[go: up one dir, main page]

WO2008146272A2 - Compositions et procédés pour traiter des maladies associées à cxcr6/cxcl16 - Google Patents

Compositions et procédés pour traiter des maladies associées à cxcr6/cxcl16 Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
cxcr6
acid sequence
amino acid
cxcl16
diseases
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IL2008/000663
Other languages
English (en)
Other versions
WO2008146272A3 (fr
Inventor
Nathan Karin
Gizi Wildbaum
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rappaport Family Institute for Research in the Medical Sciences
Original Assignee
Rappaport Family Institute for Research in the Medical Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rappaport Family Institute for Research in the Medical Sciences filed Critical Rappaport Family Institute for Research in the Medical Sciences
Publication of WO2008146272A2 publication Critical patent/WO2008146272A2/fr
Publication of WO2008146272A3 publication Critical patent/WO2008146272A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des molécules solubles. Ainsi, par exemple, l'invention concerne un polypeptide soluble comprenant une séquence d'acides aminés hétérologues conjuguée à une séquence d'acides aminés CXCR6 comprenant un domaine N terminal de CXCR6, la séquence d'acides aminés CXCR6 étant capable de lier CXCL16, et la molécule étant non immunogène dans un sujet humain. L'invention concerne également l'utilisation de telles molécules dans le traitement de maladies inflammatoires de manière générale et de la sclérose en plaques en particulier.
PCT/IL2008/000663 2007-05-31 2008-05-13 Compositions et procédés pour traiter des maladies associées à cxcr6/cxcl16 Ceased WO2008146272A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US92479907P 2007-05-31 2007-05-31
US60/924,799 2007-05-31

Publications (2)

Publication Number Publication Date
WO2008146272A2 true WO2008146272A2 (fr) 2008-12-04
WO2008146272A3 WO2008146272A3 (fr) 2009-01-29

Family

ID=39712697

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2008/000663 Ceased WO2008146272A2 (fr) 2007-05-31 2008-05-13 Compositions et procédés pour traiter des maladies associées à cxcr6/cxcl16

Country Status (1)

Country Link
WO (1) WO2008146272A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011068870A3 (fr) * 2009-12-01 2011-10-27 President And Fellows Of Harvard College Modulation de l'activité d'effecteur spécifique de l'antigène de cellules nk par la modulation de cxcr6 (cd186)
US8263064B2 (en) 2007-06-04 2012-09-11 Rappaport Family Institute For Research In The Medical Sciences Method of suppressing disease severity of multiple sclerosis using chemokine CXC11
WO2014159813A1 (fr) 2013-03-13 2014-10-02 Moderna Therapeutics, Inc. Molécules polynucléotidiques à longue durée de vie
CN112243380A (zh) * 2018-04-09 2021-01-19 儿童医疗中心有限公司 用于治疗自身免疫性疾病的方法
US20210102166A1 (en) * 2019-10-03 2021-04-08 Dana-Farber Cancer Institute, Inc. Compositions and methods for modulating and detecting tissue specific th17 cell pathogenicity
CN114460290A (zh) * 2022-03-16 2022-05-10 苏州善诺智能科技有限公司 一种苯乙烯-马来酸酐共聚物及其复合物的应用
CN116773825A (zh) * 2022-08-19 2023-09-19 天津云检医学检验所有限公司 诊断急性川崎病的血液生物标志物和方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6319675B1 (en) * 1999-11-24 2001-11-20 Millennium Pharmaceuticals, Inc. Methods for detecting and/or identifying agents which bind and/or modulate function of “bonzo” chemokine receptor
AU2003250221A1 (en) * 2002-08-19 2004-03-11 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with human cxc chemokine receptor 6(cxcr6)
EP1692161B1 (fr) * 2003-11-13 2011-12-21 Bristol-Myers Squibb Company Polypeptides du récepteur de chimiokine chimérique

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8263064B2 (en) 2007-06-04 2012-09-11 Rappaport Family Institute For Research In The Medical Sciences Method of suppressing disease severity of multiple sclerosis using chemokine CXC11
WO2011068870A3 (fr) * 2009-12-01 2011-10-27 President And Fellows Of Harvard College Modulation de l'activité d'effecteur spécifique de l'antigène de cellules nk par la modulation de cxcr6 (cd186)
US9872905B2 (en) 2009-12-01 2018-01-23 President And Fellows Of Harvard College Modulation of NK cell antigen specific effector activity by modulation of CXCR6 (CD186)
WO2014159813A1 (fr) 2013-03-13 2014-10-02 Moderna Therapeutics, Inc. Molécules polynucléotidiques à longue durée de vie
CN112243380A (zh) * 2018-04-09 2021-01-19 儿童医疗中心有限公司 用于治疗自身免疫性疾病的方法
JP2021521121A (ja) * 2018-04-09 2021-08-26 チルドレンズ メディカル センター コーポレーションＣｈｉｌｄｒｅｎ’Ｓ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ Ｃｏｒｐｏｒａｔｉｏｎ 自己免疫疾患を処置するための方法
EP3773693A4 (fr) * 2018-04-09 2022-03-16 The Children's Medical Center Corporation Méthode de traitement d'une maladie autoimmune
US20210102166A1 (en) * 2019-10-03 2021-04-08 Dana-Farber Cancer Institute, Inc. Compositions and methods for modulating and detecting tissue specific th17 cell pathogenicity
US12195725B2 (en) * 2019-10-03 2025-01-14 Dana-Farber Cancer Institute, Inc. Compositions and methods for modulating and detecting tissue specific TH17 cell pathogenicity
CN114460290A (zh) * 2022-03-16 2022-05-10 苏州善诺智能科技有限公司 一种苯乙烯-马来酸酐共聚物及其复合物的应用
CN116773825A (zh) * 2022-08-19 2023-09-19 天津云检医学检验所有限公司 诊断急性川崎病的血液生物标志物和方法
CN116773825B (zh) * 2022-08-19 2023-12-01 天津云检医学检验所有限公司 诊断急性川崎病的血液生物标志物和方法

Also Published As

Publication number Publication date
WO2008146272A3 (fr) 2009-01-29

Similar Documents

Publication Publication Date Title
JP6979423B2 (ja) キメラサイトカイン受容体
JP7010473B2 (ja) Ｌｙｍ－１およびｌｙｍ－２標的化ｃａｒ細胞免疫療法
CN112969713A (zh) 结合结构域
US20030021780A1 (en) Diagnosis and therapy of antibody-mediated inflammatory autoimmune disorders
SK288603B6 (sk) Polypeptid a protilátka viažuca sa na polypeptid
WO2007072866A1 (fr) Anticorps anti-ilt7
WO2008146272A2 (fr) Compositions et procédés pour traiter des maladies associées à cxcr6/cxcl16
US10066014B2 (en) Anti CD84 antibodies, compositions comprising same and uses thereof
CN1984672A (zh) Nkg2d的调节
JP2009514528A (ja) Ｎｋｇ２ｄ陽性ｃｄ４＋細胞による免疫応答の負の免疫調節法
US9611324B2 (en) Compositions and methods for diagnosing and treating an inflammation
US20130115217A1 (en) Antibodies against hmgb1 and fragments thereof
WO2005108428A2 (fr) Variants d'epissage de cd40 et leurs utilisations
CN112451661A (zh) α-烯醇化酶特异性抗体及其在免疫疾病治疗中的使用方法
WO2010103517A1 (fr) Compositions solubles pour le traitement de maladies associées aux ligands cxcr3
EP1601374B2 (fr) Compositions et methodes de diagnostic et de traitement d'une inflammation
WO2006120678A2 (fr) Nouvelles sequences de galectine, et compositions et procedes utilisant ces sequences pour traiter ou diagnostiquer une arthrite ou d'autres maladies inflammatoires chroniques
WO2010081787A1 (fr) Antagonisme amélioré du tnfα, prophylaxie et thérapie avec nécrose d'organe réduite
CN100352497C (zh) 应用特异性结合cd40cr(cd40 配体)的抗体抑制体液免疫
EP4538285A1 (fr) Variant de pd-1 et son utilisation
CN119866227A (zh) 用于靶向调节性t细胞以治疗化脓性汗腺炎的瓜氨酸化抗原特异性嵌合抗原受体
WO2025191260A1 (fr) Traitement d'une maladie auto-immune par co-expression de cd19-car et de bcma-car
WO2006007565A2 (fr) Nouveau variants cd40
CN120131955A (zh) PILRα/CD99通路抑制剂及其应用
US20170304436A1 (en) Novel epitope as a target for therapy of inflammatory autoimmune diseases and graft rejection

Legal Events

Date Code Title Description
DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08751352

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08751352

Country of ref document: EP

Kind code of ref document: A2