JP2009536952A - Bst2 inhibitor - Google Patents

Abstract

The present application discloses a method of preventing immune cells from binding with other cells, comprising contacting immune cells and other cells with a composition comprising a Bst2 antagonist.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to US patent application Ser. No. 11 / 471,853 filed Jun. 20, 2006, the contents of which are hereby incorporated by reference in their entirety.

1. TECHNICAL FIELD The present invention relates to molecules that inhibit cell-cell adhesion during inflammation and uses thereof. The invention also relates to the use of the Bst2 protein or Bst2 protein fragment as a decoy or Bst2-binding antibody in inhibiting cell-cell adhesion and cell activity involved in inflammation as well as small molecules. The present invention also relates to methods for searching for Bst2 ligands and Bst2 ligand inhibitors. The present invention also relates to compositions comprising the same, and methods for the prevention and treatment of inflammation-related diseases.

2. General Background and State of the Art Inflammation is the normal response of the body to protect tissues from infection, wounds or disease. The inflammatory response is initiated by the production and release of chemicals by cells in the infected tissue. Chemicals cause redness, swelling, pain, fever and loss of function. Cells within the inflamed tissue emit signals that induce leukocytes to the inflamed site. Leukocytes must adhere to the endothelial cells in order to move out of the bloodstream in the inflammatory site. In addition, leukocytes need to adhere to antigen-presenting cells so that a normal specific immune reaction occurs, and finally, appropriate targets such as lysate pathogen-infected cells and cancer cells Need to adhere to cells. The assembled leukocytes remove any infectious or harmful substances and remove damaged cell fragments from the injured tissue.

  Infiltrating leukocytes play an important role in swallowing invading microorganisms or dead cells in tissue regeneration and immune responses during normal inflammation. However, infiltrating leukocytes cause a serious and fatal condition in pathological chronic inflammation. Abnormal recognition of autologous cells as non-self (foreign), or excessive inflammation due to a persistent inflammatory response may result in diabetes, atherosclerosis, cataract, reperfusion injury, infectious meningitis, rheumatoid arthritis, asthma Causes a variety of inflammatory diseases, including sepsis, inflammatory bowel disease and multiple sclerosis.

  The interaction between leukocytes and endothelial cells is as follows.

  Leukocytes have the dual function of working in a form that circulates in the bloodstream or adheres to specific cells. Specifically, adherent leukocytes interact with endothelial cells, stabilize intercellular adhesion with antigen presenting cells, or act as effector cells to migrate to inflammatory or infected sites. For a normal specific immune response, leukocytes need to adhere to antigen-presenting cells and ultimately need to adhere to appropriate target cells such as lysate pathogen-infected cells, cancer cells, etc. is there. Massive invasion of leukocytes occurs at the site of allograft rejection, skin infection, or injury site, degenerative joint diseases like osteoarthritis, psoriasis, multiple sclerosis, asthma, rheumatoid arthritis, contact dermatitis It is also observed in various diseases including inflammatory bowel disease.

  In such diseases, more than 95% of bone marrow cells migrate to and accumulate at the site of inflammation. White blood cells are important substances in the inflammatory response and exert antibacterial, secretory and phagocytic activity. They collect in tissues that are inflamed, or that need to be inflamed by making water-soluble mediators or through specific adhesion to various cells. In fact, anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs (NSAIDs) or glucocorticoids exert a therapeutic effect by preventing leukocyte adhesion and influx. In animal models of autoimmune disease, inhibition of cell-cell adhesion reverses or prevents disease or allograft rejection. Recent clinical studies show that humanized monoclonal antibodies that inhibit LFA-1 / ICAM-1 or VLA-4 / VCAM-1 interactions are prominent against autoimmune diseases such as psoriasis, multiple sclerosis and inflammatory bowel disease It has become clear that it has good effects and good safety.

  The uncontrolled entry of leukocytes into endothelial cells, an important feature during the development of inflammation-related diseases, occurs by a multi-step process that begins with leukocyte adhesion and binding to the endothelial cell surface. Leukocyte binding to the surface of endothelial cells is mediated by cell surface molecules present on the surface of leukocytes and endothelial cells (Bevilacqua, J. Clin. Invest. 11: 767-804, 1993). Cell surface molecules are overexpressed as a result of leukocyte migration from the bloodstream.

  The interaction between leukocytes and endothelial cells is an important factor in many inflammatory diseases. For example, increased leukocyte-endothelial interactions leading to hepatic microperfusion disorders are considered as a major contributor to liver failure (Croner et al., Microvasc. Res. 67: 182-191, 2004). For example, atherosclerosis is a typical inflammatory disease, where many inflammatory cells such as T lymphocytes and activated macrophages are concentrated at the site of atherosclerosis. Monocyte accumulation and adherence in individual parts of the arterial endothelium is an early detectable event during atherogenesis and is a central feature of the development of atherosclerosis (Ross, Nature 362: 801). -809, 1993). In this area, inflammatory cytokines that control local inflammatory responses, such as interferon gamma and tumor necrosis factor alpha are abundant. Many adhesion molecules are expressed on the surface of monocytes (Valente et al., Circulation 86: III20-25, 1992), and endothelial cells on atherosclerosis express many vascular ligands ( Poston et al., Am. J. Pathol, 140: 665-673, 1992).

  Leukocyte extravasation across the endothelial barrier is an important event during the development of inflammatory diseases such as rheumatoid arthritis. Endothelial cells are involved in the basic mechanism of arthritis, whereby various inflammatory mediators such as tumor necrosis factor α and inflammation-inducing cytokines such as interleukin-1β activate endothelial cells. This leads to increased expression of endothelial cell adhesion molecules in rheumatoid arthritis, resulting in an increased interaction between leukocytes and endothelial cells. The assembly of leukocytes into vascular endothelial cells is also an important stage of asthma.

  In the airways of asthmatic patients, the number of immature macrophages with phenotypic characteristics of activated eosinophils, CD25-positive T lymphocytes and blood monocytes is increased. HLA class II expression is elevated in epithelial cells, macrophages, and other infiltrating cells (Arm et al., Adv. Immunol. 51: 323-382, 1992).

  An increased rate of leukocyte migration across the blood brain barrier is a major sign of multiple sclerosis. The interaction between tight junction proteins in leukocytes and endothelial cells contributes to leukocyte extravasation to the central nervous system under physiological conditions, and altered expression of tight junction proteins is a pathological prerequisite for multiple sclerosis (Worthylake et al., Curr. Opin. Cell Biol. 13: 569-577, 2001).

  As noted above, since leukocyte adhesion to endothelial cells is important in a variety of diseases, inhibition of cell-cell adhesion provides a therapeutic strategy for a variety of inflammatory and immune diseases. From the viewpoint of molecular biology, the following molecules are known to be involved in inflammation.

  Cytokines: Systemic inflammation, a common response to serious bacterial infections or traumatic disorders, affects tissue systems more distal than initial injury (Lush and Kvieties, Microcirculation 7: 83-101, 2000).

  Bacterial products and other inflammation-inducing mediators released from infected tissues induce the formation of inflammation-inducing mediators such as tumor necrosis factor α (TNF-α), interleukin-1β, γ interferon and interleukin-6 To do. In sepsis, vascular endothelium damage promotes the production of TNF-α and interleukin-1β. These cytokines act directly on endothelial cells and enhance leukocyte adhesion (Pober et al., J. Immunol. 137: 1893-1896, 1986; Dustin and Springer, J. Cell Biol. 107: 321-331, 1988; Cotran and Pover, J. Am. Soc. Nephrol. 1: 225-235, 1988). These cytokines also activate blood neutrophils in the blood and vascular endothelium (Arai et al., Annu Rev Biochem, 59: 783-836, 1990). For example, TNF-α induces cytokines, chemokines and protease groups via the autocrine or paracrine pathway (Ghezzi and Cerami, Methods Mol. Med. 98: 1-8.2004). Interleukin-6 induces mononuclear-endothelial cell interactions and inflammatory damage through the expression of adhesion molecules and initiates the atherosclerotic process. Increased blood levels of interleukin-6 are associated with the progression of vascular inflammation and atherosclerosis (Rader, N. Engl. J. Med. 343: 1179-1182, 2000). Interleukin-17 induces the expression of numerous inflammatory mediators and is involved in neutrophil differentiation, maturation and chemotaxis (Witowski et al., Cell Mol Life Sci. 61: 567-579, 2004). . Increased levels of interleukin-17 are associated with pathological diseases such as airway inflammation, rheumatoid arthritis, intraabdominal abscesses and adhesions, inflammatory bowel disease, allograft rejection, psoriasis, cancer and multiple sclerosis. Yes.

  Cell surface adhesion molecules: Multiple inflammatory cytokines induce the expression of endothelial cell-lymphocyte adhesion molecules (ELAMs) on the cell surface (Nortamo et al., Eur. J. Immunol. 21: 2629-2632, 1991). They are divided into two classes: ICAM-1 (intercellular molecular-1) and ELAM-1 (endothelial cell-lymphocyte adhesion molecule-1) (Staunton et al., Cell 52: 925-933, 1988). In response to various mediators, the vascular endothelium expresses specific cell surface glycoproteins. Blood leukocyte binding and extravasation is achieved by interaction with specific ligands or counter-receptors (Bevilacqua et al., 1993, 1994). Molecules involved in this process include ICAM-1 (intercellular molecular-1) as a ligand for CD18, selectins that recognize glycoconjugates on leukocyte surfaces, and other leukocyte integrin molecules of the same family. A group of immunoglobulin supramolecular groups that interact with a group (Panes et al., J. Physiol. 269: H1955-1964, 1995; Khan et al., Microcirculation 10: 351-358, 2003; Nelson et al. al., Blood 82: 3253-3258, 1993; Bevilacqua and Nelson, J. Clin. 79-387,1993). Leukocyte rolling is controlled by selectins and leukocyte migration and adhesion on endothelial cells is triggered by β2 integrin, Mac-1 (CD11b / CD18, aMb2, CR3), and LFA-1. Mac-1 and LFA-1 interact with ICAM-1, which is a counter-receptor expressed on the surface of endothelial cells.

  Prior art related to the treatment of inflammation includes the following.

  In US Pat. No. 5,367,056, polymorphonuclear leukocytes (PMNs) to endothelial cells by treatment of molecules or molecular fragments that block binding to endothelial cell-leukocyte adhesion molecules (ELAMs) as receptors or ligands. Binding inhibition has been described. The patent also describes antisense nucleotides and ribozymes that suppress ELAM expression. The patent further describes a molecule that inhibits binding of ELAM to its ligand, as well as a method for identifying antibodies to ELAM and its ligand.

  US Pat. No. 5,863,540 describes a method of inhibiting T cell activation by administering a CD44 protein peptide or derivative thereof in an amount sufficient to inhibit T cell activation. It also inhibits CD44-mediated cell adhesion or CD44-mediated monocyte IL1 production by administering a CD44 protein peptide or derivative thereof in an amount sufficient to inhibit CD44-mediated cell adhesion or monocyte IL1 production. A method is also described. In addition, there is also disclosed a method for delivering a drug or cytotoxic drug to an inflammatory site by administering a CD44 protein peptide or derivative thereof conjugated to the drug or cytotoxic drug.

  US Pat. No. 5,912,266 lists the inhibition of cell-cell adhesion mediated by the β2 integrin family of cell surface molecules. The patent discloses pharmaceutical compositions useful for inhibiting or treating inflammation and other pathological responses associated with cell adhesion. The patent also discloses a method of inhibiting or treating a pathological condition in which leukocytes and lymphocytes are causing cell or tissue damage.

  WO03026692 relates to the therapeutic use of antibodies against the CD3 antigen complex in patients suffering from chronic arthritis and rheumatoid arthritis.

  EP 1304379 relates to a humanized anti-CD18 antibody comprising part or all of an antigen-determining region capable of binding to the CD18 antigen.

  US Pat. No. 6,689,869 discloses the use of humanized anti-CD18 antibodies in inhibiting the influx of leukocytes into the lungs and other organs during sepsis and other infectious or non-infectious trauma. Humanized anti-CD18 antibodies may be used to inhibit leukocyte invasion into the lungs and other organs of patients suffering from endotoxic shock or adult respiratory distress syndrome. The antibody can be administered to treat reperfusion injury mediated by leukocytes after asthma or thrombolytic therapy. The antibodies can also be used to reduce or cure inflammation in patients administered with an anti-infective agent, or to help administer therapeutic agents to patients undergoing anti-cancer chemotherapy.

  US Pat. No. 5,821,336 discloses 160 kD molecular weight polypeptides, precursors to mediators or inflammatory mediators, derivatives such as variants and fragments thereof, and processes for their preparation. The patent also discloses nucleotide sequences encoding polypeptides and derivatives, vectors containing the nucleotide sequences, antibodies to the polypeptides or derivatives thereof and antibody derivatives. Methods of diagnosing and treating inflammatory diseases and Hodgkin lymphoma using antibodies and antibody derivatives are also disclosed.

SUMMARY OF THE INVENTION Inflammation requires at least three sequential steps to attract immune cells, including leukocytes, to the site of inflammation: (1) lymphocytes, polymorphonuclear leukocytes, natural killer cells and Immune cells, including leukocytes such as macrophages, are activated by cytokines and / or cell-cell interactions; (2) Aggregated immune cells migrate to and aggregate at inflammatory sites and are associated with endothelial cells through adhesion to endothelial cells (3) T lymphocytes and macrophages are activated and secreted cytokines such as interleukin-2 are activated to amplify the inflammatory response.

  The inventors have found that the Bst2 protein mediates allogeneic adhesion between immune cells that plays an important role in inflammation, or heterogeneous adhesion between immune cells and endothelial cells, and also that antagonists of the protein are the main components of inflammation. And is used in the prevention and treatment of inflammation-related diseases, leading to the present invention.

  In one aspect, the invention is directed to a method of inhibiting immune cell binding to other cells comprising contacting immune cells and / or other cells with a composition comprising a Bst2 antagonist. Other cells are immune cells, endothelial cells, smooth muscle cells, brain cells, spinal cord cells, peripheral nerve cells, heart cells, skeletal muscle cells, lung cells, hepatocytes, kidney cells, vascular cells, pancreatic cells, large intestine and small intestine It can be a cell, gastric cell, esophageal cell, nasoropharyngial cell, membrane cell or connective tissue cell. The Bst2 antagonist can be a Bst2 decoy. The Bst2 decoy can then be a Bst2 fragment or variant thereof that has similar or improved binding to other molecules or proteins compared to the Bst2 protein. Further, the Bst2 antagonist can be a Bst2 decoy fused to a stabilizing protein, a Bst2 decoy-Fc chimera or fusion, a Bst2-decoy-albumin chimera or fusion, or a pegylated Bst2-decoy. Further, the Bst2 antagonist can be a monoclonal antibody or antibody-like protein domain that specifically binds to Bst2 and / or mouse Damp1 protein.

  In other embodiments of the invention, the Bst2 antagonist can be a compound. In still other embodiments, the immune cells and other cells are either in the inflammatory site or a site away from inflammation, as long as they can transmit inflammation and immune cytokines or other inflammatory signals to the site of inflammation in the method described above. May be located. Furthermore, the composition may contain cell adhesion or signal transduction inhibitors or immunosuppressive agents. In preferred embodiments, the cell adhesion inhibitor is an ICAM1 antagonist or an LFA antagonist.

  In yet other embodiments, the present invention relates to Bst2 decoy-Fc chimeras. Preferably, the decoy can be fused to any domain of the immunoglobulin. In particular, a Bst2 decoy can be fused to the hinge-CH2-CH3 region of an IgG heavy chain Fc; a Bst2 fusion protein that is stabilized via an IgG kappa chain-heavy chain disulfide bond; or has other Bst2 dimers Not Bst2 decoy-IgG Fc.

  In other embodiments, the invention is directed to monoclonal antibodies specific for Bst2 and / or Bst2 homologues. The homologue can be a mouse Damp1 protein. Furthermore, a monoclonal antibody can contain two arms, one of which can contain a region that specifically binds to a protein other than Bst2 or a homologue thereof. In particular, Bst2-expressing cells to which a monoclonal antibody is bound inhibit the Bst2 ligand-Bst2 interaction or the Bst2-Bst2 interaction.

In yet another embodiment, the present invention provides:
(I) obtaining cells that bind to Bst2;
(Ii) Screening for ligands that bind to Bst2 from ligand-expressing cells:
Directed to a method for separating a ligand for Bst2 comprising:

  In another embodiment, the present invention is directed to a transgenic mouse, comprising a Damp or Bst2 gene whose somatic and germ cells are functionally disrupted, wherein the disrupted gene is mouse or embryogenic If it is homozygous for a gene that has been introduced into the ancestry of a mouse and is disrupted, it indicates an inflammation-related disease.

  In yet another embodiment, the present invention is directed to a transgenic mouse, wherein the somatic and germ cells comprise a Damp gene that has been completely or partially replaced with a Bst2 gene, wherein The Bst2 gene has been introduced into the mouse or mouse ancestry.

  In another aspect, the present invention is directed to a method of reducing inflammation in a subject comprising administering a composition comprising a Bst2 antagonist against the site of inflammation.

  In yet another aspect, the present invention is directed to a method of treating a subject with signs of inflammation-related disease comprising administering a composition comprising a Bst2 antagonist to the subject in need. The composition may contain other anti-inflammatory compounds. Applicable diseases are atherosclerosis, rheumatoid arthritis, asthma, sepsis, ulcerative colitis, type I diabetes, cataract, multiple sclerosis, acute myocardial infarction, heart failure, psoriasis, contact dermatitis , Osteoarthritis, rhinitis, Crohn's disease, autoimmune disease, cachexia, acute pancreatitis, autoimmune vasculitis, autoimmune and viral hepatitis, delayed type hypersensitivity, congestive, coronary restenosis, Glomerulonephritis, graft-versus-host rejection, uveitis, inflammatory eye diseases related to corneal transplantation, traumatic brain injury, epilepsy, bleeding, seizures, sickle cell disease, type II diabetes, obesity, age-related Macular degeneration (AMD), atopic dermatitis, dermatitis, learning / cognitive impairment, neurodegenerative disease, Parkinson's disease, Alzheimer's disease, ulcerative colitis, radiation damage, burns or electrical induced damage (e toxicity-induced injuries), poisoning causing tissue necrosis and immune cell infiltration, drug disorders, inhalation-induced disorders, radiation exposure, pulmonary aspiration-induced disorders, chemistry or Inflammation caused by radiation therapy, autoimmune disease, lupus, Sjogren's syndrome (Schogren disease), demyelinating disease including multiple sclerosis, inflammatory myopathy including multiple myositis, scleroderma, nodular polyarteritis, Sarcoidosis, local and systemic ossifying myositis, amyloid-related diseases including Alzheimer's disease, intervertebral hernia, spinal cord and nerve injury, Reye syndrome, bacterial and viral encephalitis and meningitis, prion-related diseases, Guillain Valley Symptoms, rabies, polio, cerebral hemorrhage, intracranial hemorrhage related injury, chronic fatigue syndrome, venous thrombosis, gout, granulomatous disease, nephritis including glomerulonephritis and interstitial nephritis, insect irritation allergy, anaphylaxis, aplastic anemia Bone marrow injury, multiple organ failure, thyroiditis, pancreatic insulitis, cirrhosis (chronic and acute hepatitis), pulmonary embolism, toxic and drug-induced liver disease, pancreatitis, ischemic bowel disease, acute respiratory distress syndrome, or pericarditis It can be.

  In yet another aspect, the present invention is directed to a method of assaying chemical compounds effective for inhibiting cell-cell binding through Bst2, comprising determining compounds that bind to Bst2. Furthermore, the Bst2 decoy can be expressed recombinantly in the host cell.

  These and other objects of the present invention will be more fully understood from the following description of the invention, the reference figures attached thereto and the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be more fully understood from the detailed description set forth below, and the accompanying drawings, which are given by way of illustration and method of explanation, and therefore are not limited thereto. . On this occasion,
FIG. 1 is an amino acid sequence alignment showing sequence homology between human Bst2 and mouse Damp1;
2A-2B show the placement of PCR primers used in the cloning process of human Bst2 decoy and mouse Damp1 decoy in an expression vector;
3A-3B show the results of electrophoretic analysis of human Bst2 decoy and mouse Damp1 decoy;
FIG. 4 shows the expression pattern of the Bst2 gene during homotypic aggregation of U937 cells;
FIG. 5 shows the effect of promoting Bst2 overgrowth during homotypic aggregation of U937 cells;
6A-6E show the effect of Bst2 decoy on homotypic aggregation of U937 cells;
Figures 7A-7G show the effect of Bst2 decoy on cell-cell adhesion between human vascular endothelial (HUVEC) cells and U937 cells;
Figures 8A-8F show the dose-dependent effect of Bst2 decoy on cell-cell adhesion between HUVEC and U937 cells;
Figures 9A-9G show the effect of Bst2 siRNA on cell-cell adhesion between HUVEC and U937 cells;
Figures 10A-10B show the effect of Bst2 overexpression upon aggregation of Jurkat cells and interleukin-2 (IL-2) production in Jurkat cells;
FIGS. 11A-11I show the effect of Bst2 decoy and Bst2 siRNA on Jurkat cell aggregation;
Figures 12A-12B are graphs showing the effect of Bst2 decoy on Jurkat cell aggregation and IL-2 production;
FIG. 13 shows the change in the number of immune cells precipitated upon Bst2 decoy treatment;
FIG. 14 shows decreased cytokine levels upon Bst2 decoy treatment;
Figures 15A-15D show functional similarity between human Bst2 and mouse Damp1;
Figures 16A-16D show the inhibitory effect of Bst2 and mouse Damp1 decoys in mouse ovalbumin-induced asthma;
FIG. 17 shows the PEG moiety used in the preparation of the PEG-linked form of Bst2 decoy;
FIG. 18 shows improved metabolic reduction of PEG-conjugated Bst2 decoy;
FIG. 19 shows the expression and distribution of Bst2 in inflammation-related diseases;
Figures 20A-20D show schematics of the Bst2 decoy fused to the Fc region. A, Bst2 decoy itself; B, Bst2 decoy fused to the hinge-CH2-CH3 portion of IgG heavy chain Fc; C, Bst2 fusion protein stabilized through naturally occurring IgG kappa chain-heavy chain disulfide bond; D. Bst2 decoy-IgG Fc was expressed without other Bst2 dimers;
21A-21D are representative vector maps of the Bst2 decoy-IgG Fc fusion protein of FIG. 20;
FIG. 22 shows the PCR-cloning and fusion strategy;
Figures 23A-23B show PAGE (polyacrylamide gel electrophoresis) of purified Bst2 decoy and other Fc fusions. A, Representative PAGE gel stained with Coomassie (4-12% gradient gel, Invitrogen) representing various Bst2 fusion proteins with affinity purification. B. PAGE after size exclusion chromatography;
Figures 24A-24B show Bst2 coated plates; and B, direct binding of Bst2 decoys to immune cells on BSA coated plates;
FIG. 25 shows the plasma half-life of Bst2 decoy or Fc fusion;
FIG. 26 shows the inhibitory effect of Bst2 decoy-Fc fusion on Bst2 decoy and intercellular binding;
Figures 27A-27D show the effect of Bst2 decoy-Fc fusion in a mouse model of asthma;
Figures 28A-28B show the generation of human-mouse chimeric Bst2 mice. A. Mouse (top, black) and human (bottom, gray) genomic loci. Exons are shown as rectangular boxes. The end point of the transmembrane region is indicated by an arrow and the initiating methionine (ATG) is indicated by an asterisk. The physical distance throughout the exon encoding is shown below the genomic locus. The figure is not drawn to scale. B. Production strategy of chimeric human-mouse BST2;
FIGS. 29A-29E show that endogenous Bst2 is required for atypical aggregation between endothelial cells (HUVEC) and monocyte cells (U937) after stimulation with IFNγ. A, control; B, IFNγ inflammation stimulation; C, IFNγ inflammation stimulation + control siRNA; D, IFNγ inflammation stimulation + Bst2 siRNA; E, quantitative analysis of Bst2 siRNA results of AD.
FIG. 30 shows that Bst2 siRNA treatment or ICAM1 siRNA treatment does not affect ICAM1 expression or Bst2 expression, respectively, in IFNγ-treated HUVEC. RT-PCR analysis was performed;
FIGS. 31A-31G show the combined treatment of Bst2 siRNA and ICAM1 siRNA and show the additive effect in atypical adhesion assays. A, control; B, IFNγ inflammation stimulation; C, IFNγ inflammation stimulation + control siRNA; D, IFNγ inflammation stimulation + Bst2 siRNA; E, IFNγ inflammation stimulation + ICAM1 siRNA; F, IFNγ inflammation stimulation + ICAM1 siRNA + Bst2 siRNA; G, AF Quantitative analysis of Bst2 siRNA and ICAM1 siRNA results of
Figures 32A-32M show the dose-dependent response of anti-ICAMl or Bst2 decoy in atypical adhesion assays. A shows control; B, C, D, E, and F show increasing doses of IFNγ inflammatory stimulus + ICAM-1 antibody; G shows IFNγ inflammatory stimulus + control BSA; IFNγ inflammatory stimulus + control IgG is shown; I, J, K, and L show increasing doses of IFNγ inflammatory stimulus + BST2 decoy; M is the dose of anti-ICAM1 and Bst2 decoy from AL Shows a quantitative analysis of the results of dependent reactions;
FIGS. 33A-33C show that the combination of Bst2 decoy and anti-ICAM treatment has an additive effect on cell adhesion. Sub-optimal doses of Bst2 decoy (100 ng / ml) and anti-ICAM1 (1 ug / ml) were used. Cell adhesion was completely inhibited relative to the control level when both Bst2 decoy and anti-ICAM1 were used;
FIG. 34 shows the relative expression level of Bst2 mRNA after cytokine treatment. Jurkat, HUVEC (human vascular endothelial cells), HeLa or CASMC (coronary arterial smooth muscle cells) as indicated, serum, PMA (12 or 18 hours), OKT (12 or 18 hours), TNF-α, interferon Bst2 mRNA levels (log log) were shown after treatment with γ or PGJ2. Bst2 mRNA levels were measured by real-time PCR;
FIG. 35 shows a schematic diagram of a method for causing interaction and signaling between cell A expressing a ligand for Bst2 and cell B expressing a protein or compound Y receptor. A bivalent fusion protein composed of a Bst2 decoy and a protein or compound Y can function as an adapter that causes an interaction between cells A and B. By doing so, signaling between cells A and B can be improved;
Figure 36 shows binding of phage clones to Bst2 / Damp1 decoy;
FIGS. 37A-37B show anti-Bst2 / Damp1 monoclonal antibody (A) heavy chain variable region; and (B) kappa chain variable region; and FIGS. 38A-38B are transiently expressed and purified on a PAGE gel. Anti-Bst2 monoclonal antibody produced is shown. (A) non-reducing conditions; (B) reducing conditions.

  FIG. 39 shows changes in the number of precipitated immune cells upon treatment with anti-Bst2 / Damp1 monoclonal antibody in mouse ovalbumin-induced asthma.

Detailed Description of the Preferred Forms In this application, “a” and “an” are used to refer to the singular and plural of the object.

  As used herein, “antagonist” or “blocker” refers to a substance that inhibits, blocks or reduces the activity of a protein that causes inflammation. The activity mechanism of the antagonist is not particularly limited. Examples of antagonists include organic or inorganic compounds; macromolecular compounds such as proteins, carbohydrates and lipids; and mixtures of multiple compounds. For example, “Bst2 antagonist” or “Bst2 blocker” includes a substance that inhibits, blocks, or reduces the activity of the Bst2 protein in inflammatory activity.

  As used herein, “Bst2 ligand” or “Bst L” refers to a molecule that specifically binds to Bst2.

  As used herein, a “homologue” of a protein is considered to have the same or equivalent specific activity as the comparison protein, regardless of the level of the overall homologous sequence relative to the comparison protein. Is.

  As used herein, the term “inflammatory disease” results from a host defense or infection response to adverse effects, resulting in symptoms such as redness, swelling, tenderness, pain, fever and dysfunction. It refers to any disease that becomes a condition (physical, chemical and biological).

  The term “modification” as used herein indicates a process in which a non-peptide polymer is linked to a Bst2 protein, or fragment thereof.

  The term “non-peptide polymer” as used herein refers to a biocompatible polymer in which two or more repeating units are linked to each other. Examples of non-peptide polymers include polyethylene glycol, polypropylene glycol (PPG), copoly (ethylene / propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharide, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl ethyl ether. , Polyacrylamide, polyacrylate, polycyanoacrylate, polymer lipid, chitin, hyaluronic acid, and heparin. A preferred non-peptide polymer is polyethylene glycol.

  The term “operably linked”, as used herein, refers to a nucleic acid expression control sequence and a second that encodes a target protein in a manner that causes a general function. Functional linkage between the nucleic acid sequences of For example, a promoter is operably linked to a nucleic acid sequence encoding a protein and affects the expression of the coding sequence. The operable linkage to the vector is prepared using genetic recombination techniques well known in the art, and site-specific cleavage and ligation can be performed using enzymes well known in the art.

  The term “prevention”, as used herein, means any action that inhibits or delays the development of an inflammatory disease through administration of the composition. The terms “treatment”, “treating” and “therapy” as used herein ease and help a person suffering from an inflammatory disease. It means all functions (therapeutic therapy, prophylactic therapy and preventive therapy).

  The term “siRNA” as used herein means a short double stranded RNA molecule capable of inducing RNA interference (RNAi) through cleavage of a target mRNA. The term “specific” or “specific for” as used herein means that only the target gene can be suppressed without affecting other genes in the cell. In the present invention, siRNA molecules specific for Bst2 are provided.

  As used herein, “equivalent” activity is considered to be greater than about 80% of the displayed activity measured through objectively defined parameters relative to the reference activity. Is done.

  As used herein, “low molecular weight compound or modulator” or “compound” refers to a chemical compound that is distinguished from biomolecules such as carbohydrates, polypeptides, nucleic acids, or lipids. Low molecular weight compounds or modulators include, but are not limited to, antagonists, agonists, peptidomimetics, inhibitors, ligands, and binding agents for Bst2 / Bst2L binding.

  As used herein, a “variant” is a protein or fragment thereof having a sequence that differs from the wild-type amino acid sequence of the protein by deletion, insertion, non-conservative substitution or conservative substitution, or a combination thereof. Point to. For example, in proteins and peptides, amino acid conversions that do not generally alter the activity of the protein or peptide are known in the art (H. Neuroth, RL Hill, The proteins, Academic Press, New York, 1979). The most commonly occurring transformations are bidirectional, Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

  The term “vector” as used herein refers to a vector capable of expressing a protein of interest in a suitable host cell, and the gene insertion is engineered to be expressed in the host cell. Refers to a genetic structure containing essential regulatory elements that are operably linked.

Bst2 protein During inflammation, Bst2 contributes to cell-cell adhesion. In one aspect, the present invention provides Bst2 (bone marrow stromal antigen-2 to inhibit cell-cell adhesion and activation of immune cells to endothelial cells during inflammation or cell-cell adhesion and activation between immune cells. (Bone Marrow Stroma Antigen-2) provides an antagonist of a protein.

The inventors of the present invention have (1) a homotypic aggregation model of human U937 monocytic cells to examine the effect of Bst2 upon immune cell aggregation, and (2) Bst2 upon cell-cell adhesion between immune cells and endothelial cells. Through the study using the U937 cell and HUVEC heterotypic aggregation model for examining the effect of Bst2, and (3) Jurkat T-cell model for examining the effect of Bst2 on T lymphocyte activation, It was found that leukocytes migrate, recognize extracellular matrix components that interact with cells, and are involved in inflammatory processes that adhere to cells. The inventors have further found that antagonists of the Bst2 protein can effectively inhibit such cell-cell adhesion and thus effectively treat inflammatory diseases. Bst2 protein was first identified in bone marrow stromal cells and is thought to be involved in cell differentiation and proliferation. The cDNA encoding Bst2 was cloned in 1995 and the BST-2 gene was found to be located on the human chromosome 19p13.2 (Ishikawa et al., Genomics 26: 527-534, 1995). The Bst2 gene is composed of exons and four introns. Bst2 is a 30 to 36-kD type II transmembrane protein composed of 180 amino acids (Ohtomo et al., Biochem. Biophys. Res. Commun. 258: 583-591, 1999). The Damp1 gene, a mouse homologue of the human Bst2 gene, has 45% DNA sequence identity to the human Bst2 gene, and as shown in FIG. 1, less than 40% amino acid sequence homology to human Bst2. Have Bst2 protein is mainly expressed in liver, lung, heart and placenta, and is expressed at low levels in pancreas, kidney, skeletal muscle and brain. BST-2 surface-expressed on fibroblasts accelerates stromal cell-dependent growth of murine bone marrow-derived pre-B cells. This result suggests that Bst2 controls pre-B-cell growth or plays an important role in B cell activation in rheumatoid arthritis. Bst2 is also overexpressed in certain cancers such as oral cancer, breast cancer, adenoma and cervical cancer. Referring to FIG. 1, it should be noted that the end of the transmembrane region is not limited to the arrangement shown. The transmembrane region can be plus or minus 5 amino acids at either the N- or C-terminus of the region.
Regarding Bst2 protein, isolation and expression of a gene encoding Bst2 protein (EP1033401) and use of Bst2 protein in cancer diagnosis (WO01 / 57207 and WO01 / 51513) have been reported. The Bst2 protein is divided into three regions: cytoplasm, transmembrane and extracellular domain, and the intracellular region includes cytoplasm and transmembrane region.

Inflammatory Diseases The compositions of the present invention can be used for the prevention or treatment of all types of inflammatory diseases including Bst2 overexpression. In fact, Bst2 is overexpressed in various inflammatory diseases such as asthma, atherosclerosis, rheumatoid arthritis, psoriasis, Crohn's disease, ulcerative colitis, chronic active gastritis, acute appendicitis, and lupus erythematosus (FIG. 19). Accordingly, diseases that can be prevented or treated by the composition of the present invention include, but are not limited to, atherosclerosis, rheumatoid arthritis, asthma, sepsis, ulcerative colitis, multiple sclerosis, acute myocardium Infarction, heart failure, psoriasis, contact dermatitis, osteoarthritis, rhinitis, Crohn's disease, type II diabetes, diabetic neuropathy, chronic obstructive pulmonary disease, cachexia, acute pancreatitis, autoimmune vasculitis, Autoimmune and viral hepatitis, delayed type hypersensitivity, congestion, coronary restenosis, glomerulonephritis, graft-versus-host rejection, uveitis, inflammatory eye diseases that may be associated with corneal transplantation, traumatic brain injury, Epilepsy, bleeding or seizures. Bst2 blockers may also be effective in the treatment of sickle cell disease. In sickle cell disease, recurrent inflammation and vascular disorders occur. Leukocyte adhesion to other cells and endothelium has been shown to be involved in vascular occlusion in sickle cell disease (Okpala I. Curr Opin Hematol. 2006, Jan; 13 (1): 40-4). Furthermore, the idea that activation of the inflammatory pathway could be a mechanism for obesity-related insulin resistance has recently emerged (Roytblat et al., Obes Res. 2000, 8 (9): 673-5; Straczkoski et al., Science). 1996, 271 (5249): 665-8; Hirosumi et al., Nature.2002, 420 (6913): 333-6). Bst2 blockers can be beneficial for insulin resistance, type II diabetes and obesity.

  Other inflammation-related diseases include age-related macular degeneration (AMD), atopic dermatitis, dermatitis, learning / cognitive impairment, neurodegenerative diseases, Parkinson's disease, Alzheimer's disease, ulcerative colitis, radiation damage, Includes burns or electrically induced damage, tissue necrosis and poisoning causing immune cell infiltration, drug disorders, inhalant-induced disorders, radiation exposure, lung aspiration-induced disorders, inflammation caused by chemotherapy or radiation therapy, lupus Autoimmune disease, Sjogren's syndrome, demyelinating disease including multiple sclerosis, inflammatory myopathy including multiple myositis, scleroderma, polyarteritis nodosa, sarcoidosis, local and systemic ossifying myositis, Alzheimer Amyloid-related diseases including disease, disc herniation, spinal cord and nerve injury, Reye syndrome, bacterial and viral encephalitis and meningitis, prions Continuous disease, Guillain-Barre syndrome, rabies, polio, cerebral hemorrhage, intracranial hemorrhage-related injury, chronic fatigue syndrome, venous thrombosis, gout, granulomatosis, nephritis including glomerulonephritis and interstitial nephritis, insect irritation allergy, anaphylaxis , Aplastic anemia, bone marrow injury, multiple organ failure, thyroiditis, pancreatic insulitis, cirrhosis (chronic and acute hepatitis), pulmonary embolism, toxic and drug-induced liver disease, pancreatitis, ischemic bowel disease, acute respiratory distress syndrome , And pericarditis.

Bst2 decoy A soluble form of the Bst2 protein or a fragment or variant thereof can be used as a decoy that binds completely to a molecule or site to which immune cells expressing Bst2 bind to cause inflammation. The Bst2 fragment that can be used as a decoy is not particularly limited as long as it has an anti-inflammatory effect by inhibiting cell-cell adhesion, but preferably a Bst2 protein lacking all or part of the intracellular domain It is. In a specific embodiment, the Bst2 protein fragment is a Bst2 protein comprising the amino acid sequence of SEQ ID NO: 1. The Damp1 protein fragment is a Damp1 protein fragment comprising the amino acid sequence of SEQ ID NO: 2. The Bst2 protein fragment and the Damp1 protein fragment were found to effectively inhibit the cell-cell adhesion caused by Bst2.

  It should be understood that in certain embodiments of the invention, mouse Damp1 is used in place of Bst2, and they can be used alternately. For example, it is contemplated that when a Bst2 decoy is used, a Damp1 decoy containing a Damp1 decoy chimera may be used. It is believed that Damp1 and its variants along with Bst2 can be used for the treatment and reduction of inflammation in a subject. Thus, any special use of Bst2 shown herein applies to Damp1 as well and can be similarly claimed.

  The scope of the present invention includes a protein having a wild-type amino acid sequence of Bst2 protein or a fragment or variant thereof, DNA and RNA capable of encoding a protein having an anti-inflammatory effect by inhibiting cell-cell adhesion and signal transduction. including.

  Furthermore, the protein or fragment thereof provided by the present invention may be a natural sugar chain, a sugar chain added compared to the natural type or a form having a reduced sugar chain compared to the natural type, It may be in a glycosylated form. Addition, reduction, or removal of protein sugar chains can be achieved by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Genetic engineering methods include Bst2, Bst2 decoy, Bst2 decoy Fc deletion of one or more sugar chains found in the wild-type sequence, and / or one or more missing in the native protein Includes addition of glycosylation sites.

Bst2 decoy / Bst2 decoy-Fc variant The therapeutic effect can be limited, especially when low molecular weight proteins are removed too quickly. The injected therapeutic protein can be processed by plasma proteases that bind to plasma proteins or receptors on endothelial cells or blood cells, resulting in protein uptake. Proteins that escape vascular capture can be removed in the liver or kidney glomeruli. In the kidney system, protein enters the urine and is excreted outside the body. The glomerular barrier distinguishes proteins based on both molecular size and molecular charge (Brenner et al., Am J Physiol., 1978, 234: F455). Thus, increasing molecular size or negative charge can decrease renal clearance (Wilson et al., J Gen. Physiol., 1970, 74: 495).

  General strategies for improving in vivo activity and persistence of Bst2 decoy activity used PEGylation, chemical modification to reduce clearance, protein cross-linking to albumin, multiple bonds, recombinant DNA technology Examples include direct fusion to albumin and fusion to Fc. Furthermore, glycoengineering is also applicable as a strategy for improving the in vivo activity and prolonging the activity of Bst2 decoy or Bst2 decoy-Fc. Additional N-linked oligosaccharides are added to the consensus sequence (Asn-X / Ser / Thr, where X is an amino acid other than proline) (Imperial B and Shannon KL. Biochemistry, 1991, 30: 4374). N-linked sugar chains are added even to proteins such as Epo, Mpl ligands or even leptin, which generally lacks sugar chains as a whole. Proteins obtained by glycoengineering have been shown to have substantially improved in vivo activity and sustained activity (Elliot S. et al., Nat. Biotechnol. 2003, 21: 414).

  Bst2 decoy (Bst2 decoy-Fc) mutants with stronger affinity binding to Bst2L can be produced. The dimerization domain of Bst2 may be involved in the regulation of Bst2 ligand binding affinity. Bst2 dimerization is thought to play a role in Bst2 signaling. Receptors for interleukins 2, 3, 5, and 6 and granulocyte macrophage colony stimulating factor contain two different subunits (Hatakeiyama M, et al., Science. 1989, 244 (4904). ): 551-6; Kitamura T, et al., Cell. 1991, 66 (6): 1165-74). The ligand-binding subunits of granulocyte colony stimulating factor receptor, prolactin receptor and growth hormone receptor form homodimers (Larsen A, et al., J Exp Med. 1990, 172 (6): 1559-70, Kelly PA, et al., Recent Prog Home Res. 1993, 48: 123-64). It has been shown that dimerization results in high affinity of the receptor and the beginning of the signal transduction pathway (Cunningham BC, et al. Science. 1991, 254 (5033): 821-5; Nicola NA Metcalf D, Cell. 1991, 67 (1): 1-4).

  Since Bst2 homodimerization may play a role in signaling induced by Bst2L (ligand), the Bst2 decoy (Bst2 decoy-Fc) variant that is a high affinity binding is a potential It can be produced by mutation of amino acid residues within the dimerization domain.

  SMART analysis of Bst2 predicts a coiled-coil region in the amino acid region of 96-153 (human Bst2) (or 102-149, rat Bst2) or in the corresponding region in mouse Damp1. The coiled coil region of Bst2 can be involved in Bst2 dimerization.

Determination of the Bst2 dimerization domain Dimerization of Bst2 induced by cytokines is performed in stabilized cells transfected with two differently tagged Bst2 (such as HA-Bst2 and Bst2-Flag). Alternatively, it can be shown in stabilized cells after transient transfection with an expression vector for tagged Bst2. Bst2 dimerization is indicated by co-immunoprecipitation of tagged Bst2 protein. Dimerization of the wild type Bst2 receptor can be demonstrated. If Bst2 dimerization is confirmed, information about essential residues for dimerization can be obtained after deletion analysis, alanine scanning mutation analysis, and / or site-directed mutagenesis. Mutations can be made in the entire extracellular domain or in the coiled-coil domain. Where dimerization of the wild type receptor is indicated, variants containing deletions or substitutions of residues important for dimerization will not be co-precipitated. When transiently transfected into cells containing Bst2, Bst2 mutants containing deletions or substitutions in the dimerization domain block the inflammatory response following cytokine stimulation and inhibit cell-cell adhesion. Can function as a negative mutant. When stably expressed in Damp1 − / − cells (eg, Damp1 − / − mouse fetal fibroblasts), these mutants cannot effectively exert an inflammatory response or cell-cell adhesion.

  A variety of deletion mutants, insertion mutants or substitution mutants are screened for use as high affinity Bst2 decoy or Bst2 decoy-Fc. Deletions, insertions or substitutions can be introduced at target mutation sites in the entire extracellular domain, coiled-coil domain or dimerization domain identified as described above. The position of the mutation site may be in a region having low homology in, for example, human Bst2, rat Bst2, and mouse Damp1. Deletion of the target amino acid residue, insertion of one or more amino acid residues adjacent to the target amino acid residue, or substitution of the target amino acid residue can be made. The target amino acid residue can be one or more amino acid residues. Since excessive deletion / insertion may result in complete loss of biological activity, amino acid sequence deletions or insertions can be made from 1-5 contiguous residues.

  Target amino acid residues in deletions, insertions or substitutions include those important for Bst2 dimerization identified as described above. Other affected sites are those in which the amino acid residues in the sites are similar or identical in human Bst2, rat Bst2 and mouse Damp1. Random mutagenesis can be performed for substitutional mutagenesis.

Screening for Bst2 decoy- or Bst2 decoy-Fc variants Bst2 decoy- or Bst2 decoy-Fc variants can be screened using a cell-cell adhesion assay. High affinity mutants inhibit cell-cell adhesion more effectively than the parent Bst2 decoy or Bst2 decoy-Fc protein.

  2. Variants of Bst2 decoy-Fc are screened using a solid phase adsorption assay as described herein. Plates are coated with anti-Fc antibody and incubated with Bst2 decoy-Fc variant. Cell line sources for Bst2L (see Example 29-1; Identification of cell sources rich in Bst2L in vitro) or U937 cells (see Example 20) are then radiolabeled with 3H-thymidine and added to the wells Is done. After isolation and confirmation of Bst2L (see Examples 28-34), COS7 cells transfected with an expression vector for Bst2L are radiolabeled and can be used for assays. After immobilization, the adherence of radiolabeled cells is measured.

3. The methods described herein allow one of ordinary skill in the art to identify variants with high binding affinity without the need for protein purification. Mutagen Bst2 PCR primers can be designed for random mutagenesis of selected amino acid residues or any arbitrary amino acid in the extracellular domain, coiled-coil domain or dimerization domain. The PCR product encoding the mutation is subcloned in a designed Bst2 expression vector. COS7 cells are transiently transfected with mutant Bst2 cDNA. In this method, Bst2 mutants containing mutations in the extracellular domain, coiled-coil domain or dimerization domain are expressed on the surface of cells transfected for panning. Cells can be added to a panning plate coated with purified Bst2L. Cells expressing Bst2 decoy with high affinity for Bst2L are then screened by FITC-labeled human Bst2L-Fc by indirect immunofluorescence or FACS analysis, followed by secondary antibody staining. Is called. Plasmid DNA is recovered from the cells adhered to the plate and used for the next cycle of concentration. Bst2 decoy or Bst2 decoy-Fc is modified to include a selective mutated sequence. Bst2 decoy or Bst2 decoy-Fc containing a selective mutation is tested in a cell-cell adhesion assay for functional verification.
Production of Bst2, Bst2 decoy, Bst2 decoy Fc protein, Bst2L, portions of these proteins or variants of these proteins The scope of the present invention is to express in host cells of mammalian, insect, fungal, plant or bacterial origin Methods for constructing expression vectors for Bst2, Bst2 decoy, Bst2 decoy Fc protein, Bst2L, portions of these proteins or variants of these proteins, and methods of purification of these proteins. Bst2, Bst2 decoy, Bst2 decoy Fc or Bst2L also include those derived from Bst2 and Bst2L homologs from mice, rats, rabbits, dogs, primates and other animals. For the construction of expression vectors for recombinant protein production, the corresponding genes or fragments of Bst2, Bst2 decoy, Bst2 decoy Fc, Bst2L or variants thereof having a codon-optimized nucleotide sequence for each expression system Synthesis is required. Expression vectors designed for Bst2, Bst2 decoy, Bst2 decoy Fc or Bst2L expression in mammals, insects (baculovirus, Schneider cells), fungi, plants or bacterial cells are host cells that can be present in plasmids or viruses. It is constructed by inserting a DNA fragment encoding Bst2, Bst2 decoy, Bst2 decoy Fc or Bst2L in the vicinity of a host cell-specific promoter in a specific vector. These proteins can be expressed as labeled fusion proteins in mammalian, insect, fungal, plant or bacterial cells. A label is a short protein sequence that has a high binding affinity for an antibody or a specifically modified solid support. Labels (tags) include, but are not necessarily limited to, histidine, Flag, V5, GST, and HA tags. The labeled Bst2 decoy is purified based on the affinity of the label to a solid support such as a column or bead. Additional steps such as liquid chromatography can be used to increase the purity of all Bst2-related proteins.

  Bst2, Bst2 decoy or Bst2L proteins or fragments are optionally acetylated at the N-terminal amine, amidated at the C-terminal carboxyl group, phosphorylated at serine, threonine or tyrosine residues, α-amino of lysine, arginine and histidine. It may be modified by group methylation, deamidation of glutaminyl and asparaginyl residues, hydroxylation of proline and lysine, biotinylation, palmitylation, sulfation, farnesylation and the like.

  Bst2 or Bst2L protein, Bst2 decoy, fragments thereof, or variants thereof that have anti-inflammatory effects by inhibiting cell-cell adhesion can be isolated or synthesized in nature (Merrifield, J. Amer. Chem. Soc). 85, 2149-2156, 1963), or can be prepared by recombination methods based on DNA sequences (Sambrook et. Al., Molecular Cloning, Cold Spring Harbor Press, New York, USA, 2nd Ed., 1989). . When a genetic recombination technique is used, a nucleic acid encoding a Bst2 or Bst2L protein, a fragment thereof, or a variant thereof is inserted into an appropriate expression vector, the expression vector is converted into a host cell, and the target protein is expressed. The target protein can be obtained by culturing the host cell and recovering the produced protein from the culture.

1. Preparation of recombinant Bst2, Bst2 decoy, Bst2 decoy Fc, Bst2L, parts or variants of these proteins For successful recombinant protein-based approaches, produce biologically active proteins that can be scaled up for mass production There is a need to. The compatibility of codon usage between the native gene sequence of the Bst2-related protein and the wild-type gene sequence of the expression host is an important matter to be emphasized.

  In addition to the therapeutic utility of Bst2 decoy and Bst2 decoy Fc protein, recombinant proteins of Bst2, Bst2 decoy, Bst2 decoy Fc, Bst2L, portions of these proteins or variants of these proteins may be anti-Bst2 antibodies or anti-Bst2L Required to screen for antibody variants.

  Recombinant Bst2, Bst2 decoy, and Bst2 decoy Fc proteins are also used in assays to identify Bst2L involved in binding interactions. Bst2L can be Bst2 itself, or other protein, peptide or molecule.

  Bst2, Bst2 decoy, Bst2 decoy Fc and Bst2L, portions and variants thereof can be used to screen for peptides or low molecular weight inhibitors or agonists of Bst2-Bst2L interaction. Such screening assays include high-throughput protein-protein binding assays, cell-based assays, immunoassays or biochemical screening assays of chemical libraries suitable for identifying low molecular weight drug candidates.

  Recombinant Bst2, Bst2 decoy, Bst2L, portions and variants thereof may also be useful for recombinant protein-based vaccine approaches.

1-1. Expression of Bst2, Bst2 decoy, Bst2 decoy Fc, Bst2L, portions of these proteins or variants of these proteins (various Bst2-related proteins) in mammalian cells Numerous mammalian expression vectors and host cell systems are commercially available It is. The mammalian expression system is described in Example 4.

1-2. Expression of various Bst2-related proteins in baculovirus In addition to mammalian cells, glycosylated Bst2, Bst2 decoy, Bst2L and other Bst2-related proteins include insect cells and plant cells such as Drosophila S2, Sf9 It may be derived from invertebrate cells. For baculovirus expression, the corresponding Bst2 or Bst2L sequence is fused upstream of an epitope-tagged, eg poly-his-tagged baculovirus expression vector. Bst2 decoy Fc may be used without other labels. Many baculovirus expression vectors are commercially available. Viral infection and protein expression are described in O'Reilley et al. Baculovirus expression vectors: A laboratory manual, Oxford: Oxford University Press (1994). Recombinant baculovirus is produced by co-transfecting Bst2, Bst2 decoy baculovirus vector and BaculoGold virus DNA (Pharmingen) into Sf9 cells (ATCC) using lipofectin. After incubation at 28 ° C. for 4-5 days, the released virus is collected and used for amplification.

Poly-his labeled Bst2, Bst2 decoy or Bst2L is purified by Ni ²⁺ -chelate affinity chromatography (Rupert et al. Nature, 362: 175, 1993). Purification of Bst2 decoy Fc can be performed using protein A column chromatography.

1-3. Expression of various Bst2-related proteins in Pichia pastoris Pichia pastoris is easy to carry out strictly controlled inducible expression in a medium that is easy to handle, while at the same time cloning foreign genes Is a unicellular eukaryote that has many similarities to E. coli in that it is easy (Kocken, CH et al., Infect. Immun. 67: 43-49. 1999). Being a eukaryote, Pichia pastoris can make several post-translational modifications, such as being able to form disulfide bonds that allow proper folding of the protein, and Pichia can It is also known that it may be glycosylated (Yadava A and Okkenhouse, Infect. Immun. 71: 4961, 2003).

  The genes for various Bst2-related proteins are chemically synthesized using nucleotide sequences optimized for Pichia codon usage. For example, Pichia pastoris components such as the Zeocin-selective plasmid PicZα (Invitrogen) are used for cloning and expression of Bst2-related proteins in Pichia pastoris. The plasmid contains an alcohol oxidase 1 promoter from Pichia pastoris fused to an α-mating factor from Saccharomyces cerevisia to direct the protein into the secretory pathway. Under methanol induction, the protein is expressed under the control of the alcohol oxidase 1 promoter and secreted into the medium.

  After creating PicZα expression vectors for various Bst2-related proteins, E. coli XL-1 blue cells are transformed with the construct and zeocin resistant clones are selected for insertion by PCR and restriction enzyme digestion. Positive clones are used to transform Pichia pastoris. The transformation mixture is spread on yeast-peptone-dextrose-sorbitol plates containing zeocin. For expression, positive clones are cultured in buffered glycerol medium for about 24 hours. Cells are pelleted and induced with fresh medium containing 1% methanol for an additional 24 hours. To detect various Bst2-related proteins, the supernatant is tested for expression by ELISA or Western blot. The protein expressed by Pichia is purified from the culture supernatant.

1-4. Expression of various Bst2-related proteins in yeast Codon-optimized sequences are used to create yeast expression vectors for intracellular production or secretion. For secretion, DNA encoding the yeast α-factor secretion signal / leader sequence DH2 / GAPDH promoter is used to transform Bst2, Bst2 decoy, Bst2L, portions or variants of these proteins into selected plasmids. The encoding DNA can be cloned. Yeast cells can be transformed with expression plasmids and cultured in selected fermentation media (Hsiao et al. Proc. Natl. Acad. Sci. USA, 76: 3829, 1979). Yeast supernatants are analyzed by TCA precipitation, SDS-PAGE and Coomassie blue staining. Recombinant Bst2-related protein can be separated from the concentrated supernatant using selective column chromatography.

1-5. Expression of Bst2, Bst2 decoy in E. coli Some of the aforementioned Bst2-related proteins can be produced in E. coli under certain conditions. However, not all soluble proteins produced in E. coli are correctly folded, and it is known that misfolded proteins can form insoluble aggregates in inclusion bodies (Carrio, M. M., and A. Villaverde. 2002. J. Biotechnol. 96: 3-12).

  A DNA sequence encoding a Bst2-related protein selected for expression in the E. coli system is amplified using PCR primers containing appropriate restriction enzyme sites. A wide variety of expression vectors are commercially available. The vector is digested with restriction enzymes and dephosphorylated. The PCR amplified sequence is then ligated into the vector. The ligation mixture is then used for transformation of the E. coli strain. Transformed ones are selected and plasmid DNA is isolated. Selected clones are grown in liquid medium and then used for large scale culture, during which the expression promoter works. The cell pellet can be solubilized and the solubilized Bst2-related protein is then purified using, for example, a metal chelate column if the protein is expressed from a vector containing a poly-his sequence and an enterokinase cleavage site. Is done.

2. Preparation of Bst2, Bst2 decoy, Bst2 decoy Fc, Bst2L, portions or variants of these proteins by peptide synthesis
Bst2, Bst2 decoy, Bst2 decoy Fc, Bst2L, various portions or variants thereof can be produced by direct peptide synthesis by solid phase methods or a combination of solid and liquid phase methods (Stewart et al. , Solid Phase peptide Synthesis, WH Freeman Co., San Francisco, CA, (1969); Barlos K et al. Int J Pept Protein Res. 1991; 37: 513-C. O. et al. 1978; 43: 4196-4199). In order to produce the full length of Bst2, Bst2 decoy, Bst2L or variants, the various parts of these Bst2-related proteins are chemically individually synthesized and combined using chemical or enzymological techniques. Also good. Peptide synthesis methods are also useful for producing variants (eg, phosphorylated forms) of these proteins.

  Peptides can be synthesized using L-forms or D-forms of amino acids. In particular, mammalian proteases and peptidases are unable to degrade peptides synthesized from D-amino acids. D-form Bst2 decoy or various proteins of D-form Bst2 decoy are very stable in vivo despite being small in size and are administered in drinking water or mixed with food, sprays, and / or patches sell.

  The Bst2 protein or fragment thereof provided herein that has an anti-inflammatory effect by inhibiting cell-cell adhesion or interaction and immune cell activation can be in monomeric or multimeric form. Multimers are formed by various methods well known in the art, and the method for forming multimers is not particularly limited.

  Multimers can be, but are not limited to, dimers, trimers, tetramers, pentamers, hexamers, and the like. For example, a multimer uses an isoleucine zipper (ILZ) sequence that induces trimer formation, or a sequence that induces multimer formation, such as surfactant protein-D (SP-D) that induces 12-mer formation. May be prepared. Alternatively, multimers can be prepared by joining two or more polypeptides, each made with a monomer, eg, using a linker.

  Multimers may form parallel or antiparallel structures of Bst2 protein or fragments thereof, or a combination of parallel and antiparallel structures. Bst2 is thought to function as a homodimer, but the direction of each monomer in the homodimer is unknown. For the construction of an expression vector for a multimer containing the antiparallel structure of the Bst2 protein or fragment thereof, the coding sequence for the antiparallel-structured Bst2 protein or fragment thereof is chemically synthesized using a codon-optimized nucleotide sequence. Is synthesized. In the expression vector, each Bst2 protein (or fragment) unit can be joined by a synthetic linker. Synthetic linkers include Gly / Ser-rich synthetic linkers (Berezov A et al., 2001, J Med Chem 44: 2565) or flexible Gly linkers (Kim et al. Proc. Natl. Acad. Sci. USA 96: 10092, 1999). If the Bst2 fragment unit is small enough to be synthesized directly through peptide synthesis, L and D multimers can be produced.

  A Bst2 protein, or fragment thereof, that has an anti-inflammatory effect by inhibiting cell-cell adhesion, or interaction and immune cell activation, can be modified by a non-peptide polymer.

  In a more detailed aspect, antagonists include non-peptide polymer modified Bst2 proteins or fragments thereof that have an anti-inflammatory effect by inhibiting cell-cell adhesion or interaction and immune cell activation.

  The bond between the Bst2 protein, or a fragment thereof, and the non-peptide polymer includes all types of non-covalent bonds such as covalent bonds and hydrogen bonds, ionic interactions, van der Waals forces and hydrophobic interactions. Preferably, the polymer binds to the protein through a specific reactive group. Examples of specific reactive groups in the polymer include aldehyde groups, propionic aldehyde groups, butyraldehyde groups, maleimide groups, ketone groups, vinyl sulfone groups, thiol groups, hydrazide groups, carbonyldiimidazole (CDI) groups, nitrophenyl Examples include carbonate (NPC) groups, trisylate groups, isocyanate groups, and succinimide derivatives. Non-peptide polymers react with reactive groups of the polypeptide, such as N-terminal, C-terminal and / or side chains of amino acid residues (eg, side chains of lysine, histidine or cysteine residues).

  Bst2 protein, which has an anti-inflammatory effect by inhibiting cell-cell adhesion or interaction and immune cell activation, is 1: 1 to 1:10, preferably 1: 1 to 1 in molar ratio with the non-peptide polymer. : 2 can be combined. When the Bst2 protein, or a fragment thereof, is modified with two or more non-peptide polymers, the non-peptide polymers may be the same or different. Proteins improve in vivo stability and metabolism by modification with non-peptide polymers.

  In yet another aspect, the present invention provides a Bst2 protein or fragment thereof having an anti-inflammatory effect by inhibiting cell-cell adhesion or interaction and immune cell activation as described above; inhibiting cell-cell adhesion A composition for preventing or treating inflammatory diseases, comprising one or more selected from non-peptide polymer-modified Bst2 protein or a fragment thereof having an anti-inflammatory effect.

  The composition of the present invention is applied to humans and domestic animals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs and cats whose inflammatory diseases can be inhibited or alleviated by administration of Bst2. Can be done. In this context, the inventors have found that human Bst2 and mouse Damp1 have functional similarities and act on cells with the same origin as well as different origins.

  In yet another aspect, the present invention provides a patient with one or more proteins selected from Bst2 protein or fragments thereof that have anti-inflammatory effects by inhibiting cell-cell adhesion or interaction and immune cell activation. It relates to a method for preventing or treating inflammatory diseases comprising administering.

Stabilization of decoy protein by Fc fusion The fusion of decoy Bst2 to the Fc part of an antibody is described. Given the more advantageous dosing schedule, the resulting fusion was able to prolong the therapeutic effect of the decoy Bst2 protein. Fusion to albumin has also been shown to increase the blood half-life of low molecular weight proteins. Like Bst2 decoy fusion to the Fc portion of an antibody, fusion of Bst2 decoy to albumin can increase the blood half-life of Bst2 decoy.

  Many potent therapeutic proteins, including Bst2 decoys, are smaller than 40 kDa and are therefore susceptible to renal clearance by glomerular filtration. These small proteins rarely form complete formulations. Protein redesign for longer blood half-life is an important medical and commercial goal. Since the protein must generally be administered by injection, it is preferably a therapeutic protein that minimizes the frequency of protein administration.

  In general, the effective molecular weight of a protein can be increased by fusion to a heterologous carrier protein such as albumin or the Fc region of an antibody that aids in protein purification (Capon et al. Nature. 1989 Feb 9 337 (6207): 525-31; Yeh P. et al. Proc. Natl Acad. Sci. USA 89: 1904-1908, 1992). The heterologous sequence can be any sequence, as long as the resulting chimeric protein can retain at least one of the biological activities of the Bst2 decoy.

  Bst2 is thought to exist as a homodimer on the cell surface (Ohtomo et al., Biochem Biophys Res Commun. 1999, 258 (3): 583-91). Bst2 is also thought to require dimerization for its activity. Thus, heterologous sequences that facilitate binding of Bst2 decoy monomers are prepared to form dimers, trimers and higher multimers. Construction of an Fc chimeric protein using a small protein with a molecular weight of less than 40 kDa results in a dramatic increase in blood half-life (Lo et al., PCT WO 00/40615, 2000). Bst2 decoy-Fc is a recombinant chimeric fusion protein composed of the extracellular domain of human Bst2 and the Fc region of human IgG. Bst2 decoy-Fc was produced as a dimer, to some extent as a multimer.

Rat Bst2 decoy-Fc
Rat Bst2 has 44% and 70% amino acid homology to human Bst2 and mouse Damp1, respectively (Kupzigt al., 2003, Traffic 4 (10): 694). The putative coiled-coil domain is present in the region of amino acids 96-153 (or 102-149) in human Bst2 protein and in the corresponding regions of rat Bst2 protein and mouse Damp1 protein.

  The observation that mouse Damp1 decoy inhibits cell-cell interactions between human endothelial cells and human monocyte U937 cells, and the observation that human Bst2 decoy functions in a mouse asthma model, are shown in Bst2 decoy and Bst2 decoy-Fc Indicates that it functions between different species. The effect of the mouse Damp1 decoy (Fc fusion), rat Bst2 decoy (Fc fusion) or human Bst2 decoy (Fc fusion) protein, without any species barrier, any animal such as mouse, rat, rabbit, dog or primate Even models can study each other. Alternatively, decoy Fc fusions derived from Bst2 homologs from rabbits, dogs or primates can be used. Anti-mouse Damp1 antibody or rat anti-Damp1 antibody can be used for antibody treatment in mice. For antibody treatment in rats, rabbits, dogs or primates, antibodies specific for Bst2 homologs from these animals can be used. One means for generating a monoclonal antibody panel against mouse Damp1 is to use Damp1 − / − mice. Damp1 − / − (knockout) mice are produced by a known homologous recombination method.

  A rat anti-Damp1 monoclonal antibody can be produced using rat hybridoma technology (Lebacq-Verheyden et al., Hybridoma. 1983, 2 (3): 355-8.). Similarly, antibody treatment in rats can be performed using mouse anti-rat Bst2 monoclonal antibody.

Damp1 − / − (knockout) mouse construction Damp1 − / − mice are generated by homologous recombination methods. As described above, mouse anti-Damp1 monoclonal antibodies are obtained using Damp1 − / − mice. In addition, Damp1 − / − mice are useful because disease models produce information that can be tracked using Bst2 blockers (Bst2 (Damp1) decoy, anti-Bst2 (Damp1)). Manufacturing a purified protein drug for preclinical studies is very costly and it is difficult to test many disease models. On the other hand, disease induction treatments used in animal models, such as collagen or adjuvant treatment for arthritis models, ovalbumin treatment for asthma, or other treatments commonly used in animal models can be easily done without cost. Can be done. Obtaining valuable information about which disease models can be followed with Bst2 blockers by comparing the severity of signs and disease progression in Damp1 − / − and wild type mice after various disease induction treatments it can. In this way, more processing options can be created using Bst2 blockers.

Tissue-specific Damp1 − / − (knockout) mice Transgenic expression of dominant negative protein of Damp1 or Bst2, or simple Damp1 − / − (knockout) as described above is associated with human disease or inflammation / autoimmune pathways. However, the deletion of Damp1 in a particular cell type is useful for studying the gene function of Damp1 / Bst2 and inferring the gene function of Bst2. Site-specific recombinase-systems such as the widely used CreloxP system (Lasko M et al. Proc. Natl. Acad. Sci. USA, 93: 5860, 1996; Orban P et al., Proc. Natl. Acad. Sci. USA, 89: 6861, 1992). In this system, two transgenic mouse lines are required to promote tissue specific Damp1 knockout. The first mouse line expresses Cre recombinase under the control of an optimal tissue specific promoter. Currently, nearly 40-50 Cre lines are available, and the availability and diversity of Cre lines is increasing. The second line has a loxP site around Damp1. After outcrossing, the Damp1 gene is removed from cells expressing Cre recombinase. One or both of the Damp1 genes can be targeted, for example, to examine the administration sensitivity of the Damp1 gene.

The physiological relevance of Damp1 gene function in tissue-specific, induced Damp1 − / − (knockout) mouse disease may require temporal control in addition to tissue specificity. One way to achieve inducible expression of tissue-specific Cre recombinase is the use of Cre-tuned steroid receptor ligands by fusing a mutant estrogen receptor (ER) ligand-binding domain to the C-terminus of Cre. These fusion proteins are induced by the synthetic estrogen antagonist 4-OH tamoxifen and are insensitive to endogenous β-estradiol. Three different mutant estrogen receptors, mouse ERTM (Danielian PS, Curr. Biol. 8: 1323, 1998), human ERT (Logie and Stewart, Proc. Natl. Acad. Sci. USA, 92: 5940, 1995) and human ERT2. (Feil R et al., Biochem. Biophys. Res. Commun. 237: 752, 1997) is available. By placing CreER under the control of a tissue-specific promoter, a Damp1 knockout system can be created in a tissue-specific and tamoxifen-induced manner. The second transgenic line has a loxP site around Damp1. After outcrossing, the Damp1 gene is removed from cells expressing Cre recombinase in a tamoxifen-induced manner.

  In other approaches, a tetracycline sensitive system can be used. Tetracycline binds to tTA, a tetracycline transcriptional active protein, or rtTA, a reverse tetracycline transcriptional active protein. These complexes repress or activate Damp1 expression by binding to the Tet operator (tetO). Three transgenic mice are required to obtain a Tet-induced knockout of Damp1 in a tissue specific manner. In the first line, tTA or rtTA protein is expressed under the control of a tissue-specific promoter / enhancer. The second line has a Tet-operator promoter (tetO) and Cre-recombinase. Only in the presence of tTA or rtTA and tetracycline carried in drinking water, the tetO promoter is activated and Cre recombinase is expressed. The third line has a loxP site on the Damp1 side. Cre recombinase excises the Damp1 gene in a tissue-specific manner and induced by Tet.

Transgenic Damp1 or Bst2 knockdown animals via RNAi (RNA interference) using shRNA It is difficult to apply gene knockout technology to other species other than mice, so in mice or other animals, respectively, Damp1 or RNA interference (RNAi) can be used to silence the expression of Bst2. By using short fragments of Damp1 or Bst2 siRNA in transgenic animals, the Damp1 gene in mice or the Bst2 homolog in other animals can be silenced. Tissue-specific Damp1 or Bst2 knockdown using RNA interference can be an alternative approach to creating loss of function models.

  RNA interference is sequence-specific, post-transcriptional gene silencing mediated by small double-stranded RNA (dsRNA) that is complementary to the silenced gene sequence. Mediators that degrade sequence-specific messenger RNA are 21- and 22-nucleotide small interfering RNAs (siRNAs) obtained by excision from longer dsRNAs (Bernstein E et al. Nature 409: 363, 2001). Elbashir SM et al. Nature 411: 494, 2001). These siRNAs are incorporated into multiprotein RNA-induced silencing complexes. The antisense strand directs the silencing complex to its complementary target mRNA, resulting in cleavage. A double-strand specific RNase called Dicer in the cell has been shown to be able to process short hairpin RNA structures (shRNAs), resulting in the production of microRNAs. By inhibiting translation, microRNA effectively silences gene expression and allows the gene to be targeted using only one vector. The shRNA system can be used to create transgenic animals that stably suppress gene expression.

  Damp1 or Bst2 siRNA: Damp1 or Bst2 siRNA may be designed by incorporating the corresponding sequence of human Bst2 siRNA used in FIG. 9, or siRNAs may be newly designed. In order to select potential Damp1 (Bst2) -siRNA sequences for generating Damp1 (Bst2) shRNA, in addition to different siRNA against Damp1 (Bst2), a green fluorescent protein (GFP) -Damp1 (Bst2) fusion material Simultaneous gene transfer into mammalian cells such as Cos-7 cells. Expression and knockdown of Damp1 (Bst2) -GFP fusion protein is analyzed by immunoblotting.

  Construction of Damp1 (Bst2) -shRNA expression vector: Both pol III and pol II promoters are used to synthesize short hairpin RNAs (shRNAs) for knockdown of gene expression in mammalian cells and animals. For construction of the shRNA expression vector, the most effective siRNA as selected above is then cloned into the shRNA expression plasmid, and the sense and antisense strands of the short Damp1 (Bst2) sequence in the plasmid. Is transcribed as a DNA sequence encoding Damp1 (Bst2) -shRNA, for example, in the hairpin structure under the control of the U6 promoter and processed into a functional siRNA by intracellular Dicer, a double-strand-specific RNase . The shRNA expression vector is made by cloning the corresponding DNA oligonucleotide in an shRNA expression plasmid such as pSilencer 1.0-U6 from Ambion (Austin, Texas, USA). The oligonucleotide spans the sense and antisense sequences of Damp1 (Bst2) and a 7 base pair loop, and the annealed product contains the appropriate restriction enzyme sites. This duplex is ligated into pSilencer 1.0-U6. This vector is then used to express shRNA endogenously in mammalian cells. Control RNAi vectors are constructed by inserting sequences that express siRNAs with limited homology to known sequences in the mouse or human genome.

  Generation of transgenic animals expressing Damp1 or Bst2-shRNA: Pronuclear injection method, Xia et al. (PLoS Genet. 2006; 2 (1): e10) can be used to show that shRNA transcribed from a human Pol II promoter such as the human ubiquitin C promoter can mediate gene silencing in mice . Transgenic mice are created by pronuclear injection of linearized constructs into fertilized eggs. Similarly, any type of tissue specific promoter linked to Damp1- or Bst2 shRNA may be used for the generation of transgenic mice by simple pronuclear injection.

  Alternatively, an adeno-associated virus (AAV) vector (A. Auricchio et al., Hum. Mol. Genet. 10 (2001), pp. 3075-3081) or a lentiviral vector (Golding MC) expressing Damp1- or Bst2 shRNA et al. Proc. Natl. Acad. Sci. USA 2006 Apr 4; 103 (14): 5285-90) can be used to carry transgenes into animals.

Transgenic animals expressing Bst2, Bst2L, Bst2 or Bst2L or mutations Bst2 or transgenic animals (mouse or rat) overexpressing the full length (or part thereof) of Bst2 or Bst2L are anti-Bst2, Bst2 It is useful for the development and screening of therapeutically useful reagents such as decoy, Bst2 decoy Fc and anti-Bst2L. Transgenic lines can be designed to constitutively express Bst2 or Bst2L proteins in an inducible, tissue-specific, or tissue-specific / inducible manner.

  A transgenic animal expressing Bst2 or Bst2L (or a portion thereof) may exhibit a pathological condition associated with overexpression of Bst2 or Bst2L. These animals can be treated with a Bst2 blocker and have a reduced incidence of pathological conditions and show therapeutic utility compared to untreated animals that produce the Bst2 or Bst2L transgene. If a dominant negative species of Bst2 (Damp1) or Bst2L (Damp1 L) is identified (which inhibits the function of the wild-type protein), the transgenic animals expressing the dominant negative form of these proteins are disease progression. Can be made to test whether or not. Transgenic animals expressing human Bst2 dominant negative protein can be bred with Bst2 knock-in mice prior to disease progression testing.

  The transgene expression cassette comprises a transcription unit having a Kozak consensus sequence and controls Bst2 or Bst2L, exons of parts or variants of these proteins, a stop codon (poly-A-tail) and a transgene expression Code. A number of tissue specific promoters / enhancers are available in the literature. Induction systems having a tetracycline or tamoxifen-inducible system for controlling transient expression are commercially available (see above, “Tissue-specific Damp1 − / − (knockout) mice” column). Methods for producing transgenic mice or rats are routine (US Pat. Nos. 4,736,866 and 4,870,009).

Transgenic animals expressing Bst2 decoy, Bst2 decoy-Fc and Bst2 decoy-albumin fusions Bst2 or Damp1 (or part thereof) extracellular domain, or Bst2 or Damp1 (or part thereof) fused to Fc fragment or albumin ) Can be useful for assessing the therapeutic effect of Bst2 decoy (Fc) under pathological conditions. Transgenic mice expressing these proteins can be bred with knock-in mice expressing Bst2 to evaluate the therapeutic effect of Bst2 decoy (Fc) protein in monotherapy or combination therapy under pathological conditions. Transgenic lines can be designed to constitutively express these proteins in an inducible, tissue specific, or tissue specific / inducible manner.

Generation of Knock-in Mouse-Human-Mouse Chimeric Bst2 Mice Since the amino acid sequence homology between human Bst2 and mouse Damp1 / rat Bst2 is not extensive, in murine or rat immunity, inflammatory disease models, anti-human Bst2 antibody panel The effect cannot be tested. One way to overcome this problem is to create knock-in mice that express human Bst2, in this case human-mouse chimeric Bst2. Knock-in mice may have the full-length coding region of Damp1 replaced with humans or the extracellular domain of Damp1 that is replaced with the extracellular domain of human Bst2, resulting in a chimeric protein. Although transgenic mice expressing human Bst2 can be used for this purpose, the overexpression system is not an ideal system for testing the effects of Bst2 blockers. The knock-in approach of expressing human-mouse chimeric Bst2 at physiological concentrations replaces the transgenic approach. Knock-in mice expressing human-mouse chimeric Bst2 can be performed according to standard knock-in homologous recombination protocols and performed using the exemplified construct as shown in FIG. Knock-in mice are treated to induce an immune-inflammatory condition. An anti-human Bst2 antibody is administered.

  These mice are also useful for testing the effect of combination therapy using anti-human Bst2 antibody or Bst2 decoy-Fc with various rat antibodies against the target mouse protein. For example, knock-in mice expressing human Bst2 may be treated with collagen to induce arthritis (Andren et al., J Immunol. 2006, 63 (4): 282-9), followed by rat anti-mouse. Single agent of TNFR (TNFα receptor I or II) (Abcam), rat anti-mouse IL-6 receptor (Genzyme), rat anti-mouse IL-1 receptor (Abcam) or murine CTLA4-Ig (in-house), 2 May be treated with human Bst2 decoy-Fc or anti-human Bst2 in combination with 3 agents or 4 agents. Murine CTLA4-Ig has been shown to inhibit T cell responses in rats (Shiraishi T et al. 2002, Am J Transplant 2: 223).

Animal Disease Model for Testing the Effect of Bst2 Blocker Useful animal disease models for testing the effect of Bst2 blocker are not particularly limited; rat or mouse collagen-induced arthritis model (Webb et al., Eur J Immunol. 1996, 26 (10): 2320-8; Andren et al., Scand J Immunol. 2006, 63: 282), rat or mouse adjuvant-induced arthritis model (Haruna et al., Arthritis). Rheum.2006, 54 (6): 1847-1855; Hida et al., J Autoimmun.2005 Sep; 25 (2): 93-101), an ovalbumin-induced asthma model (Sy et al., Int). mmunopharmacol. 2006, 6 (7): 1053-60), osteoarthritis model (Averbeck et al., J Rheumatol. 2004 Oct; 31 (10): 2013-20), graft versus host disease (GvHD) model (Zhang et al., Blood, 2006, 107: 2993-3001; Baliga et al., Transplantation. 1994, 58 (10): 1082-90), NOD (non-obese diabetic) mice or BB (BioBreeding) Type 1 diabetes model in rats (Yang Y, Santamaria P. Clin Sci. 2006, 110 (6): 627-39), ischemia / reperfusion model (Arumgam) tal. Nat Med. 2006 Jun; 12 (6): 621-3), septic shock model (Motobu et al. Physother Res. 2006, 20 (5): 359-63), autoimmune uveitis model (Yilmaz) et al. Curr Eye Res. 2005, 30 (9): 755-62), experimental allergic encephalomyelitis (EAE) in mice, an animal model for multiple sclerosis (Mujitaba et al., J Immunol). 2005, 175 (8): 5077-86), rat cerebral embolism model (Chapman DF, stroke. 2001, 32 (3): 748-52), mouse colitis for Crohn's disease and inflammatory bowel disease Model (Yen D et al. J Clin Invest. 2006, 116 (5): 1310-6), a concanavalin A-induced liver injury model for autoimmunity or viral hepatitis (Li et al., Hepatology. 2006 Jun; 43 (6): 1211-9), Psoriasis models (Gudjonsson JE, Elder JT. Eur J Hum Genet. 2006, 14 (1): 2-4) and rabbit corneal allograft rejection models (Shirao E, Deschenes J, Char DH. Curr Eye) Res., 1986, 5 (11): 817-22). Animal models for studying AMD are known (Dithmar et al., Arch Ophthalmol 2001, 119 (11): 1643-9; Cousins et al., Exp Eye Res. 2002, 75 (5): 543-. 53).

  Inhibition of Bst2 may suppress early acceleration of atherosclerosis by stabilizing the already occurring atherosclerosis. This hypothesis can be tested in streptozotocin-treated (diabetic) apoE-deficient mice or LDL receptor knockout mice (Jackson laboratories) (Bucciarelli et al., Circulation, 2002, 106 (22): 2827). Csaky K.K. Exp Eye Res. 2002, 75 (5): 543-53). Many patients with type II diabetes suffer from atherosclerosis. The effect of Bst2 blocker in type II diabetes and atherosclerosis can be tested in apoE deficient db / db double mutant mice.

  The concept of whether inhibition of Bst2 activity is beneficial for the treatment of antibody-mediated autoimmune disease is described in Linsley PS, Wallace PM, Johnson J, Gibson MG, Greene JL, Ledbetter JA, Singh C, Topper MA. Science. 1992, 257 (5071): 792-5, initially tested by measuring antibody responses to sheep red blood cells and keyhole limpet hemocyanin.

  Other autoimmune disease models include the lupus-like syndrome in rats (Finck et al., Science. 1994, 265 (5176): 1225-7) and the glomerulonephritis model (Nishikawa et al., Eur J Immunol. 1994, 24 (6): 1249-54.). Donor-specific transplant resistance is tested using diabetic mice that received islet cell xenografts (Lenschow et al., Science, 1992, 257 (5071): 751). Resistance is also demonstrated in the angiogenic murine heart allograft model (Larsen et al., Nature, 1996, 381 (6581): 434-8; Pearson et al., Transplantation, 1995, 59 (3): 450) and mice. Skin allograft rejection model (Tepper et al., Transplant Proc. 1994, 26 (6): 3151-4) and kidney transplant model (Laskowski IA. J Am Soc Nephrol. 2002, 13 (2): 519-27. ).

Combination Therapy Immune and inflammatory diseases are complex diseases that are mediated by a complex network of immunity and inflammatory signaling. Although these events are closely linked to each other, the underlying cellular and molecular processes can be quite different. Thus, a combination therapy is required for complete remission of an immune-inflammatory disease. In general, it has been shown that combination therapies that can change their ability to affect various inflammatory processes are superior to monotherapy.

  The idea for combination therapy with a Bst2 blocker is tested in an in vitro cell-cell adhesion assay using ICAM1 (an intercellular adhesion molecule), as in the examples (Examples 25 and 26). ICAM1 was selected because it has been shown to control numerous genes important for immunity and inflammatory pathways, and its involvement in numerous inflammation and immune diseases has been extensively studied.

  ICAM1 is a target cell counter-receptor for LFA-1 (CD11c / CD18), a lymphocyte function-related antigen that is a member of the integrin subfamily expressed in leukocytes. The interaction between these two molecules is essential to trigger an intracellular immune response. ICAM-1 is also thought to play a role in acute rejection of allograft tissue. ICAM1 and LFA1 are involved in cell-cell interactions between antigen presenting cells and T cells. ICAM1 on APC (antigen presenting cell) can bind to its receptor, LFA1, on T cells, and ICAM1 on T cells can bind to LFA1 on APC (Mackay). CR, Imhof BA, Immunol Today, 1993, 14:99). There is increasing evidence to support the idea that several molecules previously thought to be adhesion molecules can also emit costimulatory signals (eg, LFA3, LFA1 and ICAM1) for T cell activation ( McKay CR Imhof BA, Immunol Today, 1993, 14:99). Costimulatory molecules provide additional signals to T cells that lead to the onset and enhancement of proliferation (Steinman RM Young JW. 1991, Curr. Opin Immunol 3: 361).

Combination therapy for cardiovascular disease Combination therapy for cardiovascular disease can be performed with statins, ACE inhibitors, beta blockers, calcium channel blockers, leopro, clopidogrel, and renin-angiotensin inhibitors. Endothelial cell dysfunction is associated with cardiovascular diseases such as atherosclerosis, hypertension, and vascular smooth muscle cell proliferation. Bst2 expression is induced by inflammatory cytokines such as TNFα, interferon γ and histamine, indicating that Bst2 may be involved in cardiovascular disease. Thus, inhibition of Bst2 as a monotherapy or in combination with conventional therapies including statins, ACE inhibitors, beta blockers, calcium channel blockers, leopro, clopidogrel, and renin-angiotensin inhibitors, Can improve the treatment of cardiovascular disease.

  Furthermore, Bst2 is induced by inflammatory cytokines in smooth muscle cells. Smooth muscle cell proliferation can reduce the success rate of angiogenesis, a procedure that increases the diameter of atherosclerotic blood vessels, typically coronary arteries. Inhibition of Bst2 can reduce smooth muscle cell proliferation and increase the success rate of angiogenesis.

Combination therapy for rheumatoid arthritis Combination therapy for rheumatoid arthritis using a blocker of CTLA4-Ig or TNFα, IL6 or IL1. Rheumatoid arthritis (RA) is a complex inflammatory disease characterized by chronic synovitis, bone erosion and cartilage destruction. Blocking tumor necrosis factor (TNFα), one of the inflammatory cytokines, effectively inhibits arthritic processes in clinical trials. However, complete remission of the signs and symptoms of RA is rarely achieved with TNFα blockers alone, indicating that several inflammatory pathways can work independently of TNFα. In many patients who do not respond to any clinical signs of inflammation, TNFα blockade has been shown to prevent bone erosion. The effect of TNFα on bone is independent of clinical response in disease signs and symptoms. The relative role of TNFα in joint inflammation, bone erosion and cartilage destruction can therefore be different.

  Blocking the major target molecules of TNFα, interleukin-1 (IL-1), has shown numerous effects on RA. Although IL-1 receptor antagonist monotherapy did not resolve the clinical signs and symptoms of arthritis in most patients, IL-1 has been effective in cartilage damage. Complete remission of signs and symptoms of RA is hardly achieved by any monotherapy, even with TNF inhibition, but of combined inhibition of TNFα / IL-1, TNFα / RANKL or TNFα / IL-1 / RANKL in experimental models Preliminary results showed that such treatment can have an additive effect. These results reinforce the rationale for using combined inhibition of two or more inflammatory pathways for the treatment of rheumatoid arthritis.

  Recently, anti-IL6 or cytotoxic T lymphocyte associated antigen 4-Ig (CTLA4-Ig) has also been shown to be useful for the treatment of arthritis. The promoter region of the Bst2 gene has a binding site for STAT3 that mediates interleukin-6 (IL-6) responsive gene expression, indicating that Bst2 expression can be regulated by the IL6-STAT3 pathway (Ohtomo et al. al., Biochem Biophys Res Commun. 1999, 258 (3): 583-91). Blocking Bst2, a downstream target of IL6, may be beneficial for RA treatment.

  Cytotoxic T lymphocyte associated antigen 4 (CTLA4) is a T cell receptor that is elevated after T cell activation. In many cases, the T-cell receptor (TCR) -only signal is insufficient to elicit an optimal immune response, and the second, costimulatory signal, must exceed the threshold for T cell response. Needed. This enhancement of the TCR signal is provided by CD28 on T cells, and this enhancement can be triggered by B7 expressed on antigen producing cells. Once activated, T cells express a second receptor, CTLA-4, that can also bind to the same B7 molecule. Compared to CD28, CTLA-4 inhibits T-cell responses.

  CTLA4-Ig is a recombinant chimeric fusion protein composed of the extracellular domain of human CTLA4 and the Fc region of human IgG (Abatacept, Bristol-Myers Squibb). CTLA4-Ig binds to the APC (antigen-presenting cell) B7 molecule and blocks its interaction with the CD28 receptor on T cells and thus blocks costimulatory interactions with CD28 on T cells (Linsley et al. al., J Exp Med. 1991, 174 (3): 561-9). CTLA4-Ig has been shown to be effective in the treatment of rheumatoid arthritis (Moreland et al., Nat Rev Drug Discov. 2006, 5 (3): 185-6). Thus, combination therapy with Bst2 blockers and CTLA4-Ig, or TNFα, IL6 or IL1 blockers may be beneficial for the treatment of arthritis.

  A rat collagen-induced arthritis model or a rat adjuvant-induced arthritis model may be used. Mouse anti-rat Bst2 antibody, human Bst2 decoy-Fc, rat Bst2 decoy-Fc or mouse Damp1 decoy-Fc combined with mouse anti-rat TNFR, -rat IL6 receptor or -rat IL1 receptor monoclonal antibody, or murine CTLA4-Ig Can be tested. Lane et al. The murine CTLA4-Ig produced as reported by (Lane et al., Immunology, 1993, 80 (1): 56-61) has been used in rat models, as shown by other studies. (Shiraishi et al., Am J Transplant. 2002, 2 (3): 223-8). Mouse CTLA4-Ig can be made from a chimeric gene of the extracellular portion of the mouse CTLA-4 gene and the constant region of human IgG1. Human CTLA4-Ig (Abatacept, Bristol Squibb) can also be used in a rat model of collagen-induced arthritis.

  Knock-in mice expressing human Bst2 can also be used. Knock-in mice are treated with collagen or adjuvant to cause an arthritic condition, and then rat anti-mouse TNFα receptor (Abcam), mouse IL6 receptor (Genzyme) or mouse IL1 receptor (Abcam) monoclonal antibody, or mouse Treated with anti-human Bst2 antibody or human Bst2 decoy-Fc in combination with CTLA4-Ig. Anti-Bst2 treatment may also be used to treat more common pathologies of arthritis, osteoarthritis, which also have an inflammatory component.

Combination therapy for asthma A combination therapy for asthma, particularly with theophylline, glucocorticoid, TNFα blocker or anti-ICAM1, is described. Many descriptions of the pathological features of asthma include bronchial smooth muscle dilation / contraction, mucosal edema and submucosal midepithelial basement membrane and inflammatory cells, especially hypertrophy of eosinophils. These events occur subsequently and are thought to lead to the pathological features of asthma. Current therapies for asthma include: anticholinergic drugs, steroids, adenosine competitive agonists and long and short acting β2 agonists. These conventional therapies and Bst2 blocker combination therapy may be beneficial for asthma. Furthermore, our gene expression statistical data show that Bst2 is highly induced in smooth muscle cells after inflammatory stimuli such as interferon gamma (FIG. 34). These data indicate that Bst2 is involved in smooth muscle cell physiology and, in addition to the previously characterized anti-inflammatory response during asthma treatment, the Bst2 blocker may represent a further beneficial effect. . For these reasons, conventional asthma therapy and Bst2 blocker combination therapy has an additive effect.

  If asthma becomes progressively more severe or the patient does not respond to theophylline therapy, the patient is treated with corticosteroids. Bst2 blocker and corticosteroid combination therapy can reduce the dose of corticosteroid and therefore reduce its side effects.

  The role of ICAM1, α4 integrin and TNFα in the rat or primate ovalbumin-induced asthma model has been shown (Taylor et al., Am J Respir Cell Mol Biol. 1997, 17 (6): 757-66). The combination of ICAM1, TNFα and / or α4 integrin blocker and inhibition may be effective in treating asthma. For preclinical studies using murine or rat models, mouse anti-rat ICAM1 antibody, rat anti-mouse ICAM1 antibody, mouse anti-rat TNFR antibody, rat anti-mouse TNFR antibody, mouse anti-rat α4 integrin antibody and rat anti-mouse α4 integrin antibody Is commercially available. For preclinical studies using murine or rat models, mouse anti-rat ICAM1 antibody, rat anti-mouse ICAM1 antibody, mouse anti-rat TNFR antibody, rat anti-mouse TNFR antibody, mouse or rat anti-TNFα antibody, mouse anti-rat α4 integrin antibody And rat anti-mouse α4 integrin antibody is commercially available.

Combination therapy for autoimmune hepatitis The combination therapy for autoimmune hepatitis (AIH), particularly with corticosteroids, is described. Autoimmune hepatitis is a chronic, progressive liver disease. Possible triggering factors include viruses, other autoimmune diseases and drugs. The natural course of autoimmune hepatitis in untreated patients has a poor prognosis and often progresses to cirrhosis and liver failure. AIH rarely regresses spontaneously.

  Molecular mechanisms that contribute to the pathogenesis include autoantibody responses to self-antigens, cell adhesion molecules and cytokines, and the development of angiogenesis (Medina et al., Alignment Pharmacol Ther. 2003, 17 (1): 1-16). ). In patients suffering from AIH, intercellular adhesion molecule-1 (sICAM-1), vascular cellular adhesion molecule-1 (sVCAM-1), (s) E-selectin, (s) Increased serum levels of P-selectin and soluble interleukin-2 receptor (sIL-2R), IL4, LFA1, LFA3, TGFβ occur (Simpson et al., Eur J Gastroenterol Hepatol. 1995, 7 (5): 455). 60). Chronic viral hepatitis, autoimmune hepatitis, T cell-mediated immune mechanisms play an important role in the development of tissue damage (Bruck et al., Isr Med Assoc J. 2000, 2 Suppl: 74-80).

  The selective treatment for AIH patients is glucocorticoids as monotherapy or in combination with azathioprine (Czaja AJ, Drugs 57: 49-68, 1999, Cook et al., Q J Med. 1972, 40: 159. Murray-Lyon et al., Lancet, 1973, I: 735-7). Corticosteroids reduce the incidence of cirrhosis during initial treatment but progress to cirrhosis despite treatment in more than 90% of patients within 5 to 10 years (Davis et al., 1984, Gastroenterology 87: 1222-7).

  Treatment with corticosteroids is associated with well-known dose-dependent side effects (Summerskill et al. Gut 16: 876-83, 1975, Czaja AJ, In: Krawitt EL, Wiesner RH, eds. Autoimmune Liver. Disease.New York; Raven Press, 1991: 143-66). Hyperglycemia and hypertension often occur. Therefore, special attention must be given to patients with diabetes caused by liver disease and patients with metabolic bone disease.

  Combination therapy with corticosteroids and anti-Bst2 or Bst2 decoy is beneficial in maintaining disease alleviation. This combination can reduce the dose of corticosteroids and therefore reduce its side effects, giving better results than administration of corticosteroids at high doses.

  The use of anti-Bst2 or Bst2 decoy is in mice using Damp1-/-mice, rat anti-mouse Damp1 or mouse anti-Damp1 antibodies that can be made using human-, rat- or mouse Bst2 (Damp1) decoy-Fc. Concanavalin A-induced liver injury model (Kaneko et al., Biochem Biophys Res Commun. 2006, 345 (1): 85-92), or mouse anti-rat Bst2 or human-, rat- or mouse Bst2 (Damp1) decoy -Models such as the thioacetamide-induced cirrhosis model in rats using Fc (Zimmermann et al., Gastroenterol Hepatol. 2006, 21 (2): 358-66) Can be examined using

Combination Therapy for Transplantation Describes combination therapy for transplantation, particularly with cyclosporine, rapamycin, or anti-LFA1 antibody. Adhesion molecules have been shown to be very involved in transplant rejection and are obvious molecular candidates for targeted interference therapy. Adhesion molecules influence the cellular mechanism of allograft rejection by controlling the transport of host leukocytes to the allograft. Delivery of cells to the allograft is mediated by binding of adhesion molecule receptor ligand pairs between circulating leukocytes and vascular endothelium. Within allografts, adhesion molecules may also be involved in T cell recognition of target cells.

  The immunosuppressants cyclosporine or rapamycin are used as effective calcineurin inhibitors in transplantation medicine. However, patients treated with calcineurin inhibitors may have toxic effects on the kidney leading to renal failure (Miller et al., J Heart Lung Transplant, 1995, 14: S227; Vitko S, Viklicky O. Transplant Proc 2004, 36 (2 Suppl): 243S-247S). Other side effects include neurotoxicity, hyperkalemia and hypertension.

  Bst2 decoy or anti-Bst2 combination therapy with sub-threshold or moderate amounts of cyclosporine or rapamycin has a beneficial immunosuppressive effect on reducing nephrotoxicity.

  Examples of transplanted animal models for testing the effects of Bst2 blockers include mouse skin allograft rejection models (Tepper et al., Transplant Proc. 1994, 26 (6): 3151-4), graft-versus-host disease ( GvHD) model (Zhang et al., Blood, 2006, 107: 2993-3001; Baliga et al., Transplantation. 1994, 58 (10): 1082-90), rabbit corneal allograft rejection model (Shirao et al. Curr Eye Res. 1986, 5 (11): 817-22), islet cell xenograft model (Lenschow et al., Science, 1992, 257 (5071): 751), murine heart allograft. Model (Larsen et al, Nature, 1996,381 (6581):.. 434-8; Pearson et al, Transplantation, 1995,59 (3): 450) and renal transplantation model and the like.

  For preclinical studies, depending on the model, combined with different doses of cyclosporine or rapamycin, mouse anti-Damp1, rat anti-mouse Damp1, mouse anti-rat Bst2, human-, rat-, mouse Bst2 (Damp1) Decoy-Fc is used. The transplanted organ survival rate and T cell activation / proliferation are examined.

Combination therapy for multiple sclerosis A combination therapy with an α4 integrin blocker is described for multiple sclerosis. Multiple sclerosis (MS) is a common demyelinating and inflammatory disease of the central nervous system (CNS) with a probable autoimmune inflammatory cause. Antibodies that block the adhesion of activated T cells to endothelial cells can reduce the inflammatory feature of multiple sclerosis plaques. Current therapies include monoclonal antibodies to α4 integrin (Natalizumab), interferon β and galatiramel (Ropper AH, 2006, N Engl J Med. 354: 965; Rudick RA, et al., N Engl J Med. 2006, 354 ( 9): 899-910.).

  Combination therapy of anti-Bst2 or Bst2 decoy with monoclonal antibodies against α4 integrin may be beneficial. Mouse anti-Damp1 antibody, mouse anti-mouse Damp1, rat anti-mouse Damp1 or human-, rat- or mouse Bst2 (Damp1) decoy-Fc that can be made using Damp1-/-mice An experimental allergic encephalomyelitis (EAE) model can be used to investigate the use of anti-Bst2 or Bst2 decoys in combination with rat anti-mouse α4 integrin (Abcam).

Combination therapy to minimize tissue damage Tissue damage can occur as a result of ischemia, bleeding, trauma, swelling, burns, or exposure to chemicals, toxins or drugs. Cell death as a result of an inflammatory response to tissue damage often increases tissue damage. By inhibiting Bst2, tissue damage can be minimized. For example, steroids such as glucocorticoids are used to minimize brain damage after stroke. Blocking Bst2 during or immediately after a stroke in combination with steroids can minimize the spread of eventual brain damage. Similarly, blocking Bst2 during or immediately after myocardial infarction can reduce the spread of heart damage.

Combination therapy for Crohn's disease
A combination therapy with anti-α4 integrin antibody for Crohn's disease is described. Crohn's disease is a chronic debilitating disease characterized by significant helper T cell (Th) 1-induced inflammation of the colon. The role of Bst2 antagonists can be tested using a mouse model of colitis (Gonzalez-Rey et al., Gastroenterology. 2006 Jun; 130 (6): 1707-20). Combination therapy with anti-α4 integrin antibodies may be beneficial for inflammatory bowel disease.

Combination Therapy for Metabolic Syndrome A combination therapy for metabolic syndrome, particularly with metformin, TZD, statin, NSAID, ACE inhibitor and angiothecin receptor blocker is described.

  Recently, the idea that activation of the inflammatory pathway can be a mechanism for obesity-related insulin resistance has emerged. Alpha tumor necrosis factor (TNF)-is elevated in adipose tissue and blood from obese rodents, and inhibition of TNF alpha improves insulin sensitivity. Interleukin (IL) -6 and monocyte chemotactic protein (MCP-1) also cause insulin resistance, and elevated levels of TNFα, IL-6 and IL-8 are found in patients with diabetes and insulin resistance. (Roytblat et al., Obes Res. 2000, 8 (9): 673-5; Straczkowski et al., J Clin Endocrinol Metab. 2002, 87 (10): 4602-6; Hotamisligil et al. Science, 1996, 271 (5249): 665-8; Sartipy P, Losutoff DJ. Proc Natl Acad Sci USA A. 2003, 100 (12): 7265-70; l et al., J Clin Invest. 1995, 95 (5): 2409-15). Furthermore, elevated levels of C-reactive protein (CRP), an inflammatory marker, are observed in insulin resistant patients (Visser et al., JAMA. 1999, 282 (22): 2131-5). Furthermore, in obese rodents, high-dose salicylate treatment inhibits IκB kinase (IKK), a major kinase in the inflammatory pathway, and can stop impaired glucose tolerance and insulin resistance ( Yuan et al., Science. 2001, 293 (5535): 1673-7).

  Insulin resistance can promote endothelial dysfunction and anti-TNFα blockade results in a rapid recovery of endothelial function. Systemic inflammation, insulin resistance, and endothelial dysfunction are involved in the progression of cardiovascular disease. The endothelium is involved in maintaining vascular homeostasis. Under physiological conditions, the endothelium plays a role by maintaining vascular tone, blood flow, and membrane fluidity. Endothelial dysfunction that occurs in metabolic syndrome is the result of the effects of inflammatory cytokines such as TNF-α. Thus, metabolic syndrome is considered a chronic inflammatory condition that occurs with, for example, endothelial dysfunction causing increased ischemic cardiovascular events, insulin resistance and high mortality. Thus, treatments that allow inhibition of the inflammatory condition are believed to result in minimal cardiovascular risk, type II diabetes and dyslipidemia due to metabolic syndrome.

  The following drugs are widely used in the treatment of metabolic syndrome: oral antidiabetic agents such as metformin and thiazolidinedione (TZD), antihypertensive agents such as angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs). And fat-lowering statin drugs, and non-steroidal anti-inflammatory drugs (NSAIDs). These agents that have been shown to reduce the incidence and / or delay the onset of type II diabetes and atherosclerosis have been shown to have clear anti-inflammatory properties.

  Metformin has been shown to activate AMPK, which plays a central role in the regulation of energy homeostasis and metabolic addition. Metformin also inhibits tumor necrosis factor (TNF) -α-induced NF-κB activation and TNF-α-induced IκB kinase activity (IKK) in a dose-dependent manner. In addition, metformin has a variety of inflammatory and cellular properties such as vascular cell adhesion molecule-1 (VCAM1), E-selectin, intercellular adhesion molecule-1 (ICAM1), and monocyte chemotactic protein-1 (MCP1) Attenuates TNF-α-induced gene expression of adhesive molecules. Angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) reduce inflammatory markers and reduce the risk of progression of type II diabetes. Insulin sensitizers, thiazolidinediones (TZDs) are selective ligands of peroxisome proliferator activated receptor γ (PPARγ) that are widely used in the treatment of type II diabetes. PPARs are one of the nuclear hormone receptor superfamily of transcription factors and are important regulators in various pathophysiological processes related to energy metabolism, including lipid and carbohydrate metabolism, and inflammation. It is reported that PPARγ is abundantly expressed in adipose tissue and that the PPARγ signaling pathway exerts an anti-inflammatory effect by inhibiting NF-κB. Consistent with these results, both in vitro and in vivo studies provide evidence that TZDs have anti-inflammatory properties. TZDs inhibit macrophage activation and reduce inflammatory cytokine expression and release in macrophages and monocytes. In vivo, treatment with TZDs reduces the amount of circulating mononuclear cell nucleus NF-kB, while in the same cell, interleukin-1β (IL-1β), IL6, VCAM-1 which is an adhesion molecule And the expression of IkB, an NK-kB inhibitor that inhibits inflammatory mediators such as P-selectin and monocytes.

Bst2 ligand (Bst2L)
A Bst2 decoy composed of the extracellular domain of the receptor protein, Bst2, inhibits homotypic and atypical cell-cell interactions in vitro. Since the extracellular domain of any known receptor is the domain that interacts with its ligand, inhibition of cell-cell interactions mediated by Bst2 decoy indicates that: 1) it occurs naturally against Bst2 The ligand (Bst2L) is present, 2) the interaction between Bst2 and Bst2L is required for cell-cell adhesion, and 3) Bst2 decoy interacts with naturally occurring Bst2L, and in the adhesion assay Bst2L By disabling, cell-cell interactions must be inhibited, thus negatively controlling the immunoinflammatory response.

  The result that Bst2 decoy inhibits the adhesion of U937 to HUVEC indicates that Bst2L is present on the cell surface of unstimulated U937 cells. Another result that Bst2 decoy inhibits homotypic aggregation of activated T cells or activated U937 cells suggests that Bst2L can be expressed on the surface of T cells and / or U937 cells both before and after activation. To do. After activation of T cells or U937 cells, Bst2L expression may be enhanced. Thus, Bst2L is, for example, in U937 cells (or other monocyte cell lines), T cells, or primary hematopoietic cells either before or after activation, such as in a T cell activated state or LPS stimulated state. It can be expressed. Bst2L can also be expressed in B cells, dendritic cells, endothelial cells or fibroblasts.

  Bst2L can be a protein or a molecule. Bst2L can be a membrane protein or a soluble protein. There are many different Bst2L proteins or molecules that may exhibit different binding specificities and functional properties of the Bst2 receptor. Since Bst2 is known to form homodimers, it is also considered that Bst2 itself may be a potential functional ligand of Bst2. Bst2 on inflammatory cells recognizes infiltrated leukocytes and immune cells. It is possible that all Bst2L proteins or molecules are totally unrelated in terms of functional or binding properties of each other. Thus, in order to achieve the subsequent development of therapeutic targets for Bst2 decoy (Bst2 decoy-Fc) and therapeutic materials for the prevention and / or treatment of inflammatory conditions, they mediate the rate-limiting step in inflammation or immune response The functional characteristics of whether or not it has to be thoroughly tested. Other Bst2L proteins or molecules that are not present in the rate-limiting step in the inflammatory pathway may mediate other important pathways in different disease processes.

  Since Bst2L can be a target for interaction with anti-inflammatory molecules, the proof of the presence of Bst2L is an important feature of the present invention. Antibodies against Bst2L can be therapeutic antibodies for the treatment of various immune and inflammatory diseases. A chimeric molecule to the Fc of the extracellular domain of Bst2L is also beneficial. Bst2L may be involved in, for example, T cell costimulation (or inhibition) signaling for T cell activation. Bst2L binds to Bst2, but Bst2L can interact with a number of other receptors on T cells or antigen presenting cells that mediate costimulatory or coinhibitory signals. These new receptor group agonist or antagonist antibodies or Fc fusion proteins can be protein therapeutics for the treatment of various immune and inflammatory diseases.

  Furthermore, by using Bst2L, direct binding assays or binding competition assays are used to screen for Bst2 decoy- (Fc) mutants or low molecular weight modulators of Bst2. These assays allow the inventor to select Bst2 decoy mutants or low molecular weight modulators of Bst2 that inhibit or increase Bst2-Bst2L interaction.

When administration of anti-Bst2L antibody Bst2L (mouse Damp1L) enhances immunity and inflammatory response, it makes sense to produce anti-Bst2L to treat various immunity and inflammatory diseases. Combination therapy of anti-Bst2 antibody and anti-Bst2L antibody is also contemplated.

Anti-Bst2 antibody Conventional IgG antibodies are bivalent capable of binding to two antigens. This binding potential greatly increases their functional affinity and increases the retention time on many cell surface receptors and antigens. The anti-Bst2 antibody can be an antagonist or agonist antibody that inhibits or increases immune and inflammatory responses, respectively. Both antagonist and agonist anti-Bst2 antibodies can be obtained in many different examples of anti-Bst2 antibody formats described below.

  1. The anti-Bst2 antibody of the present invention may be a humanized monoclonal antibody or a human monoclonal antibody. A small portion of a murine antibody is a chimeric antibody in which only the Fc portion of the immunoglobulin molecule is human, or a humanized antibody in which only the complementarity determining region (CDR) of the immunoglobulin is murine and 90-95% of the molecule is human When added to a human immunoglobulin molecule that creates an antigenic murine monoclonal antibody (mAb) becomes humanly friendly. In one respect, fully human monoclonal antibodies can be made in transgenic mice by using conventional methods such as HuMAb-mouse (GenPharm-Mediarex) or Xeno mouse (Abgenix, Inc.) technology. Humanized antibodies include human immunoglobulins in which the recipient's CDR-derived residues are non-human species such as mice, rats or rabbits having the desired properties, affinity and biological function. In the CDR-derived residues.

  Human antibodies can also be obtained using techniques such as phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 1991, 227: 381, Marks et al., J. Mol. Biol. 1991, 222: 581). Can be manufactured. Methods for humanizing non-human antibodies are also well known. Humanization is described in Jones et al. , Nature, 1986, 321: 522; Riechmann et al. , Nature, 1988, 332: 323; and Verhoeyen et al. , Science, 1988, 239: 1534, by replacing rodent CDR sequences or CDRs with the corresponding sequences of human antibodies by Winter et al. It can be performed according to the method. Such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567). Typically, a humanized antibody is an antibody in which CDR residues are replaced by residues from analogous sites in rodent antibodies.

  2. The anti-Bst2 antibody of the present invention may be a Nanobody. Heavy chain antibodies that function without a light chain occur naturally in Nurse sharks, wobbong sharks and Camelidae (Greenberg AS. Et al. 1995, Nature 374: 168; Nut 374: 168; et al., Mol. Immunol. 2001, 38: 313; Hamers-Casterman C. et al. 1993, Nature 363: 446). Their antigen binding site extends to a single domain, the VhH domain. The variable region of a heavy chain antibody is the smallest fully functional antigen-binding fragment with a molecular mass of only 15 kDa, so this part is called a nanobody.

  Nanobodies can be a new class of therapeutic antibodies. Nanobodies have superior properties compared to conventional antibodies in that they are small, very stable, easy to mass produce and easy to convert to multivalent or multispecific proteins. Nanobodies can be administered by non-injection methods. Thus, Nanobodies provide binding affinity and antibody specificity while having small size, stability and small molecule pharmacokinetics.

  The small size of the Nanobodies makes them suitable for targeting antigens in obstructed locations, such as tumors where penetration is important, or inaccessible to conventional antibodies. Anti-Bst2 Nanobodies may be useful for in vitro diagnostic immunoassays and in vivo imaging applications. Anti-Bst2 Nanobodies are able to cross the blood brain barrier and therefore carry therapeutic Nanobodies into the brain.

  Anti-Bst2 Nanobodies can be obtained using phage display technology. As described, Nanobody libraries are constructed from immunized dromedaries (Conrath KE. Et al. Antimicrob Agents Chemother. 2001, 45: 2807). The phage display library is then used for panning of human Bst2 coated on a microtiter plate. Abundant clone selection is performed by ELISA and the clones are sequenced. Protein is purified from positive clones.

  3. The anti-Bst2 antibody of the present invention may be a bispecific antibody. Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have bitarget specificity. Bispecific antibodies are obtained from recombination of the variable regions of two antibodies with different specificities; bispecific antibodies can therefore bind to the antigens of both of their parent antibodies. In the case of anti-Bst2, one of the binding specificities is for Bst2, and the other is Bst2L or, for example, a T cell on the surface of the same cell expressing Bst2 under inflammatory or autoimmune conditions or It can be against other cell surface proteins such as receptors for other inflammatory proteins. These bispecific anti-Bst2 antibodies can function as antagonist or agonist antibodies.

  Methods for making bispecific antibodies are well known (Traunecker et al., EMBO J, 1991, 10: 3655; WO 93/08829; Suresh et al., Methods in Enzymology, 1986, 121: 210; Milstein and Cuello, 1983, Nature, 305: 537). Briefly, an antibody variable region with the desired binding specificity is fused to an immunoglobulin constant region. The fusion comprises an immunoglobulin heavy chain constant region (part of the hinge, CH2 and CH3 regions), preferably the first heavy chain constant region (CH1). DNAs encoding the immunoglobulin heavy chain fusion and the immunoglobulin light chain are inserted into separate expression vectors and co-transfected.

  4). The anti-Bst2 antibody of the present invention may be a single chain variable region fragment antibody (scFV). The recombinant approach leads to the development of single chain variable region fragment antibodies (scFv). Monomeric scFv has a molecular mass of only about 30 kDa and is expressed in diverse systems as a single VL-VH pair linked by a Gly / Ser-rich synthetic linker (Berezov A. et al. , 2001, J Med Chem 44: 2565). When expressed in bacteria or eukaryotic cells, the scFv folds into a structure similar to the corresponding region of the parent antibody. It has been shown to retain an affinity corresponding to that of Fab (Kortt et al., 1994, Eur J Biochem 221: 151). ScFv is amenable to various genetic modifications, such as humanization, and the production of fusion proteins to increase their potential as therapeutic agents. For example, Pexelizumab, a humanized scFv that binds to the C5 complement component, has been shown to reduce myocardial infarction during coronary artery bypass graft surgery (Varrier et al., 2004, JAMA 291: 2319).

  ScFvs with different specificities can also be linked together to create bispecific antibodies that bind to two different receptors on one or different cells. In the case of anti-Bst2, anti-Bst2scFv and anti-Bst2L scFv; or anti-Bst2scFv and, for example, T cells or other inflammatory proteins on the same cell surface expressing Bst2 under inflammatory or autoimmune conditions Bispecific antibody-like molecules comprising other cell surface proteins such as

  The phage display method can be used to generate anti-Bst2scFv. In this method, a broad repertoire of antibody variable region cDNAs is recovered from the combination of B cells and VH, and VL is expressed in the form of scFv on the surface of filamentous bacteriophages. The phage expressing scFv is panned from the antigen coated plate. The affinity for anti-Bst2scFv is improved by mutating the CDRs of the construct and repeating the panning procedure.

  5). The anti-Bst2 antibody of the present invention is Fab, Fab2 bispecific antibody, Fab3 trispecific antibody, bivalent minibody, trivalent trimer, or tetravalent tetramer. It is possible.

  6). The anti-Bst2 antibody of the present invention may be a monoclonal antibody. Monoclonal antibodies are prepared using the hybridoma method, as described by Kohler and Milstein (Nature, 1975, 256: 495). Mice, rats, hamsters or other host animals are immunized with the immunizing antigen so that lymphocytes producing antibodies with binding specificity for the immunizing antigen are produced. Another approach may be to immunize lymphocytes in vitro.

Monoclonal antibodies against Bst2 The use of immunotherapy has recently become well known when the protein target of the disease has been determined. The high specific targeting afforded by therapeutic antibodies causes virtually no side effects, even at relatively high doses. It also takes advantage of the natural serum stability of antibodies and forms the basis for long-acting therapeutic molecules.

  Antibody therapy is generally classified into one of two categories that are not mutually exclusive. The first category depends on the variable region of the antibody (target protein recognition part). Depending on the specific epitope recognized by the antibody, the antibody can inhibit the binding of the target protein to other proteins (suppressive or antagonistic effects), inhibit cell-cell interactions through the target protein, or Or lack the required second protein, as in the case of dimerization-dependent receptor signaling or receptor-dependent ligand mimicry, and as a result of binding to the target protein, the antibody To produce (active or agonistic effects). The second category is the antibody constant region, which determines which immune effector function, if any, is activated as a result of binding of the antibody Fc part to an allogeneic Fc receptor present on immune effector cells. Depends on (Fc part). The presence of a specific target protein on the target cell surface targets the cell for degradation by effector function.

  By developing antibodies with high Bst2 specificity, we have been able to act as a therapeutic protein that prevents cell-cell adhesion and inhibits disease-specific inflammatory responses and many properties. Therapeutic antibodies that share

  When dealing with the pathological mechanism of the mucosal immune system, antibodies can be administered orally or nasally. The mucosal immune system is unique because tolerance is preferentially induced after antigen exposure and induction of regulatory T cells is the primary mechanism of oral tolerance. Orally administered antibodies can be rapidly taken up by the gut-associated lymphoid tissue (GALT), where the antibodies exert their immune effects. Oral administration of antibodies can transmit signals to T cells in the intestine so that signals that are weak but effective in enhancing the regulatory mechanisms of T cells are transmitted. Oral administration of CD3-specific antibodies has been shown in an experimental autoimmune encephalitis (EAE) model. These studies have shown that the Fc portion of CD3-specific antibodies is not required. EAE was suppressed by oral administration of the F (ab ') 2 fragment of the CD3-specific antibody.

Antibody engineering Antibody Engineering Once a therapeutic anti-Bst2 antibody is available, the next step is to design an antigen binding domain (affinity maturation, stability) and effector function (antibody-dependent cellular cytotoxicity; ADCC ), Complement-dependent cellular cytotoxicity (CDC), and clearance rate). Another way to improve the efficacy of anti-Bst2 antibodies is to pursue antibody-toxin conjugates, bispecific antibodies, and / or to study FcR (Fc receptor) polymorphisms. is there.

  The anti-Bst2 antibody inhibits the interaction between Bst2 and Bst2L after binding to the cell to which Bst2 is bound, resulting in intracellular signal intervention. For antibody engineering, anti-Bst2 antibodies can be used when anti-Bst2 antibodies are cross-linked to use an effector function that directs intercellular signals for apoptosis, carries toxins to cells after internalization, or kills cells. It is important to characterize the antibody. All these parameters of anti-Bst2 are important in the treatment of autoimmune / inflammatory conditions.

1-1. Improvement of anti-Bst2 antibody through design of antigen-binding domain 1-1-1. Anti-Bst2 F (ab) Fragment If rapid clearance or a short half-life is required, such as in the case of ReoPro (Centocor), an anti-Bst2 F (ab) fragment may be used. Due to their small size, F (ab) fragments can penetrate more solid tissues. F (ab) fragments can be made in E. coli rather than in mammalian cells. Cross-linking of Bst2 with a bivalent full length anti-Bst2 antibody can cause apoptosis of target cells. Depending on the disease being treated, such apoptosis is either favorable or harmful. The use of F (ab) may be beneficial if cross-linking of Bst2 with full-length anti-Bst2 antibody is detrimental.

1-1-2. Affinity maturation Somatic hypermutation of immunoglobulin genes is important in producing high affinity antibodies in vivo, but somatic hypermutation occurs only after immunization. Thus, high affinity antibodies are rarely found in phage display libraries from non-immunized donors. In vitro affinity maturation is often required to improve antibodies from such libraries. Whether the anti-Bst2 antibody is from a phage library, hybridoma or other technology, antibody affinity may need to be improved. Affinity is not only important for effective inhibition of interactions, but also for dose reduction and cost effectiveness.

  However, with regard to antibody affinity, anti-Bst2 antibody with the strongest binding may not always be the best choice. One antibody may bind strongly to Bst2, but may only partially cover the Bst2L binding site on Bst2, while another antibody may not bind very strongly to Bst2, May properly cover the Bst2L binding site. The latter can be a better choice. Adams et al. Using an anti-Her2 antibody. (Cancer Res. 61: 4750, 2001), the highest affinity antibody did not show optimal penetration into solid tissues / tumors. The high affinity scFv fragment remained around the tumor, while the medium affinity antibody penetrated into the tumor. Penetration into tissues with impaired function due to therapeutic disease can be a potential concern for anti-Bst2 antibody affinity maturation.

1-1-2-1. General Methods for Affinity Maturation In affinity maturation (Levin and Weiss, Mol. BioSystem. 2:49, 2006), residues in the CDRs are altered using mutations, and the resulting mutant antibodies are bound and Selected for improved effectiveness. Several affinity maturation methods have been published. These methods include affinity maturation via phage (Gram et al. PNAS 89: 3576, 1992; Lowman et al., J. Mol. Biol., 1993, 234, 564), ribosome display (Lipovsek et al. J. Immunol. Methods 290 (2004), pp. 51-67), yeast surface display (Graff et al. Protein Eng. Des. Sel. 17 (2004), pp. 293-304), error-prone PCR (Schlapschy et al. Protein Eng.Des.Sel.17 (2004), pp.847-860), a mutagenic bacterial strain (Low et al. J. Mol. Biol. 260: 359, 1996), stepwise concentration. Stepwise focused mutations (Wu et al. PNAS 95: 6037, 1998) and saturation mutagenesis (Nishimiya et al. J. Biol. Chem. 275: 12813, 2000; Yang et al. J. Mol. Biol.254: 392, 1995; Chudhury and Pastan Nat.Biotechnol.17: 568, 1999). Other techniques often use alanine-scanning or site-directed mutagenesis to create a limited collection of specific variants.

1-1-2-2. Affinity maturation through the Look-Through Mutagenesis (LTM) method Recently, Rajpal et al. (Bioren, San Carlos, Calif.) Has developed a Look-Through Mutagenesis (LTM) technology for optimizing antibodies using a yeast display system. LTM can be applied to affinity maturation of anti-Bst2 antibodies. LTM is also useful for screening high affinity variants of Bst2 decoy (or Bst2 decoy-Fc). For affinity maturation of anti-Bst2 antibodies, Rajpal et al. The method according to this is briefly described below.

  LTM is a multidimensional mutagenesis method that results in one amino acid mutation at every position for each CDR for the purpose of rapid affinity improvement. In LTM, the targeted position is either a wild-type residue or the main side-chain chemistry—small (A), nucleophilic (S, H), hydrophobic (L, P), aromatic (Y ), Acidic (D), amide (Q), or basic (K); substituted with one of nine amino acids. LTM creates a series of mutations in the CDR where each wild type is replaced with one of nine selected amino acids.

  Initially, the anti-Bst2scFv construct is assembled by overlap PCR, using sub-cloned into yeast display vectors, using codons optimized for both S. cerevisiae and E. coli. . This initial construct becomes the template for the subsequent anti-Bst2LTM library. For the anti-Bst2LTM library construction, individual CDR oligonucleotides are synthesized to encode a mutant CDR with one target amino acid substitution at each CDR position. In order to amplify LTM-substituted CDR fragments, PCR containing a mixture of LTM oligonucleotides is used. In a triple CDR library, oligonucleotides for CDR1, CDR2 and CDR3 are combined to produce a library with three mutant CDRs (for both VH and VL regions). The corresponding antibody library is then displayed on the yeast cell surface.

  After positive selection, the clones that give higher affinity binding to Bst2 are sequenced and their beneficial mutations are mapped. In order to identify synergistic mutations for improved binding, a library of informative combinatorial mutations is generated with a mixed degenerate DNA probe. Degenerate oligonucleotides encoding selected amino acid mutations and wild-type amino acids are synthesized and collected to produce these libraries. For selection of positive clones, Bst2 (or Bst2 decoy) is biotinylated. Cells are incubated with biotinylated Bst2 and bound to streptavidin beads. A pulse chase strategy for labeling yeast cells with biotinylated Bst2 (or Bst2 decoy) and unlabeled Bst2 (or Bst2 decoy) chase is biotinylated Bst2 (or Bst2 decoy) Used to select clones that show stronger binding to. These clones can be classified by FACS. After several rounds of selection, mutations that give higher affinity can be obtained. All scFvs are then subcloned into the expression vector and secreted into E. coli. The binding affinity of the scFv antibody is measured using a BIAcore surface plasmon resonance system (BIAcore, Switzerland).

1-1-3. High affinity antibody without affinity maturation Dyax's Hoet et al. Show the synthetic diversity in naturally occurring heavy chain CDR3 and light chain sequences obtained from human donors and in the antigen contact sites in heavy chain CDR1 and CDR2. A human F (ab) library has been constructed. Using the DyaxF (ab) library, F (ab) selected for binding to the four human drug targets showed higher affinity than approved therapeutic antibodies (Hoet et al. Nature Biotechnol. 23: 344, 2005). Such an F (ab) library may provide an effective way to generate high affinity anti-Bst2 antibodies that avoid the need for affinity maturation.

1-1-4. Removal of Asn-linked glycosylation at various domains Asn-linked glycosylation in antibody variable regions can affect antigen binding (Leibiger et al. Biochem J. 338: 529, 1999). If Asn-linked glycosylation is observed in the variable region of an anti-Bst2 antibody and the carbohydrate is not required for antibody binding or biological activity, the Asn in the variable region can be changed by changing Asn to Ala, Gln or other amino acids. Can be removed.

  It has been reported that Asn-Gly or Asp-Gly sequences in CDRs form isoaspartic acid and cause natural isomerization (Cacia et al. Biochemistry 35: 1897, 1996). The formation of isoaspartic acid can weaken or eliminate antibody binding. If the CDR in the anti-Bst2 antibody contains these sequences, it would be better to replace Asn or Asp with Ala, Gln, or Glu. It can be determined whether these substitutions can maintain antibody binding and effects.

  If methionine is oxidized and it inhibits binding, the presence of methionine in the CDRs can be problematic as well. In the case of an anti-Bst2 antibody, substitution of methionine with another amino acid can be considered.

1-1-5. Increased stability of anti-Bst2 by mutation of the antigen-binding domain Anti-Bst2 stability can be improved from unstable scFv by changing special residues that affect stability as shown by Angal et al. Transfer CDRs onto a more stable scaffold (Mol. Immunol. 30: 105, 1993); or change the VH-VL junction via the introduction of disulfide bonds as shown by Schuurman et al. (Mol. Immunol. 38: 1, 2001).

1-2. Improvement of anti-Bst2 antibodies by Fc engineering Unlike low molecular weight drugs that must be able to bind to the target and affect its function, therapeutic antibodies bind to the target and effector function: antibody-dependent cytotoxicity ( The immune system can be directed to attack the target through ADCC), complement dependent cytotoxicity (CDC) and phagocytosis. However, monoclonal antibodies that function by inhibiting ligand-receptor interactions can function without the use of effector mechanisms in the case of anti-Bst2 antibodies (Agus et al. J. Clin. Oncol. 23 (2005), pp. 2534-2543; Wang et al. Angiogenesis 7 (2004), pp. 335-345).

  Nevertheless, it may be beneficial to enhance effector function in the action of anti-Bst2 antibodies. For example, all CD20 directed monoclonal antibody treatments for the treatment of autoimmunity, inflammatory conditions, particularly rheumatoid arthritis, result in temporary B cell depletion due to effector function. Infliximab (anti-TNFα) is also known to result in CDC and ADCC following binding to TNFα in vivo (Scallon et al. Cytokine, 1995).

  For anti-Bst2 antibodies, it is important to determine whether ADCC, CDC activation and / or subsequent destruction of target cells is beneficial or harmful to the treatment of the disease. One way to evaluate the effect of ADCC and CDC on the anti-Bst2 therapeutic function is to test the effect of anti-Bst2 antibodies in FcγR knockout mice.

1-2-1. Use of Bst2 knock-in FcγR knockout (double mutant) to determine if effector function is beneficial or harmful ADCC and phagocytosis are both activating and inhibitory activities; complement systems (eg C1q, CDC through interaction with proteins in C3, C4, etc.); and interaction with Fcγ receptors (FcγR) group closely related to half-life / clearance rate through binding of antibodies to neonatal Fc receptors (FcRn) Is mediated through The role of FcγR (and potential ADCC) in the mechanism of action of anti-Bst2 antibody is that mice lacking the activity of the Fc receptors FcγRI and FcγRIII, lacking the common γ chain (FcγR − / −) (Takai et al. Cell 76: 519, 1994), and mice lacking FcγRIIB (Takai et al. Nature 379: 346, 1996).

  Bst2 knock-in mice mated with FcγR knock-outs may be used. When a Bst2 knock-in is prepared in C57B1 / 6 mice, for example, an FcγR-deficient strain is crossed to C57B1 / 6 and backcrossed to obtain a syngeneic strain. This syngeneic strain is then mated with Bst2 knock-in mice to produce FcγR − / − / Bst2 / Bst2 and FcγRIIB − / − / Bst2 / Bst2 mice. This double mutant mouse is treated with a disease-inducing treatment. Mice are then treated with anti-Bst2 antibody.

1-2-2. Enhanced anti-Bst2 activity by enhancing effector function and / or stability When anti-Bst2 antibody uses ADCC for therapeutic effect, improves effector function (by improving binding to FcγR and / or complement) In doing so, designing IgG Fc can be a valuable enhancement to therapeutic antibodies. Improved binding involves changing residues in the Fc (Shields et al. J. Biol. Chem. 276: 6591, 2001), removal of the fucose moiety from the conserved sugar in the Fc (Shields et al. J. Biol. Chem. 277: 26733, 2002; Shinkawa et al. J. Biol. Chem. 276: 3466, 2003) and multiple Fc (Scallon et al., Mol. Immunol. 41: 73, 2004). Has been achieved.

1-2-2-1. Modification of amino acid residues in human IgG by amino acid modifications in Fc has been shown to enhance FcγR binding and effector function. Many such studies focused on the hinge (residues 216-230) and the lower hinge region (residues 231-236). Recently, comprehensive maps of binding sites on human IgG1 to human FcRI, FcRIIA, FcRIIB, FcRIIIA, and FcRn receptors have been published (Shields et al., J. Biol. Chem. 276: 6591, 2001 and therein). References). In this study, select IgG1 variants with improved binding to FcRIIIA showed a significant enhancement in ADCC. It has also been reported that modification of certain IgG1 residues can improve C1q binding (Idusogie et al., J. Immunol. 166 (2001), pp. 2571-2575).

  The neonatal Fc receptor (FcRn) plays a role in the clearance rate of therapeutic monoclonal antibodies (Lencer and Blumberg, Trends Cell Biol. 15 (2005), pp. 5-9). Compared to FcγR, which is a member of the immunoglobulin supramolecular group, FcRn is structurally related to MHC class I, which includes a γ chain that is non-covalently linked to α2-microglobulin (Martin et al. Mol. Cell 7 (2001), pp. 867-877).

  Information generated by Shields et al. Will help design anti-Bst2 mutants with improved binding to FcγR to enhance effector function. By changing the affinity for FcRn, the half-life of anti-Bst2 can be improved.

1-2-2-2. Defucosylation of anti-Bst2 antibody to enhance effector function One way to improve the effector function of anti-Bst2 antibody is by altering glycosylation (fucosylation or sialylation) at Asn297 in the Fc region.

  Glycosylation of IgG is essential for binding to all Fcγ receptors (Jefferis and Lund, Immunol. Lett. 82:57, 2002). In human IgG, carbohydrates that are bound to Asn297 are found in the Fc region. This complex carbohydrate is composed of a core oligosaccharide containing GlcNAc (N-acetylglucosamine) and mannose. This core also contains various additional monosaccharides such as galactose, fucose, GlcNAc, and / or galactose-sialic acid at one or both ends of N-acetylglucosamine. Over 30 different covalently attached glycans have been found at this single glycosylation site (Routier et al., J. Immunol. Methods 213: 113, 1998).

  The presence or defect of the fucose moiety has been shown to play an important role in binding to FcγR. In vitro, it has been shown that the binding of defucosylated monoclonal antibodies to FcγR is significantly increased and ADCC is increased (Shields et al., J. Biol. Chem. 277 (2002), pp. 26733). Shinkawa et al. J. Biol. Chem. 276 (2003), pp. 3466-3473; Nimmerjahn and Ravetch, Science 310: 1510, 2005; Niwa et al. Cancer Res. 64 (2004), pp. 21-27. 2133).

  Subsequently, a designed Chinese hamster ovary cell line was established, in which α-1,6-fucose transferase has been knocked out (Yamane-Ohnuki et al. Biotechnol. Bioeng. 67 (2004), pp. 199). 614-622). GlyArt (Zurich) and BioWa (Princeton, NJ) have developed techniques for designing cell lines to produce antibodies with reduced fucosylation. Antibodies produced in this cell line lacked fucose and defucosylated antibodies showed enhanced ADCC in vitro (Niwa et al. Cancer Res 64: 2127, 2004).

1-2-2-3. Fc sialylation modification in anti-Bst2 antibody to enhance effector function The Fc receptor senses the presence of IgG of both fucose and sialic acid residues. Recent studies have shown that Fc sialic acid at the Asn297 site is important in determining the interaction of IgG and Fc receptors for antibody activity (Kaneko et al. Science 313: 670, 2006). Furthermore, it supports the role of glycosylation in the immune response. Sialylation of glycans bound to Asn297 of IgG results in decreased binding affinity for the FcγR group and decreased cytotoxicity in vivo.

  Sialylation modifications in anti-Bst2 antibodies can be beneficial to improve the effectiveness of anti-Bst2 antibodies. To examine the effect of sialic acid on anti-Bst2 activity by performing a surface plasmon resonance binding assay (BIAcore analysis) using neuraminidase-treated, sialated anti-Bst2 antibody and sialic acid-containing anti-Bst2 antibody Can do. Anti-Bst2 antibodies rich in sialic acid content can be obtained by lectin affinity chromatography. The binding activity of sialylated- and sialic acid-containing anti-Bst2 antibodies to activating or inhibitory FcγR must first be compared. These antibodies may show differences in binding affinity for FcγR, but there will be no difference in binding affinity for Bst2. The effects of sialylated (neuraminidase-treated) anti-Bst2 antibodies in vivo are then tested using animal models and compared to the effects of sialylated anti-Bst2 antibodies or normal, untreated anti-Bst2 antibodies. . Since the sequence of IgG oligosaccharide is determined by the level of glycosyltransferase or glycosidase, sialylation modifications in anti-Bst2 antibodies can be achieved by cell engineering.

1-2-2-4 Addition of Xencor Fc Variant to Anti-Bst2F (ab) Addition of a new Fc variant such as Zencore to anti-Bst2F (ab) The effect of Bst2 can be enhanced.

  Lazar et al. From Zencore (Monrovia, Calif.) Used a combination of computer designed algorithms and high-throughput protein screening to modify amino acids in the Fc region that enhance or reduce responses by the immune system (Lazar et al. PNAS 103: 4005, 2006). Zencore has designed a series of Fc variants with optimized FcγR affinity and specificity. About 500-fold increase in antibody efficacy in in vitro assays when Zenco's new Fc is added to trastuzumab (Herceptin; Genentech, San Francisco, CA, USA) and rituximab (Rituxan; Genentech) And modified rituximab was also more potent in the monkey model.

1-2-3. Enhancement of anti-Bst2 activity by eliminating effector function Depending on the situation, the effector function of the anti-Bst2 antibody may be unnecessary or even detrimental. For example, anti-CD3 targeted to T cells (Xu, ML et al. Cell. Immunol. 200 (2000), pp. 16-26; Carpenter et al. J. Immunol. 165 (2000) Bolt et al. Eur. J. Immunol. 23 (1993), pp. 403-411) and anti-CD4 (Newman et al. Clin. Immunol. 98 (2001), pp. 164- 174) showed adverse side effects due to the binding of monoclonal antibodies to FcγR-producing cells resulting in T cell depletion or activation. In the case of anti-CD3, variants designed to reduce binding to FcγR alleviated the problem (Herold et al. Diabetes 54 (2005), pp. 1763-1769; Carpenter et al. Biol. Blood Marrow). Transplant. 11 (2005), pp. 465-471).

1-2-3-1. Use of IgG4 or IgG2 for anti-Bst2 When the effector function of anti-Bst2 is not required, the two subclasses lack human complementation or lack complement binding, so human IgG2 or IgG4 (Presta LG, J Allergy Clin Immunol. 2005, 116 (4): 731). Since lack of complement activation by IgG4 has been consistently reported, IgG4 may be a better choice when choosing between the use of IgG2 or IgG4. However, certain subclasses of antibodies may not be equivalent in effect of effector function (Chan et al. Mol. Immunol. 41 (2004), pp. 527-538).

1-2-3-2. Removal of Asn297-linked glycosylation from anti-Bst2 antibodies It has been reported that the lack of carbohydrate added to Asn297 of Fc in some cases reduces effector function (Leatherbarrow et al. Mol. Immunol. 22). (1985), pp. 407-415). In addition, a recent report of a phase II clinical trial of aglycosylate anti-CD3 in type 1 diabetes (Keymeulen et al. N. Engl. J. Med. 352 (2005), pp. 2598-2608) The support was shown.

1-2-3-3. Mutation of residues in anti-Bst2Fc to reduce binding to FcR Using the comprehensive map of binding sites on human IgG1 disclosed by Shields et al. (Shields et al., J. Biol. Chem. 276). : 6591, 2001 and references therein), it may be possible to design anti-Bst2 mutants with reduced binding to FcγR or FcRN.

1-2-3-4. Anti-Bst2 Fc Hinge Variants to Reduce Effector Function If effector function is not advantageous for anti-Bst2 antibodies, anti-Bst2 hinge variants are pursued. Exchange of the hinge region between IgG subclasses indicated that the hinge is important for FcγR and C1q binding. Specific mutations at the hinge (Leu235Glu) or outside the hinge (Asp265Ala) have been shown to reduce binding to FcγR (Shields et al. J. Biol. Chem. 276 (2001), pp. 6591-6604; Lund et al). FASEB J. 9 (1995), pp. 115-119; Morgan et al. Immunology 86 (1995), pp. 318-324; Clynes et al. Nat. Med. . A hinge mutant anti-CD3 monoclonal antibody with attenuated effector function is currently in clinical trials (Herold et al. Diabetes 54 (2005), pp. 1763-1769; Carpenter et al. Biol. Blood Marrow Transplant. 11). (2005), pp. 465-471).

1-3. Improving anti-Bst2 by creating bispecific antibodies Bispecific antibodies targeting Bst2 and other drug targets against inflammatory diseases expressed on the same cells more effectively ADCC and CDC Lead. Such bispecific Bst2 antibodies are more effective than antibodies that target one antigen. Bispecific antibodies targeting epidermal growth factor receptor and insulin-like growth factor receptor have been reported to be more effective than antibodies targeting a single antigen (Lu DJ Biol. Chem). 279: 2856, 2004).

1-4. Improving anti-Bst2 by making antibody conjugates with toxic substances Another way to enhance the power of antibodies is by conjugating the antibody to a toxin or radioligand. The antibody binds to a target on the cell, internalizes the toxin, carries it, and kills the cell. These toxins are added to the antibody by using a linker that is cleaved by intracellular enzymes such as cathepsins. The choice of both drug and linker is critical. When the linker is cleaved extracellularly, toxins are released into the bloodstream. Anti-Bst2 antibodies that internalize after binding to Bst2 are required for targeted delivery of toxins. Some anti-Bst2 antibodies may bind strongly to Bst2, but not at epitopes that are optimal for internalization. For this reason, in order to select the anti-Bst2 antibody that is most effectively internalized, it is necessary to develop a screening technique. Methods for screening antibodies with improved internalization have been developed (Marks JD, Methods Mol. Biol. 248: 201, 2004; Neve et al. Biochem. Biophys. Res. Commun. 280 (2001), pp. 274). -279; Heitner et al. J. Immunol. Methods 248 (2001), pp. 17-30).

1-5. Improvement of anti-Bst2 antibodies by FcR polymorphisms FcR polymorphisms appear to play an important role in many diseases including autoimmune diseases, infectious diseases, cardiovascular diseases, atherosclerosis and transplantation ecology ( van Serge et al. Tissue Antigens 61 (2003), pp. 189-202; Karassa et al. Biomed. Pharmacoather. 58 (2004), pp. 286-291; Kastbom et al. Rhematologi. −1298; van Serge et al. J. Neuroimmunol.162 (2005), pp. 157-164; Brouwer et al. J. Infect.Dis.190 (2004), pp. 1192-1 Gruel et al. Blood 104 (2004), pp. 2791-2793; Gavasso et al. Atherosclerosis 180 (2005), pp. 277-282; van der Meer et al. Thromb. Haemost. 92 (2004) p. -1276; Pawlik et al. Transplant. Proc. 36 (2004), pp. 1311-1313).

  The patient's FcγR polymorphism is associated with rituximab (anti-CD20) for cancer (Cartron et al. Blood 99 (2002), pp. 754-758; Carton et al. Blood 104 (2004), pp. 2635-2642; Treon et al.J.Clin.Oncol.23 (2005), pp.474-481; Ghielini et al.Ann.Oncol.16 (2005), pp.1675-1682), rituximab for systemic lupus erythematosus (Anolik et al. Arthritis). Rheum.48 (2003), pp.455-459), and alemtuzumab (anti-CD52) for chronic lymphocytic leukemia (Lin et al., Blood 105 (2005)). It has also been reported to affect the response to therapeutic monoclonal antibodies, such as pp.289-291).

  Thus, in diseases where FcγR polymorphisms may play a role, the design of anti-Bst2 antibodies with increased or reduced binding to FcγR may confer a new class of therapeutic anti-Bst2 monoclonal antibodies.

Hepatocyte expansion Bst2 is also thought to play a role in cell growth and proliferation to promote hematopoietic cell growth and differentiation. As a bone marrow stromal cell antigen and adhesion protein, Bst2 may play a major role in cell-cell interactions important in the hematopoiesis and differentiation of other hepatocytes.

  Multiple hematopoietic cell growth and differentiation in vivo requires direct contact with stromal cells that produce a variety of growth factors, and in some systems, direct contact between stromal cells and hematopoietic cells And required for differentiation (Daniel et al., Haematol. Blood Transfuse. 32: 172, 1989). Accordingly, bone marrow stromal cells and bone marrow stromal cell antigens are important regulators of cell survival and apoptosis.

  Bone marrow contains various types of hepatocytes. These include hematopoietic hepatocytes and mesenchymal stem cells, which are precursors of all blood cells. Mesenchymal stem cells differentiate and transform into a number of different cell types; bone cells, adipocytes, chondrocytes, tendon cells, nervous system cells and bone marrow stromal cells. Bst2 may also control mesenchymal stem cell differentiation.

  The importance of stromal cells in controlling proliferation and apoptosis is further demonstrated in the control of cell survival and apoptosis of cancer cells such as leukemia cells. For example, incubating leukemic cells with bone marrow stromal cells has been shown to protect AML leukemia from chemotherapy-induced apoptosis (Garrido et al., Exp. Hematol 29: 448, 2001; Konopleva M et al. Leukemia 16: 1713, 2002). Recent studies have shown that Bst2 directly mediates the regulatory effect of bone marrow stromal cells on leukemia cells, protecting leukemia cells from chemotherapy-induced apoptosis (Ge et al., Blood 107: 1570, 2006). Consistent with such a role of Bst2 in chemosensitivity, it is reported that Bst2 is elevated in tamoxifen-resistant breast cancer cells (Becker et al., Mol. Cancer Ther. 4: 151, 2005) and different cancers. Several potential functions of Bst2 in are shown. All of these studies indicate that Bst2 is a multifunctional protein that mediates multiple functions.

  Bst2 agonists, Bst2 peptidomimetics and Bst2 ligands can be used to stimulate hepatocyte growth / proliferation in vitro for large-scale hepatocyte preparation. Hepatocytes amplified in vitro can be used for transplantation. For example, mesenchymal stem cells cultured in vitro can be used to enhance hematopoietic hepatocyte transplantation by reestablishing the damaged bone marrow microenvironment after radiation and / or chemotherapy.

  Bst2 agonists, Bst2 peptidomimetics and Bst2 ligands can be used for ex vivo amplification of mesenchymal stem cells for gene therapy. Mesenchymal stem cells are considered promising as a vehicle for gene transfer and therapy. Cultured mesenchymal cells are present in the bone marrow after transplantation, differentiate and produce intact protein.

Bst2 Low Molecular Weight Modulator Another aspect of the present invention provides a Bst2 low molecular weight (mw) modulator for prophylactic treatment of various immune / inflammatory diseases. Bst2 modulators can affect the function and activity of Bst2 in cells and can control or affect Bst2-Bst2L interactions and signal transduction. Furthermore, Bst2 modulators can act on downstream targets and molecules that are regulated by or interact with intracellular Bst2.

  A major factor for low molecular weight compounds is that Bst2 and Bst2 and Bst2 and Bst2L interactions can be sufficiently hindered or enhanced to produce high affinity inhibitors or activators. Whether the interface interaction between Bst2L is sufficiently small. Receptor and ligand protein-protein interactions usually require large interfacial interactions. However, of these many residues, only a few in a very small region can contribute to the binding activity. Through mutational studies, protein-protein interactions are often driven by a small set of contact residues called “hot spots” whose footprints are not significantly larger than those covered by low molecular weight molecules. (Clackson T, Wells JA. Science. 1995; 267: 383-386; DeLano WL. Curr Opin Struct Biol. 2002; 12: 14-20. Wells JA. Proc Natl Aci Sci US. 1996; 93: 1-6).

  If Bst2 binds to Bst2L through a small epitope, the likelihood of finding a low molecular weight ligand may be good.

Bst2 antagonists and agonist modulators Bst2 modulators include antagonists, agonists, peptidomimetics, inhibitors, ligands, and binding agents. Antagonists neutralize, inhibit, reduce, block Bst2-Bst2L interactions and / or Bst2 protein function and / or activity in the Bst2 downstream signaling pathway. , Suppress, diminish, decrease, or eliminate compounds, materials, or drugs. Agonist modulators of Bst2 act, enhance, stimulate, increase the function and / or activity of Bst2 protein in the cell's Bst2-Bst2L interaction and / or Bst2 downstream signaling pathway ( compounds or agents that increase, augment, or amplify.

Use of Bst2 Modulators Anti-Bst2 antibodies and Bst2 decoys (Fc) may have a therapeutic role in immune / inflammatory diseases, but low molecular weight inhibition with significant affinity to inhibit Bst2 binding to Bst2 ligand Agents are also therapeutically valuable for the treatment of various immune / inflammatory diseases.

  In addition to immune / inflammatory diseases, antagonist modulators of Bst2 are also important for certain types of cancer therapy. As illustrated in a recent study by Ge Y et al., Bst2 may be involved in interactions between bone marrow stromal cells and cancer cells such as leukemia cells that lead to leukemia cell survival (Blood 107: 1570). 2006). Bst2 also plays an important role in stromal cell interactions with cancer cells for tumor progression and invasion in certain cancers such as prostate cancer or breast cancer.

  Bst2 agonists, Bst2 peptidomimetics and Bst2 ligands may be therapeutically important for the treatment of immunocompromised patients, including HIV patients or immunocompromised patients. Bst2 peptidomimetics synthesized with D-type amino acids are stable in vivo. These stable peptides may have greater therapeutic potential compared to L-type mimetics.

  Bst2 agonists, Bst2 peptidomimetics and Bst2 ligands may also play a role in the treatment of bone diseases including anemia or osteoporosis. The hematopoietic system requires help from a supportive stromal environment that allows the maintenance and differentiation of hematopoietic hepatocytes (HSC). However, only a very limited number of these stromal cell clones assist hematopoiesis without cytokine supplementation. Bst2 agonists, Bst2 peptidomimetics and Bst2 ligands may be useful for promoting hematopoiesis.

  Bst2 agonists, Bst2 peptidomimetics and Bst2 ligands can also be used to treat bone marrow cells damaged after radiation and / or chemotherapy. By restoring the bone marrow microenvironment, these Bst2 modulators may be useful in the treatment of cancer patients undergoing chemotherapy or radiation therapy.

1. High Throughput Screening (HTS) Methods for Bst2 Modulators Several high throughput screening (HTS) methods have been designed for modulator screening based on the known properties of Bst2 and / or Bst2L: Hit compounds identified by the HTS method are further evaluated by several secondary assays as shown below.

1-1. High-throughput screening of Bst2 inhibitors by detection of direct binding to Bst2 using a fluorescence thermal shift assay Most small molecules that bind to Bst2 are eg Bst2L binding sites or Bst2-Bst2 dimers Due to selective or high affinity binding to functional regions or sites on Bst2, such as dimerization sites important for body formation, Bst2 activity is modulated in some way. Screening and small molecule detection assays for the identification of small molecules that can bind to Bst2 or Bst2 peptides can be designed using thermal shift assays. For the heat shift assay, what is needed is a purified Bst2 protein and a chemical library. A fluorescence-based heat shift assay may be particularly useful when the Bst2 ligand in vivo is unknown.

  Agents or binding molecules determined by this technique are further described herein in the second screening assay to determine whether the molecule affects or modulates the function or activity of Bst2. It can be analyzed by such a method.

1-1-1. Thermal Shift Assay US Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano et al. Zimmerman, 2000, Gen. Eng. News, 20 (8); Pantoliano et al. J. et al. Bioimol Screen 6: 429, 2001; Lo MC et al. Anal Biochem. 332: 153, 2004, a fluorescence-based heat shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) Is used to identify inhibitors of target proteins from compound libraries. This is a general technique. Pantoliano et al. Describe a fluorescence-based thermal shift assay device for high-throughput drug screening.

  In this assay, to observe protein thermal denaturation, an environmentally sensitive fluorescent dye is used to measure the denaturation temperature (ΔTm) obtained in the presence of the compound as compared to that obtained in the absence of the compound. From the shift, ligand-binding affinity is assessed.

  In order to observe protein denaturation, a fluorescent dye such as Sypro orange is used. Sypro orange is an environmentally sensitive dye. The denaturation step exposes the hydrophobic region of the protein, resulting in a significant increase in fluorescence used to observe the protein-denaturation transition.

  A fully automated instrument was designed and created by Pantoliano et al. (J) to perform a miniaturized fluorescence-based thermal shift assay in a microplate format for high-throughput screening of compound libraries. Biomol.Screen 6: 429, 2001).

  The thermal shift assay is as described by Lo et al. (Anal Biochem. 332: 153, 2004) in the iCycler iQ Real Time Detection System (Bio-Rad, Hercules, CA) originally designed for PSR. You may go on. The system includes a heating / cooling device for rapid temperature control and a charge coupled device (CCD) detector for simultaneous imaging of fluorescence changes in the wells of the microplate. The reaction includes Bst2 (about 1 uM), Sypro orange, compounds (0, 10, 50, 100 uM), and buffer. The plate is heated from 25 to 89 ° C. with a heating rate of 0.5 ° C./min. The fluorescence intensity is measured at Ex / Em: 490/530 nm. Fluorescence image data is analyzed by the equation described by Pantoliano et al. (J. Biomol. Screen 6: 429, 2001). By fitting the fluorescence intensity to the equation, the temperature center point of the transition, Tm, for each well is obtained.

1-2. High-throughput screening of Bst2 modulators by detecting the Bst2-NFkB pathway using a dual luciferase assay system Bst2 overexpression results in NFkB activation in mammalian cells (Matsuda et al., Oncogene 22: 3307, 2003) . Although the detailed signaling mechanism of Bst2 in the inflammatory pathway remains unknown, previous reports by Matsuda et al. Indicate that Bst2 overexpression and activation leads to activation of transcription through NFkB through the NFkB response element It is shown.

  Using this property of Bst2, a high-throughput dual luciferase assay (Promega, Madison, Wisconsin, Paguio et al., Cell Notes 16:22, 2006) links Bst2 inhibition or activation with regulation of luciferase reporter gene transcription Is designed for screening of Bst2 modulators.

1-2-1. HTS dual luciferase assay and DNA constructs for stable cell lines In this assay, the first plasmid is selected, for example, firefly luciferase and hygromycin (Promega) linked to a tandem NFkB response element upstream of firefly luciferase Constructed to express markers. The second plasmid expresses Bst2 and other luciferase- (such as neomycin) selectable marker fusions (Promega) such as Renilla luciferase as an internal control. The dual reporter luciferase Bst2 assay has automatic control using Renilla luciferase. In each sample, firefly luciferase activity is normalized using Renilla luciferase activity.

  Cells transfected with mammalian cells and reporter constructs and double-transfected stable cell lines are then obtained for high-throughput screening assays. A control stable cell line expressing an empty vector is also obtained. Bst2 expressing stable cells will show higher luciferase activity than control stable cells containing empty vector of Renilla luciferase-neomycin fusion, as reported in a study by Matsuda et al. (Oncogene 22: 3307, 2003). ).

1-2-2. HTS dual reporter luciferase Bst2 activity screening assays are performed in 384 well format with each compound (usually 10 uM or higher concentration). 10,000 cells / well are seeded. Half of the well is stimulated with the compound and half is stimulated. Cells are harvested after several hours. Luciferase activity is determined using the Dual Glo luciferase assay system (Promega) and quantified by a luminometer. Results from a sample plate of NFkB-firefly luciferase / Bst2 selection are obtained. A hit can be determined as a reporter expression that exceeds 3-4 fold inhibition or activation over the average of the non-induced controls. Control luciferase titers obtained from control stable cells will show the highest level of inhibition. All assays are performed in quadruplicate. Induction or inhibition is calculated as firefly (NFkB) -average of stimulated LU / average of sham-stimulated RLU.

1-2-3. Titration experiments to confirm hits Double transfected stable NFkB response element / Bst2 cell lines are plated at 10,000 cells / well in 96 well plates. Each compound is serially diluted 1: 2 and added to the wells in the quadruplicate. Cells are incubated with antagonists or agonists for several hours, harvested and analyzed using the Dual Glo assay system (Promega). Luciferase activity is measured with a GloMax 96 microplate luminometer (Promega).

1-3. High-throughput screening of Bst2 expression modulators by observing the expression of Bst2 promoter / luc using a dual luciferase assay There are many precedents using promoters containing reporter constructs to identify low molecular weight therapeutic agents. For example, BMP-2, BMP-4 and BMP-7 promoters can be linked to promoters and reporter molecules of either β-galactosidase or luciferase to search for small molecules that increase reporter gene expression. Fused.

  From experiments using microarrays, it is clear that there are many therapeutically important molecules that can induce BST2 (interferon γ, TNFα, histamine, etc.). Furthermore, it is clear from literature studies that some other molecules may also be similar. Furthermore, (Blood 107: 1570, 2006; Matsuda et al. Oncogene 22: 3307, 2003; Goto et al. Blood 84: 1922, 1994) The promoter region of the Bst2 gene has been analyzed. Many important sites were found in AML, GATA1, STAT and AP1. All of these studies indicate that they are important regulatory mechanisms of intracellular Bst2 function or activity.

  HTS analysis can be designed to identify compounds that bind to regulatory sequences in the Bst2 gene. The Bst2 promoter region (about 1 kb or more) is fused upstream of the luciferase gene. Compounds selected after this assay can modulate the level of Bst2 gene expression. In the second screening assay, compounds are selected for inhibitory or stimulatory activity with respect to cell-cell adhesion and inflammatory function of Bst2.

1-3-1. DNA constructs and stable cell lines As reported by Ge et al. (Blood 107: 1570, 2006), forward (5'-ttcaggctagccccccttttcacagatagagaggctcaga-3 '(SEQ ID NO: 75) containing restriction enzyme sites for NheI and XhoI. Using the reverse (5'-ttcacctcgaggcagggagatgggtgacattgcgacactc-3 '(SEQ ID NO: 76)) primer, the Bst2 promoter region spanning 759 bp upstream of the transcription start site and 211 bp of exon 1 is PCR amplified. To construct a DNA vector containing a longer fragment of the Bst2 promoter, 1 kb or more of the Bst2 promoter region is PCR amplified. The amplified product is digested with NheI and XhoI and linked to the corresponding sites in a reporter gene vector that expresses firefly luciferase. This construct is used for high-throughput screening using a luciferase assay.

1-3-2. HTS Dual Reporter Luciferase Assay Using Bst2 Promoter / Luciferase Fusion Construct In the HTS format, mammalian cells are added to the wells of a 384 well plate and the Bst2 reporter gene construct and internal control Renilla luciferase reporter are used using Fugene 6 reagent (Roche). The gene is cotransfected. Luciferase activity is analyzed and standardized using a dual luciferase assay system (Promega).

1-4. High-throughput screening of Bst2 modulators by detecting Bst2-Bst2 interaction using fluorescence polarization technology Bst2 is thought to exist on the cell surface as a homodimer (Otomo et al., Biochem Biophys Res Commun. 1999, 258 (3): 583-91). Bst2 is also thought to require dimerization for activation. Consistent with this, Bst2 decoy protein (extracellular domain of Bst2) was expressed and secreted as a dimer (see FIG. 3, panel B).

  Furthermore, the extracellular domain of Bst2 contains a predicted coiled-coil region that plays a role in Bst2 dimerization. All these results indicate that Bst2 interacts with Bst2.

  Using this property of Bst2 homodimerization, a high-throughput competitive Bst2 binding assay for Bst2 modulators that can inhibit the interaction has been devised as shown below.

  This screening method utilizes one of the most sensitive high-throughput methods for protein-protein interaction studies, the technique of fluorescence polarization (Roehrl et al. Biochemistry 43: 16056, 2004), and the HyperCyt flow cytometry platform. To do. In this method, fluorescently labeled Bst2, Bst2 decoy, Bst2 coiled coil (Bst2CC) or fragments of these proteins are excited by polarized light. In the presence of small molecules, separation of Bst2 from fluorescently labeled Bst2, Bst2 decoy, Bst2CC or fragments of these proteins can be detected by binding competition assays in the HTS format.

1-4-1. HyperCyt
HyperCyt is a previously used automated high-throughput flow cytometry (HTFC) analysis platform whereby cell samples are rapidly aspirated from a microplate and transported to a flow cytometer (Edwards BS Molecular Pharmacology 68: 1301, 2005; Young SM et al. (2005) J Biomol Screen 10: 374-382; Arnold LA et al. Science STKE (2006) 2006: p13). This screening approach allows high-throughput protein-protein interaction assays to be performed in a non-washing homogeneous format that is not suitable with conventional fluorescent plate readers. The HyperCyt platform for HTFC screening has been shown to be a robust, sensitive and highly quantitative technique used to screen lead compound libraries (Ramirez et al., (2003) Cytometry). 53A: 55-65; Kuckuck et al., (2001) Cytometry 44: 83-90).

1-4-2. Fluorescein labeled Bst2 reagent, recombinant Bst2 protein and stable cell line Fluorescein labeled Bst2, Bst2 decoy, Bst2CC or fragments of these proteins are provided. Bst2, Bst2 decoy or Bst2 decoy Fc recombinant protein is expressed and purified. A stable cell line expressing Bst2 is created. If a Bst2 mutant that does not internalize after binding to Bst2 can be identified, use this mutant to create a stable cell line for screening of Bst2 modulators instead of wild-type Bst2. Can do.

1-4-3. HTS Fluorescence Polarization Assay by Detection of Bst2-Bst2 Interaction Fluorescence polarization assay is a fluorescent Bst2, Bst2 decoy, Bst2CC or fragment of these proteins for binding to cell membrane Bst2 or purified Bst2, Bst2 decoy or Bst2 decoy Fc Measure the ability of the test compound to compete with.

  For high throughput assays, chemical libraries are screened in 384 well format. Control wells contain only unlabeled Bst2 protein or buffer. Unlabeled Bst2 decoy, Bst2CC or a fragment of these proteins is added at a concentration greater than 100 times the concentration that completely inhibits the binding of fluorescently labeled Bst2 decoy, Bst2CC or a fragment of these proteins. Other controls that contain only buffer are also prepared. The fluorescence polarization degree of these positive and negative controls is defined as inhibition of recovery of 0% and 100% of Bst2, Bst2 decoy, Bst2CC or fragments of these proteins.

  The addition to the wells is as follows: 1) Test compounds and control reagents (usually 10 uM and above); 2) Bst2 stable cells (107 cells / ml); 3) (after incubation at 4 ° C.) Fluorescein labeled Bst2 decoy, Bst2CC or fragments of these proteins. After an additional 4 ° C incubation, the plates are analyzed by flow cytometry using the HyperCyt platform.

  In other formats, high-throughput analysis can be performed using purified Bst2, Bst2 decoy or Bst2 decoy Fc. Prior to HTS setup, the binding constant and screening concentration of Bst2 are determined. Kd values are determined after conjugation of serial dilutions of Bst2, Bst2 decoy or Bst2 decoy Fc protein to fluorescently labeled Bst2, Bst2 decoy, Bst2CC or fragments of these proteins. Binding is measured using fluorescence polarization (excitation 485 nm, emission 530 nm) using a plate reader. Data is analyzed using a program such as SigmaPlot to determine the Kd value. After determining the Kd value, test compounds are added to the wells. Bst2, Bst2 decoy or Bst2 decoy Fc protein is added, and fluorescently labeled Bst2, Bst2 decoy, Bst2CC or fragments of these proteins are added. Positive and negative controls using unlabeled Bst2, Bst2 decoy, Bst2CC or excess of fragments of these proteins, or buffer only are provided. Fluorescence polarization and fluorescence intensity are measured using a plate reader.

  Test compound inhibition of fluorescent peptide binding is calculated as described in Edwards BS et al. (Molecular Pharmacology 68: 1301, 2005): 100 × [1- (MFI test-MFI inhibition) / (MFI non-MFI). Inhibition-MFI inhibition)], where MFI is the mean fluorescence intensity of the cells in wells containing test compound, inhibition control wells and non-inhibition control wells.

  Following initial screening, compound IC50 values are determined by dose response analysis using competitive binding assays.

1-5. High-throughput screening of Bst2 modulator by detecting Bst2-Bst2L interaction using fluorescence polarization technique 1-5-1. Bst2L expressing cells The HTS assays described below require Bst2L expressing cells or purified Bst2L or Bst2L fragments. One of the Bst2L expressing cells is U937 cells, as shown in our experiments (Figs. 6, 7 and 24). The observation that Bst2 decoy inhibits U937 adhesion to interferon gamma-treated HUVECs indicates that Bst2L is present on the cell surface of unstimulated U937 cells (FIG. 7). Another observation that Bst2 decoy inhibits homotypic aggregation of activated T cells (FIG. 12) or activated U937 cells (FIG. 6) is that Bst2L is the surface of T cells and / or U937 cells both before and after activation. It suggests that it can be expressed above. To support these results, direct binding of U937 cells to purified Bst2 decoy protein is shown (FIG. 24).

  When the Bst2L protein and nucleotide sequence have been identified, purified Bst2L or a fragment thereof, or CHO or COS cells stably transfected with Bst2L can be used in place of U937 cells.

1-5-2. HTS fluorescence polarization assay by detection of Bst2-Bst2L interaction High-throughput binding competition assay to screen Bst2 modulators using the interaction of purified Bst2 decoy (or Bst2) and Bst2L expressing U937 cells (or any Bst2L expressing cells) Is designed as shown below.

  This HTS assay is based on the migration of fluorescently labeled Bst2 or Bst2 decoy from membrane Bst2L on Bst2L-expressing cells such as U937 cells. The fluorescence polarization assay measures the ability of a test compound to compete with a fluorescent Bst2 or Bst2 decoy for binding to membrane Bst2L or purified Bst2L (or fragment).

For high-throughput assays, the addition to the wells is as follows:
Test compounds are added first to the wells, followed by U937 cells. After incubation, fluorescently labeled Bst2, Bst2 decoy or a fragment thereof is added. After an additional 4 ° C. incubation, the plates are analyzed by flow cytometry using the HyperCyt platform.

  In other formats, HTS assays can be performed using purified Bst2L or fragments thereof, and fluorescently labeled Bst2, Bst2 decoys or fragments thereof. Test compounds are added to the wells, Bst2L or Bst2L fragments are added, and fluorescently labeled Bst2, Bst2 decoys or these fragments are then added. Positive and negative controls are set up as described above in the HTS fluorescence polarization assay for detection of Bst2-Bst2L interaction. Fluorescence polarization and fluorescence intensity are measured using a plate reader.

  In other formats, this high-throughput assay can be performed using purified Bst2 or Bst2 decoy and fluorescently labeled Bst2L peptide. Test compound is added to the wells, Bst2 or Bst2 decoy protein is added, and fluorescently labeled Bst2L peptide is added. Positive and negative controls are prepared. Fluorescence polarization and fluorescence intensity are measured using a plate reader.

1-6. High-throughput screening of Bst2 modulators by detecting interactions between Bst2-Bst2 peptidomimetics using fluorescence polarization technology 1-6-1. Bst2 Peptidomimetic Small peptides that bind with high affinity to Bst2 can act as peptidomimetics for Bst2. Such peptides can be identified by phage display methods as shown below. A high-throughput binding competition assay for Bst2 modulators has been devised by detecting interactions between Bst2 and Bst2 peptidomimetics using fluorescence polarization techniques.

1-6-2. Isolation of Bst2 peptidomimetics that bind with high affinity to Bst2 by phage display methods Bst2 peptidomimetics that bind with high affinity to the extracellular domain of Bst2 can be screened by phage display methods. A vast library of peptides can be generated through a combinatorial composite mixture of combinatorially synthesized oligonucleotides in a phage display vector. A linear phage display system where the expressed peptide is presented as a fusion to a phage coat protein is effective in discovering peptide ligands (Devlin et al. Science 249: 404, 1990; Greenwood et al. J. Mol). Biol. 220: 821, 1991; Scott and Smith Science 249: 386, 1990).

  The phage pool is incubated with beads coated with Bst2 decoy protein or control beads, and the positive pool is selected by magnetic separation. In order to recover peptides with binding activity, affinity purification of a population of phage particles on Bst2 decoy beads is used. By sequencing the appropriate site of the DNA of each captured phage, the primary sequence of the peptide that binds to the Bst2 decoy is determined. Bst2 peptidomimetics are further screened in functional assays to select Bst2 peptidomimetics that have activity to stimulate inflammatory responses.

1-6-3. Confirmation of the ability of the Bst2 peptidomimetic to bind to Bst2 Whether the selected peptide has the ability to bind to Bst2 is determined by labeling Bst2L such as U937 cells against immobilized Bst2 decoy or Bst2 decoy Fc protein. Confirmed in binding assays for expressed cells. Different concentrations of Bst2 peptidomimetic are added to this binding assay to measure binding competition.

  In other formats, binding assays can be prepared using immobilized Bst2L-expressing cells, such as U937 cells, and Bst2 decoy Fc or biotinylated Bst2 decoy as a probe.

In other formats, if a Bst2L protein is identified, a ¹²⁵ I-labeled Bst2L binding assay to immobilized Bst2 decoy or Bst2 decoy Fc can be performed.

1-6-4. Confirmation of Bst2 Peptidomimetic Activity in Biological Assays The biological function of Bst2 peptidomimetics can be assessed in many different assays. One such assay is as follows: HUVEC are transfected with an expression vector for Bst2 or an empty vector. 48 hours after transfection, cells are treated with Bst2 peptidomimetic or control peptide. Gene expression of inflammatory mediators and adhesion molecules is analyzed by RT-PCR and protein expression of these genes is measured by immunoblotting. Bst2 peptidomimetics stimulate inflammatory responses in Bst2-expressing HUVECs.

1-6-5. High-throughput screening of Bst2 modulators with fluorescence polarization technology using Bst2 peptidomimetics HTS analysis is performed in a manner similar to that described above. Briefly, Bst2 peptidomimetics are fluorescently labeled. An expression vector for Bst2 is stably introduced into mammalian cells. If a Bst2 variant that does not internalize after binding to Bst2 is known, this Bst2 variant is transfected into mammalian cells to screen for Bst2 modulators.

  For the HTS assay, test compounds are added to the wells. Bst2 expressing stable cells are added, followed by the fluorescently labeled Bst2 peptidomimetic. Fluorescence polarization and fluorescence intensity are measured with the plate reader described above.

  In other formats, purified Bst2 or Bst2 decoy can be used in place of Bst2 expressing stable cells. To determine if the hit is correct, the dose-dependent response of the compound is measured.

2. Second assay to confirm hits after high-throughput screening The initial hits need to be confirmed using a series of profiling analyzes in the drug discovery process. Confirmation of hits by the second assay is to determine whether inhibition or activation by low molecular weight compounds is biologically relevant. The second assay described herein is just a few examples of alternative assays that could be used to demonstrate hit compounds.

2-1. Confirmation of Hits by Bst2-Bst2L Binding Assay The activity of low molecular weight compounds is measured by Bst2-Bst2L interaction as a function of compound concentration in an ELISA format. Biotinylated Bst2 decoy (or Bst2 decoy Fc) is immobilized in the wells of a 96-well plate coated with streptavidin (or coated with anti-Fc antibody). Serial dilutions of selected lead compounds are added to a solution of Bst2L and (if possible) a Bst2L mutant that does not bind to Bst2 decoy as a control and incubated with immobilized Bst2 decoy. Unbound Bst2L is washed from the plate. Bound Bst2L is measured with an anti-Bst2L antibody labeled with horseradish peroxidase, followed by a colorimetric reaction for horseradish peroxidase.

2-2. Hit confirmation by Bst2-Bst2L binding assay using Biacore surface plasmon resonance technology
Bst2-Bst2L binding can be analyzed using Biacore's surface plasmon resonance technique in solution competition. Each compound concentration series is incubated with recombinant Bst2L and then injected onto the chip surface with the captured recombinant Bst2 decoy. Binding is measured at equilibrium and calculated as a percentage of maximum binding.

2-3. Confirmation of Hit by Glutathione S-transferase Pull-down Assay GST-Bst2 decoy protein is expressed and purified in E. coli. Radiolabeled ( ³⁵ S) -Bst2L can be obtained by using the TNT T7 transcription / translation system. Prepare serial dilutions of hit compounds in DMSO. Add 1 ul of each concentration of hit compound to the tube. Bead-containing GST-Bst2 decoy protein is added. Radiolabeled -Bst2L is then added and incubated. The pull-down assay is performed according to the manufacturer's instructions.

2-4. Confirmation of hits by cell-cell adhesion assay using fluorescently labeled cells The cell adhesion assay is performed as described by Edwards et al. (Molecular Pharmacology 68: 1301, 2005). Bst2 cells (stable cells expressing Bst2) are red fluorescent Fura-Red (Invitrogen), Bst2L cells (Bst2L expressing stable cells or U937 cells can be used) are green fluorescent 5,6-carboxyfluorescein Labeled with acetate succinimidyl ester (Invitrogen) and kept on ice throughout the experiment. Incubate 300 μl Bst2 cells (1 × 10 ⁶ cells / ml) and 300 μl Bst2L cells (3 × 10 ⁶ cells / ml) separately in the presence or absence of test compound (final 100 μM) for 5 minutes at 37 ° C. . The cells are then mixed and analyzed with a flow cytometer while the cell suspension is continuously stirred at 37 ° C. and 300 rpm with a magnetic microstir bar. Compounds are added at different concentrations after 90 seconds of agitation to determine the basal level of cell adhesion. Bst2 cells are lysed in two fractions in a flow cytometer: a singlet that is uniformly red fluorescent and a conjugate comprising red / green co-fluorescence (red fluorescent Bst2 cells that adhere to green fluorescent Bst2L cells). . At each indicated time point, the percentage of adherent Bst2 cells is calculated as 100 × (conjugate number) / (conjugate number + singlet number).

2-5. Hit confirmation by luciferase reporter assay Bst2 antagonist or agonist activity can be confirmed by a luciferase reporter assay using n-luc, a plasmid having a plurality of NFkB sites upstream of the luciferase reporter (NFkB). 293T cells are transfected with mammalian expression vectors for (NFkB) n-luc and Bst2. After 48 hours, the cells are treated with various concentrations of the selected compound. After 6 hours of incubation, a luciferase assay is performed and luminescence is measured using a luminometer.

2-6. Confirmation of Hits by Transcription Assay Transcription assays determine whether small molecules inhibit or increase Bst2-mediated signaling in the inflammatory pathway of the intracellular environment. One such assay is as follows. HUVEC are transfected with an expression vector for Bst2 or an empty vector. Forty-eight hours after transfection, cells are treated with various concentrations of hit compound. Gene expression of inflammatory mediators and adhesion molecules is analyzed by RT-PCR and protein expression of these genes is confirmed by immunoblotting or ELISA.

Bst2 and angiogenesis Angiogenesis is the growth of new capillaries. Inflammation can promote angiogenesis, and new blood vessels also enhance tissue inflammation. Thus, while angiogenesis and inflammation are co-dependent processes (Jackson et al. FASEB J 11: 457, 1997), angiogenesis and inflammation can also occur independently of each other. In particular, chronic inflammation can stimulate blood vessel growth. Angiogenesis is required for embryogenesis, tissue recovery after injury, growth and the female reproductive cycle. Angiogenesis has also been implicated in the pathology of various chronic inflammatory diseases such as cancer and psoriasis, diabetic retinopathy, rheumatoid arthritis, osteoarthritis, asthma and pulmonary fibrosis. For example, angiogenesis is required to support the growth of many solid tumors exceeding 2-3 mm in diameter. Recent studies have shown that angiogenesis inhibitors inhibit tumor progression. Furthermore, cancer is not the only disease for which the use of angiogenesis inhibitors can have an effect. Angiogenesis plays an important role in age-related macular degeneration and diabetic retinopathy. These diseases cause blindness when blood vessels infiltrate the retina, cloud it, and eventually destroy it. In fact, vascular blockers (antibodies, low molecular weight compounds) are the latest and most effective treatment for age-related macular degeneration, a major cause of blindness in over 65 people. Angiogenesis inhibitors can reduce inflammation, and chronic inflammation inhibitors can be expected that stimulation of blood vessel growth inhibits angiogenesis derived from inflammatory cells (Stogard et al. J Clin Invest. 103: 47, 1999). Bst2 induces angiogenesis and Bst2 blockers may have anti-angiogenic activity that inhibits angiogenesis.

Delivery For delivery, in addition to conventional routes of administration such as intravenous, intramuscular and intraperitoneal injection, Bst2 blockers can be administered by transdermal attachment and controlled release methods. Bst2 decoy or Bst2 binding by injecting a Bst2 inhibitor bound to a solid matrix that can be encapsulated or degraded or emptied long enough to release the Bst2 inhibitor over a longer period of time than the injection Controlled release of Bst2 inhibitors such as antibodies can be achieved locally or systemically. Bst2 inhibitors can also be applied topically in the form of creams or ointments to treat skin diseases or trauma.

  The compositions of the invention can be administered in pharmaceutically effective amounts. The term “pharmaceutically effective amount” as used herein refers to an amount sufficient for disease treatment that is commensurate with a reasonable risk-to-effect ratio applied in medicine. Effective dosage of the composition depends on the type of disease, disease severity, patient age and sex, drug activity, drug sensitivity, administration time, route of administration, drug excretion ratio, duration of treatment, combined with the composition Drug, and other factors known in the pharmaceutical arts. The compositions of the present invention may be administered as a single therapeutic agent, may be administered in combination with other therapeutic agents, or may be administered sequentially or concurrently with conventional therapeutic agents. May be. This administration may be a single dose or multiple doses. In view of all the factors, it is important to administer at the minimum dose that can give the maximum effect without causing any side effects, and the dose can be predetermined by those skilled in the art.

  The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, in addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such improvements are also within the scope of the appended claims. The following examples are provided for illustration of the invention and not for limitation.

EXAMPLES Example 1 Cell culture Human monocytic cell line U937 (ATCC, U.S; Cat.CRL- 1593.2) and, 37 ° C., in a 5% CO ₂ atmosphere, 10% fetal bovine serum (FBS; Suspension culture was carried out in RPMI-1640 (Gibco-BRL) supplemented with Gibco-BRL), 100 U / ml penicillin (Gibco-BRL) and 100 μg / ml streptomycin (Gibo-BRL).

Human umbilical vein endothelial cell line HUVEC (Cambrex, US; Cat. CC-2517A) in EGM-2 medium (Cambrex, U.S.) supplemented with 10% FBS at 37 ° C., 5% CO ₂ atmosphere. S.) was subcultured. In the examples below, cells were pretreated with 0.5% FBS instead of 10% FBS for 16 hours. According to the given conditions, the cells were pretreated with human recombinant interferon-γ (10 ng / ml, Calbiochem, US) and PMA (1 ng / ml, Cambiochem) or medium for a predetermined time.

  Mouse monocyte cell line WEHI-274.1 (ATCC, Cat. CRL-1679) and mouse endothelial cell line SVEC 4-10 (ATCC, Cat. CRL-2181) were cultured according to the same method as the human cell line. And pre-processed.

Human T lymphocyte cell line Jurkat (ATCC, TIB152 clone) was grown in RPMI-1640 (Gibco-BRL) supplemented with 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin in a 37 ° C., 5% CO ₂ atmosphere. ) In suspension culture.

Protein expression and purification was performed using CHO-S cells (Invitrogen, Cat. 11619-012). CHO-S cells were cultured in F12 / HAM (Gibco-BRL) medium (Gibco-BRL) supplemented with 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin in a 37 ° C., 5% CO ₂ atmosphere. The suspension was cultured.

Example 2 Cloning of Human Bst2 Gene and Mouse Damp1 Gene An expression vector of Bst2 labeled with histidine was constructed as follows. The full length cDNA (NM004335) of the human Bst2 gene was synthesized by Origen Technologies (USA) and amplified by PCR using Pfu ultra HF DNA polymerase (Stratagene) in a volume of 50 μl. The PCR product was cloned into pCMV HA vector (Clontech) using SalI and NotI.

  Vectors for expressing Bst2 and Damp1 decoys were constructed as follows. FIG. 2 shows the positions of the PCR primers used to clone the decoy. A DNA fragment encoding the extracellular region of human Bst2 protein was obtained by PCR, and fused to the N-terminus of the signal sequence P of tPA (tissue plasminogen activator) in order to promote extracellular secretion after expression. In addition, this DNA fragment was fused to the C-terminus of a 6-histidine label in order to measure the expression level of the protein and facilitate purification of the protein. Bst2 decoy did not contain 11 amino acid residues at the C-terminus, nor did it contain a transmembrane region or cytoplasmic region. The PCR product was treated with dimethyl sulfoxide (DMSO; Sigma) at a final concentration of 0.8%, digested with BamHI and XbaI, and further cloned into the pCDNA 3.1 vector (Invitrogen). In another experiment, the codons of the nucleotide sequence of human Bst2 decoy were optimized for mammalian expression systems, and DNA fragments were chemically synthesized.

  The full-length cDNA of mouse Damp1 gene (NM 198095) was obtained by RT-PCR using mRNA isolated from mouse liver. The RT-PCR product was digested with BamHI and XbaI and cloned into pCDNA 3.1 (Invitrogen). The decoy region was determined by amino acid sequence homology analysis between human Bst2 and mouse Damp1. As a result, a vector expressing the soluble Bst2 fragment of SEQ ID NO: 1 and another vector expressing the soluble Damp1 fragment of SEQ ID NO: 2 were obtained.

Example 3 Real-time quantitative RT-PCR
Intracellular expression levels of specific genes were analyzed by real-time quantitative RT-PCR using ABI Prism 7900HT (Applied Biosystems, Foster City, Calif.) And SYBR-Green assay kit. The primers and probes used were designed using Primer Express software (Applied Biosystems).

  Ten ng of single-stranded cDNA was placed in a reaction tube, and multiple TaqMan PCR (50 μl) was performed using TaqMan Universal PCR Master Mix. The relative amount of cDNA of interest was calculated using the comparative cycle threshold (CT) method. PCR products were analyzed by agarose gel electrophoresis.

Relative levels of specific gene A were expressed as changes relative to control samples (untransfected cells). All values are relative to normalization gene B in transfected cells (human GAPDH _{gene), 2-CT (C t1} -C t0, C t1 = C t1A -C t1B, C t0 = C t0A -Ct0B ) Obtained using the calculation method. Each value was obtained from each sample in triplicate. The above experiment was performed to quantify the expression of Bst2 gene and interferon-2.

Example 4 Expression and Purification of Soluble Bst2 Protein Fragment or Damp1 Protein Fragment To express the soluble Bst2 protein fragment or Damp1 protein fragment prepared as described above, vector DNA was transiently transferred into specific animal cells. Or introduced forever. Transient transfection was performed by calcium phosphate (CaPO ₄ ) precipitation as follows. Twenty-four hours before transfection, 7 × 10 ⁶ 293T cells (ATCC) were seeded and cultured in a 150 mm cell culture dish. One hour before transfection, the medium was replaced with IMDM medium (Cambrex) supplemented with 2% fetal calf serum (FBS; GIBCO-BRL). 1.5 ml TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0) containing 75 μg DNA and 250 mM calcium was added to 1.5 ml HEPES buffer (50 mM HEPES, 140 mM NaCl, 1.4 mM Na ₂ HPO ₄ , pH 7). .05), incubated at room temperature for about 1 minute and added to the pre-cultured cells. The cells were incubated at 37 ° C. for 6 hours in a CO ₂ incubator. After removing the DNA / calcium solution, the medium containing no serum was re-supplied to the cells, and after further culturing for 72 hours or more, the medium was collected. Separately, a permanent cell line was established using lipofectamine and dihydrofolate reductase as a selectable marker as follows. 48 hours prior to transfection, 1.35 × 10 ⁶ CHO-DUKX-B11 (dhfr ⁻ ) cells (ATCC) were seeded in 100 mm cell culture dishes and IMDM medium supplemented with 10% FBS. In culture. 0.6 ml of serum-free IMDM medium containing 18 μg DNA was mixed with 0.6 ml of serum-free IMDM medium containing 54 μl Lipofectamine 2000 (Invitrogen) and incubated at room temperature for 45 minutes. The DNA / lipofectamine mixture was supplemented with 8.8 ml of serum-free IMDM medium and added to the precultured cells. The cells were incubated at 37 ° C. for 6 hours in a CO ₂ incubator. The medium was replaced with selective medium, ie IMDM medium containing 10% dialyzed FBS. Cells were further cultured for 72 hours or longer to analyze transiently expressed proteins. The medium was then collected and passed through a 0.2 μm filter (Millipore). The resulting Bst2 decoy protein was analyzed by immunoblotting using anti-Bst2 polyclonal antibody (Roche) or anti-histidine antibody (Roche).

In order to express and purify a large amount of soluble Bst2 protein fragment or Damp1 protein fragment, a host cell line stably transfected with Bst2 or Damp1 expression vector was selected as a production cell line as follows. Expression vectors were transfected into CHO cells lacking the dihydrofolate reductase (DHFR) gene. Since the expression vector has a dhfr gene, dihydrofolate reductase was used as a selection marker. After 48 hours, transfected CHO cells were seeded in 96-well cell culture plates at a density of 1 × 10 ³ cells / well and cultured in medium containing 20 nM methotrexate (MTX) to amplify the DHFR gene. Two weeks later, the medium was collected and subjected to ELISA using an anti-Bst2 antibody, and the expression level of the Bst2 decoy protein of the clone was compared. Clones exhibiting high expression levels were selected and exposed by gradually increasing the MTX concentration to 300 nM to complete gene amplification. Thereafter, media was collected from each clone and ELISA and immunoblotting were performed to finally select the production cell line that showed the highest expression level. Since the Bst2 decoy protein is produced in the medium under serum-free conditions, the expressed protein was purified from the medium collected using a 6-histidine tag added to the C-terminus. The protein was purified by NTA chelate chromatography using a column, NTA chelate agarose CL-6B (Peptron Inc.). The purity of the purified protein was analyzed by electrophoresis and ELISA, and the amount of purified protein was measured by BCA method (Biorad, USA) and ultraviolet spectrophotometry.

  Human Bst2 decoy and mouse Damp1 decoy purified as described above were analyzed by 4-20% SDS-PAGE (FIG. 3, panel A). Treatment with 1% dithiothreitol (DTT) and N-glycosidase F (Sigma) turned the Bst2 decoy into a dimeric glycoprotein (FIG. 3, panel B). The results of the following examples were obtained using a soluble Bst2 protein fragment having the amino acid sequence of SEQ ID NO: 1 and a soluble Damp1 protein fragment having the amino acid sequence of SEQ ID NO: 2 among the prepared decoys.

Example 5-Evaluation of the effect of Bst2 protein on homotypic aggregation of U937 cells Example 5-1 Changes in expression level of Bst2 during aggregation of U937 cells Tested for expression level of Bst2 protein during aggregation of human U937 monocytes did. 1 × 10 ⁶ U937 cells were treated with PMA (2 ng / ml) and LPS (10 μg / ml) for 24 hours to induce homotypic cell aggregation of U937 cells, and a phase contrast inverted microscope (Olympus 1X71, state USA), the degree of homotypic cell aggregation was observed. In order to measure the degree of cell aggregation, the size of the formed cell aggregation was measured as pixel intensity using Adobe's Photoshop software, version 7.0. The standard deviation value shown in the figure was calculated from the average value of six arbitrarily selected aggregates. Thereafter, all used cells were collected, total RNA was isolated, and RT-PCR using a pair of primers of SEQ ID NOs: 3 and 4 was performed to evaluate the expression level of Bst2.

  One hour after U937 cells were treated with PMA and LPS to induce homotypic aggregation, intracellular Bst2 expression increased approximately 3-fold. This increase level was maintained for 24 hours. These results indicate that Bst2 gene expression increases during homotypic aggregation of U937 cells (FIG. 4).

Example 5-2 Effect of Bst2 protein on homotypic aggregation of U937 cells Cells upon overexpression of Bst2 protein to determine whether increased expression of Bst2 gene is essential for homotypic aggregation of U937 cells Aggregation was evaluated.

1 × 10 ⁶ U937 cells previously cultured under the above conditions were seeded in a 96-well cell culture dish (NUNC) for 24 hours with PMA (2 ng / ml, Calbiochem) and LPS (10 μg / ml, (Calbiochem). Next, the degree of homotypic cell aggregation was observed for these cells under a phase contrast inverted microscope (Olympus 1X71, state, USA).

  Bst2 protein itself did not induce aggregation of U937 cells, but PMA / LPS treatment stimulated homotypic aggregation of U937 cells. With transient overexpression of Bst2, the homotypic aggregation of U937 cells stimulated with PMA / LPS increased approximately 4-fold (FIG. 5). These results indicate that Bst2 expression promotes homotypic aggregation of activated monocytic leukocytes.

Example 5-3-Inhibition of homotypic aggregation of U937 cells using Bst2 decoy In order to confirm whether an increase in Bst2 gene expression is essential for homotypic aggregation of U937 cells, the action of Bst2 protein was suppressed. Cell aggregation was evaluated.

  U937 cells were pre-treated with PMA and LPS to induce cell aggregation and were treated with serial dilutions of medium containing Bst2 decoy (decoy medium) that was transiently expressed in CHO-S cells. In Bst2 decoy, it was found that U937 cell aggregation induced by PMA and LPS was reduced by 50% as compared to a culture of CHO-S cells not expressing Bst2 decoy (control medium) (FIG. 6). These results indicate that Bst2 decoy inhibits homotypic aggregation of U937 cells.

Example 6-2 Evaluation of the effect of Bst2 protein on atypical aggregation between two different cell types Example 6-1 Inhibition of aggregation between U937 and HUVECs using Bst2 decoy HUVECs (1-5 × 10 ⁴ cells / ml) was seeded in a 12-well cell culture dish. One day later, the medium was changed to low serum medium containing 0.5% FBS and the cells were pretreated with interferon-γ (IFN-γ; Calbiochem) at a final concentration of 10 ng / ml for 24 hours. The pretreated HUVECs were then cultured with U937 cells (2 × 10 ⁶ cells / ml, 500 μl) for 4 hours at 37 ° C. The co-culture was washed 3 or 4 times with phosphate buffer, and the remaining cells were fixed with 4% paraformaldehyde and observed under a microscope.

  In U937 cells, media containing Bst2 decoy was added to the cultures, showing decreased binding to IFN-γ treated HUVECs. In a control medium without Bst2 decoy, U937 cells efficiently bound to HUVECs treated with IFN-γ and formed atypical cell aggregation. Control medium or albumin treatment did not affect cell aggregation (FIG. 7). In FIG. 7, “normal medium”, HUVECs not pretreated with IFN-γ, did not bind to U937 cells. In contrast, HUVECs treated with IFN-γ bound to U937 cells and formed atypical cell aggregation. HUVECs obtained from cultures pretreated with IFN-γ and treated with medium containing Bst2 decoy protein showed reduced aggregation by U937 cells. Treatment with basal medium or albumin did not affect cell aggregation (FIG. 7). In FIG. 7, “normal medium” indicates an FBS-containing general medium, and “control medium” indicates a culture solution of cells that do not express Bst2 decoy protein. In addition, atypical cell aggregation is inhibited depending on the concentration of Bst2 decoy (FIG. 8).

Example 6-2- Inhibition of aggregation between U937 and HUVECs using Bst2 siRNA Various siRNA molecules specifically acting on Bst2 were constructed (QIAGEN). All 23 Bst2 specific siRNA molecules were constructed.

  The following test results were obtained using an siRNA composed of an antisense RNA strand complementary to the Bst2 mRNA encoded by the sequence of SEQ ID NO: 5 and a sense RNA strand complementary to the antisense strand. .

  HUVECs were transfected with an expression vector for Bst2, treated with IFN-γ or without IFN-γ, and then transfected with Bst2 siRNA. This cell was evaluated for U937 cell adhesion.

  Bst2 expressed extracellularly promoted the binding of U937 cells to HUVECs treated with or without IFN-γ. B937 siRNA treatment decreased U937 cell adhesion (FIG. 9). Combined with the data shown in FIG. 29 which shows the inhibitory effect of Bst2 siRNA on cell adhesion between untransfected HUVECs and U937 cells, these results suggest that Bst2 is involved in HUVEC-U937 adhesion The

Example 7-Evaluation of the effect of Bst2 protein on T lymphocyte homotypic aggregation and activity of aggregation Example 7-1-Effect of Bst2 overexpression on T lymphocyte homotypic aggregation and IL-2 production Human Jurkat T cells were It was induced and activated to form homotypic cell aggregation as follows.

Jurkat cells (5 × 10 ⁵ cells / ml) were incubated with anti-CD3 monoclonal antibody (OKT3: 10 μg / ml, BD Pharmingen) for 20 minutes at 4 ° C., followed by anti-mouse immunoglobulin polyclonal antibody at 37 ° C. for 1 hour. When incubated with (25 μg / ml, Zymed), cell aggregation occurred and the cells were activated and induced to produce interleukin-2 (IL-2) (FIGS. 10 and 11). Inducing green fluorescent protein (GFP) overexpression according to a similar method had no effect. In contrast, when Jurkat cells were transfected with a vector overexpressing Bst2 and induced to activate, homotypic cell aggregation increased (FIG. 10, panel A). IL-2 mRNA levels upon T cell activation were measured by real-time RT-PCR (Example 3). IL-2 mRNA expression was increased approximately 2-fold under Bst2 overexpression compared to GFP overexpression (FIG. 10, panel B).

Example 7-2-Effects of Bst2 decoy and Bst2 siRNA on homotypic aggregation of T lymphocytes and IL-2 production Jurkat cells were pretreated with Bst2 decoy 30 minutes prior to activation and using anti-CD3 monoclonal antibody And was further evaluated for inhibition of cell aggregation. The cells were treated with serial dilutions of animal cell cultures containing a relative amount of Bst2 decoy. Aggregate size was expressed as a percentage of the untreated group to aggregate size.

  Pretreatment with Bst2 decoy under activated conditions significantly reduced Jurkat cell aggregation. In addition, expression that was increased 3-fold by activation of Jurkat cells was reduced again to basal levels by Bst2 decoy treatment (FIGS. 11 and 12).

  The data presented here shows that Bst2 is important for inflammation and immunity. Blocking Bst2 function may reduce disease induced by inflammation. In patients with immune disorders, such as AIDS patients and immunocompromised patients, increased immune signals may be beneficial. A bivalent fusion protein composed of Bst2 decoy and other molecule Y, which may be a protein or compound, promotes the interaction between cells expressing Bst2 ligand and other cells expressing receptors for Y. It can act as an adapter that transmits signals between them. See FIG.

Example 8-Evaluation of Bst2 decoy cells in a mouse model of asthma Example 8-1-Induction of asthma in mice A mouse model of asthma was generated by sensitizing mice (C57B6, 8 weeks old) with ovalbumin. did. Specifically, mice were first sensitized for 5 consecutive days by injecting ovalbumin intranasally. Three weeks later, the mice were again sensitized intranasally with ovalbumin for 5 consecutive days. One week after the second sensitization, mice were challenged with ovalbumin three times every 24 hours to induce asthma. Here, Bst2 decoy was injected intravenously into mice 30 minutes prior to sensitization with ovalbumin, and was further injected into mice 30 minutes prior to the first sensitization and final ovalbumin injection. Three days after the last injection, serum samples, lung tissue, etc. were collected from the mice.

Example 8-2-Change in the number of precipitated immune cells induced by Bst2 decoy In mice sensitized with ovalbumin and treated with Bst2 decoy, the total number of infiltrating cells and each cell type (neutrophil, eosinophil) The number of spheres and lymphocytes was significantly reduced with bronchoalveolar lavage fluid (FIG. 13).

Example 8-3 Effect of Bst2 Decoy on Cytokine Production Bst2 or Dmap1 decoy was injected into a mouse model of asthma induced by sensitization and challenge with ovalbumin and cytokines (interleukin-4 (IL-4 ), Interleukin-5 (IL-5) and interleukin-13 (IL-13)) expression levels were measured as follows. After bronchoalveolar lavage, lung tissue was excised from the mouse and protein was isolated from the lung tissue. Cytosolic protein was isolated using a lysis buffer containing NP-40. Isolated proteins were separated on SDS-PAGE gels and transferred to PVDF membranes by wet transfer method. The blots were divided into several primary antibodies (anti-IL-4 antibody (Setotec Inc.), anti-IL-5 antibody (Santa Cruz Inc.), anti-IL-13 antibody (R & D Inc.), and anti-actin antibody. (Sigma Inc.)) in a 1: 1000 dilution. The bound primary antibody was detected with an HRP-conjugated secondary antibody (anti-rabbit HRP-conjugated IgG) using ECL reagent. It was found that the levels of cytokines such as IL-4, IL-5 and IL-13 were elevated in the lung tissue of asthmatic mice induced by sensitization and challenge with ovalbumin. In addition, when Bst2 decoy protein was injected into asthmatic mice sensitized with ovalbumin, cytokine levels decreased with increasing dose of decoy protein. From these results, it is shown that Bst2 decoy protein has a therapeutic effect for asthma (FIG. 14).

Example 9-Evaluation of functional similarity between human Bst2 protein and mouse Damp1 protein The human Bst2 protein and mouse Damp1 protein have about 35% amino acid similarity. In this regard, it was tested whether these two proteins show functional similarities in in vitro cell-cell adhesion assays and in vivo mouse asthma models. Human Bst2 and mouse Damp1 proteins were tested for inhibitory effects on adhesion between HUVECs and U937 cells treated with IFN-γ according to the same method as described in Example 6.

Example 10-Preparation of anti-Bst2 polyclonal antibody Purified Bst2 and Damp1 decoy protein expressed in CHO-S cells were mixed with Ribi adjuvant in a 1: 1 ratio and injected into rabbits at 2-week intervals. During immunization, blood samples were collected and tested for antibody production. Serum samples were obtained from rabbits after three immunizations. Anti-Bst2 polyclonal antibody was purified by affinity chromatography using a column in which Bst2 protein was bound to an immobilized carrier.

Example 11-Preparation of PEG-conjugated form for improved metabolism of Bst2 decoy Example 11-1-Preparation of PEG-conjugated form Two PEGs: (1) aldehyde PEG and (2) succin PEG conjugation was performed with succinimidyl carbonate PEG (FIG. 17). First, aldehyde PEG conjugation was performed as follows. 1 mg of Bst2 decoy protein was dialyzed in 0.1 mM phosphate buffer (pH 7.5) and a 30-fold molar ratio of (mPEG12000-OCH ₂ COGly-Gly) ₂ (2,4-diaminobutyric acid) -PEG ′ -After mixing with NHS, it was incubated for 2 hours at room temperature with stirring. Separately, for carbonate PEG conjugation, 1 mg of Bst2 decoy protein was dialyzed in 0.1 mM phosphate buffer (pH 5.0), mixed with 20-fold molar ratio of succinimidyl carbonate PEG, and stirred. For 2 hours at room temperature. After completion of the reaction, Bst2 decoy conjugated with PEG was isolated and purified using a size exclusion column (Superdex-200, Pharmacia), and dialyzed against 50 mM phosphate buffer (pH 7.4).

Example 11-2- Accelerating effect of PEG-conjugated form on in vivo stability of Bst2 decoy The PEG-conjugated form of Bst2 decoy prepared in Example 11-1 was transformed into a 7 week old male Sprague-Dawley. Rats were injected at a dose of 0.4-2 mg / kg into the tail vein. The negative control group was injected with an equal volume of physiological saline. An equal amount of Bst2 decoy protein was used as a positive control. Blood samples were taken before drug administration and 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours and 24 hours after drug administration from the jugular vein using a cannula. collected. The collected blood samples were analyzed by ELISA. 96 well plates were coated with anti-Bst2 decoy antibody (100 ng / ml in PBS) for more than 8 hours at 4 ° C. and blocked with albumin in PBS for 2 hours at 37 ° C. The plate was reacted with an appropriate dilution of rat serum or Bst2 decoy (standard sample) at 37 ° C. for 2 hours. The plates were then reacted for 2 hours at 37 ° C. with a monoclonal antibody (mAb, Roche Inc. conjugated with horseradish peroxidase) that recognizes the histidine label added to the C-terminus of the Bst2 decoy. After washing well, the plate was treated with peroxidase substrate and the absorbance at 450 nm was measured. Quantification of PEG-conjugated Bst2 decoy present in blood was performed using a standard sample (FIG. 18). In FIG. 18, “201B-H” indicates a human Bst2 decoy sample, and “201B-HP” indicates an aldehyde PEG-conjugated human Bst2 decoy sample.

Example 12-Expression and distribution of Bst2 in inflammation-related diseases Tissues from patients with various inflammatory diseases were obtained to examine the expression and distribution of Bst2. The obtained tissues include asthmatic lung tissue, arteriosclerotic artery, psoriatic skin lesions, Crohn's intestinal tissue, ulcer intestinal / colon tissue, chronic active gastritis There is stomach tissue, and cecal tissue of patients with acute appendicitis. Each tissue was selected as a representative lesion showing a typical inflammatory phenotype.

  For asthma, a paraffin block of lung tissue, prepared by fixing lung tissue in 10% formaldehyde and embedding the tissue in paraffin, was sliced 1.5 μm thick and placed on a glass slide. The slide was stained with hematoxylin and eosin to examine changes in lung tissue with allergen and drug administration. Histostaining was performed using the polyclonal antibody prepared in Example 10. Other tissues were prepared similarly. Compared to normal tissues, Bst2 protein was overexpressed in inflammation-related diseases. Bst2 was detected in immune cells, vascular endothelial cells, and other cell types (FIG. 19).

Example 13 Cell Culture Cell culture was performed as described in Example 1.

Example 14-Construction of expression vectors for human Bst2 decoy and Bst2 decoy Fc fusions Fusion constructs were prepared based on the expression vector pCDNA 3.1 or other commercially available dhfr vectors.

  FIG. 20 shows a schematic of Bst2 decoy and other Fc fusions. These are representative schematics of potential fusion proteins. Referring to FIG. 20, FIG. 20A shows the Bst2 decoy itself, and FIG. 20B shows the hinge of an IgG heavy chain Fc with a separate expression of Bst2 decoy so that a dimer of Bst2 decoy is formed at the head of each fusion protein. The Bst2 decoy fused to the -CH2-CH3 moiety is shown. FIG. 20C shows that Bst2-κ fusions can stably form Bst2 decoy dimers at the head of each fusion protein stabilized via native IgGκ chain-heavy chain disulfide bonds. The form expressed in coordination with the IgG Fc fusion is shown. FIG. 20D shows a form in which Bst2 decoy-IgG Fc is expressed without other Bst2 dimerization counterparts. Dimerization of the hinge-CH2-CH3 portion of the fusion occurs each time the IgG Fc portion is expressed by a natural disulfide bond between these chains.

  FIG. 21 shows a vector map of the Bst2 decoy-IgG Fc fusion protein described above. Representative expression vectors representing expression vectors for IgG1 and IgG2 Fc fusions are shown. FIG. 21A shows a Bst2 decoy (dBst2). This Bst2 decoy expression vector is obtained by PCR-cloning an XbaI site 5 ′ from the starting point of the decoy protein, an N-terminal tPA signal peptide and a C-terminal His-tag, and then a 3′-end BamHI site. This insert was cloned into pcDNA3.1 cut with XbaI and BamHI. FIG. 21B shows the dBst2-IgG1Fc fusion. The hinge-CH2-CH3 region of the IgG1 heavy chain was PCR-cloned and fused to the C-terminus of the Bst2 decoy at the 5 ′ XhoI and 3 ′ NotI sites; this insert was pcDNA3.cleaved with XhoI and NotI. Cloned in 1. FIG. 21C shows the dBST-κ fusion. The constant region of the IgG kappa light chain was PCR-cloned and fused to the C-terminus of the Bst2 decoy at the 5 ′ XhoI and 3 ′ NotI sites; the insert was in pcDNA3.1 cut with XhoI and NotI. Cloned into. (D) dBST-IgG2HC fusion. The hinge CH2-CH3 region of the IgG2 heavy chain was PCR-cloned and fused to the C-terminus of Bst2 decoy at the 5 ′ XhoI and 3 ′ NotI sites; this insert was pcDNA3.1 cut with XhoI and NotI. Cloned into.

Example 15-Construction of Vector A histidine-labeled Bst2 decoy expression vector was constructed as follows.

  Immunoglobulin gene fragments were cleaned by PCR from the following human blood cell cDNA library (Clontech): Fc region (hinge, CH1 and CH2 regions) of human IgG1 heavy chain (Genbank No: BC089417., Primers 1, 2) , Constant region of human immunoglobulin kappa chain (Genbank No: BC067092, primers 3, 4), and constant region of human IgG2 heavy chain (CH1-hinge-CH2-CH3) (Genbank No: AJ294731, primers 5, 6). The sequences of PCR primers used for fragment cloning are as follows.

Example 16-Human Bst2 decoy-Fc fusion construct (IgG1, 2, and 4)
Three different constructs of the human Bst2 decoy-Fc fusion were cloned into the expression vector pCDNA3.1 (Invitrogen). DNA encoding the extracellular region of the human Bst2 protein was obtained by PCR and fused at the N-terminus of the signal peptide sequence of tPA so as to promote extracellular secretion after expression. In addition, the Bst2 extracellular fragment was fused at the C-terminus to the IgG1, Fc region of IgG1, IgG2, and IgG4 or the constant region of the kappa chain. The overlapping PCR product was digested with XhoI and NotI and cloned into the vector pcDNA3.1 (Invitrogen). These fusion fragments were generated by overlap PCR and the primers were as follows: “pcDNA-dBST2-IgG1Fc”, “pcDNA-dBST2-kappa”, and “pcDNA-dBST-IgG2HC” or It was called pcDNA-dBST2-IgG4Fc.

Example 17-PCR cloning and fusion strategy The PCR cloning and fusion strategy is shown in FIG. The following primers were used.

Example 18-Expression of Soluble Decoy-Fc Fusion Protein Soluble Bst2 decoy-Fc fusion protein was prepared after transient transfection as described in Example 4. Stable cell lines expressing Bst2 decoy and Bst2 decoy Fc fusion protein were established as described in Example 4. Mass expression and purification were performed as described in Example 4.

Example 19-PAGE of purified Bst2 decoy and other Fc fusions
The Fc fusion protein was purified from the medium. After concentration by ultrafiltration, a two-step chromatography method was used, including Protein A affinity chromatography (Amersham Biosciences, MabSelect) and size exclusion chromatography (Amersham Biosciences, Superdex 200).

  The Fc fusion protein was loaded onto a protein A packed column pre-equilibrated with PBS buffer (1.06 mM potassium dihydrogen phosphate, 155.17 mM sodium chloride, 2.97 mM disodium hydrogen phosphate, pH 7.4). . The column was washed with excess PBS to remove contaminants. The bound antibody was eluted with a low pH buffer, such as 50 mM glycine-HCl using a step gradient and neutralized with an equal volume of 1 M Tris (pH 8.0).

  The next size exclusion chromatography step was used to remove immunoglobulin multimers. The purified antibody multimer mixture was loaded onto a Superdex 200 column pre-equilibrated with PBS (pH 7.4). The linear flow rate of the buffer was selected from speeds ranging from 50 cm / h to 150 cm / h.

  FIG. 23 shows a representative PAGE gel (4-12% gradient gel, Invitrogen) stained with Coomassie showing various Bst2 fusion proteins after affinity purification. FIG. 23B shows that high molecular weight multimeric forms can be removed by appropriate size exclusion chromatography.

Example 20-Direct binding of Bst2 decoy to immune cells Flat-bottom 96-well plates were coated with Bst2 decoy with sodium bicarbonate (100 mM, pH 9.5) for 2 hours at 37 ° C. The plate was washed with PBS (pH 7.4) and incubated with 1% bovine serum albumin (BSA) at 25 ° C. After rinsing with PBS (pH 7.4) containing 1 mM CaCl ₂ and 0.5 mM MgCl ₂ , U937 cells (1 × 10 ⁶ / ml) were added to each Bst2 coated well. After 2 hours incubation at 37 ° C., unbound cells were removed by gently washing twice with RPMI 1640 medium (Gibco-BRL), and bound cells were fixed with 2% paraformaldehyde for 20 minutes, washed, Stained with 0.5% crystal violet. After 30 minutes at 25 ° C., the plates were washed with PBS and bound cells were counted.

  FIG. 24 shows the direct binding of Bst2 decoy to U937 cells. U937 cells bound to wells containing Bst2 decoy but not BSA.

Example 21-Half-life in plasma of Bst2 decoy-Fc fusion Figure 25 shows the half-life in plasma of Bst2 decoy or Fc fusion. Bst2 decoy proteins fused to various stabilized IgG Fc regions show increased serum stability, as shown in a representative pharmacokinetic plot for two Bst2 decoy-IgG1 fusions compared to Bst2 decoy alone. It was.

  To measure the half-life in plasma of Bst2 decoy or other Fc fusions, rats (Sprague-Dawley male) were surgically implanted with an intravenous catheter. During the subsequent period, the catheter was connected to an infusion pump. Protein samples were infused by hand over 1 minute with a catheter flushed with heparinized saline to reduce the risk of clotting. The end of the infusion was called time zero. Blood samples (0.4 ml) were taken from the catheter at various time points. Plasma was centrifuged and a sandwich ELISA was performed to determine the plasma concentration of Bst2 decoy or other Fc fusion protein. The wells of a 96 well plate were coated with 5 μg / ml of rabbit anti-BST2 polyclonal antibody in 50 mM carbonate buffer (pH 9.2) (100 μl / well) and blocked with 1% BSA / PBS. Each plasma sample diluted to fall within the linear range of the standard curve was incubated at 25 ° C. for 90 minutes. After washing with PBS, the wells were incubated with goat anti-human IgG (1: 50,000 dilution, Fc specific, Sigma, Cat. No. A-0170) labeled with horseradish peroxidase for 1 hour at room temperature, Treated with TMB substrate (Pierce). The plate was read at 450 nm with a plate reader and the data was analyzed using a four-parameter curve-fitting program. In the standard curve for each different protein, each purified protein standard was used in a solution of 1% BSA, 1% rat preimmune serum at the appropriate concentration.

Example 22-Inhibition of Bst2 decoy-Fc fusion in binding of Bst2 decoy to cells Bst2 decoy-IgG Fc fusion protein inhibits U937 cell binding to Bst2 decoy coated cell culture dishes in a concentration dependent manner This indicates that the Bst2 decoy-IgG Fc fusion protein is effective.

Competitive inhibition of the Fc fusion protein in the binding between BST2 decoy and cells was measured as follows. Flat bottom 96-well plates were coated with Bst2 decoy (50 μg / ml) using sodium bicarbonate (100 mM, pH 9.5) for 2 hours at 37 ° C. The plate was washed with PBS (pH 7.4) and incubated with 1% bovine serum albumin (BSA) at 25 ° C. After rinsing with PBS (pH 7.4) containing 1 mM CaCl ₂ and 0.5 mM MgCl ₂ , U937 cells (1 × 10 ⁶ / ml) were added to each Bst2 coated well. Prior to addition, cells were pre-incubated with BST2 decoy-Fc fusion protein for 2 hours at 37 ° C. Bound cells were counted as described in Example 20.

Example 23-Effect of Bst2 decoy-Fc fusion on asthma mouse model An asthma mouse model was made as described in Example 8-1.

  The effect of Bst2 decoy-Fc fusion on immune cell invasion was evaluated in the same manner as described in Example 8-2. When mice sensitized with ovalbumin were treated with Bst2 decoy, the total number of infiltrating cells decreased, and in particular, the number of neutrophils other than macrophages, eosinophils and lymphocytes in bronchoalveolar lavage (BAL) It decreased (FIG. 27).

  IL-4, IL-5 and IL-13 expression was measured in a mouse asthma model as described in Example 8-3 after injection of Bst2 decoy or Bst2 decoy Fc fusion protein. These cytokine levels were reduced, suggesting that Bst2 decoy protein has a therapeutic effect on asthma (data not shown).

Example 24-Generation of human-mouse chimeric Bst2 mice Human-mouse chimeric Bst2 mice were generated using a type of construct as illustrated in FIG. For use in homologous recombination in mouse embryonic stem (ES) cells or other mouse cells, the extracellular domain and C-terminus of mouse BST2 (DAMP-1) were replaced with the extracellular domain and C-terminus of human BST2. The target vector is shown. Suitable homologous recombination includes homologous recombination in the flanking arm indicated by (x), and cells that have undergone appropriate homologous recombination are resistant to selection (eg, neomycin or G418 or Other selectable markers are used). Cells subjected to appropriate homologous recombination are selected by selecting with neomycin (G418), which is an example of a specific marker, and then cleaning by Southern blotting or PCR. Other selectable markers may be used. In order to remove neomycin or other markers in the target vector, recombinant ES cells may be transfected with an expression vector of Cre recombinase before producing a chimeric mouse, or a mouse expressing Cre recombinase Chimeric mice may be mated. Chimeric mice can be generated using recombinant ES cells by standard techniques for generating knockout, knock-in or other types of transformed mice. Since the extracellular portion of human-mouse chimeric BST2 is the same as the extracellular domain of human BST2, mice can be used to test human BST2 antibodies in preclinical studies. Another option is to use a strategy similar to that described herein, not only in the coding region of the extracellular domain as shown in this figure, but also in the coding region of the human BST2 gene. May replace the entire coding region.

Example 25—Experimental method for combination therapy in vitro HUVECs were cultured for 6 hours with Bst2 siRNA or control siRNA or without transfection and cultured in 12-well plates for 24 hours. , Treated with or without IFNγ. In some experiments, cells were treated with crude media containing Bst2 decoy or mouse anti-human ICAM1 antibody. After washing with PBS (phosphate buffered saline), U937 cells were resuspended in serum-free medium at 2 × 10 ⁶ cells / ml. The assay was initiated by adding 200 μl of U937 cells to HUVECs to a final volume of 1 ml. After 4 hours at 37 ° C., unbound U937 cells were removed by washing the plate 3 times with PBS. Bound cells were fixed by adding 4% paraformaldehyde in PBS, and the bound cells were counted under a microscope at various fields. All statistical analyzes were performed with Excel, and statistical significance was assessed by Student's t test. In some experiments, RNA samples were obtained from HUVECs after treatment with IFNγ and / or siRNAs, and real-time polymerase chain reaction (RT-PCR) analysis was performed.

Example 26—Results FIG. 29 shows that endogenous Bst2 is required for atypical aggregation between endothelial cells (HUVEC) and monocytes (U937) after stimulation with IFNγ. To demonstrate that endogenous Bst2 blockade is important for inhibition of heterotypic aggregation, HUVECs were treated with Bst2 siRNA to suppress endogenous expression of Bst2 prior to IFNγ treatment (10 ng / ml, 24 hours). FIG. 30 shows that Bst2 siRNA treatment or ICAM1 siRNA treatment does not affect ICAM1 expression or Bst2 expression in IFNγ-treated HUVEC, respectively. RT-PCR analysis was performed.

  As shown in FIGS. 29 and 30, although both Bst2 and ICAM1 are thought to be involved in cell adhesion, it is unclear whether these two proteins function by crosstalk in overlapping or independent non-overlapping pathways. It is. In anti-adhesion molecular therapy, combined inhibition of two adhesion proteins that function in the redundant pathway may be less effective than when two proteins that function in the non-overlapping pathway are used. When ICAM1 siRNA is added to the Bst2 siRNA reaction (B + I siRNA), ICAM1 siRNA does not further reduce Bst2 expression, suggesting that ICAM1 is not required for Bst2 expression. Similarly, when Bst2 siRNA is added to a reaction mediated by ICAM1 siRNA (I + B siRNA), ICAM1 expression does not decrease further, suggesting that Bst2 is not required for ICAM1 expression. These data indicate that Bst2 and ICAM1 may mediate cell adhesion via non-overlapping pathways.

  FIG. 31 shows that combination therapy with Bst2 siRNA and ICAM1 siRNA shows an additive effect in the heterotypic adhesion assay. Furthermore, FIG. 32 shows a quantitative analysis of the dose-dependent response of anti-ICAM1 or Bst2 decoy and the dose-dependent response of anti-ICAM1 and Bst2 decoy in atypical adhesion assays.

  Based on the siRNA experiment in FIG. 31, a cell adhesion assay was performed in the presence of mouse anti-human ICAM1 antibody or Bst2 decoy. Conditioned medium containing Bst2 decoy was used. After SDS-PAGE, the amount of Bst2 decoy in the crude cell supernatant was roughly estimated by comparing the band intensities of His-tagged Bst2 decoy and protein standards.

  FIG. 33 shows that the combined treatment of Bst2 decoy and anti-ICAM shows an additive effect on cell adhesion. Sub-optimal amounts of Bst2 decoy (100 ng / ml) and anti-ICAM1 (1 μg / ml) were used. Cell adhesion was completely inhibited to control levels using both Bst2 decoy and anti-ICAM1.

  The results shown in FIGS. 29 to 33 suggest that combined treatment with a blocker of Bst2 blocker and other immune / inflammatory mediators may be beneficial in the treatment of many immune inflammatory diseases. Such blockers used in conjunction with a BSt2 blocker include CTLA4-Ig or TNFα, IL6, IL1, LFA1, α4 integrin, ICAM1 or VCAM1 blockers. In addition, combination treatment of Bst2 decoy-Fc or anti-Bst2 with cyclosporine or glucocorticoids that suppress immune inflammatory responses is beneficial for many diseases that require transplantation or corticosteroid treatment, respectively It is suggested that there is a possibility.

  In preclinical studies in rat or mouse models, rat or mouse monoclonal antibodies against many of the rat or mouse proteins described above (TNFR, IL6R, IL1R, LFA1, α4 integrin, ICAM1, VCAM1) are commercially available (Abcam or other Company). CTLA4-Ig may have to be manufactured in-house. For protein targets for which monoclonal antibodies are not commercially available or where it is not desirable to use monoclonal antibodies, a soluble receptor decoy protein of the corresponding protein target, such as TNFR-Fc (soluble TNFR1), is used in combination therapy in animal models. May be used.

Example 27-The possibility that Bst2 is its own ligand Bst2 is known to form homodimers after activation. Consistent with that, the Bst2 decoy appears to be expressed as a dimer or more multimer. This dimerization property of Bst2 suggests that Bst2 may serve as its own ligand in cell-cell interactions.

  To test this possibility, U937 cells are incubated with anti-Bst2 antibody and U937 cells treated with the antibody are added to HUVEC after interferon treatment. In another example, U937 cells were treated with Bst2 siRNA or control siRNA, and U937 cells treated with this siRNA were added to HUVEC after interferon treatment.

  If Bst2 on U937 cells is required for cell-cell interaction, U937 cells treated with anti-Bst2 or Bst2 siRNA cannot bind to HUVEC. These results indicate that Bst2 on U937 cells interacts with Bst2 on HUVEC for adhesion to identify Bst2 as one of the Bst2L proteins.

Example 28-Identification of Bst2 L using a genomic wild type full length cDNA (GFC) array and fluorimetry Bst2 L is a GFC-array (genomic wild type full length cDNA array) (Origene Technologies, Rockville, MD). Can be screened using. The GFC-array is a set of transfection-ready (transfection-ready) cDNA plasmids of the mammalian expression vector pCMVsport6 (GIBCO) arrayed (arrayed) in a disposable 384-well plate. Each well contains 62.5 ng of single stranded lyophilized cDNA, a concentration optimized for reverse transfection into various cells. Reverse transfection involves over 24,000 transfection-ready full-length human cDNA clones. GFC arrays also provide a subset of human gene arrays, such as transmembrane protein arrays and drug discovery genes (enzyme genes / which drugs are targeted for receptors).

  These two subset arrays (or hole set arrays) can screen for binding activity to Bst2 decoy Fc. In short, individual genes are transfected into human cells such as 293T, CHO cells, COS cells or other mammalian cells by high throughput transfection methods. 20 μl of serum-free medium containing Fu gene 6 (Roche) is added to each well of a 384-well plate containing 62.5 ng of distinct cDNA. 40 microliters of 20% FBS DMEM medium containing 293T, CHO cells, COS cells or other mammalian cells is seeded in each well.

After 48 hours at 37 ° C. with 5% CO ₂ , an optimized amount of Bst2 decoy Fc is added to each well and labeled with an anti-Fc antibody labeled with FITC. Fluorescence is analyzed using microplate fluorometry (384 well format). Each well that shows positive is retested with both Bst2 decoy Fc and control Fc. After screening with the GFC-array, each positive well is confirmed by standard transfection with a specific cDNA plasmid. All cDNAs in the GFC-array can be used individually at OriGene (Rockville, MD).

Example 29-Isolation of Bst2 L by Expression Cloning Expression cloning methods are required to identify large in vitro cell sources for Bst2L for comparison with plasmid cDNA expression libraries. This cDNA expression library is screened for Bst2 L using Bst2 decoy Fc or biotinylated Bst2 decoy by the panning method.

Example 29-1-Identification of large amounts of in vitro cell source (cell source) for Bst2L Recombinant Bst2 decoy-Fc fusion protein is used to identify putative cell lines or primary cells that express a large amount of Bst2L on the surface Is done. Various cell lines and primary hematopoietic cells are screened. Cell sources for Bst2 L that can be used include, but are not limited to, human U937 cells, mouse RAW 264.7 cells, primary hematopoietic cells, B cells, dendritic cells, T cells such as endothelial cells and fibroblasts, Includes mononuclear leukocyte / macrophage cell lines. Mouse and rat cell lines are searched as wells using rat Bst2 decoy-Fc fusion protein and mouse Damp1 decoy-Fc fusion protein, respectively. Since the human Bst2 decoy and the mouse Damp1 decoy function alternately in the cell adhesion assay and the in vivo ovalbumin-induced asthma model, as shown in FIGS. 15 and 27, this cell source line is the cell line used. Or, regardless of the primary cell type, it can be screened by both mouse Damp1 decoy-Fc and human Bst2 decoy-Fc fusion protein.

  Cell line after staining with FITC-labeled human Bst2 decoy-Fc or mouse Damp1 decoy-Fc fusion protein followed by secondary antibody staining with eg goat F (ab ′) anti-human IgG secondary antibody Alternatively, primary cells are screened for the presence of Bst2L by indirect immunofluorescence or FACS (fluorescence activated cell sorter) analysis (Smith CA, Gruss HJ, Davis T, Anderson D, et al. 1993, cell). 73, 1349-1360). The cells are then analyzed by FACS.

Example 29-2-Verification of Bst2 L cell source through FACS analysis of Bst2 decoy-Fc labeled with FITC- The source cell line labeled as described above was labeled with FITC- as compared to control Ig. Bst2 shows significant and specific binding to decoy-Fc. Secondary antibody alone and purified human IgG Fc alone do not bind to the surface of the cell source strain. These results indicate that the decoy-Fc binding is due to the Bst2 or Damp1 decoy part, not the Fc part of the probe. To account for further binding specificity, binding should be suppressed by unbound Bst2 decoy rather than an irrelevant control protein.

Example 29-3- Validation of Bst2 L cell source through visualization of Bst2L with ¹²⁵ I-Bst2 decoy (Fc) For validation, the cell source is either in the presence or absence of excess non-radioactive Bst2 decoy protein, ¹²⁵ Incubate with I-labeled -Bst2 decoy or Bst2 decoy Fc. There is a description of iodination by the lactoperoxidase method (Urdal et al., 1988, J. Biol. Chem. 263: 2870-2877). The cells are then incubated with a cross-linking agent [bis (sulfosuccinimidyl) suberate].

  Proteins are solubilized with 1% Triton X-100 cocktail, exposed to SDS-PAGE and visualized by autoradiography. In finding cell line sources and evaluating as described in (Examples 29-1, 29-2 and 29-3), after many cycles by FACS analysis of the brightest stained cell source, numerous Bst2 L It would be possible to obtain a mutant cell line that expresses.

Example 29-4-Design of a plasmid expression library from a cell line source for panning For the isolation of Bst2 L, a cDNA expression library was designed from the Bst2 L cell source identified and confirmed above. To do. A directed (direct) oligo-dT primed plasmid cDNA library is designed with cell source mRNA and ligated to a mammalian expression vector (Invitrogen).

  This library is divided into pools of 1000 clones, and plasmid DNA of each pool is obtained. DNA from individual pools by the method of Seed and Aruffo (Seed B, Arufo A. Proc. Natl. Acad. Sci. USA, 1987, 84: 3365) and modifications of Lacey et al. (Cell, 93: 165, 1988). Are transfected into COS7 cells. After about 48-72 hours, stain with human Bst2 decoy-Fc fusion for about 1 hour, wash and fix to paraformaldehyde or glutaraldehyde. The culture is treated with an enzyme-linked secondary antibody, such as an alkaline phosphatase-conjugated goat anti-human IgG (Fc specific) antibody, and the immune complex is detected, for example, by assaying alkaline phosphatase activity. One positive pool was selected and plasmid DNA was transformed into E. coli. Prepare for E. coli transformation. E. E. coli transformants are used for the next enrichment cycle. By repeating this cycle, a large amount of unique cDNA encoding the Bst2 L protein can be concentrated, and a single-stranded cDNA clone can be produced. The selected plasmid DNA is then transfected into COS7 cells and immunostained with either a human IgG Fc domain, a human Bst2 decoy-Fc fusion protein or an irrelevant Fc fusion protein, and then FITC-conjugated secondary antibody and Immunostain. At this stage, only the human Bst2-Fc fusion protein can bind to the cell source. These results indicate that the cell source (or cell line) encodes Bst2 L and displays Bst2 on the cell surface.

  In another approach, panning plates are coated with anti-human IgG1 Fc polyclonal antibody (Jackson Immunoresearch) and then with Bst2 decoy-Fc. Blocking with bovine serum albumin may be essential. COS7 cells are then added to the plate as described above, and adherent cells are suspended by treatment with EGTA and EDTA. The rest of the panning method is the same as above.

Example 29-5 Isolation of Bst2 L through Expression Cloning Using Biotinylated Bst2 Decoy as a Probe and Panning In another approach, if the cell source contains very high levels of Bst2 L, Bst2 L Can be isolated using biotinylated Bst2 decoy as a probe by following the method of Harada et al. (Proc. Natl. Acad. Sci. 1990, USA 87: 857). In this method, biotinylated Bst2 decoy can be enriched by cross-linking Bst2 L expressing cells and panning Bst2 L-expressing cells on anti-biotin antibody-coated plates. It has been reported that cross-linking is essential, because without cross-linking cells do not bind to the panning plate. Design of the cDNA library and transient transfection into COS7 cells are performed as described above (see Example 29-4). After 48-72 hours, cells are detached by incubating with PBS containing 5 mM EDTA. Biotinylated Bst2 decoy is added and crosslinked with cells. Cross-linked cells are added to panning plates coated with anti-biotin antibody plasmid. DNA is recovered from the cells bound to the plate. used to transform E. coli. The amplified plasmid DNA is used for the next enrichment cycle until it yields a single stranded clone. COS7 cells transfected with one of these clones will then specifically bind to ¹²⁵ I-labeled Bst2 decoy.

Example 29-6 Isolation of Bst2 L Full-Length cDNA After Panning In expression cloning, full-length cDNA cloning is essential because the DNA insert obtained after panning can be a short cut cDNA ( Examples 29-4 and 29-5 (reference).

  A commercial cDNA library (Clontech) is searched by first using a short cDNA selected from the above procedure as a probe. Bst2L full-length cDNA is obtained by screening a cDNA library from a cell source strain using a short cDNA as a probe. Northern blot analysis of mRNA from cell source strains may indicate Bst2 L transcription. Commercially available Northern blots (Clontech) can also be used to visualize transcription.

  After obtaining full-length cDNA, nucleotide sequencing is performed. Search for signal peptide sequences, potential (potential) kinase domains, or other interesting domains. If a signal peptide is detected in the nucleotide sequence, it is tested whether Bst2 L has been released into the medium. Bst2 L is epitope labeled at the C-terminus (eg, hemagglutinin) and 293T cells are transfected with an expression vector for Bst2 L-tag (HA). Western blotting of cell extracts or aged media is probed with anti-tag (HA) antibody. When released into the aged medium, a smaller band is detected in the aged medium than is observed with the cell extract. Soluble protein affinity purification and N-terminal sequence analysis of the soluble protein indicate the cleavage site.

Example 30-Direct purification of rat Bst2 L or Damp1 L from a large amount of animal tissue source (or cell line) and search for homology to Bst2 L However, cell line (or cell culture) sources are not convenient, or It is very expensive and cannot provide sufficient samples for biochemical properties and purification. Thus, alternative tissue sources from animals can be pursued. Animal Bst2L, such as rat-, dog-, rabbit-Bst2 L or Damp1 L, can be initially identified for subsequent human homology searches. After identifying large amounts of tissue source in rats, dogs, rabbits, mice or other animals, animal Bst2 L can be identified using direct purification methods. The first step in this method is to identify a large amount of in vivo tissue source of Bst2 L in animals. Regardless of which animal species are used, the method described below is illustrated using rats.

The distribution (distribution) of Bst2-specific binding activity in rat tissues is investigated by incorporation studies of ¹²⁵ I-Bst2 decoy-BSA or RSA (rat serum albumin) (Yang et al. J. Exp. Med 174: 515 1991). A modification of the following method, the method used to isolate the receptor for the glycation end product (RAGE), is described with ¹²⁵ I-Bst2 decoy-RSA as a binding probe. Once uptake studies have described the major sites of Bst2 L, direct purification, including affinity purification steps using Bst2 decoy-BSA Sepharose 4B columns, is performed using solubilized and fractionated membrane proteins. All column fractions are performed by analyzing binding activity by solid phase Bst2 decoy binding assay (see below). At the final stage of purification, protein bands are excluded and electron elution is performed for amino acid sequence analysis. Human homologues can be searched and later cloned.

Example 30-1-BST2 Decoy In vivo tissue distribution of binding activity
By measuring the separation of ¹²⁵ I-labeled Bst2 decoy-RSA (rat serum albumin) or -BSA (bovine serum albumin), the in vivo animal tissue source of Bst2 L can be identified. For tissue distribution studies, formaldehyde modified with Bst2-RSA or Bst2-BSA is a method described in other studies by incubating RSA or BSA with formaldehyde (Horiuchi et al. J Biol Chem 261: 4962, 1986). Prepare as follows. The protein is then radioiodinated. Similarly, normal RSA or BSA is iodinated to a comparative specific activity (specific activity). Freshly born rats are labeled with ⁵¹ Cr and allowed to correct tissue associated with blood-related radioactivity.

The distribution of Bst2-specific binding activity in rat tissues is examined by a ¹²⁵ I-Bst2-RSA (BSA) uptake study. Either ¹²⁵ I-Bst2-RSA (BSA) or ¹²⁵ I-normal RSA (BSA) is injected intravenously with ⁵¹ Cr-labeled RBC (red blood cells). Blood aliquots are drowned at several intervals, various organs are removed, and radioactivity is irradiated. Specificity of Bst2 ligand uptake in organs is assessed by injecting animals with excess unlabeled Bst2-RSA (BSA) prior to administration of labeled Bst2. RBC is dissolved in water and the protein is precipitated in 20% TCA. Tissue and blood isotope rates are described in Williamson et al. Calculate according to the formula described in Diabetes 36: 813 (1987). The total organ count is corrected to a blood related count.

  The tissue accumulation of Bst2-RSA (BSA) is not affected by pre-injection of unrecognized RSA (BSA), while pre-treatment of unrecognized rats is , Bst2-RSA (BSA) accumulation is reduced. The uptake of Bst2-RSA (BSA) is retained in a small amount in other major organs with or without unlabeled competitors. Given these criteria, this organ is presented as a rich potential source for the isolation of Bst2-binding proteins.

Example 30-2 Confirmation of Bst2 L In Vivo Tissue Source through Solid Phase Binding Assay and Ligand Blotting Assay The uptake studies showed the major site of BST2 decoy protein sequestration and the potential tissue source of Bst2 L, and the tissue membrane protein of the tissue Are prepared by standard protocols specific for tissues or organs. The binding activity of tissue extracts can be explained by solid phase binding assays as described below and ligand blotting assays with ¹²⁵ I-Bst2 decoy. These assays confirm and evaluate the in vivo tissue source of Bst2 L.

Solid phase binding assay A solid phase binding assay is required to facilitate the isolation of Bst2 from tissue. The solubilized membrane proteins with detergent immobilized on ^{nitrocellulose,} and probed the ligand of specifically binding activity 125 I-Bst2 decoy -RSA or ¹²⁵ I-Bst2 decoy -Fc. This ligand binds to the ¹²⁵ I-Bst2 decoy in a dose-dependent manner in a saturated manner, and binding is inhibited by Bst2 antibodies and / or unlabeled Bst2 decoy-Fc or Bst2 decoy. Expression of Bst2 L in transfected cells also allows cells to bind to ¹²⁵ I-Bst2 decoy in a dose-dependent manner while being saturated. Using cell membrane preparations solubilized with detergents such as from other organs, the same solid phase Bst2 binding assay can behave to confirm an in vivo source of Bst2L.

Ligand Blotting Assay to Visualize Bst2 L from the Identified Tissue Source A ligand blotting assay to visualize the Bst2 L band from the identified tissue source is performed. Proteins obtained from identified tissue sources of Bst2L are electrophoretically separated on SDS-PAGE and made into nitrocellulose cell membranes incubated with ¹²⁵ I-BST2-BSA (or Bst2 decoy Fc), and ligand binding is Assessed by autoradiography.

Example 30-3-Direct Purification of Bst2 L from Solubilized Cell Membrane Preparation of In Vivo Tissue Source As described above, after identification and confirmation of an in vivo tissue source of Bst2 L, direct purification of Bst2 L increases Bst2 L activity. It can be performed using a solid phase Bst2 decoy binding assay (see Example 30-2) as monitored. Cell membrane preparations from animal tissues (Example 30-1) or Bst2 L cell source strain (human or other species) (Example 29-1) are used. A variety of purification steps can be used including column chromatography and affinity chromatography. It is preferred to employ a Bst2 (Bst2 decoy) -BSA Sepharose 4B column for affinity purification after one or two crude purification steps such as DEAE column or Sephadex column. The protein bound to the affinity column is eluted, concentrated and analyzed for ¹²⁵ I-Bst2 (Bst2 decoy) -BSA binding activity. Subsequently, a preparatory electrophoresis method is performed. The protein band is removed and electrophoretic elution is performed for N-terminal amino acid sequence analysis. Human homologues can be identified based on rat, dog or rabbit Bst2 L sequence or mouse Damp1L sequence.

Example 31-Isolation of Bst2L through the yeast two-hybrid system Isolate using a yeast two-hybrid system that relies on reconstitution of the GAL4 transcriptional activator in S. cerevisiae (Fields S and Song OK, 1989, Nature 340: 245-246). For example, a commercially available library obtained from activated human T cells (Clontech) can be screened with a bait containing the extracellular domain of Bst2 using a commercially available yeast two-hybrid kit.

Example 32-Validation of isolated Bst2 L through in vitro binding assay The above isolated Bst2 L (Examples 28-31) specifically binds Bst2 (Bst2 decoy) in vitro. Bst2 (Bst2 decoy) -Bst2 L interaction can be determined by a number of different assays, and various examples of such assays are described below. In some embodiments, COS7 cells are transfected with an expression vector comprising a full-length cDNA and the presence of unlabeled Bst2 decoy (Bst2 decoy-Fc) or an unrelated protein (unrelated protein-Fc). Incubated with various concentrations of ¹²⁵ I-labeled Bst2 decoy-Fc in the absence or absence.

  Unlabeled Bst2 decoy (Bst2 decoy Fc) completely inhibits the binding of radiolabeled Bst2 decoy-Fc. These results may suggest Bst2 L that specifically binds to physically active Bst2, Bst2 decoy or Bst2 decoy-Fc. Subsequently, the binding data is analyzed to determine the affinity and number of sites per cell as described above (Munson PJ, Roddard D, 1980, Anal. Biochem. L 107: 220-239).

  In other embodiments, the Bst2-Bst2L interaction can be determined by FACS analysis. 293 cells, CHO cells or COS cells are transiently transfected with Bst2 L. After 24-48 hours, the cells are incubated with recombinant biotinylated Bst2 decoy Fc for 1 hour. The cells are further incubated with phycoerythrin-conjugated streptavidin (Gibco BRL) for 30 minutes and analyzed by fluorescence activated cell sorting (FACS).

  In other aspects, Bst2-Bst2 L interaction can be determined by co-immunoprecipitation assay. Purified Bst2 L is incubated with Bst2 decoy Fc and immunoprecipitated with protein A sepharose. The precipitate is lysed by SDS-PAGE and visualized by immunoblotting with anti-Bst2 L.

In other embodiments, recombinant Bst2 L is produced, eg, E. coli. E. coli and ¹²⁵ I-labeled Bst2 L were immobilized on a nylon filter after SDS-PAGE, Bst2, Bst2 decoy or Bst2 decoy-Fc wild type, deletion mutant and non-reducing. Exposed to the control protein. ¹²⁵ I-labeled Bst2 L recognizes the Bst2 protein. This assay confirms direct binding of Bst2-Bst2 L in vitro. When various deletion mutants of Bst2, Bst2 decoy or Bst2 decoy Fc protein are employed, the binding domain of Bst2 that binds to Bst2L is also determined.

Example 33-In vitro function of isolated Bst2 L Cells treated with recombinant Bst2 L can lead to an inflammatory response. Cells containing HUVECS are treated with recombinant Bst2 L, inflammatory cytokines including interferon gamma or a combination of Bst2 L and cytokines.

  The cytokine production of these cells and U937 adhesion to these cells is measured. Bst2 alone or in combination with inflammatory cytokines enhances the inflammatory response and cell-cell adhesion, and Bst2 decoy or Bst2 decoy-Fc is expected to inhibit these sclerosis in vitro. Similarly, T cell activity and proliferation assays can be used for in vitro functional testing of Bst2 L. These data suggest that Bst2 L mediates cell-cell interactions directly, and that Bst2 and Bst2 L are key regulators of the immune inflammatory response. These assays can be continued using rat or mouse cells to see if human Bst2 L functions in the rat or mouse system. These data suggest that Bst2 L mediates cell-cell interactions directly, and that Bst2 and Bst2 L are key regulators of the immune-inflammatory response.

Example 34-In vivo function of isolated Bst2 L Mice or rats are injected with recombinant Bst2L, Damp1L or rat Bst2 L. After injection, in vivo inflammatory parameters such as cytokine release are assessed. It is expected that Bst2 L (Damp1L) injection will cause an inflammatory response. These inflammatory responses are inhibited by injection of Bst2 (Damp1) decoy Fc or anti-Bst2 (Damp1) antibody. In other approaches, anti-Bst2 L antibodies also exhibit anti-inflammatory effects. Such anti-Bst2 L antibodies can be used as other therapeutic agents that inhibit Bst2-Bst2 L interaction.

Example 35-Biochemical and biological properties of Bst2 ligand Isolated Bst2 L meets the following biological criteria:

Measurement of the binding properties of full-length Bst2 L protein COS7 cells was performed by transfecting with an expression vector containing full-length cDNA and incubating with various concentrations of ¹²⁵ I-labeled Bst2 decoy-Fc to determine the radioactivity of the cell binding. Measured. Competition with unrecognized Bst2 or Bst2 decoy-Fc, irrelevant protein or irrelevant protein-Fc, completely inhibits the binding of radiolabeled Bst2 decoy-Fc. These results suggest that Bst2 L specifically binds to physically active Bst2, Bst2 decoy or Bst2 decoy-Fc. Binding data was analyzed and Munson PJ, Rodbard D, 1980, Anal. Biochem. Determine the affinity and number of sites per cell as described in 107: 220-239.

Determination of the ligand-binding domain of Bst2 using ¹²⁵ I-labeled Bst2 L as a probe Recombinant Bst2 L is produced, for example E. coli and ¹²⁵ I-labeled Bst2 L can be used as described in Chen et al. (Chen et al., 1995; J. Biol. Chem. 270: 2874-2878). It is exposed to a control protein immobilized on a nylon filter after non-reducing.

Example 36-Construction of Bst2 / Damp1 Oriented Fab Library Human Bst2-decoy or mouse Damp1-decoy proteins expressed in CHO cells are added to adjuvant (RIBI's) until saturation of antibody titers specific for Bst2 / Damp1 antigen. Or rabbits (New Zealand White) were inoculated by injection of the appropriate amount of Freund's incomplete / complete. The inoculated rabbit antibody titer was determined by enzyme immunoassay using a horseradish peroxidase (HRP) -conjugated anti-His antibody that recognizes a histidine tag at the C-terminus of the decoy protein.

For preparation of the Fab-phage display library, total RNA was prepared from rabbit bone marrow and spleen inoculated with TRI reagent. First-strand cDNA was synthesized using Superscript II First-strand synthesis system using oligo (dT) priming (Invitrogen). First-strand cDNA from each rabbit was subjected to the first PCR using the Expand High Fidelity PCR System (Roche Molecular System), 10 primer combinations and rabbit VH coding for amplification of the rabbit _VL coding sequence. A 4-primer combination for sequence amplification was used. Human Cκ and C _H 1 coding sequences were amplified from Fab. Antisense primer consists of human and V _L and V _H coding sequence of the rabbit to C _k, CH1 coding sequence and the hybrid rabbits designed to fuse / human sequence. In the second round of PCR, the first variable region rabbit V _H overlapped with human constant CH1, and the first variable region rabbit VL overlapped with human constant Cκ. In the third round of PCR, the chimeric light chain product and the chimeric heavy chain fragment were joined by overlap extension PCR.

  The PCR product resulting from digestion with SfiI was ligated to the phagemid vector pComb3X (gene bank AF268281) and transformed into XL1-Blue / F '. After absorption of helper phage VSCM13, followed by addition of PEG and NaCl, the phage library was obtained from the overnight culture medium.

Example 37-Panning of Fab library of anti-Bst2 or anti-Damp1 antibody A total of a fourth panning was performed. For high affinity antibody clones to Bst2 and Damp1, a dynal bead (DYNAL, Cat. No. 143.01) panning method using the resulting chimeric Fab phage library was used.

Dynalbead M270 epoxy was coated with Bst2 decoy, Damp1 decoy or bovine serum albumin (BSA) for 6-24 hours at 37 ° C. Beads coated with Bst2 decoy were washed with PBS (1.06 mM primary potassium phosphate, 155.17 mM sodium chloride, 2.97 mM dibasic sodium phosphate, pH 7.4) and 0.5% tween in PBS. 0.5% BSA was suspended in PBS. To remove non-specific binding, pre-incubation was performed with beads coated with BSA from the Bst2 phage library. The pre-washed phage pool was incubated with Bst2-beads for 2 hours at room temperature and washed several times with 0.5% Tween 20 in PBS by magnetic separation to remove non-specifically bound phage. Specific binding phage were eluted by incubation with 0.1 M sodium citrate (pH 3.0, 0.45 ml) twice for 10 minutes, and 1 M Tris-HCl (pH 9.5, 0.1 ml) was added. Neutralized. The eluted phages were infected with exponentially growing XL1-Blue F ′ and amplified with helper phage VSCM13 overnight. Phages were prepared by precipitation with 4% PEG and 3% NaCl (w / v) and suspended in 1% BSA and 0.02% NaN ₃ in PBS buffer. Each number of output phage pools was anti-HA-horseradish peroxidase (Roche, Cat No 2 013 819). Was monitored by phage ELISA. The Damp1 decoy specific phage pool was selected with the same protocol as the Bst2 specific above.

Example 38-Screening of Fab libraries of antibodies specific for both Bst2 and Damp1 For selection to react with clones with both Bst2 and Damp1, single-stranded phage clones were 30 g / ml tetracycline, 50 ug / ml. Inoculated in 2xYT broth containing carbenicillin and 1% glucose and cultured overnight at 37 ° C. The culture suspension was subcultured on a 96 deep well plate with a culture medium containing 30 g / ml tetracycline, 50 g / ml carbenicillin 2xYT and amplified using helper phage VSCM13 and kanamycin. After overnight culture, phage suspensions were obtained by centrifugation at 3000 rpm for 30 minutes and used in a Bst2 / Damp1 binding assay in an ELISA format. Each well on a 96-well maxisorp plate (Nunc) was coated overnight with 4 g Bst2 decoy or 1 g Damp1 decoy at 4 ° C., 37 ° C. for 2 hours, TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Inhibited by incubation with 5% BSA. Subsequently, 100 μl of phage suspension was added for 1 hour at 37 ° C. Each well was washed with 0.05% Tween 20 in TBS (7.4 pH) and added with 100 μl of horseradish peroxidase conjugated anti-HA antibody at 37 ° C. for 1 hour. After the washing, 200 l OPD (o-phenylenediamine dihydrochloride, 0.4 mg / ml, Sigma) solution was added, and then 50 ul of 3M sulfuric acid (50 μl) as a stop solution was added. The results are shown in FIG.

Example 39-Expression of selected antibodies The positive phage clones obtained above were analyzed by DNA sequencing and selected based on sequence alignment. See FIG.

  For expression in the overall IgG1 form, each phage Fab DNA fragment was cloned into the expression vectors pCDH and pCDK from pCDNA3.1 (Invitrogen). pCDH is an intermediate cloning vector for expression of full-length IgG heavy chains. After digestion with EcoR1 and Not1 restriction enzymes, amplification was performed from a cDNA library of whole blood cells (Clontech) using primers R1-CH1 and CH3-Not1 cloned into EcoR1 and Not1 sites of pcDNA3.1. The secreted full-length IgG heavy chain was reconstructed by fusing the secretory signal for tPA5 'to the heavy chain variable region through overlapping PCR cloning by first PCR of the tPA signal.

  An initial PCR is performed on the tPA signal peptide using primers R1-tPA5 and tPA3 from the library used above and Fab_CH1_Rev and Fab-specific primers (Ra_Hv_Fw1 to Ra_Hv_Fw9) are used for the variable region. By fusing the secretory signal for tPA5 ′ to the heavy chain variable region through overlapping PCR cloning by performing PCR on the variable region and CH1 derived from phagemid used to express the fragment A secreted full-length IgG heavy chain was constructed; these two PCR fragments were then fused through an overlapping PCR reaction with primers R1-tPA5 and Heavy_CH1_Rev, and E Digested with oR1 and Age1, cloned into pCDH digested with the same enzymes.

  pCDK is made by cloning the light chain with primers H3-light and light-Xba1, digesting the PCR product with HindIII and XbaI, and cloning into pcDNA3.1 digested with the same enzymes It is a vector for expression of lgG light chain. Initial PCR is performed on the tPA signal peptide using primers R3-tPA5 and tPA3 from the library used above, using specific primer pairs for the variable regions (Ra_Kp_F1 to 6 and Ra_Kp_Rva to d). Fusing the secretory signal for tPA5 ′ to the light chain variable region through overlapping PCR cloning by performing PCR on the variable region and CK from phamidid used to express the Fab fragment Secreted full-length IgG light chain was constructed; these two PCR fragments were then fused through an overlap PCR reaction with primer H3-tPA5 and a specific light chain 3 ′ primer, and HinDIII and Digested with siWI, it has been cloned into pCDK that has been digested with the same enzyme.

Each phage Fab DNA fragment was cloned into the expression vector pCDNA3.1 (Invitrogen) for expression in the whole IgG1 form. In order to express the selected monoclonal antibody (mAb, IgG1), vector DNA was transiently or stably introduced into mammalian cells. Transient transfection was performed by precipitating calcium phosphate (CaPO ₄ ) as follows. One day before transfection, 293T (ATCC) 7 × 10 ⁶ cells were seeded and cultured on 150-mm cell culture plates. One day before transfection, the medium was replaced with IMDM medium (Cambrex) supplemented with 2% fetal bovine serum (GIBCO-BRL).

A buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0) containing 75 μg DNA and 250 mM calcium in 1.5 ml and an equal volume of HEPES buffer (50 mM HEPES, 140 mM NaCl, 1.4 mM Na ₂ HPO ₄ , pH 7.05). ). The mixture was incubated at room temperature for about 1 minute and applied to pre-cultured cells. The cells were incubated at 37 ° C. for 6 hours in a CO ₂ incubator.

  After removing the DNA / calcium solution, the cells were added to a serum-free medium and further cultured for 72 hours or more, and the medium was collected. Each mAb was purified from the culture medium using protein affinity chromatography (Amersham Biosciences, MabSelect). The cultured medium was pre-loaded onto a protein-packed column equilibrated with PBS buffer (1.06 mM primary potassium phosphate, 155.17 mM sodium chloride, 2.97 mM dibasic sodium phosphate, pH 7.4). The column was washed with PBS buffer to remove approximately 20 column volumes of contamination. Bound antibody was eluted with a low pH buffer such as 50 mM glycine-HCl using a step gradient and neutralized with a volume equivalent to 1 M Tris (pH 8.0). The purified protein sample was subjected to gel electrophoresis in 4-20% native PAGE (4-20% native PAGE, Invitrogen). See FIG. 38 for purified protein in gel.

Example 40-Comparative Binding Assay (In Vitro)
Comparative inhibition of mAbs specific to Bst2 or Damp1 in binding between BST2 decoy and cells was measured as in Example 22.

Example 41 Effect of mAbs on Asthma Mouse Model An asthma mouse model was prepared as described in Example 8-1. The effect of anti-Bst2 / Damp1 antibody on immune cell invasion was evaluated as described in Example 8-2. In mice sensitized with ovalbumin and mice treated with the respective mAb, the total number of invasive cells increased in bronchoalveolar lavage (BAL) after treatment with anti-Bst2 / Damp1 antibody (FIG. 39). Anti-Damp1 antibody 2-15 did not significantly inhibit immune cell invasion. One possibility is that the 2-15 monoclonal antibody binds strongly to the Damp1 decoy and does not accurately cover the potential Damp1L binding site.

Example 42-Diagnostic Method for Measuring Inflammatory Conditions Bst2 mRNA expression is decreased in inflammatory conditions. Quantitative PCR in cells and tissues isolated from objects of interest Measurement of Bst2 mRNA levels using real-time PCR or Northern blots yields valuable information on the inflammatory status of these cells and tissues. Immunoblotting using antibodies specific to Bst2, or alternatively using immunofluorescence microscopy and FACS (fluorescence activated cell sorter) using fluorescently-labeled antibodies that can bind to Bst2 on the cell membrane Measurement of Bst2 protein by epidemic blotting can also yield information about the inflammatory status of these cells. Frequently cell membrane proteins such as Bst2 are anchored, yielding soluble Bst2 fragments that circulate in the body. Bst2 such as serum and urine circulating in the body is quantified with specific antibodies to circulate Bst2 fragments using commonly used methods such as radioimmunoassay (RIA) and ELISA. it can. Quantification to circulate Bst2 fragments reflects the host's inflammatory status and may be useful for diagnostic and therapeutic purposes. All citations cited herein are incorporated by reference throughout the specification.

  Those skilled in the art will recognize and explore at least routine experimentation and have equivalents to the embodiments of the invention described herein. Such equivalents are included within the scope of this claim.

FIG. 1 is an amino acid sequence alignment showing sequence homology between human Bst2 and mouse Damp1. 2A-2B show the arrangement of PCR primers used in the process of cloning human Bst2 decoy and mouse Damp1 decoy in an expression vector. 3A-3B show the results of electrophoretic analysis of human Bst2 decoy and mouse Damp1 decoy. FIG. 4 shows the expression pattern of the Bst2 gene during homotypic aggregation of U937 cells. FIG. 5 shows the effect of promoting Bst2 overgrowth during homotypic aggregation of U937 cells. 6A-6E show the effect of Bst2 decoy on homotypic aggregation of U937 cells. Figures 7A-7G show the effect of Bst2 decoy on intercellular adhesion between human vascular endothelial (HUVEC) cells and U937 cells. Figures 8A-8F show the effect of dose-dependent Bst2 decoy on cell-cell adhesion between HUVEC and U937 cells. Figures 9A-9G show the effect of Bst2 siRNA on cell-cell adhesion between HUVEC and U937 cells. FIGS. 10A-10B show the effects of Bst2 overexpression upon Jurkat cell aggregation and interleukin-2 (IL-2) production in Jurkat cells. FIGS. 11A-11I show the effect of Bst2 decoy and Bst2 siRNA on Jurkat cell aggregation. 12A-12B are graphs showing the effect of Bst2 decoy on Jurkat cell aggregation and IL-2 production. FIG. 13 shows changes in the number of immune cells precipitated during Bst2 decoy treatment. FIG. 14 shows decreased cytokine levels upon Bst2 decoy treatment. Figures 15A-15D show the functional similarity between human Bst2 and mouse Damp1. FIGS. 16A-16D show the inhibitory effect of Bst2 decoy and mouse Damp1 decoy in mouse ovalbumin-induced asthma. FIG. 17 shows the PEG moiety used in the preparation of the PEG-linked form of Bst2 decoy. FIG. 18 shows the improved metabolic decline of PEG-conjugated Bst2 decoy. FIG. 19 shows the expression and distribution of Bst2 in inflammation-related diseases. Figures 20A-20D show schematics of the Bst2 decoy fused to the Fc region. A, Bst2 decoy itself; B, Bst2 decoy fused to the hinge-CH2-CH3 portion of IgG heavy chain Fc; C, Bst2 fusion protein stabilized through naturally occurring IgG kappa chain-heavy chain disulfide bond; D, Bst2 decoy-IgG Fc was expressed without other Bst2 dimers. 21A-21B are representative vector maps of the Bst2 decoy-IgG Fc fusion protein of FIG. 21C-21D are representative vector maps of the Bst2 decoy-IgG Fc fusion protein of FIG. FIG. 22 shows the PCR-cloning and fusion strategy. Figures 23A-23B show PAGE (polyacrylamide gel electrophoresis) of purified Bst2 decoy and other Fc fusions. A, Representative PAGE gel stained with Coomassie (4-12% gradient gel, Invitrogen) representing various Bst2 fusion proteins with affinity purification. B. PAGE after size exclusion chromatography. Figures 24A-24B show the direct binding of Bst2 decoys to immune cells on Bst2 coated plates; and B, BSA coated plates. FIG. 25 shows the plasma half-life of Bst2 decoy or Fc fusion. FIG. 26 shows the inhibitory effect of Bst2 decoy-Fc fusion on binding between Bst2 decoy and cells. Figures 27A-27D show the effect of Bst2 decoy-Fc fusion in a mouse model of asthma. Figures 28A-28B show the generation of human-mouse chimeric Bst2 mice. A. Mouse (top, black) and human (bottom, gray) genomic loci. Exons are shown as rectangular boxes. The end point of the transmembrane region is indicated by an arrow and the initiating methionine (ATG) is indicated by an asterisk. The physical distance throughout the exon encoding is shown below the genomic locus. The figure is not drawn to scale. B. Production strategy of chimeric human-mouse BST2. FIGS. 29A-29E show that endogenous Bst2 is required for atypical aggregation between endothelial cells (HUVEC) and monocyte cells (U937) after stimulation with IFNγ. A, control; B, IFNγ inflammation stimulation; C, IFNγ inflammation stimulation + control siRNA; D, IFNγ inflammation stimulation + Bst2 siRNA; E, quantitative analysis of Bst2 siRNA results of AD. FIG. 30 shows that Bst2 siRNA treatment or ICAM1 siRNA treatment does not affect ICAM1 expression or Bst2 expression, respectively, in IFNγ-treated HUVEC. RT-PCR analysis was performed. FIGS. 31A-31G show the combined treatment of Bst2 siRNA and ICAM1 siRNA and show the additive effect in atypical adhesion assays. A, control; B, IFNγ inflammation stimulation; C, IFNγ inflammation stimulation + control siRNA; D, IFNγ inflammation stimulation + Bst2 siRNA; E, IFNγ inflammation stimulation + ICAM1 siRNA; F, IFNγ inflammation stimulation + ICAM1 siRNA + Bst2 siRNA; G, AF Quantitative analysis of Bst2 siRNA and ICAM1 siRNA results. Figures 32A-32M show the dose-dependent response of anti-ICAMl or Bst2 decoy in atypical adhesion assays. A shows control; B, C, D, E, and F show increasing doses of IFNγ inflammatory stimulus + ICAM-1 antibody; G shows IFNγ inflammatory stimulus + control BSA; IFNγ inflammation stimulation + control IgG is shown; I, J, K, and L indicate increasing doses of IFNγ inflammation stimulation + BST2 decoy. M shows a quantitative analysis of the results of a dose-dependent response of anti-ICAM1 and Bst2 decoy from AL. FIGS. 33A-33C show that the combination of Bst2 decoy and anti-ICAM treatment has an additive effect on cell adhesion. Sub-optimal doses of Bst2 decoy (100 ng / ml) and anti-ICAM1 (1 ug / ml) were used. Cell adhesion was completely inhibited relative to the control level when both Bst2 decoy and anti-ICAM1 were used. FIG. 34 shows the relative expression level of Bst2 mRNA after cytokine treatment. Jurkat, HUVEC (human vascular endothelial cells), HeLa or CASMC (coronary arterial smooth muscle cells) as shown, serum, PMA (12 or 18 hours), OKT (12 or 18 hours), TNF-α, interferon Bst2 mRNA levels (log log) were shown after treatment with γ or PGJ2. Bst2 mRNA levels were measured by real-time PCR. FIG. 35 shows a schematic diagram of a method for causing interaction and signaling between cell A expressing a ligand for Bst2 and cell B expressing a protein or compound Y receptor. A bivalent fusion protein composed of a Bst2 decoy and a protein or compound Y can function as an adapter that causes an interaction between cells A and B. By doing so, signal transmission between the cell A and the cell B can be improved. FIG. 36 shows the binding of phage clones to Bst2 / Damp1 decoy. FIG. 37A shows the heavy chain variable region of the anti-Bst2 / Damp1 monoclonal antibody. FIG. 37B shows the anti-Bst2 / Damp1 monoclonal antibody κ chain variable region. 38A-38B show transiently expressed and purified anti-Bst2 monoclonal antibody on a PAGE gel. (A) non-reducing conditions; (B) reducing conditions. FIG. 39 shows changes in the number of precipitated immune cells upon treatment with anti-Bst2 / Damp1 monoclonal antibody in mouse ovalbumin-induced asthma.

Claims (26)

  A method of inhibiting immune cells from binding to other cells, comprising contacting immune cells and / or other cells with a composition comprising a Bst2 antagonist.   Said other cells are immune cells, endothelial cells, smooth muscle cells, brain cells, spinal cord cells, peripheral nerve cells, heart cells, skeletal muscle cells, lung cells, hepatocytes, kidney cells, vascular cells, pancreatic cells, colon and The method according to claim 1, which is a small intestine cell, gastric cell, esophageal cell, nasopharyngeal cell, membrane cell or connective tissue cell.   2. The method of claim 1, wherein the Bst2 antagonist is a Bst2 decoy.   4. The method of claim 3, wherein the Bst2 decoy is a Bst2 fragment or a variant thereof, wherein the binding to other molecules or proteins is similar or improved to the Bst2 protein.   Bst2 decoy, Bst2 decoy-Fc chimera or fusion construct, Bst2-decoy-albumin chimera or fusion construct, or peg Bst2-decoy, or other stabilizing protein fused to a stabilizing protein The method of claim 1, wherein the method is a Bst2 decoy fused to.   2. The method of claim 1, wherein the Bst2 antagonist is a monoclonal antibody or antibody-like protein domain that specifically binds to Bst2 and / or mouse Damp1 protein.   2. The method of claim 1, wherein the Bst2 antagonist is a compound.   The immune cells and other cells are located at sites of inflammation or remote from inflammation, but may transmit inflammation and immune cytokines or other inflammatory signals to the site of inflammation, The method of claim 1.   The method of claim 1, wherein the composition further comprises a compound or immunosuppressant that inhibits cell adhesion and signal transduction.   10. The method according to claim 9, wherein the compound that inhibits cell adhesion is an ICAM1 antagonist or an LFA antagonist.   Bst2 decoy with anti-inflammatory activity.   Bst2 decoy-immunoglobulin Fc chimera.   12. The Bst2 decoy-Fc fusion of claim 11, wherein the decoy is fused to an immunoglobulin domain.   Monoclonal antibodies specific for Bst2 and / or Bst2 homologues.   15. The monoclonal antibody of claim 14, comprising two arms, one of which is specific for a protein other than Bst2 or a homologue thereof.   15. The monoclonal antibody according to claim 14, wherein the homolog is mouse Damp1 protein.   The monoclonal antibody according to claim 14, wherein the Bst2-expressing cell to which the monoclonal antibody is bound inhibits the Bst2 ligand-Bst2 interaction or the Bst2-Bst2 interaction. (I) obtaining cells that bind to Bst2;
(Ii) screening for ligands that bind to Bst2 from cells expressing the ligand, thereby isolating the ligand for Bst2;
A method for isolating a ligand for Bst2 comprising:   Damp or Bst2 gene in which somatic and germ cells are functionally disrupted, the disrupted gene is introduced into the ancestors of mice or mice during embryogenesis, and if the disrupted gene is isomorphic it indicates inflammation-related disease A transgenic mouse comprising   A transgenic mouse comprising a Damp gene in which somatic cells and germ cells are completely or partially replaced with the Bst2 gene, wherein the Bst2 gene is introduced into the ancestors of the mouse or embryonic mouse.   A method for reducing inflammation in a subject, comprising administering a composition comprising a Bst2 antagonist for an inflammation site.   A method of treating a subject with signs of inflammation-related disease comprising administering to a subject in need a composition comprising a Bst2 antagonist.   21. The method of claim 20, wherein the composition comprises other anti-inflammatory compounds.   The diseases are: atherosclerosis, rheumatoid arthritis, asthma, sepsis, ulcerative colitis, type I diabetes, cataract, multiple sclerosis, acute myocardial infarction, heart failure, psoriasis, contact dermatitis, degenerative joint Disease, rhinitis, Crohn's disease, autoimmune disease, cachexia, acute pancreatitis, autoimmune vasculitis, autoimmune and viral hepatitis, delayed hypersensitivity, congestion, coronary restenosis, glomerulonephritis, graft pair Host rejection, uveitis, inflammatory eye disease associated with corneal transplantation, traumatic brain injury, epilepsy, bleeding, stroke, sickle cell disease, type II diabetes, obesity, age-related macular degeneration (AMD), Causes atopic dermatitis, dermatitis, learning / cognitive impairment, neurodegenerative disease, Parkinson's disease, Alzheimer's disease, ulcerative colitis, radiation damage, burns or electrically induced damage, tissue necrosis and immune cell infiltration Demyelination including poisoning, drug damage, inhalation-induced damage, radiation exposure, lung aspiration-induced damage, inflammation resulting from chemotherapy or radiation therapy, autoimmune disease, lupus, Sjogren's syndrome, multiple sclerosis Diseases, inflammatory myopathy including polymyositis, scleroderma, polyarteritis nodosa, sarcoidosis, local and systemic ossifying myositis, amyloid-related diseases including Alzheimer's disease, herniated disc, spinal cord and nerve injury, Reye syndrome, bacterial and viral encephalitis and meningitis, prion related diseases, Guillain-Barre syndrome, rabies, polio, cerebral hemorrhage, intracranial hemorrhage related injury, chronic fatigue syndrome, venous thrombosis, gout, granulomatosis, glomerulus Nephritis including nephritis and interstitial nephritis, insect irritation allergy, anaphylaxis, aplastic anemia, bone marrow injury, multiple organ failure, thyroiditis 23. The method of claim 22, selected from isletitis, cirrhosis (chronic and acute hepatitis), pulmonary embolism, toxic and drug-induced liver disease, pancreatitis, ischemic bowel disease, acute respiratory distress syndrome, and pericarditis Method.   A method of assaying a compound effective for inhibiting cell-cell binding mediated by Bst2, comprising determining a compound that binds to Bst2.   A method for producing a Bst2 decoy comprising expressing the Bst2 decoy in a host cell by a recombinant technique.

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