WO2006102408A2 - Methods and compositions for the modulation of immune responses and autoimmune diseases - Google Patents

Methods and compositions for the modulation of immune responses and autoimmune diseases Download PDF

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Publication number
WO2006102408A2
WO2006102408A2 PCT/US2006/010398 US2006010398W WO2006102408A2 WO 2006102408 A2 WO2006102408 A2 WO 2006102408A2 US 2006010398 W US2006010398 W US 2006010398W WO 2006102408 A2 WO2006102408 A2 WO 2006102408A2
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Prior art keywords
activator
tlr4
disease
expression
condition
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PCT/US2006/010398
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French (fr)
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WO2006102408A3 (en
Inventor
Christopher L. Karp
Senad Divanovic
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Children's Hospital Medical Center
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Publication of WO2006102408A2 publication Critical patent/WO2006102408A2/en
Publication of WO2006102408A3 publication Critical patent/WO2006102408A3/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0325—Animal model for autoimmune diseases

Definitions

  • the present invention relates to regulation of inflammation and immune responses.
  • the present invention relates to a method of treating a condition
  • RP105 as a specific inhibitor of TLR4 signaling and as a physiological regulator of TLR4 signaling for the treatment of TLR4-mediated inflammation and TLR4-driven
  • TLR Toll-like receptor
  • hyper-inflammatory responses that characterize sepsis provide a paradigmatic example, as do the more localized inappropriate inflammatory processes
  • the eleven known members of the mammalian TLR family are characterized structurally by an extracellular leucine-rich repeat (LRR) domain, a conserved pattern of juxtamembrane cysteine residues, and an intracytoplasmic signaling domain
  • LRR leucine-rich repeat
  • TIR Toll/IL-1 resistance
  • the TLR-like molecule RP 105 was originally cloned as a B cell-specific molecule able to drive B cell proliferation (9, 10).
  • RP 105 has a conserved extracellular LRR domain and a TLR-like pattern of
  • RP105 lacks a TIR
  • RP 105 is actually a specific homologue of TLR4. Further, RP 105 is not B cell-specific as originally proposed: we have found that its expression directly mirrors that of TLR4 on antigen-presenting cells.
  • Toll and TLR4 mutation of the conserved juxtamembrane cysteine residues, or deletion of the extracellular portion altogether, results in a constitutively active molecule (18-20). This suggests that the activation of Toll/TLRs is normally restrained through extracellular protein/protein interactions, likely through the LRR domain (21).
  • Toll/TLRs can yield inactive or dominant negative molecules (18, 22, 23).
  • RP 105 has the structure of an inhibitory TLR4.
  • RP 105 specifically inhibits TLR4 signaling when co-expressed in cells
  • RP 105 is a physiological regulator of TLR4 signaling and of TLR4-driven proinflammatory responses in vivo. Although the activation of proinflammatory responses through TLRs is critical for both innate and adaptive immunity, excessive or inappropriate inflammation can itself be maladaptive. RP 105 inhibits TLR signaling and this regulation of TLR expression provides a point of control (48-50) since RP 105 acts specifically to inhibit TLR4 signaling.
  • the present invention now provides methods of using RP105 as a specific inhibitor of TLR4 signaling to treat TLR4-mediated pathogenic processes and
  • the present invention relates to the modulation of inflammation and immune responses.
  • Methods and compositions for suppressing a pathological inflammatory or immune response are disclosed.
  • the methods involve administering an effective amount of an agent that upregulates RP105-mediated inhibition of TLR inhibition, through either upregulating RP 105 expression or activity.
  • the methods are useful in the treatment of diseases marked by excessive or inappropriate inflammatory
  • autoimmune diseases such as rheumatoid arthritis, juvenile
  • the present invention provides for methods and compositions for upregulating the expression of RP 105, or for the engagement of or signaling through RP 105, in order to inhibit Toll-like receptor signaling. More specifically, the patent relates to the use of RP 105 as a specific inhibitor of TLR4 signaling and as a
  • This invention also relates to treating an animal having a disease or condition associated with Toll-like receptor 4 by agonists of RP 105.
  • the invention involves a method for treating a subject having a disease or condition associated with Toll-like receptor 4 comprising administering to the subject a therapeutically or prophylactically effective amount of one or more compounds capable of activating RP 105 so that the action and/or expression of Toll-
  • the present invention provides a method of modulating an immune response comprising modulating the expression or activity of RP 105.
  • the present invention provides a method of suppressing an immune response comprising administering an effective amount of an activator of RP 105 to an animal in need of such treatment.
  • proinflammatory responses through TLRs are treated by modulation of RP105.
  • the present invention provides a method of preventing or treating an autoimmune disease comprising administering an effective
  • an activator of RP 105 to an animal having, suspected of having, or susceptible to having an autoimmune disease.
  • the invention also includes pharmaceutical compositions containing one or more activators of RP 105 for use in inducing tolerance in autoimmune, inflammatory
  • the RP 105 In preferred embodiments of the above methods and compositions, the RP 105
  • activator is an antibody that binds RP 105, a small molecule that signals through
  • RP 105 or a compound that upregulates RP 105 expression.
  • the activator of RP105 is administered in combination a
  • the invention also includes pharmaceutical compositions containing an RP 105
  • compositions can include other molecules that can suppress the immune response such as activators of RP 105.
  • the invention further includes screening assays for identifying substances that
  • the invention also includes diagnostic kits and methods for detecting conditions associated with increased, decreased or abnormal expression of RP105.
  • the method further comprises
  • the present invention relates to methods for inhibiting Toll-like receptor-4
  • TLR-4 Toll-like receptor 4 induced inflammatory bowel disease and related gastrointestinal pathologies.
  • the present invention concerns the treatment of Toll-like receptor 4 induced inflammatory bowel disease, including ulcerative colitis, Crohn's disease, indeterminate colitis, infectious colitis, drug or chemical-induced colitis, diverticulitis, and ischemic colitis.
  • the present methods provide for the treatment of Toll-like receptor 4 -dependent colitis.
  • TLR-4 biological activity of TLR-4, as, for example, by inhibiting its expression or signaling.
  • the Toll-like receptor 4 induced disease is one or more of systemic lupus erythematosis, sceleroderma, Sjogren's syndrome, multiple sclerosis
  • rheumatoid arthritis juvenile rheumatoid arthritis, myocarditis, uveitis, Reiter's syndrome, gout, osteoarthritis, polymyositis, primary biliary cirrhosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis,
  • vascular diseases include atherosclerosis, transplant atherosclerosis, vein-graft atherosclerosis, stent restenosis, and angioplasty restenosis.
  • vascular diseases include atherosclerosis, transplant atherosclerosis, vein-graft atherosclerosis, stent restenosis, and angioplasty restenosis.
  • Such patients include those suffering from, for example, angina pectoris and its subtypes (e.g., unstable angina and variant angina); ischemias affecting organs such as the brain, heart, bone, and intestines, and conditions associated with the ischemias, such as stroke, transient ischemic attacks, heart attack,
  • angina pectoris and its subtypes e.g., unstable angina and variant angina
  • ischemias affecting organs such as the brain, heart, bone, and intestines
  • conditions associated with the ischemias such as stroke, transient ischemic attacks, heart attack
  • osteonecrosis osteonecrosis, colitis, poor kidney function, and congestive heart failure; poor blood
  • such diseases include, for example, vascular disease such as atherosclerosis and thrombosis, restenosis after angioplasty and/or stenting, and vein-graft disease after bypass surgery.
  • vascular disease such as atherosclerosis and thrombosis
  • restenosis after angioplasty and/or stenting and vein-graft disease after bypass surgery.
  • the invention involves a method for treating a condition involving a cytokine-induced diseases or pathology by administering to a patient an
  • TLR Toll-like receptor
  • the cytokine-mediated disease is selected from acquired
  • ARDS adult respiratory distress syndrome
  • Alzheimer's disease Alzheimer's disease, aphthous ulcers, arthritis, asthma, atherosclerosis, atherosclerosisatopic dermatitis, bone resorption diseases, cachexia, chronic obstructive pulmonary disease, congestive heart failure, contact dermatitis, Crohn's disease, dermatoses with acute inflammatory components, diabetes, endotoxemia, glomerulonephritis, graft versus host disease, granulocyte transfusion, Guillain-Barre syndrome, inflammatory bowel disease, leprosy, leukopherisis, malaria, multiple organ injury secondary to trauma, multiple sclerosis,
  • the present invention provides for methods of treating a disease mediated by cytokines which comprises administering to a patient in need of such treatment a therapeutically effective amount of one or more activators of RP 105.
  • the present invention provides for methods of treating a gastrointestinal disorder in a patient in need thereof comprising administering to the
  • the gastrointestinal disorder is an inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, ulcerative colitis, a peptic ulcer, a stress ulcer, a bleeding ulcer, gastric hyperacidity, dyspepsia, gastroparesis, Zollinger-Ellison syndrome, gastroesophageal reflux disease, abacterial infection, short-bowel (anastomosis) syndrome, a hypersecretory state associated with systemic mastocytosis or basophilic leukemia or hyperhistaminemia.
  • the cytokine involved in the TLR-4-mediated disease or pathology is a proinflammatory cytokine.
  • the proinflammatory cytokine is a proinflammatory cytokine.
  • cytokine is selected from the group consisting of TNF- ⁇ , EL-I, EL-l ⁇ , BL-6, and EL-8.
  • modulating an immune response comprising modulating the expression or activity of RP105.
  • the present invention provides a method of suppressing an
  • the present invention provides a method of inducing immune suppression or tolerance to a transplanted organ or tissue in a recipient animal comprising administering an effective amount of an activator of RP 105 activation or expression to the recipient animal prior to the transplantation of the organ or tissue.
  • the present invention provides a method of preventing or inhibiting graft versus host disease in a recipient animal receiving an organ or tissue transplant comprising administering an effective amount of an activator of
  • RP 105 activation or expression to the organ or tissue prior to the transplantation in the recipient animal.
  • the present invention provides a method of
  • preventing or inhibiting fetal loss comprising administering an effective amount of an activator of RP 105 activation or expression to an animal in need thereof.
  • the present invention provides a method of preventing or treating an autoimmune disease comprising administering an effective amount of an activator of RP 105 activation or expression to an animal having,
  • the present invention provides a method of
  • the invention also includes pharmaceutical compositions containing one or more activators of RP 105 activation or expression for use in inducing tolerance in transplantation, allergy or autoimmune disease or for preventing or treating fetal loss.
  • the RP 105 activator is an antibody that binds RP 105 or an antisense oligonucleotide that activates
  • the inhibitor of RP 105 is administered in combination with an MD-I protein or a nucleic acid
  • activating RP 105 can be used to induce immune suppression.
  • inhibiting RP 105 can be used in preventing immune suppression or
  • the present invention provides a method of immune suppression comprising administering an effective amount of an RP 105 protein or a nucleic acid sequence encoding an RP 105 protein to an animal in need thereof.
  • the invention also includes pharmaceutical compositions containing an RPl 05
  • compositions can include other molecules that can activate
  • the immune response such as activators of MD-I activity or expression.
  • the invention further includes screening assays for identifying substances that modulate RP 105 expression or activity. Such substances maybe useful in the therapeutic methods and compositions of the invention.
  • the invention also includes diagnostic kits and methods for detecting conditions associated with increased, decreased or abnormal expression of RP 105.
  • the present invention provides for a method for screening a test compound for the potential to prevent, stabilize, or treat an autoimmune or inflammatory disease comprising the steps of: a) contacting a first cell sample from a first subject that has, or is at risk for developing, an autoimmune or inflammatory
  • RP 105 inhibition of TLR4 is measured by the ability of the lipopolysaccharide (LPS) signaling complex to bind LPS.
  • LPS lipopolysaccharide
  • RP 105 activation or expression is measured.
  • autoimmune or inflammatory disease comprising the steps of: a) contacting a population of cells from a subject that has, or is at risk for developing, an autoimmune
  • the cells are autoimmune cells.
  • the cells are leukocytes.
  • the present invention provides for a method for
  • the second subject does not have, or is not at risk for
  • autoimmune disease developing, the autoimmune disease; c) contacting the first cell sample and the second cell sample with a compound that preferentially increases the activation or expression of RP 105, wherein both of the first and second samples are contacted with the compound in the same manner; and d) measuring the activation or expression of RP 105 in the first cell sample and in the second cell sample, wherein a decrease in activation or expression of RP 105 in the first sample relative to the activation or expression of RP 105 in the second sample indicates that the first subject has, or is
  • the autoimmune disease predisposed to developing, the autoimmune disease.
  • the present invention provides for a method for diagnosing an autoimmune disease, or a predisposition to the disease, in a subject
  • autoimmune cells from a subject that has an autoimmune disease, or is at risk for developing an autoimmune disease, with the test compound; b) contacting a second cell element from the subject with the test compound, wherein the autoimmune cells and the second cell element are contacted with the compound in the same manner; and c) measuring the activation of RP 105 of
  • the autoimmune cells wherein the subject has, or is at risk for developing, the autoimmune disease if the activation or expression of RP 105 of the autoimmune cells
  • the present invention provides for a method for the stratification of a human patient into a therapeutic subgroup for an autoimmune disease comprising the steps of: a) contacting a cell sample from the patient with a compound that preferentially decreases the activation of RP 105 of leukocytes; b) measuring the activation of RP 105 of the leukocytes; and c) placing the patient into a therapeutic subgroup based on the amount of decrease of the activation or expression ofRP105.
  • the present invention provides for a method for monitoring a therapy for a human that has an autoimmune disease, or is at risk for developing the disease, comprising the steps of: i) obtaining a first cell sample from the patient and contacting the first cell sample with a compound that preferentially decreases the activation of RP 105 of leukocytes; ii) measuring the activation of
  • the patient and contacting the second cell sample with the compound, wherein the second cell sample is obtained at least 12 hours after obtaining the first cell sample;
  • the present invention provides for a pharmaceutical composition for use in suppressing an immune response comprising an activator of RP105/MD-1 complex formation in admixture with a suitable diluent or carrier.
  • the activator is an antibody that binds MD-I, RPl 05, or both.
  • the present invention provides for a screening method
  • test compound is an immunostimulatory
  • the present invention provides for a screening method for identifying an immunostimulatory compound, comprising: contacting a functional RP 105 with a test compound under conditions which, in presence of a reference
  • immunostimulatory compound permit a reference response mediated by a RP105 signal transduction pathway; detecting a test response mediated by the RP 105 signal
  • test compound is an immunostimulatory
  • test response equals or exceeds the reference response.
  • the present invention provides for a screening method
  • immunostimulatory compound under conditions which, in presence of the reference immunostimulatory compound alone, permit a reference response mediated by a RP 105 signal transduction pathway; detecting a test-reference response mediated by the RP 105 signal transduction pathway; determining the test compound is an agonist of RP 105 signaling activity when the test-reference response exceeds the reference response; and determining the test compound is an antagonist of RP 105 signaling activity when the reference response exceeds the test-reference response.
  • the present invention provides for a screening method for identifying species specificity of an immunostimulatory compound, comprising:
  • the screening method is performed on a plurality of test compounds, hi another embodiment, the response mediated by the RP 105 signal transductkm pathway is measured quantitatively. In another embodiment, the
  • functional RP 105 is expressed in a cell.
  • the cell is an isolated
  • the cell is an isolated mammalian cell that does not
  • the cell is a human fibroblast.
  • the cell comprises an expression vector comprising an isolated nucleic acid which encodes a reporter construct selected from the group of interleuldns, NF- kappaB, interferons, and TNFs.
  • the functional RP 105 is part of a cell-free system.
  • the reference immunostimulatory compound is a nucleic acid.
  • the nucleic acid is a CpG nucleic acid.
  • the reference immunostimulatory compound is a small molecule.
  • the test compound is a part of a combinatorial
  • test compound is a nucleic acid.
  • the nucleic acid is a CpG nucleic acid.
  • test compound is a small molecule. In another embodiment, the test compound is a small molecule.
  • the response mediated by a RP 105 signal transduction pathway is induction of a reporter gene under control of a
  • the response mediated by a RP 105 signal transduction pathway is selected from the group consisting of (a) induction of a reporter gene under control of a minimal promoter responsive to a transcription factor selected from the group consisting of API, NF-ka ⁇ aB, ATF2, IRF3, and IRF7; (b) secretion of a chemoMne;
  • RP 105 signal transduction pathway is secretion of a type 1 BFN.
  • the response mediated by a RP 105 signal transduction pathway is
  • test compound with a test compound further comprises, for each test compound, contacting with the test compound at each of a plurality of concentrations.
  • the e detecting is performed 6-12 hours following the contacting.
  • the detecting is performed 16-24 hours following the contacting.
  • the present compounds may also be used in co-therapies, partially or completely, in place of other conventional anti-inflammatories, such as together with steroids, cyclooxygenase-2 inhibitors, NSAIDs, DMARDS, antibiotics, immunosuppressive agents, 5-lipoxygenase inhibitors, LTB 4 antagonists and LTA4 hydrolase inhibitors and anti-cell adhesion molecules such as anti E-selectin.
  • this aspect additionally involves administering a therapeutic steroid to the patient.
  • a therapeutic steroid for example, therapeutic
  • steroids may include, for example, corticoids, glucocorticoids, dexamethasone,
  • prednisone prednisalone
  • betamethasone betamethasone
  • These methods may employ the compounds of this invention in a monotherapy or in combination with an anti-inflammatory or immunosuppressive agent.
  • Such combination therapies include administration of the agents in a single dosage form or in multiple dosage forms administered at the same time or at different times.
  • Figure 2 RP105 expression by human peripheral blood leukocytes. Flow cytometric analysis of PBMC from healthy human donors, (a) B cells; (b) monocytes; (c) myeloid DC; (d) plasmacytoid DC. Myeloid DC were
  • n 3 for the other cell types.
  • Figure 3 RP 105 expression by murine leukocytes. Flow cytometric analysis of cell populations, (a) splenic B cells (representative of an experimental n >
  • splenic plasmacytoid DC (CD19 + B22O+CD1 Ic + GR-I + )
  • FIG. 4 Suppression of TLR4 signaling in HEK293 cells by RP 105 expression, (a) Lack of activity of RP 105 as a signaling receptor for LPS in HEK293 cells.
  • HEK293 cells stably expressing CD14 were transiently transfected with cDNA encoding MD-I, MD-2, TLR4, empty vector control
  • TLR4 were transiently transfected with MD-I and MD-2 along with the
  • HEK293 cells stably expressing MD-2 and TLR4
  • plasmid a TK-renilla luciferase reporter plasmid and MD-I, along with EV (open bars) or RP 105 (filled bars).
  • EV open bars
  • RP 105 filled bars.
  • HEK293 cells stably expressing CD14 and TLR2 were transiently transfected with MD-I and EV (open bars) or MD-I and RP105 (filled bars)
  • Figure 7 Altered cytokine production by dendritic cells from RP105 "A mice following in vitro stimulation with TLR4-specific LPS. Bone marrow-derived
  • FIG. 8 Ability of heterologous TLR4 signaling to overcome RP105- mediated inhibition of TLR4 signaling.
  • Bone marrow-derived DC from wild type U] or RP 1O5 'A [•] mice were stimulated with: (a) non-repurified E. coli K235 LPS; or (b) repurified E, coli K235 LPS plus varying concentrations of Pam 3 Cys. *p ⁇ 0.05, $p ⁇ 0.001. Means + SE of triplicate cultures in a
  • Wild type mice n - 17 (open bars) or RP105 'A mice (n -
  • Figure 10 Genotyping and phenotyping of RP105 ⁇ ' ⁇ mice. FACS analysis of peripheral blood cells.
  • TLRs as inferred by sequence alignment using ClustalW software.
  • activation means a mechanism wherein a compound upregulates RP105-
  • administration includes systemic use, as by injection (especially parenterally), intravenous infusion, suppositories and oral administration
  • Oral administration is particularly preferred in the present invention.
  • “Ameliorate” or “amelioration” means a lessening of the detrimental effect or severity of the cell adhesion disorder in the subject receiving therapy, the severity of ⁇ the response being determined by means that are well known in the art.
  • Chemokines are chemotactic cytokines that are released by a wide variety of cells to attract macrophages, T-cells, eosinophils, basophils, neutrophils and endothelial cells to sites of inflammation and tumor growth.
  • CXC-chemokines include interleukin-8 (IL-8), neutrophil-activatmg protein- 1 (NAP-I), neutrophil-activating
  • NAP-2 protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • GRO ⁇ protein-2
  • ENA-78 GCP-2
  • IP-IO IP-IO
  • MIG MIG
  • PF4 PF4
  • chemokines include RANTES, MIP- l ⁇ , MIP-2 ⁇ , monocyte chemotactic protein- 1
  • MCP-I MCP-2
  • MCP-3 MCP-3
  • eotaxin MCP-I
  • compositions which comprise the present invention are capable of being commingled without interacting in a manner which would substantially decrease the efficacy of the pharmaceutically active compound under ordinary use conditions.
  • corticosteroid is meant any naturally occurring or synthetic steroid
  • Synthetic corticosteriods may be
  • Corticosteroids may have glucocorticoid and/or mineralocorticoid activity.
  • corticosteroids include, for example,, dexamethasone, betamethasone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide, beclomethasone, dipropionate, beclomethasone dipropionate monohydrate, flumethasone pivalate, diflorasone diacetate, fluocinolone acetonide, fluorometholone, fluorometholone acetate, clobetasol propionate, desoximethasone, fluoxymesterone, fluprednisolone, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone sodium phosphate, hydrocortisone
  • prednisolone acetate prednisolone sodium phosphate, prednisolone tebutate, clocortolone pivalate, flucinolone, dexamethasone 21 -acetate, betamethasone 17- valerate, isoflupredone, 9-fluorocortisone, 6-hydroxydexamethasone, dichlorisone, meclorisone, flupredidene, doxibetasol, halopredone, halometasone, clobetasone, diflucortolone, isoflupredone acetate, fluorohydroxyandrostenedione, beclomethasone, flumethasone, diflorasone, fluocinolone, clobetasol, cortisone, paramethasone, clocortolone, prednisolone 21-hemisuccinate free acid, prednisolone
  • low dose corticosteroid is meant a dose that is less than a dose that would typically be given to a patient for treatment of inflammation.
  • exemplary low dose corticosteroid is meant a dose that is less than a dose that would typically be given to a patient for treatment of inflammation.
  • corticosteroids 12 mg/day
  • cortisone 15 mg/day
  • prednisone 3 mg/day
  • methylprednisolone 2.5 mg/day
  • triamcinolone 2.5 mg/day
  • betamethasone 250 ⁇ g/day
  • dexamethasone 450 ⁇ g/day
  • hydrocortisone 9 mg/day.
  • “Pharmaceutically-acceptable” shall mean that the pharmaceutically active compound and other ingredients used in the pharmaceutical compositions and methods defined herein are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like,
  • prodrug indicates a therapeutic agent that is prepared in an inactive
  • an active form i.e., drug
  • an active form i.e., drug
  • safe and effective amount means a sufficient amount of pharmaceutically active compound to effect the activation of RP 105 and inhibition of TLR-4.
  • the required dosage of a pharmaceutically active agent or of the pharmaceutical composition containing that active agent will vary with the severity of the condition being treated, the duration of
  • Toll-like receptors or "TLRs” are type I transmembrane proteins containing repeated leucine-rich motifs in their extracellular domains, At least eight mammalian TLR proteins have been identified, Toll-like receptors 1-8. Ligand engagement of the TLRs results in activation of NF- ⁇ B and induction of the cytokines and co-stimulatory molecules required for the activation of the adaptive immune response.
  • Human Toll- like receptor 4 also known as TLR4 and hToll
  • Toll was cloned from a human fetal liver/spleen library, characterized, and mapped to chromosome 9q32-33. Toll-like receptor 4 mRNA expression can be detected in the cells of the immune system: monocytes,
  • Treatment refers to any curative therapy, prophylactic therapy, ameliorative therapy and preventative therapy
  • bacterial infections that occur in mammals, fish and birds as well as disorders related to bacterial infections that may be treated or prevented by administering
  • antibiotics such as the compounds of the present invention.
  • Such bacterial infections include bacterial infections
  • Streptococcus pneumonia Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, or Peptostreptococcus spp.; pharynigitis, rheumatic fever, and
  • Streptococcus agalactiae Streptococcal groups C-F (minute-colony streptococci), viridans streptococci, Corynebacterium minutissimum, Clostridium spp., or Bartonella
  • aureus food poisoning and Toxic shock syndrome
  • Groups A, B, and C streptococci ulcers related to infection by Helicobacter pylori; systemic febrile syndromes related to infection by Borrelia recurrentis; Lyme disease related to infection by Borrelia burgdorferi; conjunctivitis, keratitis, and dacrocystitis related to infection by Chlamydia trachomatis, Neisseria gonorrhoeae, S. aureus, S. pneumoniae, S. pyogenes, H. influenzae, or Listeria spp.;
  • MAC Mycobacterium avium complex
  • Mycobacterium avium, or Mycobacterium intracellulare gastroenteritis related to
  • Bacterial infections and protozoa infections and disorders related to such infections include the following: bovine respiratory disease related to infection by P. haem., P. multocida, Mycoplasma bovis, or Bordetella spp.; cow enteric disease related to infection by E.
  • coli i.e., coccidia, Cryptosporidia, etc.
  • Enterococcus spp. Enterococcus spp.; swine respiratory disease related to infection by A. pleuro., P. multocida, or Mycoplasma spp.; swine enteric disease related to infection by E coli,
  • cow metritis related to infection by E coli cow metritis related to infection by E coli
  • nodosus cow pink-eye related to infection by Moraxella bovis; urinary tract infection in dogs and cats related to infection by E coli; skin and soft tissue infections in dogs and cats related to infection by Staph, epidermidis, Staph, intermedius, coagulase neg. Staph, or P. multocida; and dental or mouth infections in dogs and cats related to infection by Alcaligenes spp., Bacteroides spp., Clostridium spp., Enterobacter spp.,
  • Eubacterium Peptostreptococcus, Porphyromonas, or Prevotella.
  • Other bacterial infections and disorders related to such infections that may be treated or prevented in
  • the invention provides a method of treating a condition associated
  • cytokine production in a mammal comprising administering a pharmaceutically
  • the invention also provides the use of an activator of RP 105 in the manufacture of a medicament for use in the treatment of a condition associated with cytokine production
  • the cytokine is TNF, preferably TNF ⁇ .
  • the cytokine is IL-I, preferably IL-I ⁇ .
  • the cytokine-associated condition maybe any one of sepsis, septic shock, inflammation, rheumatoid arthritis or Crohn's disease.
  • the cytokine-associated condition maybe irritable bowel disease (IBD).
  • the cytokine-associated condition maybe ulcerative colitis.
  • the cytokine-associated condition is induced by a
  • TRR Toll-related receptor
  • LPS lipopolysaccharide
  • the invention also provides a pharmaceutical formulation comprising a pharmaceutically acceptable carrier and an activator of RP 105 identifiable by a method of the invention.
  • the invention also provides the use of an activator of RP 105 to study the
  • TRR-ligand preferably LPS
  • the invention provides a method of treating sepsis or septic shock in a subject administering a pharmaceutically effective amount of an activator of RP 105.
  • the invention provides a method of treating inflammation in a subject comprising administering a pharmaceutically effective amount of an activator of RP105.
  • the invention provides a RP105 activator for use as a medicament.
  • the invention provides a RP 105 activator for use in the manufacture of a medicament to
  • the invention provides a method or
  • activator of the invention wherein the activator is a vaccine, an antibody or a fragment thereof which is capable of binding RP 105 or a fragment thereof.
  • the invention provides a method of treating sepsis, septic shock and/or
  • the invention provides amethod of treating rheumatoid arthritis in a subject comprising administering a pharmaceutically effective amount of an activator of a member of RP 105.
  • the invention provides a method of treating Crohn's disease in a subject comprising administering a pharmaceutically effective amount of an activator of a member of RP 105.
  • the invention provides a method of treating a condition induced by a TRR ligand in a subject comprising administering a pharmaceutically effective amount of an activator of a member of RP 105.
  • the invention provides a method of treating a condition induced by a TLR4 ligand in a subject comprising administering a pharmaceutically effective amount of
  • the invention provides a method of treating a condition induced by a pathogen in a subject comprising administering a
  • invention provides a method of treating a condition induced by bacteria, or a factor derived there from in a subject comprising administering a pharmaceutically effective
  • RP 105 is an immune modulating molecule that has utility in a wide range of applications. Accordingly, the present invention includes all uses that relate to the realization of the immune modulatory properties of RP 105 including, but not limited to, the development of therapeutic and diagnostic assays and compositions as well as the preparation and/or isolation of other molecules that modulate RP 105 that maybe useful in the therapeutic
  • the present invention provides a method of suppressing an immune response comprising administering an effective amount of an activator of RP 105 to an animal in need of such treatment.
  • the invention includes a use of an effective amount of an activator of RP 105 to suppress an immune response or to
  • an activator of RP 105" means any molecule or compound that can activate the expression of the RP 105 gene or that can activate the activity of the
  • the activator of RP 105 is an RP 105 specific antibody.
  • Antibodies to RP 105 may be prepared using techniques known in the art such as those described by Kohler and Milstein, Nature 256, 495 (1975) and in U.S. Pat. Nos. RE
  • antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab').sub.2) and recombinantly produced binding partners.
  • the therapeutic methods of the invention can be used to treat any condition wherein it is desirable to modulate RP 105 expression or activity. Such conditions
  • autoimmune disease include, but are not limited to, , autoimmune disease, , inflammatory conditions, septic
  • neurodegenerative diseases e.g., Alzheimer's disease
  • the method of the present invention may also be used to treat or prevent autoimmune disease. Ih an autoimmune disease, the immune system of the host fails to recognize a particular antigen as "self and an immune reaction is
  • the present invention provides a method of preventing or treating an autoimmune disease comprising administering an effective amount of an activator of RP 105 to an animal having, suspected of having, or susceptible to having an autoimmune disease.
  • the invention includes a use of an effective amount of an activator of RP 105 to prevent or activate an autoimmune disease or to prepare a medicament to prevent or activate an autoimmune disease.
  • Autoimmune diseases that may be treated or prevented according to the present invention include, but are not limited to, type 1 insulin-dependent diabetes mellitus, adult respiratory distress syndrome, inflammatory bowel disease, dermatitis, meningitis, thrombotic thrombocytopenic purpura, Sjogren's syndrome, encephalitis,
  • lupus erythematosus polymyositis, sarcoidosis, granulomatosis, vasculitis, pernicious anemia, CNS inflammatory disorder, antigen-antibody complex mediated diseases, autoimmune haemolytic anemia, Hashimoto's thyroiditis, Graves disease, habitual spontaneous abortions, Reynard's syndrome, glomerulonephritis, dermatomyositis, chronic active hepatitis, celiac disease, autoimmune complications of AIDS, atrophic
  • RP 105 activators maybe further enhanced by co-administering other immune
  • modulators including but not limited to RP 105, anti-fgl2, anti-B7, anti-CD80 or anti-
  • the RP 105 activators are co-administered with an RP 105 protein or a nucleic acid molecule encoding an RP 105 protein as described in the PCT
  • RP 105 protein includes the full-length RP 105 protein as well as fragments or portions of the protein. Preferred fragments or portions of the protein are those that are sufficient to induce an immune response or prevent immune suppression.
  • the RP 105 protein or the nucleic acid encoding the RP 105 protein can
  • the RP 105 protein or nucleic acid may be modified from the known sequences to make it more useful in the methods of the present invention.
  • the RP105 protein is prepared as a soluble fusion protein.
  • the fusion protein may contain the extracellular domain of RP 105 linked to an immunoglobulin (Ig) Fc Region.
  • the RP 105 fusion maybe prepared using techniques known in the art. Generally, a DNA sequence encoding the extracellular domain of RP105 is linked to a DNA sequence encoding the Fc of the Ig and expressed in an appropriate expression system where the RP105-Fclg fusion protein is produced.
  • the RP 105 protein may be obtained from known sources or prepared using recombinant DNA techniques. The protein may have any of the known published sequences for RP 105. For example, the sequences can be obtained from GenBank as described above. The protein may also be modified to contain amino acid substitutions,
  • Non- conserved substitutions involve replacing one or more amino acids of the RP 105 amino acid sequence with one or more amino acids that possess dissimilar charge, size, and/or hydrophobicity characteristics.
  • Administration of an "effective amount" of the RP105 protein and nucleic acid of the present invention is defined as an amount effective, at dosages and for periods
  • the effective amount of the RP105 protein or nucleic acid of the invention may vary according to factors such as the disease state, age, sex, and weight of the animal. Dosage regime maybe adjusted to provide the optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced as indicated by the
  • the present invention provides a method of inducing fetal loss comprising administering an effective amount of an RP 105 protein or a nucleic acid sequence encoding an RP 105 protein to an animal in need thereof.
  • RP 105 Modulators
  • the present invention also includes the isolation and/or identification of
  • substances to modulate RP105 expression or activity Such substances or RP105
  • RP 105 modulators may be useful in the above-described therapeutic methods.
  • Two examples of RP 105 modulators include antibodies and antisense molecules that are described in detail above.
  • Other RP 105 modulators may be identified, for example, using the
  • Substances that affect RP 105 activity can be identified based on their ability to bind to RP105.
  • Substances that can bind with the RP105 of the invention may be identified by
  • RP105 reacting the RP105 with a substance that potentially binds to RP105, and assaying for complexes, for free substance, or for non-complexed RP 105, or for activation of RP105.
  • the invention provides a method of identifying substances that
  • RP105 can bind with RP105, comprising the steps of: (a) reacting RP105 and a test substance, under conditions that allow for formation of a complex between the RP 105 and the test substance, and (b) assaying for complexes of RP 105 and the test substance, for free substance or for non complexed RP 105, wherein the presence of complexes indicates that the test substance is capable of binding RP 105.
  • Conditions that permit the formation of substance and RP 105 complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.
  • isolation techniques for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption,
  • labeled RP 105 or a labeled substance maybe utilized.
  • the antibodies, proteins, or substances may be labeled with a detectable substance as described above.
  • RP105 or the substance used in the method of the invention maybe insolubilized.
  • RP 105 or substance may be bound to a suitable carrier.
  • suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the carrier maybe in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
  • the insolubilized protein or substance maybe prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for.example, cyanogen bromide coupling.
  • the proteins or substance may also be expressed on the surface of a cell in the above assay.
  • the invention also contemplates assaying for an antagonist or agonist of the action of RP 105.
  • the agonists and antagonists that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding
  • the invention also makes it possible to screen for antagonists that activate the
  • the invention may be used to assay for a
  • the present invention also includes peptide mimetics of the RP105 of the invention.
  • a peptide derived from a binding domain of RP 105 will interact directly or indirectly with an associated molecule in such a way as to mimic the native binding domain.
  • Such peptides may include competitive activators, enhancers, peptide mimetics, and the like. All of these peptides as well as molecules substantially homologous, complementary or otherwise functionally or structurally equivalent to these peptides may be used for purposes of the present invention.
  • Peptide mimetics are structures that serve as substitutes for peptides in interactions between molecules (See Morgan et al. (1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimetics include synthetic structures that may or
  • Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide of the invention.
  • Peptide mimetics may be designed based on information obtained by
  • the mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues maybe used to constrain amino acid residues to particular conformational states.
  • the mimetics can also include mimics of activator peptide secondary structures. These structures can model the 3- dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N- substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.
  • Peptides of the invention may also be used to identify lead compounds for
  • the structure of the peptides described herein can be readily determined by a number of methods such as NMR and X-ray crystallography.
  • comparison of the structures of peptides similar in sequence, but differing in the biological activities they elicit in target molecules can provide information about the structure-activity relationship of the target.
  • Information obtained from the examination of structure-activity relationships can be used to design either modified peptides, or other small molecules or lead compounds that can be tested for predicted properties as related to the target molecule.
  • the activity of the lead compounds can be evaluated using assays similar to those described herein.
  • a peptide with a desired activity is crystallized in association with a target molecule, and the X-ray structure of the complex is determined. The structure can then be compared to the structure of the target molecule in its native state, and information from such a comparison maybe used to design compounds expected to possess.
  • the invention enables a method for screening candidate compounds for their ability to increase or decrease the activity of a RP 105 protein.
  • the method comprises providing an assay system for assaying RP 105 activity, assaying the activity in the presence or absence of the candidate or test
  • the present invention provides a method for identifying a compound that affects RP 105 protein activity or expression comprising: (a) incubating a test compound with a RP 105 protein or a nucleic acid encoding a RP 105 protein; and (b) determining an amount of RP 105 protein activity or expression and comparing with a control (i.e. in the absence of the test substance), wherein a change in the RP 105 protein activity or expression as compared to the control indicates that the test
  • the invention enables a method for
  • the method comprises putting a cell with a candidate compound, wherein the cell includes a regulatory region of a RP 105 gene operably joined to a reporter gene coding region, and detecting a change in expression of the reporter gene.
  • the present invention enables culture systems in which cell lines which express the RP 105 gene, and thus RP 105 protein products, are incubated with candidate compounds to test their effects on RP 105 expression.
  • Such culture systems can be used to identify compounds that upregulate or downregulate
  • Such compounds can be selected from protein compounds, chemicals and various drugs that are added to the culture medium. After a period of incubation in the presence of a selected test compound(s), the expression of RP 105 can be examined by quantifying the levels of RP 105 mRNA using standard Northern blotting procedure to determine any changes in expression as a result of the test compound. Cell lines transfected with constructs expressing RP 105 can also be used to test the function of compounds developed to modify the protein expression. In addition, transformed cell lines expressing a normal RP 105 protein could be mutagenized by the use of mutagenizing agents to produce an altered phenotype in which the role of mutated RP105 can be studied in order to study structure/function relationships of the protein products and their physiological effects.
  • the present invention provides a method, for identifying a compound that affects the binding of an RP105 protein and an RP105 binding protein comprising: (a) incubating (i) a test compound; (ii) an RP 105 protein and (iii) an RP105 binding protein under conditions which permit the binding of RP105 protein to the RP 105 binding protein; and (b) assaying for complexes of RP 105 protein and the RP 105 binding protein and comparing to a control (i.e. in the absence of the test substance), wherein a reduction of complexes indicates that the compound has an
  • the invention also includes pharmaceutical compositions containing substances that activate RP 105 activators for use in immune suppression as well as
  • Substances that activate RP 105 include substances that activate RP105 gene expression as well as substances that activate RP105 activity. Such substances include antisense molecules to RP 105, antibodies to RP 105 as well as other substances or RP 105 antagonists identified using the screening assays described herein. [00130] Substances that enhance RP105 include substances that enhance RP105 expression and/or activity. Such substances include nucleic acid molecules encoding RP 105, RP 105 proteins and other substances or RP 105 agonists identified using the screening assays described herein.
  • compositions can be for intralesional, intravenous,
  • composition can be in
  • liquid, solid or semisolid form for example pills, tablets, creams, gelatin capsules,
  • capsules for example, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets, solutions or suspensions.
  • compositions of the invention can be intended for any topical application.
  • Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration.
  • compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically
  • compositions may be prepared from a suitable pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • the pharmaceutical compositions may be provided in a suitable pH and iso-osmotic with the physiological fluids.
  • the pharmaceutical composition for use in inducing is a pharmaceutical composition for use in inducing
  • immune tolerance comprises an effective amount of an activator of RP 105 in
  • oligonucleotide is preferably an antisense oligonucleotide to RP 105 or an antibody that binds to
  • compositions may also contain other active agents such as other immune modulators including, but not limited to RP 105, fgl2, B7, CD80 or
  • compositions further contain an RP 105 protein or a nucleic acid molecule encoding an RP 105 protein.
  • compositions comprising nucleic acid molecules may be directly introduced into cells or tissues in vivo using delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors. They may also be introduced into cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as co-precipitation and incorporation of
  • DNA into liposomes may also be delivered in the form of an aerosol or by lavage.
  • the nucleic acid molecules of the invention may also be applied
  • the pharmaceutical composition for use in preventing for use in preventing
  • immune suppression comprises an effective amount of an RP 105 protein or a nucleic acid encoding an RP 105 protein in admixture with a pharmaceutically acceptable diluent or carrier.
  • Such compositions may be administered either alone or in combination with other active agents such as RP 105 activators.
  • Such conditions include, but are not . limited to, habitual fetal loss,
  • autoimmune diseases erythematosus erythematosus .
  • allergies immune deficiency diseases
  • graft rejection inflammatory conditions
  • wound healing neurodegenerative diseases
  • stroke spinal injury and conditions that lead to septic shock and organ dysfunction in critically ill patients.
  • the present invention provides a method of detecting a condition
  • RP 105 expression or activity comprising assaying a sample for (a) a nucleic acid molecule encoding a RP 105 protein or a fragment thereof or (b) an RP 105 protein or a fragment thereof and comparing the amount of RP 105 nucleic acid or protein detected with a suitable
  • Nucleotide probes can be prepared based on the sequence of RP105 for use in the detection of nucleotide sequences encoding RP 105 or fragments thereof in samples, preferably biological samples such as cells, tissues and bodily fluids.
  • the probes can be useful in detecting the presence of a condition associated with RP 105 or monitoring the progress of such a condition.
  • the present invention provides a method for detecting a nucleic acid molecules encoding RP 105 comprising contacting the sample with a nucleotide probe capable of hybridizing with the nucleic acid molecule to form a hybridization product, under conditions which permit the formation of the hybridization product, and assaying for the hybridization product.
  • a nucleotide probe may be labeled with a detectable substance such as a radioactive label, which provides for an adequate signal and has sufficient half-life
  • An appropriate label maybe selected having regard to the rate of hybridization and binding of the probe to the nucleic acid to be detected and the
  • Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et ⁇ l, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleotide probes maybe used to detect genes, preferably in human cells, that hybridize to the nucleic acid molecule of the present invention preferably, nucleic acid molecules which hybridize to the nucleic acid molecule encoding RP 105 under stringent hybridization conditions as described herein.
  • Nucleic acid molecules encoding a RP 105 protein can be selectively amplified in a sample using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleotide sequence of RP 105 for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using oligonucleotide primers and standard PCR
  • cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al, Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase
  • the RP 105 protein may be detected in a sample using antibodies that bind to the protein as described in detail above. Accordingly, ' the present invention provides a
  • a method for detecting a RP 105 protein comprising contacting the sample with an antibody that binds to RP 105 which is capable of being detected after it becomes bound to the RP 105 in the sample.
  • Antibodies specifically reactive with RP 105, or derivatives thereof, such as enzyme conjugates or labeled derivatives, maybe used to detect RP 105 in various biological materials, for example they may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of RP105, and
  • the antibodies are used to detect and quantify RP 105 in a sample in order to determine its role in
  • the antibodies of the invention may be used in immuno- histochemical analyses, for example, at the cellular and sub-subcellular level, to detect RP 105, to localize it to particular cells and tissues and to specific subcellular locations, and to quantitate the level of expression.
  • an antibody of the invention maybe labeled with a detectable substance and RP 105 maybe localized in
  • detectable substances include various enzymes, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase,
  • biotin, alkaline phosphatase, b-galactosidase, or acetylcholinesterase examples include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include radioactive iodine 1-125, 1-131 or 3-H.
  • Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualized by electron microscopy.
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against RP 105.
  • a second antibody having specificity for the antibody reactive against RP 105.
  • the antibody having specificity against RP 105 is a rabbit IgG antibody
  • the second antibody maybe goat
  • RP 105 may be localized by autoradiography. The results of autoradiography may be quantitated by
  • TLR Toll-like receptor
  • RP 105 is a TLR homolog, thought to be largely B cell-specific, which
  • RP 105 expression is wide, directly mirroring that of TLR4 on antigen presenting cells.
  • RP 105 is a specific inhibitor of TLR4 signaling in HEK293 cells, a function conferred by its extracellular domain.
  • RP 105 and its helper molecule, MD-I interact directly with the TLR4 signaling complex, inhibiting the ability of this complex to bind microbial ligand.
  • MD-I helper molecule
  • TLR signaling by conserved microbial molecular signatures promotes the induction of both innate and adaptive immune responses. It has long been clear that such immune responses need to be kept under tight control. Responses that are delayed or of insufficient vigor can lead to a failure to control infection. On the other hand, excessive or inappropriate inflammation can be harmful or even fatal.
  • the hyper-inflammatory responses that characterize sepsis provide a paradigmatic example, as do the more localized inappropriate inflammatory processes leading to inflammatory bowel disease and arthritis.
  • Mammalian TLRs are characterized structurally by an extracellular leucine- rich repeat (LRR) domain, a conserved pattern of juxtamembrane cysteine residues,
  • TIR intracytoplasmic signaling domain
  • RP 105 has a conserved extracellular LRR domain and a
  • RP 105 is a specific homolog of TLR4.
  • RP 105 is not B cell-specific as originally proposed: RP 105 protein expression directly mirrors that of TLR4 on antigen ⁇ presenting cells, hi Toll and TLR4, mutation of the conserved juxtamembrane cysteine residues, or significant deletion of the extracellular portion, has been shown to result in a constitutively active molecule. This suggests that Toll/TLR activation is normally restrained through extracellular
  • RP 105 has the apparent structure of an inhibitory TLR4 and
  • RP 105 is a physiological regulator of TLR4 signaling.
  • RP 105 is a specific inhibitor of TLR4 signaling, a function conferred by its extracellular domain. Without wishing to be bound by theory in any way, it is believed that the RP105/MD-1 complex interacts directly with TLR4/MD-2, inhibiting the ability of this LPS signaling complex to bind LPS.
  • RP105 is a physiological regulator of TLR4 signaling in primary dendritic cells,
  • RP 105 a TLR4 homolog whose expression mirrors that of TLR4 on antigen presenting cells, is a negative regulator of TLR4 signaling. RP 105 specifically inhibits TLR4 signaling when co-expressed in HEK293 cells. Further, RP105 is a physiological regulator of endotoxin-driven TLR4 signaling, as well as of endotoxicity
  • RP105-mediated counter-regulation is relevant in the context of infection with a well-described model (and human) pathogen: RP105 modulates the course of L. major infection, restraining both innate and adaptive immune responses. Although the activation of proinflammatory responses through TLRs is critical for
  • RP 105 joins a growing list of molecules and processes that have been shown to inhibit
  • RP105 stands out for its apparent specificity for inhibition of TLR4 signaling. This specificity, together with the structural homologies between RP105
  • TLR4 provides a mechanism of negative regulation of TLR4 signaling by RP105: interference with TLR4 signaling through direct interactions of RP105/MD-1 with the
  • TLR4/MD-2 cell surface signaling complex.
  • Co-immunoprecipitation experiments revealed direct physical association between RP105/MD-1 and TRL4/MD-2, an association that inhibits LPS binding to this signaling complex. The exact
  • RP 105 regulates LPS responses differently in myeloid cells and B cells. As noted, treatment with antibodies to RP 105 does not activate human
  • TLR4 cell surface molecular partners in these different cell types. Widely differing levels of expression of TLR4 (significant on myeloid cells; hardly detectable on B cells) may provide the key. TLR4 multimerization appears to be necessary for signaling. While TLR4/MD-2 would be expected to have a higher affinity for homodimerization than for heterodimerization with RP105/MD-1, data presented here suggest the likelihood that both homo- and heterodimers can multimerize with further TLR4/MD-2 complexes.
  • RPl 05 irnmunoregulation by RPl 05 leads to augmentation of humoral immunity (through effects on B cells) along with concomitant inhibition of cellular immune responses (through effects on DCs and macrophages).
  • TLR4 and other TLRs are able to effect reversal RP105-mediated inhibition of TLR4 signaling.
  • TLR4 is notable for leading to a more robust and complex response, both in
  • TLR4 may also stand out among the TLRs for its ability to signal in response to a
  • RP105-mediated modulation of the in vivo response to L. major infection may not be a function of the expression of yet-to-be discovered leishmanial ligands for TLR4, but of the inflammation-induced expression of host- derived TLR ligands. More generally, such considerations raise the possibility that RP 105 may well be of special importance in down-modulation of the injurious inflammatory responses observed in the systemic inflammatory response syndrome
  • PCR primers were designed to amplify the entire coding
  • HSV TK-Renilla luciferase reporter plasmid (pRL-TK) was from
  • CD14/TLR2 were cultured in RPMI 1640 (Gibco), supplemented with 10% FCS
  • HEK293 cells stably expressing TLR4/MD-2 were cultured
  • gentamicin 10 ⁇ g/ml ciprofloxacin (Bayer), and 0.5 mg/ml geneticin (Gibco).
  • HEK293 cells stably expressing RP105/CD14/TLR4 were generated by transfecting linearized RPl 05 cDNA into CD14/TLR4 HEK293 cells.
  • pRL-TK 0.1 ⁇ g plasmids for 24 h. After change of the culture media, cells were stimulated (or mock stimulated) for 5 h. Cells were subsequently lysed in passive lysis
  • luciferase activity was quantified on a Monolight 3010 luminometer (Pharmingen) using the dual luciferase reporter assay system (Promega).
  • Firefly luciferase activity was normalized to Renilla luciferase activity.
  • mice [00162] Mice. RP105 "A mice, on a C57BL/6 background (> 10 generations) have been described previously (27). Mice were genotyped using standard PCR techniques. Mice were also phenotyped for RP 105 surface expression on peripheral blood B cells by flow cytometry, as previously described (27) using B220-FITC mAb (Becton Dickinson) and RPl 05-PE mAb (e-Biosciences), along with isorype control antibodies from the respective companies (Fig. 10).
  • B220-FITC mAb Becton Dickinson
  • RPl 05-PE mAb e-Biosciences
  • mice were housed in a specific pathogen free facility in high-efficiency
  • Bone marrow-derived DC generated using standard protocols (28), were cultured in 48-well plates at 2 x 10 6 cells/ml (500
  • DC were stimulated with TLR ligands (or mock stimulated) for 24
  • phenotype was analyzed by flow cytometry before and after stimulation (see below).
  • mice were challenged i.p. with 25 ⁇ g of re-purified E. coli K235 LPS. One h later,
  • PBMC Human leukocytes.
  • PBMC peripheral blood mononuclear cells
  • derived DC were generated by standard protocols through culturing in media containing GM-CSF (500 UM) and IL-4 (1000 UM) [both from Peprotech] for 7-8
  • Flow Cytometry Surface and intracellular protein expression by HEK293 cells was quantified by FACS techniques described previously (29). For surface protein expression, cells were harvested, washed in FACS staining buffer (PBS with 1% FCS and 0.05% sodium azide [Mallinckrodt]) and blocked in PBS containing 10% human AB serum for 30 min at 4 0 C. After a further wash, cells were incubated with directly labeled mAb for 30 min, washed, and fixed in 4% paraformaldehyde in PBS for 10-15 min. After further washing, cells were resuspended in FACS staining buffer. For intracellular protein expression, harvested cells were washed in FACS staining
  • RP 105 expression (RP 105-PE [e-Bioscience]) was analyzed on mouse splenic
  • B-cells B220-FITC [Becton-Dickinsonj), BMDDC, and resident peritoneal macrophages (F4/80-allophycocyanin (APC) [e-Bioscience]).
  • RP 105 expression was
  • CD4 CD8 ⁇ , B220, CD19 and GR-I (all from Becton-Dickinson).
  • I-A b CD80, CD86
  • RP105 expression by human leukocytes was analyzed using RP105-PE (e-
  • RP105 expression was examined on peripheral blood B cells (CD19- APC), myeloid DC (lineage mAb-FITC cocktail [CD3, CD14, CD19, CD20, CD56] negative; HLA-DR-PerCP and CDl Ic-APC positive), and plasmacytoid DC (lineage negative; HLA-DR-PerCP and BDCA4-APC positive) in PBMC. All mAb for these studies were from Becton-Dickinson, with the exception of that for BDCA4-APC
  • TLR4 (2) TLR3; (3) TLR5; (4) TLR7, TLR8, TLR9; and (5) TLR 1 (2).
  • Similar analytic techniques unequivocally place RP 105 in the TLR4 subfamily (Fig. 1 ; Fig. 11).
  • the domain structure of the 641 amino acid mature RP105 protein has previously been commented upon (10, 12, 13, 16).
  • the extracellular portion of this type I transmembrane protein contains 22 leucine-rich repeats (repeats 7-10 being non ⁇
  • domain of RP 105 contains 1-2 tyrosine residues, it is devoid of recognized motifs that would suggest phosphorylation activity.
  • RP 105 expression mirrors TLR4 expression on APC.
  • RP 105 has been thought to be a B cell-specific molecule in mice (10). Further, despite the fact that RP 105 has been noted to be expressed at the rnRNA level in primary human monocytic cells (11- 13), the only published examination of RP 105 protein expression in monocytic cells (by western blot analysis) was reported to be negative (13).
  • mice express RP 105 on resident peritoneal macrophages, splenic
  • RPl 05 suppresses TLR4 mediated signaling in HEK293 cells.
  • HEK293 cells lack expression of endogenous TLR2, TLR4, TLR9, MD-2 and CD14 (26), as
  • RP105-mediated suppression of proinflammatory signaling was subsequently examined. As shown in Figure 5, RP105 does not inhibit BL-I- or , TLR2-driven HL-8 production. Indeed, RP 105 expression led to variable augmentation of TLR2-mediated IL-8 production in some experiments (shown). Thus, RPl 05- mediated suppression is TLR4-specific.
  • RPl 05 regulates cytokine production in DC.
  • Expression studies in cell lines have considerable utility. They also have obvious drawbacks; principally, that overexpression may drive non-physiological interactions and processes.
  • RP105 '7 mice generated by targeted disruption of exon 3 of RP105 (27), backcrossed for more than 10 generations onto the C57BL/6 background.
  • bone marrow-derived DC were generated by standard techniques (47) from age-matched RPIOS " '' " mice and
  • mice produced significantly more TNF- ⁇ , IL-12p70 and DIr 6 than did
  • LPS is induced in HEK293 cells transfected with TLR2 [data not shown]). Notably, stimulation of DC with these higher doses of "dirty" LPS led to similar amounts of
  • TLR2 agonists are able to effect functional reversal of RP105-mediated inhibition of TLR4 signaling (Fig. 8b).
  • RP105-mediated suppression of TLR4 signaling is not merely an overexpression artifact in cell lines
  • LPS-driven cytokine production was characterized in vivo in order to examine whether RP 105 plays a role in regulating systemic cytokine productioa RP 105 mice and wild type littermate controls were challenged intraperitoneally with re-purified E.
  • mice produce significantly more TNF- ⁇ in response to LPS
  • RP 105 is a negative regulator of TLR4 signaling in vivo.
  • RP 105 is also of special importance in down-modulation of the injurious inflammatory responses observed in autoimmune diseases and the systemic inflammatory response syndrome (66).
  • RP105 a novel B cell surface molecule implicated in B cell activation
  • RP 105 is associated with MD- 1 and transmits an activation signal in human B cells. Blood 92:2815-2822.
  • MD-2-CD14 complex in a process that is distinct from the initiation of signal
  • the toll-like receptor protein RP 105 regulates lipopolysaccharide signaling in B cells. J Exp Med 192:23-29.
  • LPS- TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM
  • TLR Toll-like receptor
  • the LPS receptor generates inflammatory signals from
  • TLR3 toll-like receptor 3
  • TLR4 intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun 68:7010-7017.
  • IRAK-M is a negative regulator of Toll-like receptor signaling. Cell 110:191-202.
  • IgM-induced apoptosis a role for Fc receptor coligation. J Exp Med 184:113- 120.
  • TLRs Differential adapter utilization by Toll-like receptors mediates TLR-specific
  • heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol 164:558-561.

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Abstract

The present invention relates to a regulation of inflammation and immune responses. The present invention relates to a method of treating a condition comprising administering a pharmaceutically effective amount of an activator of RP 105. The condition is typically associated with TLR-4 activation and cytokine production. Conditions addressed by the invention include sepsis, septic shock, inflammation, rheumatoid arthritis and Crohn's disease. The invention also provides the use of an activator of RP 105 in the manufacture of a medicament for use in the treatment of a condition associated with cytokine production and methods for identifying an activator of RP 105, which is also suitable for use in the treatment of a condition associated with stimulus-induced cytokine production. More specifically the patent relates to the use of RP105 as a specific inhibitor of TLR4 signaling and as a physiological regulator of TLR4 signaling for the treatment of TLR4-mediated inflammation and immune-related diseases. This invention also relates to treating an animal having a disease or condition associated with Toll-like receptor (4).

Description

METHODS AND COMPOSITIONS FOR THE MODULATION OF IMMUNE RESPONSES AND AUTOIMMUNE DISEASES
FIELD OF THE INVENTION
[0001] The present invention relates to regulation of inflammation and immune responses. The present invention relates to a method of treating a condition
comprising administering a pharmaceutically effective amount of a compound that upregulates RP105-mediated inhibition of TLR inhibition, through either upregulating RP 105 expression or activity. More specifically the patent relates to the use of RP 105 as a specific inhibitor of TLR4 signaling and as a physiological regulator of TLR4 signaling for the treatment of TLR4-mediated inflammation and TLR4-driven
immune-processes.
BACKGROUND OF THE INVENTION
[0002] The field of innate immunity has undergone a recent renaissance, fueled largely by the molecular identification of critical receptors and signaling pathways involved in pathogen recognition. Study of the Toll-like receptor (TLR) family has led , the way. Activation of TLR signaling by conserved microbial molecular signatures promotes the induction of both innate and adaptive immune responses (1, 2). It has long been clear that such immune responses need to be kept under tight control. Responses that are delayed or of insufficient vigor can lead to a failure to control infection. On the other hand, excessive or inappropriate inflammation can be harmful
or even fatal. The hyper-inflammatory responses that characterize sepsis provide a paradigmatic example, as do the more localized inappropriate inflammatory processes
leading to inflammatory bowel disease and arthritis (3-7).
[0003] The eleven known members of the mammalian TLR family are characterized structurally by an extracellular leucine-rich repeat (LRR) domain, a conserved pattern of juxtamembrane cysteine residues, and an intracytoplasmic signaling domain
(Toll/IL-1 resistance [TIR]) that is highly conserved across the TLRs as well as the
receptors for IL-I and IL- 18 (2, 8). The TLR-like molecule RP 105 was originally cloned as a B cell-specific molecule able to drive B cell proliferation (9, 10). Like
TLRs, RP 105 has a conserved extracellular LRR domain and a TLR-like pattern of
V juxtamembrane cysteines (9-13). Unlike the TLRs, however, RP105 lacks a TIR
domain, containing a mere 6 to 11 intracytoplasmic amino acids. In parallel with TLR4, whose surface expression and signaling depends upon co-expression of the
molecule MD-2, surface expression of RP105 is dependent upon the co-expression of the MD-2 homologue, MD-I (14-17).
[0004] Phylogenetic analysis demonstrates that RP 105 is actually a specific homologue of TLR4. Further, RP 105 is not B cell-specific as originally proposed: we have found that its expression directly mirrors that of TLR4 on antigen-presenting cells. In Toll and TLR4, mutation of the conserved juxtamembrane cysteine residues, or deletion of the extracellular portion altogether, results in a constitutively active molecule (18-20). This suggests that the activation of Toll/TLRs is normally restrained through extracellular protein/protein interactions, likely through the LRR domain (21). On the other hand, deletions or mutations in the TIR domain of
Toll/TLRs can yield inactive or dominant negative molecules (18, 22, 23). Thus,
RP 105 has the structure of an inhibitory TLR4.
[0005] RP 105 specifically inhibits TLR4 signaling when co-expressed in cells and
RP 105 is a physiological regulator of TLR4 signaling and of TLR4-driven proinflammatory responses in vivo. Although the activation of proinflammatory responses through TLRs is critical for both innate and adaptive immunity, excessive or inappropriate inflammation can itself be maladaptive. RP 105 inhibits TLR signaling and this regulation of TLR expression provides a point of control (48-50) since RP 105 acts specifically to inhibit TLR4 signaling.
[0006] The present invention now provides methods of using RP105 as a specific inhibitor of TLR4 signaling to treat TLR4-mediated pathogenic processes and
diseases.
BRIEF SUMMARY OF THE INVENTION
[0Q07] The present invention relates to the modulation of inflammation and immune responses. Methods and compositions for suppressing a pathological inflammatory or immune response are disclosed. The methods involve administering an effective amount of an agent that upregulates RP105-mediated inhibition of TLR inhibition, through either upregulating RP 105 expression or activity. The methods are useful in the treatment of diseases marked by excessive or inappropriate inflammatory
processes, including autoimmune diseases such as rheumatoid arthritis, juvenile
rheumatoid arthritis, spondyloarthropathy, multiple sclerosis and inflammatory bowel
disease; pathological systemic responses to a variety of injuries, such as systemic
inflammatory response syndrome and adult respiratory distress syndrome; and
localized and systemic infectious processes such as sepsis and meningitis..
[0008] Specifically, the present invention provides for methods and compositions for upregulating the expression of RP 105, or for the engagement of or signaling through RP 105, in order to inhibit Toll-like receptor signaling. More specifically, the patent relates to the use of RP 105 as a specific inhibitor of TLR4 signaling and as a
physiological regulator of TLR4 signaling for the treatment of TLR4-driven inflammatory processes and TRLR4-driven immune-immune pathology. This invention also relates to treating an animal having a disease or condition associated with Toll-like receptor 4 by agonists of RP 105.
[0009] In another aspect, the invention involves a method for treating a subject having a disease or condition associated with Toll-like receptor 4 comprising administering to the subject a therapeutically or prophylactically effective amount of one or more compounds capable of activating RP 105 so that the action and/or expression of Toll-
like receptor 4 is inhibited.
[0010] Consequently, broadly stated, the present invention provides a method of modulating an immune response comprising modulating the expression or activity of RP 105. In one aspect, the present invention provides a method of suppressing an immune response comprising administering an effective amount of an activator of RP 105 to an animal in need of such treatment.
[0011] In one embodiment, excessive or inappropriate inflammatory or
proinflammatory responses through TLRs are treated by modulation of RP105.
[0012] In a further embodiment, the present invention provides a method of preventing or treating an autoimmune disease comprising administering an effective
amount of an activator of RP 105 to an animal having, suspected of having, or susceptible to having an autoimmune disease.
[0013] The invention also includes pharmaceutical compositions containing one or more activators of RP 105 for use in inducing tolerance in autoimmune, inflammatory
or infectious disease
[0014] In preferred embodiments of the above methods and compositions, the RP 105
activator is an antibody that binds RP 105, a small molecule that signals through
RP 105, or a compound that upregulates RP 105 expression. Also in a preferred embodiment of the invention, the activator of RP105 is administered in combination a
nucleic acid sequence encoding an RP 105 protein. [0015] The invention also includes pharmaceutical compositions containing an RP 105
protein or a nucleic acid sequence encoding an RP 105 protein for use in suppressing
an immune response. Such compositions can include other molecules that can suppress the immune response such as activators of RP 105.
[0016] The invention further includes screening assays for identifying substances that
modulate RP 105 expression or activity. Such substances may be useful in the therapeutic methods and compositions of the invention. The invention also includes diagnostic kits and methods for detecting conditions associated with increased, decreased or abnormal expression of RP105.
[0017] Further provided are methods for treating a condition involving Toll-like receptor-induced diseases or pathology comprising administering to a patient an v
effective amount of an agent that activates RP105. Preferably, the Toll-like receptor is Toll-like receptor 4. Ih another embodiment, the method further comprises
administering a therapeutic steroid. In another embodiment, the agent that down-
regulates the Toll-like receptor decreases the endogenous amount of intracellular or
extracellular cytokine.
[0018] The present invention relates to methods for inhibiting Toll-like receptor-4
("TLR-4") activity and treatment of Toll-like receptor 4 induced inflammatory bowel disease and related gastrointestinal pathologies. In a further aspect, the present invention concerns the treatment of Toll-like receptor 4 induced inflammatory bowel disease, including ulcerative colitis, Crohn's disease, indeterminate colitis, infectious colitis, drug or chemical-induced colitis, diverticulitis, and ischemic colitis. In another embodiment, the present methods provide for the treatment of Toll-like receptor 4 -dependent colitis.
[0019] It is one object of the present invention to provide methods for inhibiting the
biological activity of TLR-4, as, for example, by inhibiting its expression or signaling.
It is a further object of the invention to provide methods of treating those diseases in which inhibiting TLR-4 would have a beneficial effect.
[0020] In one embodiment, the Toll-like receptor 4 induced disease is one or more of systemic lupus erythematosis, sceleroderma, Sjogren's syndrome, multiple sclerosis
and other demyelinating diseases, rheumatoid arthritis, juvenile rheumatoid arthritis, myocarditis, uveitis, Reiter's syndrome, gout, osteoarthritis, polymyositis, primary biliary cirrhosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis,
aplastic anemia, Addison's disease, insulin-dependent diabetes mellitus, and other diseases.
[0021] In. an alternate embodiment, methods of the present invention are used to
inhibit the Toll-like receptor 4 induced disease including atherosclerosis, transplant atherosclerosis, vein-graft atherosclerosis, stent restenosis, and angioplasty restenosis, and to thereby treat the cardiovascular diseases that atherosclerosis causes (hereinafter "vascular diseases"). These methods may be used in any patient who could benefit from reducing atherosclerosis that is already present, from inhibiting atherosclerosis that has yet to form, or from both reducing existing atherosclerosis and inhibiting new atherosclerosis. Such patients include those suffering from, for example, angina pectoris and its subtypes (e.g., unstable angina and variant angina); ischemias affecting organs such as the brain, heart, bone, and intestines, and conditions associated with the ischemias, such as stroke, transient ischemic attacks, heart attack,
osteonecrosis, colitis, poor kidney function, and congestive heart failure; poor blood
circulation to the extremities and the complications of poor blood circulation, such as slow wound healing, infections, and claudication; atherosclerosis itself, including
restenosis following angioplasty or stenting of atherosclerotic lesions; vein-graft atherosclerosis following bypass surgery; transplant atherosclerosis; and other diseases caused by or associated with atherosclerosis.
[0022] hi another embodiment, such diseases include, for example, vascular disease such as atherosclerosis and thrombosis, restenosis after angioplasty and/or stenting, and vein-graft disease after bypass surgery.
[0023] hi another aspect, the invention involves a method for treating a condition involving a cytokine-induced diseases or pathology by administering to a patient an
effective amount of an agent that down-regulates a Toll-like receptor (TLR). In one embodiment, the agent that down-regulates the Toll-like receptor does so by
decreasing the endogenous amount of intracellular or extracellular cytokines.
[0024] In one embodiment, the cytokine-mediated disease is selected from acquired
immune deficiency syndrome, acute and chronic pain, acute purulent meningitis, adult respiratory distress syndrome (ARDS), Alzheimer's disease, aphthous ulcers, arthritis, asthma, atherosclerosis, atherosclerosisatopic dermatitis, bone resorption diseases, cachexia, chronic obstructive pulmonary disease, congestive heart failure, contact dermatitis, Crohn's disease, dermatoses with acute inflammatory components, diabetes, endotoxemia, glomerulonephritis, graft versus host disease, granulocyte transfusion, Guillain-Barre syndrome, inflammatory bowel disease, leprosy, leukopherisis, malaria, multiple organ injury secondary to trauma, multiple sclerosis,
myocardial infarction, necrotizing enterocolitis and syndromes associated with
hemodialysis, osteoarthritis, osteoporosis, psoriasis, reperfusion injury, restenosis
following percutaneous transluminal coronary angioplasty, rheumatoid arthritis, sarcoidosis, scleroderma, sepsis, septic shock, stroke, systemic lupus erythrematosis, thermal injury, toxic shock syndrome, traumatic arthritis, and ulcerative colitis.
[0025] Li another embodiment, the present invention provides for methods of treating a disease mediated by cytokines which comprises administering to a patient in need of such treatment a therapeutically effective amount of one or more activators of RP 105. Ih another embodiment, the present invention provides for methods of treating a gastrointestinal disorder in a patient in need thereof comprising administering to the
patient a therapeutically effective amount of one or more activators of RP 105. In an additional embodiment, the gastrointestinal disorder is an inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, ulcerative colitis, a peptic ulcer, a stress ulcer, a bleeding ulcer, gastric hyperacidity, dyspepsia, gastroparesis, Zollinger-Ellison syndrome, gastroesophageal reflux disease, abacterial infection, short-bowel (anastomosis) syndrome, a hypersecretory state associated with systemic mastocytosis or basophilic leukemia or hyperhistaminemia.
[0026] In another aspect, the cytokine involved in the TLR-4-mediated disease or pathology is a proinflammatory cytokine. In still another aspect, the proinflammatory
cytokine is selected from the group consisting of TNF-α, EL-I, EL-lβ, BL-6, and EL-8. [0027] Consequently, broadly stated, the present invention provides a method of
modulating an immune response comprising modulating the expression or activity of RP105.
[0028] In one aspect, the present invention provides a method of suppressing an
immune response comprising administering an effective amount of a modulator of
RP 105 activation or expression to an animal in need of such treatment.
[0029] In one embodiment, the present invention provides a method of inducing immune suppression or tolerance to a transplanted organ or tissue in a recipient animal comprising administering an effective amount of an activator of RP 105 activation or expression to the recipient animal prior to the transplantation of the organ or tissue.
[0030] In another embodiment, the present invention provides a method of preventing or inhibiting graft versus host disease in a recipient animal receiving an organ or tissue transplant comprising administering an effective amount of an activator of
RP 105 activation or expression to the organ or tissue prior to the transplantation in the recipient animal.
[0031] Ih yet another embodiment, the present invention provides a method of
preventing or inhibiting fetal loss comprising administering an effective amount of an activator of RP 105 activation or expression to an animal in need thereof.
[0032] In a further embodiment, the present invention provides a method of preventing or treating an autoimmune disease comprising administering an effective amount of an activator of RP 105 activation or expression to an animal having,
suspected of having, or susceptible to having an autoimmune disease.
[0033] In yet a further embodiment, the present invention provides a method of
preventing or treating an allergy comprising administering an effective amount of an
activator of RP 105 activation or expression to an animal having or suspected of having an allergy.
[0034] The invention also includes pharmaceutical compositions containing one or more activators of RP 105 activation or expression for use in inducing tolerance in transplantation, allergy or autoimmune disease or for preventing or treating fetal loss.
[0035] In preferred embodiments of the above methods and compositions, the RP 105 activator is an antibody that binds RP 105 or an antisense oligonucleotide that activates
the expression of RP 105. Also in a preferred embodiment of the invention, the inhibitor of RP 105 is administered in combination with an MD-I protein or a nucleic
acid sequence encoding an MD-I protein.
[0036] As stated above, activating RP 105 can be used to induce immune suppression.
Consequently, inhibiting RP 105 can be used in preventing immune suppression or
inducing an immune response.
[0037] Therefore, in another aspect, the present invention provides a method of immune suppression comprising administering an effective amount of an RP 105 protein or a nucleic acid sequence encoding an RP 105 protein to an animal in need thereof. [0038] The invention also includes pharmaceutical compositions containing an RPl 05
protein or a nucleic acid sequence encoding an RP 105 protein for use in suppressing
an immune response. Such compositions can include other molecules that can activate
the immune response such as activators of MD-I activity or expression.
[0039] The invention further includes screening assays for identifying substances that modulate RP 105 expression or activity. Such substances maybe useful in the therapeutic methods and compositions of the invention. The invention also includes diagnostic kits and methods for detecting conditions associated with increased, decreased or abnormal expression of RP 105.
[0040] Ih one embodiment, the present invention provides for a method for screening a test compound for the potential to prevent, stabilize, or treat an autoimmune or inflammatory disease comprising the steps of: a) contacting a first cell sample from a first subject that has, or is at risk for developing, an autoimmune or inflammatory
disease with the test compound; b) contacting a second cell sample from a second
subject that does not have, or is not predisposed to developing, an autoimmune or
inflammatory disease with the test compound, wherein the first and second subjects
are of the same species and the first and second cell samples are contacted with the test compound in the same manner; and c) measuring RP 105 inhibition of TLR4 in the first and second samples, wherein the compound is determined to have the potential if TLR4 inhibition in the first sample is decreased relative to the second sample. In one embodiment, RP 105 inhibition of TLR4 is measured by the ability of the lipopolysaccharide (LPS) signaling complex to bind LPS. In another embodiment, RP 105 activation or expression is measured. [0041] In another embodiment, the present invention provides for a method for
screening a test compound for the potential to prevent, stabilize, or treat an
autoimmune or inflammatory disease comprising the steps of: a) contacting a population of cells from a subject that has, or is at risk for developing, an autoimmune
or inflammatory disease with the test compound; b) contacting a second cell element from the subject with the test compound, wherein the cells and the second cell element are contacted with the test compound in the same manner; and c) measuring RP 105 activation or expression of the cells, wherein the test compound is determined to have the potential if the RP 105 activation or expression of the cell increases relative to the second cell element. In another embodiment, the cells are autoimmune cells. Ih another embodiment, the cells are leukocytes.
[0042] In another embodiment, the present invention provides for a method for
diagnosing an autoimmune or inflammatory disease, or a predisposition to the disease,
in a subject comprising the steps of: a) obtaining a first cell sample from a first
subject; b) obtaining a second cell sample from a second subject of the same species
as the first subject, wherein the second subject does not have, or is not at risk for
developing, the autoimmune disease; c) contacting the first cell sample and the second cell sample with a compound that preferentially increases the activation or expression of RP 105, wherein both of the first and second samples are contacted with the compound in the same manner; and d) measuring the activation or expression of RP 105 in the first cell sample and in the second cell sample, wherein a decrease in activation or expression of RP 105 in the first sample relative to the activation or expression of RP 105 in the second sample indicates that the first subject has, or is
predisposed to developing, the autoimmune disease.
[0043] In another embodiment, the present invention provides for a method for diagnosing an autoimmune disease, or a predisposition to the disease, in a subject
comprising the steps of: a) contacting autoimmune cells from a subject that has an autoimmune disease, or is at risk for developing an autoimmune disease, with the test compound; b) contacting a second cell element from the subject with the test compound, wherein the autoimmune cells and the second cell element are contacted with the compound in the same manner; and c) measuring the activation of RP 105 of
the autoimmune cells, wherein the subject has, or is at risk for developing, the autoimmune disease if the activation or expression of RP 105 of the autoimmune cells
decreases relative to the second cell element.
[0044] In another embodiment, the present invention provides for a method for the stratification of a human patient into a therapeutic subgroup for an autoimmune disease comprising the steps of: a) contacting a cell sample from the patient with a compound that preferentially decreases the activation of RP 105 of leukocytes; b) measuring the activation of RP 105 of the leukocytes; and c) placing the patient into a therapeutic subgroup based on the amount of decrease of the activation or expression ofRP105.
[0045] In another embodiment, the present invention provides for a method for monitoring a therapy for a human that has an autoimmune disease, or is at risk for developing the disease, comprising the steps of: i) obtaining a first cell sample from the patient and contacting the first cell sample with a compound that preferentially decreases the activation of RP 105 of leukocytes; ii) measuring the activation of
RP 105 of leukocytes in the first cell sample; iii) obtaining a second cell sample from
the patient and contacting the second cell sample with the compound, wherein the second cell sample is obtained at least 12 hours after obtaining the first cell sample;
iv) measuring the activation of RP 105 of leukocytes in the second cell sample; and v)
determining the efficacy of the therapy based on leukocyte RP 105 activation or
expression, wherein an increase in the activation or expression indicates that the therapy is efficacious.
[0046] In another embodiment, the present invention provides for a pharmaceutical composition for use in suppressing an immune response comprising an activator of RP105/MD-1 complex formation in admixture with a suitable diluent or carrier. In
one embodiment, the activator is an antibody that binds MD-I, RPl 05, or both.
[0047] Ih another embodiment, the present invention provides for a screening method
for identifying an immunostimulatory compound, comprising: contacting a functional
RP 105 with a test compound under conditions which, in absence of the test
compound, permit a negative control response mediated by a RP 105 signal
transduction pathway; detecting a test response mediated by the RP 105 signal transduction pathway; and determining the test compound is an immunostimulatory
compound when the test response exceeds the negative control response.
[0048] Ih another embodiment, the present invention provides for a screening method for identifying an immunostimulatory compound, comprising: contacting a functional RP 105 with a test compound under conditions which, in presence of a reference
immunostimulatory compound, permit a reference response mediated by a RP105 signal transduction pathway; detecting a test response mediated by the RP 105 signal
transduction pathway; and determining the test compound is an immunostimulatory
compound when the test response equals or exceeds the reference response.
[0049] In another embodiment, the present invention provides for a screening method
for identifying a compound that modulates RP 105 signaling activity, comprising:
contacting a functional RP 105 with a test compound and a reference
immunostimulatory compound under conditions which, in presence of the reference immunostimulatory compound alone, permit a reference response mediated by a RP 105 signal transduction pathway; detecting a test-reference response mediated by the RP 105 signal transduction pathway; determining the test compound is an agonist of RP 105 signaling activity when the test-reference response exceeds the reference response; and determining the test compound is an antagonist of RP 105 signaling activity when the reference response exceeds the test-reference response.
[0050] In another embodiment, the present invention provides for a screening method for identifying species specificity of an immunostimulatory compound, comprising:
measuring a first species-specific response mediated by a RP 105 signal transduction pathway when a functional RP 105 of a first species is contacted with a test compound; measuring a second species-specific response mediated by the RP 105 signal transduction pathway when a functional RP 105 of a second species is contacted with the test compound; and comparing the first species-specific response with the second species-specific response.
[0051] hi one embodiment, the screening method is performed on a plurality of test compounds, hi another embodiment, the response mediated by the RP 105 signal transductkm pathway is measured quantitatively. In another embodiment, the
functional RP 105 is expressed in a cell. In another embodiment, the cell is an isolated
mammalian cell that naturally expresses the functional RP 105.
In another embodiment, the cell is an isolated mammalian cell that does not
naturally express the functional RP 105, and wherein the cell comprises an expression
vector for RP105. In another embodiment, the cell is a human fibroblast. In another embodiment, the cell comprises an expression vector comprising an isolated nucleic acid which encodes a reporter construct selected from the group of interleuldns, NF- kappaB, interferons, and TNFs. In another embodiment, the functional RP 105 is part of a cell-free system. In another embodiment, the reference immunostimulatory compound is a nucleic acid. In another embodiment, the nucleic acid is a CpG nucleic acid. In another embodiment, the reference immunostimulatory compound is a small molecule. In another embodiment, the test compound is a part of a combinatorial
library of compounds. In another embodiment, the test compound is a nucleic acid. In
another embodiment, the the nucleic acid is a CpG nucleic acid. In another
embodiment, the test compound is a small molecule. In another embodiment, the test
compound is a polypeptide. In another embodiment, the response mediated by a RP 105 signal transduction pathway is induction of a reporter gene under control of a
promoter response element selected from the group consisting of ISRE, JL-6, TL-S, IL- 12 ρ40, IFN-alpha, IFN-beta, IFN-omega, TNF, IP-10, and I-TAC. In another embodiment, the response mediated by a RP 105 signal transduction pathway is selected from the group consisting of (a) induction of a reporter gene under control of a minimal promoter responsive to a transcription factor selected from the group consisting of API, NF-kaρρaB, ATF2, IRF3, and IRF7; (b) secretion of a chemoMne;
and (c) secretion of a cytokine. In another embodiment, the response mediated by a
RP 105 signal transduction pathway is secretion of a type 1 BFN. In another embodiment, the response mediated by a RP 105 signal transduction pathway is
secretion of a chemokine. hi another embodiment, the contacting a functional RP 105
with a test compound further comprises, for each test compound, contacting with the test compound at each of a plurality of concentrations. In another embodiment, the e detecting is performed 6-12 hours following the contacting. In another embodiment, the detecting is performed 16-24 hours following the contacting.
[0053] The present compounds may also be used in co-therapies, partially or completely, in place of other conventional anti-inflammatories, such as together with steroids, cyclooxygenase-2 inhibitors, NSAIDs, DMARDS, antibiotics, immunosuppressive agents, 5-lipoxygenase inhibitors, LTB4 antagonists and LTA4 hydrolase inhibitors and anti-cell adhesion molecules such as anti E-selectin.
[0054] In one embodiment, this aspect additionally involves administering a therapeutic steroid to the patient. By way of non-limiting example, therapeutic
steroids may include, for example, corticoids, glucocorticoids, dexamethasone,
prednisone, prednisalone, and betamethasone.
[0055] These methods may employ the compounds of this invention in a monotherapy or in combination with an anti-inflammatory or immunosuppressive agent. Such combination therapies include administration of the agents in a single dosage form or in multiple dosage forms administered at the same time or at different times. [0056] The above summary of the present invention is not intended to describe each
embodiment or every implementation of the present invention. Advantages and
attainments, together with a more complete understanding of the invention, will
become apparent and appreciated by referring to the following detailed description
and claims taken in conjunction with the accompanying drawings.
[0057] Throughout this document, all temperatures are given in degrees Celsius, and all percentages are weight percentages unless otherwise stated. All publications
mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the compositions and methodologies, which are described in the publications which might be used in connection with the presently described invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such a disclosure by virtue
of prior invention.
BRBEF DESCRIPTION OF TBDE FIGURES
[0058] Figure 1. RP 105 phytogeny. Phylogenetic relationships among the human
TLRs as inferred by sequence alignment using ClustalW software. Similar results were obtained with PiIeUp software. Horizontal branch lengths are proportional to the degree of inferred evolutionary change.
[0059] Figure 2. RP105 expression by human peripheral blood leukocytes. Flow cytometric analysis of PBMC from healthy human donors, (a) B cells; (b) monocytes; (c) myeloid DC; (d) plasmacytoid DC. Myeloid DC were
identified as lineage negative (CD37CD147CD197CD207CD56"), HLA-DR+, CDl Ic+. Plasmacytoid DC were identified as lineage negative, HLA-DR+, CDl Ic", BDC A-4". Data are representative of an experimental n > 15 for
monocytes; n = 3 for the other cell types.
[0060] Figure 3. RP 105 expression by murine leukocytes. Flow cytometric analysis of cell populations, (a) splenic B cells (representative of an experimental n >
50). (b) resident peritoneal macrophages (n = 5). (c) splenic CDl Ic+CDl Ib+
CD4- and CDl Ic+CDl lb+CD4+DC in = 3). (d) splenic CDl Ic+CDl Ib"
CDSa+DC (n = 3). (e) splenic plasmacytoid DC (CD19+B22O+CD1 Ic+GR-I+)
[n = 2]. (J) bone marrow-derived DC. (n = 6). Analysis of splenic DC subsets was performed after 1O d of in vivo treatment with flt3L.
[0061] Figure 4. Suppression of TLR4 signaling in HEK293 cells by RP 105 expression, (a) Lack of activity of RP 105 as a signaling receptor for LPS in HEK293 cells. HEK293 cells stably expressing CD14 were transiently transfected with cDNA encoding MD-I, MD-2, TLR4, empty vector control
cDNA (EV) and/or RP 105. Cells were subsequently stimulated with re-
purified E. coli K235 LPS (10 ng/ml). (b) Dose-dependent suppression of
LPS-driven TL-S production by RP105. HEK293 cells stably expressing CD14
and TLR4 were transiently transfected with MD-I and MD-2 along with the
indicated concentrations of RP 105 and/or EV cDNA, and secondarily
stimulated with LPS (10 ng/ml). (c) RP105-mediated suppression of LPS-
induced NF-κB activation. HEK293 cells stably expressing MD-2 and TLR4
were transiently co-transfected with an NF-κB-firefiy luciferase reporter
plasmid, a TK-renilla luciferase reporter plasmid and MD-I, along with EV (open bars) or RP 105 (filled bars). Cells were stimulated with the indicated concentrations of LPS. * p < 0.03, % p < 0.004, compared with RP105- deficient cells. Means +/- SE of triplicate cultures in a single experiment are depicted, representative of an experimental n = 4 (α and b); n = 2 (c). NS: no stimulation.
Figure 5. Specificity of RP 105-mediated suppression, (a) Lack of suppression of IL-IR signaling. HEK293 cells stably expressing CD14 and TLR4 (open bars) or CD14, TLR4 and RP105 (filled bars) were transiently transfected with MD-I and MD-2, and subsequently stimulated with re-purified E. coli K235
LPS (10 ng/ml) or IL-lβ (100 ng/ml). (b) Lack of suppression of TLR2
signaling. HEK293 cells stably expressing CD14 and TLR2 were transiently transfected with MD-I and EV (open bars) or MD-I and RP105 (filled bars)
and subsequently stimulated with Zymosan A (10 μg/ml) or IL-lβ (100 ng/ml). ). *p < 0.0001, %p = 0.05, compared with RP105-deficie.it cells.
Means +/- SE of triplicate cultures in a single experiment are depicted,
representative of an experimental n — 2-4.
[0063] Figure 6. Necessity of MD-I for RP105-mediated suppression of TLR4
signaling, (a) MD-I dependence of RP105 mediated suppression. HEK293
cells stably expressing CD 14 and TLR4 were transiently transfected with MD-
2; along with EV, MD-I and/or RP105 as indicated. Cells were subsequently stimulated with re-purified E. coli K235 LPS (10 ng/ml). (b) Association of
MD-I expression with surface expression of RP105. HEK293 cells were further analyzed for surface and intracellular RP105 expression by FACS. *p < 0.008, compared with RP105-deficient cells. Means +/- SE of triplicate cultures in a single experiment are depicted, representative of an experimental n = 4 (a); n = 2 (έ).
[0064] Figure 7. Altered cytokine production by dendritic cells from RP105"A mice following in vitro stimulation with TLR4-specific LPS. Bone marrow-derived
DC from wild type [if] or RP 105^ [•] mice were stimulated with repurified E.
coli K235 LPS. (a) TNF-α. (b) EL-12p70. (c) IL-6. *p < 0.05, %p < 0.001, §
p < 0.01. Means + SE of triplicate cultures in a single experiment, representative of an n = 8 (a); n = 4 (b and c).
[0065] Figure 8. Ability of heterologous TLR4 signaling to overcome RP105- mediated inhibition of TLR4 signaling. Bone marrow-derived DC from wild type U] or RP 1O5'A [•] mice were stimulated with: (a) non-repurified E. coli K235 LPS; or (b) repurified E, coli K235 LPS plus varying concentrations of Pam3Cys. *p < 0.05, $p < 0.001. Means + SE of triplicate cultures in a
single experiment; representative of an experimental n - A.
[0066] Figure 9. Elevated production of TNF-α by RP 105";" mice following LPS
challenge in vivo. Wild type mice (n - 17) (open bars) or RP105'A mice (n -
18) (tilled bars) were challenged intraperitoneally with 25 μg of re-purified E,
coli K235 LPS. Serum was harvested 60 min later. *p < 0.01.
[0067] Figure 10. Genotyping and phenotyping of RP105 ~'~ mice. FACS analysis of peripheral blood cells.
[0068] Figure 11. RP 105 phylogeny. Phylogenetic relationships among the mouse
TLRs as inferred by sequence alignment using ClustalW software.
DETAILED DESCRIPTION OF THE INVENTION
[0069] Before the present device and methods for tissue augmentation is described, it
is to be understood that this invention is not limited to the specific methodology,
devices, formulations, and compositions described as such may, of course, vary. It is
also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.
[0070] It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwisβi Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now described.
[0071] As used herein, the following terms shall have the definitions given below.
[0072] The term "activation" as used herein to describe the mechanism of action of an activator of RP105, means a mechanism wherein a compound upregulates RP105-
mediated inhibition of TLR activity, through upregulating RP 105 expression, increasing engagement of or signaling activity with TLR and/or other means that provide for a decrease in the activity of TLR. [0073] The term "administration" of the pharmaceutically active compounds and the pharmaceutical compositions defined herein includes systemic use, as by injection (especially parenterally), intravenous infusion, suppositories and oral administration
thereof, as well as topical application of the compounds and compositions. Oral administration is particularly preferred in the present invention.
[0074] "Ameliorate" or "amelioration" means a lessening of the detrimental effect or severity of the cell adhesion disorder in the subject receiving therapy, the severity of ■ the response being determined by means that are well known in the art.
[0075] "Chemokines" are chemotactic cytokines that are released by a wide variety of cells to attract macrophages, T-cells, eosinophils, basophils, neutrophils and endothelial cells to sites of inflammation and tumor growth. There are two main classes of chemokines, the CXC-chemokines and the CC-chemokines. The class depends on whether the first two cysteines are separated by a single amino acid (CXC-
chemokmes) or are adjacent (CC-chemokines). The CXC-chemokines include interleukin-8 (IL-8), neutrophil-activatmg protein- 1 (NAP-I), neutrophil-activating
protein-2 (NAP-2), GROα, GROβ, GROγ, ENA-78, GCP-2, IP-IO, MIG and PF4. CC
chemokines include RANTES, MIP- lα, MIP-2β, monocyte chemotactic protein- 1
(MCP-I), MCP-2, MCP-3 and eotaxin.
[0076] By "compatible" herein is meant that the components of the compositions which comprise the present invention are capable of being commingled without interacting in a manner which would substantially decrease the efficacy of the pharmaceutically active compound under ordinary use conditions. [0077] By "corticosteroid" is meant any naturally occurring or synthetic steroid
hormone that can be derived from cholesterol and is characterized by a hydrogenated
cyclopentanoperhydrophenanthrene ring system. Naturally occurring corticosteriods
are generally produced by the adrenal cortex. Synthetic corticosteriods may be
halogenated. Functional groups required for activity include a double bond at Δ4, a C3
ketone, and a C20 ketone. Corticosteroids may have glucocorticoid and/or mineralocorticoid activity.
[0078] Exemplary corticosteroids include, for example,, dexamethasone, betamethasone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide, beclomethasone, dipropionate, beclomethasone dipropionate monohydrate, flumethasone pivalate, diflorasone diacetate, fluocinolone acetonide, fluorometholone, fluorometholone acetate, clobetasol propionate, desoximethasone, fluoxymesterone, fluprednisolone, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone sodium phosphate, hydrocortisone
sodium succinate, hydrocortisone cypionate, hydrocortisone probutate, hydrocortisone valerate, cortisone acetate, paramethasone acetate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, prednisolone,
prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, clocortolone pivalate, flucinolone, dexamethasone 21 -acetate, betamethasone 17- valerate, isoflupredone, 9-fluorocortisone, 6-hydroxydexamethasone, dichlorisone, meclorisone, flupredidene, doxibetasol, halopredone, halometasone, clobetasone, diflucortolone, isoflupredone acetate, fluorohydroxyandrostenedione, beclomethasone, flumethasone, diflorasone, fluocinolone, clobetasol, cortisone, paramethasone, clocortolone, prednisolone 21-hemisuccinate free acid, prednisolone
metasulphobenzoate, prednisolone terbutate, and triamcinolone acetonide 21-
palmitate. By "low dose corticosteroid" is meant a dose that is less than a dose that would typically be given to a patient for treatment of inflammation. Exemplary low
doses of corticosteroids are as follows: Cortisol: 12 mg/day; cortisone: 15 mg/day;
prednisone: 3 mg/day; methylprednisolone: 2.5 mg/day; triamcinolone: 2.5 mg/day;
betamethasone: 250 μg/day; dexamethasone: 450 μg/day; hydrocortisone: 9 mg/day.
[0079] The term "patient" or "subject," as used herein, is intended to encompass any
mammal, animal or human subject, which may benefit from treatment with the compounds, compositions and methods of the present invention.
[0080] "Pharmaceutically-acceptable" shall mean that the pharmaceutically active compound and other ingredients used in the pharmaceutical compositions and methods defined herein are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like,
commensurate with a reasonable benefit/risk ratio.
[0081] The term "prodrug" indicates a therapeutic agent that is prepared in an inactive
form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
[0082] The phrase "safe and effective amount" means a sufficient amount of pharmaceutically active compound to effect the activation of RP 105 and inhibition of TLR-4. Within the scope of sound medical judgment, the required dosage of a pharmaceutically active agent or of the pharmaceutical composition containing that active agent will vary with the severity of the condition being treated, the duration of
the treatment, the nature of adjunct treatment, the age and physical condition of the patient, the specific active compound employed, and like considerations discussed more fully hereinafter. In arriving at the "safe and effective amount" for a particular
compound, these risks must be taken into consideration, as well as the fact that the
compounds described herein provide pharmaceutical activity at lower dosage levels than conventional compounds.
[0083] "Toll-like receptors" or "TLRs" are type I transmembrane proteins containing repeated leucine-rich motifs in their extracellular domains, At least eight mammalian TLR proteins have been identified, Toll-like receptors 1-8. Ligand engagement of the TLRs results in activation of NF-κB and induction of the cytokines and co-stimulatory molecules required for the activation of the adaptive immune response. Human Toll- like receptor 4 (also known as TLR4 and hToll), the human homolog to the Drosophilae protein known as Toll, was cloned from a human fetal liver/spleen library, characterized, and mapped to chromosome 9q32-33. Toll-like receptor 4 mRNA expression can be detected in the cells of the immune system: monocytes,
macrophages, dendritic cells, γΔ T-cells, ThI and Th2 αβ T-cells, and B-cells. Expression has also been detected in the cardiac myocytes and placenta.
[0084] "Treat," "treating," "treatment," and "therapy" as used herein refer to any curative therapy, prophylactic therapy, ameliorative therapy and preventative therapy
of a subject. "Ameliorate" or "amelioration" means a lessening of the detrimental
effect or severity of a disease or pathology in the subject receiving therapy, the severity of the response being determined by means that are well known in the art. As used herein, unless otherwise indicated, the terms "bacterial infection(s)"
include bacterial infections that occur in mammals, fish and birds as well as disorders related to bacterial infections that may be treated or prevented by administering
antibiotics such as the compounds of the present invention. Such bacterial infections,
and disorders related to such infections, include the following: pneumonia, otitis
media, sinusitus, bronchitis, tonsillitis, and mastoiditis related to infection by Streptococcus pneumonia, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, or Peptostreptococcus spp.; pharynigitis, rheumatic fever, and
glomerulonephritis related to infection by Streptococcus pyogenes, Groups C and G streptococci, Clostridium diptheriae, or Actinobacillus haemolyticum; respiratory tract
infections related to infection by Mycoplasma pneumoniae, Legionella pneumophila, Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydia pneumoniae; uncomplicated skin and soft tissue infections, abscesses and osteomyelitis, and puerperal fever related to infection by Staphylococcus aureus, coagulase-positive staphylococci (i.e., S. epidermidis, S. hemolyticus, etc.), Streptococcus pyogenes,
Streptococcus agalactiae, Streptococcal groups C-F (minute-colony streptococci), viridans streptococci, Corynebacterium minutissimum, Clostridium spp., or Bartonella
henselae; uncomplicated acute urinary tract infections related to infection by Staphylococcus saprophyticus or Enterococcus spp.; urethritis and cervicitis; and sexually transmitted diseases related to infection by Chlamydia trachomatis, Haemophilus ducreyi, Treponema pallidum, Ureaplasma urealyticum, or Neiserria gonorrheae; toxin diseases related to infection by S. aureus (food poisoning and Toxic shock syndrome), or Groups A, B, and C streptococci; ulcers related to infection by Helicobacter pylori; systemic febrile syndromes related to infection by Borrelia recurrentis; Lyme disease related to infection by Borrelia burgdorferi; conjunctivitis, keratitis, and dacrocystitis related to infection by Chlamydia trachomatis, Neisseria gonorrhoeae, S. aureus, S. pneumoniae, S. pyogenes, H. influenzae, or Listeria spp.;
disseminated Mycobacterium avium complex (MAC) disease related to infection by
Mycobacterium avium, or Mycobacterium intracellulare; gastroenteritis related to
infection by Campylobacter jejuni; odontogenic infection related to infection by
viridans streptococci; persistent cough related to infection by Bordetella pertussis; gas gangrene related to infection by Clostridium perfringensor Bacteroides spp.; and atherosclerosis related to infection by Helicobacter pylori or Chlamydia pneumoniae. Bacterial infections and protozoa infections and disorders related to such infections that may be treated or prevented in animals include the following: bovine respiratory disease related to infection by P. haem., P. multocida, Mycoplasma bovis, or Bordetella spp.; cow enteric disease related to infection by E. coli (i.e., coccidia, Cryptosporidia, etc.); dairy cow mastitis related to infection by Staph, aureus, Strep, uberis, Strep, agalactiae, Strep, dysgalactiae, Klebsiella spp., Corynebacterium, or
Enterococcus spp.; swine respiratory disease related to infection by A. pleuro., P. multocida, or Mycoplasma spp.; swine enteric disease related to infection by E coli,
Lawsonia intracellularis, Salmonella, or Serpulina hyodyisinteriae; cow footrot related
to infection by Fusobacterium spp.; cow metritis related to infection by E coli; cow
hairy warts related to infection by Fusobacterium necrophorum or Bacteroides
nodosus; cow pink-eye related to infection by Moraxella bovis; urinary tract infection in dogs and cats related to infection by E coli; skin and soft tissue infections in dogs and cats related to infection by Staph, epidermidis, Staph, intermedius, coagulase neg. Staph, or P. multocida; and dental or mouth infections in dogs and cats related to infection by Alcaligenes spp., Bacteroides spp., Clostridium spp., Enterobacter spp.,
Eubacterium, Peptostreptococcus, Porphyromonas, or Prevotella. Other bacterial infections and disorders related to such infections that may be treated or prevented in
accord with the method of the present invention.
[0086] Accordingly the invention provides a method of treating a condition associated
with cytokine production in a mammal comprising administering a pharmaceutically
effective amount of an activator of a member of RP105. The invention also provides the use of an activator of RP 105 in the manufacture of a medicament for use in the treatment of a condition associated with cytokine production
[0087] In one embodiment the cytokine is TNF, preferably TNFα. In another embodiment the cytokine is IL-I, preferably IL-I β. The cytokine-associated condition maybe any one of sepsis, septic shock, inflammation, rheumatoid arthritis or Crohn's disease. The cytokine-associated condition maybe irritable bowel disease (IBD). The cytokine-associated condition maybe ulcerative colitis.
[0088] In a preferred embodiment the cytokine-associated condition is induced by a
Toll-related receptor (TRR) ligand. m another preferred embodiment the cytokine-
associated condition is induced by lipopolysaccharide (LPS). Gram-negative bacteria
may induce the condition. Gram-positive bacteria may induce the condition. In another preferred embodiment the cytokine-associated condition is induced by zymosan. The condition may induced by yeast. The TRR ligand may be a microbial factor. [0089] The invention also provides a pharmaceutical formulation comprising a pharmaceutically acceptable carrier and an activator of RP 105 identifiable by a method of the invention.
[0090] The invention also provides the use of an activator of RP 105 to study the
inhibition of sepsis, septic shock and/or inflammation caused by a TRR-ligand, preferably LPS, in vitro.
[0091] The invention provides a method of treating sepsis or septic shock in a subject administering a pharmaceutically effective amount of an activator of RP 105.
[0092] The invention provides a method of treating inflammation in a subject comprising administering a pharmaceutically effective amount of an activator of RP105. The invention provides a RP105 activator for use as a medicament. The invention provides a RP 105 activator for use in the manufacture of a medicament to
treat sepsis, septic shock and/or inflammation. The invention provides a method or
activator of the invention wherein the activator is a vaccine, an antibody or a fragment thereof which is capable of binding RP 105 or a fragment thereof.
[0093] The invention provides a method of treating sepsis, septic shock and/or
inflammation in a subject comprising administering a pharmaceutically effective amount of an activator of a member of RP105. The invention provides amethod of treating rheumatoid arthritis in a subject comprising administering a pharmaceutically effective amount of an activator of a member of RP 105. The invention provides a method of treating Crohn's disease in a subject comprising administering a pharmaceutically effective amount of an activator of a member of RP 105. The invention provides a method of treating a condition induced by a TRR ligand in a subject comprising administering a pharmaceutically effective amount of an activator of a member of RP 105.
[0094] The invention provides a method of treating a condition induced by a TLR4 ligand in a subject comprising administering a pharmaceutically effective amount of
an activator of a member of RP105. The invention provides a method of treating a condition induced by a pathogen in a subject comprising administering a
pharmaceutically effective amount of an activator of a member of RP 105. The
invention provides a method of treating a condition induced by bacteria, or a factor derived there from in a subject comprising administering a pharmaceutically effective
amount of an activator of a member of RP 105.
[0095] All of the results of the inventors demonstrate that RP 105 is an immune modulating molecule that has utility in a wide range of applications. Accordingly, the present invention includes all uses that relate to the realization of the immune modulatory properties of RP 105 including, but not limited to, the development of therapeutic and diagnostic assays and compositions as well as the preparation and/or isolation of other molecules that modulate RP 105 that maybe useful in the therapeutic
and diagnostic assays and compositions of the invention.
Inducing Immune Suppression
[0096] In one aspect, the present invention provides a method of suppressing an immune response comprising administering an effective amount of an activator of RP 105 to an animal in need of such treatment. The invention includes a use of an effective amount of an activator of RP 105 to suppress an immune response or to
prepare a medicament to suppress an immune response.
[0097] The term "an activator of RP 105" means any molecule or compound that can activate the expression of the RP 105 gene or that can activate the activity of the
RP 105 protein.
[0098] In another embodiment, the activator of RP 105 is an RP 105 specific antibody.
Antibodies to RP 105 may be prepared using techniques known in the art such as those described by Kohler and Milstein, Nature 256, 495 (1975) and in U.S. Pat. Nos. RE
32,011; 4,902,614; 4,543,439; and 4,411,993, which are incorporated herein by reference. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kenneft, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference). Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab').sub.2) and recombinantly produced binding partners.
[0099] The therapeutic methods of the invention can be used to treat any condition wherein it is desirable to modulate RP 105 expression or activity. Such conditions
include, but are not limited to, , autoimmune disease, , inflammatory conditions, septic
shock, organ dysfunction, neurodegenerative diseases (e.g., Alzheimer's disease),
stroke and spinal injury. [00100] As stated previously, the method of the present invention may also be used to treat or prevent autoimmune disease. Ih an autoimmune disease, the immune system of the host fails to recognize a particular antigen as "self and an immune reaction is
mounted against the host's tissues expressing the antigen. Normally, the immune
system is tolerant to its own host's tissues and autoimmunity can be thought of as a breakdown in the immune tolerance system.
[00101] Accordingly, in a further embodiment, the present invention provides a method of preventing or treating an autoimmune disease comprising administering an effective amount of an activator of RP 105 to an animal having, suspected of having, or susceptible to having an autoimmune disease. The invention includes a use of an effective amount of an activator of RP 105 to prevent or activate an autoimmune disease or to prepare a medicament to prevent or activate an autoimmune disease.
[00102] Autoimmune diseases that may be treated or prevented according to the present invention include, but are not limited to, type 1 insulin-dependent diabetes mellitus, adult respiratory distress syndrome, inflammatory bowel disease, dermatitis, meningitis, thrombotic thrombocytopenic purpura, Sjogren's syndrome, encephalitis,
uveitic, leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever, Reiter's
syndrome, psoriatic arthritis, progressive systemic sclerosis, primary biniary cirrhosis,
pemphigus, pemphigoid, necrotizing vasculitis, myasthenia gravis, multiple sclerosis,
lupus erythematosus, polymyositis, sarcoidosis, granulomatosis, vasculitis, pernicious anemia, CNS inflammatory disorder, antigen-antibody complex mediated diseases, autoimmune haemolytic anemia, Hashimoto's thyroiditis, Graves disease, habitual spontaneous abortions, Reynard's syndrome, glomerulonephritis, dermatomyositis, chronic active hepatitis, celiac disease, autoimmune complications of AIDS, atrophic
gastritis, ankylosing spondylitis and Addison's disease.
[00103] The above described methods for suppressing an immune response using
RP 105 activators maybe further enhanced by co-administering other immune
modulators including but not limited to RP 105, anti-fgl2, anti-B7, anti-CD80 or anti-
CD86. Preferably, the RP 105 activators are co-administered with an RP 105 protein or a nucleic acid molecule encoding an RP 105 protein as described in the PCT
application no. WO00/12130 and U.S. Pat. No. 5,780,609, which are incorporated herein by reference in their entirety.
Preventing Immune Suppression
[00104] The term "RP 105 protein" includes the full-length RP 105 protein as well as fragments or portions of the protein. Preferred fragments or portions of the protein are those that are sufficient to induce an immune response or prevent immune suppression. The RP 105 protein or the nucleic acid encoding the RP 105 protein can
be readily obtained by one of skill in the art. The RP 105 protein or nucleic acid may be modified from the known sequences to make it more useful in the methods of the present invention.
[00105] Li one embodiment, the RP105 protein is prepared as a soluble fusion protein.
The fusion protein may contain the extracellular domain of RP 105 linked to an immunoglobulin (Ig) Fc Region. The RP 105 fusion maybe prepared using techniques known in the art. Generally, a DNA sequence encoding the extracellular domain of RP105 is linked to a DNA sequence encoding the Fc of the Ig and expressed in an appropriate expression system where the RP105-Fclg fusion protein is produced. The RP 105 protein may be obtained from known sources or prepared using recombinant DNA techniques. The protein may have any of the known published sequences for RP 105. For example, the sequences can be obtained from GenBank as described above. The protein may also be modified to contain amino acid substitutions,
insertions and/or deletions that do not alter the immunomodulatory properties of the protein. Conserved amino acid substitutions involve replacing one or more amino
acids of the RP 105 amino acid sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made
the resulting analog should be functionally equivalent to the RP 105 protein. Non- conserved substitutions involve replacing one or more amino acids of the RP 105 amino acid sequence with one or more amino acids that possess dissimilar charge, size, and/or hydrophobicity characteristics.
[00106] Administration of an "effective amount" of the RP105 protein and nucleic acid of the present invention is defined as an amount effective, at dosages and for periods
of time necessary to achieve the desired result. The effective amount of the RP105 protein or nucleic acid of the invention may vary according to factors such as the disease state, age, sex, and weight of the animal. Dosage regime maybe adjusted to provide the optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced as indicated by the
exigencies of the therapeutic situation.
[00107] Li one embodiment, the present invention provides a method of inducing fetal loss comprising administering an effective amount of an RP 105 protein or a nucleic acid sequence encoding an RP 105 protein to an animal in need thereof. RP 105 Modulators
[00108] The present invention also includes the isolation and/or identification of
substances to modulate RP105 expression or activity. Such substances or RP105
modulators may be useful in the above-described therapeutic methods. Two examples of RP 105 modulators include antibodies and antisense molecules that are described in detail above. Other RP 105 modulators may be identified, for example, using the
screening assays described below.
Substances that Bind RP 105
[00109] Substances that affect RP 105 activity can be identified based on their ability to bind to RP105.
[00110] Substances that can bind with the RP105 of the invention may be identified by
reacting the RP105 with a substance that potentially binds to RP105, and assaying for complexes, for free substance, or for non-complexed RP 105, or for activation of RP105.
[00111] Accordingly, the invention provides a method of identifying substances that
can bind with RP105, comprising the steps of: (a) reacting RP105 and a test substance, under conditions that allow for formation of a complex between the RP 105 and the test substance, and (b) assaying for complexes of RP 105 and the test substance, for free substance or for non complexed RP 105, wherein the presence of complexes indicates that the test substance is capable of binding RP 105. [00112] Conditions that permit the formation of substance and RP 105 complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.
[00113] The substance-protein complex, free substance or non-complexed proteins
may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption,
poiyacrylamide gel electrophoresis, agglutination, or combinations thereof. To
facilitate the assay of the components, antibody against RP105 or the substance, or
labeled RP 105, or a labeled substance maybe utilized. The antibodies, proteins, or substances may be labeled with a detectable substance as described above.
[00114] RP105, or the substance used in the method of the invention maybe insolubilized. For example, RP 105 or substance may be bound to a suitable carrier. Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier maybe in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insolubilized protein or substance maybe prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for.example, cyanogen bromide coupling. The proteins or substance may also be expressed on the surface of a cell in the above assay. The invention also contemplates assaying for an antagonist or agonist of the action of RP 105. [00115] It will be understood that the agonists and antagonists that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding
sites, non-competitive antagonist binding sites or allosteric sites.
[00116] The invention also makes it possible to screen for antagonists that activate the
effects of an agonist of RP 105. Thus, the invention may be used to assay for a
substance that competes for the same binding site of RP 105.
Peptide Mimetics
[00117] The present invention also includes peptide mimetics of the RP105 of the invention. For example, a peptide derived from a binding domain of RP 105 will interact directly or indirectly with an associated molecule in such a way as to mimic the native binding domain. Such peptides may include competitive activators, enhancers, peptide mimetics, and the like. All of these peptides as well as molecules substantially homologous, complementary or otherwise functionally or structurally equivalent to these peptides may be used for purposes of the present invention.
[00118] "Peptide mimetics" are structures that serve as substitutes for peptides in interactions between molecules (See Morgan et al. (1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimetics include synthetic structures that may or
may not contain amino acids and/or peptide bonds but retain the structural and
functional features of a peptide, or enhancer or activator of the invention. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide of the invention.
[00119] Peptide mimetics may be designed based on information obtained by
systematic replacement of L-amino acids by D-amino acids, replacement of side
chains with groups having different electronic properties, and by systematic
replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for
activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues maybe used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of activator peptide secondary structures. These structures can model the 3- dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N- substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.
[00120] Peptides of the invention may also be used to identify lead compounds for
drug development. The structure of the peptides described herein can be readily determined by a number of methods such as NMR and X-ray crystallography. A
comparison of the structures of peptides similar in sequence, but differing in the biological activities they elicit in target molecules can provide information about the structure-activity relationship of the target. Information obtained from the examination of structure-activity relationships can be used to design either modified peptides, or other small molecules or lead compounds that can be tested for predicted properties as related to the target molecule. The activity of the lead compounds can be evaluated using assays similar to those described herein.
[00121] Information about structure-activity relationships may also be obtained from
co-crystallization studies. In these studies, a peptide with a desired activity is crystallized in association with a target molecule, and the X-ray structure of the complex is determined. The structure can then be compared to the structure of the target molecule in its native state, and information from such a comparison maybe used to design compounds expected to possess.
Drug Screening Methods
[00122] In accordance with one embodiment, the invention enables a method for screening candidate compounds for their ability to increase or decrease the activity of a RP 105 protein. The method comprises providing an assay system for assaying RP 105 activity, assaying the activity in the presence or absence of the candidate or test
compound and deteπnining whether the compound has increased or decreased RP 105 activity.
[00123] Accordingly, the present invention provides a method for identifying a compound that affects RP 105 protein activity or expression comprising: (a) incubating a test compound with a RP 105 protein or a nucleic acid encoding a RP 105 protein; and (b) determining an amount of RP 105 protein activity or expression and comparing with a control (i.e. in the absence of the test substance), wherein a change in the RP 105 protein activity or expression as compared to the control indicates that the test
compound has an effect on RP 105 protein activity or expression.
[00124] In accordance with a further embodiment, the invention enables a method for
screening candidate compounds for their ability to increase or decrease expression of a RP 105 protein. The method comprises putting a cell with a candidate compound, wherein the cell includes a regulatory region of a RP 105 gene operably joined to a reporter gene coding region, and detecting a change in expression of the reporter gene.
[00125] hi one embodiment, the present invention enables culture systems in which cell lines which express the RP 105 gene, and thus RP 105 protein products, are incubated with candidate compounds to test their effects on RP 105 expression. Such culture systems can be used to identify compounds that upregulate or downregulate
RP 105 expression or its function, through the interaction with other proteins.
[00126] Such compounds can be selected from protein compounds, chemicals and various drugs that are added to the culture medium. After a period of incubation in the presence of a selected test compound(s), the expression of RP 105 can be examined by quantifying the levels of RP 105 mRNA using standard Northern blotting procedure to determine any changes in expression as a result of the test compound. Cell lines transfected with constructs expressing RP 105 can also be used to test the function of compounds developed to modify the protein expression. In addition, transformed cell lines expressing a normal RP 105 protein could be mutagenized by the use of mutagenizing agents to produce an altered phenotype in which the role of mutated RP105 can be studied in order to study structure/function relationships of the protein products and their physiological effects. [00127] Accordingly, the present invention provides a method, for identifying a compound that affects the binding of an RP105 protein and an RP105 binding protein comprising: (a) incubating (i) a test compound; (ii) an RP 105 protein and (iii) an RP105 binding protein under conditions which permit the binding of RP105 protein to the RP 105 binding protein; and (b) assaying for complexes of RP 105 protein and the RP 105 binding protein and comparing to a control (i.e. in the absence of the test substance), wherein a reduction of complexes indicates that the compound has an
effect on the binding of the RP 105 protein to an RP 105 binding protein.
[00128] All testing for novel drug development is well suited to defined cell culture
systems, which can be manipulated to express RP 105 and study the result of RP 105 protein modulation. Animal models are also important for testing novel drugs and thus may also be used to identify any potentially useful compound affecting RP 105
expression and activity and thus physiological function.
Compositions
[00129] The invention also includes pharmaceutical compositions containing substances that activate RP 105 activators for use in immune suppression as well as
pharmaceutical compositions containing substances that enhance RP 105 for use in
preventing immune suppression. Substances that activate RP 105 include substances that activate RP105 gene expression as well as substances that activate RP105 activity. Such substances include antisense molecules to RP 105, antibodies to RP 105 as well as other substances or RP 105 antagonists identified using the screening assays described herein. [00130] Substances that enhance RP105 include substances that enhance RP105 expression and/or activity. Such substances include nucleic acid molecules encoding RP 105, RP 105 proteins and other substances or RP 105 agonists identified using the screening assays described herein.
[00131] Such pharmaceutical compositions can be for intralesional, intravenous,
topical, rectal, parenteral, local, inhalant or subcutaneous, intradermal, intramuscular, intrathecal, transperiton al, oral, and intracerebral use. The composition can be in
liquid, solid or semisolid form, for example pills, tablets, creams, gelatin capsules,
capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets, solutions or suspensions.
[00132] The pharmaceutical compositions of the invention can be intended for
administration to humans or animals. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration.
[00133] The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
[00134] On this basis, the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically
acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The pharmaceutical compositions may
additionally contain other agents such as immunosuppressive drugs or antibodies to enhance immune tolerance or irnmunostimulatory agents to enhance the immune response.
[00135] In one embodiment, the pharmaceutical composition for use in inducing
immune tolerance comprises an effective amount of an activator of RP 105 in
admixture with a pharmaceutically acceptable diluent or carrier. The RP 105 activator
is preferably an antisense oligonucleotide to RP 105 or an antibody that binds to
RP 105. The pharmaceutical compositions may also contain other active agents such as other immune modulators including, but not limited to RP 105, fgl2, B7, CD80 or
CD86 including antagonists, agonists and modulators thereof. Preferably the compositions further contain an RP 105 protein or a nucleic acid molecule encoding an RP 105 protein.
[00136] Pharmaceutical compositions comprising nucleic acid molecules may be directly introduced into cells or tissues in vivo using delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors. They may also be introduced into cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as co-precipitation and incorporation of
DNA into liposomes. Recombinant molecules may also be delivered in the form of an aerosol or by lavage. The nucleic acid molecules of the invention may also be applied
extracellularly such as by direct injection into cells.
[00137] In another aspect, the pharmaceutical composition for use in preventing
immune suppression comprises an effective amount of an RP 105 protein or a nucleic acid encoding an RP 105 protein in admixture with a pharmaceutically acceptable diluent or carrier. Such compositions may be administered either alone or in combination with other active agents such as RP 105 activators.
Diagnostic Assays
[00138] The finding by the present inventors that RP 105 is involved in immune regulation allows the detection of conditions involving an increase or decrease in RP 105 activity or expression resulting in an aberrant or inappropriate immune
response. Such conditions include, but are not. limited to, habitual fetal loss,
autoimmune diseases, allergies, immune deficiency diseases, graft rejection, inflammatory conditions, wound healing, neurodegenerative diseases, stroke, spinal injury and conditions that lead to septic shock and organ dysfunction in critically ill patients.
[00139] Accordingly, the present invention provides a method of detecting a condition
associated with increased or decreased RP 105 expression or activity (including an absence) comprising assaying a sample for (a) a nucleic acid molecule encoding a RP 105 protein or a fragment thereof or (b) an RP 105 protein or a fragment thereof and comparing the amount of RP 105 nucleic acid or protein detected with a suitable
control.
Nucleic Acid Molecules
[00140] Nucleotide probes can be prepared based on the sequence of RP105 for use in the detection of nucleotide sequences encoding RP 105 or fragments thereof in samples, preferably biological samples such as cells, tissues and bodily fluids. The probes can be useful in detecting the presence of a condition associated with RP 105 or monitoring the progress of such a condition. Accordingly, the present invention provides a method for detecting a nucleic acid molecules encoding RP 105 comprising contacting the sample with a nucleotide probe capable of hybridizing with the nucleic acid molecule to form a hybridization product, under conditions which permit the formation of the hybridization product, and assaying for the hybridization product.
[00141] A nucleotide probe may be labeled with a detectable substance such as a radioactive label, which provides for an adequate signal and has sufficient half-life
such as 32P, 3H, 14C or the like. Other detectable substances that may be used
include antigens that are recognized by a specific labeled antibody, fluorescent
compounds, enzymes, antibodies specific for a labeled antigen, and
chemiluminescence. An appropriate label maybe selected having regard to the rate of hybridization and binding of the probe to the nucleic acid to be detected and the
amount of nucleic acid available for hybridization. Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et αl, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). The nucleotide probes maybe used to detect genes, preferably in human cells, that hybridize to the nucleic acid molecule of the present invention preferably, nucleic acid molecules which hybridize to the nucleic acid molecule encoding RP 105 under stringent hybridization conditions as described herein.
[00142] Nucleic acid molecules encoding a RP 105 protein can be selectively amplified in a sample using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleotide sequence of RP 105 for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using oligonucleotide primers and standard PCR
amplification techniques. The amplified nucleic acid can be cloned into an appropriate vector and characterized by DNA sequence analysis. cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al, Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase
available from Gfrbco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, FIa.).
Proteins
[00143] The RP 105 protein may be detected in a sample using antibodies that bind to the protein as described in detail above. Accordingly,'the present invention provides a
method for detecting a RP 105 protein comprising contacting the sample with an antibody that binds to RP 105 which is capable of being detected after it becomes bound to the RP 105 in the sample.
[00144] Antibodies specifically reactive with RP 105, or derivatives thereof, such as enzyme conjugates or labeled derivatives, maybe used to detect RP 105 in various biological materials, for example they may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of RP105, and
the antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassays {e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination and histochemical tests. Thus, the antibodies maybe used to detect and quantify RP 105 in a sample in order to determine its role in
particular cellular events or pathological states, and to diagnose and treat such pathological states,
[00145] In particular, the antibodies of the invention may be used in immuno- histochemical analyses, for example, at the cellular and sub-subcellular level, to detect RP 105, to localize it to particular cells and tissues and to specific subcellular locations, and to quantitate the level of expression.
[00146] Cytochemical techniques known in the art for localizing antigens using light
and electron microscopy may be used to detect RP105. Generally, an antibody of the invention maybe labeled with a detectable substance and RP 105 maybe localized in
tissue based upon the presence of the detectable substance. Examples of detectable substances include various enzymes, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase,
biotin, alkaline phosphatase, b-galactosidase, or acetylcholinesterase; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include radioactive iodine 1-125, 1-131 or 3-H. Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualized by electron microscopy.
[00147] Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against RP 105. By way of example, if the antibody having specificity against RP 105 is a rabbit IgG antibody, the second antibody maybe goat
anti-rabbit gamma-globulin labeled with a detectable substance as described herein.
[00148] Where a radioactive label is used as a detectable substance, RP 105 may be localized by autoradiography. The results of autoradiography may be quantitated by
determining the density of particles in the autoradiographs by various optical methods, or by counting the grains.
[00149] The preferred embodiments are exemplified by the following examples.
EXAMPLES
[00150] Activation of Toll-like receptor (TLR) signaling by microbial signatures is
critical to the induction of immune responses. Such responses demand tight regulation. RP 105 is a TLR homolog, thought to be largely B cell-specific, which
lacks a signaling domain. The present invention demonstrates that RP 105 expression is wide, directly mirroring that of TLR4 on antigen presenting cells. Furthermore, RP 105 is a specific inhibitor of TLR4 signaling in HEK293 cells, a function conferred by its extracellular domain. Notably, RP 105 and its helper molecule, MD-I, interact directly with the TLR4 signaling complex, inhibiting the ability of this complex to bind microbial ligand. Finally, we demonstrate that RP 105 is a physiological regulator of TLR4 signaling in dendritic cells, and of endotoxicity aadLeishmania major infection in vivo.
[00151] Activation of TLR signaling by conserved microbial molecular signatures promotes the induction of both innate and adaptive immune responses. It has long been clear that such immune responses need to be kept under tight control. Responses that are delayed or of insufficient vigor can lead to a failure to control infection. On the other hand, excessive or inappropriate inflammation can be harmful or even fatal. The hyper-inflammatory responses that characterize sepsis provide a paradigmatic example, as do the more localized inappropriate inflammatory processes leading to inflammatory bowel disease and arthritis.
[00152] Mammalian TLRs are characterized structurally by an extracellular leucine- rich repeat (LRR) domain, a conserved pattern of juxtamembrane cysteine residues,
and an intracytoplasmic signaling domain (ToML- 1 resistance [TIR]) that is highly conserved across the TLRs as well as the receptors for EL-I and BL-18. The TLR-like molecule RP 105 was originally cloned as a B cell-specific molecule able to drive B
cell proliferation. Like TLRs, RP 105 has a conserved extracellular LRR domain and a
TLR-like pattern of juxtamembrane cysteines. Unlike the TLRs, however, RP 105
lacks a TIR domain, containing a mere 6-11 intracytoplasmic amino acids (depending
upon the prediction algorithm). In parallel with TLR4, whose surface expression and signaling depends upon co-expression of the secreted extracellular protein MD-2, surface expression of RP 105 is dependent upon the co-expression of the MD-2
homolog, MD-I .
[00153] Here, we show that RP 105 is a specific homolog of TLR4. We further show that RP 105 is not B cell-specific as originally proposed: RP 105 protein expression directly mirrors that of TLR4 on antigen^presenting cells, hi Toll and TLR4, mutation of the conserved juxtamembrane cysteine residues, or significant deletion of the extracellular portion, has been shown to result in a constitutively active molecule. This suggests that Toll/TLR activation is normally restrained through extracellular
protein/protein interactions, likely through the LRR domain. On the other hand, deletions or mutations in the TIR domain of Toll/TLRs can yield inactive or dominant negative molecules. Thus, RP 105 has the apparent structure of an inhibitory TLR4 and
RP 105 is a physiological regulator of TLR4 signaling. RP 105 is a specific inhibitor of TLR4 signaling, a function conferred by its extracellular domain. Without wishing to be bound by theory in any way, it is believed that the RP105/MD-1 complex interacts directly with TLR4/MD-2, inhibiting the ability of this LPS signaling complex to bind LPS. RP105 is a physiological regulator of TLR4 signaling in primary dendritic cells,
and of responses to endotoxin as well as Leishmania infection in vivo.
] RP 105, a TLR4 homolog whose expression mirrors that of TLR4 on antigen presenting cells, is a negative regulator of TLR4 signaling. RP 105 specifically inhibits TLR4 signaling when co-expressed in HEK293 cells. Further, RP105 is a physiological regulator of endotoxin-driven TLR4 signaling, as well as of endotoxicity
in vivo. Finally, RP105-mediated counter-regulation is relevant in the context of infection with a well-described model (and human) pathogen: RP105 modulates the course of L. major infection, restraining both innate and adaptive immune responses. Although the activation of proinflammatory responses through TLRs is critical for
host defense, excessive or inappropriate inflammation can itself be maladaptive. RP 105 joins a growing list of molecules and processes that have been shown to inhibit
TLR signaling. RP105 stands out for its apparent specificity for inhibition of TLR4 signaling. This specificity, together with the structural homologies between RP105
and TLR4 provides a mechanism of negative regulation of TLR4 signaling by RP105: interference with TLR4 signaling through direct interactions of RP105/MD-1 with the
TLR4/MD-2 cell surface signaling complex. Co-immunoprecipitation experiments revealed direct physical association between RP105/MD-1 and TRL4/MD-2, an association that inhibits LPS binding to this signaling complex. The exact
stoichiometry of RP105/MD-1/MD-2/TLR4 interactions remains to be defined. We
show that RP 105 regulates LPS responses differently in myeloid cells and B cells. As noted, treatment with antibodies to RP 105 does not activate human
monocyte/macrophages, failing, for example, to drive proinflammatory cytokine production. As for LPS-driven B cell responses, while LPS-induced murine B cell proliferation is strictly dependent upon TLR4, B cells from RP 105 knockout mice have reduced LPS-driven proliferative responses as well as diminished humoral
immune responses when LPS is used as an adjuvant for vaccination with T cell- dependent antigens. It is reasonable to suspect that the dichotomous effects of RP105
on TLR4 signaling in B cells and myeloid cells are due to differential interactions with
cell surface molecular partners in these different cell types. Widely differing levels of expression of TLR4 (significant on myeloid cells; hardly detectable on B cells) may provide the key. TLR4 multimerization appears to be necessary for signaling. While TLR4/MD-2 would be expected to have a higher affinity for homodimerization than for heterodimerization with RP105/MD-1, data presented here suggest the likelihood that both homo- and heterodimers can multimerize with further TLR4/MD-2 complexes. This suggests a model whereby: (a) when TLR4/MD-2 is highly expressed (e.g., on myeloid cells), lower affinity heterodimeric interactions inhibit TLR4 multimerization and signaling; but (b) when TLR4 is limiting (e.g., on B cells), such
heterodimeric interactions serve to facilitate further TLR4 recruitment and signaling. The fact that RP 105 appears to promote B cell activation, while inhibiting DC
activation, suggests the possibility that, overall, irnmunoregulation by RPl 05 leads to augmentation of humoral immunity (through effects on B cells) along with concomitant inhibition of cellular immune responses (through effects on DCs and macrophages).
] The apparent TLR4-specificity of RP 105 raises another issue. Concurrent
stimulation of TLR4 and other TLRs is able to effect reversal RP105-mediated inhibition of TLR4 signaling. Clearly, most microbes that express ligands that
stimulate TLR4 signaling also express ligands that stimulate signaling through other
TLRs. TLR4 is notable for leading to a more robust and complex response, both in
terms of signaling and in terms of subsequent gene expression, than the other TLRs. TLR4 may also stand out among the TLRs for its ability to signal in response to a
variety of endogenous "danger signals". The ability of TLR4 to recognize endogenous heat shock proteins and extracellular matrix components unmasked by tissue injury
suggests the possibility RP105-mediated modulation of the in vivo response to L. major infection may not be a function of the expression of yet-to-be discovered leishmanial ligands for TLR4, but of the inflammation-induced expression of host- derived TLR ligands. More generally, such considerations raise the possibility that RP 105 may well be of special importance in down-modulation of the injurious inflammatory responses observed in the systemic inflammatory response syndrome
-/- and in autoimmune diseases, hi this regard, it is notable that RP105 mice
spontaneously develop splenomegaly with age (data not shown). Materials and Methods
[00156] Reagents, Zymosan A and E, coli K235 lipopolysaccharide (LPS) were from Sigma. Re-pυrified E. coli K235 LPS, free of contaminating TLR2 ligands, was generated as described previously (24). (Pani3) Cys-Ser-(Lys)4 [PamaCys] was from EMC Microcollections. All restriction enzymes were from New England Biolabs. All culture media and reagents coming into contact with cultured cells were endotoxin- free to the limits of detection of the Limulus amebocyte lysate assay (Bio-Whittaker)
at the concentrations employed, unless otherwise stated.
[00157] Cloning and Expression Constructs. Human RPl 05 and MD-I were cloned from primary human monocytes isolated by countercurrent elutriation (25) using RT- PCR-mediated cloning techniques. Harvested RNA was treated with RNase-free DNase (Roche), and cDNA was synthesized using Superscript II RNase BT reverse
transcriptase (Gibco). PCR primers were designed to amplify the entire coding
regions of LY64 and LY86, the genes encoding human RP105 and MD-I, respectively. Fragments of the anticipated size were amplified arid cloned into pCR2.1-TOPO (Invitrogen). Nucleotide sequencing revealed 100% identity with the previously published sequences (11-13, 16). For expression in mammalian cells, MD-I and RP 105 were subcloned into pcDNA3.1 and pcDNA4/Myc-His (Invitrogen).
[00158] The HSV TK-Renilla luciferase reporter plasmid (pRL-TK) was from
Promega; the NF-κB-Firefly Luciferase reporter plasmid (p-ELAM) has previously
been described (26). cDNA for TLR4, a gift from A. Visintin, was subcloned into pcDNA3.1. The pEFBOS expression vector encoding MD-2 was a gift of K. Miyake. [00159] Cell lines, HEK293 cell lines stably expressing CD14, CDUfTLRA, and
CD14/TLR2 were cultured in RPMI 1640 (Gibco), supplemented with 10% FCS
(Gibco), 2 mM L-glutamine (Sigma), 50 μg/ml gentamicin (Cellgro) and 5 μg/ml
puromycin (Calbiochem). HEK293 cells stably expressing TLR4/MD-2 were cultured
in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 50 μg/ml
gentamicin, 10 μg/ml ciprofloxacin (Bayer), and 0.5 mg/ml geneticin (Gibco).
HEK293 cells stably expressing RP105/CD14/TLR4 were generated by transfecting linearized RPl 05 cDNA into CD14/TLR4 HEK293 cells. RP105-expressing clones
were selected with 250 μg/ml Zeocin (Invitrogen) plus puromycin.
[00160] Transient transfections were performed using the PolyFect transfection reagent
(Qiagen) according to the manufacturer's instructions. All plasmid DNA was isolated using the EndoFree plasmid maxi kit (Qiagen). Construct expression was quantified
by flow cytometry (see below) using TLR4-PE, TLR2-F1TC, RP105-PE mAb, (e-
Bioscience) and CD14-FITC mAb (Becton Dickinson).
[00161] In vitro stimulation. HEK293 cells were cultured in 6-well plates (Costar) at
3 x 105 cells/ml (2 ml/well) for 24 h prior to transient transfection. 24 h after transfection, the culture media was changed and cells were stimulated (or mock stimulated) for an additional 24 h. Cell-free supernatants were collected and stored at
-200C until assayed for IL-8 production by ELISA (Pharmingen). To assay NF-κB-
driven luciferase expression (26), cells were co-transfected with pELAM (0.5 μg) and
pRL-TK (0.1 μg) plasmids for 24 h. After change of the culture media, cells were stimulated (or mock stimulated) for 5 h. Cells were subsequently lysed in passive lysis
buffer (Promega), and luciferase activity was quantified on a Monolight 3010 luminometer (Pharmingen) using the dual luciferase reporter assay system (Promega).
Firefly luciferase activity was normalized to Renilla luciferase activity.
[00162] Mice. RP105"A mice, on a C57BL/6 background (> 10 generations) have been described previously (27). Mice were genotyped using standard PCR techniques. Mice were also phenotyped for RP 105 surface expression on peripheral blood B cells by flow cytometry, as previously described (27) using B220-FITC mAb (Becton Dickinson) and RPl 05-PE mAb (e-Biosciences), along with isorype control antibodies from the respective companies (Fig. 10).
[00163] Resident peritoneal macrophages were harvested using standard techniques, and analyzed for RP 105 expression by FACS (see below). Primary mouse DC
subpopulations in spleen were analyzed by FACS after in vivo amplification of DC numbers through daily i.p. administration of 10 μg of flt3 ligand (Immunex/Amgen).
[00164] Mice were housed in a specific pathogen free facility in high-efficiency
particulate-filtered laminar flow hoods with free access to food and water. Animal
care was provided in accordance with National Institutes of Health guidelines. These studies were approved by the Cincinnati Children's Hospital Medical Center IACUC.
[00165] Ex vivo stimulation. Bone marrow-derived DC (BMDDC) generated using standard protocols (28), were cultured in 48-well plates at 2 x 106 cells/ml (500
μl/well) in RPMI 1640 supplemented with 10% FCS5 2 mM L-glutamine, and 50
μg/ml gentamicin. DC were stimulated with TLR ligands (or mock stimulated) for 24
h. Cell-free supernatants were collected and stored at -200C until assay by ELISA for TNF-α (Becton Dickinson), IL-12ρ70 and EL-6 (R&D Systems). DC surface
phenotype was analyzed by flow cytometry before and after stimulation (see below).
[00166] In vivo stimulation. 6-8 week old RP 105'A and wild type litteπnate control
mice were challenged i.p. with 25 μg of re-purified E. coli K235 LPS. One h later,
serum was collected and stored at -2O0C until analysis of TNF-α concentrations by ELISA.
[00167] Human leukocytes. PBMC were isolated from healthy volunteers by sedimentation over Ficoll/Hypaque gradients. Monocytes were purified by
countercurrent elutriation from PBMC isolated by leukapheresis (25). Monocyte-
derived DC were generated by standard protocols through culturing in media containing GM-CSF (500 UM) and IL-4 (1000 UM) [both from Peprotech] for 7-8
d. These studies were approved by the Cincinnati Children's Hospital Medical Center
and the University of Cincinnati College of Medicine IRBs.
[00168] Flow Cytometry. Surface and intracellular protein expression by HEK293 cells was quantified by FACS techniques described previously (29). For surface protein expression, cells were harvested, washed in FACS staining buffer (PBS with 1% FCS and 0.05% sodium azide [Mallinckrodt]) and blocked in PBS containing 10% human AB serum for 30 min at 40C. After a further wash, cells were incubated with directly labeled mAb for 30 min, washed, and fixed in 4% paraformaldehyde in PBS for 10-15 min. After further washing, cells were resuspended in FACS staining buffer. For intracellular protein expression, harvested cells were washed in FACS staining
buffer and fixed and permeabilized using Fix&Perm (Caltag) according to the
manufacturer's instructions, followed by incubation with mAb for 30 min. Cells were subsequently washed in permeabilizatiόn buffer and resuspended in staining buffer.
All incubations were done on ice. Flow cytometric analysis was performed using a FACSCalibur along with CellQuest Software (Becton Dickinson). At least 20,000
events were acquired for each data point.
[00169] RP 105 expression (RP 105-PE [e-Bioscience]) was analyzed on mouse splenic
B-cells (B220-FITC [Becton-Dickinsonj), BMDDC, and resident peritoneal macrophages (F4/80-allophycocyanin (APC) [e-Bioscience]). RP 105 expression was
also quantified on splenic DC subsets using directly labeled niAb to CDl Ic, CDl Ib,
CD4, CD8α, B220, CD19 and GR-I (all from Becton-Dickinson). I-Ab, CD80, CD86
and CD40 expression were quantified on BMDDC using directly labeled mAb (I-Ab from Becton-Dickinson; the rest from e-Bioscience). Cell death was monitored by FACS using propidium iodide (Sigma). FACS techniques were as above, except for a
blocking step with mAb 24G2.
[00170] RP105 expression by human leukocytes was analyzed using RP105-PE (e-
Biosciences). RP105 expression was examined on peripheral blood B cells (CD19- APC), myeloid DC (lineage mAb-FITC cocktail [CD3, CD14, CD19, CD20, CD56] negative; HLA-DR-PerCP and CDl Ic-APC positive), and plasmacytoid DC (lineage negative; HLA-DR-PerCP and BDCA4-APC positive) in PBMC. All mAb for these studies were from Becton-Dickinson, with the exception of that for BDCA4-APC
(Miltenyi Biotech).
[00171] Phylogenetic and domain analysis. For phylogenetic analysis, sequence alignments were performed using the ClustalW (30) and Pileup (31) programs. MEMSAT (32), SOSUI (33)and SABLE (34) software were used to predict the boundaries of the transmembrane domain of RP 105.
[00172] Statistical analysis. Data analysis was performed using the unpaired Student's t test, or ANOVA with post-hoc analysis by Student's t test, as appropriate.
Results.
[00173] Phylogenetic analysis of the TLR family has revealed 5 TLR subfamilies: (1)
TLR4; (2) TLR3; (3) TLR5; (4) TLR7, TLR8, TLR9; and (5) TLR 1 (2). Similar analytic techniques unequivocally place RP 105 in the TLR4 subfamily (Fig. 1 ; Fig. 11). The domain structure of the 641 amino acid mature RP105 protein has previously been commented upon (10, 12, 13, 16). The extracellular portion of this type I transmembrane protein contains 22 leucine-rich repeats (repeats 7-10 being non¬
homologous), along with a pattern of juxtamembrane cysteines that is conserved among the Toll and TLR families. Prediction algorithms disagree on the C-terminal
boundary of the transmembrane domain. Although the 6-11 amino acid intracellular
domain of RP 105 contains 1-2 tyrosine residues, it is devoid of recognized motifs that would suggest phosphorylation activity.
[00174] RP 105 expression mirrors TLR4 expression on APC. RP 105 has been thought to be a B cell-specific molecule in mice (10). Further, despite the fact that RP 105 has been noted to be expressed at the rnRNA level in primary human monocytic cells (11- 13), the only published examination of RP 105 protein expression in monocytic cells (by western blot analysis) was reported to be negative (13). We examined RP 105 expression by FACS in human and murine monocytic cells. In neither species is RP 105 expression limited to B cells. In humansrRP105 is also expressed by human
monocytes, as well as myeloid DC (Fig. 2a-c), cells that express considerably more TLR4 than do B cells (3.5) (and data not shown). Interestingly, RP105 is not expressed
by human plasmacytoid DC (Fig. 2d), cells that also fail to express TLR4 (35-38) (and data not shown).
[00175] Similarly, mice express RP 105 on resident peritoneal macrophages, splenic
DC subsets, as well as bone marrow derived DC (Fig. 3). Interestingly, unlike those found in human peripheral blood, splenic plasmacytoid DC from flt3L-treated mice express both RP105 and TLR4 (Fig 3e; and data not shown). RP105 thus joins an
elect list of "failed" B cell-specific molecules, including CD40 and NF-κB. Further,
the expression of RP 105 appears to directly mirror that of TLR4 on macrophages and DC.
[00176] RPl 05 suppresses TLR4 mediated signaling in HEK293 cells. HEK293 cells lack expression of endogenous TLR2, TLR4, TLR9, MD-2 and CD14 (26), as
well as RP 105 and MD-I (data not shown). Their TLR signaling machinery is fully functional, however. As a result, HEK293 cells have been extensively employed for
the in vitro analysis of TLR function (26, 39-46). Given the homology of RP105 to TLR4, we first examined whether RP105/MD-1 could act as a signaling receptor for LPS in HEK293 cells. HEK293 cells stably expressing CD14 were transiently transfected with cDNA encoding MD-I, MD-2, RP105, and/or TLR4. As shown in Figure 4a, while TLR4/MD-2 expression confers LPS-sensitivity, with resultant LPS-
driven EL-8 production, RP105/MD-1 expression does not. As has been found previouslv, in vitro overexpression of TLR4/MD-2 in HEK293 cells leads to baseline
IL-8 production in the absence of stimulation.
[00177] The effects of RP 105/MD- 1 expression on LPS-driven TLR4 signaling were
subsequently examined. Notably, RP 105 expression inhibits LPS-driven lL-8
production by HEK293 cells in a dose-dependent manner (Fig. Ab). As might be expected, RP105-mediated inhibition of LPS-driven IL-8 production is associated
with inhibition of LPS-driven NF-κB activation (Figure Ac).
[00178] The generality of RP 105-mediated suppression of proinflammatory signaling was subsequently examined. As shown in Figure 5, RP105 does not inhibit BL-I- or , TLR2-driven HL-8 production. Indeed, RP 105 expression led to variable augmentation of TLR2-mediated IL-8 production in some experiments (shown). Thus, RPl 05- mediated suppression is TLR4-specific.
[00179] Finally, we formally examined the necessity for MD-I expression in this
system. As shown in Figure 6a, in the absence of MD-I co-expression, RP 105 does
not inhibit TLR4 signaling. As previously reported (16), such MD-I expression is required for surface expression of RP 105 (Fig. 6b).
[00180] RPl 05 regulates cytokine production in DC. Expression studies in cell lines have considerable utility. They also have obvious drawbacks; principally, that overexpression may drive non-physiological interactions and processes. We thus examined RP105'7" mice, generated by targeted disruption of exon 3 of RP105 (27), backcrossed for more than 10 generations onto the C57BL/6 background. To define the effects of genetic deletion of RP 105 on APC function, bone marrow-derived DC were generated by standard techniques (47) from age-matched RPIOS"''" mice and
wild-type littermate controls. No differences in baseline expression of CD80, CD86 or MHC class π were apparent in DC generated from mice of the different genotypes (data not shown). DC were subsequently stimulated with repurified E, coli LPS. DC
from RPl 05"A mice produced significantly more TNF-α, IL-12p70 and DIr 6 than did
DC from wild type controls (Fig. 7).
] The biphasic dose-response for TNF-α, IL-12ρ70 and TL-6 production
observed in DC from both wild type and knockout mice (Fig. 7) was of interest, as such biphasic dose response curves do not appear to have been reported previously in human or murine DC (or monocyte/macrophages) either by us or by others. Flow
cytometric analysis revealed that this lower cytokine production at higher doses of
LPS was not associated with greater DC apoptosis (data not shown). The use of non-
repurified LPS, the usual reagent employed in both in vitro and in vivo studies, was
revealing, however. Commercial, non-purified LPS is contaminated with lipopeptide ligands for TLR2 (24). As shown in Figure 8a, such LPS continues to drive increasing
TNF-α production at higher doses (the very doses at which IL-8 production by "dirty"
LPS is induced in HEK293 cells transfected with TLR2 [data not shown]). Notably, stimulation of DC with these higher doses of "dirty" LPS led to similar amounts of
TNF-α production from RP 105 sufficient and deficient DC (Fig. 8a). Indeed,
stimulation with combinations of purified TLR4 and TLR2 agonists revealed that TLR2 agonists are able to effect functional reversal of RP105-mediated inhibition of TLR4 signaling (Fig. 8b). These data indicate that: (a) as with many biological agonistic responses, the response to a pure TLR4 ligand is biphasic, something modifiable by secondary agonists; and (b) signaling through other TLRs can overcome
RP105-mediated modulation of TLR4 signaling. These data further indicate that
RP105-mediated suppression of TLR4 signaling is not merely an overexpression artifact in cell lines,
[00182] RPl 05 regulates proinflammatory cytokine production in vivo. Finally,
LPS-driven cytokine production was characterized in vivo in order to examine whether RP 105 plays a role in regulating systemic cytokine productioa RP 105 mice and wild type littermate controls were challenged intraperitoneally with re-purified E.
coli LPS. RP1O5"7" mice produce significantly more TNF-α in response to LPS
challenge (Fig. 9). Thus, RP 105 is a negative regulator of TLR4 signaling in vivo.
Discussion
[00183] These results demonstrate that RP 105, a TLR4 homologue whose expression
mirrors that of TLR4 in antigen presenting cells, is a negative regulator of TLR4 signaling.
[00184] Without wishing to be bound by theory in any way, this specificity, along with the structural homologies between RP 105 and TLR4, suggests an attractive hypothesis for the mechanism of negative regulation of TLR4 signaling by RP 105: direct interference with TLR4 homoaggregation through the formation of RP105/TLR4 heterodimers. Other obvious mechanistic possibilities include more mediate interference with TLR4 aggregation via MD-l/MD-2 interactions, or signal transduction through the diminutive intracellular portion of RP 105. [00185] Mechanism aside, the fact that RP 105 promotes B cell activation while
inhibiting DC activation such that, immunoregulation by RP 105 leads to
augmentation of humoral immunity (through effects on B cells) along with
concomitant inhibition of cellular immune responses (through effects on DCs).
[00186] RP 105 is also of special importance in down-modulation of the injurious inflammatory responses observed in autoimmune diseases and the systemic inflammatory response syndrome (66).
[00187] The description fully satisfies the objects, aspects and advantages set forth.
While the invention has been set forth in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in the light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and
variations which fall within the spirit and scope of the following claims.
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Claims

CLAIMS What is claimed is:
1. A method of using of an activator of RP 105 to prepare a medicament to
suppress an immune response.
2. The method according to claim 1 wherein the medicament is used to prevent or treat an autoimmune disease.
3. A use according to claims 1 or 2 wherein the activator of RP105 is an ' oligonucleotide that activates the expression of RP 105.
4. A use according to any one of claims 1 or 2 wherein the activator of RP105 is an antibody that binds to RP105.
5. A use according to any one of claims 1 to 4 wherein the medicament further
comprises an ORP 105 protein or a nucleic acid molecule encoding an RP 105
protein.
6. A use of an RP 105 protein or a nucleic acid sequence encoding an RP 105
protein to prepare a medicament to inhibit an immune response.
7. A pharmaceutical composition for use in suppressing an immune response comprising an activator of RP 105 in admixture with a suitable diluent or carrier.
8. A composition according to claim 7 wherein the activator is an antibody that binds RP105.
9. A composition according to claim 7 wherein the activator is an oligonucleotide that is complimentary to a nucleic acid sequence from an RP 105 gene.
10; A pharmaceutical composition for use in preventing immune suppression
comprising an RP 105 protein or a nucleic acid encoding an RP 105 protein in
admixture with a suitable diluent or carrier.
11. A method of identifying substances that can activate RP 105, comprising the steps of: (a) reacting RP 105 and a test substance, under conditions which
allow for formation of a complex between the RP 105 and the test substance, and (b) assaying for complexes of RP 105 and the test substance, for free substance or for activation of RP 105.
12. A method of identifying substances that can activate RP 105, comprising the steps of: (a) reacting RP 105 and a test substance, under conditions which
allow for the activation of the RP105, and (b) assaying for activation of RP105.
13. A method of identifying substances that can activate RP 105, comprising the
steps of: (a) reacting RP 105 and a test substance, under conditions which allow for the activation of the RP 105, and (b) assaying for the inhibition of the
activity or expression level of TLR4.
14. A method of using a compound identified according to claim 11 to prepare a
medicament to modulate an inflammatory or immune response.
15. A method of using a compound identified according to claiml2 to prepare a medicament to suppress an inflammatory or immune response.
16. A method of using a compound identified according to claim 13 to prepare a medicament to suppress an inflammatory or immune response.
17. A method of treating a TLR-4 mediated disease or pathology in a subject
comprising administering a pharmaceutically effective amount of an activator
of RP 105 to the subject.
18. The method according to claim 17, wherein the TLR-4 mediated disease or
pathology is selected from the group consisting of psoriasis, atopic dermatitis, asthma, COPD, adult respiratory disease, arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, septic shock, endotoxic shock,
gram negative sepsis, toxic shock syndrome, stroke, cardiac and renal reperfusion injury, glomerulonephritis, thrombosis, Alzheimer's disease, graft vs. host reaction, allograft rejections, malaria, acute respiratory distress syndrome, delayed type hypersensitivity reaction, atherosclerosis, cerebral and cardiac ischemia, osteoarthritis, multiple sclerosis, restinosis, angiogenesis, osteoporosis, gingivitis, respiratory viruses, herpes viruses, hepatitis viruses.,
HIV, Kaposi's sarcoma associated virus, meningitis, cystic fibrosis, pre-teπn labor, cough, pruritis, multi-organ dysfunction, trauma, strains, sprains, contusions, psoriatic arthritis, herpes, encephalitis, CNS vasculitis, traumatic brain injury, CNS tumors, subarachnoid hemorrhage, post surgical trauma,
interstitial pneumonitis, hypersensitivity, crystal induced arthritis, acute and chronic pancreatitis, acute alcoholic hepatitis, necrotizing enterocolitis, chronic sinusitis, angiogenic ocular disease, ocular inflammation, retinopathy of prematurity, diabetic retinopathy, macular degeneration with the wet type preferred and corneal neovascularization, polymyositis, vasculitis, acne, gastric and duodenal ulcers, celiac disease, esophagitis, glossitis, airflow obstruction,
airway hyperresponsiveness, bronchiectasis, bronchiolitis, bronchiolitis obliterans, chronic bronchitis, cor pulmonae, cough, dyspnea, emphysema,
hypercapnea, hyperinflation, hypoxemia, hyperoxia-induced inflammations,
hypoxia, surgical lung volume reduction, pulmonary fibrosis, pulmonary
hypertension, right ventricular hypertrophy, peritonitis associated with continuous ambulatory peritoneal dialysis (CAPD), granulocytic ehrlichiosis,
sarcoidosis, small airway disease, ventilation-perfusion mismatching, wheeze, colds, gout, alcoholic liver disease, lupus, burn therapy, periodontitis and early transplantation.
19. The method according to claim 17, wherein the compounds of the present invention are administered in conjunction with one or more drugs, agents or therapeutics selected from the group consisting of: glucocorticoids, 5-
lipoxygenase inhibitors, β-2 adrenoreceptor agonists, muscarinic Ml and M3
antagonists, muscarinic M2 agonists, NK3 antagonists, LTB4 antagonists, cysteinyl leukotriene antagonists, bronchodilators, PDE4 inhibitors, PDE inhibitors, elastase inhibitors, MMP inhibitors, phospholipase A2 inhibitors,
phospholipase D inhibitors, histamine Hl antagonists, histamine H3 antagonists, dopamine agonists, adenosine A2 agonists, NKl and NK2 antagonists, GABA-b agonists, nociceptin agonists, expectorants, mucolytic agents, decongestants, antioxidants, anti-IL-8 anti-bodies, anti-IL-5 antibodies, anti-IgE antibodies, anti-TNF antibodies, EL-IO, adhesion molecule inhibitors, and growth hormones.
20. The method according to claim 17, wherein the compounds of the present
invention are administered in conjunction with one or more therapeutic
steroids.
21. The method according to claim 17, wherein the one or more therapeutic
steroids are selected from the group consisting of corticoids, glucocorticoids, dexamethasone, prednisone, prednisalone, and betamethasone.
22. A method of treating a condition associated with cytokine production in a
subject comprising administering a pharmaceutically effective amount of an activator of RP 105 to the subject.
23. A method of using an activator of RP 105 in the manufacture of a medicament for the treatment of a condition associated with cytokine production, wherein the condition is rheumatoid arthritis or a condition resulting from an infection.
24. The method according to claim 22 wherein the condition is a tumor necrosis
factor (TNF) associated condition.
25. The method according to claim 24 wherein the TNF is TNF-α.
26. The method according to claim 22 wherein the condition is an interleukin-1
(IL-I) associated condition.
27. The method according to claim 26 wherein the IL-I is IL-lβ.
28. The method according to claim 22 wherein the condition is sepsis.
29. The method according to claim 22 wherein the condition is septic shock.
30. The method according to claim 22 wherein the condition is systemic
inflammatory response syndrome
31. The method according to claim 17 or 22 wherein the condition is induced by a
Toll Related Receptor (TRR) ligand.
32. The method according to claim 17 or 22 wherein the condition is induced by
lipopolysaccharide (LPS),
33. The method according to claim 17 or 22 wherein the condition is induced by Gram-negative bacteria.
34. The method according to claim 17 or 22 wherein the activator is specific for RP105.
35. The method according to claim 17 or 22 wherein the activator is a chemical activator.
36. The method according to claim 17 or 22 wherein the activator is an antibody or a fragment thereof that is capable of binding specifically to RP 105 or a fragment thereof.
37. The method according to claim 17 or 22 wherein the activator is a nucleic acid
capable of increasing the expression of RP 105.
38. A method for identifying an activator of RP105, which activator is suitable for
use in the treatment of a condition associated with cytokine production
wherein the condition s marked by pathogenic inflammatory or immune responses, the method comprising: (a) providing, as a first component, a cell capable of TLR4 or TLR ligand-induced cytokine production; (b) providing, as a second component, a TLR ligand or TLR4 activator; (c) contacting the first and second components in the presence of a test agent; (d) determining whether the test agent is able to increase RP105-mediated downregulation of
TLR4 activity; thereby to determine whether the test agent acts as an inhibitor
of cytokine production.
39. The method according to claim 38 further comprising the step of determining
whether the activator is specific for RP 105.
40. The activator of TLR4-induced expression of cytokines identified or identifiable by a method as defined in claim 38.
41. A pharmaceutical formulation comprising a pharmaceutically acceptable carrier and an activator of RP 105 identifiable by a method according to claim
38.
42. A method of using an activator of RP 105 to study the inhibition of sepsis and/or septic shock caused by a TLR4, in vitro.
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US10443099B2 (en) 2005-04-15 2019-10-15 Becton, Dickinson And Company Diagnosis of sepsis
US11578367B2 (en) 2005-04-15 2023-02-14 Becton, Dickinson And Company Diagnosis of sepsis
US8404656B2 (en) 2007-08-09 2013-03-26 Children's Hospital Medical Center Methods of treatment and prevention of diet-induced obesity and sequelae thereof
WO2009089399A2 (en) * 2008-01-10 2009-07-16 Bausch & Lomb Incorporated Compositions comprising toll-like receptor or coreceptor antagonists and methods for ocular neuroprotection
WO2009089399A3 (en) * 2008-01-10 2009-10-08 Bausch & Lomb Incorporated Compositions comprising toll-like receptor or coreceptor antagonists and methods for ocular neuroprotection
US9708661B2 (en) 2008-04-03 2017-07-18 Becton, Dickinson And Company Advanced detection of sepsis
US9885084B2 (en) 2008-04-03 2018-02-06 Becton, Dickinson And Company Advanced detection of sepsis
US10221453B2 (en) 2008-04-03 2019-03-05 Becton, Dickinson And Company Advanced detection of sepsis
WO2017176319A1 (en) * 2016-04-06 2017-10-12 University Of Washington Therapeutic vaccine for hepatitis b virus (hbv) using the hbv core antigen
US11324819B2 (en) 2016-04-06 2022-05-10 University Of Washington Therapeutic vaccine for Hepatitis b virus (HBV) using the HBV core antigen
US11883488B2 (en) 2016-04-06 2024-01-30 Abacus Bioscience, Inc. Therapeutic vaccine for Hepatitis B virus (HBV) using the HBV core antigen
WO2024010959A1 (en) * 2022-07-07 2024-01-11 University Of Pittsburgh - Of The Commonwealth System Of Higher Education C-glycosides as anti-inflammatory agents

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