JP5041464B2 - Method for treating cytokine-mediated diseases - Google Patents

Description

  The present invention relates to a therapeutic composition and a therapeutic method for cytokine-mediated diseases by suppressing the interaction between Sema7A and VLA-1.

  The semaphorin family consists of a number of systematically conserved soluble and transmembrane proteins, many of which play diverse roles in axon guidance, organogenesis, angiogenesis, angiogenesis, tumorigenesis and immune responses ( 1-3). Sema7A, whose expression is observed in both the nervous system and the immune system, is the only GPI-anchored semaphorin, which was identified in studies on vertebrate homologues of semaphorins that were initially encoded by the virus. Although Sema7A has been shown to bind plexin-C1 (5), it has recently been suggested that Sema7A promotes axon outgrowth by β1-integrin in a plexin-C1-dependent manner (6). However, the manner and extent to which Sema7A is involved in the immune response remains unclear.

Therefore, an object of the present invention is to provide a method for treating inflammatory diseases by administering to a patient a composition that inhibits Sema7A-VLA-1 interaction.
Still another object of the present invention is to (1) contact a cell containing Sema7A protein and VLA-1 protein with a putative regulatory compound, and (2) inhibit Sema7A interaction with VLA-1 It is to provide a method for identifying a compound that controls the interaction of Sema7A with VLA-1 activity in a cell, characterized by evaluating the ability of a putative regulatory compound.
Yet another object of the present invention is to provide a composition that controls the interaction of Sema7A with VLA-1 activity in cells, which composition is therapeutically beneficial for treating inflammatory diseases. .

In a first general embodiment, a method of treating a cytokine-mediated disease is provided by administering to a patient a composition that inhibits the interaction between Sema7A and VLA-1.
We have identified that Sema7A, which is expressed on the cell surface of activated T cells, is critically involved in human monocyte activation. As shown in FIG. 1, mouse Sema7A (Sema7A-Fc) fused with the Fc portion of human IgG1 induced the production of inflammatory cytokines not only in mouse bone marrow-derived macrophages but also in human monocytes. Sema7A-Fc also enhanced LPS-induced production of cytokines in these cells (amino acid identity between mouse and human Sema7A is 90%). In addition, Sema7A binding to human monocytes was inhibited by soluble α1β1-integrin as well as anti-β1Ab and / or anti-α1Ab (FIG. 2). Furthermore, the production of IL-6 induced by Sema7A was significantly blocked by Ab (FIG. 2). Taken together, our findings not only indicate a role for Sema7A in the immune system, but also identify the involvement of α1β1-integrin (VLA-1) as a receptor for Sema7A in monocyte activation. Therefore, the interaction between Sema7A and α1β1-integrin (VLA-1) may be a potential therapeutic target for various inflammatory diseases.
FIG. 1 shows that Sema7A is a potent monocyte stimulator. (A) and (B) Human monocytes or mouse bone marrow derived macrophages were cultured on Sema7A-Fc coated plates or human IgG1 coated plates with or without LPS (100 ng / ml) for 24 hours. Production of the indicated cytokines in the culture supernatant was measured by ELISA. The result was the average of 3 replicate measurements.
Method: Human monocytes were isolated from peripheral blood of volunteers by using RosseteSep Human Monocyte Enrichment Cocktail (Stem Cell Technologies). Mouse bone marrow derived macrophages were obtained from 5 day cultures of bone marrow cells in the presence of G-CSF (50 ng / ml; peprotech). Cells were plated on flat bottom 96 well plates coated with the indicated concentrations of Sema7A-Fc or human IgG1 (1 × 10 ⁵ cells / well in 200 μl RPMI 1640 medium supplemented with 10% FCS) and incubated for 24 hours. The concentration of cytokine in the culture supernatant was measured by ELISA kit (R & D Systems).

FIG. 2 shows that α1β1 integrin / VLA-1 is a functional receptor for Sema7A.
(A) Attachment of THP-1 cells, a human monocytic cell line, to Sema7A coated plates was blocked with both anti-β1 integrin Ab and anti-α1 integrin Ab. THP-1 cells were preincubated with the indicated antibody (25 μg / ml) and subjected to an adhesion assay. The number of cells attached to Sema7A coated wells was quantified as previously described (6,7).
(B) THP-1 cell attachment to immobilized Sema7A was inhibited in the presence of soluble α1β1 integrin protein. Sema7A coated wells were preincubated with the indicated soluble integrin protein, after which THP-1 cells were applied.
(C) Sema7A-induced cytokine production by human monocytes was suppressed by anti-β1 integrin Ab and anti-α1 integrin Ab. Human monocytes were pretreated with 10 μg / ml of the indicated Ab and then inoculated into Sema7A coated plates. The production of IL-6 in the culture supernatant was measured by ELISA.
Method: Tissue culture plates (96 wells, suspension culture treatment, Sumilon) are coated overnight with 100 μl / well of 10 nM fusion protein solution, followed by blocking with 10 mg / ml BSA in PBS buffer (200 μl / well) did. Cells are added at 5 × 10 ⁴ cells / well in 200 μl of Tyrode's buffer (135 mM NaCl, 5.4 mM KCl, 1.0 mM MgCl ₂ , 5 mM NaOH-Hepes (pH 7.4), 10 mM glucose, 10 mg / ml BSA) at room temperature For 1 hour. The wells were then washed 3 times in pre-warmed PBS buffer. The number of adherent cells was determined by CyQUANT cell proliferation assay kit (molecular probe). For the antibody blocking assay, THP-1 cells were incubated with 25 μg / ml of the indicated anti-integrin antibody in Tyrode's buffer for 30 minutes on ice. The soluble form of the integrin protein was used for pretreatment of Sema7A-Fc coated wells at 1 μM in Tyrode's buffer and the plates were incubated for 30 minutes at room temperature.

One embodiment of the invention is (1) contacting a cell with a putative regulatory compound (the cell comprises Sema7A protein and VLA-1 protein) and (2) inhibiting Sema7A interaction with VLA-1 The present invention relates to a method for identifying a compound that controls the interaction of Sema7A with VLA-1 activity in a cell, characterized by evaluating the ability of the putative regulatory compound. The evaluation step preferably involves studying the attachment of THP-1 cells to Sema7A or an equivalent method known in the art.
The term “modulate” refers to controlling the activity and / or biological function of a molecule, eg, enhancing or decreasing such activity or function.
The term “patient” includes both human and non-human mammals.
The term “treat” or “treatment” means treatment of a patient's condition,
(i) prevent such symptoms from occurring in patients, especially if such patients are genetically or otherwise foreseen for the symptoms but have not yet been diagnosed as having it;
(ii) suppress or ameliorate the patient's symptoms, i.e. stop or delay its occurrence, or
(iii) alleviate the patient's symptoms, i.e., cause regression or cure of symptoms.
Yet another embodiment of the invention relates to an antibody or antibody binding site that binds Sema7A, VLA-1, or a fragment thereof. Embodiments of the present invention further include polyclonal and monoclonal antibodies. Preferred embodiments of the invention include monoclonal antibodies, such as anti-VLA-1 monoclonal antibodies. The antibody or antibody binding site that binds Sema7A or VLA-1 suppresses Sema7A binding by VLA-1.
Yet another embodiment of the present invention relates to a biotherapeutic agent comprising Sema7A or VLA-1 protein or fragments thereof, which biotherapeutic agent is useful for treating inflammatory diseases.

The term “composition” as referred to herein encodes a putative compound or a substantially pure protein selected from Sema7A and VLA-1 or fragments thereof, Sema7A, VLA-1 or fragments thereof An antibody or antibody binding site that binds Sema7A and VLA-1 or a fragment thereof to an expression vector, a fusion protein comprising Sema7A, VLA-1 or a fragment thereof. In an antibody binding site embodiment, the antibody binding site may be specifically immunoreactive with a mature protein selected from the group consisting of Sema7A and VLA-1; purified or recombinantly produced human or mouse May be produced against Sema7A or VLA-1; may be in a monoclonal antibody, Fab, or F (Ab) 2; may be immunoreactive with denatured antigen; or labeled antibody May be inside. In some embodiments, the antibody binding site contacts a binding agent having affinity for Sema7A or VLA-1 with the biological sample; the binding agent is incubated with the biological sample and the binding agent: Sema7A or VLA-1 protein A complex is produced; detected in a biological sample by a method that detects the complex. In a preferred embodiment, the biological sample is human and the binding agent is an antibody.
The putative compounds referred to herein include, for example, compounds that are the product of rational drug design, natural products, and compounds that have partially specified signal transduction modulating properties. Putative compounds are protein based compounds, carbohydrate based compounds, lipid based compounds, nucleic acid based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotype antibodies and / or It may be a catalytic antibody or a fragment thereof. A putative regulatory compound is, for example, a library of natural or synthetic compounds, in particular a chemical library or a combinatorial library (ie a library of compounds of different sequence or size but having the same building blocks; eg Rutter And Santi, U.S. Pat. Nos. 5,010,175 and 5,266,684, which are incorporated herein by reference in their entirety, or by rational drug design.

In rational drug design operations, the three-dimensional structure of a compound, such as a signaling molecule, can be analyzed by, for example, nuclear magnetic resonance (NMR) or X-ray crystallography. This three-dimensional structure can then be used to predict the structure of potential compounds, such as putative regulatory compounds by computer modeling, for example. The predicted compound structure can then be generated, for example, by chemical synthesis, recombinant DNA techniques, or by isolating mimetopes from natural sources (eg, plants, animals, bacteria and molds). Potential modulatory compounds also use SELEX technology as described, for example, in PCT Publication Nos. WO 91/19813; WO 92/02536 and WO 93/03172, which are hereby incorporated by reference in their entirety. Can be identified.
In particular, naturally occurring intracellular signaling molecules can be modified based on analysis of their structure and function to produce suitable regulatory compounds. For example, compounds capable of modulating Sema7A or VLA-1 can include compounds having a structure similar to the amino acid residues in their respective binding domains. Such compounds can include peptides, polypeptides or small organic molecules.
Putative regulatory compounds can also include molecules designed to interfere with Sema7A or VLA-1. For example, mutants of VLA-1 are created that interfere with protein coupling with Sema7A. Putative modulatory compounds can include agonists and antagonists of Sema7A or VLA-1. Such agonists and antagonists are selected based on the structure of naturally occurring ligands to these proteins.
Techniques for generating monoclonal antibodies are known. In general, immortal cell lines (typically myeloma cells) are fused to lymphocytes (typically spleen cells) from mammals immunized with whole cells expressing a given antigen, eg, Sema7A or VLA-1. The resulting culture supernatant of hybridoma cells is screened for antibodies to the antigen. See generally Kohler et al., 1975, Nature 265: 295-497, “Continuous cultures of fusion cells secreting antibodies of a previously specified specificity”.

Immunization can be accomplished using routine procedures. The unit dose and immunization regimen will depend on the species of mammal being immunized, its immunity, the weight of the mammal, etc. Typically, the immunized mammal is bled and the serum from each blood sample is assayed for a particular antibody using an appropriate screening assay. For example, anti-integrin antibodies can be identified by immunoprecipitation of ¹²⁵ I-labeled cell lysates from integrin-expressing cells. For example, antibodies, including anti-VLA-1 antibodies, can also be identified by flow cytometry, for example, by measuring fluorescent staining of antibody-expressing cells incubated with antibodies suspected of recognizing VLA-1 molecules. Lymphocytes used for the generation of hybridoma cells are typically isolated from immunized mammals whose serum has already been tested positive for the presence of anti-VLA-1 antibodies using such screening assays.
Typically, immortal cell lines (eg, myeloma cell lines) are derived from the same mammalian species as the lymphocytes. A preferred immortal cell line is a mouse myeloma cell line that is sensitive to medium containing hypoxanthine, arninopterin and thymidine (“HAT medium”). Typically, HAT-sensitive mouse myeloma cells are fused to mouse spleen cells using 1500 molecular weight polyethylene glycol (“PEG1500”). The hybridoma cells resulting from the fusion are then selected using HAT medium (which kills unfused and non-proliferating fused myeloma cells) (unfused spleen cells die after a few days. Because they are not transformed). Hybridomas producing the desired antibody are detected by screening the hybridoma culture supernatant. For example, hybridomas prepared to produce anti-VLA-1 antibodies may be screened by testing hybridoma culture supernatants for secreted antibodies that have the ability to bind to recombinant VLA-1 expressing cell lines.

For example, hybridoma cells that have been tested positive in such a screening assay to produce antibody homologs within the scope of the present invention, including anti-VLA-1 antibody homologs that are intact immunoglobulins, The cells were cultured in nutrient medium for conditions and for a time sufficient to allow the cells to secrete monoclonal antibodies into the medium. Tissue culture techniques and media suitable for hybridoma cells are known. The adjusted hybridoma culture supernatant may be collected, and the anti-VLA-1 antibody may be further purified by a known method if necessary.
Alternatively, the desired antibody may be generated by injecting hybridoma cells into the peritoneal cavity of an unimmunized mouse. Hybridoma cells grow in the peritoneal cavity and secrete antibodies that accumulate as ascites. The antibody may be recovered by removing ascites from the abdominal cavity with a syringe.
For example, a fully human monoclonal antibody homologue of Sema7A or VLA-1 is another preferred binding agent that can block antigens in the methods of the invention. In their intact form, these are as described in Boerner et al., 1991, J. Immunol. 147: 86-95, “Generation of antigen-specific human monoclonal antibodies from human spleen cells sensitized in vitro”. It may be prepared using human spleen cells sensitized in vitro.
They are also described in Persson et al., 1991, Proc. Nat. Acad. Sci. USA 88: 2432-2436, “Generation of diverse high affinity human monoclonal antibodies by repertoire cloning” and Huang and Stollar, 1991, J. Immunol. Methods. 141: 227-236, “Construction of a representative immunoglobulin variable region cDNA library from human peripheral blood lymphocytes without in vitro stimulation”. US Pat. No. 5,798,230 (August 25, 1998, “Methods for preparing human monoclonal antibodies and their use”) describes the preparation of human monoclonal antibodies from human B cells. According to this method, human antibody-producing B cells are immortalized by infection with Epstein-Barr virus, or Epstein-Barr virus nuclear antigen 2 (EBNA2) derivatives thereof. EBNA2 function (which is required for immortalization) is subsequently stopped, leading to increased antibody production.

In yet another method for generating fully human antibodies, US Pat. No. 5,789,650 (August 4, 1998, “Transgenic non-human animals to generate heterologous antibodies”) can generate heterologous antibodies. Described are transgenic non-human animals that can be produced and transgenic non-human animals that have inactivated endogenous immunoglobulin genes. Endogenous immunoglobulin genes are repressed by anti-sense polynucleotides and / or antisera induced against endogenous immunoglobulins. Heterologous antibodies are encoded by immunoglobulin genes not normally found in the genome of that species of non-human animals. One or more transgenes containing sequences of untranslated heterologous human immunoglobulin heavy chains are introduced into a non-human animal, thereby functionally translocating the transgenic immunoglobulin sequences, and various isoforms encoded by the human immunoglobulin genes. Transgenic animals are formed that are capable of producing a repertoire of types of antibodies. Such heterologous human antibodies are produced in B cells, after which they are heterologous by fusing with an immortal cell line such as, for example, myeloma, or by manipulating such B cells by other techniques. It is immortalized by perpetuating cell lines that are capable of producing fully human monoclonal antibody homologs.
The conditions under which the cells of the invention are contacted with a putative regulatory compound, for example by mixing, are such that the cell exhibits Sema7A or VLA-1 activity in the absence of other regulatory compounds that interfere with such activity. This is a condition that can be shown. Obtaining such conditions is within the purview of those skilled in the art and includes effective media in which the cells can be cultured such that the cells can exhibit Sema7A or VLA-1 activity. For example, for mammalian cells, an effective medium is an aqueous medium typically containing RPMI 1640 medium containing 10% fetal calf serum.
The cells of the present invention can be cultured in a variety of vessels including, but not limited to, tissue culture flasks, test tubes, microtiter dishes, and petri dishes. Culturing is performed at a temperature, pH and carbon dioxide content suitable for the cells. Such culture conditions are also within the skill of the art. For example, for Ramos cells, culturing can be performed at 37 ° C. in a 5% CO ₂ environment.

The protocols allowed to contact cells with the putative regulatory compound in an effective manner are the number of cells per container contacted, the concentration of one or more putative regulatory compounds administered to the cell, the putative estimate with the cell. It includes the incubation time of the modulating compound, the concentration of the ligand and / or intracellular initiator molecule administered to the cell, and the incubation time of the ligand and / or intracellular initiator molecule with the cell. The determination of such a protocol is based on variables such as the size of the container, the volume of liquid in the container, the type of cell being tested, and the chemical composition (ie, size, charge, etc.) of the putative regulatory compound being tested. Can be done by a vendor.
In one embodiment of the method of the present invention, a suitable number of cells are added to a 96 well tissue culture dish in medium. The preferred number of cells includes the number of cells that allows detecting changes in VLA-1 or Sema7A activity using the detection methods of the invention (described in detail below). A more preferred number of cells comprises about 1-1 × 10 ⁶ cells per well of a 96 well tissue culture dish. After adding the cells to the tissue culture dish, the cells can be preincubated at 37 ° C. with 5% CO ₂ for about 0 hours to about 24 hours.
A suitable amount of one or more putative regulatory compounds suspended in the medium is added to the cells that are sufficient to modulate the activity of Sema7A or VLA-1 protein in the cells, so that regulation is achieved according to the present invention. The detection method can be used. Preferred amounts of the putative one or more modulatory compounds comprise from about 1 nM to about 10 mM of the putative one or more modulatory compounds per well of a 96 well plate. The cells are incubated for a length of time suitable to allow the putative regulatory compound to enter the cell and interact with the Sema7A or VLA-1 protein. A preferred incubation time is from about 1 minute to about 48 hours.
In another embodiment of the method of the present invention, cells suitable for use in the present invention are stimulated with a stimulating substance capable of binding to the Sema7A or VLA-1 protein of the present invention to initiate a signal transduction pathway, and a cellular response Produce. It is preferred that the cell is stimulated with a stimulatory molecule after contacting the putative regulatory compound with the cell. Suitable stimulating molecules include, for example, antibodies that specifically bind to Sema7A or VLA-1 protein. The amount of stimulatory molecule suitable for addition to the cell depends on factors such as the type of ligand used (eg, monomer or multimer; permeability, etc.) and the amount of Sema7A or VLA-1 protein present. Preferably, about 1.0 nM to about 1 mM of ligand is added to the cell.

The methods of the invention include determining whether the composition can modulate Sema7A or VLA-1 protein activation. Such methods include the assays detailed in the methods section. The method of the present invention may further comprise the step of performing a toxicity test to determine the toxicity of the composition.
Another aspect of the invention includes a kit for identifying a composition capable of modulating Sema7A or VLA-1 protein activity in a cell. Such kits include (1) cells containing Sema7A or VLA-1 protein, and (2) means for detecting modulation of Sema7A or VLA-1 protein. Such means for detecting modulation of Sema7A or VLA-1 protein include methods and reagents known to those skilled in the art, eg, VLA-1 protein activity, eg, activity described below Can be detected using an assay. Means for detecting modulation of Sema7A or VLA-1 protein also include methods and reagents known to those skilled in the art. Suitable cells for use with the kits of the invention include the cells described in detail herein. Preferred cells for use with the kit include human cells.

Methods of therapeutic use As noted above, the inventors have found that Sema7A has a role in the immune system and that α1β1-integrin (VLA-1) as a receptor for Sema7A in monocyte activation. The involvement was identified. Therefore, the interaction between Sema7A and α1β1-integrin (VLA-1) would be a potential therapeutic target for various diseases mediated by cytokines, such as inflammatory diseases.
Therefore, the present invention provides a method for treating cytokine-mediated diseases by administering to a patient a composition that inhibits Sema7A and VLA-1 interaction.
A composition that blocks the interaction between Sema7A and VLA-1 will block the production of inflammatory cytokines from the cells. Inhibition of cytokine production is an attractive means to prevent and treat various cytokine-mediated diseases or conditions associated with excessive cytokine production, for example, diseases and pathological conditions involving inflammation, autoimmune response or bone resorption. is there. Thus, the composition is useful for the treatment of diseases and conditions including:
Osteoarthritis, atherosclerosis, contact dermatitis, bone resorption disease including osteoporosis, reperfusion injury, asthma, multiple sclerosis, Guillain-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft versus host Disease, systemic lupus erythematosus and insulin-dependent diabetes mellitus, rheumatoid arthritis, toxic shock syndrome, Alzheimer's disease, diabetes, inflammatory bowel disease, acute and chronic pain, as well as symptoms of inflammation and cardiovascular disease, stroke, myocardial infarction Heat damage, adult respiratory distress syndrome (ARDS), multiple organ damage secondary to trauma, acute nephritis, dermatitis due to acute inflammatory components, acute suppurative meningitis or other centers Nervous system disorders, syndromes associated with hemodialysis, leukopherisis, granulocyte infusion-related syndromes, and necrotizing enterocolitis, reperfusion after percutaneous transluminal coronary angioplasty Complications including stenosis, traumatic arthritis, sepsis, chronic obstructive pulmonary disease and congestive heart failure. The composition may also be beneficial for anticoagulants or thrombolytic agents (and diseases or conditions associated with such treatment).
Anti-cytokine activity can be demonstrated by using methods known in the art. See, for example, Branger et al. (2002) J Immunol. 168: 4070-4077, and 46 references cited therein, each of which is incorporated herein by reference in its entirety.
The compositions of the present invention may also be useful for treating cancer diseases. These diseases include solid tumors such as breast cancer, respiratory tract cancer, brain cancer, genital cancer, digestive tract cancer, urethral cancer, eye cancer, liver cancer, skin cancer, head and neck cancer. Thyroid cancer, parathyroid cancer and their distant metastases, but are not limited to. These disorders also include lymphoma, sarcoma, and leukemia.

Examples of breast cancer include, but are not limited to, erosive ductal carcinoma, eroded lobular carcinoma, in situ ductal carcinoma, and in situ lobular carcinoma.
Examples of respiratory tract cancers include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma and mesothelioma.
Examples of brain cancer include brain stem, eye and subglial glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as pituitary, neuroectodermal and pineal tumors However, it is not limited to these.
Examples of peripheral nervous system tumors include, but are not limited to, neuroblastoma, ganglion neuroblastoma, and peripheral nerve sheath tumor.
Examples of endocrine and exocrine tumors include, but are not limited to, thyroid cancer, adrenocortical carcinoma, pheochromocytoma, and carcinoma.
Male genital tumors include, but are not limited to, prostate cancer and testicular cancer.
Female genital tumors include, but are not limited to, uterine sarcomas, as well as cancers of the endometrium, cervix, ovary, vagina, and vulva.
Digestive tract tumors include, but are not limited to, anus, colon, colorectal, esophagus, gallbladder, stomach, pancreas, rectum, small intestine, and salivary gland cancer.
Urethral tumors include, but are not limited to, bladder, penis, kidney, kidney disc, ureter, and urethral cancer.
Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.
Examples of liver cancer include hepatocellular carcinoma (hepatocellular carcinoma with or without a fiber layer variant), hepatoblastoma, cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma, It is not limited to these.

Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
Head and neck cancers include, but are not limited to, laryngeal / hypopharyngeal / nasopharyngeal / oropharyngeal cancer, and lip cancer and oral cancer.
Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma, and central nervous system lymphoma.
Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, Ewing sarcoma, malignant fibrous histiocytoma, lymphosarcoma, angiosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
Plasma cell cachexia includes, but is not limited to, multiple myeloma and Waldenstrom macroglobulinemia.
These disorders have been well characterized in males, but also exist in other mammals with similar etiology and can be treated with the pharmaceutical compositions of the present invention.
For therapeutic use, the composition may be administered in any conventional dosage form in any conventional manner. Routes of administration include, but are not limited to, intravenous, intramuscular, subcutaneous, intrabursal, infusion, sublingual, transdermal, oral, topical or inhalation. The preferred modes of administration are oral and intravenous.
The compositions may be administered alone or increase the stability of inhibitors, facilitate the administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, and increase inhibitory activity. May be administered in combination with an adjuvant (including other active ingredients) such as providing adjuvant treatment. Advantageously, such combination therapies utilize low doses of conventional therapeutics, thus avoiding possible toxicity and adverse side effects that are induced when these drugs are used as a single therapeutic. The composition may be physically combined with conventional therapeutic agents or other adjuvants into a single pharmaceutical composition. Advantageously, the compositions may then be administered together in a single dosage form. In certain embodiments, a pharmaceutical composition comprising such a combination of compositions comprises at least about 5% (w / w), more preferably at least about 20% of the composition or combination thereof. The optimum% (w / w) of the composition of the present invention may vary and is within the skill of the art. The compositions may also be administered separately (sequentially or in parallel). Separate dosing allows for greater flexibility in dosing regimens.

As noted above, the dosage forms of the compositions described herein include a pharmaceutically acceptable carrier and an adjuvant known to those skilled in the art. These carriers and adjuvants include, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, buffer substances, water, salts or electrolytes and cellulose based substances. Preferred dosage forms include tablets, capsules, caplets, liquids, solutions, suspensions, emulsions, lozenges, syrups, renewable powders, granules, suppositories and transdermal patches. Methods for preparing such dosage forms are known (see, eg, HC Ansel and NG Popovish, “Pharmaceutical Dosage Forms and Drug Delivery Systems”, Volume 5, Lea and Febiger (1990)). Dosage levels and requirements are well recognized in the art and may be selected by those skilled in the art from available methods and techniques appropriate to the particular patient. In certain embodiments, the dosage level ranges from about 1-1000 mg / dose for a 70 kg patient. One dose daily may be sufficient, but up to 5 doses may be given daily. For oral dosing, up to 2000 mg / day may be required. As those skilled in the art will appreciate, lower or higher doses may be required depending on the particular factors. For example, the particular dose and treatment regimen will depend on factors such as the patient's overall health profile, the severity and course of the patient's disorder or disposition, and the judgment of the treating physician.
All documents (both literature and patents) cited herein are hereby incorporated by reference in their entirety.

Literature
1) Kolodkin, AL, Matthes, DJ, and Goodman, CS (1993) .The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules.Cell 75, 1389-1399.
2) Pasterkamp, RJ, and Kolodkin, AL (2003). Semaphorin junction: making tracks toward neural connectivity. Curr Opin Neurobiol 13, 79-89.
3) Kikutani, H., and Kumanogoh, A. (2003). Semaphorins in interactions between T cells and antigen-presenting cells. Nat Rev Immunol 3, 159-167.
4) Xu, X., Ng, S., Wu, ZL, Nguyen, D., Homburger, S., Seidel-Dugan, C., Ebens, A. and Luo, Y. (1998). Human semaphorin K1 is glycosylphosphatidylinositol-linked and defines a new subfamily of viral-related semaphorins.J Biol Chem 273, 22428-22434.
5) Tamagnone, L., Artigiani, S., Chen, H., He, Z., Ming, GI, Song, H., Chedotal, A., Winberg, ML, Goodman, CS, Poo, M. et al (1999). Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates.Cell 99, 71-80.
6) Pasterkamp, RJ, Peschon, JJ, Spriggs, MK, and Kolodkin, AL (2003). Semaphorin 7A promotes axon outgrowth through integrins and MAPKs. Nature 424, 398-405.
7) Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S (2002) Identification of a factor that links apoptotic cells to phagocytes. Nature 417 182-7.

Mouse Sema7A fused with the Fc portion of human IgG1 (Sema7A-Fc) induced the production of inflammatory cytokines in human monocytes as well as mouse bone marrow derived macrophages. Sema7A binding to human monocytes was inhibited by soluble α1β1-integrin as well as anti-β1Ab and / or anti-α1Ab. Also shown is the production of IL-6 induced by Sema7A blocked by Ab.

Claims (1)

  (1) contacting cells containing Sema7A protein and VLA-1 protein with a putative regulatory compound, and (2) evaluating the ability of the putative regulatory compound to inhibit Sema7A interaction with VLA-1. A method for identifying a compound that suppresses the interaction of Sema7A with VLA-1 activity in a cell.

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