WO2008147938A2 - Wnt5a utilise en tant que marqueur des maladies inflammatoires - Google Patents

Wnt5a utilise en tant que marqueur des maladies inflammatoires Download PDF

Info

Publication number
WO2008147938A2
WO2008147938A2 PCT/US2008/064624 US2008064624W WO2008147938A2 WO 2008147938 A2 WO2008147938 A2 WO 2008147938A2 US 2008064624 W US2008064624 W US 2008064624W WO 2008147938 A2 WO2008147938 A2 WO 2008147938A2
Authority
WO
WIPO (PCT)
Prior art keywords
wnt5a
expression
wnt5
gene
antibody
Prior art date
Application number
PCT/US2008/064624
Other languages
English (en)
Other versions
WO2008147938A3 (fr
Inventor
Chong Huang
Xilin Li
Farhat Syed
Original Assignee
Centocor, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centocor, Inc. filed Critical Centocor, Inc.
Publication of WO2008147938A2 publication Critical patent/WO2008147938A2/fr
Publication of WO2008147938A3 publication Critical patent/WO2008147938A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines

Definitions

  • the invention relates to method of using WNT5A gene expression detection, WNT5 A gene product detection, and modulators of WNT5 A or cellular receptors of WNT5A in the diagnosis, treatment, or prevention of diseases and conditions in which WNT5 A plays a causative or mediating role such as asthma, COPD, ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, psoriatic arthritis and other immune mediated inflammatory diseases.
  • the invention further relates to modulators of WNT5A which are antagonists of bioactivities related to WNT5A gene expression.
  • WNT5 A Human wingless-type mouse mammary tumor virus (MMTV) integration site family member 5 A (herein referred to as WNT5 A (Entrez Gene ID 7474, refseq NM_003392) is located on chromosome 3pl4-p21, flanked by CAST (Entrez Gene ID 26059) and CACNA2D3 (Entrez Gene ID 55799). It was initially cloned as a proto-oncogene that belonged to the Wnt gene family and detected only in human neonatal heart and lung (Clark et al. 1993. Genomics 18 (2), 249-260) .
  • the mature WNT5A protein contains 343 residues and maintains certain features of the Wnt family with a hydrophobic signal sequence, the CKCHGVSGSC motif, 24 cysteine residues, 4 N-linked glycosylation sites, and a tyrosine sulfation site.
  • WNT5 A aberrant expression has been reported in many types of cancers, such as lung, prostate (Iozzo et al. 1995), colon (Smith et al. 1999), gastric, esophageal (Saitoh and Katoh 2003), squamous carcinoma (Taki et al. 2003) and thyroid carcinomas (Kremenevskaja et al. 2005).
  • increased WNT5A expression in human melanoma biopsies directly correlates with increasing tumor grade (Weeraratna 2002).
  • low or abolished expression of WNT5A has been associated with aggressiveness of other types of cancer, such as invasive ductal carcinomas (Jonsson et al. 2002; Dejmek et al.
  • WNT5A transfection of a thyroid carcinoma cell line clearly demonstrates a decreased proliferation, migration, invasiveness and clonogenic activity. WNT5 A may also serve as an antagonist to the canonical Wnt-signaling pathway with tumor suppressor activity.
  • WNT5A signaling on B-cells inhibits cell proliferation, by negatively regulating the response of B-cells to IL-7 and suppressing cyclin Dl expression (Liang et al. 2003. Cancer Cell. 4(5):349-60).
  • WNT5A and its receptor, FZD5 have been reported present on human PBMC, in lung granulomatous lesions of tuberculosis patients, and to regulate IL 12 produced by antigen presenting cells and IFNgamma from T-cells in response to mycobacterial antigen (Blumenthal et al. 2006 Blood 108: 965- 973).
  • neoplastic disease As various pathologies dependent upon cell proliferation are not limited to neoplastic disease but include pathophysiology related to aberrant response to injury, such as fibrosis and restenosis, and immune-mediated inflammatory disease, such as psoriasis, it is important to understand and be able to intervene in the underlying mechanisms of the disease process. Understanding the role of WNT5A in nonneoplastic disease lends itself to the production of new diagnostic methods and therapeutics for such intervention. SUMMARY OF THE INVENTION
  • the invention is based on a previously undisclosed role of WNT5 A as an important mediator of immune mediated inflammatory diseases.
  • novel WNT5 A functions disclosed permit the generation of compositions and methods for the diagnosis and treatment of asthma, COPD, ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, psoriatic arthritis and other immune mediated inflammatory diseases.
  • compositions may comprise one or more protein isoforms, immunogenic portions thereof, or polynucleotide that encode such portions.
  • a therapeutic composition may comprise an antigen-presenting cell that expresses WNT5 A protein, or a T-cell that is specific for cells expressing a polypeptide encoded by the gene, or other type of agonists; and antagonistic agents such as neutralizing monoclonal antibodies (mAb) or small molecule compounds to any portion of WNT5A DNA, RNA or protein.
  • mAb monoclonal antibodies
  • These compositions may be used, for example, for the prevention and treatment of a range of immune mediated inflammatory diseases.
  • FIG. 1 shows a graphic representation of the changes in WNT5A differential gene expression in cultured HASMC expressed as normalized microarray intensity (average intensity and standard error) for four sample groups: asthmatic and non-asthmatic subjects and 2 culture conditions, with serum from atopic or nonatopic individuals; (asthmatic atopic Filled Square, asthmatic non-atopic Open Squares, non- asthmatic atopic Filled Circle, and non-asthmatic non-atopic Open Circles), sampled at 6 time points (15 min, 30 min, 2 hr, 4 hr, 8 hr, and 24 hr) and prior to treatment (cntl).
  • the expression level of WNT5A is higher in all asthmatic samples compared to non- asthmatic samples.
  • Abs antibodies polyclonal or monoclonal; Ig immunoglobulin; Mab monoclonal antibody; human h; interleukin-12 IL 12; interferon IFN; Frizzled FZD; PBMC peripheral blood mononuclear cell.
  • an "activity,” a biological activity, and a functional activity refers to an activity exerted by a gene product in response to its specific interaction with another protein or molecule as determined in vivo, in situ, or in vitro, according to standard techniques.
  • activities can be a direct activity, such as an association with or an enzymatic activity on a second protein, or an indirect activity, such as a cellular process mediated by interaction of the protein with a second protein, such as a membrane bound receptor protein, or a series of interactions as in intracellular signaling or the coagulation cascade that results from binding of the gene product protein to one or more other proteins.
  • activity may be measured directly as by measuring the binding of a gene product protein, e.g.
  • WNT5A protein to another protein or measured indirectly as biological activity, for example y measuring cell proliferation or cell apoptosis which results from a series of protein-protein interactions and protein-nucleic acid interactions.
  • antibody herein is used in the broadest sense. As used herein, an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof.
  • the antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity-determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein, which can be incorporated into an antibody of the present invention.
  • CDR complementarity-determining region
  • the term "antibody” is further intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof.
  • Functional fragments include antigen-binding fragments to a preselected target.
  • binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH, domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH, domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • CDR complementarity determining region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al.1988 Science 242:423-426, and Huston et al. 1988 Proc. Natl. Acad Sci. USA 85:5879-5883.
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.
  • array or “microarray” or “biochip” or “chip” as used herein refer to articles of manufacture or devices comprising a plurality of immobilized target elements, each target element comprising a “clone,” “feature,” “spot” or defined area comprising a particular composition, such as a biological molecule, e.g., a nucleic acid molecule or polypeptide, immobilized to a solid surface, as discussed in further detail, below.
  • a biological molecule e.g., a nucleic acid molecule or polypeptide
  • nucleic acids polymers of nucleotides, wherein a nucleotide comprises a base linked to a sugar which sugars are in turn linked one to another by an interceding at least bivalent molecule, such as phosphoric acid.
  • the sugar is either 2'-deoxyribose (DNA) or ribose (RNA).
  • Unnatural poly- or oligonucleotides contain modified bases, sugars, or linking molecules, but are generally understood to mimic the complementary nature of the naturally occurring nucleic acids after which they are designed.
  • An example of an unnatural oligonucleotide is an antisense molecule composition that has a phosphorothiorate backbone.
  • oligonucleotide generally refers to a nucleic acid molecule having less than 30 nucleotides.
  • siRNA means a double stranded short interfering RNA molecule of no larger than about 23 nucleotides in length.
  • shRNA means a short interfering RNA.
  • WNT5A and WNT5A gene also known as homo sapiens wingless- type MMTV integration site family member 5 A, are used herein interchangeably and refer to the gene WNT5A (Entrez Gene ID 7474, refseq NM_003392) located on chromosome 3pl4-p21 with coding a protein (residues 319-1461). It was initially cloned as a proto-oncogene that belonged to the Wnt gene family (Clark et al. 1993. Genomics 18 (2), 249-260) and detected human neonatal heart and lung. EF028086 represents WNT5A variant 2 complete cds.
  • WNT5A gene product and "WNT5A protein” are used interchangeably for the polypeptide and mature protein coded for by the WNT5 A gene and represented by the NCBI curated sequence record NP 003383.3 for the 380 amino acid precursor protein and protein Wnt-5a (P41221) for a 365 amino acid preprotein.
  • the active or mature WNT5A sequence is 343 amino acid long (23 -365 aa of P41221 or 38 -380 of NP 003383).
  • the C-terminal 313 amino acids represent a protein domain called "wnt domain" that is conserved in all WNT protein family members. Determination of a WNT5A-associated Inflammatory Disease
  • Microarray technology is a powerful tool since it enables analysis of the expression of thousands of genes simultaneously and can also be automated allowing for a high-throughput format.
  • multifactorial diseases such as asthma
  • microarray results can provide a gene expression profile which can provide insight into the design and use of therapeutics.
  • these types of data can be used to examine relationships between genes or patterns of gene expression.
  • the present invention is based on a known gene, WNT5A, which has differential expression patterns known to be associated with cancer, now found by microarray analysis of samples derived from human subjects to be associated with two types of immune mediated inflammatory diseases, asthma and ulcerative colitis. Moreover, the expression of WNT5A in ulcerative colitis patients being treated with an anti-inflammatory cytokine (TNFalpha) antibody was reduced in patients responding to therapy while not in patients who did not respond clinically to therapy. Recent clinical studies (ACT 1 and 2) reported by Rutgeerts et al. (N Engl J Med, 2005. 353(23): p.
  • infliximab is effective when administered at weeks 0, 2, 6 and every 8 weeks thereafter in achieving and maintaining clinical response and remission in patients with moderate -to-severe, active UC who have had an inadequate response or failed to tolerate conventional therapy.
  • ACT 1 ACT 1 study who had significant treatment effects of infliximab
  • global gene expression changes in the biopsies were observed supporting a crucial role of TNFa in UC pathogenesis. WNT5A reduction in expression level was among the genes which could be correlated with a response.
  • Infliximab treatment responders were patients who showed a marked clinical improvement following infliximab therapy as defined by a decrease from baseline in the total Mayo score by at least 3 points and at least 30 percent with an accompanying decrease in rectal bleeding sub-score of at least 1 point or an absolute rectal bleeding sub-score of 0 or 1 (Rutgeerts supra).
  • WNT5A mediates disease processes in several pathologies associated with inflammatory mechanisms and mediated by the immune system, in particular, pathologies which are classified as having an autoimmune component.
  • WNT5A expression was down-regulated in skin biopsies of patients treated with infliximab. These results indicate that WNT5A can be viewed as a marker for TNFa bioactivity in psoriasis.
  • WNT5 A gene expression is elevated in cultured PBMC from asthmatic but not non-asthmatic patients. PBMC treated with inflammatory cytokines, IL-4 and IL- 13, also display a large increase in WNT5A expression. Therefore, WNT5 A is associated with inflammatory disease processes and recovery or response to anti-inflammatory treatment in human subjects.
  • WNT5A is a biomarker for disease and therapy of diseases such as asthma, COPD, ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, psoriatic arthritis and other immune mediated inflammatory diseases.
  • cells from patients suffering from respiratory disorders such as COPD or Asthma, can be used to diagnose an asthmatic genotype based on an elevated level of WNT5A compared to cells from a population of normal patient due to the finding that WNT5A is elevated in subjects previously diagnosed with asthma compared to normal subjects.
  • the elevation of WNT5A by greater than 1.5-fold is indicative of the condition of asthma.
  • the cells may be taken from bronchial scrapings, PBMCs, or sputum of patients.
  • WNT5A expression from PBMCs of patients with COPD or asthma can be monitored in a patient receiving anti-inflammatory therapy and compared to its baseline, or pretreatment, expression level.
  • a decrease in the level of WNT5 A expression is expected in response to anti-inflammatory therapy such as anti- IL- 13 therapy.
  • a whole blood sample is taken at baseline from a patient receiving anti-IL-13 therapy.
  • PBMCs are isolated and then total RNA is isolated using Qiagen RNeasy kit and the expression of WNT5A mRNA is assessed using Real Time PCR using primer probe sets from Applied Biosystems that are specific to WNT5 A.
  • the expression of WNT5A in the baseline PBMC sample is compared to the PBMC sample taken from the same patient post treatment at week 8.
  • a reduction in expression of greater than 2 fold upon anti-IL-13 therapy is indicative of a response and therefore patient should be maintained on therapy.
  • a whole blood sample is taken at baseline from a patient receiving anti-IL-13 therapy.
  • PBMCs are isolated using Ficoll gradient and then total RNA is isolated using Qiagen RNeasy kit and the expression of WNT5 A mRNA is assessed using Real Time PCR using primer probe sets from Applied Biosystems that are specific to WNT5A.
  • the expression of WNT5A in the baseline PBMC sample is compared to the PBMC sample taken from the same patient post treatment at week 8.
  • a reduction in expression of greater than 2 fold upon anti-IL-13 therapy is indicative of a response and therefore patient should be maintained on therapy.
  • WNT5A expression in tissue from biopsies of patients with ulcerative colitis can be used to monitor response to therapy, such as anti-TNFalpha therapy, in a patient.
  • therapy such as anti-TNFalpha therapy
  • a baseline or pretreatment expression level is determined using inflamed tissue biopsy samples, PBMC, or sloughed cells.
  • PBMC inflamed tissue biopsy samples
  • sloughed cells a second similar sample is processed.
  • the WNT5 A levels in the pretreatment sample are compared to the post-treatment level.
  • a decrease in the level of WNT5 A expression is indicative of a response to anti-TNF therapy.
  • the sample is a colon biopsy. The biopsy is flash frozen to preserve the integrity of the RNA.
  • Total RNA is isolated using Qiagen RNeasy kit and the expression of WNT5A mRNA is assessed using Real Time PCR using primer probe sets from Applied Biosystems that are specific to WNT5A.
  • the expression of WNT5A in the baseline biopsy sample is compared to the biopsy taken from the same patient post treatment at week 8. A reduction in expression of greater than 2 fold upon anti-TNF therapy is indicative of a response and therefore the patient should be maintained on therapy.
  • a biosensor capable of specifically detecting WNT5 A protein or mRNA. Automatic isolation and amplification of RNA could be used for on- site analysis of WNT5 A mRNA expression. Expression values can be used with a WNT5 A biosensor or the biosensor may have integrated technology capable of performing the steps of mRNA isolation and amplification as well as detection. Thus, patients' WNT5A level may be assessed before and after treatment.
  • Biosensor instrumentation incorporates surfaces capable of detecting specific binding, such as by an anti-WNT5A antibody to WNT5A protein or a WNT5A oligonucleotide probe binding to WNT5 A mRNA.
  • a detectable signal generated by the binding pair may be by surface plasmon resonance or may incorporate a quartz crystal resonator onto which the molecular target is coupled.
  • a potential binding partner is passed over the surface.
  • the refractive index of the surface changes at the interface between the surface and a solution flowing over the surface, altering the angle at which the reduced-intensity light is reflected from the metal- (e.g., gold) coated glass.
  • the change in angle, caused by binding or dissociation of molecules from the sensor surface is proportional to the mass of bound material.
  • changes in vibrational frequency indicate changes in mass and therefore biomolecular interactions
  • the WNT gene family consists of structurally related genes which encode secreted proteins capable of promoting receptor signaling. These proteins have been implicated in oncogenesis and in several developmental processes, including regulation of cell fate and patterning during embryogenesis.
  • the WNT5A gene encodes a protein which shows 98%, 98% and 87% amino acid identity to the mouse, rat and the xenopus WNT5A protein, respectively.
  • Human frizzled-5 (hFz5) is a receptor for the WNT5A ligand, however, Fz proteins represent a multi-member family of proteins to which Wnt proteins can bind.
  • WNT5A binds to members of the Frizzled (Fzd) family of seven-transmembrane domain receptors on the cell surface, and triggers a series of intracellular events that ultimately regulate gene transcription. These intracellular events are grouped according to two known signaling pathways, the canonical Wnt/ ⁇ -catenin pathway and the Wnt/Ca++ pathway.
  • WNT5A-mediated release of intracellular Ca++ leads to the phosphorylation of both protein kinase C (PKC) and calcium/calmodulin dependent protein kinase II (CamK II), and the activation of these two kinases blocks the Wnt/ ⁇ -catenin signaling cascade via phosphorylation of dishevelled (dvl) (by PKC) or Lymphoid Enhancer-binding Factor (LEF-I) (by CamK II).
  • the signaling pathway that is induced may be governed by the cell surface Fzd receptor to which WNT5 A binds.
  • transactivation of receptor signal pathways by Wnt proteins, including ERBBl by WNT5A has been described.
  • a "WNT5A antagonist” prevents the biological functions or bioactivity associated with WNT5A or WNT5A in cells that display WNT5A or WNT5A receptors.
  • Antagonists included within the scope of the present invention include antibodies, synthetic or native sequence peptides, nucleotides, and small molecule antagonists, which bind WNT5A or which prevent the binding of WNT5A with its cognate receptor(s) and thereby inhibit WNT5A biological functions.
  • an inhibitor refers to substances including antagonists which bind WNT5A protein (e.g., an antibody, a mutant of a natural ligand, small molecular weight organic molecules, other competitive inhibitors of WNT5A protein binding), and substances which inhibit WNT5A receptor function without binding thereto (e.g., an anti-idiotypic antibody).
  • WNT5A protein e.g., an antibody, a mutant of a natural ligand, small molecular weight organic molecules, other competitive inhibitors of WNT5A protein binding
  • substances which inhibit WNT5A receptor function without binding thereto e.g., an anti-idiotypic antibody
  • a suitable WNT5A antagonist can affect WNT5A gene or WNT5A protein function in a variety of ways, such as, but not limited to, inhibition of RNA, DNA or protein synthesis; WNT5 A release, WNT5 A receptor signaling, WNT5 A binding to receptor, WNT5A production, synthesis, or secretion.
  • WNT5 A protein activity can be blocked by preventing its interaction with its cognate receptor, for example hFz5. Therefore, antibodies capable of neutralizing WNT5A are useful antagonists of WNT5A initiated receptor signaling.
  • Antibodies of the invention can be generated by methods well known in the art.
  • the antibody sequence is substantially of human origin. Preparation of human antibodies that are specific for human WNT5A protein or fragments thereof, such as isolated WNT5 A protein or a portion thereof (including synthetic molecules, such as synthetic peptides), can be performed using any suitable technique known in the art.
  • the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et Io al.
  • Suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK; Biolnvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma, Berkeley, CA; Ixsys. See, e.g., EP 368,684, PCT/GB91/01134; PCT/GB92/01755; PCT/GB92/002240;
  • a peptide or protein library e.g., but not limited to, a bacteriophage,
  • An antibody useful in the methods of the invention specifically recognizes at least the "wnt" domain (C-terminal 313 AA of human WNT5A) and may neutralize the biological activities associated with WNT5A.
  • the antibody may be generated through any of the methods mentioned above and can be used as to detect WNT5 A or, in a therapeutic setting, to antagonize WNT5 A function through blocking of its interaction with its cellular receptor.
  • an antibody useful in a therapeutic setting is an antibody which specifically recognizes extracellular domain of the receptor hFz5 and neutralizes the biological activities associated with hFz5 when bound by WNT5 A.
  • Inhibition of WNT5A expression can be effective in reducing ambient levels of WNT5A activity.
  • Gene expression can be modulated in several different ways, including by the use of siRNAs, shRNAs, antisense molecules and DNAzymes.
  • SiRNAs and shRNAs both work via the RNAi pathway and have been successfully used to suppress the expression of genes.
  • RNAi was first discovered in worms and the phenomenon of gene silencing related to dsRNA was first reported in plants by Fire and Mello (Fire et al., 1998. Nature 391 : 806) and is thought to be a way for plant cells to combat infection with RNA viruses.
  • RISC RNA induced silencing complex
  • Synthetic siRNAs can be designed to specifically target one gene and they can easily be delivered to cells in vitro or in vivo.
  • Nucleic acids known as shRNAs are the DNA equivalents of siRNA molecules and have the advantage of being incorporated into the cells' genome and then being replicated during every mitotic cycle.
  • DNAzymes have also been used to modulate gene expression.
  • DNAzymes are catalytic DNA molecules that cleave single-stranded RNA. They are highly selective for the target RNA sequence and as such can be used to down-regulate specific genes through targeting of the messenger RNA.
  • RNA interference refers to the process of sequence- specific post- transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al, 2000, Cell, 101, 25-33; Fire et al, 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13:139-141; and Strauss, 1999, Science, 286, 886).
  • siRNAs short interfering RNAs
  • dsRNAs The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293).
  • Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001, Nature, 409, 363).
  • Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Genes Dev., 15, 188).
  • Dicer has also been implicated in the excision of 21- and 22- nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834).
  • RNA-induced silencing complex RISC
  • siRNAs are double stranded RNAs that include the target sequence and its complement. Two uridine residues are added to the 3' end of the RNAs (Elbashir et al. 2001 Nature 411 :494-498).
  • RNA interference is now being used routinely in mammalian cells to study the functional consequences of reducing the expression of specific genes.
  • RNAi is induced by transfecting small interfering RNAs (siRNAs), comprising double- stranded RNA molecules approximately 21 nt in length with 2 nt 3' overhangs (Elbashir et al. 2001 supra), or hairpin- forming 45-50mer (shRNA) molecules (Paddison, PJ, et al., 2002. Genes & Development 16:948-958), that are complementary to the gene of interest.
  • siRNA expression vectors When transfected into mammalian cells, siRNA expression vectors and have been shown to reduce the levels of both exogenous and endogenous gene products.
  • siRNA vectors can provide longer term reduction in target gene expression when coexpressed with a selectable marker (Brummelkamp, TR, et al., 2002. Science 296:550-553).
  • SEQ ID NO: 1-3 were generated using E-RNAi software (Arziman et. al. 2005, Nucleic Acids Research, Vol. 33, W582- W588) based on the input sequence given in the NCBI record Accession No. NM_003392.3.
  • the starting nucleotide position within WNT5A gene for each RNAi is given in the right column as "position”.
  • the software is available to public online at a website provided by the German Cancer Reseach Center (dkfz) and the version used was Boutros lab, E-RNAi-Version 2.0, Database- Version 2.0. Only those graded by the software as a 10, on a scale of 1 -10, were selected.
  • the implemented algorithm estimates the efficiency of siRNAs using eight criteria: (i) low GC content (30-52%), (ii) at least three AAJ bases at positions 15- 19,
  • siRNA fulfills criteria (i), (iii), (v) and (vi)
  • one point is added to its score.
  • For criterion (iv), potential hairpin structures of siRNAs are calculated using RNA fold. If the melting temperature of the potential hairpin region is 20° C or less, one point is added the score. Other sequences with lower scores can be readily generated using this information.
  • sequences can be synthesized using techniques known to those skilled in the art and tested using methods known in the art or as described herein. In general, multiple sequences, such as 20, will be tested to determine the optimal level of WNT5 A knock-down in a cell which is normally capable of expressing WNT5 A prior to or after being stimulated. Stimulation of the cells for WNT5A expression may include, but is not limited to, exposure to inflammatory cytokines, such as TNF alpha, IL4 or IL13.
  • the invention includes methods for preparing pharmaceutical compositions for modulating the transcription, expression, or activity of a WNT5 A gene or protein. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent that modulates expression or activity of a WNT5 A gene or protein. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent that modulates expression or activity of a WNT5 A gene or protein and one or more additional active compounds.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant expression or activity of a WNT5 A gene or protein and/or in which a WNT5 A gene or protein is involved.
  • the present invention provides a method for modulating or treating at least one WNT5 A gene or protein related disease or condition, in a cell, tissue, organ, animal, or patient, as known in the art or as described herein, using at least one WNT5 A gene or protein antagonist.
  • Compositions of a WNT5 A gene or protein antagonist may find therapeutic use in the treatment of inflammatory diseases, conditions and disorders. These methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a WNT5 A gene or polypeptide.
  • the method involves administering an agent, or combination of agents that modulate (e.g., up-regulates or down-regulates) expression or activity of WNT5A.
  • agents that modulate e.g., up-regulates or down-regulates
  • Inhibition of activity is desirable in situations in which activity or expression is abnormally high or up-regulated and/or in which decreased activity is likely to have a beneficial effect.
  • Inflammatory disorders particularly amenable to treatment using a WNT5 A gene or protein antagonist are those which involve the elaboration of TNF alpha as further described elsewhere in this disclosure.
  • TNF alpha neutralization, as by an anti-TNF antibody has been shown to be effective in treating a variety of immune mediated diseases characterized by inflammatory processes.
  • the findings disclosed in the present invention demonstrate that inflammatory disorders in which the subject experiences elevated levels of cytokines IL-4 and IL- 13 can be recognized by the increased expression of WNT5A in certain cells, particularly PBMC.
  • intervention in such patients to reduce IL-4 and IL- 13 levels may prove effective in moderating such an inflammatory disease and which moderation can be monitored through the analysis of WNT5A levels.
  • treatment of the patient with an agent which diminishes, moderates, or ameliorates the WNT5A expression or activity may likewise provide effective therapy for such patients.
  • the invention provides a method for at least substantially preventing in a subject, a disease or condition associated with an aberrant expression or activity of a WNT5A gene or protein, by administering to the subject an agent that modulates expression or at least one activity of the gene and, therefore, the polypeptide.
  • Subjects at risk for a disease that is caused or contributed to by aberrant expression or activity of a WNT5 A gene or protein can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the disease or disorder so that it is prevented or, alternatively, delayed in its progression. The timing and amount of agent to deliver can be determined based on screening assays described herein.
  • the present invention also provides a method for preventing or treating at least one immune -mediated or inflammatory disease or disorder in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/ admireer's granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures, allergic/atopic diseases, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis
  • WNT5A antagonists can be used alone or in combination with other anti-inflammatory compounds or inflammatory pathway modulators to prevent or treat pathologic conditions.
  • the WNT5 A antagonist such as an anti-WNT5 A antibody, or other agent capable of inhibiting preformed WNT5A protein activity may be administered to a subject in need thereof in a pharmaceutically acceptable composition.
  • a method of the present invention can comprise a method for treating an immune-mediated disorder or inflammatory disorder as described above, comprising administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-WNT5 A antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • Such a method can optionally further comprise co-administration or combination therapy for treating such diseases or disorders, wherein the administering of the anti-WNT5A antibody, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from an immunomodulation drug (such as a TNF antagonist e.g., but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist); an antirheumatic (e.g., methotrexate, auranofm, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine); a non-steroid antiinflammatory drug (NSAID); an analgesic; an anesthetic; a sedative; a local anesthetic; a neuromuscular blocker; an immunosuppressive
  • Such drugs are well known in the art, including formulations, indications, dosing and administration for each presented herein (see., e.g., Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; Pharmcotherapy Handbook, Wells et al, ed., Appleton & Lange, Stamford, CT, each entirely incorporated herein by reference).
  • treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one anti-WNT5 A antibody composition that totals, on average, a range from at least about 0.01 to 500 milligrams of at least one anti-WNT5A antibody per kilogram of patient per dose, and preferably from at least about 0.1 to 100 milligrams antibody/kilogram of patient per single or multiple administration, depending upon the specific activity of contained in the composition.
  • the effective serum concentration can comprise about 0.1- 5000 ug/ml serum concentration per single or multiple administration.
  • Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.
  • the WNT5 A nucleic acid antagonist molecules can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054- 3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • Either the naked nucleic acid antagonist molecules or the engineered vectors comprising the antagonist sequences of the invention can be encapsulated for administration to a subject.
  • the encapsulated form may be a microparticle that is comprised of a wall forming material.
  • the encapsulated form is a lipid vesicle, e.g., a liposome.
  • microparticle is synonymous with and includes the terms “microsphere” and "microcapsule.”
  • the microparticle composition is substantially dry or powder- like, i.e., liquid has not been added to the composition. Some minor amounts of liquid may however remain with the microparticles.
  • the polymeric matrix material of the microparticles present invention can be composed of a biocompatible and biodegradable polymeric material.
  • biocompatible material is defined as a polymeric material which is not toxic to an animal and not carcinogenic.
  • the matrix material is preferably biodegradable in the sense that the polymeric material should degrade by bodily processes in vivo to products readily disposable by the body and should not accumulate in the body.
  • the microparticles of the present invention usually have a spherical shape, although irregularly-shaped microparticles are possible.
  • the microparticles vary in size, ranging in diameter from about 0.1 microns to 250 microns, more preferably, from about 10 or 20 microns to 75 microns and, most preferably, from about 30 microns to 70 microns.
  • sustained-release encompasses the term "controlled-release” and means that the biologically active agent is released from the microparticle polymeric matrix over an extended period of time so as to give continuing or delayed dosage of the treated organism.
  • the controlled-release period can be from a few hours to about 1 to 500 days or longer and, preferably, is from about 3 to 60 days.
  • Suitable wall-forming materials for use in microcapsules include, but are not limited to, poly(dienes) such as poly(butadiene) and the like; poly(alkenes) such as polyethylene, polypropylene, and the like; poly(acrylics) such as poly(acrylic acid) and the like; poly(methacrylics) such as poly(methyl methacrylate), poly(hydroxyethyl methacrylate), and the like; poly(vinyl ethers); poly(vinyl alcohols); poly( vinyl ketones); poly(vinyl halides) such as poly(vinyl chloride) and the like; poly( vinyl nitrites), poly( vinyl esters) such as poly(vinyl acetate) and the like; poly( vinyl pyridines) such as poly(2 -vinyl pyridine), poly(5-methyl-2-vinyl pyridine) and the like; poly(styrenes); poly(carbonates); poly(esters); poly(orthoesters);
  • a preferred group of wall-forming materials includes biodegradable polymers, such as poly(lactide), poly(glycolide), poly(caprolactone), poly(hydroxybutyrate), and copolymers thereof including, but not limited to, poly(lactide-co- glycolide), poly(lactide-co-caprolactone) and the like. Again, these polymers may be cross-linked.
  • the copolymers may be block, random or regular copolymers. The duration of release of the active agent from the microparticle can be adjusted from less than a week to several months or longer by manipulation of various parameters.
  • the amount (level) of biologically active agent released can also be controlled.
  • the parameters include the polymer composition of the controlled-release material, the polymer molecular weight, the polymer:bioactive agent ratio, microparticle diameter and the presence/absence of a release rate modifier in the composition.
  • Other parameters include bound/unbound drug (with respect to a polymer matrix), hydrophobicity of the drug and/or polymer composition and porosity of the polymer matrix.
  • Liposomes as well as other micellar lipid vesicles are included in the methods of the invention for incorporation of the WNT5A nucleic acid antagonist in order to act as drug delivery vehicles.
  • the methods of preparation and drug loading procedures for liposomes and the others are well-known in the art.
  • Liposomes can store both nonpolar and polar compounds via interactions with the biocompatible and biodegradable lipid bilayer, or within the aqueous core, respectively.
  • Lipids suitable for use in the composition of the present invention include those vesicle-forming lipids.
  • Such a vesicle-forming lipid is one which (a) can form spontaneously into unilamellar or bilayer vesicles in water, as exemplified by the diglycerides and phospholipids, or (b) is stably incorporated into lipid structures including unilammellar, bilayered, or rafts.
  • the vesicle-forming lipids of this type typically have two hydrocarbon chains, usually acyl chains, and a head group, either polar or nonpolar.
  • lipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin, where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin
  • the above-described lipids and phospholipids whose acyl chains have varying degrees of saturation can be obtained commercially or prepared according to published methods.
  • suitable lipids include glycolipids, cerebrosides and sterols, such as cholesterol.
  • Cationic lipids are also suitable for use in the liposomes of the invention, where the cationic lipid can be included as a minor component of the lipid composition or as a major or sole component.
  • Such cationic lipids typically have a lipophilic ligand, such as a sterol, an acyl or diacyl chain, and where the lipid has an overall net positive charge. Typicallly, the head group of the lipid carries the positive charge.
  • Exemplary cationic lipids include l,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[I- (2,3 ,-ditetradecyloxy)propyl]-N,N-dimethyl-N- hydroxyethylammonium bromide (DMRIE); N-[l-(2,3,-dioleyloxy)propyl]-N,N- dimethyl-N-hydroxy ethylammonium bromide (DORIE); N-[l-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3 [N-(N',N'- dimethylaminoethane)carbamoly]cholesterol (DC-Choi); and dimethyldioctadecylammonium (DDAB) .
  • DOTAP l,2-dioleyloxy-3-(trimethylamino) propane
  • DMRIE N
  • the cationic vesicle-forming lipid may also be a neutral lipid, such as dioleoylphosphatidyl ethanolamine (DOPE) or an amphipathic lipid, such as a phospholipid, derivatized with a cationic lipid, such as polylysine or other polyamine lipids.
  • DOPE dioleoylphosphatidyl ethanolamine
  • an amphipathic lipid such as a phospholipid
  • a cationic lipid such as polylysine or other polyamine lipids.
  • the neutral lipid (DOPE) can be derivatized with polylysine to form a cationic lipid.
  • the vesicle-forming lipid is selected to achieve a specified degree of fluidity or rigidity, to control the stability of the liposome in serum, to control the conditions effective for insertion of the targeting conjugate, as will be described, and to control the rate of release of the entrapped agent in the liposome.
  • Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer are achieved by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g., up to 60° C.
  • a relatively rigid lipid e.g., a lipid having a relatively high phase transition temperature, e.g., up to 60° C.
  • Rigid, i.e., saturated, lipids contribute to greater membrane rigidity in the lipid bilayer.
  • Other lipid components, such as cholesterol are also known to contribute to membrane rigidity in lipid bilayer structures.
  • lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g., at or below room temperature.
  • the pre-formed liposomes also include a vesicle-forming lipid derivatized with a hydrophilic polymer.
  • a vesicle-forming lipid derivatized with a hydrophilic polymer As has been described, for example in U.S. Pat. No. 5,013,556, including such a derivatized lipid in the liposome composition causes the formation of a surface coating of hydrophilic polymer chains around the liposome. The surface coating of hydrophilic polymer chains is effective to increase the in vivo blood circulation lifetime of the liposomes when compared to liposomes lacking such a coating by presentation of a non- immunogenic outer surface.
  • Such liposomes are also structurally stabilized and are known as sterically-stabilized liposomes
  • Vesicle-forming lipids suitable for derivatization with a hydrophilic polymer include any of those lipids listed above, and, in particular, phospholipids, such as distearoyl phosphatidylethanolamine (DSPE).
  • Hydrophilic polymers suitable for derivatization with a vesicle-forming lipid include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences.
  • the polymers may be employed as homopolymers or as block or random copolymers.
  • An exemplary hydrophilic polymer chain is polyethyleneglycol (PEG) having a molecular weight between 500-10,000 daltons, more typically between 1,000- 5,000 daltons.
  • PEG polyethyleneglycol
  • Methoxy or ethoxy- capped analogues of PEG are also useful hydrophilic polymers, commercially available in a variety of polymer sizes, e.g., 120- 20,000 daltons.
  • vesicle-forming lipids derivatized with hydrophilic polymers has been described, for example in U.S. Pat. No. 5,395,619.
  • liposomes including such derivatized lipids has also been described, where typically, between 1-20 mole percent of such a derivatized lipid is included in the liposome formulation.
  • the liposomes are composed of distearoylphosphatidylcholine (DSPC): cholesterol (52:45 molar ratio), and contain additionally 3 mol % PEG(2000)-DSPE compared to total lipid.
  • DSPC distearoylphosphatidylcholine
  • the liposomes are prepared by freeze-thaw cycles and extrusion as described (Huwyler, et al. (1996) Proc Natl Acad Sci USA 93: 14164-14169). Essentially, lipids are first dissolved in chloroform or chloroform/methanol 2:1 vol/vol. A lipid film is prepared by vacuum evaporation using a Rotavapor (B ⁇ chi, Switzerland).
  • Dried lipid films are hydrated at 40 0 C in 0.01 M PBS or 65o in 0.3 M citrate (pH4.0), such that a final lipid concentration of 10 mM is achieved.
  • Lipids are subjected to five freeze-thaw cycles, followed by extrusion (5 times) at 20 0 C through a 100 nm pore-size polycarbonate membrane employing an extruder (Avanti Polar Lipids, Alabaster, AL). Extrusion is repeated 9 times using a 50 nm polycarbonate membrane. This procedure produces
  • biotinylated loaded liposomes may be prepared by substituting a portion of the PEG-DSPE with linker lipid (biotin-PEG-DSPE) and dye or drug may encapsulated by adding the active at the hydration step.
  • SNALP Stable nucleic acid-lipid particles
  • EXAMPLE 1 WNT5A IN HUMAN AIRWAY SMOOTH MUSCLE CELLS The role of WNT5 A in immune mediated pathological conditions was examined using human airway smooth muscle cells from normal and asthmatic human subjects and sera from normal subjects and those classified as atopic (hyper-sensitive to an antigen or allergen).
  • HASMC human airway smooth muscle cells
  • the quiescent control HASMC were grown for 4 weeks (6 passages) in 10% FBS/DMEM, quiescent in 1% FBS/DMEM for 24 hours and in 0% FBS/DMEM for an additional 24 hours.
  • HASMC were exposed to atopic sera (from individuals that were hyper-sensitive to an antigen or allergen) or non-atopic sera for up to 24 hours.
  • Batch 1 2490 female (F) -50 year old (y) lung transplant for hemorrhage; 2723 F 44y asthmatic lung biopsy.
  • Batch 2 2599 male (M) 5Oy lung transplant for emphysema; 2724 M 2Oy non-asthmatic lung biopsy.
  • Batch 3 2204 F 47y lung resection for large cell CA; 2702 F 47y asthmatic lung biopsy.
  • the times of sampling were 0 (untreated, control), 15 minutes, 30 minutes, 2 hours, 4 hours, 8 hours, and 24 hours for most samples.
  • Number of Samples 76.
  • the samples were processed and analyzed using a cDNA microarray on a solid phase (DNA Chip) the composition of which was developed internally and known as TargetA l and TargetA_2. Each array is comprised of more than 8100 cDNA clones.
  • the hybridization of sample nucleic acid to cDNA on the solid phase is quantitated as fluorescence intensity using an Agilent Image Scanner (Palo Alto, CA).
  • the times of sampling were 0 (untreated), 15 minutes, 30 minutes, 2 hours, 4 hours, 8 hours, and 24 hours.
  • the 76 samples are more or less evenly distributed across the 4 treatment groups.
  • the data was grouped to improve statistical power.
  • the results, shown graphically in Fig. 1, demonstrate no statistically significant differences between times or treatments for WNT5A expression among the asthmatic samples and, similarly, for the non-asthmatic samples. However, the expression level of WNT5A is higher in all asthmatic samples compared to all non-asthmatic samples.
  • PBMC peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • IL-13 10 ng/ml
  • IL-4 10 ng/ml
  • Total RNA was isolated from the cells using the RNeasy kit (Qiagen). The quality and quantity of RNA was assessed using the BioAnalyzer (Agilent) and microarray analysis was performed using the Affymetrix platform.
  • PBMCs from each individual were treated under the 3 conditions: PBS (untreated), IL-13, IL-4.
  • a cDNA microarray, GeneChip Human Genome Ul 33 Plus 2.0 arrays (Affymetrix), which comprises more than 54,000 probe sets capable of analysis of the expression level of over 47,000 transcripts and variants, including 38,000 well- characterized human genes.
  • the Affymetrix design represents each gene by a collection of probes called a probe set. Each probe set contains multiple probe pairs; the current design uses 11 probe pairs per gene. Each probe pair consists of two probes — one called a perfect match (PM) and the other called a mismatch (MM). Sequences used in the design of the array were selected from GenBank®, dbEST, and RefSeq.
  • sequence clusters were created from the UniGene database (Build 133, April 20, 2001) and then were refined by analysis and comparison with a number of other publicly available databases including the Washington University EST trace repository and the University of California, Santa Cruz Golden-Path human genome database (April 2001 release).
  • the intensity of each probe set was normalized to the median intensity of that probe set in the control group.
  • the control groups in this study were the 8 untreated samples.
  • a probe set was regarded as reliably detected if it was called Present or Marginally present at least once among the 24 samples.
  • 54,675 probe sets on a chip only 36,357 probe sets passed the filtering and were analyzed further.
  • Replicate samples were grouped according to their experimental conditions. The average of normalized intensities was used to represent each condition.
  • WNT5A gene is represented by 3 probe sets (205990_s_at, 213425_at, 231227_at) on the Affymetrix HG Ul 33 Plus 2 microarray (Data Sheet Human Genome Ul 33 Plus 2 Array from Affymetrix, available over the internet at affymetrix.com).
  • the intensity value is the average of 8 data points from 8 healthy volunteers in this study. Both raw and normalized intensities of each of probeset are presented, as well as the ratio of the intensity between treated and untreated samples.
  • a probe set is a group of probe pairs that together detect the transcripts from a single gene. More than one probe set can be used to target one gene.
  • probe set has different thermal dynamic properties towards it unique targeted sequences
  • absolute intensity value of probe sets that targeted the same gene can be different. They should, however, produce similar trends and be well correlated in general. Thus, expression changes detected by multiple probe sets with a similar trend are more reliable than changes detected by a single probe set.
  • WNT5A expression was found to be up-regulated by IL- 13 or IL-4 in cultured normal human PBMCs based on the analysis of the microarray data (Table 2 and 3). WNT5A, which is represented on the Ul 33 Plus 2.0 chip by 3 different probesets, was consistently up-regulated by the stimulation of either IL- 13 or IL-4 in human PBMC cultures.
  • the mean RNA quantity after normalization to GAPDH represents 8 data points from 8 subjects. The ratio of the intensity between treated and untreated samples was also calculated.
  • WNT5A expression is up-regulated by IL- 13 or IL-4 in cultured normal human PBMCs.
  • IL- 13 and IL-4 are inflammatory cytokines and therefore, PBMC are highly responsive to these cytokines.
  • Colon biopsies from ulcerative colitis patients treated with infliximab were examined for WNT5A expression using a DNA chip analysis method. WNT5A expression was down-regulated by infliximab in colon biopsies of treated responders.
  • ulcerative colitis All eligible patients had an established diagnosis of ulcerative colitis. Eligible patients had active ulcerative colitis with a Mayo score of 6 to 12 points (scores can range from 0 to 12, with higher scores indicating more severe disease activity) and moderate-to-severe active disease on sigmoidoscopy (Mayo endoscopic subscore of at least 2). Rectally administered corticosteroids or medications containing 5- aminosalicylates were not permitted within two weeks before screening. Patients previously exposed to infliximab or any other anti-TNF agent were excluded (Rutgeerts P, Sandborn WJ, Feagan BG, et al. N Engl J Med 2005; 353:2462-76).
  • RNAlaterTM buffer at -8O 0 C (Ambion Inc., Austin, TX) until being extracted using RNeasyTM mini kit according to the manufacturer's instructions (Qiagen Inc., Valencia, CA) for global gene expression analysis.
  • RNeasyTM mini kit according to the manufacturer's instructions (Qiagen Inc., Valencia, CA) for global gene expression analysis.
  • a set of samples that include 3 time points at day 0 (baseline), week 8 and week 30 and two dosing regiments at 5 mg/kg and 10 mg/kg were available for comparisons.
  • clinical responsiveness was also captured and used in assessment of the impact of the treatment.
  • a responder was defined as a patient who had a decrease in the Mayo score of at least 3 points and at least 30%, together with a decrease in the rectal-bleeding subscore of at least 1 point, or an absolute rectal-bleeding subscore of 0 to 1.
  • the number of samples was 123.
  • Solid phase cDNA microarrays, Affymetrix GeneChip Human Genome Ul 33 Plus 2.0 arrays, as described in Example 2 were used.
  • WNT5A gene is represented by 3 probe sets (205990_s_at, 213425_at, 231227_at) on the Affymetrix HG Ul 33 Plus 2 microarray (see Data Sheet Human Genome Ul 33 Plus 2 Array from Affymetrix).
  • the intensity value is the average of data points from all subjects in the group where the negative sign "-" indicates down-regulation or a reduction in amount of mRNA.
  • Significant differential expression was represented with * (p-value ⁇ 0.05).
  • WNT5A expression in colon biopsies from the responder group at both post-treatment time points and both doses, was down-regulated.
  • infliximab treatment had little effect on WNT5A in the non-responder group. This can be seen by noting the similarity in fold-change between the responder group versus baseline (time 0) and responder versus the non-responder group.
  • the non-responders versus baseline comparison was not presented here, but the responder versus non-responders comparison is listed in Table 4.
  • WNT5A The gene expression level of WNT5A was relatively consistent across patients prior to treatment (data not shown). Post anti-TNFa treatment, WNT5A expression in colon biopsies from the responder group, at both time points and both doses, was down-regulated. Interestingly, infliximab treatment had little effect on WNT5A in the non-responder group. Therefore, the results indicate that WNT5A can be used as a marker of TNFa bioactivity in a subset of patients, the treatment responders.
  • RNAlaterTM buffer at - 8O 0 C (Ambion Inc., Austin, TX). Total RNA was isolated using the RNeasyTM mini kit according to the manufacturer's instructions (Qiagen Inc., Valencia, CA). Briefly, the skin biopsy samples were lysed and homogenized in the presence of 600 ⁇ L of RNAlater buffer.
  • RNA quality and quantity were assessed using a 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA) according to the manufacturer's guidelines. RNA samples with acceptable quality were used for microarray and TaqMan ® analysis.
  • Solid phase cDNA microarrays Affymetrix GeneChip Human Genome Ul 33 Plus 2.0 arrays, as described in Example 2 were used as described above. Samples which did not meet the quality control check were removed from analysis. For the week 10 samples, there were 12 placebo, 16 from 3mg/kg Responders, 15 from 5mg/kg Responders, and only 4 total non-responder samples. Partial responder samples were not analyzed.
  • the intensity value is the average of data points from all subjects in the group where the negative sign, "-" indicates down-regulation or a reduction in amount ofmRNA.
  • WNT5 A expression was down-regulated in skin biopsies of psoriatic patients who responded to anti-TNFa mAb, infliximab, at both dose levels 10 wks post-treatment. These results indicate that WNT5 A can be viewed as a marker for TNFa bioactivity in psoriasis but may not be indicative of a clinically relevant response at week 10 post-treatment.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Selon l'invention, l'expression de WNT5A et les niveaux d'ARNm associés constituent des biomarqueurs permettant de prédire la réaction à un traitement anti-TNFα dans des maladies inflammatoires à médiation immunitaire, telles que la colite ulcéreuse, et peuvent influer sur la production de TNFα dans ces maladies. L'expression de WNT5A dans des cellules des muscles lisses des voies respiratoires est un biomarqueur de l'asthme. Des inhibiteurs de WNT5A sont utilisés pour traiter des affections caractérisées par une surexpression de WNT5A.
PCT/US2008/064624 2007-05-24 2008-05-23 Wnt5a utilise en tant que marqueur des maladies inflammatoires WO2008147938A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US93996207P 2007-05-24 2007-05-24
US60/939,962 2007-05-24

Publications (2)

Publication Number Publication Date
WO2008147938A2 true WO2008147938A2 (fr) 2008-12-04
WO2008147938A3 WO2008147938A3 (fr) 2009-02-19

Family

ID=40075739

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/064624 WO2008147938A2 (fr) 2007-05-24 2008-05-23 Wnt5a utilise en tant que marqueur des maladies inflammatoires

Country Status (1)

Country Link
WO (1) WO2008147938A2 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012012725A2 (fr) 2010-07-23 2012-01-26 President And Fellows Of Harvard College Méthodes de dépistage de maladies ou d'affections à l'aide de cellules phagocytaires
CN110373457A (zh) * 2019-06-20 2019-10-25 镇江市第一人民医院 一种用于溃疡性结肠炎诊断的mRNA标志物及其应用
US10494675B2 (en) 2013-03-09 2019-12-03 Cell Mdx, Llc Methods of detecting cancer
US10626464B2 (en) 2014-09-11 2020-04-21 Cell Mdx, Llc Methods of detecting prostate cancer
US10934589B2 (en) 2008-01-18 2021-03-02 President And Fellows Of Harvard College Methods of detecting signatures of disease or conditions in bodily fluids
US10961578B2 (en) 2010-07-23 2021-03-30 President And Fellows Of Harvard College Methods of detecting prenatal or pregnancy-related diseases or conditions
US11111537B2 (en) 2010-07-23 2021-09-07 President And Fellows Of Harvard College Methods of detecting autoimmune or immune-related diseases or conditions
EP4015651A1 (fr) * 2020-12-17 2022-06-22 Koninklijke Philips N.V. Prédiction de traitement et efficacité d'un traitement anti-tnf alpha chez les patients atteints de la maladie intestinale inflammatoire
US11585814B2 (en) 2013-03-09 2023-02-21 Immunis.Ai, Inc. Methods of detecting prostate cancer
WO2023174116A1 (fr) * 2022-03-15 2023-09-21 浙江大学 Régulateur wnt5a et son utilisation
EP4303584A2 (fr) 2010-07-23 2024-01-10 President and Fellows of Harvard College Procédés de détection de signatures de maladies ou pathologies dans des liquides biologiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050037011A1 (en) * 2002-11-21 2005-02-17 Jones Stephen N. Diagnosing and treating hematopoietic cancers
US20050107399A1 (en) * 2003-09-11 2005-05-19 Kemia, Inc. Cytokine inhibitors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050037011A1 (en) * 2002-11-21 2005-02-17 Jones Stephen N. Diagnosing and treating hematopoietic cancers
US20050107399A1 (en) * 2003-09-11 2005-05-19 Kemia, Inc. Cytokine inhibitors

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CATRINA ET AL.: 'Evidence that anti-tumor necrosis factor therapy with both etanercept and infliximab induces apoptosis in macrophages, but not lymphocytes, in rheumatoid arthritis joints: extended report' ARTHRITIS RHEUM. vol. 51, no. 1, January 2005, pages 61 - 72 *
PUKROP ET AL.: 'Wnt 5a Signaling is critical for macrophage-induced invasion of breast cancer cell lines' PROC. NATL. ACAD. SCI. USA vol. 103, no. 14, 04 April 2006, pages 5454 - 5459 *
SEN M. ET AL.: 'Expression and function of wingless and frizzled homologs in rheumatoid arthritis' PROC. NATL. ACAD. SCI. USA vol. 97, no. 6, 14 March 2000, pages 2791 - 2796, XP002970274 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10934589B2 (en) 2008-01-18 2021-03-02 President And Fellows Of Harvard College Methods of detecting signatures of disease or conditions in bodily fluids
US10934588B2 (en) 2008-01-18 2021-03-02 President And Fellows Of Harvard College Methods of detecting signatures of disease or conditions in bodily fluids
US11001894B2 (en) 2008-01-18 2021-05-11 President And Fellows Of Harvard College Methods of detecting signatures of disease or conditions in bodily fluids
EP4303584A2 (fr) 2010-07-23 2024-01-10 President and Fellows of Harvard College Procédés de détection de signatures de maladies ou pathologies dans des liquides biologiques
WO2012012725A2 (fr) 2010-07-23 2012-01-26 President And Fellows Of Harvard College Méthodes de dépistage de maladies ou d'affections à l'aide de cellules phagocytaires
US10961578B2 (en) 2010-07-23 2021-03-30 President And Fellows Of Harvard College Methods of detecting prenatal or pregnancy-related diseases or conditions
US11111537B2 (en) 2010-07-23 2021-09-07 President And Fellows Of Harvard College Methods of detecting autoimmune or immune-related diseases or conditions
US11585814B2 (en) 2013-03-09 2023-02-21 Immunis.Ai, Inc. Methods of detecting prostate cancer
US10494675B2 (en) 2013-03-09 2019-12-03 Cell Mdx, Llc Methods of detecting cancer
US10626464B2 (en) 2014-09-11 2020-04-21 Cell Mdx, Llc Methods of detecting prostate cancer
CN110373457B (zh) * 2019-06-20 2023-03-14 镇江市第一人民医院 一种用于溃疡性结肠炎诊断的mRNA标志物及其应用
CN110373457A (zh) * 2019-06-20 2019-10-25 镇江市第一人民医院 一种用于溃疡性结肠炎诊断的mRNA标志物及其应用
WO2022128708A1 (fr) * 2020-12-17 2022-06-23 Innosign B.V. Prédiction de traitement et efficacité d'un traitement anti-tnf alpha chez des patients atteints d'ibd
EP4015651A1 (fr) * 2020-12-17 2022-06-22 Koninklijke Philips N.V. Prédiction de traitement et efficacité d'un traitement anti-tnf alpha chez les patients atteints de la maladie intestinale inflammatoire
WO2023174116A1 (fr) * 2022-03-15 2023-09-21 浙江大学 Régulateur wnt5a et son utilisation

Also Published As

Publication number Publication date
WO2008147938A3 (fr) 2009-02-19

Similar Documents

Publication Publication Date Title
WO2008147938A2 (fr) Wnt5a utilise en tant que marqueur des maladies inflammatoires
JP6309450B2 (ja) セロトニン関連の医学的状態、アドレナリン関連の医学的状態、ノルアドレナリン関連の医学的状態、グルタミン酸関連の医学的状態および副腎皮質刺激ホルモン放出ホルモン関連の医学的状態の処置および診断のためのミクロｒｎａおよび該ミクロｒｎａを含む組成物
JP6437946B2 (ja) 線維症の検出および処置
WO2015149056A1 (fr) Inhibition de tyrosine kinase de bruton (btk) dans le poumon pour traiter de l'inflammation pulmonaire grave et des blessures pulmonaires graves
RU2674147C2 (ru) Способы лечения колоректального рака
US10048250B2 (en) Molecular targets and compounds, and methods to identify the same, useful in the treatment of fibrotic diseases
KR20140019303A (ko) 루푸스의 치료 또는 예방을 위한 조성물 및 방법
TW200922626A (en) Cancer-related genes, CDCA5, EPHA7, STK31 and WDHD1
JP2013519869A (ja) 筋肉成長のための方法および化合物
WO2017070198A1 (fr) Polymérase q utilisée comme cible dans des cancers déficients en rh
JP2012517822A (ja) 炎症を包含する疾患の診断及び治療に有用な同定方法及び化合物
US20140315973A1 (en) Parp-1 inhibitors
CN111166867B (zh) Pd-1泛素化激动剂的功能与用途
EP2165710A1 (fr) Récepteur TYRO3 de la tyrosine kinase en tant que cible thérapeutique dans le traitement d'une tumeur de la vessie
JP2005528113A (ja) 診断標的および治療標的としてのＳｇｋおよびＮｅｄｄ
KR102132222B1 (ko) 항암제 내성 진단 마커로서의 miR-143-3p 및 miR-373-5p, 및 이의 이용
US7160681B2 (en) Method for regulating cell growth and assays related thereto
JP2016518812A (ja) 線維症治療において有用な分子標的及び前記標的のインヒビター
KR20200028982A (ko) 시냅스 기능을 증진시키기 위한 hdac2-sp3 복합체 표적화
US9709552B2 (en) Use of inhibitors of leukotriene B4 receptor BLT2 for treating asthma
CN114908158B (zh) Cdk1在晚期胃肠间质瘤的诊断和治疗中的应用
US20220364184A1 (en) Compositions and methods for treatment of a poor prognosis subtype of colorectal cancer
US11013717B1 (en) Methods and compositions for treating cancer using SERCA pump inhibitors
Dabral Role of Notch signaling in pulmonary hypertension
CN118019548A (zh) 用于治疗癌症的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08756165

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08756165

Country of ref document: EP

Kind code of ref document: A2