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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4726—Lectins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to novel galectin sequences and use thereof in treatment or diagnosis of arthritis and other chronic inflammatory diseases.
• Rheumatoid Arthritis is a chronic, systemic inflammatory disease primarily involving the joints. It affects 1-3 % of the population with a female to male ratio of 3:1. Constitutional symptoms may include malaise, fever and weight loss. The disease characteristically begins in the small joints of the hands and feet, usually progressing in a centripetal and symmetric fashion. Deformation of joints and disability are very common, but the disease may also decrease expected life span by an average of ⁇ 9 years in the more active forms.
• Extra-articular manifestations of the rheumatoid process may cause significant morbidity and include vasculitis, atrophy of the skin and muscle, subcutaneous nodules, lymphadenopathy, splenomegaly and leukopenia, neuropathy, cervical myelopathy, ophthalmic complications, Sjogren syndrome, lung and heart involvement and amyloidosis.
• the inflammation of the synovial joints characterizing RA is the result of hype ⁇ lasia of synovial fibroblasts and infiltration of lymphocytes, macrophages and plasma cells, all of which manifest signs of activation. These cells proliferate abnormally, invade bone and cartilage, produce an elevated amount of pro-inflammatory cytokines, metalloproteinases and trigger osteoclast formation and activation. Some of the pathophysiological consequences of the disease are explained by inadequate apoptosis, which may promote the survival of autoreactive T cells, macrophages or synovial fibroblasts [Rabinovich G. A., Mem Inst Oswaldo Cruz., 95:225, (2000)].
• pathological cells such as inflammatory cells
• Differences in cell surface molecules or signal transduction pathways between pathological cells and their normal counterparts can be used as a handle for selective destruction of the former.
• the existence of such discrete target entities in pathological cells is not an obvious phenomenon.
• TCR T cell receptor
• MHC major histocompatibility
• a different tactic is to target pro-inflammatory molecules that are involved in more progress stage of the process, when there is a non-specific influx of leukocytes, attracted by chemokines and activated by cytokines.
• autoimmune diseases can be controlled by targeting inflammation- supportive molecules, such as selectins, integrins, IL-12, TNF ⁇ and NFkB, using specific antibodies or anti- sense oligonucleotide [Taylor P. C, Curr. Pharm. Des., 9:1095, (2003); Kevorkov N. N. and Futlik D. M., Russ. J. Immunol., 5:5, (2000); Louie S.G., et al. Am. J. Health Syst. Pharm. 60:346, (2003)].
• inflammation- supportive molecules such as selectins, integrins, IL-12, TNF ⁇ and NFkB
• Galectins comprise a family of proteins that are widely expressed in mammals. All galectins share the ability to bind ⁇ -galactosides through an evolutionariry conserved sequence motif in the carbohydrate-binding site (1). They have a cytoplasmic or nuclear localization and despite of lack of signal peptide, they can be externalized as soluble proteins by non-classical secretory mechanisms (2,3). In mammals, ten galectins have been characterized (3,4). Two groups of galectins are distinguished according to the number of lectin domains. Most of them, including galectin- 1, 2, 3, 5, 7 and 10, have a single lectin domain. Galectin-4, 6, 8 and 9 belong to the second group and are characterized by two lectin domains linked by a hinged peptide (4- 7).
• galectins The exact role(s) of galectins is not clearly established, but they are suspected to modulate several cellular functions such as physiological (8) and malignant cell adhesion, cell proliferation (9), apoptosis (10), metastasis seeding (11) and immune function
• compositions and methods containing these novel galectins may be utilized for treatment and diagnostics of RA, or restricted targeting of inflammation- supportive molecules.
• galectin-8 is capable of reducing the inflammatory response in human synovial tissue.
• compositions and methods containing this agent can be utilized for treating RA and other chronic inflammatory diseases.
• a pharmaceutical composition comprising, as an active ingredient, galectin-8 or a functional equivalent or derivative thereof and at least one type of a disease-modifying antirheumatic drug (DMARD) and a pharmaceutically acceptable carrier.
• DMARD disease-modifying antirheumatic drug
• a pharmaceutical composition comprising, as an active ingredient, galectin-8 or a functional equivalent or derivative thereof and at least one type of a nonsteroidal anti- inflammatory drug (NTHE) and a pharmaceutically acceptable carrier.
• NTHE nonsteroidal anti- inflammatory drug
• an isolated polypeptide comprising a galectin-8 sequence being devoid of the amino acid sequence region encompassed by amino acid coordinates 115-150 of SEQ ID NO: 4.
• a pharmaceutical composition comprising, as an active ingredient, a galectin-8 sequence devoid of the amino acid sequence region encompassed by amino acid coordinates 115- 150 of SEQ ID NO: 4 and a pharmaceutically acceptable carrier.
• an article-of-manufacture comprising packaging material and a pharmaceutical composition identified for reducing inflammatory response, being contained within the packaging material, the pharmaceutical composition including, as an active ingredient, a galectin-8 or a variant or a derivative thereof and a pharmaceutically acceptable carrier.
• a method of reducing inflammatory response in an individual comprising providing to the individual a galectin-8 polypeptide or a variant or derivative thereof, thereby reducing the inflammatory response in the individual.
• the galectin-8 is set forth in SEQ ID NO: 4.
• the galectin-8 variant is set forth in SEQ ID NO: 6 or 8.
• providing is effected by expressing the polypeptide within cells of the individual.
• the cells of the individual are synovial fluid cells.
• providing is effected by administration of a pharmaceutical composition comprising a galectin-8 or a variant or a derivative thereof.
• administration is oral, injection or topical administration.
• administration by injection is direct injection into a joint of the individual.
• providing is effected by: (i) isolating synovial fluid cells from the individual; (ii) transforming the synovial fluid cells with an expression construct capable of expressing the galectin-8 polypeptide or the variant or derivative thereof in the synovial fluid cells; and (iii) administering the synovial fluid cells resultant from step (b) into the individual.
• a population of cells comprising synovial fluid cells transformed to express galectin-8 or a functional equivalent or a variant or derivative thereof.
• an isolated polynucleotide encoding a polypeptide comprising a galectin-8 sequence being devoid of the amino acid sequence region encompassed by amino acid coordinates 115-150 of SEQ
• polynucleotide is set forth in SEQ ID NO: 5 or 7.
• nucleic acid construct comprising the polynucleotide.
• nucleic acid construct further comprising a promoter for regulating expression of the polynucleotide.
• a cell comprising the nucleic acid construct.
• a method of detecting an inflammatory response in an individual comprising identifying in biological sample obtained from the individual a transcription and/or translation product of a galectin-8 variant, wherein a presence of the transcription and/or translation product is indicative of an inflammatory response.
• a method of diagnosing an individual having an inflammatory disease comprising: (a) obtaining a biological sample from the individual; and (b) detecting in the biological sample a transcription and/or translation product of a galectin-8 variant, wherein a presence of the transcription and/or translation product indicates presence of an inflammatory disease.
• a pair of oligonucletides capable of directing PCR amplification of a polynucleotide sequence encoding a galectin-8.
• the pair of oligonucletides are set forth SEQ ID NO: 1 and 2.
• a kit for diagnosing RA patients comprising the pair of nucleotide of claim 33 and reagents for detecting a PCR product.
• a polynucleotide probe comprising a nucleic acid sequence specifically hybridizable with a polynucleotide sequence encoding a galectin-8 variant.
• kits for diagnosing RA patients comprising the polynucleotide probe of claim 37 and reagents for detecting a hybridization product.
• the polynucleotide probe is labeled with a detectable moiety.
• the detectable moiety is selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a light-emitting moiety and a radioactive moiety.
• an antibody comprising an antigen recognition sequence capable of specifically binding a galectin-8 variant.
• the galectin-8 variant is set forth in SEQ ID NO: 6 or 8.
• kits for diagnosing RA patients comprising the antibody of claim 42 and reagents for detecting antibody binding.
• the polynucleotide probe is labeled with a detectable moiety.
• the detectable moiety is selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a light-emitting moiety and a radioactive moiety.
• FIG. 1 is a bar graph which demonstrates the effect of galectin-8 (GAL) on IL-l ⁇ levels found in synovial tissue culture medium supplemented with 3 ⁇ g/ml lipopolysaccharide (LPS).
• GAL galectin-8
• LPS lipopolysaccharide
• the graph shows decrease of IL-l ⁇ levels in media of synovial tissue cultures supplemented with galectin-8 as compared with IL-l ⁇ level in medium of cells with no galectin-8 addition (LPS).
• Addition of 0.125 ⁇ M (GAL 0.125 ⁇ M), 0.25 ⁇ M (GAL 0.25 ⁇ M) and 0.5 ⁇ M (GAL 0.5 ⁇ M) galectin-8 leads to approximately 9%, 42% and 67% decrease of IL-l ⁇ levels respectively, illustrating that the effect of galectin-8 on IL-l ⁇ levels is dose dependent.
• FIG. 2 is a bar graph which demonstrates the effect of galectin-8 (GAL) on TNF ⁇ levels in media of synovial tissue cultures supplemented with 3 ⁇ g/ml lipopolysaccharide (LPS).
• GAL galectin-8
• LPS lipopolysaccharide
• the graph shows decrease of TNF ⁇ levels in media of synovial tissue cultures supplemented with galectin-8 as compared with TNF ⁇ level in medium of cells with no galectin-8 addition (LPS).
• Addition of 0.125 ⁇ M (GAL 0.125 ⁇ M), 0.25 ⁇ M (GAL 0.25 ⁇ M), and 0.5 ⁇ M (GAL 0.5 ⁇ M) galectin-8 leads to approximately 2%, 43% and 74% decrease of TNF ⁇ levels respectively, illustrating that the effect of galectin-8 on TNF ⁇ levels is dose dependent.
• FIG. 3 is a bar graph which demonstrates the effect of galectin-8 (GAL) on IL-lra levels found in media of synovial tissue culture medium supplemented with 3 ⁇ g/ml lipopolysaccharide (LPS).
• GAL galectin-8
• LPS lipopolysaccharide
• the graph shows increase of IL-lra levels in media of synovial tissue cultures supplemented with galectin-8 as compared with IL-lra level in medium of cells with no galectin-8 addition (LPS 3 ⁇ g/ml).
• Addition of 0.25 ⁇ M (GAL 0.25 ⁇ M), 0.5 ⁇ M (GAL 0. 5 ⁇ M), and l ⁇ M (GAL l ⁇ M) galectin-8 leads to approximately 4-fold, 9-fold and 22-fold increase of IL-lra levels respectively, illustrating that the presence of LPS in the medium dramatically increases the effect of galectin-8 on IL-lra levels.
• FIGs. 4a-f are plot graphs demonstrating the effect of galectin-8 on the development of apoptosis in synovial fluid cells from RA patients.
• the plot graphs represent flow cytometric analysis of synovial fluid cells from RA patients incubated for 24 h with various concentrations of galectin-8 addition ( Figures 4b-e), no galectin-8 addition ( Figure 4a) or doxorubicine addition ( Figure 4f) followed by staining with Phosphatidyl Serine Detection Kit (IOP-116F FITC-conjugated, IQ Products, The Netherlands) using the manufacturer instructions).
• Phosphatidyl Serine Detection Kit IOP-116F FITC-conjugated, IQ Products, The Netherlands
• FIG. 4a is a plot presenting flow cytometric analysis of synovial fluid cells from RA patients incubated for 24 h with no galectin-8 addition. The plot shows red and green fluorescence of annexin V/PI stained cells, indicating the apoptotic state of untreated synovial fluid cells from RA patients after 24 h incubation. The plot shows 19% of the total cells population in the apoptotic area.
• FIG. 4b is a plot presenting flow cytometric analysis of synovial fluid cells from RA patients incubated for 24 h with 0.03 ⁇ M galectin-8 addition.
• the plot shows red and green fluorescence of annexin N/PI stained cells similar to that of the untreated control.
• the plot shows 17% of the total cells population ' in the apoptotic area, illustrating that addition of 0.03 ⁇ M galectin-8 does not change significantly the apoptosis development in synovial fluid cells from RA patients.
• Figure 4c is a plot presenting flow cytometric analysis of synovial fluid cells from RA patients incubated for 24 h with O.l ⁇ M galectin-8 addition.
• the plot shows red and green fluorescence of annexin N/PI stained cells similar to that of the untreated control.
• the plot shows 20% of the total cells population in the apoptotic area, illustrating that addition of O.l ⁇ M galectin-8 does not change significantly the apoptosis development in synovial fluid cells from RA patients.
• Figure 4d is a plot presenting flow cytometric analysis of synovial fluid cells from RA patients incubated for 24 h with 0.3 ⁇ M galectin-8 addition.
• the plot shows red and green fluorescence of annexin N/PI stained cells similar to that of the untreated control.
• FIG. 4e is a plot presenting flow cytometric analysis of synovial fluid cells from RA patients incubated for 24 h with 1 ⁇ M galectin-8 addition.
• the plot shows red and green fluorescence of annexin V/PI stained cells concentrated toward the apoptotic area compared with untreated control.
• the plot shows 58% of the total cells population in the apoptotic area, illustrating that addition of l ⁇ M galectin-8 resulted in a 3-fold increase in apoptosis development in synovial fluid cells from RA patients.
• Figure 4f is a plot presenting flow cytometric analysis of synovial fluid cells from RA patients incubated for 24 h with doxorubicine (150 ⁇ g/ml). The plot shows red and green fluorescence of annexin V/PI stained cells. The plot shows 80% of the total cells population in the necrotic area, illustrating good quality of the experimental staining procedure. DESCRIPTION OF THE PREFERRED EMBODIMENTS
• the present invention is of galectin-8 and novel variants thereof, which can be used in treatment, diagnosis and prognosis of chronic inflammatory diseases, such as rheumatoid arthritis (RA).
• RA rheumatoid arthritis
• RA is a chronic inflammatory and destructive joint disease, which commonly leads to significant disability and a consequent reduction in quality of life [Gabriel, S. E., Rheum. Dis. Clin. North Am. 27:269, (2001)].
• NSAIDs non-steroidal anti-inflammatory drugs
• DMARDs disease-modifying antirheumatic drugs
• COXs cyclooxygenases
• DMARDs impede both the inflammatory and destructive processes of RA and as such, effective DMARDs treatment does not require additional symptomatic therapy [Smolen, J. S. and G. Steiner, Nat. Rev. Drug Discovery, 2:473 (2003)].
• remission occurs in only 20-25% of DMARDs treated patients, continued DMARD treatment is advised since discontinuation of treatment significantly increases the risk of flares [ten Wolde, S. et al. Lancet 347:347, (1996)].
• DMARDs presently used in RA therapy are drugs based on inhibition of pro-inflammatory cytokines.
• the pro-inflammatory role of cytokines, and the involvement of different cell types and their surface molecules in the pathogenesis of RA, provides the rationale for the development of highly specific therapeutics to target these molecules [Smolen, J. S. and G. Steiner, Nat. Rev. Drug Discovery, 2:473 (2003)].
• TNF Tumor Necrosis Factor
• IL-1 Interlaukin-1
• infliximab and adalimumab which are are antibodies against TNF, and etanercept which is a fusion protein of the TNF receptor II. All of these agents exhibited a therapeutic effect on RA patients, however, the response to treatment in over half the patients participating in clinical trials of these drugs were less than satisfactory. In addition, although remissions were rare in patients who responded to treatment, many side effects were prevalent in such patients [Smolen, J. S. and G. Steiner, Nat. Rev. Drug Discovery, 2:473 (2003)].
• Interleukin 1 is another pro-inflammatory cytokine which has been implicated in the progression of RA. Binding of IL-1 to IL-1 receptor I (IL-IRI) allows the engagement of the IL-IR accessory protein (IL-lRacP) and subsequent cell signaling and activation.
• the natural inhibitor IL-1 receptor antagonist (IL-lra) which belongs to the IL- 1 family, competes with IL-1 for its receptor but does not allow engagement of IL-lRacP, thereby blocking activation of signal transduction mechanisms [Arend, W. P. et al, Annu. Rev. Immunol. 16:27 (1998)].
• IL-lra The importance of the role of IL-lra in regulating the inflammatory response is exemplified by the occurrence of a destructive arthritis in IL- lra- deficient animals [Horai, R. et al, J. Exp. Med. 191:313, (2000)].
• Anakinra a recombinant form of IL-lra, is an RA drug which targets IL-1 -blockage [Smolen, J. S. and G. Steiner, Nat. Rev. Drug Discovery, 2:473 (2003)].
• galectin-8 and novel variants thereof which were identified as a part of the present study, are capable of reducing the inflammatory response in human synovial tissue due to their ability to inhibit the release of the pro-inflammatory cytokines TNF- ⁇ and IL-l ⁇ , to increase the production of the anti-inflammatory cytokine IL-lra and to induce apoptosis in synovial fluid cells.
• inflammatory response refers to an immune response which results in inflammation, typically occurring as a result of injurious stimuli including infection, burns, trauma, neoplasia, autoimmune signals and exposure to chemicals, heat or cold or any other harmful stimulus.
• An inflammatory response according to the present invention refers to an acute phase response and a chronic inflammation.
• the term "individual” refers to an individual, who may benefit from the present invention such as a mammal (e.g., canine, feline, ovine, porcine, equine, bovine, human), preferably a human individual.
• the method of this aspect of the present invention is effected by identifying in a biological sample obtained from the individual, a transcription and/or translation product of a galectin-8 variant, wherein the presence of said transcription and/or translation product is indicative of an inflammatory response.
• transcription and/or translation product of a galectin-8 variant refers to a gene expression product (i.e., RNA or protein) of a galectin-8 variant.
• variants refers to splice variants and allelic variants of galectin-8.
• splice variant refers to alternative forms of RNA transcribed from a galectin-8 gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence.
• the term splice variant is also used herein to denote a polypeptide encoded by a splice variant of an mRNA transcribed from a gene.
• allelic variant refers to two or more alternative forms of a galectin-8 gene occupying the same chromosomal locus.
• allelic variation arises naturally through mutation, and may result in phenotypic polymo ⁇ hism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
• allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
• homologues of the sequences described hereinabove are also utilizable by the present invention.
• Such homologues include for example a polynucleotide which encode a polypeptide having a galectin-8 sequence which is devoid of the amino acid sequence coordinates 115-150 of SEQ ID NO: 4.
• the polypeptide has an amino acid sequence at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 87 %, at least 89 %, at least 91 %, at least 93 %, at least 95 % or more say 100 % homologous to SEQ ID NO: 6 or 8, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.
• NCBI National Center of Biotechnology Information
• the polynucleotide has a nucleic acid sequence at least 50 %, at least 55 %, at least 60 %, at least 65 •%, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 87 %, at least 89 %, at least 91 %, at least 93 %, at least 95 % or more say 100 % identical to SEQ ID NO: 5 or 7, as determined using BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters.
• NCBI National Center of Biotechnology Information
• biological sample refers to a sample of tissue or fluid isolated from an individual, including, but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, synovial cell fluid, tumors, organs such as synovial tissue and also samples of in vivo cell culture constituents (e.g., synovial fluid cells).
• the biological sample is a synovial tissue or a synovial fluid.
• Procedures for obtaining biological samples i.e., biopsying
• biological samples i.e., biopsying
• Such procedures include, but are not limited to, bone biopsy, lymph node biopsy, pleural biopsy, skin biopsy, thyroid biopsy, CT-guided biopsy, joint biopsy, needle aspiration biopsy and breast biopsy.
• a joint biopsy refers to joint or synovial biopsy.
• a sample of the joint lining or synovial membrane or fluid is taken.
• the procedure is effected in a clinical facility by a surgeon.
• a number of approaches are available to perform this biopsy: such as through an incision in the joint; with a scope inserted in the joint; or, more typically, by the insertion of a sha ⁇ instrument through the skin.
• the sample can be taken from any joint, typically the examined joint is the knee.
• a sha ⁇ instrument (trocar) is pushed into the joint space.
• a needle with an attached syringe is inserted into the joint to withdraw fluid for laboratory analysis.
• the surgeon may instill analgesic compounds into the joint and along the needle track before the needle is withdrawn.
• the trocar and then the biopsy needle is inserted and specimens taken. After the specimen is taken, both the trocar and the biopsy needle are removed. Regardless of the procedure employed, once the biological sample is obtained, the presence of the galectin-8 variant
• determination of the level of galectin-8 variant in the biological sample can be effected at the transcriptional level (i.e., mRNA) using an oligonucleotide probe, which is capable of specifically hybridizing to the variant sequence (e.g., SEQ ID Nos: 5 or 7).
• Hybridization of oligonucleotide probes can be detected using a variety of methods known to those of skill in the art (e.g., colorimetric assays, amplification assays and the like).
• 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.
• Oligonucleotides which hybridize specifically with galectin-8 variants, are preferably those directed at unique nucleotide sequences, which are not shared by wild type galectin-8 (e.g., SEQ ID NO: 3).
• nucleotide targets encompassed by the polynucleotide sequence encoding amino acid sequence coordinates 115-150 of SEQ ID NO: 4.
• oligonucleotides designed to hybridize to nucleotide coordinates 193-261 of SEQ ID NO: 5 can be used to detect the presence of galectin-8 RA1 variant (SEQ ID NO: 5) in the biological sample.
• Oligonucleotides designed to hybridize to exon junctions of SEQ ID NO: 7, such as those set forth in SEQ ID NOs: 9 •and 10, PLEASE INSERT, can be used to detect the presence of galectin-8 RA2 variant (SEQ ID NO: 7) in the biological sample.
• 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.
• oligonucleotide of the present invention is of at least 17, at least 18, at least
• 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. Such modifications can oftentimes facilitate oligonucleotide uptake and resistivity to intracellular conditions.
• Specific examples of preferred oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non- natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat.
• 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, fhionophosphoramidates, 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'.
• 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.
• 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-substiruted
• 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 r been shown to mcrease nucleic acid duplex stability by 0.6-1.2°C. [Sanghvi YS et al. (1993) Antisense Research and Applications, CRC Press, Boca Raton 276-278] and are presently preferred base substitutions, even jnore particularly when combined with 2'-O-methoxyethyl sugar modifications.
• Hybridization of short nucleic acids (below 200 bp in length, e.g.
• Dete ⁇ nination of hybridization complexes is well known in the art and may be achieved by any one of several approaches. These approaches are generally based on the detection of a label or marker, such as any 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 inco ⁇ orating 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.
• labeled 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.
• PCR polymerase chain reaction
• RT-PCR RT-PCR-based methods
• galectin-8 variants a pair of oligonucleotides is used, which is specifically hybridizable with the galectin-8 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.
• an oligonucleotide pair of primers which can hybridize with the galectin-8 variants of the present invention is set forth in SEQ ID NOs: 1 and 2 (see Example 1 of the Examples section).
• Such primers will amplify galectin 8 polynucleotides and variants which share flanking sequences (i.e., SEQ ID NOs. 3, 5 and 7).
• identification of each variant can be effected by, for example, sequencing or fine electrophoresis.
• 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 °C.
• Tm melting temperatures
• Methods for detecting minor polynucleotide sequence alterations e.g., such as insertion of T at nucleotide coordinate 494 of SEQ ID NO: 7 can also be used according to this aspect of the present invention.
• Such methods include, but are not limited to, single-strand conformational polymo ⁇ hism (SSCP), denaturing gradient-gel electrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavage analysis (CCM), ribonuclease cleavage (RNAase), and direct sequencing of the target. More direct methods of mutation analysis include but are not limited to allele-specific amplification (ASA), oligonucleotide ligation assay (OLA), primer extension, artificial introduction of restriction sites (AIRS), allele-specific oligonucleotide hybridization (ASO), and variations of these procedures [Cotton, R. G. H. (1993) Mutat Res, 285, 125-144; Forrest, S.
• ASA allele-specific amplification
• OVA oligonucleotide ligation assay
• ASO allele-specific oligonucleotide hybridization
• Detection of galectin-8 variants can also be effected at the protein level.
• a number of protein detection methods are known in the art. Examples include, but are not limited to, chromatography and electrophoretic methods which are preferably used to detect polypeptides mainly based on molecular weight variation and immunodetection assays such as ELISA and western blot analysis, immunohistochemistry and the like, which may be effected using antibodies specific to the galectin-8 variants of the present invention.
• antibodies which specifically interact with the galectin-8 variants of the present invention.
• Such antibodies are directed to, for example, the unique sequence portions of the polypeptide variants of the present invention (e.g., amino acid coordinates 65-87 of SEQ ID NO: 6 and amino acid coordinates 162-170 of SEQ ID NO: 8) or to unique sequences, which bridge the galectin-8 common portion and the unique sequence regions.
• Specific peptides chosen for antibody generation are preferably selected immunogenic (i.e., capable of stimulating an antibody response). Parameters for testing peptide immunogenicity are well known in the art including, but not limited to, foreginess, molecular size, chemical composition and heterogeneity and susceptibility to antigen processing and presentation. .
• sequence analysis software applications are known in the art, which provide an immunogenicity index according to, for example, the Jameson-Wolf algorithm.
• sequence analysis software applications include, but are not limited to, Sciprot (available from www.asiaonline.net.hk/ ⁇ twcbio/ DOCS/1/scPrtein.htm) and Macvector (available from www.accelrys.com/ products/macvector/) as well as the widely utilized GCG package (Genetics Computer Group, Wisconsin).
• antibody as used in this invention includes whole antibody molecules as well as functional fragments thereof, such as Fab, F(ab') , and Fv that are capable of binding with antigenic portions of the target polypeptide.
• functional antibody fragments constitute preferred embodiments of the present invention, and are defined as follows:
• Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
• Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
• (Fab') 2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;
• Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
• SCA Single chain antibody
• Purification of serum immunoglobulin antibodies can be accomplished by a variety of methods known to those of skill in the art including, precipitation by ammonium sulfate or sodium sulfate followed by dialysis against, saline, ion exchange chromatography, affinity or immunoaffinity chromatography as well as gel filtration, zone electrophoresis, etc. (see Goding in, Monoclonal Antibodies: Principles and Practice, 2nd ed., pp. 104-126, 1986, Orlando, Fla., Academic Press). Under normal physiological conditions antibodies are found in plasma and other body fluids and in the membrane of certain cells and are produced by lymphocytes of the type denoted B cells or their functional equivalent.
• Antibodies of the IgG class are made up of four polypeptide chains linked together by disulfide bonds.
• the four chains of intact IgG molecules are two identical heavy chains referred to as H- chains and two identical light chains referred to as L-chains.
• Additional classes include IgD, IgE, IgA, IgM and related proteins.
• a recombinant galectin-8 polypeptide of the present invention may be used to generate antibodies in vitro. More preferably, the recombinant galectin-8 of the present invention is used to elicit antibodies in vivo.
• a suitable host animal is immunized with the recombinant Galectin-8 of the present invention.
• the animal host used is a mouse of an inbred strain.
• Animals are typically immunized with a mixture comprising a solution of the recombinant Galectin-8 of the present invention in a physiologically acceptable vehicle, and any suitable adjuvant, which achieves an enhanced immune response to the immunogen.
• the primary immunization conveniently may be accomplished with a mixture of a solution of the recombinant Galectin-8 of the present invention and Freund's complete adjuvant, said mixture being prepared in the form of a water in oil emulsion.
• the immunization will be administered to the animals intramuscularly, intradermaliy, subcutaneously, intraperitoneally, into the footpads, or by any appropriate route of administration.
• the immunization schedule of the immunogen may be adapted as required, but customarily involves several subsequent or secondary immunizations using a milder adjuvant such as Freund's incomplete adjuvant.
• Antibody titers and specificity of binding to the Galectin-8 can be determined during the immunization schedule by any convenient method including by way of example radioimmunoassay, or enzyme linked immunosorbant assay, which is known as the ELISA assay.
• ELISA assay enzyme linked immunosorbant assay
• lymphocytes may be obtained in large numbers from the spleens of immunized animals, but they may also be retrieved from the circulation, the lymph nodes or other lymphoid organs. Lymphocytes are then fused with any suitable myeloma cell line, to yield hybridomas, as is well known in the art. Alternatively, lymphocytes may also be stimulated to grow in culture, and may be immortalized by methods known in the art including the exposure of these lymphocytes to a virus, a chemical or a nucleic acid such as an oncogene, according to established protocols.
• the hybridomas are cultured under suitable culture conditions, for example in multi-well plates, and the culture supematants are screened to identify cultures containing antibodies that recognize the hapten of choice.
• Hybridomas that secrete antibodies that recognize the recombinant Galectin-8 of the present invention are cloned by limiting dilution and expanded, under appropriate culture conditions.
• Monoclonal antibodies are purified and characterized in terms of immunoglobulin type and binding affinity.
• 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 cell culture or other protein expression systems) of DNA encoding the fragment.
• E. coli or mammalian cells e.g. Chinese hamster ovary cell culture or other protein expression systems
• Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
• antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
• This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
• an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
• Fv fragments comprise an association of V H and VL chains. This association may be noncovalent, as described in Inbar et al. (Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise V H and V L chains connected by a peptide linker.
• sFv single-chain antigen binding proteins
• 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 V domains.
• Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426, 1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat. No. 4,946,778, all of which are hereby inco ⁇ orated, by reference, in entirety.
• CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick and Fry Methods, 2: 106-10, 1991).
• Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
• Humanized antibodies may also comprise residues, which are 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 CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al, Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)].
• Fc immunoglobulin constant region
• 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 import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co- workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• humanized antibodies are chimeric antibodies (U.S. Pat. No.. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
• the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al, J. Immunol., 147(l):86-95 (1991)].
• human monoclonal antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
• Inflammatory diseases include, but are not limited to, chronic inflammatory diseases and acute inflammatory diseases. Inflammatory diseases associated with hypersensitivity
• 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 Histopafhol 2000 Jul;15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998;17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al, Clin Diagn Lab Immunol.
• myasthenic diseases myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine JC. and Honnorat J.
• 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.
• delayed type hypersensitivity examples include, but are not limited to, contact dermatitis and drug eruption.
• T lymphocyte mediating hypersensitivity examples 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 l 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: SI 35), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al, Wien Klin Klin Klin Klinschr 2000 Aug 25;112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S.
• 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 Eisenbarfh 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.
• 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 Immunopafhol 1990 Mar;54 (3):382), primary biliary cirrhosis (Jones DE. Cliii Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et al, Eur J Gasfroenterol Hepatol. 1999 Jun; 11 (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326).
• 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.
• autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al, Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al, Immunol Rev 1999 Jun;169:107).
• infectious diseases 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, 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.
• Cancerous 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 cancerous diseases but are not limited to:
• Myeloid leukemia such as Chronic myelogenous leukemia. Acute myelogenous leukemia with maturation. Acute promyelocytic leukemia, Acute nonlymphocytic leukemia with increased basophils, Acute monocytic leukemia. Acute myelomonocytic leukemia with eosinophilia; Malignant lymphoma, such as Birkitt's Non-Hodgkin's; Lymphoctyic leukemia, such as Acute lumphoblastic leukemia. Chronic lymphocytic leukemia;
• Myeloproliferative diseases such as Solid tumors Benign Meningioma, Mixed tumors of salivary gland, Colonic adenomas; Adenocarcinomas, such as Small cell lung cancer,
• Ewing's tumor other include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, breast, skin, prostate, and ovarian.
• galectin 8 is capable of reducing inflammatory response, probably due to its ability to inhibit the release of the pro-inflammatory cytokines TNF- ⁇ and IL-l ⁇ , to increase the production of the anti -inflammatory cytokine IL-lra and to induce apoptosis in synovial fluid cells, rendering it a valuable tools in a number of therapeutic applications.
• a method of reducing an inflammatory response in an individual is effected by providing to the individual a galectin-8 polypeptide or a variant (as described above) or derivative thereof to thereby reduce the inflammatory response in the individual.
• galectin-8 polypeptide refers to human galectin-8 (SEQ ID NO: 1
• galectin-8 derivative refers to a fragment of the galectin-8 polypeptides and variants and polypeptides having mutations such as deletion, insertion or substitution of one or more amino acids, either naturally occurring or man-induced either randomly or in a targeted fashion. Provision of the above-described polypeptide can be effected by administering it to the individual.
• polypeptide encompasses native polypeptides (either degradation products, synthetically synthesized polypeptides or recombinant polypeptides) and peptidomimetics (typically, synthetically synthesized polypeptides), as well as as peptoids and semipeptoids which are polypeptide analogs, which may have, for example, modifications rendering the polypeptides 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 Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is inco ⁇ orated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
• Natural aromatic amino acids, T ⁇ , Tyr and Phe may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or 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 unusual 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.
• the polypeptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing polypeptide solubility due to their hydroxyl-containing side chain.
• polypeptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with polypeptide characteristics, cyclic forms of the polypeptide can also be utilized.
• the polypeptides 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, classical solution synthesis. These methods are preferably used when the polypeptide 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) and therefore involves different chemistry. Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
• Synthetic polypeptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing.
• Recombinant techniques are preferably used to generate the polypeptides of the present invention since these techniques are better suited for generation of relatively long polypeptides (e.g., longer than 20 amino acids) and large amounts thereof.
• Such recombinant techniques are 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 310:511-514, Takamatsu et al. (1987) EMBO J. 3:17-311, Coruzzi et al. (1984) EMBO J.
• a polynucleotide encoding a polypeptide of the present invention is ligated into a nucleic acid expression vector, which includes the polynucleotide sequence under the transcriptional control of a promoter sequence suitable for directing constitutive tissue specific or inducible transcription in the host cells.
• Polynucleotide sequences which can be used to express the polypeptides of the present invention, include isolated polynucleotides which encode the polynucleotides described above (see Table 1, and SEQ ID NOs: 3, 5 and 7) and homologues thereof (described above).
• an isolated polynucleotide refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
• complementary polynucleotide sequence refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
• genomic polynucleotide sequence refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
• composite polynucleotide sequence refers to a sequence, which is at least partially complementary and at least partially genomic.
• a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences inte ⁇ osing therebetween.
• the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
• Such isolated polynucleotides of the present invention can be qualified using hybridization assays.
• the isolated polynucleotides of the present invention are preferably hybridizable with SEQ ID NO: 5 or 7 under moderate to stringent hybridization conditions.
• Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 10° " cpm 32 P labeled probe, at 65 °C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65°C and whereas moderate hybridization is effected using a hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 10 6 cpm 32 P labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.
• a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 10° " cpm 32 P labeled probe, at 65 °C
• moderate hybridization is effected using
• polynucleotide sequences of the present invention are inserted into expression vectors to enable expression of the recombinant polypeptide.
• the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
• Typical cloning vectors contain transcription and translation initiation sequences (e.g., promoters, enhances) and transcription and translation terminators (e.g., polyadenylation signals). Detailed description of expression vectors is provided hereinbelow.
• prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of the present invention.
• These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide 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 polypeptide coding sequence.
• Mammalian expression systems can also be used to express the polypeptide of the present invention. Bacterial systems are preferably used to produce recombinant polypeptides since they enable a high production volume at low cost.
• a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. For example, when large quantities of polypeptide are desired, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified may be desired. Certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide may also be desirable. Such vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al, Methods in Enzymol. 185:60-89 (1990)].
• vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. Application No: 5,932,447.
• vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.
• the expression of the polypeptide coding sequence can be driven by a number of promoters.
• viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al, Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al, EMBO J. 3:17-311 (1987)] can be used.
• plant promoters can be used such as, for example, the small subunit of RUBISCO [Coruzzi et al, EMBO J. 3:1671- 1680 (1984); and Brogli et al, Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al, Mol. Cell. Biol. 6:559-565 (1986)].
• RUBISCO Reaset al, EMBO J. 3:1671- 1680 (1984); and Brogli et al, Science 224:838-843 (1984)
• heat shock promoters e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al, Mol. Cell. Biol. 6:559-565 (1986)].
• These constructs can be introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors,
• the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.
• 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 polypeptide 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 petti 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 polypeptides of the present invention may either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane. Following a predetermined time in culture, recovery of the recombinant polypeptide is effected.
• polypeptides 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.
• 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.
• the expressed coding sequence can be engineered to encode the polypeptide of the present invention and fused cleavable moiety.
• a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety.
• the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al, Immunol. Lett. 19:65-70 (1988); and Gardella et al, J. Biol. Chem. 265:15854-15859 (1990)].
• polypeptide of the present invention is preferably retrieved in "substantially pure” form.
• the phrase "substantially pure” refers to a purity that allows for the effective use of the protein in the applications described herein.
• polypeptide of the present invention can also be synthesized using in vitro expression systems. These methods are well known in the art and the components of the system are commercially available.
• Example 1 Production of a galectin-8 polypeptide using recombinant DNA technology is illustrated in Example 1 of the Examples section which follows.
• polypeptides of the present invention can be provided to the individual per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically 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 pu ⁇ ose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• active ingredient refers to the polypeptide or antibody preparation, which is accountable for the biological effect.
• physiologically acceptable carrier refers to the phrases "physiologically acceptable carrier" and
• pharmaceutically acceptable carrier refers 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).
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in
• Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and inframeduUary injections as well as infrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
• the preparation may be directly injected into a joint of an RA patient by intra-articular administration.
• 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.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art.
• the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the compounds of the invention 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 if 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, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this pu ⁇ ose, concentrated sugar solutions may be used which may optionally contain gum arable, 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 fo ⁇ n 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. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
• a suitable vehicle e.g., sterile, pyrogen-free water based solution
• the preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended pu ⁇ ose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
• the therapeutically effective amount or dose can be estimated initially from in vitro assays.
• a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l].
• 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.
• compositions 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 including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• 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 may also be accommodated by a notice associated with the container 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 or human or veterinary administration.
• Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product
• polypeptides of the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in-vivo gene therapy).
• Systemic administration of various factors e.g., cytotoxic T lymphocyte antigen CTLA4 and CIA
• RA patients was successfully achieved using viral delivery systems such as described below.
• 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).
• an appropriate gene delivery vehicle/method transfection, transduction, homologous recombination, etc.
• 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).
• polynucleotides encoding galectin-8 and variants thereof can be introduced into a population of synovial cells. Briefly, synovial tissue is removed by joint surgery and cells are expanded and infected in vitro prior to being re-introduced into the joint space. Synovial tissue may be removed several days later by joint arthoplasty for expression analysis. This approach was successfully used in a clinical trial for RA involving nine patients, aiming to express IL-lRa. Evidence for IL-lRa expression was provided both at the transcriptional level and at the protein level. This study of local ex vivo retroviral gene therapy of RA demonstrated that safe and effective transgene expression can be achieved [Evans (1999) Arthritis Rheum 42: SI 70].
• galectin-8 polypeptides of the present invention are ligated into nucleic acid expression constructs under the transcriptional control of a promoter sequence suitable for directing constitutive, tissue specific or inducible transcription in the cells.
• Constitutive promoters suitable for use with the present invention include sequences which are functional (i.e., capable of directing transcription) under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and CMV.
• CMV cytomegalovirus
• Rous sarcoma virus (RSV).
• Tissue specific promoters suitable for use with the present invention include sequences which are functional in specific cell population, example include, but are not limited to promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes
• pancreas-specific promoters [Edlunch et al. (1985) Science
• mammary gland-specific promoters such as the milk whey promoter
• Inducible promoters suitable for use with the present invention include for example the tetracycline- inducible promoter (Srour, M.A., et al., 2003. Thromb. Haemost. 90: 398-405).
• the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
• Typical cloning vectors contain transcription and translation initiation sequences (e.g., promoters, enhances) and transcription and translation terminators (e.g., polyadenylation signals).
• Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
• the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
• the other upstream promoter elements determine the rate at which transcription is initiated.
• Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is inco ⁇ orated herein by reference.
• CMV cytomegalovirus
• Polyadenylation sequences can also be added to the expression vector in order to increase the translation effeciency of a polypeptide expressed from the expression vector of the present invention.
• Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
• Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40.
• the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA.
• a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated ' episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
• the vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
• the expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
• IRS internal ribosome entry site
• mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, pZeoSV2(+/-), P SecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
• SV40 vectors include pSVT7 and pMT2.
• Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
• exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
• Viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types.
• the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
• the type of vector used by the present invention will depend on the cell type transformed.
• the ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
• bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I).
• Recombinant viral vectors are useful for in vivo expression of the polypeptides of the present invention since they offer advantages such as lateral infection and targeting specificity.
• Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired pu ⁇ ose is to introduce a specified gene into only a localized number of targeted cells.
• AAV adeno associated virus
• polypeptides of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.
• measures e.g., dosing and selection of the complementary agent
• adverse side effects are taken to adverse side effects which may be associated with combination therapies.
• Administration of such combination therapy can be simulataneous, such as in a single capsule having a fixed ration of these active agents, or in multiple capsules for each agent.
• polypeptides of the present invention can be administered along with nonsteroidal anti-inflammatory drugs (NSAID), disease-modifying antirheumatic drugs (DMARDS), corticosteroids, analgesics, Fibromyalgia medications, chemotherapeutic agents and others such as listed in Table 4, below.
• NSAID nonsteroidal anti-inflammatory drugs
• DARDS disease-modifying antirheumatic drugs
• RNA of mononuclear cells (MNC) derived from synovial fluids of RA patients was extracted using the Qiagen RNeasy kit (Qiagen, USA). RT-PCR l ⁇ g of the extracted RNA was reverse transcribed, and PCR-amplified using M- MuLV reverse transcriptase (first strand cDNA synthesis kit, Pharmacia) and Taq DNA polymerase (2.5units) (Gibco-BRL).
• the reactions were carried out in a DNA thermal cycler 480 (Perkin Elmer Cetus) in a final volume of 50 ⁇ l using oligonucleotide primers complementary to a translated region of human galectin-8: [sense primer 5'- AAGAATTCGCCGCCACCATGATGTTGTCCTTAAAC-3' (SEQ ID NO:l), antisense primer 5 '-AATCTAGACTACCAGCTCCTTACTTC-3 ' (SEQ ID NO:2)].
• the above described reaction mixture was subjected to an amplification program of 1 min at 94 °C, 1 min at 60 °C and 2 min at 72 °C for 30 cycles.
• PCR products were gel-purified and sequenced (ABI PRISM 310, PerkinElmer,
• the PCR product corresponding to galectin-8 encoding sequence (SEQ ID NO:3) was subcloned into pGEM vector (Promega) to generate pGEM-galectin-8 and purified with a commercial kit (Promega).
• the plasmid, pGEM-galectin-8 was used as a template for a second PCR reaction generating galectin-8 PCR product (SEQ ID NO: 3).
• r-galectin-8 In order to express recombinant-galectin-8 (r-galectin-8) in E. coli, the PCR product (SEQ ID NO:3), was digested with EcoRI (SEQ ID NO: 1, underlined sequence) and Xbal (SEQ ID NO: 2, underlined sequence), gel-purified and ligated into a pET-3a expression plasmid (Novagen, USA). The expression plasmid generated (pET-3a- galectin-8) was verified via sequencing, purified with a commercial kit (Promega) and then introduced into a pLyaS bacterial host. The transformed bacteria were cultured in 0.5 liter of LB medium until the absorbance at 600 nm was 0.5. The expression of r-galectin-8 was induced with 5mM isopropyl- 1-thio- ⁇ -D-galactopy-ranoside for 4h.
• Bacterial pellet was isolated by centrifugation, resuspended in 30 ml buffer I
• Synovial tissues were obtained from patients undergoing total knee or hip replacement for osteoarthritis.
• synovial tissues were cut into small pieces, of a few millimeters in diameter.
• Synovial tissue pieces (approx. 80mg) were cultured in 2ml RPMI 1640 supplemented with 2mM L-glutamine, lOOU/ml penicillin and 100 ⁇ g/ml streptomycin sulphate (Biological Industries) with test materials (r-galectin-8: 0.125 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M and l ⁇ M) and lipopolysaccharide (LPS, 3 ⁇ g/ml).
• IL-lra production was tested with and without the addition of lipopolysaccharide (LPS, 3 ⁇ g/ml) to the synovial tissues culture media.
• LPS lipopolysaccharide
• IL-l ⁇ , IL-lra and TNF- ⁇ secreted into culture media were determined by quantified human immunoassay technique (R&D Systems, Inc., USA), with detection limits of 1 pg/ml and 14pg/ml, respectively. £ell Viability and Toxicity Determination
• Galectin-8 (0.125 ⁇ M, 0.25 ⁇ M, and 0.5 ⁇ M) inhibited TNF- ⁇ and IL-l ⁇ release in the media of human synovial tissue as compared with LPS treated control in a dose dependent fashion ( Figures 1 and 2).
• Galectin-8 (0.25 ⁇ M, 0.5 ⁇ M and l ⁇ M) increased the IL-lra production in the media of human synovial tissue as compared with untreated control in a dose dependent fashion (Figure 3).
• TNF and IL-1 are mediators of the innate immune system, and they enhance inflammation and destruction in various ways, mostly through effects on endothelial cells, synovial fibroblasts, osteoclasts and cartilage. The inhibition of these cytokines was shown to ameliorate the symptoms and joint destruction of RA [Pope R. M., Nat. Rev. Immun., 2:527, (2002)].
• IL-1-receptor antagonist (IL- lRa) is produced in healthy subjects and helps to protect against the adverse effects associated with IL-1 overexpression.
• Administration of IL-lRa (Anakinra) for RA patients was shown ameliorate inflammatory conditions [Louie S. G., et al, Am. J. Health Syst. Pharm., 60:346, (2003); Wendling, D. and Jorgensen C, Rev. Med. Interne., 23:1006(2002)].
• Galectin-8 combines the anti-inflammatory effect of three factors. It reduces the production TNF ⁇ and IL-l ⁇ while increases the production of IL-lra. Each of these effects was shown to reduce inflammatory response in RA patients and therefore galectin- 8 that combines these effects presents a new advantageous therapeutic agent for RA.
• RA variant 2 (SEQ ID NO: 7) products.
• Galectins PCR products of 2464 bp, 657bp and 669bp were gel-purified and sequenced (ABI).
• PCR products were subcloned into pGEM vector (Promega). Plasmids from positive clones (termed pGEM-galectin RA 1; pGEM- galectin RA 2, respectively) were purified with a commercial kit (Promega), digested with EcoRI and Xbal and cloned into the mammalian expression vector pcDNA3.1 (Invitrogen, Paisley, UK) to generate two expression vectors (termed pcDNA3.1 -galectin RA 1; pcDNA3.1 -galectin RA 1, respectively). RESULTS
• RA galectins variants identification Two novel isoforms of human galectins were discovered in the synovial fluid cells of RA patients by RT-PCR of galectin transcripts. Sequencing of those transcripts revealed transcripts with product sizes of 2464bp, 657bp and 669bp in 10 RA patients.
• the 2464 bp product was the sequence encoding galectin-8 (GenBank Accession No: AF074000), while the other two transcripts designated galectin RA variant 1 [(SEQ ID NO:5 and SEQ ID NO:6) nucleotide and amino acid sequences, respectively] and galectin RA variant 2 [(SEQ ID NO:7 and SEQ ID NO:8) nucleotide and amino acid sequences, respectively] were both new galectin-8 variants.
• the three products were detected in the synovial fluid cells of all patients whose RT-PCR products were sequenced. When the same RT-PCR reaction was performed on the peripheral blood, only the transcript of 2464 bp of galectin-8 was detected, suggesting local expression of the new galectin variants in synovial fluids of RA patients.
• the assay was performed by Phosphatidyl Serine Detection Kit (IOP-116F FITC- conjugated, IQ Products, The Netherlands) using the manufacturer instructions.
• Synovial fluid cells (10 6 ) were incubated (RPMI 1640) for 24 h with various concentrations of galectin-8 (0.03 ⁇ M, O.l ⁇ M, 0.3 ⁇ M, l ⁇ M), with serum free medium alone (negative control) or with doxorubicine (150 ⁇ g/ml; positive control).
• annexin V Specific binding of annexin V was performed by the incubation of cells for 20 minutes on ice in a binding calcium buffer containing a saturating concentration of annexin V-fluorescein isothiocyanate (FITC) and propdium iodide (PI). After incubation, the cells were pelleted, and analyzed in FACS analyzer (Beckton Dickinson, San Jose, CA). Annexin V + /PI + cells were defined as necrotic, while annexin VYPI " cells were defined as apoptotic. The percent of the apoptotic cells of the total cells was determined.
• FITC annexin V-fluorescein isothiocyanate
• PI propdium iodide
• RA synovial tissue contains few neutrophils, whereas macrophages, synovial fibroblasts and lymphocytes are abundant. Insufficient apoptosis possibly contributes to the increased numbers of synovial fibroblasts and chronic inflammatory cells in RA joints [Pope R. M., Nat. Rev. Immun., 2:527, (2002)].
• Galectin- 1 was shown to gain therapeutic advantage in the disease when used to induce apoptosis in rheumatoid joints [Rabinovich G. A., Mem Inst Oswaldo Cruz., 95:225, (2000)].
• galectin-8 By inducing apoptosis in human synovial fluid cells, galectin-8, is shown to have therapeutic effect on chronic inflammatory cells.

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