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Title:  Osteopontin-related compositions and methods
United States Patent: 
7,282,490
Issued: 
October 16, 2007

Inventors: 
Chabas; Dorothee (Paris, FR), Steinman; Lawrence (Stanford, CA)
Assignee: 
The Board of Trustees of the Leland Stanford Junior University (Palo Alto, CA)
Appl. No.: 
10/495,893
Filed: 
November 21, 2002
PCT Filed: 
November 21, 2002
PCT No.: 
PCT/US02/37466
371(c)(1),(2),(4) Date: 
December 17, 2004
PCT Pub. No.: 
WO03/046135
PCT Pub. Date: 
June 05, 2003


 

Pharm Bus Intell & Healthcare Studies


Abstract

This invention provides a method for reducing the amount of osteopontin in an osteopontin-expressing cell comprising introducing into the cell a nucleic acid which specifically inhibits osteopontin expression in the cell. This invention also provides methods for inhibiting the onset of, and treating, osteopontin-related disorders, as well as compositions for practicing the same. This invention further provides methods for determining the amount of osteopontin in a sample, and a kit for practicing the same. This invention also provides methods for determining whether an agent reduces the amount of osteopontin in an osteopontin-expressing cell. Finally, this invention provides methods for treating a subject afflicted with a disorder mediated by an endogenous protein.

SUMMARY OF THE INVENTION

The invention provides methods for preventing or treating a patient suffering or at risk of a disorder in which expression of osteopontin contributes to the pathogenesis. These methods comprise administering to the subject an effective amount of a nucleic acid comprising a segment encoding osteopontin, whereby the nucleic acid is expressed in the patient to produce osteopontin, and the osteopontin induces an immune response that reduces the level of osteopontin in the patient. In some methods, the immune response includes formation of antibodies. In some methods, the patient is suffering from or at risk of raft versus host disease, epilepsy, a granulomatous disorder, herpes simplex keratisits, bacterial arthritis, or an autoimmune disease. Examples of autoimmune diseases include multiple sclerosis, rheumatoid arthritis, type I diabetes.

The nucleic acid can be DNA, in which case the nucleic acid further comprises a promoter and optionally an enhancer in operable linkage to the segment encoding the osteopontin. The promoter can be constitutive or cell-type specific. Alternatively, the nucleic acid can be RNA. In some methods, the nucleic acid is administered intramuscularly. In some methods, the the subject is a human.

Some methods, further comprise monitoring a decrease in the level of osteopontin responsive to the administering step. In some methods, the level of osteopontin is monitored in a cell of the patient selected from the group consisting of a neuron, a macrophage, a vascular endothelial cell, an astrocyte and a microglial cell. In some methods, the patient has the disorder and the method further comprises monitoring a decrease in the symptoms of the patient responsive to the administering.

The invention further provides a composition comprising a nucleic acid encoding osteopontin and a pharmaceutically acceptable carrier.

The invention further provides methods for preventing or treating a patient suffering from or at risk of a disorder in which expression of osteopontin contributes to the pathogenesis. These methods comprise administering to the patient an effective amount of osteopontin, whereby the osteopontin induces an immune response that reduces the level of osteopontin in the patient. In some such methods, the the osteoponin is administered with an adjuvant. In some methods, the immune response comprises formation of antibodies to osteopontin.

In some methods, the patient is suffering from or at risk of graft versus host disease, epilepsy, a granulomatous disorder, herpes simplex keratisits, bacterial arthritis or an autoimmune disease as described above. In some methods the patient is a human.

Some methods further comprise monitoring a decrease in the level of osteopontin responsive to the administering step. In some methods, the level of osteopontin is monitored in a cell of the patient selected from the group consisting of a neuron, a macrophage, a vascular endothelial cell, an astrocyte and a microglial cell. In some methods, the the patient has the disease and the method further comprises monitoring a decrease in the symptoms of the patient responsive to the administering.

The invention further provides a composition comprising osteoponin and an adjuvant.

This invention provides a method for reducing the amount of osteopontin in disorders in which osteopontin is produced.

This invention also provides a first method for inhibiting the onset of an osteopontin-related disorder in a subject comprising administering to the subject a prophylactically effective amount of a nucleic acid which specifically reduces levels of osteopontin.

In addition, this invention provides a first method for treating a subject afflicted with an osteopontin-related disorder in a subject comprising administering to the subject a therapeutically effective amount of a nucleic acid which specifically inhibits the expression of osteopontin in the subject's osteopontin-expressing cells.

This invention further provides a second method for inhibiting the onset of an osteopontin-related disorder in a subject comprising administering to the subject a prophylactically effective amount of an anti-osteopontin antibody or antigen-binding portion thereof.

This invention further provides a second method for treating a subject afflicted with an osteopontin-mediated disorder comprising administering to the subject a therapeutically effective amount of an anti-osteopontin antibody or antigen-binding portion thereof.

This invention further provides two compositions. The first composition comprises a nucleic acid which specifically inhibits the expression of osteopontin in an osteopontin-expressing cell and a pharmaceutically acceptable carrier. The second composition comprises an anti-osteopontin antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

This invention further provides a first method for determining the amount of osteopontin in a sample comprising (a) contacting the sample with an anti-osteopontin antibody or antigen-binding portion thereof under suitable conditions, (b) determining the amount of antibody or antigen-binding portion thereof bound to the sample, and (c) comparing the amount so determined to a known standard, thereby determining the amount of osteopontin in the sample.

This invention further provides a second method for determining the amount of osteopontin in a sample comprising (a) contacting the sample under suitable conditions with a nucleic acid which specifically hybridizes to osteopontin-encoding mRNA, (b) determining the amount of nucleic acid so hybridized, (c) comparing the amount of nucleic acid so determined to a known standard so as to determine the amount of osteopontin-encoding mRNA in the sample, and (d) comparing the amount of mRNA so determined to a known standard, thereby determining the amount of osteopontin in the sample.

This invention provides a kit for practicing the first and second quantitative methods comprising (a) an agent selected from the group consisting of (i) an anti-osteopontin antibody or antigen-binding portion thereof and (ii) a nucleic acid which specifically hybridizes with osteopontin-encoding mRNA, and (b) instructions for use.

This invention also provides two methods ("assays") for determining whether an agent reduces the amount of osteopontin in an osteopontin-expressing cell. The first assay comprises (a) contacting the cell with the agent under suitable conditions, (b) determining the amount of osteopontin in the cell, and (c) comparing the amount so determined to the amount of osteopontin in a comparable cell in the absence of the agent, thereby determining whether the agent reduces the amount of osteopontin in the cell.

The second assay comprises the steps of (a) contacting the agent with osteopontin under suitable conditions, (b) determining the activity of osteopontin in the presence of the agent, and (c) comparing the activity so determined to the activity of osteopontin in the absence of the agent, thereby determining whether the agent reduces the activity of osteopontin.

This invention further provides a method of treating a subject afflicted with multiple sclerosis comprising administering to the subject a therapeutically effective amount of an expressible nucleic acid encoding osteopontin. Also provided is a method of inhibiting the onset of multiple sclerosis in a subject comprising administering to the subject a prophylactically effective amount of an expressible nucleic acid encoding osteopontin.

This invention further provides kits for treating or preventing an osteopontin-related disorder. The first kit comprises a nucleic acid which specifically inhibits the expression of osteopontin in an osteopontin-expressing cell, and instructions for using the nucleic acid in the treatment or prophylaxis of an osteopontin-related disorder. The second kit comprises an anti-osteopontin antibody or antigen-binding portion thereof, and instructions for using the antibody or antigen-binding portion thereof in the treatment or prophylaxis of an osteopontin-related disorder.

Finally, this invention provides two methods for treating a subject afflicted with a disorder mediated by an endogenous protein. The first method comprises administering to the subject (a) osteopontin and (b) the endogenous protein or an antigenic portion thereof, wherein the osteopontin and endogenous protein or antigenic portion thereof are administered in amounts effective to treat the subject. The second method for treating a subject afflicted with a disorder mediated by an endogenous protein comprises administering to the subject (a) an expressible osteopontin-encoding nucleic acid and (b) an expressible nucleic acid encoding the endogenous protein or an antigenic portion thereof, wherein the nucleic acids are administered in amounts effective to treat the subject.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for reducing the amount of osteopontin in an osteopontin-expressing cell comprising introducing into the cell a nucleic acid which specifically inhibits osteopontin expression in the cell. In one embodiment, this method further reduces the amount of osteopontin secreted by an osteopontin-secreting cell.

In this method, the nucleic acid can be, for example, DNA or RNA. In the preferred embodiment, the nucleic acid is DNA.

In addition, the nucleic acid can be an anti-sense nucleic acid that hybridizes to osteopontin-encoding mRNA, or a catalytic nucleic acid that cleaves osteopontin-encoding mRNA. In the preferred embodiment, the nucleic acid is an expressible nucleic acid encoding an anti-sense nucleic acid that hybridizes to osteopontin-encoding mRNA, and/or encoding a catalytic nucleic acid that cleaves osteopontin-encoding mRNA.

Osteoponin expression can also be inhibited using zinc finger proteins or nucleic acids encoding the same as described in WO 00/00409. Alternatively, inhibition of expression can be achieved using siRNAs as described by WO 99/32619, Elbashir, EMBO J. 20, 6877-6888 (2001) and Nykanen et al., Cell 107, 309-321 (2001); WO 01/29058.

In these methods, the osteopontin-expressing cell can be, for-example, a neuron, a macrophage, a vascular endothelial cell, an astrocyte or a microglial cell. In the preferred embodiment, the cell is a neuron.

This invention also provides a first method for inhibiting the onset of an osteopontin-related disorder in a subject comprising administering to the subject a prophylactically effective amount of a nucleic acid which specifically inhibits the expression of osteopontin in the subject's osteopontin-expressing cells.

In addition, this invention provides a first method for treating a subject afflicted with an osteopontin-related disorder in a subject comprising administering to the subject a therapeutically effective amount of a nucleic acid which specifically inhibits the expression of osteopontin in the subject's osteopontin-expressing cells.

In these first methods of prophylaxis and treatment, the nucleic acid can be, for example, DNA or RNA. In the preferred embodiment, the nucleic acid is DNA.

In addition, the nucleic acid can be an anti-sense nucleic acid that hybridizes to osteopontin-encoding mRNA, or a catalytic nucleic acid that cleaves osteopontin-encoding mRNA. In the preferred embodiment, the nucleic acid is an expressible nucleic acid encoding an anti-sense nucleic acid that hybridizes to osteopontin-encoding mRNA, and/or encoding a catalytic nucleic acid that cleaves osteopontin-encoding mRNA.

Also in these first methods of prophylaxis and treatment, the subject's cells in which the amount of osteopontin is reduced can be, for example, neurons, macrophages, vascular endothelial cells, astrocytes or microglial cells.

This invention further provides a second method for inhibiting the onset of an osteopontin-related disorder in a subject comprising administering to the subject a prophylactically effective amount of an anti-osteopontin antibody or antigen-binding portion thereof.

This invention further provides a second method for treating a subject afflicted with an osteopontin-mediated disorder comprising administering to the subject a therapeutically effective amount of an anti-osteopontin antibody or antigen-binding portion thereof.

In the preferred embodiment of the first and second methods of prophylaxis and treatment, the osteopontin-related disorder is multiple sclerosis. Preferably, the subject is a human.

This invention further provides two compositions. The first composition comprises a nucleic acid which specifically inhibits the expression of osteopontin in an osteopontin-expressing cell and a pharmaceutically acceptable carrier. The second composition comprising an anti-osteopontin antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

Determining a therapeutically or prophylactically effective amount of the instant compositions can be done based on animal data using routine computational methods. In one embodiment, the therapeutically or prophylactically effective amount contains between about 0.1 mg and about 1 g of nucleic acid or protein, as applicable. In another embodiment, the effective amount contains between about 1 mg and about 100 mg of nucleic acid or protein, as applicable. In a further embodiment, the effective amount contains between about 10 mg and about 50 mg of the nucleic acid or protein, as applicable.

In this invention, administering the instant compositions can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, via implant, transmucosally, transdermally, intramuscularly, intrathecally, and subcutaneously. The following delivery systems, which employ a number of routinely used pharmaceutical carriers, are only representative of the many embodiments envisioned for administering the instant compositions.

Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone. Osteopontin or nucleic acids of the invention can also be administered attached to particles using a gene gun.

Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).

Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

This invention further provides a first method for determining the amount of osteopontin in a sample comprising (a) contacting the sample with an anti-osteopontin antibody or antigen-binding portion thereof under suitable conditions, (b) determining the amount of antibody or antigen-binding portion thereof bound to the sample, and (c) comparing the amount so determined to a known standard, thereby determining the amount of osteopontin in the sample.

In one embodiment of this first quantitative method, the agent is an anti-osteopontin antibody or antigen-binding portion thereof. Preferably, the antibody or antigen-binding portion thereof is labeled with a detectable marker.

This invention further provides a second method for determining the amount of osteopontin in a sample comprising (a) contacting the sample under suitable conditions with a nucleic acid which specifically hybridizes to osteopontin-encoding mRNA, (b) determining the amount of nucleic acid so hybridized, (c) comparing the amount of nucleic acid so determined to a known standard so as to determine the amount of osteopontin-encoding mRNA in the sample, and (d) comparing the amount of mRNA so determined to a known standard, thereby determining the amount of osteopontin in the sample.

In one embodiment of this second quantitative method, the nucleic acid is labeled with a detectable marker.

In the first and second quantitative methods, the sample can be any sample containing or suspected of containing osteopontin alone, or in the presence of osteopontin-producing cells. In one embodiment, the sample is a tissue sample. Tissue samples include, without limitation, bodily fluid samples such as cerebrospinal fluid and blood and its component parts, and sections of solid tissue such as brain and spinal cord. Tissue samples can comprise, for example, neurons, macrophages, vascular endothelial cells, astrocytes or microglial cells. In one embodiment, the tissue sample is from a subject afflicted with or suspected of being afflicted with an osteopontin-related disorder, preferably multiple sclerosis.

In a further embodiment, the first and second quantitative methods comprise the step of determining the location of osteopontin within the tissue sample. Steps (a) and (b) of these methods can be performed, for example, either in vivo or ex vivo.

This invention provides a kit for practicing the first and second quantitative methods comprising (a) an agent selected from the group consisting of (i) an anti-osteopontin antibody or antigen-binding portion thereof and (ii) a nucleic acid which specifically hybridizes with osteopontin-encoding mRNA, and (b) instructions for use.

This invention also provides two methods ("assays") for determining whether an agent reduces osteopontin activity or the amount of osteopontin in an osteopontin-expressing cell. The first assay comprises (a) contacting the cell with the agent under suitable conditions, (b) determining the amount of osteopontin in the cell, and (c) comparing the amount so determined to the amount of osteopontin in a comparable cell in the absence of the agent, thereby determining whether the agent reduces the amount of osteopontin in the cell.

In the first assay, the osteopontin-expressing cell can be, for example, a neuron, a macrophage, a vascular endothelial cell, an astrocyte and a microglial cell. Preferably, the cell is a human cell. In step (b) of the first assay, the amount of osteopontin in the cell is determined using an anti-osteopontin antibody or antigen-binding portion thereof. Alternatively, in step (b), the amount of osteopontin in the cell is determined using a nucleic acid which specifically hybridizes with osteopontin-encoding mRNA.

The second assay comprises the steps of (a) contacting the agent with osteopontin under suitable conditions, (b) determining the activity of osteopontin in the presence of the agent, and (c) comparing the activity so determined to the activity of osteopontin in the absence of the agent, thereby determining whether the agent reduces the activity of osteopontin.

In one embodiment of the second assay, the osteopontin is in a cell. This cell can be, for example, a neuron, a macrophage, a vascular endothelial cell, an astrocyte and a microglial cell. Preferably, the cell is a human cell.

This invention further provides methods for treating a subject afflicted with a disorder mediated by an endogenous protein. One method comprises administering to the subject (a) osteopontin and (b) the endogenous protein or an antigenic portion thereof, wherein the osteopontin and endogenous protein or antigenic portion thereof are administered in amounts effective to treat the subject.

In this method, the osteopontin and endogenous protein or antigenic portion thereof can be administered simultaneously. Alternatively, the osteopontin and endogenous protein or antigenic portion thereof are administered separately.

A second method for treating a subject afflicted with a disorder mediated by an endogenous protein comprises administering to the subject (a) an expressible osteopontin-encoding nucleic acid and (b) an expressible nucleic acid encoding the endogenous protein or an antigenic portion thereof, wherein the nucleic acids are administered in amounts effective to treat the subject.

In this second method, the osteopontin-encoding nucleic acid and nucleic acid encoding the endogenous protein or antigenic portion thereof can be administered simultaneously, either on the same vector or separate vectors. Alternatively, the osteopontin-encoding nucleic acid and nucleic acid encoding the endogenous protein or antigenic portion thereof are administered separately.

In one embodiment of the first and second methods for treating a subject afflicted with a disorder mediated by an endogenous protein, the disorder is an autoimmune disorder. In another embodiment, the disorder is multiple sclerosis, insulin-dependent diabetes mellitus, rheumatoid arthritis, autoimmune uveitis, primary billiary cirrhosis or Alzheimer's disease. Preferably, the disorder is multiple sclerosis.

In the first and second methods where the disorder treated is multiple sclerosis, the endogenous protein can be, for example, myelin basic protein, proteolipid protein, myelin-associated glycoprotein, cyclic nucleotide phosphodiesterase, myelin-associated oligodendrocytic basic protein, or alpha-B-crystalin.

Table 1 (see Original Patent) sets forth examples of disorders treatable by the instant methods, and their corresponding endogenous proteins.

Finally, this invention provides kits for treating or preventing an osteopontin-related disorder. The first kit comprises a nucleic acid which specifically inhibits the expression of osteopontin in an osteopontin-expressing cell, and instructions for using the nucleic acid in the treatment or prophylaxis of an osteopontin-related disorder. The second kit comprises an anti-osteopontin antibody or antigen-binding portion thereof, and instructions for using the antibody or antigen-binding portion thereof in the treatment or prophylaxis of an osteopontin-related disorder.

Methods of Treatment Using Osteoponin or Nucleic Acids Encoding Osteononin

The invention provides methods of treating osteopontin related disorders using osteopontin, epitopic fragments thereof or nucleic acids encoding either of these, graft versus host disease. These methods are useful for treating a variety of disease for which downregulation of type 1 immune response and or upregulation of type 2 immune response is required. Such diseases include autoimmune diseases, host versus graft disease and graft versus host disease, granulomatous disorder, herpes simplex keratisits, bacterial arthritis and epilepsy. Autoimmune diseases include multiple sclerosis, rheumatoid arthritis, and type I diabetes. The methods can be used to treat or prevent such disorders in patients having or at risk of such disorders. Patients having a disorder include those who are currently experiencing clinical symptoms, and patients who experience symptoms intermittently who may be symptomatic or asymptomatic at any particular time. These methods are particularly effective for treating a subject afflicted with multiple sclerosis comprising administering to the subject a therapeutically effective amount of an expressible nucleic acid encoding osteopontin. Also provided is a method of inhibiting the onset of multiple sclerosis in a subject comprising administering to the subject a prophylactically effective amount of an expressible nucleic acid encoding osteopontin.

1. Nucleic Acids Encoding Osteopontin

The nucleic acids used in these methods encodes osteopontin or an epitopic fragment thereof. The nucleic acids are transcribed and translated (DNA) or transcribed (mRNA) in situ, and the translation product generates an immune response. DNA immunization is described by WO 99/28471, Chowdhury et al., PNAS 95, 669-674 (1998) and J. Immunol. Methods 231, 83-91 (1999)).

DNA immunization can be performed with or without an adjuvant. The adjuvant, if present, can be one that is typically used with a protein antigen (see below), or it can be an adjuvant that is specifically chosen to associate with DNA, such as the positively charged detergent CTAB. The DNA can be administered naked or complexed with colloidal materials. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam.TM. and Lipofectin.TM.). Cationic and neutral lipids that are suitable for efficient lipofection of polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.

Optionally, the DNA is dissolved in a pharmaceutical carrier in a solution that is sterile and substantially isotonic.

The nucleic acid used as an immunogen contains a segment encoding osteopontin and other segments encoding one or more regulatory sequences that ensure translation and transcription (in the case of DNA) of the immunogen. Regulatory sequences include a promoter, enhancer, transcription termination site, ribosome binding site, and intronic sites. The promoter can be constitutive or inducible or tissue specific, in which case the promoter is preferably specific for antigen presenting cells. For expression in blood cells, as is desirable for induction of an immune response, promoter and enhancer elements from light or heavy chain immunoglobulin genes or the CMV major intermediate early promoter and enhancer are suitable to direct expression. Optionally, DNA immunogens is present as a component of a vector. In some instances, the vector encodes proinflammatory cytokines to attract immune cells to the site of injection. In some instances, the DNA encodes a fusion protein, comprising an antigenic component to which antibodies are desired and a T-cell antigen, such as tetanus toxoid, or other adjuvant such as C3d (see Dempsey et al., Science 271, 348-50 (1996)). The DNA can encode a full length protein or a desired epitopic fragment thereof.

In a further variation, the nucleic acid can be incorporated into the genome of a virus or a bacteria. Optionally, the nucleic acid is incorporated in such a manner that the immunogenic peptide is expressed as a secreted protein or as a fusion protein with an outer surface protein of a virus or a transmembrane protein of a bacteria so that the peptide is displayed. Viruses or bacteria used in such methods should be nonpathogenic or attenuated. Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitis virus and other alpha viruses, vesicular stomatitis virus, and other rhabdo viruses, vaccinia and fowl pox. Suitable bacteria include Salmonella and Shigella. Fusion of an immunogenic peptide to HBsAg of HBV is particularly suitable.

2. Administration of Osteopontin

The invention also provides methods in which osteopontin or an epitopic fragment thereof is administered to a patient. The osteopontin generates an immune response which lowers levels of osteopontin in the patient in the same manner as described for nucleic acids encoding osteopontin administration. Osteopontin can be administered alone or fused as a component of a longer protein. Optionally such a fusion protein can include a heterologous amino acid sequence that induces a helper T-cell response against the heterologous amino acid sequence and thereby a B-cell response against osteopontin.

Epitopic fragments of osteopontin suitable for use in the methods can be initially screened by standard computer programs that identify regions of probable immunogenicity. Fragments are then tested for activity in animal models as described in the Examples.

3. Adjuvants

A variety of adjuvants can be used in combination with osteopontin or epitopic fragments thereof or nucleic acids encoding the same to elicit an immune response. Preferred adjuvants include aluminum hydroxide and aluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPL.TM.) (see GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Mont., now part of Corixa). Stimulon.TM. QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540),(Aquila BioPharmaceuticals, Framingham, Mass.). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (WO 98/40100).

4. Therapeutic and Prophylactic Regimes for Generating an Immune Response

In prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, an osteoponin related disorder in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. In therapeutic applications, compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient immune response has been achieved. An effective regime comprises a combination of an effective dosage and frequency of administration. Typically, the immune response is monitored and repeated dosages are given if the immune response starts to wane.

Effective doses of the compositions of the present invention vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy. The amount of immunogen depends on whether adjuvant is also administered, with higher dosages being required in the absence of adjuvant. The amount of immunogen depends on whether adjuvant is also administered, with higher dosages being required in the absence of adjuvant. The amount of an immunogen for administration sometimes varies from 1-500 .mu.g per patient and more usually from 5-500 .mu.g per injection for human administration. Occasionally, a higher dose of 1-2 mg per injection is used. Typically about 10, 20, 50 or 100 .mu.g is used for each human injection. The timing of injections can vary significantly from once a day, to once a year, to once a decade. A typical regimen consists of an immunization followed by booster injections at time intervals, such as 6 week intervals. Another regimen consists of an immunization followed by booster injections 1, 2 and 12 months later. Another regimen entails an injection every two months for life. Alternatively, booster injections can be on an irregular basis as indicated by monitoring of immune response.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1 g, 100 ng to 100 mg, 1 .mu.g to 10 mg, or 30-300 .mu.g DNA per patient.

 

Claim 1 of 10 Claims

1. A method for treating multiple sclerosis in a patient, the method comprising administering to the patient intramuscularly an effective amount of a nucleic acid encoding osteopontin, whereby the nucleic acid is expressed in the patient to produce osteopontin, and the osteopontin induces an immune response wherein the immune response comprises formation of antibodies to osteopontin that reduces the level of osteopontin in the patient, thereby treating multiple sclerosis.

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