use of human IgGl immunoglobulin sequences is preferred (e.g., as set forth in SEQ ID NOs. 5 and 6).
  • a major advantage of using IgGl is that IgGl can be purified efficiently on immobilized protein A.
  • other structural and functional properties of immunoglobulins should be considered when choosing the Ig fusion partner for a particular chimera construction.
  • the IgG3 hinge is longer and more flexible, so it can accommodate larger CXCR6 amino acid sequences that may not fold or function properly when fused to IgGl.
  • IgG are bivalent homodimers, whereas Ig subtypes like IgA and IgM may give rise to dimeric or pentameric structures, respectively, of the basic Ig homodimer unit.
  • IgA and IgM may give rise to dimeric or pentameric structures, respectively, of the basic Ig homodimer unit.
  • Other considerations in selecting the immunoglobulin portion of the chimeric molecules of this aspect of the present invention are described in U.S. Pat. No. 6, 77,196.
  • heterologous amino acid sequences commonly used in fusion protein construction include, but are not limited to galactosidase, glucuronidase, glutathione-S-transferase (GST), carboxy terminal peptide (CTP) from chorionic gonadotrophin (CG ⁇ ) and chloramphenicol acetyltransferase (CAT).
  • GST glutathione-S-transferase
  • CTP carboxy terminal peptide
  • CG ⁇ chorionic gonadotrophin
  • CAT chloramphenicol acetyltransferase
  • the isolated soluble molecule of this aspect of the present invention is encoded by a nucleic acid sequences as set forth in SEQ ID NO: 7.
  • molecules of this aspect of the present invention may comprise heterologous amino acid sequences, as described above.
  • CXCR6 amino acid sequences of the present invention may be attached to a non-proteinaceous moiety, such molecules are preferably selected non-immunogenic in a subject.
  • an isolated soluble molecule comprising a CXCR6 amino acid sequence (as described above) attached to a non-proteinaceous moiety.
  • Such a molecule is highly stable (resistant to in-vivo proteaolytic activity probably due to steric hindrance conferred by the non-proteinaceous moiety) and may be produced using common solid phase synthesis methods which are inexpensive and highly efficient, as further described hereinbelow.
  • recombinant techniques may still be used, whereby the recombinant peptide product is subjected to in-vitro modification (e.g., PEGylation as further described hereinbelow).
  • non-proteinaceous moiety refers to a molecule not including peptide bonded amino acids that is attached to the above-described CXCR6 amino acid sequence.
  • the non-proteinaceous moiety of this aspect of the present invention is a polymer or a co- polymer (synthetic or natural).
  • Non-limiting examples of the non-proteinaceous moiety of the present invention include polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), divinyl ether and maleic anhydride copolymer (DIVEMA; see for example, Kaneda Y, et al., 1997, Biochem. Biophys. Res. Commun. 239: 160-5) and poly(styrene comaleic anhydride) (SMA; see for example, Mu Y, et al., 1999, Biochem Biophys Res Commun. 255: 75-9).
  • PEG polyethylene glycol
  • PVP Polyvinyl pyrrolidone
  • non-proteinaceous moieties may be also attached to the above mentioned fusion molecules (i.e., which comprise a heterologous amino acid sequence) to promote stability and possibly solubility of the molecules.
  • Bioconjugation of such a non-proteinaceous moiety confers the CXCR6 amino acid sequence with stability (e.g., against protease activities) and/or solubility (e.g., within a biological fluid such as blood, digestive fluid) while preserving its biological activity and prolonging its half-life.
  • Bioconjugation is advantageous particularly in cases of therapeutic proteins which exhibit short half-life and rapid clearance from the blood.
  • the increased half-lives of bioconjugated proteins in the plasma results from increased size of protein conjugates (which limits their glomerular filtration) and decreased proteolysis due to polymer steric hindrance.
  • the more polymer chains attached per peptide the greater the extension of half-life.
  • measures are taken not to reduce the specific activity of the CXCR6 amino acid sequence of the present invention (i.e., CXCR6 ligand binding).
  • Bioconjugation of the CXCR6 amino acid sequence with PEG can be effected using PEG derivatives such as N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, monomethoxyPEG 2 -NHS, succinimidyl ester of carboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxy PEG-NPC), PEG aldehydes, PEG-orthopyridyl-disuMde, carbonyldimidazol- activated PEGs, PEG-thiol, PEG-maleimide.
  • PEG derivatives such as N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, monomethoxyPEG 2 -NHS, succinimidyl ester of carboxymethylated PEG (SCM-P
  • PEG derivatives are commercially available at various molecular weights [See, e.g., Catalog, Polyethylene Glycol and Derivatives, 2000 (Shearwater Polymers, Inc., Huntsvlle, Ala.)]. If desired, many of the above derivatives are available in a monofunctional monomethoxyPEG (mPEG) form.
  • mPEG monofunctional monomethoxyPEG
  • the PEG added to the CXCR6 amino acid sequence of the present invention should range from a molecular weight (MW) of several hundred Daltons to about 100 kDa (e.g., between 3-30 kDa). Larger MW PEG may be used, but may result in some loss of yield of PEGylated peptides.
  • PEG purity of larger PEG molecules should be also watched, as it may be difficult to obtain larger MW PEG of purity as high as that obtainable for lower MW PEG. It is preferable to use PEG of at least 85 % purity, and more preferably of at least 90 % purity, 95 % purity, or higher. PEGylation of molecules is further discussed in, e.g., Hermanson, Bioconjugate Techniques, Academic Press San Diego, Calif. (1996), at Chapter 15 and in Zalipsky et al., "Succinimidyl Carbonates of Polyethylene Glycol," in Dunn and Ottenbrite, eds., Polymeric Drugs and Drug Delivery Systems, American Chemical Society, Washington, D.C. (1991).
  • PEG can be attached to a chosen position in the CXCR6 amino acid sequence by site-specific mutagenesis as long as the activity of the conjugate is retained (i.e., CXCR6 ligand binding).
  • a target for PEGylation could be any Cysteine residue at the N-terminus or the C-terminus of the CXCR6 amino acid sequence.
  • other Cysteine residues can be added to the CXCR6 amino acid sequence (e.g., at the N-terminus or the C-terminus) to thereby serve as a target for PEGylation.
  • Computational analysis may be effected to select a preferred position for mutagenesis without compromising the activity.
  • activated PEG such as PEG-maleimide, PEG-vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide
  • Methods of preparing activated PEG molecules are known in the arts.
  • PEG-VS can be prepared under argon by reacting a dichloromethane (DCM) solution of the PEG-OH with NaH and then with di-vinylsulfone (molar ratios: OH 1: NaH 5: divinyl sulfone 50, at 0.2 gram PEG/mL DCM).
  • DCM dichloromethane
  • PEG- AC is made under argon by reacting a DCM solution of the PEG-OH with acryloyl chloride and triethylamine (molar ratios: OH 1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM).
  • acryloyl chloride and triethylamine molar ratios: OH 1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM.
  • Such chemical groups can be attached to linearized, 2-arm, 4-arm, or 8-arm PEG molecules.
  • cysteine residues While conjugation to cysteine residues is one convenient method by which the CXCR6 amino acid of the present invention can be PEGylated, other residues can also be used if desired.
  • acetic anhydride can be used to react with NH 2 and SH groups, but not COOH, S-S, or -SCH 3 groups
  • hydrogen peroxide can be used to react with -SH and -SCH 3 groups, but not NH 2 .
  • Reactions can be conducted under conditions appropriate for conjugation to a desired residue in the peptide employing chemistries exploiting well-established reactivities.
  • the terminal COOH-bearing PVP is synthesized from N-vinyl-2- pyrrolidone by radical polymerization in dimethyl formamide with the aid of 4,4'- azobis-(4-cyano valeric acid) as a radical initiator, and 3-mercaptopropionic acid as a chain transfer agent.
  • Resultant PVPs with an average molecular weight of Mr 6,000 can be separated and purified by high-performance liquid chromatography and the terminal COOH group of synthetic PVP is activated by the N- hydroxysuccinimide/dicyclohexyl carbodiimide method.
  • the CXCR6 amino acid sequence is reacted with a 60-fold molar excess of activated PVP and the reaction is stopped with amino caploic acid (5-fold molar excess against activated PVP), essentially as described in Haruhiko Kamada, et al., 2000, Cancer Research 60: 6416- 6420, which is fully incorporated herein by reference.
  • Resultant conjugated CXCR6 molecules e.g., PEGylated or PVP-conjugated CXCR6 are separated, purified and qualified using e.g., high-performance liquid chromatography (HPLC).
  • purified conjugated molecules of this aspect of the present invention may be further qualified using e.g., in vitro assays in which the binding specificity of CXCR6 ligand to its receptor (e.g., CXCR6) is tested in the presence or absence of the CXCR6 conjugates of the present invention, essentially as described for other chemokines [e.g., MIP- l ⁇ , see for example, Hesselgesser J, 1998 (Supra), which is fully incorporated herein by reference].
  • chemokines e.g., MIP- l ⁇ , see for example, Hesselgesser J, 1998 (Supra), which is fully incorporated herein by reference.
  • Molecules of this aspect of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence, such as a "Tag” further described hereinbelow) and therefore involve different chemistry.
  • the polypeptides of the present invention can be generated using recombinant techniques such as described by Bitter et al. (1987) Methods in Enzymol. 153:516- 544; Studier et al. (1990) Methods in Enzymol. 185:60-89; Brisson et al. (1984) Nature 310:51 1-514; Takamatsu et al. (1987) EMBO J. 3:17-311; Coruzzi et al. (1984) EMBO J. 3: 1671-1680; Brogli et al. (1984) Science 224:838-843; Gurley et al.
  • an expression construct i.e., expression vector
  • an isolated polynucleotide i.e., isolated from a naturally occurring source thereof, e.g., SEQ ID NO: 1, 3
  • SEQ ID NO: 1 an isolated polynucleotide which comprises a nucleic acid sequence encoding the CXCR6 amino acid sequence (optionally in frame fused to a nucleic acid sequence encoding the heterologous amino acid sequence e.g., SEQ ID NO: 5) of the present invention positioned under the transcriptional control of a regulatory element, such as a promoter, is introduced into host cells.
  • a nucleic acid sequence encoding a CXCR6 polypeptide of the present invention is ligated in frame to an immunoglobulin cDNA sequence (e.g., SEQ ID NO: 5).
  • an immunoglobulin cDNA sequence e.g., SEQ ID NO: 5
  • ligation of genomic immunoglobulin fragments can also be used.
  • fusion requires the presence of immunoglobulin regulatory sequences for expression.
  • cDNAs encoding IgG heavy- chain constant regions can be isolated based on published sequence from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybridization or by polymerase chain reaction (PCR) techniques.
  • the nucleic acid sequences encoding the CXCR6 amino acid sequence and immunoglobulin can be ligated in tandem into an expression construct (vector) that directs efficient expression in the selected host cells, further described hereinbelow.
  • an expression construct vector
  • pRK5 -based vectors Scholl et al., Cell, 61:361-370 (1990)
  • CDM8-based vectors Seed, Nature, 329:840 (1989)
  • junction can be created by removing the extra sequences between the designed junction codons using oligonucleotide- directed deletional mutagenesis [Zoller et al, Nucleic Acids Res., 10:6487 (1982); Capon et al., Nature, 337:525-531 (1989)].
  • Synthetic oligonucleotides can be used, in which each half is complementary to the sequence on either side of the desired junction; ideally, these are 1 1 to 48-mers.
  • PCR techniques can be used to join the two parts of the molecule in-frame with an appropriate vector.
  • Methods of introducing the expression construct into a host cell include, electroporation, lipofection and chemical transformation (e.g., calcium phosphate). See also Example 3 of the Examples section which follows.
  • the "transformed" cells are cultured under suitable conditions, which allow the expression of the chimeric molecule encoded by the nucleic acid sequence.
  • the expressed chimeric molecule is recovered from the cell or cell culture, and purification is effected according to the end use of the recombinant polypeptide.
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like, can be used in the expression vector [see, e.g., Bitter et al., (1987) Methods in Enzymol. 153:516- 544].
  • the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or toxicity of the expressed fusion protein.
  • a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the fusion protein coding sequence.
  • microorganisms such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the chimera coding sequence; yeast transformed with recombinant yeast expression vectors containing the chimera coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the chimera coding sequence.
  • Mammalian expression systems are preferably used to express the chimera of the present invention. The choice of host cell line for the expression of the molecules depends mainly on the expression vector.
  • Eukaryotic expression systems are preferred (e.g., mammalian and insects) since they allow post translational modifications (e.g., glyccosylation). Another consideration is the amount of protein that is required. Milligram quantities often can be produced by transient transfections.
  • the adenovirus EIA-transformed 293 human embryonic kidney cell line can be transfected transiently with pRK5-based vectors by a modification of the calcium phosphate method to allow efficient expression.
  • CDM8-based vectors can be used to transfect COS cells by the DEAE-dextran method (Aruffo et al., Cell, 61 : 1303-1313 (1990); Zettmeissl et al., DNA Cell Biol.
  • the molecules can be expressed after stable transfection of a host cell line (see Example 1 of the Examples section). It will be appreciated that the presence of a hydrophobic leader sequence at the N-terminus of the molecule will ensure processing and secretion of the molecule by the transfected cells.
  • transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An effective medium refers to any medium in which a cell is cultured to produce the recombinant chimera molecule of the present invention.
  • Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • resultant proteins of the present invention may either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.
  • recovery of the recombinant protein refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification.
  • Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • Molecules of the present invention are preferably retrieved in "substantially pure” form.
  • substantially pure refers to a purity that allows for the effective use of the protein in the applications, described hereinbelow.
  • Recombinant molecules of the present invention can be conveniently purified by affinity chromatography.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain that is used in the chimera.
  • Protein A can be used to purify chimeric molecules that are based on human ⁇ l, ⁇ 2, or ⁇ 4 heavy chains [Lindmark et al., J. Immunol. Meth., 62:1-13 (1983)].
  • Protein G is preferably used for all mouse isotypes and for human ⁇ 3 [Guss et al., EMBO J., 5:1567-1575 (1986)].
  • the solid support to which the affinity ligand is attached is most often agarose, but other solid supports are also available.
  • chimeric molecules of this aspect of the present invention allows for faster flow rates and shorter processing times than can be achieved with agarose.
  • the conditions for binding the chimeric molecules to the protein A or G affinity column are dictated entirely by the characteristics of the Fc domain; that is, its species and isotype. Generally, when the proper ligand is chosen, efficient binding occurs directly from unconditioned culture fluid.
  • One distinguishing feature of chimeric molecules of this aspect of the present invention is that, for human .gamma.1 molecules, the binding capacity for protein A is somewhat diminished relative to an antibody of the same Fc type.
  • Bound chimeric molecules of this aspect of the present invention can be efficiently eluted either at acidic pH (at or above 3.0), or in a neutral pH buffer containing a mildly chaotropic salt.
  • This affinity chromatography step can result in a chimeric molecule preparation that is > 95 % pure. Medical grade purity is essential for therapeutic applications.
  • the present invention provides for numerous configurations of soluble molecules which are capable of binding CXCR6 ligands and neutralize signaling therefrom.
  • CXCR6/CXCL16 Another agent for reducing the activity or expression of CXCR6/CXCL16 can also be a neutralizing antibody directed at CXCR6/CXCL16 (such as by binding to the CXCR6 binding site on CXCL 16 or by binding to the CXCL 16 binding site on CXCR6) or an effector thereof.
  • Neutralizing antibodies for CXCR6/CXCL16 are known in the art, for example, U.S. Pat. No. 7,038,018, which is hereby incorporated by reference in its entirety.
  • a preferred neutralizing antibody according to the teachings of the present invention preferably binds the N-ter domain of CXCR6 as set forth in SEQ ID NO: 4. This antibody is expected to compete with CXCL 16 on receptor binding.
  • the term "antibody” refers to a substantially intact antibody molecule.
  • antibody fragment refers to a functional fragment of an antibody that is capable of binding to an antigen.
  • Suitable antibody fragments for practicing the present invention include, inter alia, a complementarity-determining region (CDR) of an immunoglobulin light chain
  • light chain a CDR of an immunoglobulin heavy chain
  • variable chain a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single-chain Fv, an Fab, an Fab', and an F(ab')2.
  • Fv defined as a genetically engineered fragment consisting of the variable region of the light chain and the variable region of the heavy chain expressed as two chains;
  • scFv single-chain Fv
  • Fab a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain, which consists of the variable and CHl domains thereof
  • Fab' a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab' fragments are obtained per antibody molecule);
  • F(ab')2 a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab' fragments held together by two disulfide bonds).
  • 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 (Orlandi, R. et al. (1989). Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci USA 86, 3833-3837; and Winter, G. and Milstein, C. (1991). Man-made antibodies, Nature 349, 293-299), or generation of monoclonal antibody molecules by continuous cell lines in culture.
  • haptens can be coupled to antigenically neutral carriers such as keyhole limpet hemocyanin (KLH) or serum albumin (e.g., bovine serum albumin (BSA)) carriers (see, for example, US. Pat. Nos. 5,189,178 and 5,239,078).
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Coupling a hapten to a carrier can be effected using methods well known in the art. For example, direct coupling to amino groups can be effected and optionally followed by reduction of the imino linkage formed.
  • the carrier can be coupled using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents.
  • Condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents.
  • Linker compounds can also be used to effect the coupling; both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, Illinois, USA.
  • the resulting immunogenic complex can then be injected into suitable mammalian subjects such as mice, rabbits, and others. 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 or monoclonal antibodies may be obtained, as described hereinabove.
  • Antibody fragments may be obtained using methods well known in the art.
  • 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 systems) of DNA encoding the fragment.
  • E. coli or mammalian cells e.g., Chinese hamster ovary (CHO) cell culture or other protein expression systems
  • antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • an (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 3.5 S 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 (see, for example, Inbar, D. et al. (1972). Localization of antibody-combining sites within the variable portions of heavy and light chains. Proc Natl Acad Sci USA 69, 2659-2662). 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 in the literature of the art (see, e.g.: Whitlow, M. and Filpula, D. (1991). Single-chain Fv proteins and their fusion proteins.
  • Isolated complementarity-determining region peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes may be prepared, for example, by RT-PCR of the mRNA of an antibody-producing cell. Ample guidance for practicing such methods is provided in the literature of the art (e.g., Larrick, J. W. and Fry, K. E. (1991). PCR Amplification of Antibody Genes. METHODS: A Companion to Methods in Enzymology 2(2), 106-110). It will be appreciated that for human therapy or diagnostics, 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 (see, for example: Jones, P. T. et al. (1986). Replacing the complementarity- determining regions in a human antibody with those from a mouse. Nature 321, 522- 525; Riechmann, L. et al. (1988). Reshaping human antibodies for therapy. Nature 332, 323-327; Presta, L. G. (1992b). Curr Opin Struct Biol 2, 593-596; and Presta, L. G. (1992a). Antibody engineering. Curr Opin Biotechnol 3(4), 394-398).
  • 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 essentially as described (see, for example: Jones et al. (1986); Riechmann et al. (1988); Verhoeyen, M. et al. (1988). Reshaping human antibodies: grafting an antilysozyme activity. Science 239, 1534-1536; and U.S. Pat. No. 4,816,567), by substituting human CDRs with corresponding rodent CDRs.
  • 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 (Hoogenboom, H. R. and Winter, G. (1991). By-passing immunization. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J MoI Biol 227, 381-388; Marks, J. D. et al. (1991). By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J MoI Biol 222, 581-597; Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96; and Boerner, P. et al. (1991).
  • Humanized antibodies can also be created by introducing sequences encoding human immunoglobulin loci into transgenic animals, e.g., into mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon antigenic challenge, human antibody production is observed in such animals which closely resembles that seen in humans in all respects, including gene rearrangement, chain assembly, and antibody repertoire. Ample guidance for practicing such an approach is provided in the literature of the art (for example, refer to: U.S. Pat. Nos.
  • 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 agent of this aspect of the present invention can be a chemical, which is designed to specifically inhibit the activity or expression of CXCR6 or an effector thereof.
  • Chemical inhibitors directed at CXCR6 effectors are well known in the art. Examples include, but are not limited to, pertussis toxin (Gj inhibitor), wortmannin or LY294002 (PI3K inhibitors) [Chandrasekar et al., supra] and curcumin (NF-kappa B inhibitor) [Thaloor et al., Am J Physiol Cell Physiol (1999) 277:C320-C329]. Signal transduction inhibitors are available from a number of chemical companies including Calbiochem (San Diego, CA, USA) and Sigma- Aldrich Corp. (St Louis, MO, USA).
  • siRNA small interfering RNA
  • the first step which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with 2-nucleotide 3' overhangs [Hutvagner and Zamore, Curr. Opin.
  • the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC.
  • the active RISC targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore, Curr. Opin. Genetics and Development (2002) 12:225-232; Hammond et al., Nat. Rev. Gen.
  • RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al., Nat. Rev. Gen. 2: 110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore, Curr. Opin. Genetics and Development 12:225-232 (2002)]. For more information on RNAi see the following reviews Tuschl, ChemBiochem. 2:239-245 (2001); Cullen, Nat. Immunol. 3:597-599 (2002); and Brantl, Biochem. Biophys. Act.
  • RNAi molecules suitable for use with the present invention can be effected as follows. First, a CXCR6/CXCL16 mRNA sequence, for example, is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl, ChemBiochem. 2:239-245].
  • UTRs untranslated regions
  • siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).
  • sequence alignment software e.g., human, mouse, rat etc.
  • sequence alignment software such as the BLAST software available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/).
  • Putative target sites which 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.
  • 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.
  • DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of interest.
  • DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology (1995) 2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA (1997) 943:4262)
  • a general model (the "10-23" model) for the DNAzyme has been proposed.
  • DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine :pyrimidine junctions [Santoro and Joyce, Proc. Natl. Acad. Sci. USA (1997) 94(9):4262-6; for rev of DNAzymes see Khachigian, Curr Opin MoI Ther (2002) 4:1 19-21].
  • DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL).
  • CML Chronic Myelogenous Leukemia
  • ALL Acute Lymphoblastic Leukemia
  • Reducing CXCR6/CXCL16 or an effector thereof can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the proteins of interest.
  • Design of antisense molecules which can be used to efficiently down-regulate a gene product of interest must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
  • antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin MoI Ther 1:372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz, Curr Opin MoI Ther 1 :297-306 (1999)].
  • Another agent capable of reducing the expression of CXCR6/CXCL16 or an effector thereof is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding this gene product.
  • Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)].
  • the possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
  • ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)].
  • ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.
  • Several ribozymes are in various stages of clinical trials.
  • ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials.
  • ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway.
  • Ribozyme Pharmaceuticals, Inc. as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models.
  • HEPTAZYME a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - WEB home page).
  • TFOs triplex forming oligonuclotides
  • triplex forming sequence preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.
  • Transfection of cells for example, via cationic liposomes
  • TFOs Transfection of cells (for example, via cationic liposomes) with TFOs, and formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific downregulation of gene expression.
  • Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res.
  • TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest (2003) 112:487-94).
  • Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn. Additional description of oligonucleotide agents is further provided hereinbelow. It will be appreciated that therapeutic oligonucleotides may further include base and/or backbone modifications which may increase bioavailability therapeutic efficacy and reduce cytotoxicity. Such modifications are described in Younes (2002) Current Pharmaceutical Design 8:1451-1466.
  • the oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
  • oligonucleotides are those modified in either backbone, internucleoside linkages or bases, as is broadly described hereinunder.
  • oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms can also be used.
  • modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts, as disclosed in U.S. Pat. Nos.
  • oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for complementation with the appropriate polynucleotide target.
  • An example for such an oligonucleotide mimetic includes peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • a PNA oligonucleotide refers to an oligonucleotide where the sugar-backbone is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Oligonucleotides of the present invention may also include base modifications or substitutions.
  • "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted urac
  • 5-substituted pyrimidines include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 0 C. [Sanghvi YS et al. (1993) Antisense Research and Applications, CRC Press, Boca Raton 276-278] and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • Example 5 of the Examples section which follows the present inventors were able to suppress an ongoing MS, using agents of the present invention, substantiating the use of same in therapy.
  • Agents of the present invention can be administered to the subject per se, or as part of a pharmaceutical composition, which also includes a physiologically acceptable carrier.
  • a pharmaceutical composition refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • the term "active ingredient" refers to the preparation accountable for the intended biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media [Mutter et al. (1979)].
  • the bioconjugated polymer e.g., the PEGylated CXCR6 polypeptide of the present invention
  • the pharmaceutically acceptable carrier e.g., the PEGylated CXCR6 polypeptide of the present invention
  • a preferred embodiment of this dual use is a liposomal vehicle, e.g., PEG-conjugated liposomes, as described e.g., in U.S. Pat. Appl. No. 20030186869 to Poiani, George et al., which is fully incorporated herein by reference.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries as desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, and sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form.
  • suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.
  • compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a "therapeutically effective amount” means an amount of active ingredients (e.g., a nucleic acid construct) effective to prevent, alleviate, or ameliorate symptoms of a disorder (e.g., ischemia) or prolong the survival of the subject being treated.
  • a therapeutically effective amount means an amount of active ingredients (e.g., a nucleic acid construct) effective to prevent, alleviate, or ameliorate symptoms of a disorder (e.g., ischemia) or prolong the survival of the subject being treated.
  • the dosage or the therapeutically effective amount can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and 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.
  • Dosage amount and administration intervals may be adjusted individually to provide sufficient plasma or brain levels of the active ingredient to induce or suppress the biological effect (i.e., minimally effective concentration, MEC).
  • MEC minimally effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, 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, etc.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser device may also be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may include labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as further detailed above.
  • nucleic acid agents can be administered to the individual employing any suitable mode of administration, described hereinbelow (i.e., in-vivo gene therapy).
  • the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex-vivo gene therapy).
  • nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3 1 LTR or a portion thereof.
  • Other vectors can be used that are non- viral, such as cationic lipids, polylysine, and dendrimers.
  • Treatment of Multiple Sclerosis according to the present invention may be combined with other treatment methods known in the art (i.e., combination therapy). These include, but are not limited to, Interferon Beta Ia, Interferon Beta Ib, Glatiramer Acetate, Mitoxantrone, MethylPrednisolone, Prednisone, Prednisolone, Dexamethasone, Adreno-corticotrophic Hormone (ACTH) and Corticotropin.
  • Interferon Beta Ia Interferon Beta Ib
  • Glatiramer Acetate Mitoxantrone
  • MethylPrednisolone MethylPrednisolone
  • Prednisone Prednisone
  • Prednisolone Prednisolone
  • Dexamethasone Dexamethasone
  • Adreno-corticotrophic Hormone (ACTH) Adreno-corticotrophic Hormone
  • Corticotropin Adreno-corticotroph
  • the present invention also envisages a novel method for diagnosis of MS.
  • the term “diagnosing” refers to classifying a disease or a symptom as Multiple Sclerosis, determining a severity of a Multiple Sclerosis, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • the method is effected by detecting CXCR6 in cells obtained from the subject, wherein each of the levels determined can be correlated with progression of Multiple Sclerosis in the subject, to thereby diagnose presence of Multiple Sclerosis in the subject
  • the term “level” refers to expression levels of RNA and/or protein of CXCR6.
  • a level correlatable with presence or absence of Multiple Sclerosis can be a level of CXCR6 in a Multiple Sclerosis derived cells which is increased as compared to the level of the same in a normal healthy cells obtained from a similar tissue or cellular origin.
  • cells refers to cells derived from a tissue or from a fluid, preferably cellular sample, isolated from a subject, such as but not limited to, for example, spinal fluid and peripheral blood.
  • tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of each of the above-described proteins in the subject. Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy.
  • the level of CXCR6 can be determined and a diagnosis can thus be made. Determining a level of CXCR6 can be effected using various biochemical and molecular approaches used in the art for determining gene amplification, and/or level of gene expression.
  • oligonucleotide refers to a single stranded or double stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally- occurring bases, sugars and covalent internucleoside linkages (e.g., backbone) as well as oligonucleotides having non-naturally-occurring portions which function similarly to respective naturally-occurring portions.
  • Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art, such as enzymatic synthesis or solid phase synthesis.
  • Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, "Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes HII Ausubel, R. M., ed.
  • the oligonucleotide of the present invention is of at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the examined mRNA.
  • Hybridization based assays which allow the detection of a DNA or RNA of interest in a biological sample rely on the use of oligonucleotide which can be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides.
  • Hybridization of short nucleic acids can be effected using the following examplery hybridization protocols which can be modified according to the desired stringency; (i) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 ⁇ g/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 0 C below the T m , final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 0 C below the T m ; (U) hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1
  • hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.
  • labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art.
  • a label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample (target).
  • oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent.
  • biotinylated dNTPs or rNTP or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs)
  • streptavidin e.g., phycoerythrin-conjugated streptavidin
  • fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, Calif] can be attached to the oligonucleotides.
  • Traditional hybridization assays include PCR, RT-PCR, RNase protection, in- situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis.
  • wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.
  • standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
  • samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.
  • PCR-based methods may be used to identify the presence of mRNA of interest.
  • a pair of oligonucleotides is used, which is specifically hybridizable with the polynucleotide sequences described hereinabove in an opposite orientation so as to direct exponential amplification of a portion thereof (including the hereinabove described sequence alteration) in a nucleic acid amplification reaction.
  • the pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7 °C, preferably less than 5 °C, more preferably less than 4 °C, most preferably less than 3 °C, ideally between 3 °C and 0 0 C.
  • Tm melting temperatures
  • Hybridization to oligonucleotide arrays may be also used to determine the presence of the mRNA or DNA of interest.
  • nucleic acid sample which includes the candidate region to be analyzed is isolated, amplified and labeled with a reporter group.
  • This reporter group can be a fluorescent group such as phycoerythrin.
  • the labeled nucleic acid is then incubated with the probes immobilized on the chip using a fluidics station.
  • Manz et al. (1993) Adv in Chromatogr 1993; 33:1-66 describe the fabrication of fluidics devices and particularly microcapillary devices, in silicon and glass substrates.
  • the chip is inserted into a scanner and patterns of hybridization are detected.
  • the hybridization data is collected, as a signal emitted from the reporter groups already incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.
  • the level of CXCR6 may also be determined at the protein level.
  • Numerous protein detection assays are known in the art, examples include, but are not limited to, chromatography, electrophoresis, immunodetection assays such as ELISA and western blot analysis, immunohistochemistry and the like, which may be effected using antibodies such as described hereinabove. Note, however, that such antibodies need not be neutralizing antibodies.
  • Diagnosis of Multiple Sclerosis using the above-described methodology can be further supported by other diagnostic methods for Multiple Sclerosis which are well known in the art to thereby provide a more accurate diagnosis.
  • diagnostic methods include, but are not limited to, evidence of two or more neurological signs (e.g., lesions) that are localized to the central nervous system (e.g., brain or spinal cord) and are disseminated in time and space (e.g., occur in different parts of the central nervous system at least three months apart).
  • Such lesions can be diagnosed by magnetic resonance imaging (MRI) with gadolinium contrast, especially during or following a first attack, and again at least three months after the initial attack to identify new lesions and provide evidence of dissemination over time.
  • MRI magnetic resonance imaging
  • kits can include a solid phase for attaching multiple cell samples packaged with appropriate buffers and preservatives and used for diagnosis.
  • CXCL 16 binding on CXCR6 has allowed the generation of polypeptide agents as described above (e.g., SEQ ID NO: 4 and 8). These can be used for the treatment of a variety of diseases in which CXCR6 or CXCL 16 is associated with disease onset or progression (as used herein CXCR6/CXCL16-associated diseases).
  • CXCR6/CXCL16 associated disease refers to a medical condition (e.g., disease, syndrome or condition), which depends on the interaction between a CXCR6 and it's ligand CXCL 16 for onset or progression.
  • autoimmune diseases such as such as Grave's disease [Aust et al., Eur J Endocrinol. (2005) 152(4):635-43]
  • inflammatory diseases such as rheumatoid arthritis [Ruth et al., supra; van der Voort et al. supra]
  • vascular disease vascular disease [Charo and Taubman, Circulation Res.
  • inflammatory disorder includes but is not limited to chronic inflammatory diseases and disorders and acute inflammatory diseases and disorders.
  • inflammatory diseases include, but are not limited to, conditions summarized in the list infra.
  • hypersensitivity examples include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.
  • Type I or immediate hypersensitivity such as asthma.
  • Type II hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 JuI; 15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998; 17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y.
  • paraneoplastic neurological diseases cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de Ia Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan;156 (1):23); neuropathies, dysimmune neuropathies (Nobile-Orazio E.
  • vasculitises necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May;151 (3): 178); antiphospholipid syndrome (Flamholz R. et al, J Clin Apheresis 1999;14 (4):171); heart failure, agonist-like beta-adrenoceptor antibodies in heart failure (Wallukat G. et al, Am J Cardiol.
  • Type IV or T cell mediated hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18;91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998;7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves' disease (Sakata S.
  • Examples of delayed type hypersensitivity include, but are not limited to, contact dermatitis and drug eruption.
  • Examples of types of T lymphocyte mediating hypersensitivity include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.
  • helper T lymphocyte-mediated hypersensitivity examples include, but are not limited to, T h I lymphocyte mediated hypersensitivity and T h 2 lymphocyte mediated hypersensitivity.
  • Autoimmune diseases include, but are not limited to, T h I lymphocyte mediated hypersensitivity and T h 2 lymphocyte mediated hypersensitivity.
  • cardiovascular diseases include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
  • autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A.
  • autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18 ;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189).
  • autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome, diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J.
  • autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan;23 (1):16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16;138 (2):122), colitis, ileitis and Crohn's disease.
  • autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
  • autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al, Clin Immunol Immunopathol 1990 Mar;54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999
  • autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al, J Neuroimmunol 2001 Jan 1;112 (1-2):1), Alzheimer's disease (Oron L. et al, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83; Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci.
  • autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al, Int Arch Allergy Immunol 2000 Sep;123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234).
  • autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140).
  • autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al, Lupus 1998;7 Suppl 2:S107-9).
  • autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al, Cell Immunol 1994 Aug;157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al, Ann N Y Acad Sci 1997 Dec 29;830:266).
  • autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998;17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al, Clin Diagn Lab Immunol. 1999 Mar;6
  • infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases.
  • diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.
  • allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.
  • cancer examples include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancerous diseases include but are not limited to: Myeloid leukemia such as Chronic myelogenous leukemia. Acute myelogenous leukemia with maturation. Acute promyelocytic leukemia, Acute nonlymphocytic leukemia with increased basophils, Acute monocytic leukemia.
  • Acute myelomonocytic leukemia with eosinophilia Malignant lymphoma, such as
  • Lymphoctyic leukemia such as Acute lumphoblastic leukemia.
  • Chronic lymphocytic leukemia Myeloproliferative diseases, such as Solid tumors Benign Meningioma, Mixed tumors of salivary gland, Colonic adenomas;
  • Adenocarcinomas such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder,
  • Rhabdomyosarcoma (alveolar), Extraskeletel myxoid chondrosarcoma, Ewing's tumor; other include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, breast, skin, prostate, and ovarian.
  • the present invention also envisages the use of the present polypeptides for research and laboratory applications.
  • CXCR6 peptide of the present invention allows use thereof in purification and detection of CXCR6 ligands.
  • an isolated soluble polypeptide comprising a tag attached to a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCL 16 (as described above).
  • the term "tag” refers to a moiety which is specifically recognized by a binding partner such as an antibody, a chelator or an avidin (biotin) molecule.
  • the tag can be placed C-terminally or N-terminally of the CXCR6 peptide, as long as it does not interfere with a biological activity thereof (e.g., ligand binding).
  • a tag polypeptide has enough residues to provide an epitope (i.e., epitope tag) against which an antibody thereagainst can be made, yet is short enough such that it does not interfere with biological activity of the CXCR6 peptide.
  • the epitope tag preferably also is fairly unique so that the antibody thereagainst does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8-50 amino acid residues
  • poly-histidine sequences which bind nickel, allowing isolation of the tagged protein by Ni-NTA chromatography as described (Lindsay et al. Neuron 17:571-574 (1996)], for example.
  • Such epitope-tagged forms of the CXCR6 are desirable, as the presence thereof can be detected using a labeled antibody against the tag polypeptide. Also, provision of the epitope tag enables the CXCR6 peptide of the present invention to be readily purified by affinity purification using the anti-tag antibody. Affinity purification techniques and diagnostic assays involving antibodies are described later herein.
  • Tag polypeptides and their respective antibodies are well known in the art. Examples include the flu HA tag polypeptide and its antibody 12CA5 (Field et al., MoI. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology, 5:3610- 3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody. Paborsky et al., Protein Engineering, 3(6):547-553 (1990). Other tag polypeptides have been disclosed. Examples include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194
  • an antibody thereto can be generated using methods which are well known in the art. Such antibodies are commercially available such as from Sigma, St. Louis. USA.
  • a method of isolating a CXCR6 ligand from a biological sample or detecting the presence of CXCR6 ligands therein refers to a biological material, such as cells, tissues and fluids such as blood, serum, plasma, lymph, bile fluid, urine, saliva, sputum, synovial fluid, semen, tears, cerebrospinal fluid, bronchioalveolar large fluid, ascites fluid, pus, conditioned medium and the like in which CXCR6 ligand is present.
  • Isolation of CXCR6 ligand according to this aspect of the present invention is effected by contacting the biological sample with the molecule of this aspect of the present invention, such that CXCR6 ligand and the molecule form a complex (using buffer, temperature conditions which allow binding of the molecule to CXCR6 ligand, see for Example Datta-Mannan and Stone 2004, supra); and isolating the complex to thereby isolate CXCR6 ligand from the biological sample.
  • the molecule is preferably immobilized on a solid support.
  • solid support refers to a non-aqueous matrix to which a reagent of interest (e.g., the molecule of this aspect of the present invention) can adhere.
  • solid supports include, but are not limited to, solid supports formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid support can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
  • detectable molecules can be used to detect the levels of CXCR6 ligand in biological samples.
  • molecules typically will be labeled with a detectable moiety.
  • the detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, a fluorescent or chemiluminescent compound, or a tag (such as described hereinabove and to which a labeled antibody can bind).
  • the molecules of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. [Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987)].
  • the molecules of this aspect of the present invention can be included in a diagnostic kit, in which the molecule and optionally solid support and imaging reagents (e.g., antibodies, chromogenic substrate etc.) can be packaged in suitable containers with appropriate buffers and preservatives and used for diagnosis.
  • a diagnostic kit in which the molecule and optionally solid support and imaging reagents (e.g., antibodies, chromogenic substrate etc.) can be packaged in suitable containers with appropriate buffers and preservatives and used for diagnosis.
  • EAE Encephalomyelitis
  • RNA Extraction (myelin oligodendrocyte glycoprotein, SEQ ID NO: 19) as was previously described [Kassiotis and Kollias, J Exp Med (2001) 193(4):427-434]. On day 18, spinal cords of EAE induced mice or na ⁇ ve mice were removed for further analysis. RNA Extraction
  • RNA Five ⁇ g mRNA were synthesized into a double stranded cDNA, in a reaction mixture that included: RT-first strand reaction buffer, 1 nM dNTPs (NEB), 1 nmole random hexamers (Fermentas International INC, Canada), 10 u/ml RNasin (pharmaBiotech), 25 units/ml Superscript MuLUV-RT (NEB), CXCR6 Sense primer: ATGGATGATGGGCATCAAGAGTCAGCTCT, SEQ ID NO: 20 and CXCR6 anti- sense primer: CTACAATTGGAACATACTGGTGGTCTCTACA SEQ ID NO: 21.
  • the mixture was incubated at 37 °C for 2 hours.
  • CXCR6 expressing T helper cells are present in spinal cords of EAE induced mice
  • spleen, cervical lymph nodes (CLN) and spinal cord (SC) of EAE induced mice were collected 14 days after induction.
  • Cells were isolated by tissue pulverization and washed with PBS. Red blood cells were lysed by RBC (Red Blood Cell) Lysis buffer, washed and removed from cell mixture. The remaining white blood cells were double stained with rat anti-mouse CXCR6 (R&D, Minneapolis, MN) and rat anti-mouse CD4-FITC antibodies (BD Pharmingen, BD Biosciences). Cells were washed and stained by anti-rat PE (Jackson ImmunoResearch Laboratories, Inc, PA, USA). Results were read and analyzed in a flow cytometer.
  • FIG. 3A-B Construction of the nucleic acid vector encoding the CXCR ⁇ -Ig fusion protein of the present invention is schematically illustrated in Figures 3A-B.
  • Murine CXCR6 GenBank Accession No. NM_030712, SEQ ID NO: 12
  • SEQ ID NO: 14 primers complementary to the extracellular N-terminal (N-ter amino acid coordinates, amino acid 1-39 of CXCR6; SEQ ID NO: 14) domain of the receptor as follow: Sense, cccaagcttatggatgatgggcatcaaga (SEQ ID NO. 15); antisense: ccgctcgagcttgaactttaggaagcgtt (SEQ ID NO. 16).
  • the pSec-CXCR6-IgG plasmid was co-transfected into DG44 Chinese hamster ovary (CHO) cells that have a double deletion for the dihydrofolate reductase (DHFR) gene (DG44 CHO DHFR 7" cells ATCC Accession No. CRL-9096), with CHO DHFR minigene vector, which transfects DHFR-deficient CHO cells with high efficiency, using jet PEI (Polypluse transfection - Illkirch Cedex, France) according the manufacturer's protocol.
  • DHFR dihydrofolate reductase
  • Stably transfected cells were selected in a culture medium (MEM-alpha) containing hygromycine (200 ⁇ g/ml) and increasing doses of methotrixate (2.5 nM to 0.1 mM).
  • the CXCRo-IgG fusion protein was purified from DHFR-positive transfectant supernatants by a Ni-NTA column (Qiagen) and verified by coomassie staining (Figure 4B) and by western blot analysis ( Figure 4A) using mouse anti-myc (Santa Cruz Biotechnology) as primary antibody and goat anti mouse HRP (Jackson immuno research) secondary antibody.
  • the fusion polypeptide CXCRo-IgG is a 42Kd polypeptide the higher band is a polypeptide dimmer.
  • the binding specificity of the murine CXCRo-IgG fusion protein to various commercially available murine recombinant chemokines was determined by direct ELISA as follows: 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated with 20 ng/ml of murine recombinant chemokines overnight and blocked with 1% BSA in PBS. Wells were washed and incubated with 100 ⁇ g/ml CXCRo-IgG overnight.
  • mice Two groups of C57/B mice (3 mice in each group) were subjected to active induction of EAE by MOGp35-55 (myelin oligodendrocyte glycoprotein). Beginning one day after the onset of disease (day 12), these mice were treated with repeated intravenous administrations (every other day) of 300 ⁇ g/mouse of either a CXCR6- IgG or isotype matched IgG. On day 22, spinal cords were removed from these mice and from naive mice, which were not subjected to EAE induction.
  • MOGp35-55 myelin oligodendrocyte glycoprotein
  • spinal cords Histochemistry of spinal cords
  • the spinal cords as detailed above were fixed in 10 % NBF, dehydrated in ethanol and chloroform and blocked in paraffin.
  • the spinal cords were then cut in a microtome, mounted on a microscope slide, stained with hematoxylin and eosin, and examined using an Olympus microscope.
  • mice Three groups of C57/B mice (three mice in each group) were subjected to active induction of EAE by MOGp35-55 (myelin oligodendrocyte glycoprotein) as was previously described [Kassiotis and Kollias, J Exp Med (2001) 193(4):427-434].
  • MOGp35-55 myelin oligodendrocyte glycoprotein
  • mice Beginning one day after the onset of disease (day 12), these mice were treated with repeated intravenous administrations (every other day) of 300 ⁇ g/mouse of either a
  • CXCR ⁇ -IgG isotype matched IgG or PBS.
  • the splenocytes were harvested and were restimulated for 72 hours with 50 ⁇ g/ml MOGp35-55 or with cell medium.
  • the supernatants were analyzed by ELISA for cytokine production.
  • mice Two groups of C57/B mice (three mice in each group) were subjected to active induction of EAE by MOGp35-55 (myelin oligodendrocyte glycoprotein) as was previously described [Kassiotis and Kollias, J Exp Med (2001) 193(4):427-434].
  • MOGp35-55 myelin oligodendrocyte glycoprotein
  • Splenocytes were isolated and washed with PBS. Red blood cells were lysed using RBC Lysis buffer. Splenocytes were stained with rat anti-mouse CDl IB-FITC antibody (Serotec) and analyzed by FACS.
  • FIGS 9A-B clearly show there was no significant difference in relative number of CDl Ib+ cells in mice treated with CXCRo-IgG compared to control mice (34 % compared to 26 %, respectively).
  • One of the suggested mechanisms by which CXCRo-IgG exerts its therapeutic effect is by deleting the relative number of CDl lb+ macrophages/dendritic cells which present antigens to T cells. The present inventors showed that CXCRo-IgG does not lead to such elimination, either directly, or indirectly.
  • Spleen cells were isolated from naive C57BL/6 mice and incubated for 1 hour with MOGp35-55 alone or along with 1 ⁇ g CXCRo-IgG. Splenocytes were stimulated for 5 hours with 30 nM PMA and 1 ⁇ M ionomycin. Splenocytes were double stained with rat anti-mouse CXCR6 (R&D, Minneapolis, MN) and rat anti- mouse CD4-FITC antibodies (BD Pharmingen, BD Biosciences).
  • Splenocytes were then subjected to intracellular staining of IL-4 and IFN- ⁇ using anti-mouse IL-4 and anti-mouse IFN- ⁇ (Pharmingen, BD Immunocytometry Systems) according to the protocol previously, described [Goldberg et al., J Immunol (2004) 173:6465-6471]. Results were analyzed by FACS.
  • Figures 10A-D show a significantly reduced number of IFN- ⁇ hlgh IL-4 low producing T cells in cultures supplemented with CXCRo-IgG. Results indicate that the number of IFN- ⁇ hlgh IL-4 low producing T cells decreased from 14.5 % in splenocytes treated by MOGp35-55 alone ( Figure 10A) to 4.5 % in cultures supplemented with CXCR6-IgG ( Figure 10C). Similar results were documented for CXCR6 expressing T cells.

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Abstract

Soluble molecules are provided. Thus, for example, provided is a soluble polypeptide comprising a heterologous amino acid sequence conjugated to a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCL16, and wherein the molecule is non-immunogenic in a human subject. Use of such molecules in the treatment of inflammatory diseases in general and Multiple Sclerosis in particular is also envisaged.

Description

COMPOSITIONS AND METHODS FOR TREATING CXCR6/CXCL16
ASSOCIATED DISEASES
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to novel molecules and, more particularly, to methods of using same for treating CXCR6/CXCL16 associated diseases, such as autoimmune inflammatory diseases.
Chemokines are small (-8-14 kDa), structurally cytokine-like, secreted proteins that regulate cell trafficking. They are produced and secreted by a wide variety of cell types in response to early inflammatory mediators, such as IL- lβ or TNF-α, and in response to bacterial or viral infection. Chemokines function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils and other effector cells from the blood to sites of infection or damage. They can be released by many different cell types (e.g. macrophages) and can mediate a range of pro-inflammatory effects on leukocytes, such as triggering of chemotaxis, degranulation, synthesis of lipid mediators, and integrin activation.
Chemokines can be subdivided into four classes, the C-C, C-X-C, C and C— X3-C chemokines, depending on the location of the first two cysteines in their protein sequence. The interaction of these soluble proteins with their specific receptors, which belong to the superfamily of seven-transmembrane domain G-protein-coupled receptors (GPCRs), mediate their biological effects resulting in, among other responses, rapid increase in intracellular calcium concentration, changes in cell shape, increased expression of cellular adhesion molecules, degranulation and promotion of cell migration.
In the last several years, the key role of chemokines as important mediators in inflammatory and autoimmune disorders and diseases has been well established. Chemokines have been indicated as important mediators in multiple sclerosis (MS), allergic responses, asthma, atherosclerosis, glomerulonephritis, pancreatitis, restenosis, rheumatoid arthritis (RA), diabetic nephropathy, pulmonary fibrosis, transplant rejection and in cancer.
The CXC Chemokine Receptor 6 (CXCR6, NM 006564), which former nomenclature included STRL33, BONZO, and TYMSTR, is expressed on T-cells, including T helper, T-cytotoxic and natural killer T (NKT) cells [Aust et al., Eur J Endocrinol (2005) 152(4):635-43; Matloubian et al., Nat Immunol (2000) 1(4):298-
304]. CXCR6 binds specifically to its chemokine ligand CXCL 16 [Wilbanks et al., J Immunol (2001) 166(8):5145-54]. CXCL16 is produced by several cells, including dendritic cells [Matloubian et al. supra] and macrophages [van der Voort et al., Arthritis Rheum (2005) 52(5):1381-91], following induction by the inflammatory cytokines IFN-γ and TNF-α [Abel et al., J Immunol (2004) 172(10):6362-72]. CXCL 16 is an unusually large chemokine (254 amino acids) which is composed of a CXC chemokine domain, a mucin-like stalk, a transmembrane domain and a cytoplasmic tail containing a potential tyrosine phosphorylation site. AU of these are unusual features for a chemokine which allow CXCLl 6 to be a soluble chemokine or to be expressed as a cell surface bound molecule. Surface-expressed CXCL 16 functions as an adhesion molecule by binding to its receptor CXCR6 expressed on activated T-cells and bone marrow plasma cells [Nakayama et al., J Immunol (2003) 170:1136-1 140; Shimaoka et al., J Leukoc Biol (2004) 75:267-274]. However, CXCL 16 also exists as a soluble molecule, inducing chemotaxis of CXCR6 expressing lymphocytes, including T helper, T-cytotoxic and NKT cells [Matloubian et al. supra; Wilbanks et al. supra; Nakayama et al. supra].
Recent studies have demonstrated the significance of CXCR6/CXCL16 axis in inflammatory diseases such as Rheumatoid Arthritis (RA). Ruth et al. have demonstrated elevated levels of CXCL 16 in RA synovial fluid (SF) samples [Ruth et al., Arthritis Rheum. (2006) 54(3): 765-78] while van der Voort et al. provided evidence that enhanced production of CXCL 16 in RA synovia leads to recruitment of CXCR6 expressing memory T cells, thereby contributing to the inflammatory cascade associated with RA pathology [van der Voort et al. supra]. Taken together, these results point to the potential therapeutic value of targeting CXCL 16, its receptor CXCR6 or a down-stream signaling pathways of inflammatory diseases. However, neither publication addressed a direct connection between CXCR6 and/or CXCL 16 and Multiple Sclerosis (MS), even more so, a therapeutic connection between MS and the CXCR6/CXCL16 axis has not been achieved. Various approaches for blocking CXCR6/CXCL16 activation have been attempted, some are summarized infra.
U.S. Pat. No. 7,038,018 discloses CXCR6 (Bonzo) chemokine receptor- specific monoclonal antibodies (mAb) and ligands. CXCR6 monoclonal antibodies compete for receptor binding thereby blocking natural responses by interfering with ligand-receptor interactions. The contemplated uses of these antibodies include treating chronic inflammatory diseases, such as multiple sclerosis. Furthermore, the inventors have disclosed a CXCR6 ligand [e.g., Spleen Extracted Chemokine (SExCkine)] which could potentially be administered to recruit killer T cells (expressing CXCR6) to solid tumors or sites of infection. This invention has the disadvantages of using mAb: 1) they may induce anti idiotypic responses in the host; 2) they compete for receptor binding yet they do not neutralize the ligands which may still transmit activating signals. Furthermore, the use of CXCR6 ligand does not block CXCR6 activity but rather enhances it. The inventors of this application teach a full size CXCR6 protein, containing an N-terminal Hemagglutinin (HA) epitope, and contemplate the use of CXCR6 protein functional variants, such as those having deletions (N-terminal, C-terminal or internal deletions) which retain their binding activity, a signaling activity and/or ability to stimulate a cellular response, yet such proteins have not been disclosed, probably since the ligand binding site of CXCR6 has not been discovered.
PCT Publication No. WO04019046 discloses a human CXCR6 fusion protein and CXCR6 specific monoclonal antibodies. The use of such antibodies was contemplated for diagnostics and therapeutics of several diseases including hematological diseases, cardiovascular diseases, disorders of the peripheral and central nervous system, genitourinary diseases, cancer and respiratory diseases. This invention has the disadvantages of using mAb as described hereinabove. The fusion protein disclosed in this application is a full-length CXCR6 protein. Although PCT Publication No. WO04019046 contemplates the possibility of creating a CXCR6 soluble fusion protein, it in fact teaches away from soluble chemokine receptors by specifically mentioning that transmembrane (TM) domains of such receptors are crucial for ligand binding. As described in details in PCT Publication No. WO04019046, the ligand binding sites of some G-protein coupled receptors (GPCRs), possibly including CXCR6, are believed to comprise hydrophilic sockets formed by several GPCR transmembrane domains (including TM3, TM5, TM6, TM6 and TM7).
PCT Publication No. WO03066830 discloses monoclonal antibodies against membrane proteins including CXCR6. This invention enables generation of high affinity monoclonal antibodies against peptide fragments of chemokine receptors by using fusion proteins as an immunogen that contains a peptide fragment derived from the membrane protein. Administering these antibodies to a host may offer a wide variety of therapeutic and diagnostic applications, including treatment of cancer and other diseases associated with abnormal chemokine activities. This invention has the disadvantages of using mAb as described hereinabove. Extracellular fragments of the CXCR6 receptor are contemplated for antibody production but the N terminal domain is not mentioned, again, probably due to the fact that it was not recognized essential for ligand binding.
PCT Publication No. WO05049799 discloses chimeric chemokine receptors. This invention teaches chimeric chemokine receptors comprising N terminal and TM region of a first chemokine receptor, such as CCR3, and the intracellular C terminus of a second chemokine receptor, such as CCR2. The first or second chemokine receptors may comprise CXCR6 domains. PCT Publication No. WO05049799 teaches away soluble receptors by specifically mentioning that GPCRs retain all of their known ligand binding regions within the extracellular regions and TM domains 2 through 7.
U.S. Publication No. 20060257359 discloses means of modulating phenotypes of macrophage related cells for the treatment of diseases, such as multiple sclerosis. Modulating the cellular phenotype is accomplished by introducing to macrophage related cells effectors, such as a protein, an antibody or a RNA molecule (e.g., a short interfering RNA), thereby altering gene expression and cell phenotype (e.g., secretion of cytokines or cell migration). Specifically mentioned are, CXCR6 and CXCR6 ligand, thus in fact teaching away from down-regulating this pathway for treating MS.
There is thus a widely recognized need and it would be highly advantageous to have therapeutic modalities which target the CXCR6/CXCL16 axis and which can be used in the treatment of a myriad of inflammatory and autoimmune diseases which pathogenicity involves these proteins.
SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided an isolated soluble polypeptide comprising a heterologous amino acid sequence conjugated to a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCL 16, and wherein the molecule is non-immunogenic in a human subject.
According to another aspect of the present invention there is provided an isolated soluble polypeptide comprising an amino acid sequence which comprises an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCLl 6.
According to yet another aspect of the present invention there is provided an isolated soluble polypeptide comprising at least two CXCR6 amino acid sequences each being capable of binding CXCL 16. According to still another aspect of the present invention there is provided an isolated soluble polypeptide comprising a tag attached to a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCL 16.
According to an additional aspect of the present invention there is provided an isolated soluble polypeptide comprising an amino acid sequence as set forth in SEQ ID NO. 4 or 8.
According to yet an additional aspect of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding any of the soluble polypeptides. According to still an additional aspect of the present invention there is provided a pharmaceutical composition comprising the soluble polypeptide and a pharmaceutically acceptable carrier.
According to a further aspect of the present invention there is provided a method of treating a CXCR6/CXCL16 associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the molecule, thereby treating the CXCR6/CXCL16 associated disease in the subject.
According to yet a further aspect of the present invention there is provided a use of the soluble polypeptide, for the manufacture of a medicament identified for treating a CXCR6/CXCL16 associated disease.
According to still a further aspect of the present invention there is provided a method of isolating CXCR6 ligand from a biological sample, the method comprising: (a) contacting the biological sample with the soluble polypeptide such that the CXCR6 ligand and the soluble polypeptide form a complex; and (b) isolating the complex to thereby isolate the CXCR6 ligand from the biological sample.
According to further features in preferred embodiments of the invention described below, the isolated soluble polypeptide is attached to a non-proteinaceous moiety.
According to still further features in the described preferred embodiments the soluble polypeptide is non-immunogenic in a human subject.
According to still further features in the described preferred embodiments the binding affinity of the CXCR6 to the CXCL 16 ligand is above K0=IO"6 M. According to still further features in the described preferred embodiments the heterologous amino acid sequence comprises an immunoglobulin amino acid sequence.
According to still further features in the described preferred embodiments the CXCR6 amino acid sequence is as set forth in SEQ ID NO: 4. According to still further features in the described preferred embodiments the
CXCR6 amino acid sequence is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 3 or 7.
According to still further features in the described preferred embodiments the CXCR6 amino acid sequence is no longer than 50 amino acids in length. According to still further features in the described preferred embodiments the
CXCR6/CXCL16 associated disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, hematological diseases, cardiovascular diseases, disorders of the peripheral and central nervous system, genitourinary diseases, respiratory diseases and cancer. According to still further features in the described preferred embodiments the tag is an epitope tag.
According to still further features in the described preferred embodiments the soluble polypeptide is attached to a solid support.
According to still further features in the described preferred embodiments the non-proteinaceous moiety is selected from the group consisting of polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA). According to still a further aspect of the present invention there is provided a method of treating Multiple Sclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent capable of reducing activity and/or expression of CXCR6/CXCL16 or of an effector thereof, thereby treating Multiple Sclerosis in the subject.
According to still a further aspect of the present invention there is provided the use of an agent capable of reducing activity and/or expression of CXCR6/CXCL16 or of an effector thereof, for the manufacture of a medicament identified for treating Multiple Sclerosis. According to still a further aspect of the present invention there is provided a method of diagnosing Multiple Sclerosis in a subject, the method comprising, detecting expression and/or activity of CXCR6 in cells of the subject, wherein expression and/or activity of the CXCR6 in the cells of the subject above a predetermined threshold is indicative of Multiple Sclerosis in the subject. According to still further features in the described preferred embodiments detecting expression of the CXCR6 is effected by detecting CXCR6 protein.
According to still further features in the described preferred embodiments detecting expression of the CXCR6 protein is effected via an assay selected from the group consisting of immunohistochemistry, ELISA, RIA, Western blot analysis, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.
According to still further features in the described preferred embodiments detecting expression of the CXCR6 is effected by detecting CXCR6 mRNA.
According to still further features in the described preferred embodiments detecting expression of the CXCR6 mRNA is effected via an assay selected from the group consisting of PCR, RT-PCR, chip hybridization, RNase protection, in-situ hybridization, primer extension, Northern blot and dot blot analysis.
According to a further aspect of the present invention there is provided a pharmaceutical composition comprising as an active ingredient an anti-Multiple
Sclerosis drug and an agent capable of reducing activity and/or expression of CXCR6/CXCL16 and/or of an effector thereof and a pharmaceutically acceptable carrier.
According to still further features in the described preferred embodiments the agent is selected from the group consisting of: (i) an oligonucleotide directed to an endogenous nucleic acid sequence expressing the CXCR6/CXCL16 or the effector thereof; (ii) a chemical inhibitor directed to the CXCR6 or the effector thereof; (iii) a neutralizing antibody directed at CXCR6/CXCL16 or the effector thereof; and (iv) a non-functional derivative of the CXCR6 or the effector thereof. According to still further features in the described preferred embodiments the non-functional derivative of the CXCR6/CXCL16 comprises any of the soluble polypeptides.
According to still further features in the described preferred embodiments the anti-Multiple Sclerosis drug is selected from the group consisting of Interferon Beta Ia, Interferon Beta Ib, Glatiramer Acetate, Mitoxantrone, MethylPrednisolone,
Prednisone, Prednisolone, Dexamethasone, Adreno-corticotrophic Hormone (ACTH) and Corticotropin.
The present invention successfully addresses the shortcomings of the presently known configurations by providing novel molecules and methods of using same for treating CXCR6/CXCL16 associated diseases, such as autoimmune inflammatory diseases.
As used herein, the terms "comprising" and "including" or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms
"consisting of and "consisting essentially of.
The phrase "consisting essentially of or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings:
FIG. 1 is a photograph depicting the presence of CXCR6 mRNA in spinal cords of EAE induced mice. cDNA from the spinal cord of EAE induced mouse (day 18, middle lane) or from a naϊve mouse (left lane) was amplified by PCR using specific primers for the entire murine CXCR6 gene coding region. A water sample was amplified as a negative control (right lane). GADPH was used as a positive control (bottom panel).
FIGs. 2A-C are FACS analysis results depicting CXCR6 expressing T helper cells in spinal cords of EAE induced mice at the peak of disease. Figure 2A shows spleen cells of an EAE induced mouse; Figure 2B shows cervical lymph node (LN) cells of an EAE induced mouse; and Figure 2C shows spinal cord (SC) cells of an EAE induced mouse. Results were read and analyzed by FACS.
FIGs. 3A-B are schematic illustrations depicting the generation of the expression construct encoding the murine CXCRo-IgG peptide of the present invention (SEQ ID NO: 17). Figure 3 A shows insertion of the CXCR6 cDNA sequence encoding the extracellular N-terminal region of murine CXCR6 (SEQ ID NO: 13) to pSecTag vector along with a sequence encoding a part of murine IgGl heavy chain (SEQ ID NO: 5); Figure 3B shows the resulting chimeric protein of CXCR6 and IgGl. FIGs. 4A-B are images depicting purification of the soluble receptor CXCR6-
IgG from DHFR-positive DG44 transfectant supernatants by a Ni-NTA column. Figure 4A shows Western blots analysis with mouse anti-myc antibody; and Figure 4B shows Coomassie staining. For both Figures 4 A and 4B, lane no. 1 is of a size marker (kDa), lane no. 2 is of medium in which DG44-CXCR6IgG transfected cells were grown, lanes no. 3-7 are of five sequential elution fractions and lane no. 8 is of the flow through medium. Of note, the soluble receptor CXCRo-IgG is a 42Kd polypeptide.
FIG. 5 is a bar graph depicting the binding specificity of the murine CXCR6- IgG fusion protein to various commercially available murine chemokines. Specific binding was determined by direct ELISA. Where indicated, the soluble receptor CCR2-IgG was added instead of CXCRo-IgG. Results are shown as O.D. reading at 450 nm. Bars are average of triplicates. The data shown represent one of three experiments completed. Error bars: ± standard deviation. FIG. 6 is a graph depicting the ability of CXCRo-IgG to suppress ongoing
Experimental Autoimmune Encephalomyelitis (EAE) in mice. In brief, two groups of C57/B mice (six mice in each group) were subjected to active induction of EAE by MOGp35-55 (on day 0) and Pertussis toxin (on days 0 and 2). Beginning one day after the onset of disease (day 12), the mice were treated with repeated intravenous administrations (every other day) of 300 μg/mouse of either a CXCRo-IgG (indicated by diamonds) or isotype matched IgG (indicated by X). An observer blind to the experimental procedure scored EAE daily for clinical manifestation of disease. Error bars indicates standard error. The data are representative of three independent experiments. FIGs. 7A-F are images depicting histochemistry of spinal cords of naive mice and EAE induced mice left untreated or treated with the composition of the present invention. Figures 7A-B show the lumbar spinal cords of a naϊve mouse (xlOO and x400 magnifications, respectively); Figures 7C-D show the lumbar spinal cords of an EAE induced mouse that was treated with control isotype matched (IgGl) antibody (xlOO and x400 magnifications, respectively); and Figures 7E-F show an EAE induced mouse that was treated with the soluble receptor CXCRo-IgG (xlOO and x400 magnifications, respectively). Spinal cords were removed 22 days after MOG immunization. The stained slides were pictured under an Olympus microscope. FIGs. 8A-D are bar graphs depicting ex- vivo cytokine secretion by EAE derived splenocytes treated by CXCRo-IgG. In brief, three groups of C57/B mice (three mice in each group) were subjected to active induction of EAE by MOGp35- 55. Mice were treated with the soluble receptor CXCR6, an isotype matched IgG (mlgG) or PBS (control). On day 9 the splenocytes were harvested and restimulated for 72 hours with 50 μg/ml MOGp35-55 (solid bars) or cell medium (striped bars). The supernatants were analyzed by ELISA for cytokine production. Figure 8A is a graph showing IL-2 secretion; Figure 8B is a graph showing IFN-γ secretion; Figure 8C is a graph showing TNF-α secretion; and Figure 8D is a graph showing IL- 12 secretion. Bars indicate average of triplicate wells. Error bars represent ±SD.
FIGs. 9A-B are histograms depicting the macrophage levels in CXCR6 treated mice. In brief, two groups of C57/B mice (three mice in each group) were subjected to active induction of EAE by MOGp35-55. Figure 9A shows mice that were not treated (control); and Figure 9B shows mice that were treated with the soluble receptor CXCR6. On day 16 the splenocytes were harvested and stained anti-CD 1 Ib-FITC and analyzed by FACS. The percentage of CDl Ib positive cells is indicated above the peak of each appropriate histogram.
FIGs. 10A-D are FACS analysis results depicting a decrease in IFNγ producing cells in vitro. Splenocytes from C57/B mice were harvested, washed and incubated with MOGp35-55 alone (Figures 10A-B) or MOGp35-55 and CXCRoIgG (Figures 1 OC-D). Cells were stimulated with 30 nM PMA and 1 μM ionomycin for 5 hours and stained with anti CD4-PERCP and anti CXCR6-PE. After permeabilization, the cells were stained intracellularly with anti-IL-4-APC and anti IFNγ-FITC. Figures 1OA and 1OC show analysis of total lymphocytes; and Figures 1OB and 1OD show analysis of CXCR6+ gated lymphocytes.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is of compositions of down-regulating signaling through CXCLl 6 and uses of same for treating a myriad of medical conditions such as Multiple Sclerosis.
The principles and operation of the method according to the present invention may be better understood with reference to the drawings and accompanying descriptions. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS), characterized by various symptoms of neurological dysfunction. MS and its animal model, experimental autoimmune encephalomyelitis (EAE), are believed to result from autoimmune mediated activated immune cells, such as T- and B-lymphocytes as well as macrophages and microglia, and is considered to be an inflammatory neurodegenerative disease. Pathologically, MS is characterized by perivenous infiltration of lymphocytes and macrophages into the CNS parenchyma, resulting in demyelinative lesions termed plaques. These plaques, which are the hallmark of MS, are associated with oligodendrocytes death, axonal damage and neuronal loss. The etiology of MS has not yet been fully elucidated and it is attributed to both genetic and environmental causes, yet factors which regulate leukocyte entry into the CNS may play a role in MS development as well as in lesion pathogenesis. While reducing the present invention to practice, the present inventors have discovered that the CXCR6/CXCL16 axis is directly involved in MS pathogenesis and therefore, the present inventors envision therapeutics of MS by suppressing signaling through the CXCR6/CXCL16 pathway.
While further reducing the present invention to practice, the present inventors have surprisingly discovered that the N-terminus domain of CXCR6 (N-ter, amino acid coordinates 1-32 of GenBank Accession No. NP_006555; SEQ ID NO: 4) is sufficient for binding CXCL 16. This finding is crucial for the generation of new therapeutic tools for treating diseases which involve the CXCR6/CXCL16 axis such as MS. As is illustrated herein below and the Examples section which follows, the present inventors have demonstrated that CXCR6 is selectively expressed by T helper (Th) cells in spinal cords of E AE- induced mice (Examples 1-2 of the Examples section which follows), suggesting that CXCR6 may be involved in MS pathogenesis. To test this, the present inventors have constructed soluble CXCR6 fusion polypeptides and expressed them in mammalian cell systems (see Example 3 of the Examples section which follows). Functionality of the fusion was shown by binding of CXCRo-IgG to CXCL 16 as was demonstrated in Example 4. The fusion polypeptide was proven therapeutic for the treatment of MS as was manifested by suppression of ongoing encephalomyelitis (EAE) in vivo (see Examples 5 and 6) and ex vivo by suppression of pro-inflammatory cytokine production by splenocytes (see Example 7). Additionally, the present results illustrate that CXCRo-IgG treatment leads to a decrease in IFN-gamma producing cells and does not cause depletion in Macrophages (see Examples 8-9).
Altogether the present findings portray a pathogenic role for the CXCR6/CXCL16 axis in MS development and suggest that down regulation of this pathway may be used for the treatment of same.
Thus, according to one aspect of the present invention there is provided a method of treating MS in a subject in need thereof. The method comprising administering to the subject a therapeutically effective amount of an agent capable of reducing activity and/or expression of CXCR6/CXCL16 or of an effector thereof, thereby treating MS in the subject.
As used herein the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of Multiple Sclerosis.
As used herein the phrase "Multiple Sclerosis" refers to the inflammatory, demyelinating disease of the central nervous system (CNS) which is typically characterized by various symptoms of neurological dysfunction. Multiple sclerosis according to the present teachings refers to any type of multiple sclerosis (e.g., stage, severity) as outlined infra.
Relapsing-remitting - Relapsing-remitting describes the initial course of 85 % to 90 % of individuals with MS. This subtype is characterized by unpredictable attacks (relapses) followed by periods of months to years of relative quiet (remission) with no new signs of disease activity. Deficits suffered during the attacks may either resolve or may be permanent. When deficits always resolve between attacks, this is referred to as "benign" MS. Secondary progressive - Secondary progressive describes around 80 % of those with initial relapsing-remitting MS, who then begin to have neurological decline between their acute attacks without any definite periods of remission. This decline may include new neurological symptoms, worsening cognitive function, or other deficits. Secondary progressive is the most common type of MS and causes the greatest amount of disability.
Primary progressive - Primary progressive describes the approximately 10 % of individuals who never have remission after their initial MS symptoms. Decline occurs continuously without clear attacks. The primary progressive subtype tends to affect people who are older at disease onset.
Progressive relapsing - Progressive relapsing describes those individuals who, from the onset of their MS, have a steady neurological decline but also suffer superimposed attacks; and is the least common of all subtypes.
Special cases of the disease with non-standard behavior have also been described although many researchers believe they are different diseases. These cases are sometimes referred to as borderline forms of multiple sclerosis and are: Neuromyelitis optica (NMO), BaIo concentric sclerosis, Schilder disease, Marburg multiple sclerosis, acute disseminated encephalomyelitis (ADEM) and autoimmune variants of peripheral neuropathies. As used herein the phrase "a subject in need thereof refers to a mammal, preferably a human subject who has been diagnosed with probable or definite multiple sclerosis, e.g., a subject who experienced one neurological attack affecting the CNS and accompanied by demyelinating lesions on brain magnetic resonance imaging (MRJ). The neurological attack can involve acute or sub-acute neurological symptomatology (attack) manifested by various clinical presentations like unilateral loss of vision, vertigo, ataxia, incoordination, gait difficulties, sensory impairment characterized by paresthesia, dysesthesia, sensory loss, urinary disturbances until incontinence, diplopia, dysarthria, various degrees of motor weakness until paralysis, cognitive decline either as a monosymptomatic or in combination. The symptoms usually remain for several days to few weeks, and then partially or completely resolve.
The diagnosis MS can also include laboratory tests involving evaluation of IgG synthesis and oligoclonal bands (immunoglobulins found in 85-95 % of subjects diagnosed with definite MS) in the cerebrospinal fluid (CSF, obtained by e.g., lumbar puncture) which provide evidence of chronic inflammation of the central nervous system. Combined with MRI of the brain and spinal cord and clinical data, the presence of oligoclonal bands can help make a definite diagnosis of MS. As mentioned hereinabove, the method, according to this aspect of the present invention, is effected by administering to the subject a therapeutically effective amount of an agent capable of reducing activity and/or expression of CXCR6/CXCL16 or of an effector thereof, thereby treating MS in the subject.
As used herein the term "CXCR6" refers to a CXCR6 gene product (i.e., protein or mRNA) such as set forth in GenBank Accession Nos. NM 006564 or NP 006555.
As used herein CXCR6 activity refers to cell signaling activity (e.g., G protein signaling, NF-kappa B signaling), chemokine binding activity (CXCL 16), viral replication and/or co-receptor for SIM and HIV, cell adhesion, cell proliferation or chemotaxis.
As used herein CXCR6 expression refers to expression of the chemokine receptor CXCR6 either at the protein level or at the mRNA level. Typically, CXCR6 is expressed on T cells, such as without limitation, T helper, T-cytotoxic and NKT cells, as well as on bone marrow plasma cells. As used herein the term "CXCL 16" refers to CXCL 16 gene product (i.e., protein or mRNA) such as set forth in GenBank Accession Nos. NM 022059 or NP_071342.
As used herein CXCL 16 expression refers to expression of CXCL 16 chemokine either at the protein or mRNA level. CXCL 16 is expressed as a soluble chemokine or as a cell surface bound molecule, such as for example without limitation, on monocytes and dendritic cells.
As used herein an effector of CXCR6/CXCL16 refers to a molecule which mediates signaling via CXCR6. Thus, a CXCR6/CXCL16 effector can be, for example, heterotrimeric G proteins (e.g., Gi proteins, GenBank Accession Nos. NM 002069 and NP 002060), focal adhesion signaling components (e.g., PB-K [Chandrasekar et al., J Biol Chem. (2004) 279(5):3188-96]), down stream signaling components (e.g., kinases such as PDK-I GenBank Accession Nos. NP 002601 and NM_002610, and Akt GenBank Accession Nos. NM 001014431, NMJ)Ol 014432, or NM 005163) and NF-kappa B signaling pathway components (e.g., NF-kappa B
GenBank Accession Nos. NP 078804 and NM_024528 and I kappa B kinase GenBank Accession Nos. NP 001547.1 and NM 001556.1) [Chandrasekar et al., supra]. A number of agents can be used in accordance with this aspect of the present invention to reduce/down-regulate the activity or expression of CXCR6/CXCL16 or an effector thereof. Assays for qualifying such agents will be apparent to those of skill in the art. Thus, any in vitro or in vivo activity assay (e.g., signaling, reduction in EAE score) can be used such as described in the Examples section which follows. Thus, for example the agent can be a non-functional derivative of CXCR6 or an effector thereof.
As used herein the phrase "non-functional derivative of CXCR6" refers to a CXCR6 amino acid sequence which competes with native CXCR6 (e.g., on ligand or effector molecule binding) but is designed so as to block signaling from same. A non- functional derivative is also referred to in the art of signaling as a "dominant negative molecule".
Thus, a non-functional derivative of CXCR6, for example, is the N-terminal domain (amino acid coordinates 1-32 of GenBank Accession No. NP 006555, SEQ ID NOs: 3 and 4) of CXCR6 being capable of binding a ligand (e.g., CXCL 16) without transmitting a signal. As is explicated in the Examples section which follows, the present inventors have uncovered that the N-terminal domain of CXCR6 is sufficient to bind CXCL 16 (see Example 4).
Thus, the present invention provides for an isolated soluble polypeptide comprising a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCL 16, and wherein the molecule is non-immunogenic in a human subject (for maximizing therapeutic efficacy).
As used herein the term "non-immunogenic" refers a substance which is substantially incapable of producing an immune response in a subject administered therewith. For example, non-immunogenic in a human means that upon contacting the molecule of this aspect of the present invention with the appropriate tissue of a human, no state of sensitivity or resistance to the molecule is demonstrable upon the second administration of the molecule after an appropriate latent period (e.g., 8 to 14 days). Such molecules are preferably devoid of other CXCR6 extracellular domains which are not necessary for ligand binding. Such domains are listed infra.
Table 1
Key r From To Length I Description
CHAIN I 1 342 342 I C-X-C chemokine receptor type 6.
TRANSMEM I 33 59 27 \ 1 (Potential).
TOPO DOM I 60 68 9 I Cytoplasmic (Potential).
TRANSMEM 69 89 21 I 2 (Potential).
TOPO DOM 90 103 I 14 Extracellular (Potential).
TRANSMEM 104 125 I 22 3 (Potential).
TOPO DOM 126 143 I 18 Cytoplasmic (Potential).
TRANSMEM 144 164 ] 21 4 (Potential).
TOPO DOM 165 187 I 23 Extracellular (Potential).
TRANSMEM 188 215 I 28 5 (Potential).
TOPO DOM 216 231 I 16 Cytoplasmic (Potential).
TRANSMEM 232 259 I 28 6 (Potential).
TOPO DOM 260 275 ) 16 Extracellular (Potential).
TRANSMEM 276 293 I 18 7 (Potential).
Although a soluble N-terminal sequence of CXCR6 (as described above) may be used per se, it may be modified to improve its availability and pharmacokinetics. Such modifications are provided below. As used herein the term "soluble" refers to the ability of the molecules of the present invention to dissolve in a physiological aqueous solution (pH about 7, e.g., solubility level in aqueous media of >100 μg/ml) without substantial aggregation. Thus, it is readily understood that soluble CXCR6 are preferably devoid of any hydrophobic transmembrane domains (listed in Table 1 above). As used herein the phrase "CXCR6 amino acid sequence" refers to a peptide portion of a mammalian (e.g., human) chemokine C-X-C receptor 6 protein having binding affinity for CXCR6 ligands (e.g., CXCL 16). Binding affinity refers to a minimal KD value of at least 10"6 M., 10"7 M, lO"8 M, 10"9 M, 10'10 M. Methods of assaying ligands for qualified affinity are well known in the art and include Scatchard plotting. It should be noted that a single CXCR6 amino acid sequence may be included in the molecules of the present invention, but inclusion of at least two CXCR6 amino acid sequences (e.g., of similar affinity), each being capable of binding CXCR6 ligand (preferably with high affinity) may be preferred. Due to increased avidity, these polypeptides may be used as potent inhibitors of CXCR6 ligand activity and lower dosages may be administered. An example of a CXCR6 amino acid sequence which can be used in accordance with the teachings of the present invention is set forth in SEQ ID NO: 4 or 8. Such CXCR6 amino acid sequences may be encoded by a nucleic acid sequences as set forth in SEQ ID NO. 3 or 7. Due to the soluble nature of the molecules of the present invention, it would be preferred that the CXCR6 amino acid sequence is no longer than 50 amino acids in length.
The CXCR6 amino acid sequence of the present invention comprises the N terminus (N-ter) part of the receptor. As shown in Example 4 of the Examples section which follows, binding of CXCR6 to its ligand CXCL 16 is mediated by the N-ter domain of CXCR6.
The term "polypeptide" as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides, or recombinant peptides), peptidomimetics (typically, synthetically synthesized peptides), and the peptide analogues peptoids and semipeptoids, and may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to: N-terminus modifications; C-terminus modifications; peptide bond modifications, including but not limited to CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH, and CF=CH; backbone modifications; and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Ramsden, C. A., ed. (1992), Quantitative Drug Design, Chapter 17.2, F. Choplin Pergamon Press, which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinbelow. Peptide bonds (-C0-NH-) within the peptide may be substituted, for example, by N-methylated bonds (-N(CH3)-CO-); ester bonds (-C(R)H-C-O-O-C(R)-N-); ketomethylene bonds (-CO-CH2-); α-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl group, e.g., methyl; carba bonds (-CH2-NH-); hydroxyethylene bonds (- CH(0H)-CH2-); thioamide bonds (-CS-NH-); olefinic double bonds (-CH=CH-); retro amide bonds (-NH-C0-); and peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time. Natural aromatic amino acids, Tip, Tyr, and Phe, may be substituted for synthetic non-natural acids such as, for instance, tetrahydroisoquinoline-3-carboxylic acid (TIC), naphthylelanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe, and o-methyl-Tyr. In addition to the above, the polypeptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g., fatty acids, complex carbohydrates, etc.).
The term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other less common amino acids, including but not limited to 2- aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine, and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.
Tables 2 and 3 below list naturally occurring amino acids (Table 2) and non- conventional or modified amino acids (Table 3) which can be used with the present invention.
Table 2
Figure imgf000020_0001
Table 3
Figure imgf000020_0002
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Table 3 Cont.
Generation of peptide mimetics (e.g., which comprise a CXCR6 amino acid sequence with various natural and/or synthetic alterations but which still display dominant negative activity) , as described hereinabove, can be effected using various approaches, including, for example, display techniques.
Thus, the present invention contemplates a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 5, at least 7, at least 11, at least 15, at least 20, at least 25 consecutive amino acids derived from polypeptide sequences of the N-ter domain of CXCR6 (e.g., SEQ ID NO: 4).
Peptide mimetics can also be uncovered using computational biology.
As mentioned, the soluble polypeptides of the present invention preferably includes a heterologous amino acid sequence
As used herein the phrase "heterologous amino acid sequence" refers to a non- immunogenic amino acid sequence which does not form a part of the CXCR6 amino acid sequence. This sequence preferably confers solubility to the molecule of this embodiment of the present invention, preferably increasing the half-life of the chimeric molecule in the serum.
The heterologous amino acid sequence is generally localized at the amino- or carboxyl- terminus of the CXCR6 peptide of the present invention.
As mentioned, the at least one heterologous amino acid sequence can be conjugated to the CXCR6 amino acid sequence of the present invention. For example, the at least one CXCR6 amino acid sequence may be embedded between two heterologous sequences, such as described Hoogenboom (1991) MoI. Immunol. 28: 1027-1037. The heterologous amino acid sequence may be attached to the CXCR6 amino acid sequence by any of peptide or non-peptide bond. Attachment of the CXCR6 amino acid sequence to the heterologous amino acid sequence may be effected by direct covalent bonding (peptide bond or a substituted peptide bond) or indirect binding such as by the use of a linker having functional groups. Functional groups include, without limitation, a free carboxylic acid (C(=O)OH), a free amino group (NH2), an ester group (C(=O)OR, where R is alkyl, cycloalkyl or aryl), an acyl halide group (C(=O)A, where A is fluoride, chloride, bromide or iodide), a halide
(fluoride, chloride, bromide or iodide), a hydroxyl group (OH), a thiol group (SH), a nitrile group (C≡N), a free C-carbamic group (NR"-C(=O)-OR', where each of R' and
R" is independently hydrogen, alkyl, cycloalkyl or aryl).
An example of a heterologous amino acid sequence which may be used in accordance with this aspect of the present invention is an immunoglobulin amino acid sequence, such as the hinge and Fc regions of an immunoglobulin heavy domain (see U.S. Pat. No. 6,777,196). The immunoglobulin moiety in the chimeras of this aspect of the present invention may be obtained from IgGl, IgG2, IgG3 or IgG4 subtypes,
IgA, IgE, IgD or IgM, as further discussed hereinbelow.
Chimeras constructed from a receptor sequence linked to an appropriate immunoglobulin constant domain sequence (immunoadhesins) are known in the art. Immunoadhesins reported in the literature include fusions of the T cell receptor
[Gascoigne et al., Proc. Natl. Acad. Sci. USA, 84: 2936-2940 (1987)]; CD4 [Capon et al., Nature 337: 525-531 (1989); Traunecker et al., Nature, 339: 68-70 (1989);
Zettmeissl et al., DNA Cell Biol. USA, 9: 347-353 (1990); Byrn et al., Nature, 344:
667-670 (1990)]; L-selectin (homing receptor) [(Watson et al., J. Cell. Biol., 110:2221-2229 (1990); Watson et al., Nature, 349: 164-167 (1991)]; CD44 [Aruffo et al., Cell, 61: 1303-1313 (1990)]; CD28 and B7 (Linsley et al., J. Exp. Med., 173: 721-
730(1991)]; CTLA-4 [Lisley et al., J. Exp. Med. 174: 561-569 (1991)]; CD22
[Stamenkovic et al., Cell, 66:1133-1144 (1991)]; TNF receptor [Ashkenazi et al., Proc.
Natl. Acad. Sci. USA, 88: 10535-10539 (1991); Lesslauer et al., Eur. J. Immunol., 27: 2883-2886 (1991); Peppel et al., J. Exp. Med., 174:1483-1489 (1991)]; NP receptors
[Bennett et al., J. Biol. Chem. 266:23060-23067 (1991)]; and IgE receptor α [Ridgway et al., J. Cell. Biol., 1 15: abstr. 1448 (1991)].
Typically, in such fusions the chimeric molecule will retain at least functionally active hinge and CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions can also be generated to the C-terminus of the
Fc portion of a constant domain, or immediately N-terminal to the CHl of the heavy chain or the corresponding region of the light chain. The exact site at which fusion (conjugation) between the heterologous sequence and the CXCR6 amino acid sequence is not critical. Particular sites are well known in the art and may be selected in order to optimize the biological activity, secretion or binding characteristics of the chimeric molecules of this aspect of the present invention (see Example 3 of the Example section which follows).
Though it may be possible to conjugate the entire heavy chain constant region to the CXCR6 amino acid sequence of the present invention, it is preferable to fuse shorter sequences. For example, a sequence beginning in the hinge region just upstream of the papain cleavage site, which defines IgG Fc chemically; residue 216, taking the first residue of heavy chain constant region to be 114, or analogous sites of other immunoglobulins, is used in the fusion. In a particularly preferred embodiment, the CXCR6 amino acid sequence is fused to the hinge region and CH2 and CH3, or to the CHl, hinge, CH2 and CH3 domains of an IgGl, IgG2, or IgG3 heavy chain (see U.S. Pat. No. 6,777,196). The precise site at which the fusion is made is not critical, and the optimal site can be determined by routine experimentation.
As mentioned, the immunoglobulin sequences used in the construction of the chimeric molecules of this aspect of the present invention may be from an IgG immunoglobulin heavy chain constant domain. The use of human IgGl immunoglobulin sequences is preferred (e.g., as set forth in SEQ ID NOs. 5 and 6). A major advantage of using IgGl is that IgGl can be purified efficiently on immobilized protein A. However, other structural and functional properties of immunoglobulins should be considered when choosing the Ig fusion partner for a particular chimera construction. For example, the IgG3 hinge is longer and more flexible, so it can accommodate larger CXCR6 amino acid sequences that may not fold or function properly when fused to IgGl. Another consideration may be valency; IgG are bivalent homodimers, whereas Ig subtypes like IgA and IgM may give rise to dimeric or pentameric structures, respectively, of the basic Ig homodimer unit. Other considerations in selecting the immunoglobulin portion of the chimeric molecules of this aspect of the present invention are described in U.S. Pat. No. 6, 77,196. Further examples of heterologous amino acid sequences commonly used in fusion protein construction include, but are not limited to galactosidase, glucuronidase, glutathione-S-transferase (GST), carboxy terminal peptide (CTP) from chorionic gonadotrophin (CGβ) and chloramphenicol acetyltransferase (CAT). According to a preferred embodiment of this aspect of the present invention, the isolated soluble molecule of this aspect of the present invention is as set forth in SEQ ID NO: 8.
The isolated soluble molecule of this aspect of the present invention is encoded by a nucleic acid sequences as set forth in SEQ ID NO: 7.
Thus, molecules of this aspect of the present invention may comprise heterologous amino acid sequences, as described above.
Additionally or alternatively as mentioned hereinabove CXCR6 amino acid sequences of the present invention may be attached to a non-proteinaceous moiety, such molecules are preferably selected non-immunogenic in a subject.
Thus, according to a preferred embodiment of this aspect of the present invention, there is provided an isolated soluble molecule comprising a CXCR6 amino acid sequence (as described above) attached to a non-proteinaceous moiety.
Such a molecule is highly stable (resistant to in-vivo proteaolytic activity probably due to steric hindrance conferred by the non-proteinaceous moiety) and may be produced using common solid phase synthesis methods which are inexpensive and highly efficient, as further described hereinbelow. However, it will be appreciated that recombinant techniques may still be used, whereby the recombinant peptide product is subjected to in-vitro modification (e.g., PEGylation as further described hereinbelow).
The phrase "non-proteinaceous moiety" as used herein refers to a molecule not including peptide bonded amino acids that is attached to the above-described CXCR6 amino acid sequence. According to presently preferred embodiments the non- proteinaceous moiety of this aspect of the present invention is a polymer or a co- polymer (synthetic or natural). Non-limiting examples of the non-proteinaceous moiety of the present invention include polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), divinyl ether and maleic anhydride copolymer (DIVEMA; see for example, Kaneda Y, et al., 1997, Biochem. Biophys. Res. Commun. 239: 160-5) and poly(styrene comaleic anhydride) (SMA; see for example, Mu Y, et al., 1999, Biochem Biophys Res Commun. 255: 75-9).
It will be appreciated that such non-proteinaceous moieties may be also attached to the above mentioned fusion molecules (i.e., which comprise a heterologous amino acid sequence) to promote stability and possibly solubility of the molecules.
Bioconjugation of such a non-proteinaceous moiety confers the CXCR6 amino acid sequence with stability (e.g., against protease activities) and/or solubility (e.g., within a biological fluid such as blood, digestive fluid) while preserving its biological activity and prolonging its half-life. Bioconjugation is advantageous particularly in cases of therapeutic proteins which exhibit short half-life and rapid clearance from the blood. The increased half-lives of bioconjugated proteins in the plasma results from increased size of protein conjugates (which limits their glomerular filtration) and decreased proteolysis due to polymer steric hindrance. Generally, the more polymer chains attached per peptide, the greater the extension of half-life. However, measures are taken not to reduce the specific activity of the CXCR6 amino acid sequence of the present invention (i.e., CXCR6 ligand binding).
Bioconjugation of the CXCR6 amino acid sequence with PEG (i.e., PEGylation) can be effected using PEG derivatives such as N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, monomethoxyPEG2-NHS, succinimidyl ester of carboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxy PEG-NPC), PEG aldehydes, PEG-orthopyridyl-disuMde, carbonyldimidazol- activated PEGs, PEG-thiol, PEG-maleimide. Such PEG derivatives are commercially available at various molecular weights [See, e.g., Catalog, Polyethylene Glycol and Derivatives, 2000 (Shearwater Polymers, Inc., Huntsvlle, Ala.)]. If desired, many of the above derivatives are available in a monofunctional monomethoxyPEG (mPEG) form. In general, the PEG added to the CXCR6 amino acid sequence of the present invention should range from a molecular weight (MW) of several hundred Daltons to about 100 kDa (e.g., between 3-30 kDa). Larger MW PEG may be used, but may result in some loss of yield of PEGylated peptides. The purity of larger PEG molecules should be also watched, as it may be difficult to obtain larger MW PEG of purity as high as that obtainable for lower MW PEG. It is preferable to use PEG of at least 85 % purity, and more preferably of at least 90 % purity, 95 % purity, or higher. PEGylation of molecules is further discussed in, e.g., Hermanson, Bioconjugate Techniques, Academic Press San Diego, Calif. (1996), at Chapter 15 and in Zalipsky et al., "Succinimidyl Carbonates of Polyethylene Glycol," in Dunn and Ottenbrite, eds., Polymeric Drugs and Drug Delivery Systems, American Chemical Society, Washington, D.C. (1991).
Conveniently, PEG can be attached to a chosen position in the CXCR6 amino acid sequence by site-specific mutagenesis as long as the activity of the conjugate is retained (i.e., CXCR6 ligand binding). A target for PEGylation could be any Cysteine residue at the N-terminus or the C-terminus of the CXCR6 amino acid sequence. Additionally or alternatively, other Cysteine residues can be added to the CXCR6 amino acid sequence (e.g., at the N-terminus or the C-terminus) to thereby serve as a target for PEGylation. Computational analysis may be effected to select a preferred position for mutagenesis without compromising the activity.
Various conjugation chemistries of activated PEG such as PEG-maleimide, PEG-vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide can be employed. Methods of preparing activated PEG molecules are known in the arts. For example, PEG-VS can be prepared under argon by reacting a dichloromethane (DCM) solution of the PEG-OH with NaH and then with di-vinylsulfone (molar ratios: OH 1: NaH 5: divinyl sulfone 50, at 0.2 gram PEG/mL DCM). PEG- AC is made under argon by reacting a DCM solution of the PEG-OH with acryloyl chloride and triethylamine (molar ratios: OH 1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM). Such chemical groups can be attached to linearized, 2-arm, 4-arm, or 8-arm PEG molecules.
While conjugation to cysteine residues is one convenient method by which the CXCR6 amino acid of the present invention can be PEGylated, other residues can also be used if desired. For example, acetic anhydride can be used to react with NH2 and SH groups, but not COOH, S-S, or -SCH3 groups, while hydrogen peroxide can be used to react with -SH and -SCH3 groups, but not NH2. Reactions can be conducted under conditions appropriate for conjugation to a desired residue in the peptide employing chemistries exploiting well-established reactivities.
For bioconjugation of the CXCR6 amino acid sequence of the present invention with PVP, the terminal COOH-bearing PVP is synthesized from N-vinyl-2- pyrrolidone by radical polymerization in dimethyl formamide with the aid of 4,4'- azobis-(4-cyano valeric acid) as a radical initiator, and 3-mercaptopropionic acid as a chain transfer agent. Resultant PVPs with an average molecular weight of Mr 6,000 can be separated and purified by high-performance liquid chromatography and the terminal COOH group of synthetic PVP is activated by the N- hydroxysuccinimide/dicyclohexyl carbodiimide method. The CXCR6 amino acid sequence is reacted with a 60-fold molar excess of activated PVP and the reaction is stopped with amino caploic acid (5-fold molar excess against activated PVP), essentially as described in Haruhiko Kamada, et al., 2000, Cancer Research 60: 6416- 6420, which is fully incorporated herein by reference.
Resultant conjugated CXCR6 molecules (e.g., PEGylated or PVP-conjugated CXCR6) are separated, purified and qualified using e.g., high-performance liquid chromatography (HPLC). In addition, purified conjugated molecules of this aspect of the present invention may be further qualified using e.g., in vitro assays in which the binding specificity of CXCR6 ligand to its receptor (e.g., CXCR6) is tested in the presence or absence of the CXCR6 conjugates of the present invention, essentially as described for other chemokines [e.g., MIP- lα, see for example, Hesselgesser J, 1998 (Supra), which is fully incorporated herein by reference]. Molecules of this aspect of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence, such as a "Tag" further described hereinbelow) and therefore involve different chemistry.
Solid phase peptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). Synthetic peptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N. Y.] and the composition of which can be confirmed via amino acid sequencing.
In cases where large amounts of the peptides of the present invention are desired, the polypeptides of the present invention can be generated using recombinant techniques such as described by Bitter et al. (1987) Methods in Enzymol. 153:516- 544; Studier et al. (1990) Methods in Enzymol. 185:60-89; Brisson et al. (1984) Nature 310:51 1-514; Takamatsu et al. (1987) EMBO J. 6:307-311; Coruzzi et al. (1984) EMBO J. 3: 1671-1680; Brogli et al. (1984) Science 224:838-843; Gurley et al.
(1986) MoI. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.
Briefly, an expression construct (i.e., expression vector), which includes an isolated polynucleotide (i.e., isolated from a naturally occurring source thereof, e.g., SEQ ID NO: 1, 3) which comprises a nucleic acid sequence encoding the CXCR6 amino acid sequence (optionally in frame fused to a nucleic acid sequence encoding the heterologous amino acid sequence e.g., SEQ ID NO: 5) of the present invention positioned under the transcriptional control of a regulatory element, such as a promoter, is introduced into host cells.
For example, a nucleic acid sequence encoding a CXCR6 polypeptide of the present invention (e.g., SEQ ID NO: 1 or 3) is ligated in frame to an immunoglobulin cDNA sequence (e.g., SEQ ID NO: 5). It will be appreciated that, ligation of genomic immunoglobulin fragments can also be used. In this case, fusion requires the presence of immunoglobulin regulatory sequences for expression. cDNAs encoding IgG heavy- chain constant regions can be isolated based on published sequence from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybridization or by polymerase chain reaction (PCR) techniques. The nucleic acid sequences encoding the CXCR6 amino acid sequence and immunoglobulin can be ligated in tandem into an expression construct (vector) that directs efficient expression in the selected host cells, further described hereinbelow. For expression in mammalian cells, pRK5 -based vectors [Schall et al., Cell, 61:361-370 (1990)]; and CDM8-based vectors [Seed, Nature, 329:840 (1989)] can be used. The exact junction can be created by removing the extra sequences between the designed junction codons using oligonucleotide- directed deletional mutagenesis [Zoller et al, Nucleic Acids Res., 10:6487 (1982); Capon et al., Nature, 337:525-531 (1989)]. Synthetic oligonucleotides can be used, in which each half is complementary to the sequence on either side of the desired junction; ideally, these are 1 1 to 48-mers. Alternatively, PCR techniques can be used to join the two parts of the molecule in-frame with an appropriate vector. Methods of introducing the expression construct into a host cell are well known in the art and include, electroporation, lipofection and chemical transformation (e.g., calcium phosphate). See also Example 3 of the Examples section which follows. The "transformed" cells are cultured under suitable conditions, which allow the expression of the chimeric molecule encoded by the nucleic acid sequence.
Following a predetermined time period, the expressed chimeric molecule is recovered from the cell or cell culture, and purification is effected according to the end use of the recombinant polypeptide.
Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like, can be used in the expression vector [see, e.g., Bitter et al., (1987) Methods in Enzymol. 153:516- 544].
Other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the chimera), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or toxicity of the expressed fusion protein. A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the fusion protein coding sequence. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the chimera coding sequence; yeast transformed with recombinant yeast expression vectors containing the chimera coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the chimera coding sequence. Mammalian expression systems are preferably used to express the chimera of the present invention. The choice of host cell line for the expression of the molecules depends mainly on the expression vector. Eukaryotic expression systems are preferred (e.g., mammalian and insects) since they allow post translational modifications (e.g., glyccosylation). Another consideration is the amount of protein that is required. Milligram quantities often can be produced by transient transfections. For example, the adenovirus EIA-transformed 293 human embryonic kidney cell line can be transfected transiently with pRK5-based vectors by a modification of the calcium phosphate method to allow efficient expression. CDM8-based vectors can be used to transfect COS cells by the DEAE-dextran method (Aruffo et al., Cell, 61 : 1303-1313 (1990); Zettmeissl et al., DNA Cell Biol. US, 9:347-353 (1990)]. If larger amounts of protein are desired, the molecules can be expressed after stable transfection of a host cell line (see Example 1 of the Examples section). It will be appreciated that the presence of a hydrophobic leader sequence at the N-terminus of the molecule will ensure processing and secretion of the molecule by the transfected cells.
It will be appreciated that the use of bacterial or yeast host systems may be preferable to reduce cost of production. However since bacterial host systems are devoid of protein glycosylation mechanisms, a post production glycosylation may be needed. In any case, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce the recombinant chimera molecule of the present invention. Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.
Following a predetermined time in culture, recovery of the recombinant protein is effected. The phrase "recovering the recombinant protein" refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
Molecules of the present invention are preferably retrieved in "substantially pure" form. As used herein, "substantially pure" refers to a purity that allows for the effective use of the protein in the applications, described hereinbelow.
Recombinant molecules of the present invention can be conveniently purified by affinity chromatography. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain that is used in the chimera. Protein A can be used to purify chimeric molecules that are based on human γl, γ2, or γ4 heavy chains [Lindmark et al., J. Immunol. Meth., 62:1-13 (1983)]. Protein G is preferably used for all mouse isotypes and for human γ3 [Guss et al., EMBO J., 5:1567-1575 (1986)]. The solid support to which the affinity ligand is attached is most often agarose, but other solid supports are also available. Mechanically stable solid supports such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. The conditions for binding the chimeric molecules to the protein A or G affinity column are dictated entirely by the characteristics of the Fc domain; that is, its species and isotype. Generally, when the proper ligand is chosen, efficient binding occurs directly from unconditioned culture fluid. One distinguishing feature of chimeric molecules of this aspect of the present invention is that, for human .gamma.1 molecules, the binding capacity for protein A is somewhat diminished relative to an antibody of the same Fc type. Bound chimeric molecules of this aspect of the present invention can be efficiently eluted either at acidic pH (at or above 3.0), or in a neutral pH buffer containing a mildly chaotropic salt. This affinity chromatography step can result in a chimeric molecule preparation that is > 95 % pure. Medical grade purity is essential for therapeutic applications.
Other methods known in the art can be used in place of, or in addition to, affinity chromatography on protein A or G to purify chimeric molecules which include an immunoglobulin portion. Such chimeric molecules behave similarly to antibodies in thiophilic gel chromatography [Hutchens et al., Anal. Biochem., 159:217-226 (1986)] and immobilized metal chelate chromatography [Al-Mashikhi et al., J. Dairy Sci., 71:1756-1763 (1988)]. In contrast to antibodies, however, their behavior on ion exchange columns is dictated not only by their isoelectric points, but also by a charge dipole that may exist in the molecules due to their chimeric nature.
Thus, the present invention provides for numerous configurations of soluble molecules which are capable of binding CXCR6 ligands and neutralize signaling therefrom.
Another agent for reducing the activity or expression of CXCR6/CXCL16 can also be a neutralizing antibody directed at CXCR6/CXCL16 (such as by binding to the CXCR6 binding site on CXCL 16 or by binding to the CXCL 16 binding site on CXCR6) or an effector thereof. Neutralizing antibodies for CXCR6/CXCL16 are known in the art, for example, U.S. Pat. No. 7,038,018, which is hereby incorporated by reference in its entirety. A preferred neutralizing antibody according to the teachings of the present invention preferably binds the N-ter domain of CXCR6 as set forth in SEQ ID NO: 4. This antibody is expected to compete with CXCL 16 on receptor binding. As used herein, the term "antibody" refers to a substantially intact antibody molecule.
As used herein, the phrase "antibody fragment" refers to a functional fragment of an antibody that is capable of binding to an antigen.
Suitable antibody fragments for practicing the present invention include, inter alia, a complementarity-determining region (CDR) of an immunoglobulin light chain
(referred to herein as "light chain"), a CDR of an immunoglobulin heavy chain
(referred to herein as "heavy chain"), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single-chain Fv, an Fab, an Fab', and an F(ab')2.
Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:
(i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain and the variable region of the heavy chain expressed as two chains;
(ii) single-chain Fv ("scFv"), a genetically engineered single-chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker. (iii) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain, which consists of the variable and CHl domains thereof; (iv) Fab', a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab' fragments are obtained per antibody molecule); and
(v) F(ab')2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab' fragments held together by two disulfide bonds).
Methods of generating monoclonal and polyclonal antibodies are well known in the art. 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 (Orlandi, R. et al. (1989). Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci USA 86, 3833-3837; and Winter, G. and Milstein, C. (1991). Man-made antibodies, Nature 349, 293-299), 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 (Kohler, G. and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497; Kozbor, D. et al. (1985). Specific immunoglobulin production and enhanced tumorigenicity following ascites growth of human hybridomas. J Immunol Methods 81, 31-42; Cote RJ. et al. (1983). Generation of human monoclonal antibodies reactive with cellular antigens. Proc Natl Acad Sci USA 80, 2026-2030; and Cole, S. P. et al. (1984). Human monoclonal antibodies. MoI Cell Biol 62, 109-120).
In cases where target antigens are too small to elicit an adequate immunogenic response when generating antibodies in vivo, such antigens (referred to as "haptens") can be coupled to antigenically neutral carriers such as keyhole limpet hemocyanin (KLH) or serum albumin (e.g., bovine serum albumin (BSA)) carriers (see, for example, US. Pat. Nos. 5,189,178 and 5,239,078). Coupling a hapten to a carrier can be effected using methods well known in the art. For example, direct coupling to amino groups can be effected and optionally followed by reduction of the imino linkage formed. Alternatively, the carrier can be coupled using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents. Linker compounds can also be used to effect the coupling; both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, Illinois, USA. The resulting immunogenic complex can then be injected into suitable mammalian subjects such as mice, rabbits, and others. 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 or monoclonal antibodies may be obtained, as described hereinabove. 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.) For example, 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 systems) of DNA encoding the fragment.
Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. As described hereinabove, an (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 3.5 S Fab' monovalent fragments. Alternatively, 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. Pat. Nos. 4,036,945 and 4,331,647; and Porter, R. R. (1959). The hydrolysis of rabbit γ- globulin and antibodies with crystalline papain. Biochem J 73, 119-126). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments retain the ability to bind to the antigen that is recognized by the intact antibody.
As described hereinabove, an Fv is composed of paired heavy chain variable and light chain variable domains. This association may be noncovalent (see, for example, Inbar, D. et al. (1972). Localization of antibody-combining sites within the variable portions of heavy and light chains. Proc Natl Acad Sci USA 69, 2659-2662). 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.
Preferably, 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 in the literature of the art (see, e.g.: Whitlow, M. and Filpula, D. (1991). Single-chain Fv proteins and their fusion proteins. METHODS: A Companion to Methods in Enzymology 2(2), 97- 105; Bird, R. E. et al. (1988). Single-chain antigen-binding proteins. Science 242, 423-426; Pack, P. et al. (1993). Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli. Biotechnology (N. Y.) 11(11), 1271-1277; and U.S. Pat. No. 4,946,778).
Isolated complementarity-determining region peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes may be prepared, for example, by RT-PCR of the mRNA of an antibody-producing cell. Ample guidance for practicing such methods is provided in the literature of the art (e.g., Larrick, J. W. and Fry, K. E. (1991). PCR Amplification of Antibody Genes. METHODS: A Companion to Methods in Enzymology 2(2), 106-110). It will be appreciated that for human therapy or diagnostics, 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. In some instances, 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. In general, 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 (see, for example: Jones, P. T. et al. (1986). Replacing the complementarity- determining regions in a human antibody with those from a mouse. Nature 321, 522- 525; Riechmann, L. et al. (1988). Reshaping human antibodies for therapy. Nature 332, 323-327; Presta, L. G. (1992b). Curr Opin Struct Biol 2, 593-596; and Presta, L. G. (1992a). Antibody engineering. Curr Opin Biotechnol 3(4), 394-398).
Methods for humanizing non-human antibodies are well known in the art. Generally, 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 essentially as described (see, for example: Jones et al. (1986); Riechmann et al. (1988); Verhoeyen, M. et al. (1988). Reshaping human antibodies: grafting an antilysozyme activity. Science 239, 1534-1536; and 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. In practice, 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 (Hoogenboom, H. R. and Winter, G. (1991). By-passing immunization. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J MoI Biol 227, 381-388; Marks, J. D. et al. (1991). By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J MoI Biol 222, 581-597; Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96; and Boerner, P. et al. (1991). Production of antigen-specific human monoclonal antibodies from in vitro- primed human splenocytes. J Immunol 147, 86-95). Humanized antibodies can also be created by introducing sequences encoding human immunoglobulin loci into transgenic animals, e.g., into mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon antigenic challenge, human antibody production is observed in such animals which closely resembles that seen in humans in all respects, including gene rearrangement, chain assembly, and antibody repertoire. Ample guidance for practicing such an approach is provided in the literature of the art (for example, refer to: U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks, J. D. et al. (1992). Bypassing immunization: building high affinity human antibodies by chain shuffling. Biotechnology (N. Y.) 10(7), 779-783; Lonberg et al., 1994. Nature 368:856-859; Morrison, S. L. (1994). News and View: Success in Specification. Nature 368, 812- 813; Fishwild, D. M. et al. (1996). High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat Biotechnol 14, 845- 851; Neuberger, M. (1996). Generating high-avidity human Mabs in mice. Nat Biotechnol 14, 826; and Lonberg, N. and Huszar, D. (1995). Human antibodies from transgenic mice. Int Rev Immunol 13, 65-93).
After antibodies have been obtained, they may be tested for activity, for example via enzyme-linked immunosorbent assay (ELISA).
Alternatively, the agent of this aspect of the present invention can be a chemical, which is designed to specifically inhibit the activity or expression of CXCR6 or an effector thereof. Chemical inhibitors directed at CXCR6 effectors are well known in the art. Examples include, but are not limited to, pertussis toxin (Gj inhibitor), wortmannin or LY294002 (PI3K inhibitors) [Chandrasekar et al., supra] and curcumin (NF-kappa B inhibitor) [Thaloor et al., Am J Physiol Cell Physiol (1999) 277:C320-C329]. Signal transduction inhibitors are available from a number of chemical companies including Calbiochem (San Diego, CA, USA) and Sigma- Aldrich Corp. (St Louis, MO, USA).
Another agent capable of reducing the expression of CXCR6/CXCL16 or effectors thereof is a small interfering RNA (siRNA) molecule. siRNA for CXCR6/CXCL16 are known in the art, for example, U.S. Publication No. 20060257359, which is hereby incorporated by reference in its entirety. RNA interference is a two-step process. The first step, which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with 2-nucleotide 3' overhangs [Hutvagner and Zamore, Curr. Opin. Genetics and Development 12:225- 232 (2002); and Bernstein, Nature 409:363-366 (2001)]. In the effector step, the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore, Curr. Opin. Genetics and Development (2002) 12:225-232; Hammond et al., Nat. Rev. Gen. (2001) 2: 1 10-119; and Sharp, Genes. Dev. (2001) 15:485-90]. Although the mechanism of cleavage is still to be elucidated, research indicates that each RISC contains a single siRNA and an RNase [Hutvagner and Zamore, Curr. Opin. Genetics and Development (2002) 12:225-232]. Because of the remarkable potency of RNAi, an amplification step within the
RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al., Nat. Rev. Gen. 2: 110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore, Curr. Opin. Genetics and Development 12:225-232 (2002)]. For more information on RNAi see the following reviews Tuschl, ChemBiochem. 2:239-245 (2001); Cullen, Nat. Immunol. 3:597-599 (2002); and Brantl, Biochem. Biophys. Act. 1575:15-25 (2002). Synthesis of RNAi molecules suitable for use with the present invention can be effected as follows. First, a CXCR6/CXCL16 mRNA sequence, for example, is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl, ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).
Second, 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 which 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.
For better evaluation of the selected siRNAs, 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. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
Another agent capable of down-regulating CXCR6/CXCL16 or an effector thereof is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of interest. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology (1995) 2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA (1997) 943:4262) A general model (the "10-23" model) for the DNAzyme has been proposed. "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine :pyrimidine junctions [Santoro and Joyce, Proc. Natl. Acad. Sci. USA (1997) 94(9):4262-6; for rev of DNAzymes see Khachigian, Curr Opin MoI Ther (2002) 4:1 19-21].
Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 2002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL).
Reducing CXCR6/CXCL16 or an effector thereof can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the proteins of interest. Design of antisense molecules which can be used to efficiently down-regulate a gene product of interest must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
The prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Luft, J MoI Med 76: 75-6 (1998); Kronenwett et al., Blood 91: 852-62 (1998); Rajur et al., Bioconjug Chem 8: 935-40 (1997); Lavigne et al., Biochem Biophys Res Commun 237: 566-71 (1997) and Aoki et al., Biochem Biophys Res Commun 231: 540-5 (1997)].
In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the energetic of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et al., Biotechnol Bioeng 65: 1-9 (1999)].
Such algorithms have been successfully used to implement an antisense approach in cells. For example, the algorithm developed by Walton et al. enabled scientists to successfully design antisense oligonucleotides for rabbit beta-globin
(RBG) and mouse tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group has more recently reported that the antisense activity of rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gpl30) in cell culture as evaluated by a kinetic PCR technique proved effective in almost all cases, including tests against three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries.
In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et al., Nature Biotechnology 16: 1374 - 1375 (1998)].
Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin MoI Ther 1:372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz, Curr Opin MoI Ther 1 :297-306 (1999)].
More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60 (2001)].
Thus, the current consensus is that recent developments in the field of antisense technology which, as described above, have led to the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for down-regulating expression of known sequences without having to resort to undue trial and error experimentation. Another agent capable of reducing the expression of CXCR6/CXCL16 or an effector thereof is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding this gene product. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - WEB home page).
An additional method of reducing the expression of a CXCR6/CXCL16 gene or effectors thereof in cells is via triplex forming oligonuclotides (TFOs). Recent studies have shown that TFOs can be designed which can recognize and bind to polypurine/polypirimidine regions in double-stranded helical DNA in a sequence- specific manner. These recognition rules are outlined by Maher III, L. J., et al., Science,1989;245:725-730; Moser, H. E., et al., Science,1987;238:645-630; Beal, P. A., et al, Science,1992;251:1360-1363; Cooney, M., et al., Science, 1988;241:456- 459; and Hogan, M. E., et al., EP Publication 375408. Modification of the oligonuclotides, such as the introduction of intercalators and backbone substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review see Seidman and Glazer, J Clin Invest 2003; 112:487-94).
However, it has been shown that the A-AT and G-GC triplets have the greatest triple helical stability (Reither and Jeltsch, BMC Biochem, 2002, Septl2, Epub). The same authors have demonstrated that TFOs designed according to the A-AT and G-
GC rule do not form non-specific triplexes, indicating that the triplex formation is indeed sequence specific.
Thus for any given sequence a triplex forming sequence may be devised. Triplex-forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.
Transfection of cells (for example, via cationic liposomes) with TFOs, and formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific downregulation of gene expression. Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27: 1176-81, and Puri, et al, J Biol Chem, 2001;276:28991-98), and the sequence- and target specific downregulation of expression of the Ets2 transcription factor, important in prostate cancer etiology (Carbone, et al, Nucl Acid Res. 2003;31:833-43), and the pro-inflammatory ICAM-I gene (Besch et al, J Biol Chem, 2002;277:32473-79). In addition, Vuyisich and Beal have recently shown that sequence specific TFOs can bind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).
Additionally, TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest (2003) 112:487-94). Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn. Additional description of oligonucleotide agents is further provided hereinbelow. It will be appreciated that therapeutic oligonucleotides may further include base and/or backbone modifications which may increase bioavailability therapeutic efficacy and reduce cytotoxicity. Such modifications are described in Younes (2002) Current Pharmaceutical Design 8:1451-1466.
For example, the oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
Preferably used oligonucleotides are those modified in either backbone, internucleoside linkages or bases, as is broadly described hereinunder.
Specific examples of preferred oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat. NOs: 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466, 677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.
Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms can also be used.
Alternatively, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.
Other oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example for such an oligonucleotide mimetic, includes peptide nucleic acid (PNA). A PNA oligonucleotide refers to an oligonucleotide where the sugar-backbone is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No: 6,303,374.
Oligonucleotides of the present invention may also include base modifications or substitutions. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Further bases include those disclosed in U.S. Pat. No: 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. , ed., CRC Press, 1993. Such bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 0C. [Sanghvi YS et al. (1993) Antisense Research and Applications, CRC Press, Boca Raton 276-278] and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
As shown in Example 5 of the Examples section which follows, the present inventors were able to suppress an ongoing MS, using agents of the present invention, substantiating the use of same in therapy.
Agents of the present invention can be administered to the subject per se, or as part of a pharmaceutical composition, which also includes a physiologically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism. As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. As used herein, the term "active ingredient" refers to the preparation accountable for the intended biological effect.
Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media [Mutter et al. (1979)].
It will be appreciated that the bioconjugated polymer (e.g., the PEGylated CXCR6 polypeptide of the present invention) can be used in, and as a part of, the pharmaceutically acceptable carrier, and thus serves as a carrier molecule for delivery of the CXCR6 amino acid sequence, while at the same time serving as a component of the delivery vehicle. A preferred embodiment of this dual use is a liposomal vehicle, e.g., PEG-conjugated liposomes, as described e.g., in U.S. Pat. Appl. No. 20030186869 to Poiani, George et al., which is fully incorporated herein by reference.
Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
Alternately, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body.
Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries as desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, and sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.
The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a "therapeutically effective amount" means an amount of active ingredients (e.g., a nucleic acid construct) effective to prevent, alleviate, or ameliorate symptoms of a disorder (e.g., ischemia) or prolong the survival 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 provided herein. For any preparation used in the methods of the invention, the dosage or the therapeutically effective amount can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and 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. (See, e.g., Fingl, E. et al. (1975), "The Pharmacological Basis of Therapeutics," Ch. 1, p.l.)
Dosage amount and administration intervals may be adjusted individually to provide sufficient plasma or brain levels of the active ingredient to induce or suppress the biological effect (i.e., minimally effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, 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, etc.
Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser device may also be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may include labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as further detailed above.
It will be appreciated that nucleic acid agents (as described above) can be administered to the individual employing any suitable mode of administration, described hereinbelow (i.e., in-vivo gene therapy). Alternatively, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex-vivo gene therapy).
Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation 14(1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 31 LTR or a portion thereof. Other vectors can be used that are non- viral, such as cationic lipids, polylysine, and dendrimers.
It will be appreciated that treatment of Multiple Sclerosis according to the present invention may be combined with other treatment methods known in the art (i.e., combination therapy). These include, but are not limited to, Interferon Beta Ia, Interferon Beta Ib, Glatiramer Acetate, Mitoxantrone, MethylPrednisolone, Prednisone, Prednisolone, Dexamethasone, Adreno-corticotrophic Hormone (ACTH) and Corticotropin.
Since the present inventors have uncovered an elevated expression of CXCR6 and CXCR6 expressing Th cells in spinal cords of EAE induced mice (see Examples 1-2), the present invention also envisages a novel method for diagnosis of MS.
Thus, according to another aspect of the present invention there is provided a method of diagnosing Multiple Sclerosis in a subject.
As used herein the term "diagnosing" refers to classifying a disease or a symptom as Multiple Sclerosis, determining a severity of a Multiple Sclerosis, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The method is effected by detecting CXCR6 in cells obtained from the subject, wherein each of the levels determined can be correlated with progression of Multiple Sclerosis in the subject, to thereby diagnose presence of Multiple Sclerosis in the subject As used herein, the term "level" refers to expression levels of RNA and/or protein of CXCR6.
A level correlatable with presence or absence of Multiple Sclerosis can be a level of CXCR6 in a Multiple Sclerosis derived cells which is increased as compared to the level of the same in a normal healthy cells obtained from a similar tissue or cellular origin.
As used herein the term "cells" refers to cells derived from a tissue or from a fluid, preferably cellular sample, isolated from a subject, such as but not limited to, for example, spinal fluid and peripheral blood.
Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of each of the above-described proteins in the subject. Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy.
Regardless of the procedure employed, once a biopsy is obtained the level of CXCR6 can be determined and a diagnosis can thus be made. Determining a level of CXCR6 can be effected using various biochemical and molecular approaches used in the art for determining gene amplification, and/or level of gene expression.
Typically, detection of a nucleic acid of interest in a biological sample is effected by hybridization-based assays using an oligonucleotide probe. The term "oligonucleotide" refers to a single stranded or double stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally- occurring bases, sugars and covalent internucleoside linkages (e.g., backbone) as well as oligonucleotides having non-naturally-occurring portions which function similarly to respective naturally-occurring portions.
Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art, such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes HII Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988) and "Oligonucleotide Synthesis" Gait, M. J., ed. (1984) utilizing solid phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting and purification by for example, an automated trityl-on method or HPLC.
The oligonucleotide of the present invention is of at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the examined mRNA.
Hybridization based assays which allow the detection of a DNA or RNA of interest in a biological sample rely on the use of oligonucleotide which can be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides.
Hybridization of short nucleic acids (below 200 bp in length, e.g. 17-40 bp in length) can be effected using the following examplery hybridization protocols which can be modified according to the desired stringency; (i) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 0C below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 0C below the Tm; (U) hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 2 - 2.5 °C below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm, final wash solution of 6 x SSC, and final wash at 22 °C; (Hi) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature. The detection of hybrid duplexes can be carried out by a number of methods.
Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Such labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. A label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample (target).
For example, oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent. Alternatively, when fluorescently-labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, Calif] can be attached to the oligonucleotides. Traditional hybridization assays include PCR, RT-PCR, RNase protection, in- situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis.
Those skilled in the art will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays. For instance, samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.
Polymerase chain reaction (PCR)-based methods may be used to identify the presence of mRNA of interest. For PCR-based methods a pair of oligonucleotides is used, which is specifically hybridizable with the polynucleotide sequences described hereinabove in an opposite orientation so as to direct exponential amplification of a portion thereof (including the hereinabove described sequence alteration) in a nucleic acid amplification reaction.
The polymerase chain reaction and other nucleic acid amplification reactions are well known in the art and require no further description herein. The pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7 °C, preferably less than 5 °C, more preferably less than 4 °C, most preferably less than 3 °C, ideally between 3 °C and 0 0C. Hybridization to oligonucleotide arrays may be also used to determine the presence of the mRNA or DNA of interest. Such screening has been undertaken in the BRCAl gene and in the protease gene of HIV-I virus [see Hacia et al., (1996) Nat Genet 1996; 14(4):441-447; Shoemaker et al., (1996) Nat Genet 1996;14(4):450-456; Kozal et al., (1996) Nat Med 1996;2(7):753-759]. The nucleic acid sample which includes the candidate region to be analyzed is isolated, amplified and labeled with a reporter group. This reporter group can be a fluorescent group such as phycoerythrin. The labeled nucleic acid is then incubated with the probes immobilized on the chip using a fluidics station. For example, Manz et al. (1993) Adv in Chromatogr 1993; 33:1-66 describe the fabrication of fluidics devices and particularly microcapillary devices, in silicon and glass substrates.
Once the reaction is completed, the chip is inserted into a scanner and patterns of hybridization are detected. The hybridization data is collected, as a signal emitted from the reporter groups already incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.
It will be appreciated that when utilized along with automated equipment, the above described detection methods can be used to screen multiple samples for multiple sclerosis both rapidly and easily. The level of CXCR6 may also be determined at the protein level. Numerous protein detection assays are known in the art, examples include, but are not limited to, chromatography, electrophoresis, immunodetection assays such as ELISA and western blot analysis, immunohistochemistry and the like, which may be effected using antibodies such as described hereinabove. Note, however, that such antibodies need not be neutralizing antibodies.
Diagnosis of Multiple Sclerosis using the above-described methodology can be further supported by other diagnostic methods for Multiple Sclerosis which are well known in the art to thereby provide a more accurate diagnosis. Examples of such diagnostic methods include, but are not limited to, evidence of two or more neurological signs (e.g., lesions) that are localized to the central nervous system (e.g., brain or spinal cord) and are disseminated in time and space (e.g., occur in different parts of the central nervous system at least three months apart). Such lesions can be diagnosed by magnetic resonance imaging (MRI) with gadolinium contrast, especially during or following a first attack, and again at least three months after the initial attack to identify new lesions and provide evidence of dissemination over time.
It will be appreciated that the diagnostic reagents described hereinabove can also be included in kits. For example a kit for diagnosing presence of Multiple Sclerosis in a subject can include a solid phase for attaching multiple cell samples packaged with appropriate buffers and preservatives and used for diagnosis.
Mapping the CXCL 16 binding on CXCR6 has allowed the generation of polypeptide agents as described above (e.g., SEQ ID NO: 4 and 8). These can be used for the treatment of a variety of diseases in which CXCR6 or CXCL 16 is associated with disease onset or progression (as used herein CXCR6/CXCL16-associated diseases).
As used herein the term "CXCR6/CXCL16 associated disease" refers to a medical condition (e.g., disease, syndrome or condition), which depends on the interaction between a CXCR6 and it's ligand CXCL 16 for onset or progression. Examples of art references for CXCR6 and/or CXCLl 6 associated diseases include, but are not limited to, autoimmune diseases such as such as Grave's disease [Aust et al., Eur J Endocrinol. (2005) 152(4):635-43], inflammatory diseases such as rheumatoid arthritis [Ruth et al., supra; van der Voort et al. supra], vascular disease [Charo and Taubman, Circulation Res. (2004) 95:858], liver disease [Charo and Ransohoff, N Engl J Med (2006) 354:610-21], cardiovascular diseases [Lehrke et al., J Am Coll Cardiol. (2007) 49(4):442-9], disorders of the peripheral and central nervous system [Ie Blanc et al., Neurosci Lett. (2006) 397(1-2): 145-8], respiratory diseases [Morgan et al, Clin Exp Allergy (2005) 35(12): 1572-1580(9)], cancer [Wagsater et al., Int J MoI Med. (2004) 14(l):65-9].
As used herein the phrase "inflammatory disorder" includes but is not limited to chronic inflammatory diseases and disorders and acute inflammatory diseases and disorders. Various examples of inflammatory diseases which are included in the scope of the present invention include, but are not limited to, conditions summarized in the list infra.
Inflammatory diseases associated with hypersensitivity
Examples of hypersensitivity include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.
Type I or immediate hypersensitivity, such as asthma. Type II hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 JuI; 15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998; 17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al, Clin Diagn Lab Immunol. 1999 Mar;6 (2):156); Chan OT. et al, Immunol Rev 1999 Jun;169:107), glandular diseases, glandular autoimmune diseases, pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), thyroid diseases, autoimmune thyroid diseases, Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339), thyroiditis, spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15;165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al, Nippon Rinsho 1999 Aug;57 (8): 1810), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8): 1759); autoimmune reproductive diseases, ovarian diseases, ovarian autoimmunity (Garza KM. et al, J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti-sperm infertility (Diekman AB. et al, Am J Reprod Immunol. 2000 Mar;43 (3):134), repeated fetal loss (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), neurodegenerative diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis (Cross AH. et al, J Neuroimmunol 2001 Jan 1;112 (1-2):1), Alzheimer's disease (Oron L. et al, J
Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83), motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191), Guillain-Barre syndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234), myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de Ia Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan;156 (1):23); neuropathies, dysimmune neuropathies (Nobile-Orazio E. et al, Electroencephalogr CHn Neurophysiol Suppl 1999;50:419); neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al, Ann N Y Acad Sci. 1998 May 13;841:482), cardiovascular diseases, cardiovascular autoimmune diseases, atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S107-9), granulomatosis, Wegener's granulomatosis, arteritis, Takayasu's arteritis and Kawasaki syndrome (Praprotnik S. et al, Wien Klin Wochenschr 2000 Aug 25 ; 112 ( 15- 16):660); anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al, Semin Thromb Hemost.2000;26 (2): 157); vasculitises, necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May;151 (3): 178); antiphospholipid syndrome (Flamholz R. et al, J Clin Apheresis 1999;14 (4):171); heart failure, agonist-like beta-adrenoceptor antibodies in heart failure (Wallukat G. et al, Am J Cardiol. 1999 Jun 17;83 (12A):75H), thrombocytopenic puφura (Moccia F. Ann Ital Med hit. 1999 Apr-Jun;14 (2):114); hemolytic anemia, autoimmune hemolytic anemia (Efremov DG. et al, Leuk Lymphoma 1998 Jan;28 (3-4):285), gastrointestinal diseases, autoimmune diseases of the gastrointestinal tract, intestinal diseases, chronic inflammatory intestinal disease (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122), autoimmune diseases of the musculature, myositis, autoimmune myositis, Sjogren's syndrome (Feist
E. et al, Int Arch Allergy Immunol 2000 Sep;123 (1):92); smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 Jun;53 (5-6):234), hepatic diseases, hepatic autoimmune diseases, autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326) and primary biliary cirrhosis (Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999 Jun;l l (6):595).
Type IV or T cell mediated hypersensitivity, include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18;91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998;7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves' disease (Sakata S. et al., MoI Cell Endocrinol 1993 Mar;92 (1):77); ovarian diseases (Garza KM. et al, J Reprod Immunol 1998 Feb;37 (2):87), prostatitis, autoimmune prostatitis (Alexander RB. et al, Urology 1997 Dec;50 (6):893), polyglandular syndrome, autoimmune polyglandular syndrome, Type I autoimmune polyglandular syndrome (Hara T. et al, Blood. 1991 Mar 1;77 (5): 1127), neurological diseases, autoimmune neurological diseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom M. et al, J Neurol Neurosurg Psychiatry 1994 May;57 (5):544), myasthenia gravis (Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), stiff-man syndrome (Hiemstra HS. et al, Proc Natl Acad Sci U S A 2001 Mar 27;98 (7):3988), cardiovascular diseases, cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al, J Clin Invest 1996 Oct 15;98 (8): 1709), autoimmune thrombocytopenic purpura (Semple JW. et al, Blood 1996 May 15;87 (10):4245), anti- helper T lymphocyte autoimmunity (Caporossi AP. et al, Viral Immunol 1998; 11 (1):9), hemolytic anemia (Sallah S. et al, Ann Hematol 1997 Mar;74 (3):139), hepatic diseases, hepatic autoimmune diseases, hepatitis, chronic active hepatitis (Franco A. et al, Clin Immunol Immunopathol 1990 Mar;54 (3):382), biliary cirrhosis, primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551), nephric diseases, nephric autoimmune diseases, nephritis, interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140), connective tissue diseases, ear diseases, autoimmune connective tissue diseases, autoimmune ear disease (Yoo TJ. et al, Cell Immunol 1994 Aug;157 (1):249), disease of the inner ear (Gloddek B. et al, Ann N Y Acad Sci 1997 Dec 29;830:266), skin diseases, cutaneous diseases, dermal diseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
Examples of delayed type hypersensitivity include, but are not limited to, contact dermatitis and drug eruption. Examples of types of T lymphocyte mediating hypersensitivity include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.
Examples of helper T lymphocyte-mediated hypersensitivity include, but are not limited to, ThI lymphocyte mediated hypersensitivity and Th2 lymphocyte mediated hypersensitivity. Autoimmune diseases
Include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases. Examples of autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al, Wien Klin Wochenschr 2000 Aug 25;112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al, Semin Thromb Hemost.2000;26 (2): 157), necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing and crescentic glomerulonephritis (Noel, LH. Ann Med Interne (Paris). 2000 May;151 (3): 178), antiphospholipid syndrome (Flamholz R. et al, J Clin Apheresis 1999;14 (4):171), antibody-induced heart failure (Wallukat G. et al, Am J Cardiol. 1999 Jun 17;83 (12A): 75H), thrombocytopenic purpura (Moccia F. Ann Ital Med hit. 1999 Apr- Jun; 14 (2): 114; Semple JW. et al, Blood 1996 May 15;87 (10):4245), autoimmune hemolytic anemia (Efremov DG. et al, Leuk Lymphoma 1998 Jan;28 (3-4):285; Sallah S. et alt. Ann Hematol 1997 Mar;74 (3):139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al, J Clin Invest 1996 Oct 15;98 (8): 1709) and anti-helper T lymphocyte autoimmunity (Caporossi AP. et al, Viral Immunol 1998;11 (1):9).
Examples of autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18 ;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189).
Examples of autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome, diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339; Sakata S. et al, MoI Cell Endocrinol 1993 Mar;92 (1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15;165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al, Nippon Rinsho 1999 Aug;57 (8):1810), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8): 1759), ovarian autoimmunity (Garza KM. et al, J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti-sperm infertility (Diekman AB. et al, Am J Reprod Immunol. 2000 Mar;43 (3): 134), autoimmune prostatitis (Alexander RB. et al, Urology 1997 Dec;50 (6):893) and Type I autoimmune polyglandular syndrome (Hara T. et al, Blood. 1991 Mar 1;77 (5):1127). Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan;23 (1):16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16;138 (2):122), colitis, ileitis and Crohn's disease.
Examples of autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
Examples of autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al, Clin Immunol Immunopathol 1990 Mar;54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999
Jun;l 1 (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326).
Examples of autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al, J Neuroimmunol 2001 Jan 1;112 (1-2):1), Alzheimer's disease (Oron L. et al, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83; Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191); Guillain-Barre syndrome and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204); paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiff-man syndrome (Hiemstra HS. et al, Proc Natl Acad Sci units S A 2001 Mar 27;98 (7):3988); non-paraneoplastic stiff man syndrome, progressive cerebellar atrophies, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de Ia Tourette syndrome and autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156 (1):23); dysimmune neuropathies (Nobile-Orazio E. et al, Electroencephalogr Clύi Neurophysiol Suppl 1999;50:419); acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al, Ann N Y Acad Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M. et al, J Neurol Neurosurg Psychiatry 1994 May;57 (5):544) and neurodegenerative diseases.
Examples of autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al, Int Arch Allergy Immunol 2000 Sep;123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234).
Examples of autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140). Examples of autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al, Lupus 1998;7 Suppl 2:S107-9).
Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al, Cell Immunol 1994 Aug;157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al, Ann N Y Acad Sci 1997 Dec 29;830:266).
Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998;17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al, Clin Diagn Lab Immunol. 1999 Mar;6
(2):156); Chan OT. et al, Immunol Rev 1999 Jun;169:107).
Infectious diseases
Examples of infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases.
Graft rejection diseases
Examples of diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.
Allergic diseases
Examples of allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.
Cancerous diseases
Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Particular examples of cancerous diseases but are not limited to: Myeloid leukemia such as Chronic myelogenous leukemia. Acute myelogenous leukemia with maturation. Acute promyelocytic leukemia, Acute nonlymphocytic leukemia with increased basophils, Acute monocytic leukemia.
Acute myelomonocytic leukemia with eosinophilia; Malignant lymphoma, such as
Birkitt's Non-Hodgkin's; Lymphoctyic leukemia, such as Acute lumphoblastic leukemia. Chronic lymphocytic leukemia; Myeloproliferative diseases, such as Solid tumors Benign Meningioma, Mixed tumors of salivary gland, Colonic adenomas;
Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder,
Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovial sarcoma,
Rhabdomyosarcoma (alveolar), Extraskeletel myxoid chondrosarcoma, Ewing's tumor; other include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, breast, skin, prostate, and ovarian.
The present invention also envisages the use of the present polypeptides for research and laboratory applications.
The affinity of the CXCR6 peptide of the present invention to CXCR6 ligands (e.g., CXCL16) allows use thereof in purification and detection of CXCR6 ligands.
According to an embodiment of this aspect of the present invention, there is provided an isolated soluble polypeptide comprising a tag attached to a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, the CXCR6 amino acid sequence being capable of binding CXCL 16 (as described above).
As used herein the term "tag" refers to a moiety which is specifically recognized by a binding partner such as an antibody, a chelator or an avidin (biotin) molecule. The tag can be placed C-terminally or N-terminally of the CXCR6 peptide, as long as it does not interfere with a biological activity thereof (e.g., ligand binding).
For example, a tag polypeptide has enough residues to provide an epitope (i.e., epitope tag) against which an antibody thereagainst can be made, yet is short enough such that it does not interfere with biological activity of the CXCR6 peptide. The epitope tag preferably also is fairly unique so that the antibody thereagainst does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8-50 amino acid residues
(preferably between about 9-30 residues). Preferred are poly-histidine sequences, which bind nickel, allowing isolation of the tagged protein by Ni-NTA chromatography as described (Lindsay et al. Neuron 17:571-574 (1996)], for example.
Such epitope-tagged forms of the CXCR6 are desirable, as the presence thereof can be detected using a labeled antibody against the tag polypeptide. Also, provision of the epitope tag enables the CXCR6 peptide of the present invention to be readily purified by affinity purification using the anti-tag antibody. Affinity purification techniques and diagnostic assays involving antibodies are described later herein.
Tag polypeptides and their respective antibodies are well known in the art. Examples include the flu HA tag polypeptide and its antibody 12CA5 (Field et al., MoI. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology, 5:3610- 3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody. Paborsky et al., Protein Engineering, 3(6):547-553 (1990). Other tag polypeptides have been disclosed. Examples include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194
(1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)]. Once the tag polypeptide has been selected, an antibody thereto can be generated using methods which are well known in the art. Such antibodies are commercially available such as from Sigma, St. Louis. USA.
According to an embodiment of this aspect of the present invention, there is provided a method of isolating a CXCR6 ligand from a biological sample or detecting the presence of CXCR6 ligands therein. As used herein the phrase "biological sample" refers to a biological material, such as cells, tissues and fluids such as blood, serum, plasma, lymph, bile fluid, urine, saliva, sputum, synovial fluid, semen, tears, cerebrospinal fluid, bronchioalveolar large fluid, ascites fluid, pus, conditioned medium and the like in which CXCR6 ligand is present. Isolation of CXCR6 ligand according to this aspect of the present invention is effected by contacting the biological sample with the molecule of this aspect of the present invention, such that CXCR6 ligand and the molecule form a complex (using buffer, temperature conditions which allow binding of the molecule to CXCR6 ligand, see for Example Datta-Mannan and Stone 2004, supra); and isolating the complex to thereby isolate CXCR6 ligand from the biological sample.
In order to isolate the complex, the molecule is preferably immobilized on a solid support. As used herein the phrase "solid support" refers to a non-aqueous matrix to which a reagent of interest (e.g., the molecule of this aspect of the present invention) can adhere. Examples of solid supports, include, but are not limited to, solid supports formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid support can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
Alternatively, such molecules can be used to detect the levels of CXCR6 ligand in biological samples. For diagnostic applications, molecules typically will be labeled with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, a fluorescent or chemiluminescent compound, or a tag (such as described hereinabove and to which a labeled antibody can bind). The molecules of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. [Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987)].
The molecules of this aspect of the present invention can be included in a diagnostic kit, in which the molecule and optionally solid support and imaging reagents (e.g., antibodies, chromogenic substrate etc.) can be packaged in suitable containers with appropriate buffers and preservatives and used for diagnosis.
As used herein the term "about" refers to ± 10 %.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531;
5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 ; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
EXAMPLE 1 CXCR6 is expressed in spinal cords of Experimental Autoimmune
Encephalomyelitis (EAE) induced mice
MATERIALS AND EXPERIMENTAL PROCEDURES
Induction of EAE in mice and collection of spinal cord samples Six CSIfB mice were subjected to active induction of EAE by MOGp35-55
(myelin oligodendrocyte glycoprotein, SEQ ID NO: 19) as was previously described [Kassiotis and Kollias, J Exp Med (2001) 193(4):427-434]. On day 18, spinal cords of EAE induced mice or naϊve mice were removed for further analysis. RNA Extraction
Total RNA was extracted using Tri-Zol reagent (Life Technologies) according to the manufacturer's protocol. The sediment (total RNA) was dried and dissolved in 1 % DEPC-ddH2O. Total RNA was quantified using a spectrophotometer (260 run wavelength).
RT-PCR
Five μg mRNA were synthesized into a double stranded cDNA, in a reaction mixture that included: RT-first strand reaction buffer, 1 nM dNTPs (NEB), 1 nmole random hexamers (Fermentas International INC, Canada), 10 u/ml RNasin (pharmaBiotech), 25 units/ml Superscript MuLUV-RT (NEB), CXCR6 Sense primer: ATGGATGATGGGCATCAAGAGTCAGCTCT, SEQ ID NO: 20 and CXCR6 anti- sense primer: CTACAATTGGAACATACTGGTGGTCTCTACA SEQ ID NO: 21. The mixture was incubated at 37 °C for 2 hours. The RT-PCR cycle profile used: denaturation at 95 °C for 60 seconds, annealing at 55 °C for 60 seconds and elongation at 72 °C for 60 seconds.
RESULTS Results indicated that CXCR6 mRNA is present only in spinal cords of EAE induced mice and not in spinal cords of naive (EAE-not induced) mice (Figure 1). Positive control, amplified GADPH was observed in both EAE induced and naive mice spinal cords.
EXAMPLE 2
CXCR6 expressing T helper cells are present in spinal cords of EAE induced mice
MATERIALS AND EXPERIMENTAL PROCEDURES
Induction of EAE in mice and sample collection Three C57/B mice were subjected to active induction of EAE by MOGp35-55
(myelin oligodendrocyte glycoprotein) as was previously described [Kassiotis and Kollias, J Exp Med (2001) 193(4):427-434]. On day 14, spleen, cervical lymph nodes and spinal cords of EAE induced mice were removed for further analysis. FACS staining and analysis
Flow cytometry analysis was performed according to the protocol previously described elsewhere [Schif-Zuck et al., J Immunol (2006) 177:8241-8247]. Briefly, spleen, cervical lymph nodes (CLN) and spinal cord (SC) of EAE induced mice were collected 14 days after induction. Cells were isolated by tissue pulverization and washed with PBS. Red blood cells were lysed by RBC (Red Blood Cell) Lysis buffer, washed and removed from cell mixture. The remaining white blood cells were double stained with rat anti-mouse CXCR6 (R&D, Minneapolis, MN) and rat anti-mouse CD4-FITC antibodies (BD Pharmingen, BD Biosciences). Cells were washed and stained by anti-rat PE (Jackson ImmunoResearch Laboratories, Inc, PA, USA). Results were read and analyzed in a flow cytometer.
RESULTS Results clearly show a selective accumulation of CXCR6+ CD4+ T cells in spinal cords of EAE induced mice (10%, Figure 2C) in comparison to draining lymph nodes (3%, Figure 2B) or spleen (2%, Figure 2A), and may therefore suggest that CXCR6+ CD4+ T cells are associated with the manifestation of disease.
EXAMPLE 3
Generation of recombinant murine CXCRό-Fc fusion protein (CXCR6~IgG) and stable expression in cell lines
MATERIALS AND EXPERIMENTAL PROCEDURES Generation of murine CXCRo-IgG construct
Construction of the nucleic acid vector encoding the CXCRό-Ig fusion protein of the present invention is schematically illustrated in Figures 3A-B. Murine CXCR6 (GenBank Accession No. NM_030712, SEQ ID NO: 12) was subcloned from LPS activated murine PBMC using primers complementary to the extracellular N-terminal (N-ter amino acid coordinates, amino acid 1-39 of CXCR6; SEQ ID NO: 14) domain of the receptor as follow: Sense, cccaagcttatggatgatgggcatcaaga (SEQ ID NO. 15); antisense: ccgctcgagcttgaactttaggaagcgtt (SEQ ID NO. 16). Following sequence verification the amplified PCR product was cloned into a pSec-Tag2 vector (Invitrogen, San Diego, CA). A sequence of IgGl, from the Hinge-CH2-CH3 region to the C-terminal end of the chain (SEQ ID NO: 6) was ligated to the plasmid down stream of the CXCR6 to create a fusion protein CXCRo-IgG (SEQ ID NO: 18).
Generation of stable murine CXCR6-IgG-expressing cell lines
The pSec-CXCR6-IgG plasmid was co-transfected into DG44 Chinese hamster ovary (CHO) cells that have a double deletion for the dihydrofolate reductase (DHFR) gene (DG44 CHO DHFR7" cells ATCC Accession No. CRL-9096), with CHO DHFR minigene vector, which transfects DHFR-deficient CHO cells with high efficiency, using jet PEI (Polypluse transfection - Illkirch Cedex, France) according the manufacturer's protocol. Stably transfected cells were selected in a culture medium (MEM-alpha) containing hygromycine (200 μg/ml) and increasing doses of methotrixate (2.5 nM to 0.1 mM). The CXCRo-IgG fusion protein was purified from DHFR-positive transfectant supernatants by a Ni-NTA column (Qiagen) and verified by coomassie staining (Figure 4B) and by western blot analysis (Figure 4A) using mouse anti-myc (Santa Cruz Biotechnology) as primary antibody and goat anti mouse HRP (Jackson immuno research) secondary antibody. Of note, the fusion polypeptide CXCRo-IgG is a 42Kd polypeptide the higher band is a polypeptide dimmer.
EXAMPLE 4 CXCRo-IgG specifically binds CXCL16
MATERIALS AND EXPERIMENTAL PROCEDURES ELISA
The binding specificity of the murine CXCRo-IgG fusion protein to various commercially available murine recombinant chemokines (R&D Systems, Minneapolis, NM including CXCL 16, MCP-I, MIP- lα (CCL3), MIP- lβ (CCL4) and RANTES (CCL5) was determined by direct ELISA as follows: 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated with 20 ng/ml of murine recombinant chemokines overnight and blocked with 1% BSA in PBS. Wells were washed and incubated with 100 μg/ml CXCRo-IgG overnight. Wells were washed and the presence of CXCR6- IgG was detected with mouse anti-myc and goat anti-mouse IgG - HRP (Jackson ImmunoResearch). Results are shown as O.D. reading at 450 nm. Where indicated, the soluble receptor CCR2-IgG was added instead of CXCRo-IgG.
RESULTS
Binding of the N-terminal domain of CXCRo-IgG to several murine chemokines was established. As shown in Figure 5, only CXCL 16 bound specifically to CXCRo-IgG. The soluble receptor CCR2IgG was added as a positive control.
EXAMPLE 5
CXCRo-IgG treatment suppresses ongoing Experimental Autoimmune Encephalomyelitis (EAE) in mice
MATERIALS AND EXPERIMENTAL PROCEDURES
Induction of EAE in mice and suppression of the ongoing disease with CXCRo-IgG. Two groups of C57/B mice (6 mice in each group) were subjected to active induction of EAE by MOGp35-55 (myelin oligodendrocyte glycoprotein) in CFA (heat-killed Mycobacterium tuberculosis) on day 0 and received Pertussis toxin on days 0 and 2 to induce disease. One day following the onset of disease (day 12), these mice were treated with repeated intravenous administrations (every other day) of 300 μg/mouse of either a CXCRβ-IgG or isotype matched IgG. An observer blind to the experimental procedure scored EAE daily for clinical manifestation of disease.
RESULTS
Administration of CXCRo-IgG to EAE induced mice initiated EAE remission without residual sign of disease while mice treated with control IgG developed severe EAE (Figure 6). EXAMPLE 6 CXCRo-IgG treatment alters spinal cord histology of EAE induced mice
MATERIALS AND EXPERIMENTAL PROCEDURES
Induction of EAE in mice and spinal cord sample collection
Two groups of C57/B mice (3 mice in each group) were subjected to active induction of EAE by MOGp35-55 (myelin oligodendrocyte glycoprotein). Beginning one day after the onset of disease (day 12), these mice were treated with repeated intravenous administrations (every other day) of 300 μg/mouse of either a CXCR6- IgG or isotype matched IgG. On day 22, spinal cords were removed from these mice and from naive mice, which were not subjected to EAE induction.
Histochemistry of spinal cords The spinal cords as detailed above were fixed in 10 % NBF, dehydrated in ethanol and chloroform and blocked in paraffin. The spinal cords were then cut in a microtome, mounted on a microscope slide, stained with hematoxylin and eosin, and examined using an Olympus microscope.
RESULTS
As shown in Figures 7A-B, lumbar spinal cords of a naϊve mice and EAE- induced mice were subjected to histological analysis. Histochemistry of lumbar spinal cords of EAE induced mice showed that no treatment (control isotype matched antibody, IgGl, Figures 7C-D) resulted in a large amount of infiltrating cells while treatment with the soluble receptor CXCR6-IgG resulted in only a few infiltrating cells. EXAMPLE 7
CXCRo-IgG treatment of EAE induced mice alters the cytokine profile of EAE splenocytes ex-vivo
MATERIALS AND EXPERIMENTAL PROCEDURES
Induction of EAE in mice and EAE splenocyte reactivation ex-vivo for cytokine production.
Three groups of C57/B mice (three mice in each group) were subjected to active induction of EAE by MOGp35-55 (myelin oligodendrocyte glycoprotein) as was previously described [Kassiotis and Kollias, J Exp Med (2001) 193(4):427-434].
Beginning one day after the onset of disease (day 12), these mice were treated with repeated intravenous administrations (every other day) of 300 μg/mouse of either a
CXCRβ-IgG, isotype matched IgG or PBS. On day 9 the splenocytes were harvested and were restimulated for 72 hours with 50 μg/ml MOGp35-55 or with cell medium.
The supernatants were analyzed by ELISA for cytokine production.
ELISA
Supernatants were assayed for the protein secretion level of IL-2, IFN-γ, TNF- α and IL- 12 using the commercially available ELISA kits according to the manufacturer's protocols (Biolegend).
RESULTS
As clearly shown in Figures 8A-D, primary T cells isolated from mice treated with CXCRo-IgG show a significantly reduced production of IL-2 in response to the target autoimmune antigen (MOGp35-55, Figure 8A, pO.OOl). Similar results were recorded for IFN-γ production (Figure 8B, p<0.001), TNF-α production (Figure 8C, pO.OOl) as well as IL- 12 production (Figure 8D, pO.OOl). During a CD4+ T cell mediated immune response, vast majority of IL-2 is produced by the effector inflammatory ThI cells [Abbas et al., Nature (1996) 383:787-93]. TNF-α and IL-12 are key pro-inflammatory cytokines in autoimmune inflammatory disorders
[Feldmann et al., Int Rev Immunol (1998) 17:217-28]. Thus, the significant reduction in IL-2 production (Figure 8A) together with the reduction in IFN-γ (Figure 8B) may explain, in part, the protective competence of CXCRo-IgG.
EXAMPLE 8
CXCRo-IgG treatment of EAE induced mice does not initiate macrophage depletion
MATERIALS AND EXPERIMENTAL PROCEDURES
Induction of EAE in mice and sample collection
Two groups of C57/B mice (three mice in each group) were subjected to active induction of EAE by MOGp35-55 (myelin oligodendrocyte glycoprotein) as was previously described [Kassiotis and Kollias, J Exp Med (2001) 193(4):427-434].
On day 16, spleens from CXCRo-IgG treated mice or untreated mice were removed for further analysis.
FACS staining and analysis
Splenocytes were isolated and washed with PBS. Red blood cells were lysed using RBC Lysis buffer. Splenocytes were stained with rat anti-mouse CDl IB-FITC antibody (Serotec) and analyzed by FACS.
RESULTS
Figures 9A-B clearly show there was no significant difference in relative number of CDl Ib+ cells in mice treated with CXCRo-IgG compared to control mice (34 % compared to 26 %, respectively). One of the suggested mechanisms by which CXCRo-IgG exerts its therapeutic effect is by deleting the relative number of CDl lb+ macrophages/dendritic cells which present antigens to T cells. The present inventors showed that CXCRo-IgG does not lead to such elimination, either directly, or indirectly. EXAMPLE 9
Addition ofCXCR6-IgG ex-vivo to primary splenocytes proliferating in response to PMA decreases the number oflFN-gamma producing T cells
MATERIALS AND EXPERIMENTAL PROCEDURES
FACS staining and analysis
Spleen cells were isolated from naive C57BL/6 mice and incubated for 1 hour with MOGp35-55 alone or along with 1 μg CXCRo-IgG. Splenocytes were stimulated for 5 hours with 30 nM PMA and 1 μM ionomycin. Splenocytes were double stained with rat anti-mouse CXCR6 (R&D, Minneapolis, MN) and rat anti- mouse CD4-FITC antibodies (BD Pharmingen, BD Biosciences). Splenocytes were then subjected to intracellular staining of IL-4 and IFN-γ using anti-mouse IL-4 and anti-mouse IFN-γ (Pharmingen, BD Immunocytometry Systems) according to the protocol previously, described [Goldberg et al., J Immunol (2004) 173:6465-6471]. Results were analyzed by FACS.
RESULTS
Figures 10A-D show a significantly reduced number of IFN-γhlgh IL-4low producing T cells in cultures supplemented with CXCRo-IgG. Results indicate that the number of IFN-γhlgh IL-4low producing T cells decreased from 14.5 % in splenocytes treated by MOGp35-55 alone (Figure 10A) to 4.5 % in cultures supplemented with CXCR6-IgG (Figure 10C). Similar results were documented for CXCR6 expressing T cells. Thus, the number of IFN-γ11'811 IL-4low cells decreased from 47 % in cultures treated by MOGp35-55 alone (Figure 10B) to 22 % in cultures supplemented with CXCRo-IgG (Figure 1 OD).
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications and GenBank Accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application or GenBank Accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:
1. An isolated soluble polypeptide comprising a heterologous amino acid sequence conjugated to a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, said CXCR6 amino acid sequence being capable of binding CXCL16, and wherein the molecule is non-immunogenic in a human subject.
2. An isolated soluble polypeptide comprising an amino acid sequence which comprises an N-terminus domain of CXCR6, said CXCR6 amino acid sequence being capable of binding CXCL 16.
3. The isolated soluble polypeptide of claim 1 or 2 being attached to a non-proteinaceous moiety.
4. An isolated soluble polypeptide comprising at least two CXCR6 amino acid sequences each being capable of binding CXCL 16.
5. An isolated soluble polypeptide comprising a tag attached to a CXCR6 amino acid sequence comprising an N-terminus domain of CXCR6, said CXCR6 amino acid sequence being capable of binding CXCLl 6.
6. An isolated soluble polypeptide comprising an amino acid sequence as set forth in SEQ ID NO. 4 or 8.
7. An isolated polynucleotide comprising a nucleic acid sequence encoding any of the soluble polypeptides of claim 1, 2, 4, 5 or 6.
8. A pharmaceutical composition comprising the soluble polypeptide of claims 1, 2, 3, 4 or 6 and a pharmaceutically acceptable carrier.
9. A method of treating a CXCR6/CXCL16 associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the molecule of claim 1, 2, 3, 4 or 6, thereby treating the CXCR6/CXCL16 associated disease in the subject.
10. Use of the soluble polypeptide of claim 1, 2, 3, 4 or 6, for the manufacture of a medicament identified for treating a CXCR6/CXCL16 associated disease.
11. A method of isolating CXCR6 ligand from a biological sample, the method comprising:
(a) contacting the biological sample with the soluble polypeptide of claim 1, 2, 3, 4, 5, 6, or 7 such that the CXCR6 ligand and the soluble polypeptide form a complex; and
(b) isolating said complex to thereby isolate the CXCR6 ligand from the biological sample.
12. The soluble polypeptides, pharmaceutical compositions, methods and uses of any of claims 2, 3, 4, 6, 8, 9 or 10, wherein the soluble polypeptide is non- immunogenic in a human subject.
13. The soluble polypeptides, pharmaceutical compositions, methods and use of any of claims 1, 2, 3, 4, 6, 8, 9 or 10, wherein a binding affinity of said CXCR6 to said CXCL16 ligand is above K0=IO"6 M.
14. The soluble polypeptides, pharmaceutical compositions, methods and use of any of claims 1, 3, 7, 8, 9 or 10, wherein said heterologous amino acid sequence comprises an immunoglobulin amino acid sequence.
15. The soluble polypeptides, pharmaceutical compositions, methods and use of any of claims 1, 2, 3, 4, 5, 7, 8, 9, or 10, wherein said CXCR6 amino acid sequence is as set forth in SEQ ID NO: 4.
16. The soluble polypeptides, pharmaceutical compositions, methods and use of claim 15, wherein said CXCR6 amino acid sequence is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 3 or 7.
17. The soluble polypeptides, pharmaceutical compositions, methods and use of any of claims 1, 2, 3, 4, 5, 7, 8, 9, or 10, wherein said CXCR6 amino acid sequence is no longer than 50 amino acids in length.
18. The method or use of any of claims 9 or 10, wherein the CXCR6/CXCL16 associated disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, hematological diseases, cardiovascular diseases, disorders of the peripheral and central nervous system, genitourinary diseases, respiratory diseases and cancer.
19. The soluble polypeptide or method of any of claims 5 or 11, wherein said tag is an epitope tag.
20. The soluble polypeptide or method of any of claims 5 or 11, wherein the soluble polypeptide of claim 5 is attached to a solid support.
21. The soluble polypeptide , pharmaceutical composition, method or use of any of claims 3, 8, 9, or 10, wherein said non-proteinaceous moiety is selected from the group consisting of polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA).
22. A method of treating Multiple Sclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent capable of reducing activity and/or expression of CXCR6/CXCL16 or of an effector thereof, thereby treating Multiple Sclerosis in the subject.
23. Use of an agent capable of reducing activity and/or expression of CXCR6/CXCL16 or of an effector thereof, for the manufacture of a medicament identified for treating Multiple Sclerosis.
24. A method of diagnosing Multiple Sclerosis in a subject, the method comprising, detecting expression and/or activity of CXCR6 in cells of the subject, wherein expression and/or activity of said CXCR6 in said cells of the subject above a predetermined threshold is indicative of Multiple Sclerosis in the subject.
25. The method of claim 24, wherein said detecting expression of said CXCR6 is effected by detecting CXCR6 protein.
26. The method of claim 25 wherein said detecting expression of said CXCR6 protein is effected via an assay selected from the group consisting of immunohistochemistry, ELISA, RIA, Western blot analysis, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.
27. The method of claim 24, wherein said detecting expression of said CXCR6 is effected by detecting CXCR6 niRNA.
28. The method of claim 27, wherein said detecting expression of said CXCR6 mRNA is effected via an assay selected from the group consisting of PCR, RT-PCR, chip hybridization, RNase protection, in-situ hybridization, primer extension, Northern blot and dot blot analysis.
29. A pharmaceutical composition comprising as an active ingredient an anti-Multiple Sclerosis drug and an agent capable of reducing activity and/or expression of CXCR6/CXCL16 and/or of an effector thereof and a pharmaceutically acceptable carrier.
30. The method, use or pharmaceutical composition of claim 22, 23 or 29, wherein said agent is selected from the group consisting of: (i) an oligonucleotide directed to an endogenous nucleic acid sequence expressing said CXCR6/CXCL16 or said effector thereof;
(ii) a chemical inhibitor directed to said CXCR6 or said effector thereof; (iii) a neutralizing antibody directed at CXCR6/CXCL16 or said effector thereof; and (iv) a non-functional derivative of said CXCR6 or said effector thereof.
31. The method, use, pharmaceutical composition of claim 30, wherein said non-functional derivative of said CXCR6/CXCL16 comprise any of the soluble polypeptides of claim 1, 2, 3, 4 or 6.
32. The pharmaceutical composition of claim 29, wherein said anti- Multiple Sclerosis drug is selected from the group consisting of Interferon Beta Ia, Interferon Beta Ib, Glatiramer Acetate, Mitoxantrone, MethylPrednisolone, Prednisone, Prednisolone, Dexamethasone, Adreno-corticotrophic Hormone (ACTH) and Corticotropin.
PCT/IL2008/000663 2007-05-31 2008-05-13 Compositions and methods for treating cxcr6/cxcl16 associated diseases Ceased WO2008146272A2 (en)

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