Methods and kits for treating inflammatory conditions are described that include modulating kallikrein 6 protease activity.

Description of the Invention

SUMMARY

The invention is based on the discovery that modulators of kallikrein 6 (K6) can alter pathogenesis of inflammatory cell mediated diseases both within the central nervous system (CNS) and in the periphery, and as a result, can aid in the treatment and prevention of inflammatory conditions such as MS, rheumatoid arthritis, lupus, and asthma. As described herein, an antibody having specific binding affinity for K6 reduced the degree of demyelination and reduced behavioral deficits in animal models of multiple sclerosis.

The invention features a method for treating an inflammatory condition in a mammal. The method includes administering to the mammal an amount of a K6 modulator effective to treat the inflammatory condition, and can further include monitoring the inflammatory condition in the mammal. The inflammatory condition can be selected from the group consisting of multiple sclerosis, rheumatoid arthritis, lupus, and asthma. The method is particularly useful for multiple sclerosis. The K6 modulator can be an antibody having specific binding affinity for K6. The antibody can be polyclonal or monoclonal, and can inhibit the enzyme activity of K6. The K6 modulator can be an antisense nucleic acid that inhibits the expression of K6. In some embodiments, the K6 modulator is a peptide nucleic acid that inhibits the expression of K6. The K6 modulator also can be a serine protease inhibitor.

In another aspect, the invention features an antibody that specifically binds to human K6 and inhibits the enzymatic activity of K6 and kits containing such an antibody. The antibody can be polyclonal or monoclonal. A kit further can include a label or package insert indicating that the antibody is useful for treating an inflammatory condition.

The invention also features a method for screening a subject for an inflammatory condition. The method includes detecting the level of K6 protein or RNA present in a biological sample from the subject; and comparing the level of K6 protein or RNA in the sample to the corresponding level in a control population, wherein an increase in the level of K6 protein or RNA in the subject relative to that of the control population is indicative of the inflammatory condition in the subject.

A method for monitoring therapy for an inflammatory condition also is featured. The method includes detecting the level of K6 protein or RNA present in a biological sample from a subject undergoing treatment for the inflammatory condition; and comparing the level of K6 protein or RNA in the sample to a baseline level of K6 present in the subject, wherein a decrease in the level of K6 protein or RNA in the subject relative to that of the control population is indicative of a positive response to the therapy in the subject. The inflammatory condition can be selected from the group consisting of multiple sclerosis, rheumatoid arthritis, lupus, and asthma. The biological sample can be selected from the group consisting of serum and cerebrospinal fluid.

The level of K6 protein can be detected immunologically. For example, the level of K6 protein can be detected using a monoclonal antibody. The level of K6 also can be detected using a capture antibody and a detection antibody, wherein the detection antibody includes a label (e.g., a fluorophore such as fluorescein, fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), or peridinin chlorophyll protein (PerCP); biotin; an enzyme; or a radioisotope. The capture antibody can be attached to a solid substrate (e.g., a bead or a microtiter plate). The capture antibody can be a polyclonal antibody.

In another aspect, the invention features an antisense oligonucleotide that inhibits the expression of K6, wherein the oligonucleotide is at least 8 nucleotides in length. The oligonucleotide can be at least 15 nucleotides in length.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION

In general, the invention provides methods for treating inflammatory conditions in mammals using modulators of K6 as well as methods of detecting the presence of an inflammatory condition and monitoring inflammatory disease state by detecting the level of K6 protein or a ribonucleic acid encoding K6 in biological samples from the mammals. The term "K6" as used herein refers to mammalian kallikrein 6 (e.g., from mice, rat, and humans). It should be noted that human K6 also is referred to as protease M, neurosin, zyme, and myelencephalon-specific protease (MSP). In the mouse, K6 also is referred to as brain and skin serine protease (BSSP) or brain serine protease (BSP). The nucleic acid sequence encoding human K6 can be found in GenBank under Accession Nos. AF013988, AF149289, and D78203. K6 is expressed in the CNS and within the CNS, is most abundant in the hippocampus, substantia nigra, basal ganglia, and spinal cord. K6 exhibits a limited distribution in non-neural tissues. Within normal white matter, K6 expression is almost exclusively associated with oligodendroglia. K6 levels are up-regulated in both neural and glial elements following injurious events, such as glutamate-receptor mediated excitotoxic injury.

Without being bound by a particular mechanism, K6 is localized within both macrophages and T cell subsets at sites of CNS inflammation and demyelination and can degrade myelin-specific and extracellular matrix proteins, and when present in excess, negatively effect oligodendrocyte process outgrowth and integrity. K6 may facilitate transendothelial migration of inflammatory cells into, and within, the CNS.

Methods of Treating Inflammatory Conditions

The term "inflammatory condition" as used herein refers to any inflammatory cell mediated disease within the CNS or within the periphery, including infectious (bacterial or viral) and autoimmune diseases. Non-limiting examples of inflammatory conditions affecting the nervous system include MS; all types of encephalitis and meningitis; acute disseminated encephalomyelitis; acute transverse myelitis; neuromyelitis optica; focal demyelinating syndromes (e.g., Balo's concentric sclerosis and Marburg variant of MS); progressive multifocal leukoencephalopathy; subacute sclerosing panencephalitis; acute haemorrhagic leucoencephalitis (Hurst's disease); human T-lymphotropic virus type-1-associated myelopathy/tropical spactic paraparesis; Devic's disease; human immunodeficiency virus encephalopathy; human immunodeficiency virus vacuolar myelopathy; peipheral neuropathies; Guillanin-Barre Syndrome and other immune mediated neuropathies; and myasthenia gravis. Non-limiting examples of non-nervous system inflammatory conditions include rheumatoid arthritis; osteoarthritis; infectious arthritis; psoriatic arthritis; polychondritis; periarticular disorders; colitis; pancreatitis; system lupus erythematosus; conjunctivitis; diabetes type II; dermatitis; asthma; systemic sclerosis (scleroderma); Sjogren's syndrome; Behcet's Syndrome; vasculitis sarcoidosis amyloidosis; allergies; anaphylaxis; systemic mastocytosis; and infectious diseases of the internal organs such as hepatitis or ulcers.

Typically, a K6 modulator is administered to a mammal such as a human patient that has been diagnosed with an inflammatory condition (e.g., MS). Suitable modulators can decrease the expression of a nucleic acid encoding K6, decrease levels of the K6 protein, or inhibit K6 activity. K6 modulators that can be used include, for example, antibodies having specific binding affinity for K6, antisense K6 molecules, selective serine protease inhibitors, and pharmaceutically acceptable salts thereof. K6 modulators also can be administered prophylactically in patients at risk for developing inflammatory conditions to prevent development of symptoms of the disease from occurring, delaying onset of symptoms, or lessening the severity of subsequently developed disease symptoms. As described herein, immunization with K6 in an autoimmune model of MS (experimental allergic encephalomyelitis (EAE) model) inhibited the development of clinical signs of EAE. In either case, an amount of a K6 modulator effective to treat the inflammatory condition is administered to the patient. Treatment of an inflammatory condition can include reducing the severity of the disease or slowing progression of the disease. As used herein, the term "effective amount" refers to an amount of a K6 modulator that reduces the deleterious effects of the inflammatory condition without inducing significant toxicity to the host. Effective amounts of K6 modulators can be determined by a physician, taking into account various factors that can modify the action of drugs such as overall health status, body weight, sex, diet, time and route of administration, other medications, and any other relevant clinical factors.

A K6 modulator can be administered by any route, including, without limitation, oral or parenteral routes of administration such as intravenous, intramuscular, intraperitoneal, subcutaneous, intrathecal, intraarterial, nasal, transdermal (e.g., as a patch), or pulmonary absorption. A K6 modulator can be formulated as, for example, a solution, suspension, or emulsion with pharmaceutically acceptable carriers or excipients suitable for the particular route of administration, including sterile aqueous or non-aqueous carriers. Aqueous carriers include, without limitation, water, alcohol, saline, and buffered solutions. Examples of non-aqueous carriers include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Preservatives, flavorings, sugars, and other additives such as antimicrobials, antioxidants, chelating agents, inert gases, and the like also may be present.

For oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods known in the art. Preparations for oral administration can also be formulated to give controlled release of the compound.

Nasal preparations can be presented in a liquid form or as a dry product. Nebulised aqueous suspensions or solutions can include carriers or excipients to adjust pH and/or tonicity.

In some embodiments, anti-inflammatory agents are administered in combination with a modulator of K6. For example, a non-steroidal anti-inflammatory agent such as acetaminophen, ibuprofen, or nabumetone or a steroid such as prednisolone can be administered to a subject. A modulator of K6 also can be administered with an immunomodulator such as interferon .beta. (e.g., Betaseron.RTM. (recombinant interferon .beta.-1.beta.) and Avonex.RTM. (recombinant interferon .beta.-1.alpha.): glatiramer acetate (Copaxone.RTM.) for relapsing-remitting MS; or Novantrone.RTM..

Methods of the invention can include monitoring the inflammatory condition to, for example, determine if the inflammatory condition is improving with treatment. Any method can be used to monitor an inflammatory condition. For example, for multiple sclerosis patients, lower extremity function, upper extremity function, vision, and cognitive function can be monitored. Magnetic resonance imaging (e.g., fluid-attenuated inversion recovery) can be performed to examine lesions and to differentiate old lesions from new or active lesions. Evoked potential tests can be performed to monitor nerve transmission. For example, visual evoked potentials can be used to detect optic neuritis. Brain stem auditory evoked potentials can be used to detect abnormalities in patients with demyelinating lesions in the brainstem that can cause delays in the transmission of sound. Somatosensory evoked potentials can be used to detect disruptions in the pathways from the arms and legs to the brain at very specific positions of the CNS. Cerebrospinal fluid can be examined for myelin breakdown products, oligoclonal bands, or IgG antibodies (e.g., IgG index). In addition, as discussed below, levels of K6 protein or ribonucleic acid (RNA) can be monitored.

Anti-K6 Antibodies

Antibodies having specific binding affinity for K6 can be used to modulate K6 (e.g., decrease activity). As used herein, the terms "antibody" or "antibodies" include intact molecules as well as fragments thereof that are capable of binding to an epitopic determinant of K6 (e.g., human K6). The term "epitope" refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes generally have at least five contiguous amino acids (a continuous epitope), or alternatively can be a set of noncontiguous amino acids that define a particular structure (e.g., a conformational epitope). The terms "antibody" and "antibodies" include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab).sub.2 fragments. Polyclonal antibodies are heterogenous populations of antibody molecules that are contained in the sera of the immunized animals. Monoclonal antibodies are homogeneous populations of antibodies to a particular epitope of an antigen.

Antibody fragments that have specific binding affinity for K6 can be generated by known techniques. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule; Fab fragments can be generated by reducing the disulfide bridges of F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed. See, for example, Huse et al., Science, 246:1275 (1989). Once produced, antibodies or fragments thereof are tested for recognition of K6 by standard immunoassay methods including ELISA techniques, radioimmunoassays, and Western blotting. See, Short Protocols in Molecular Biology, Chapter 11, Green Publishing Associates and John Wiley & Sons, Edited by Ausubel, F. M et al., 1992.

Antibodies having specific binding affinity for K6 can be produced through standard methods. In general, a K6 polypeptide can be recombinantly produced, or can be purified from a biological sample, and used to immunize animals. As used herein, the term "polypeptide" refers to a polypeptide of at least five amino acids in length. To produce a recombinant K6 polypeptide, a nucleic acid sequence encoding a K6 polypeptide can be ligated into an expression vector and used to transform a bacterial or eukaryotic host cell. Nucleic acid constructs typically include a regulatory sequence operably linked to a K6 nucleic acid sequence. Regulatory sequences do not typically encode a gene product, but instead affect the expression of the nucleic acid sequence. In bacterial systems, a strain of Escherichia coli such as BL-21 can be used. Suitable E. coli vectors include the pGEX series of vectors that produce fusion proteins with glutathione S-transferase (GST). Transformed E. coli are typically grown exponentially, then stimulated with isopropylthiogalactopyranoside (IPTG) prior to harvesting. In general, such fusion proteins are soluble and can be purified easily from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

Mammalian cell lines that stably express a K6 polypeptide can be produced by using expression vectors with the appropriate control elements and a selectable marker. For example, the eukaryotic expression vector pCDNA.3.1+ (Invitrogen, San Diego, Calif.) is suitable for expression of a K6 polypeptide in, for example, COS cells, Chinese hamster ovary (CHO), or HEK293 cells. Following introduction of the expression vector by electroporation, DEAE dextran, or other suitable method, stable cell lines are selected. Alternatively, K6 can be transcribed and translated in vitro using wheat germ extract or rabbit reticulocyte lysase.

In eukaryotic host cells, a number of viral-based expression systems can be utilized to express a K6 polypeptide. A nucleic acid encoding a K6 polypeptide can be introduced into a SV40, retroviral or vaccinia based viral vector and used to infect host cells. Alternatively, a nucleic acid encoding a K6 polypeptide can be cloned into, for example, a baculoviral vector and then used to transfect insect cells. For example, the cDNA encoding the sequence for the mature form of K6 can be inserted into the pBAC3 transfer vector (Novagen, Madison, Wis.) immediately 3' to the enterokinase (EK) recognition sequence of (Asp)4Lys. This results in a 44 amino acid synthetic prosequence (ending in the EK recognition sequence) leading into the amino-terminal Val-Val-His-Gly (SEQ ID NO:1) sequence of the mature form of K6. Expression of K6 in a pBAC3 transfer vector can use the BacVector transfection system (Novagen, Madison, Wis.). The Sf9 insect cell line, in conjunction with sf-900 II serum-free media (Life Technologies, Rockville, Md.), can be used for preparation of high-titer (i.e., >10.sup.9 pfu/mL) viral stock. The TN5 (High5, Invitrogen Corp., Carlsbad, Calif.) insect cell line can be used for production of expressed protein by the viral stock. Recombinant K6 protein can be purified in a single step utilizing the His-tag fusion and nickel affinity resin (Ni-NTA). The eluted K6 fraction can be pooled and extensively dialyzed versus 40 mM Tris-HCl, pH 7.5 (or 40 mM sodium acetate, pH 4.5), using 6-8 kDa molecular mass cutoff dialysis tubing (Spectrum Laboratories, Rancho Dominguez, Calif.).

Various host animals can be immunized by injection of the K6 polypeptide. Host animals include rabbits, chickens, mice, guinea pigs and rats. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Monoclonal antibodies can be prepared using a K6 polypeptide and standard hybridoma technology. In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described by Kohler, G. et al., Nature, 256:495 (1975), the human B-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72 (1983); Cole et al., Proc. Natl. Acad. Sci USA, 80:2026 (1983)), and the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc., pp. 77-96 (1983)). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the monoclonal antibodies of the invention can be cultivated in vitro and in vivo.

In some embodiments, antibodies of the invention can inhibit the enzymatic activity of K6. In vitro assays can be used to monitor K6 activity after incubation in the presence of an antibody. Typically, K6 can be incubated with an antibody (e.g, polyclonal or monoclonal), then the ability of K6 to cleave a substrate such as myelin basic protein or an arginine-specific fluorogenic substrate can be assessed at 37.degree. C. in a suitable buffer (e.g., Tris buffer). Depending on the substrate, cleavage can be monitored using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) or a spectrophotometer.

Antisense Oligonucleotides

Antisense oligonucleotides can be used to modulate K6 by decreasing levels of K6 protein. The antisense oligonucleotides in accordance with this invention are at least 8 nucleotides in length. For example, a nucleic acid can be about 8, 9, 10-20 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length), 15 to 20, 18-25, or 20-50 nucleotides in length. In other embodiments, antisense molecules can be used that are greater than 50 nucleotides in length, including the full-length sequence of a K6 mRNA. As used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogs thereof. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of a nucleic acid. Modifications at the base moiety include substitution of deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. Other examples of nucleobases that can be substituted for a natural base include 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other useful nucleobases include those disclosed, for example, in U.S. Pat. No. 3,687,808.

Modifications of the sugar moiety can include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone (e.g., an aminoethylglycine backbone) and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone. See, for example, U.S. Pat. Nos. 4,469,863, 5,235,033, 5,750,666, and 5,596,086 for methods of preparing oligonucleotides with modified backbones.

Antisense oligonucleotides of the invention also can be modified by chemical linkage to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties (e.g., a cholesterol moiety); cholic acid; a thioether moiety (e.g., hexyl-S-tritylthiol); a thiocholesterol moiety; an aliphatic chain (e.g., dodecandiol or undecyl residues); a phospholipid moiety (e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate); a polyamine or a polyethylene glycol chain; adamantane acetic acid; a palmityl moiety; or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. The preparation of such oligonucleotide conjugates is disclosed in, for example, U.S. Pat. Nos. 5,218,105 and 5,214,136.

Methods for synthesizing antisense oligonucleotides are known, including solid phase synthesis techniques. Equipment for such synthesis is commercially available from several vendors including, for example, Applied Biosystems (Foster City, Calif.). Alternatively, expression vectors that contain a regulatory element that directs production of an antisense transcript can be used to produce antisense molecules.

Antisense oligonucleotides can bind to a nucleic acid encoding K6, including DNA encoding K6 RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, under physiological conditions (i.e., physiological pH and ionic strength). The nucleic acid sequence encoding human K6 can be found in GenBank under Accession Nos. AF013988, AF149289, and D78203. The nucleic acid sequence encoding rat K6 can be found in GenBank under Accession No. AF016269. For example, an antisense oligonucleotide can hybridize under physiological conditions to the nucleotide sequence set forth in GenBank Accession Nos. AF013988 (FIG. 25; SEQ ID NO:2 (see Original Patent)), AF149289, D78203, or AF016269.

It is understood in the art that the sequence of an antisense oligonucleotide need not be 100% complementary to that of its target nucleic acid to be hybridizable under physiological conditions. Antisense oligonucleotides hybridize under physiological conditions when binding of the oligonucleotide to the K6 nucleic acid interferes with the normal function of the K6 nucleic acid, and non-specific binding to non-target sequences is minimal.

Target sites for K6 antisense oligonucleotides include the regions encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. In addition, the ORF has been targeted effectively in antisense technology, as have the 5' and 3' untranslated regions. Furthermore, antisense oligonucleotides have been successfully directed at intron regions and intron-exon junction regions. Further criteria can be applied to the design of antisense oligonucleotides. Such criteria are well known in the art, and are widely used, for example, in the design of oligonucleotide primers. These criteria include the lack of predicted secondary structure of a potential antisense oligonucleotide, an appropriate G and C nucleotide content (e.g., approximately 50%), and the absence of sequence motifs such as single nucleotide repeats (e.g., GGGG runs). The effectiveness of antisense oligonucleotides at modulating expression of a K6 nucleic acid can be evaluated by measuring levels of the K6 mRNA or protein (e.g., by Northern blotting, RT-PCR, Western blotting, ELISA, or immunohistochemical staining).

Identifying Modulators of K6

The invention provides methods for identifying K6 modulators that are suitable for treating one or more inflammatory conditions in mammals. In vitro or in vivo models of inflammatory conditions can be used to identify suitable modulators of K6. In vitro cell lines, including CG4 OLG cell line, or cultured explants or cultures (e.g., purified cultures of OLG progenitors) from an animal model, can be used to identify suitable K6 modulators. Such cells can be treated with a test compound over a period of time (e.g., days, weeks, or longer) then samples (e.g., cells and cell medium) can be collected and examined, for example, for OLG process stability and outgrowth. As a control, the effect of the test compound can be compared with cultures treated with a serine protease inhibitor (positive control) and to untreated cultures (negative control). Other assays for identifying K6 modulators include contacting immune cell cultures with a test compound and determining transmigration ability of the cells in vitro. See Example 16 for such an assay. In addition, K6 can be incubated with a test compound and ability to cleave a substrate can be monitored. See Example 15 for such an assay.

Once a test compound is determined to be effective in vitro, the test compound can be tested in vivo. For example, a test compound can be administered to an animal model of multiple sclerosis, such as the Theiler's murine encephalomyelitis virus (TMEV) model or the EAE model (autoimmune model). Samples (e.g., cerebrospinal fluid, blood, serum, or tissue) can be collected over a period of time and assayed. For example, cerebrospinal fluid can be assayed for myelin breakdown products. Spinal cord pathology can be examined for the degree of demyelination and inflammation. Animals also can be examined for behavioral deficits.

The invention provides methods for designing, modeling, and identifying compounds that can bind to K6 and inhibit K6 activity. Such compounds also can be referred to as "ligands" or "inhibitors." Compounds designed, modeled, and identified by methods of the invention typically have a binding affinity of at least 1 .mu.M (e.g., at least 500 nM, at least 100 nM, at least 50 nM, or at least 10 nM) for K6.

Compounds identified by methods of the invention can be polypeptides such as, for example, serine protease inhibitors or antibodies. Alternatively, a compound can be any suitable type of molecule that can specifically bind to K6.

By "modeling" is meant quantitative and/or qualitative analysis of K6-inhibitor structure/function based on three-dimensional structural information and K6-inhibitor interaction models. This includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure-based constraint models. Modeling typically is performed using a computer and may be further optimized using known methods.

Methods of designing ligands that bind specifically (i.e., with high affinity) to K6 typically are computer-based, and involve the use of a computer having a program capable of generating an atomic model. Computer programs that use X-ray crystallography data are particularly useful for designing ligands that can interact with K6. Programs such as RasMol, for example, can be used to generate a three-dimensional model of K6 and/or determine the structures involved in ligand binding. Computer programs such as INSIGHT (Accelrys, Burlington, Mass.), GRASP (Anthony Nicholls, Columbia University), Dock (Molecular Design Institute, University of California at San Francisco), and Auto-Dock (Accelrys) allow for further manipulation and the ability to introduce new structures.

Methods of the invention can include, for example, providing to a computer the atomic structural coordinates for amino acid residues within K6 or a portion of K6, using the computer to generate an atomic model of K6 or a portion of K6, further providing the atomic structural coordinates of a candidate compound and generating an atomic model of the compound optimally positioned to interact with K6, and identifying the candidate compound as a ligand of interest if the compound interacts with K6. By "optimally positioned" is meant positioned to optimize hydrophobic interactions between the candidate compound and K6.

Alternatively, a method for designing a ligand having specific binding affinity for K6 can utilize a computer with an atomic model stored in its memory. The atomic coordinates of a candidate compound then can be provided to the computer, and an atomic model of the candidate compound optimally positioned can be generated.

Compounds of the invention also may be interactively designed from structural information of the compounds described herein using other structure-based design/modeling techniques (see, e.g., Jackson (1997) Seminars in Oncology 24:L164-172; and Jones et al. (1996) J. Med. Chem. 39:904-917).

Compounds and polypeptides of the invention also can be identified by, for example, identifying candidate compounds by computer modeling as interacting spatially and preferentially (i.e., with high affinity) with K6, and then screening those compounds in vitro or in vivo for the ability to reduce K6 activity or decrease inflammation and/or demyelination. Suitable methods for such in vitro and in vivo screening include those described herein.

Methods of Using K6 as a Marker for Inflammatory Conditions

Levels of K6 protein or RNA can be used to monitor therapy of inflammatory conditions, screen for the presence of an inflammatory condition, or to monitor the disease state (e.g., relapses of MS). In general, methods of the invention include detecting the level of K6 protein or a RNA encoding K6 in a biological sample from a patient (e.g., a human patient) and comparing the level of K6 protein or RNA to that from a control population (e.g., the average level of K6 from a plurality of subjects without an inflammatory condition). Methods for detecting levels of K6 protein and RNA are described below. Suitable biological samples for measuring K6 levels include, for example, blood (including whole blood, plasma, and serum), urine, and cerebrospinal fluid (CSF). Serum and CSF are particularly useful biological samples.

The presence of an inflammatory condition can be determined based on the level of K6 protein or RNA relative to the control population. Thus, it is determined if K6 protein or RNA levels are increased, decreased, or the same as that of the control population. An increase in K6 levels relative to that of the control population is indicative of an inflammatory condition. Additional factors that can be considered when diagnosing an inflammatory condition include, for example, patient history, family history, genetic factors, and/or altered neurologic examination (e.g., for MS or other neurological inflammatory condition).

The levels of K6 protein or RNA in a subject also can be used to monitor treatment. Typically, the subject's baseline level of K6 protein or RNA is obtained (e.g., before treatment) and compared to the level of K6 at various time points after or between treatments (e.g., one or more days, weeks, or months after treatment). A decrease in K6 protein or RNA levels relative to the baseline level is indicative of a positive response to treatment. Similarly, disease state in a subject can be monitored (e.g., for relapse of disease) by comparing levels of K6 protein or RNA in the subject to the subject's baseline level of K6 protein or RNA.

Detecting K6 Protein

K6 can be detected, for example, immunologically using one or more antibodies. In immunological assays, an antibody having specific binding affinity for K6 or a secondary antibody that binds to such an antibody can be labeled, either directly or indirectly. Suitable labels include, without limitation, radionuclides (e.g., .sup.125I, .sup.131I, .sup.35S, .sup.3H, .sup.32P, .sup.33P, or .sup.14C), fluorescent moieties (e.g., fluorescein, FITC, PerCP, rhodamine, or PE), luminescent moieties (e.g., Qdot.TM. nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.), compounds that absorb light of a defined wavelength, or enzymes (e.g., alkaline phosphatase or horseradish peroxidase). Antibodies can be indirectly labeled by conjugation with biotin then detected with avidin or streptavidin labeled with a molecule described above. Methods of detecting or quantifying a label depend on the nature of the label and are known in the art. Examples of detectors include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers. Combinations of these approaches (including "multi-layer" assays) familiar to those in the art can be used to enhance the sensitivity of assays.

Immunological assays for detecting K6 can be performed in a variety of known formats, including sandwich assays, competition assays (competitive RIA), or bridge immunoassays. See, for example, U.S. Pat. Nos. 5,296,347; 4,233,402; 4,098,876; and 4,034,074. Methods of detecting K6 generally include contacting a biological sample with an antibody that binds to K6 and detecting binding of K6 to the antibody. For example, an antibody having specific binding affinity for K6 can be immobilized on a solid substrate by any of a variety of methods known in the art and then exposed to the biological sample. Binding of K6 to the antibody on the solid substrate can be detected by exploiting the phenomenon of surface plasmon resonance, which results in a change in the intensity of surface plasmon resonance upon binding that can be detected qualitatively or quantitatively by an appropriate instrument, e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden). Alternatively, the antibody can be labeled and detected as described above. A standard curve using known quantities of K6 can be generated to aid in the quantitation of K6 levels.

In other embodiments, a "sandwich" assay in which a capture antibody is immobilized on a solid substrate is used to detect the level of K6. The solid substrate can be contacted with the biological sample such that any K6 in the sample can bind to the immobilized antibody. The level of K6 bound to the antibody can be determined using a "detection" antibody having specific binding affinity for K6 and the methods described above. It is understood that in these sandwich assays, the capture antibody should not bind to the same epitope (or range of epitopes in the case of a polyclonal antibody) as the detection antibody. Thus, if a monoclonal antibody is used as a capture antibody, the detection antibody can be another monoclonal antibody that binds to an epitope that is either completely physically separated from or only partially overlaps with the epitope to which the capture monoclonal antibody binds, or a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture monoclonal antibody binds. If a polyclonal antibody is used as a capture antibody, the detection antibody can be either a monoclonal antibody that binds to an epitope that is either completely physically separated from or partially overlaps with any of the epitopes to which the capture polyclonal antibody binds, or a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture polyclonal antibody binds. Sandwich assays can be performed as sandwich ELISA assays, sandwich Western blotting assays, or sandwich immunomagnetic detection assays.

Suitable solid substrates to which an antibody (e.g., a capture antibody) can be bound include, without limitation, microtiter plates, tubes, membranes such as nylon or nitrocellulose membranes, and beads or particles (e.g., agarose, cellulose, glass, polystyrene, polyacrylamide, magnetic, or magnetizable beads or particles). Magnetic or magnetizable particles can be particularly useful when an automated immunoassay system is used.

Alternative techniques for detecting K6 include mass-spectrophotometric techniques such as electrospray ionization (ESI), and matrix-assisted laser desorption-ionization (MALDI). See, for example, Gevaert et al., Electrophoresis 22(9):1645-51, 2001; Chaurand et al., J Am Soc Mass Spectrom 10(2):91-103, 1999. Mass spectrometers useful for such applications are available from Applied Biosystems (Foster City, Calif.); Bruker Daltronics (Billerica, Mass.) and Amersham Pharmacia (Sunnyvale, Calif.).

Detecting K6 Ribonucleic Acid

K6 RNA can be detected, for example, by polymerase chain reaction (PCR) assays or RNA blotting techniques (e.g., Northern blotting). For example, K6 RNA can be detected in peripheral blood mononuclear cells. In general, PCR refers to amplification of a target nucleic acid, using sequence information from the ends of the region of interest or beyond to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Primers are typically 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. PCR is described, for example in PCR Primer: A Laboratory Manual. Ed. by Dieffenbach, C. and Dveksler, G., Cold Spring Harbor Laboratory Press, 1995. Nucleic acids also can be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification. See, for example, Lewis, R., Genetic Engineering News, 12(9):1 (1992); Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874-1878 (1990); and Weiss, R., Science, 254:1292 (1991).

For example, the levels of K6 mRNA can be detected using reverse transcription-polymerase chain reaction (RT-PCR). Real-time quantitative PCR can be performed using, for example, the ABI PRISM 7700 Sequence Detection System and Taqman fluorogenic probes, or the LightCycler.TM. instrument from Roche.

Articles of Manufacture

Antibodies having specific binding affinity for K6 can be combined with packaging material and sold as a kit for detecting K6 from biological samples, treating inflammatory conditions, monitoring therapy of inflammatory conditions, or monitoring disease relapses (e.g., of MS). Antisense oligonucleotides that inhibit expression of K6 also can be combined with packaging material and sold as a kit for treating inflammatory conditions. Components and methods for producing articles of manufactures are well known. The articles of manufacture may combine one or more anti-K6 antibodies or fragments thereof or one or more antisense oligonucleotides as described herein. In addition, the articles of manufacture may further include reagents such as secondary antibodies, buffers, indicator molecules, solid phases (e.g., beads), additional anti-inflammatory agents, and/or other useful reagents for detecting K5 from biological samples, treating inflammatory conditions, monitoring therapy of inflammatory conditions, or monitoring disease relapses. The anti-K6 antibody or antisense oligonucleotide can be in a container, such as a plastic, polyethylene, polypropylene, ethylene, or propylene vessel that is either a capped tube or a bottle. In some embodiments, the anti-K6 antibody can be included on a solid phase such as a handheld device for bedside testing. Instructions describing how the various reagents are effective for treating inflammatory conditions, monitoring therapy of inflammatory conditions, or monitoring disease relapses also may be included in such kits.

Claim 1 of 4 Claims

1. A method for treating multiple sclerosis in a mammal, said method comprising administering to said mammal an amount of an antibody having specific binding affinity for kallikrein 6 (K6) effective to treat said multiple sclerosis, wherein said antibody inhibits the enzyme activity of K6.

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