WO2009104388A1 - Prophylactic/therapeutic agent for collagenosis - Google Patents

Abstract

Disclosed is a prophylactic/therapeutic agent for collagenosis, which can inhibit the function of TREM-1 to exhibit a prophylactic/therapeutic effect on collagenosis. Also disclosed is a method for the screening of a prophylactic/therapeutic agent for collagenosis which can inhibit the function of TREM-1 to prevent or treat collagenosis. Specifically, the prophylactic/therapeutic agent for collagenosis comprises a TREM-1 inhibitor. Preferred examples of the TREM-1 inhibitor include soluble TREM-1 and a vector capable of expressing soluble TREM-1.

Description

Preventive and therapeutic agents for collagen disease

The present invention relates to a preventive / therapeutic agent for collagen disease and a screening method thereof.

Collagen diseases are rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), dermatomyositis (DM), multiple myositis (PM), rheumatic fever (RF), nodular polyposis. It is a general term for a series of diseases such as arteritis (PN) and mixed connective tissue disease (mixed connective tissue disease; MCTD). Collagen disease is an autoimmune disease, in which autoantibodies in the body's blood react with the nuclei of their own cells to form immune complexes and deposit in tissues, such as joints, blood vessels, and internal organs throughout the body. It is thought to be caused by an attack.

Rheumatoid arthritis, a type of collagen disease, is accompanied by cartilage degeneration and bone erosion, and eventually loses joint function. Rheumatoid arthritis is said to be caused by a large amount of inflammatory cells, including lymphocytes that produce autoantibodies, infiltrating the joint and hyperplasia of the periosteum. More specifically, rheumatoid arthritis is caused by T cells recognizing an unknown self-antigen causing inflammation in synovial tissue, followed by local assembly of macrophages and lymphocytes in the synovial tissue, It is said to be caused by further activation at the site of inflammation (Non-patent Document 1). In particular, macrophages greatly contribute to the progression of rheumatoid arthritis because they produce various inflammatory molecules such as inflammatory cytokines, prostaglandins, and nitric oxide. The production of these inflammatory molecules is controlled by the activation of cell surface molecules such as cytokine receptors, complement receptors and ITAM (Immunoreceptor-tyrosine-based activation-motif) -related receptors through their respective ligand involvement. Some receptors, such as those mentioned above that are involved in the regulation of the immune system, are associated non-covalently with membrane anchored adapter molecules. In many cases, these receptors cannot be stably expressed on the cell surface without their adapter molecules, and signal transduction into cells via these adapter molecules. .

TREM-1 (Triggering receptor expressed on myeloidcells-1) has been identified as a transmembrane receptor on bone marrow cells associated with activation of DAP12 (DNAX activationprotein 12), an adapter molecule having ITAM (Non-patent Document 2, 3). TREM-1 is expressed mainly in monocytes / macrophages and neutrophils and is induced by various stimuli such as TLR ligands (LPS, LTA, etc.) and inflammatory cytokines (TNFα, IL-1β, etc.) Patent Documents 2 to 4) have not yet identified their natural ligands. Although the detailed function of TREM-1 is not yet clear, treatment of neutrophils and monocytes with an agonistic monoclonal antibody against TREM-1 causes various inflammation in neutrophils and monocytes. It is known that production of sex cytokines and expression of cell surface molecules are caused (Non-patent Document 2). Further, soluble TREM-1 (sTREM-1) is known as a substance that inhibits the function of TREM-1 (Non-patent Document 5). This sTREM-1 is a soluble polypeptide obtained by proteolytic cleavage of membrane-bound TREM-1, and has a property of inhibiting the function of TREM-1 by competing with TREM-1 itself.

Bacterial infection is known as a disease involving the action of TREM-1. Several experiments have shown that TREM-1 plays an important role in the pathophysiology of bacterial infections. For example, administration of a “sTREM-1-Fc recombinant fusion protein” or “sTREM-1 synthetic peptide” that competes with TREM-1 to septic shock model mice results in administration of lethal amounts of LPS or septic bacteria. It was confirmed that mice were protected from infection (Non-Patent Documents 6 and 7). Inflammatory colitis is a disease in which bacterial damage in the intestine is known to play an important role in its pathogenesis, and a TREM-1 antagonist peptide was administered to experimental inflammatory colitis model mice. As a result, it was confirmed that experimental inflammatory colitis was reduced as a result of blocking signal transmission from TREM-1 (Non-patent Document 8). Thus, TREM-1 is known to play an important role in disease-related bacterial infections.
However, nothing has been known about the presence or absence of an association between TREM-1 and other pathological conditions and diseases (particularly collagen disease).

An object of the present invention is to provide a preventive / therapeutic agent for collagen disease that inhibits the function of TREM-1 and exerts a preventive / therapeutic effect on collagen disease. Another object of the present invention is to provide a screening method for a prophylactic / therapeutic agent for collagen disease for preventing / treating collagen disease by inhibiting the function of TREM-1.

As a result of intensive investigations to achieve the above-mentioned object, the present inventors introduced the adenovirus-sTREM-1-Ig gene to inhibit the function of TREM-1, thereby causing the progression of collagen-induced arthritis (CIA) It was found that the onset was significantly suppressed without impairing the immune response of T cells or B cells, and the present invention was completed based on this finding.

That is, the present invention relates to (1) a preventive / therapeutic agent for collagen disease containing a TREM-1 inhibitor, and (2) the TREM-1 inhibitor is a soluble TREM-1 or a soluble TREM-1 expression vector The preventive / therapeutic agent for collagen disease according to (1) above, (3) a polypeptide comprising the amino acid sequence represented by amino acid numbers 1 to 194 in SEQ ID NO: 1, and the amino acid number in SEQ ID NO: 5 The above (2), which is a polypeptide consisting of the amino acid sequence shown by 1 to 200, a polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 3 or 7, or a polypeptide comprising the amino acid sequence shown by SEQ ID NO: 3 or 7 (4) Soluble TREM-1 is represented by amino acid numbers 1 to 194 in SEQ ID NO: 1. Substitution or deletion of one or several amino acids in the amino acid sequence, the amino acid sequence shown by amino acid numbers 1 to 200 in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 3 or 7; The preventive / therapeutic agent for collagen disease according to (2) above, which is a polypeptide having an inserted or added amino acid sequence and having TREM-1 inhibitory activity, and (5) a TREM-1 inhibitor, The preventive / therapeutic agent for collagen disease according to (1) above, which is an immunoglobulin binding-soluble TREM-1 (sTREM-1-Ig), or (6) an immunoglobulin binding-soluble TREM-1 (sTREM-1-Ig) ) Is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 5, the preventive / therapeutic agent for collagen disease according to (5) above, or (7) soluble TR The M-1 expression vector is a polynucleotide consisting of the nucleotide sequence shown in nucleotide numbers 1 to 582 in SEQ ID NO: 2, a polynucleotide consisting of the nucleotide sequence shown in nucleotide numbers 1 to 600 in SEQ ID NO: 6, SEQ ID NO: 4 or 8 The prevention of collagen disease according to (2) above, which is a recombinant vector comprising a polynucleotide consisting of the nucleotide sequence shown in (2) and a polynucleotide selected from the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 4 or 8. A therapeutic agent, or (8) a soluble TREM-1 expression vector is a nucleotide sequence represented by nucleotide numbers 1 to 582 in SEQ ID NO: 2, a nucleotide sequence represented by nucleotide numbers 1 to 600 in SEQ ID NO: 6, and SEQ ID NO: 4 Or the number 8 A recombinant vector comprising a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a sequence complementary to a nucleotide sequence selected from a nucleotide sequence and encodes a polypeptide having TREM-1 inhibitory activity The preventive / therapeutic agent for collagen disease according to (2) above, or (9) the soluble TREM-1 expression vector is a recombinant viral vector expressing soluble TREM-1 And (10) the preventive / therapeutic agent for collagen disease according to any one of the above (1) to (9), wherein the collagen disease is rheumatoid arthritis.

The present invention also includes (11) a screening method for a prophylactic / therapeutic agent for collagen disease comprising a step of searching for a TREM-1 inhibitor, and (12) a step of searching for a TREM-1 inhibitor. The method according to (11) above, which is a step of culturing cells containing TREM-1 in the presence or absence of a substance, measuring the TNFα concentration in the culture supernatant, and comparing the TNFα concentrations. The present invention relates to a screening method for a preventive / therapeutic agent for collagen disease.

A: It is a figure which shows the result of having measured the sTREM-1 density | concentration in the synovial fluid of a rheumatoid arthritis (RA) or osteoarthritis (OA) patient by specific ELISA. B: It is a figure which shows the result of having double-stained the synovial cell of RA patient about TREM-1 and CD14 (macrophage marker). C: TREM-1 of all synovial cells of RA patients is stimulated with an anti-TREM-1 agonist antibody for 24 hours, and the inflammatory cytokine (TNFα) concentration in the culture supernatant is measured by ELISA. . Note that * indicates p <0.01, and ** indicates p <0.001. AC: Results of immunohistochemical analysis of synovial cells of CIA mice. A is a diagram stained with hematoxylin and eosin, B is a diagram stained with a normal rabbit IgG antibody as an isotype-matched control antibody, and C is a diagram stained with a rat anti-mouse TREM-1 antibody. D: It is a figure which shows the result of having double-stained the synoviocyte of a CIA mouse | mouth about TREM-1 and CD11b. A: It is a figure which shows the result of having measured sTREM-1 density | concentration in the serum of the mouse | mouth which administered AxCA-sTREM-1-Ig over time using ELISA. B: What effect does sTREM-1-Ig, an expression product of AxCA-sTREM-1-Ig, have on TNFα production by resident peritoneal macrophages (RPM) stimulated with an anti-TREM-1 agonist antibody? It is a figure which shows the result of having investigated. A: It is a figure which shows the time-dependent transition of the histological score of arthritis at the time of introduce | transducing AxCA-sTREM-1-Ig to the CIA mouse | mouth after performing the 2nd immunization. B: It is a figure which shows the time-dependent transition of the ankle width | variety of a mouse | mouth when AxCA-sTREM-1-Ig is introduce | transduced into the CIA mouse | mouth after performing the 2nd immunization. C: It is a figure which shows the time-dependent transition of the thickness of a hind limb when AxCA-sTREM-1-Ig is introduce | transduced into the CIA mouse | mouth after performing the 2nd immunization. Note that * indicates p <0.05, and ** indicates p <0.01. A: It is a figure which shows the result of having dye | stained the section | slice of the knee joint of the mouse | mouth into which CIA induction | guidance | derivation and AxCA-LacZ (control) were introduce | transduced with hematoxylin and eosin. B: It is a figure which shows the result of having dye | stained the section | slice of the knee joint of the mouse | mouth into which CIA induction | guidance | derivation and AxCA-sTREM-1-Ig were introduce | transduced with hematoxylin and eosin. C: shows histological scores of arthritis of knee joints of mice introduced with CIA induction and AxCA-LacZ (control), and knee joints of mice introduced with CIA induction and AxCA-sTREM-1-Ig . A: Proliferation of T cells in spleen cells of mice introduced with CIA and AxCA-sTREM-1-Ig. B: It is a figure which shows the result analyzed about the anti- CII antibody (IgG1, IgG2a, and IgG2b) in the serum of the mouse | mouth which introduce | transduced AxCA-sTREM-1-Ig.

The preventive / therapeutic agent for collagen disease of the present invention is not particularly limited as long as it is a composition containing a TREM-1 inhibitor. Here, the “TREM-1 inhibitor” refers to a TREM-1 A substance having an activity of inhibiting all signal transduction, such as TREM-1 inhibitory substance, soluble TREM-1, a recombinant vector expressing soluble TREM-1 (sTREM-1), and a TREM-1 antagonist Can be preferably exemplified. These TREM-1 inhibitors inhibit the function of TREM-1 (signal transduction via TREM-1). As a result, TREM-1 inhibitors are advantageous as active ingredients for preventive and therapeutic agents for collagen disease. Can be used. The origin of TREM-1 is not particularly limited, but is preferably derived from animals, such as human, mouse, rat, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc. More preferably, it is derived from a mammal, and more preferably, it is derived from a mammal targeted for prevention / treatment of collagen disease. TREM-1 in these mammals is mouse TREM-1 (Genbank accession No. AK089439), human TREM-1 (Genbank accession No. AF287008), bovine TREM-1 (Genbank accession No. AY525122), porcine TREM-1 ( Obtain a polynucleotide encoding TREM-1 using a genetic engineering technique (PCR, Southern hybridization, etc.) using sequence information such as Genbank accession No. AY382476) and express the polynucleotide using an appropriate expression system. By doing so, various origins of TREM-1 can be obtained.

As the above-mentioned soluble TREM-1,
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3 or a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3 (mouse LP17);
(2) a polypeptide (mouse sTREM-1) comprising an amino acid sequence represented by amino acid numbers 1 to 194 in SEQ ID NO: 1;
(3) including the amino acid sequence represented by amino acid numbers 1-194 in SEQ ID NO: 1 or the amino acid sequence represented by SEQ ID NO: 3, wherein one or several amino acid substitutions, deletions, insertions, or additions are added. And a polypeptide having a TREM-1 inhibitory activity (mouse sTREM-1 mutant);
(4) Amino acid substitutions, deletions, insertions or additions of one or several amino acids in the amino acid sequences shown in amino acid numbers 1-102 and 120-194 among amino acid numbers 1-194 in SEQ ID NO: 1 A polypeptide comprising a sequence and having TREM-1 inhibitory activity (mouse sTREM-1 mutant);
(5) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 7 or a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 7 (human LP17);
(6) a polypeptide comprising the amino acid sequence represented by amino acid numbers 1 to 200 in SEQ ID NO: 5 (human sTREM-1);
(7) including the amino acid sequence represented by amino acid numbers 1 to 200 in SEQ ID NO: 5 or the amino acid sequence represented by SEQ ID NO: 7 in which one or several amino acid substitutions, deletions, insertions, or additions are added And a polypeptide having a TREM-1 inhibitory activity (human sTREM-1 mutant);
(8) Among amino acid numbers 1 to 200 in SEQ ID NO: 5, one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequences represented by amino acid numbers 1 to 102 and 120 to 200 A polypeptide comprising a sequence and having TREM-1 inhibitory activity (human sTREM-1 variant);
Among them, mouse sTREM-1 and human sTREM-1 can be preferably exemplified.

From the viewpoint of improving the stability, a linker sequence (amino acid sequence represented by amino acid numbers 195 to 199 in SEQ ID NO: 1; amino acid numbers 201 to 205 in SEQ ID NO: 5 is provided on the C-terminal side of these soluble TREM-1s. And a polypeptide to which an IgG1 Fc region (amino acid sequence represented by amino acid numbers 200 to 430 in SEQ ID NO: 1; amino acid sequence represented by amino acid numbers 206 to 436 in SEQ ID NO: 5) is added is preferable. It can be illustrated. Therefore, mouse or human immunoglobulin-binding-soluble TREM-1 (sTREM-1-Ig) can also be advantageously used as a preventive / therapeutic agent for collagen disease. Soluble TREM-1 is thought to inhibit the function of TREM-1 by trapping the natural ligand of TREM-1, and is a mouse LP17 polypeptide (polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3). ) Inhibits the function of mouse TREM-1 is disclosed in Non-Patent Document 7.

The “several” in the sTREM-1 mutant is, for example, 2 to 60, preferably 2 to 40, more preferably 2 to 30, further preferably 2 to 20, and even more preferably 2 to 10. Examples thereof are 2 to 5, particularly preferably 2 to 5, and most preferably 2 to 3. The amino acid sequence of the sTREM-1 variant is 50% or more, preferably 60% of the amino acid sequence represented by amino acid numbers 1 to 194 in SEQ ID NO: 1 or 5 or the amino acid sequence represented by SEQ ID NO: 3 or 7. Examples thereof include amino acid sequences having homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably about 95% or more.

The above-mentioned soluble TREM-1 can be prepared by deleting a part of the C-terminal of TREM-1 (membrane type TREM-1). For example, in the case of mouse TREM-1, soluble TREM-1 (sTREM-1) can be produced by deleting the C-terminal 36 amino acids, and in the case of human TREM-1, the C-terminal 34 amino acids Soluble TREM-1 (sTREM-1) can be produced by deletion. The sTREM-1 mutant can also be prepared by chemical synthesis, or by applying any method known to those skilled in the art, such as genetic engineering techniques and mutagenesis. Specifically, a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 1 to 582 in SEQ ID NO: 2 (polynucleotide encoding mouse sTREM-1), or the nucleotide sequence represented by nucleotide numbers 1 to 600 in SEQ ID NO: 6 A polynucleotide comprising (a polynucleotide encoding human sTREM-1), a method of contacting a sTREM-1 polynucleotide derived from another organism with a drug that is a mutagen, a method of irradiating ultraviolet rays, a genetic engineering method A sTREM-1 mutant can be obtained by introducing mutations into these polynucleotides using various techniques and expressing the mutant polynucleotides using an appropriate expression system.

The above TREM-1 antagonist can be obtained from an appropriate compound library by using, for example, the screening method for a preventive / therapeutic agent for collagen disease of the present invention described later. In addition, since there is a report (Blood. 110: 1029-35) that suggests that a TREM-1 antagonist is present on the plasma plate, a substance expressed on the plasma plate can also be used as a candidate substance.

The soluble TREM-1 expression vector is not particularly limited as long as it can express the above-mentioned soluble TREM-1 in the living body of any animal, but the above-mentioned soluble TREM-1 expression vector in the living body of any mammal A recombinant vector expressing TREM-1 can be mentioned, preferably a recombinant viral vector. Specifically, as recombinant vectors expressing these soluble TREM-1,
(1) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 4 or a recombinant vector (mouse LP17 vector) in which a polynucleotide containing the nucleotide sequence is integrated;
(2) a recombinant vector (mouse sTREM-1 vector) in which a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 1 to 582 in SEQ ID NO: 2 is integrated;
(3) hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence shown in SEQ ID NO: 4 or a nucleotide sequence complementary to the nucleotide sequence shown in nucleotide numbers 1 to 582 in SEQ ID NO: 2, and TREM- A recombinant vector in which a polynucleotide encoding a polypeptide having 1 inhibitory activity is integrated (mouse mutant sTREM-1 vector);
(4) a recombinant vector in which a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 is integrated (stable mouse sTREM-1-Ig vector);
(5) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 8, or a recombinant vector (human LP17 vector) in which a polynucleotide containing the nucleotide sequence is integrated;
(6) a recombinant vector (human sTREM-1 vector) in which a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 1 to 600 in SEQ ID NO: 6 is integrated;
(7) hybridizes under stringent conditions with a nucleotide sequence represented by SEQ ID NO: 8 or a polynucleotide comprising a sequence complementary to the nucleotide sequence represented by nucleotide numbers 1 to 600 in SEQ ID NO: 6, and TREM- A recombinant vector integrated with a polynucleotide encoding a polypeptide having 1 inhibitory activity (human mutant sTREM-1 vector);
(8) a recombinant vector in which a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 6 is integrated (stable human sTREM-1-Ig vector);
Among them, mouse sTREM-1 vector, stable mouse sTREM-1-Ig vector, human sTREM-1 vector, and stable human sTREM-1-Ig vector can be preferably exemplified.

The above-mentioned “polynucleotide hybridizing under stringent conditions” uses a polynucleotide such as DNA or RNA as a probe, and uses a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Specifically, using a filter on which a polynucleotide derived from a colony or plaque or a fragment thereof is immobilized, the polynucleotide is obtained at 65 ° C. in the presence of 0.7 to 1.0 M NaCl. After hybridization, the filter is washed under conditions of 65 ° C. using an SSC solution of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Identifiable poly Kureochido can be mentioned. Hybridization can be performed according to the method described in Molecular Cloning 3rd edition and the like.

That is, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Specifically, polynucleotides having a homology of 50 to 70% or more (in particular, DNA) are hybridized and polynucleotides having lower homology (particularly DNA) are not hybridized with each other, or are washing conditions for normal Southern hybridization at 65 ° C., 1 × SSC, 0.1% SDS Or a condition of hybridizing at a salt concentration corresponding to 0.1 × SSC and 0.1% SDS. For example, the polynucleotide that can hybridize under stringent conditions can include a polynucleotide having a certain homology with the base sequence of the polynucleotide used as a probe, for example, 60% or more, preferably Is preferably a polynucleotide having a homology of 70% or more, more preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more. It can be illustrated.

The recombinant vector of the present invention, preferably a recombinant virus vector, can be constructed by appropriately integrating the aforementioned polynucleotide encoding sTREM-1 into an expression vector. The expression vector is preferably one that can replicate autonomously in the animal cell to be administered, or one that can be integrated into the chromosome of the aforementioned animal cell, and a promoter or enhancer at a position where sTREM-1 can be expressed. Those containing a control sequence such as a terminator can be preferably used. As an expression vector, an expression vector for animal cells, for example, an adenovirus vector (Science, 252, 431-434, 1991) used for transient expression in all cells including non-sorted cells (other than blood cells) And retroviral vectors (Microbiology 導入 and Immunology, 158, 1-23, 1992) used for long-term expression in dividing cells and adeno-associated viruses that can be introduced into non-pathogenic, non-dividing cells and used for long-term expression Virus vectors such as vectors (Curr. Top. Microbiol. Immunol., 158, 1992 97-129, 1992) can be mentioned, among which adenovirus vectors can be preferably exemplified. As a procedure and method for constructing an expression vector that can be used in the present invention, those commonly used in the field of genetic engineering can be used.

The preventive / therapeutic agent for collagen disease of the present invention may be a TREM-1 inhibitor alone, or may be formulated with a physiologically recognized carrier such as an adjuvant for promoting intake. . Further, a prophylactic / therapeutic agent for collagen disease other than a TREM-1 inhibitor may be used in combination.
The dosage form of the prophylactic / therapeutic agent for collagen disease of the present invention is not particularly limited, but is an oral agent such as a tablet, capsule, powder, syrup, water or other pharmaceutically acceptable liquid. An injection such as a sterile solution or suspension can be exemplified, and when the TREM-1 inhibitor is a substance expressing TREM-1 or soluble TREM-1, the injection can be preferably exemplified it can. As a dosage form of the injection, intravenous administration and subcutaneous administration can be preferably exemplified.

The preventive / therapeutic agent for collagen disease of the present invention includes, for example, the above-described TREM-1 inhibitor in the present invention, a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, It can be produced by mixing in a unit dosage form that is generally required for the practice of the formulation together with a binder and the like. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained. Examples of additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid, Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry can be used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type material. The injection can be prepared by dissolving, suspending or emulsifying the polypeptide of the present invention in a sterile aqueous or oily liquid usually used for injection. As an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like can be used. Further, suitable solubilizers such as alcohol (eg, ethanol), Alcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adductadof hydrogenated castor oil)] or the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like can be used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. Buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), A preservative (for example, benzyl alcohol, phenol, etc.), an antioxidant and the like may be blended. The prepared injection solution can usually be filled into a suitable ampoule. The dose varies depending on the type of TREM-1 inhibitor, the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose.

Preferred examples of the target for the administration of a prophylactic / therapeutic agent for collagen disease include mammals such as humans, mice, rats, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc. Can do.

The collagen disease to be targeted by the preventive / therapeutic agent for collagen disease of the present invention is not particularly limited as long as it is a collagen disease, but rheumatoid arthritis (RA), gout, systemic lupus erythematosus (SLE), Sjogren's syndrome (SjS), Systemic sclerosis (SSc), polymyositis (PM), dermatomyositis (DM), polyarteritis nodosa (PN), rheumatic fever (RF), mixed connective tissue disease (mixed connective tissue disease; MCTD) , Microscopic polyangitis (MPA), Wegener's granulomatosis (WG), allergic granulomatous vasculitis (AGA), hypersensitivity vasculitis, Behcet's disease, Kogan syndrome RS3PE can be exemplified, among which rheumatoid arthritis, systemic lupus erythematosus (SLE), and Sjogren's syndrome (SjS) can be preferably exemplified.

The screening method for a preventive / therapeutic agent for collagen disease of the present invention comprises a step of searching for a TREM-1 inhibitor. As a step of searching for a TREM-1 inhibitor, for example, cells containing TREM-1 are cultured in the presence or absence of a test substance, the TNFα concentration in the culture supernatant is measured, and the TNFα concentrations are compared. The process of performing can be illustrated. When the TNFα concentration in the presence of the test substance is lower than the TNFα concentration in the absence of the test substance, the test substance can be evaluated as a TREM-1 inhibitor.

The collagen disease prevention / treatment method of the present invention comprises a step of administering the collagen disease prevention / treatment agent of the present invention to an animal. As the animal, the aforementioned mammals can be preferably exemplified. Further, a preventive / therapeutic agent for collagen disease other than the preventive / therapeutic agent for collagen disease of the present invention may be used in combination.

Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples.

Examples 2 to 7 described later are as follows. ~ 15. The materials and methods described in 1 were used.
[Materials and methods]
1. Reagents Rat anti-mouse TREM-1 antibody, mouse anti-human TREM-1 antibody, phycoerythrin (PE) -labeled anti-mouse TREM-1 antibody, PE-labeled anti-human TREM-1 monoclonal antibody (mAb), anti-mouse IgG ₁ antibody, anti-rat IgG antibodies, mouse TREM-1 duo set, and mouse tumor necrosis factor α (TNFα) duo set were obtained from R & D (Minneapolis, MN). Fluorescein isothiocyanate (FITC) labeled anti-human CD14 monoclonal antibody was purchased from Beckman-Coulter (Fullerton, Calif.). Allophycocyanin (APC) labeled anti-mouse CD11b monoclonal antibody was purchased from e-Bioscience (San Diego, Calif.). Rabbit anti-rat IgG antibody, streptavidin-horseradish peroxidase (HRP), and diaminobenzidine (Envision kit) were purchased from Dako Cytomation Co. Ltd. (Kyoto). Reagents for specific enzyme immunoassay (ELISA) used for human TNFα were obtained from Biosource International (Camarillo, CA). LPS and collagenase A were purchased from Sigma (St. Louis, MO). Rabbit anti-mouse IgG1, IgG2a, and IgG2b antibodies labeled with HRP were purchased from Zymed (Burlingame, Calif.).

2. Cells Human synovial tissue was obtained from patients with rheumatoid arthritis undergoing total joint replacement or synovectomy at Shinshu University and Shimoshizu National Hospital. The patient signed a consent form before surgery. Rheumatoid arthritis was diagnosed according to the criteria of the American College of Rheumatology.
On the other hand, mouse synovial tissue was separated from the knee joint of collagen arthritis (CIA) mice.
Human and mouse synovial cells were prepared by the method described in the literature (Arthritis Rheum. 54: 2074-2083; J Exp Med. 203: 325-335). That is, the synovial tissue was excised and digested with 1 mg / ml collagenase A at 37 ° C. for 1 hour in serum-free DMEM. This synovial tissue digest was suspended and filtered through a nylon mesh. Cells obtained by separation were washed three times in serum-free DMEM.
In addition, resident peritoneal macrophages (RPM) were isolated from DBA1 / J male mice (6 to 8 weeks old) by the method described in Non-Patent Document 4.
In addition, human PBMC was isolated by performing Ficoll Park density gradient centrifugation on heparin-added peripheral blood provided by a healthy person.

3. Determination of sTREM-1 concentration in synovial fluid Synovial fluid (SF) samples were obtained from inflamed joints of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) at Kitasato University. All samples were provided with informed consent. The sTREM-1 concentration in the synovial fluid was measured by a conventional enzyme immunoassay (ELISA).

4). Flow cytometry For human synovial cells or PBMC, it was double-stained with FITC-labeled anti-human CD14 monoclonal antibody and PE-labeled anti-human TREM-1 monoclonal antibody and analyzed for TREM-1 expression in gated CD14 cells did.
As for mouse synovial cells, they were double-stained with APC-labeled anti-mouse CD11b monoclonal antibody and PE-labeled anti-mouse TREM-1 monoclonal antibody, and the expression of TREM-1 in the gated CD11b cells was analyzed.
Appropriate isotype control monoclonal antibodies labeled with FITC, PE or APC were used in all flow cytometry experiments. Data were collected using a FACSCalibur and analyzed using CellQuest software (BDBiosciences Immunocytometry Systems, San Jose, Calif.).

5). Quantitative real-time PCR
Quantitative real-time PCR was performed by the method described in Non-Patent Document 4. That is, total RNA was extracted from cell lysates using RNeasy Mini kit (Qiagen, Tokyo). This RNA was treated with DNase I (Qiagen), and cDNA was synthesized using Omniscript reverse transcriptase (Qiagen). HTREM-1 and rRNA were examined by quantitative real-time PCR (TaqMan) using specific oligonucleotide primers and probes. TaqMan PCR was performed by QuantiTect Probe PCR (Qiagen). PCR amplification was monitored in real time by quantitative analysis of emitted fluorescence using a 7900 sequence detection system. The amount of control sample was 1 arbitrary unit, and each mRNA sample was quantified relative to the amount of control sample.

6). Immunohistochemical analysis Immunohistochemical analysis was performed using sections of frozen synovial membrane samples embedded in CMC (carboxymethylcellulose). That is, an 8 μm thick cryostat section was fixed in 4% paraformaldehyde for 20 minutes, and then the sample was immersed in PBS three times for 5 minutes each. The tissue sections in the sample were then treated for 15 minutes in PBS containing 1.5% H ₂ O ₂ and then in PBS containing 5% normal rabbit serum for 30 minutes. Serial sections were incubated overnight at 4 ° C. in 5% normal rabbit serum / PBS containing 25 μg / ml primary antibody of rabbit anti-TREM-1 antibody and normal rabbit IgG as isotype matched control antibody. The samples were then washed in PBS for 5 minutes twice and treated with diaminobenzidine according to the manufacturer's manual. The sections were then counterstained with hematoxylin for 5 seconds and washed with tap water for 10 minutes.

7). Cytokine production assay 5 μg / ml of anti-human TREM-1 monoclonal antibody, anti-mouse TREM-1 monoclonal antibody, or isotype matched control antibody (IgG antibody) was precoated at 4 ° C. overnight at 4 ° C. Cells washed with PBS were added to each well of the flat bottom plate at 1 × 10 ⁵ cells / well, and this was centrifuged at 1200 rpm for a short time to bind TREM-1 to the antibody. After incubation for 24 hours, the culture medium was collected by centrifugation and stored at −20 ° C. until the TNFα concentration in the culture medium supernatant was measured by specific ELISA.
The above-mentioned anti-human TREM-1 monoclonal antibody and anti-mouse TREM-1 monoclonal antibody also have agonistic properties against human TREM-1 and mouse TREM-1, respectively. Also referred to as “TREM-1 agonist antibody”.

8). A replication-deficient adenovirus containing a recombinant adenovirus mouse sTREM-1-Ig gene (SEQ ID NO: 1) (hereinafter also referred to as “AxCA-sTREM-1-Ig”) or a replication-deficient adenovirus containing a LacZ gene (hereinafter referred to as “AxCA-sTREM-1-Ig”) “AxCA-LacZ”) was prepared by the method described in the literature (Arthritis Rheum. 54: 2074-2083). That is, a recombinant adenovirus was grown in HEK293 cells to prepare a high-titer recombinant adenovirus, and the recombinant adenovirus was purified from HEK239 cells by cesium chloride density gradient centrifugation.

9. Bioassay for TREM-1 Inhibitory Activity In order to show that sTREM-1-Ig expressed by AxCA-sTREM-1-Ig has biological activity, it is infected with AxCA-sTREM-1-Ig or AxCA-LacZ The inhibitory activity of TREM-1 was examined using the culture supernatant of NIH / 3T3 cells. The supernatant of these cells was collected 3 days after infection. Each supernatant was serially diluted and 50 μl of each dilution was incubated at 37 ° C. in separate wells precoated with anti-mouse TREM-1 monoclonal antibody (anti-TREM-1 agonist antibody). After 15 minutes from the start of incubation, RPM (TREM-1-expressing cells) was added to each well at 1 × 10 ⁵ cells. After incubation for 24 hours from the start of RPM addition, a culture medium was obtained, and the TNFα concentration in the culture medium supernatant was measured by ELISA.

10. STREM-1 Concentration in Serum To examine the sTREM-1 concentration in serum after intravenous administration of AxCA-sTREM-1-Ig, on day 0, 10 ⁹ pfu of AxCA-sTREM-1-Ig or AxCA- LacZ was administered to non-immunized mice. Blood samples were collected from mice at 0, 2, 4, and 7 days after adenovirus administration and stored at -20 ° C. The stored blood sample was subjected to ELISA, and the sTREM-1 concentration in the serum was measured.

11. Clinical evaluation of CIA induction and arthritis DBA / 1J male mice were purchased from Charles River Breathing Laboratories, Japan (Japan) and were bred at the animal experiment facility of Tokyo Medical and Dental University. CFA (complete Freund's adjuvant) containing 200 μg of bovine collagen type II (CII) was administered into the skin of the tail base of 8-week-old DBA / 1J male mice (primary immunization). 21 days after the first immunization, these mice were similarly immunized a second time (day 0), and the disease severity of each limb of the mice was recorded according to the following scoring method.
Scoring method: 0 = Normal: 1 = Mild swelling in one joint: 2 = Mild swelling in two joints: 3 = Severe swelling in the foot or finger: 4 = Severe swelling in the entire foot and finger .
Joint swelling was quantified by measuring the thickness of the hind limb and the width of the ankle with a micrometer (Okazaki Corporation, Tokyo).
The collagen arthritis (CIA) mice were then sacrificed 14 days after the second immunization for histological examination of the mouse joints. The knee joint of this mouse was excised, fixed in 10% buffered formalin, decalcified with 10% EDTA, and embedded in paraffin. The embedded knee joint sections (4 μm thick) were stained with hematoxylin and eosin and examined histologically. Inflammatory cell infiltration, transformation of synovial lining, cartilage destruction, and pannus formation were each scored from 0 to 3 according to the scoring method described above. The highest histological score was 12 points.

12 Gene transfer in vivo 2 days and 9 days after the second immunization, 10 ⁸ pfu or 10 ⁹ pfu of AxCA-sTREM-1-Ig (sample number: n = 8) or AxCA-LacZ (sample number: n = Mice were intravenously administered with 100 μl of PBS containing 8).

13. Detection of bovine type II collagen (CII) specific antibody CII specific antibody in mouse serum was measured by ELISA. Specifically, it measured by the following methods.
Flat bottom plates were coated with 2 μg / ml bovine type II collagen denatured by boiling in phosphate buffered saline (PBS). The plate was washed in PBS containing 0.05% Tween 20 (PBST), blocked with PBS containing 2% bovine serum albumin (BSA), and incubated with 1000-fold diluted mouse serum. Positive reactions were detected by incubation with rabbit anti-mouse IgG1, IgG2a, IgG2b antibodies labeled with horseradish peroxidase (Zymed, Burlingame, Calif.) In PBST containing 2% BSA. The final reaction was visualized using the TMB Microwell Peroxidase Substrate System (KPL, Gaithersburg, MD). Absorbance was measured at 450 nm. CII-specific antibody units were measured using standard sera prepared from pooled sera obtained from arthritic or non-arthritic mice. A 40-fold dilution of sera from arthritic mice was 1000 units / ml.

14 Proliferation of CII-specific T cells Splenocytes were isolated from each group of mice 14 days after the second immunization. These spleen cells (5 × 10 ⁵ cells) were cultured in RPMI 1640 medium supplemented with penicillin / streptomycin and 10% fetal calf serum while being stimulated with denatured CII (0-100 μg / ml). Cultures were cultured with [ ³ H] thymidine (0.037 MBq / well) for the last 16 hours of the 72 hour culture period and splenocytes were collected on a micro 96 harvester (PerkinElmer, Yokohama) to proliferate. evaluated. Incorporated radiation was measured by a microplate beta counter (“MicroβPlus”; PerkinElmer).

15. Statistical method The results of each experiment were expressed as mean ± standard deviation (SD). For statistical analysis, Mann-Whitney U test was used, and p <0.05 was considered significant.

[Detection of human sTREM-1 in synovial fluid and human membrane-bound TREM-1 in synovial cells of rheumatoid arthritis]
(1) Results of measurement of sTREM-1 concentration in synovial fluid There are several documents reporting that sTREM-1 and membrane-bound TREM-1 are found in large amounts in bacterial infections (Nature.410: 1103). -1107; N Engl J Med. 350: 451-458), the expression of sTREM-1 and TREM-1 in rheumatoid arthritis was not shown. Therefore, in order to examine the sTREM-1 concentration in rheumatoid arthritis, the sTREM-1 concentration in the synovial fluid of 23 rheumatoid arthritis (RA) patients and 9 osteoarthritis (OA) patients was described in Example 1 above. It was measured by ELISA according to the method of “Measurement of sTREM-1 concentration in synovial fluid”. The result is shown in FIG. As can be seen from the results in FIG. 1A, the sTREM-1 concentration in the synovial fluid of RA patients was 1763.2 ± 1180.2 pg / ml, whereas the sTREM-1 concentration in the synovial fluid of OA patients Was 162.2 ± 220.2 pg / ml, and the sTREM-1 concentration in the synovial fluid of RA patients was significantly higher than that of OA patients. This indicates that sTREM-1 is highly expressed in RA with inflammation than OA without inflammation.

(2) Results of flow cytometry in human synoviocytes Next, in order to examine the presence or absence of TREM-1 expression in 3 synovial cells among RA patients in Example 2 (1) above, According to the method of “flow cytometry” described in 1, flow cytometry analysis was performed. Specifically, a single cell suspension of synovial cells collected from RA patients was double-stained for TREM-1 and CD14 (macrophage marker), and the synovial cells expressing TREM-1 were analyzed. It was. The result is shown in FIG. As can be seen from the results in FIG. 1B, macrophages expressing CD14 in inflammatory synovial tissue of RA patients showed expression of TREM-1. This showed that TREM-1 was expressed in infiltrating macrophages in synovial tissue.

(3) Results of cytokine production assay in human synovial cells In order to directly examine the results considered to be attributable to the expression of TREM-1, TREM-1 of all synovial cells of RA patients in Example 2 (1) above was According to the method of “cytokine production assay” described in Example 1 above, stimulation with an anti-TREM-1 monoclonal antibody (anti-TREM-1 agonist antibody) for 24 hours, and the concentration of inflammatory cytokine (TNFα) in the culture supernatant Was measured by ELISA. The result is shown in FIG. TNFα secretion itself has already been detected in synovial cells obtained from RA inflamed joints, but as can be seen from the results in FIG. 1C, TNFα crosslinking by anti-TREM-1 agonist antibody Secretion was significantly higher than when stimulated with IgG antibody (control antibody). From this, it was shown that the disease in patients with rheumatoid arthritis worsens and tissue destruction progresses by increasing the inflammatory process via TREM-1.

[Expression of mouse TREM-1 in synovial cells of collagen arthritis (CIA) mice]
(1) Results of immunohistochemical analysis on synovial cells of CIA mice In order to examine whether TREM-1 expression in bone marrow cells contributes to inflammatory reaction in synovial tissue, in synovial tissue of CIA mice The expression of mouse TREM-1 was examined by the “immunohistochemical analysis” method described in Example 1 above. The results are shown in FIGS. As can be seen from the results of FIGS. 2A to 2C, cells expressing TREM-1 (mononuclear cells) were abundantly observed in the inflamed synovial tissue (FIG. 2C).

(2) Results of flow cytometry analysis on synovial cells of CIA mice Next, in order to examine whether TREM-1 is expressed in the inflamed periosteal tissue of CIA mice, the “CIA induction” described in Example 1 above was conducted. And CIA-induced mice according to the method of “Clinical evaluation of arthritis” were subjected to flow cytometry analysis according to the method of “flow cytometry”. Specifically, a single cell suspension of synovial cells collected from CIA mice was double-stained for TREM-1 and CD11b (macrophage marker), and synovial cells expressing TREM-1 were analyzed. It was. The result is shown in FIG. As can be seen from the results in FIG. 2D, most of the macrophages expressing CD11b in the inflamed synovial tissue of CIA mice showed TREM-1 expression. In particular, the expression level (Day 7) of TREM-1 in the early inflammatory joint (7 days after the second collagen immunization) was higher than that in the chronic inflammatory joint (16 days after the second collagen immunization) (Day 16). This indicates that TREM-1 plays a more important role in early inflammatory joints than in chronic inflammatory joints.

[Expression level of sTREM-1 in vivo and biological activity of AxCA-sTREM-1-Ig in vitro]
(1) Result of measurement of sTREM-1 concentration in mouse serum In order to examine the therapeutic effect of inhibiting TREM-1 in CIA, first, a recombinant adenovirus containing sTREM-1-Ig gene (AxCA-sTREM-1 -Ig) was constructed according to the "recombinant adenovirus" method described in Example 1 above.
Next, in order to examine whether sTREM-1 is actually expressed in vivo when AxCA-sTREM-1-Ig is administered to mice, the “sTREM-1 concentration in serum” described in Example 1 above is used. According to the method, the concentration of sTREM-1 in mouse serum was measured by ELISA. The result is shown in FIG. As can be seen from the results in FIG. 3A, the sTREM-1 concentration reached its maximum value 2 days after intravenous administration of AxCA-sTREM-1-Ig, and gradually decreased until the 7th day thereafter.
On the other hand, in mice administered with AxCA-LacZ instead of AxCA-sTREM-1-Ig, no increase in serum sTREM-1 concentration was observed (data not shown).
From these results, it was shown that sTREM-1-Ig was efficiently produced in vivo by intravenous administration of AxCA-sTREM-1-Ig.

(2) Results of bioassay of TREM-1 inhibitory activity In order to confirm whether sTREM-1-Ig expressed by AxCA-sTREM-1-Ig has biological activity, it was described in Example 1 above. Experiments were performed according to the method of “Bioassay for TREM-1 Inhibitory Activity”. That is, the culture supernatant containing sTREM-1-Ig in NIH / 3T3 cells infected with AxCA-sTREM-1-Ig was transformed into TNFα by resident peritoneal macrophages (RPM) stimulated with an anti-TREM-1 agonist antibody. The effect on production was investigated. The result is shown in FIG. As can be seen from the results in FIG. 3B, when the culture supernatant of cells infected with AxCA-LacZ (without sTREM-1-Ig) was added, TNFα production was inhibited regardless of the amount added. Was not. In contrast, when the culture supernatant of NIH / 3T3 cells infected with AxCA-sTREM-1-Ig (including sTREM-1-Ig) was added, the production of TNFα was remarkably inhibited, and the inhibition The degree was dependent on the amount of culture supernatant added, and TNFα production was completely inhibited when the concentration of sTREM-1 was 320 pg / ml.
From these results, it was shown that sTREM-1-IG, which is an expression product of AxCA-sTREM-1-Ig, has biological activity (inhibition effect on inflammation).

[Suppression of arthritis by sTREM-1-Ig gene introduction in collagen arthritis (CIA)]
(1) Results of clinical evaluation of CIA induction and arthritis In order to examine the pathophysiological function of TREM-1 in CIA, mice were treated according to the method of “CIA evaluation of CIA induction and arthritis” described in Example 1 above. By inducing CIA and further inhibiting the involvement of TREM-1 by introducing AxCA-sTREM-1-Ig into the mouse according to the method of “in vivo gene transfer” described in Example 1 above The effect of CIA on the pathological condition was examined. The time course of the histological score of arthritis in CIA mice is shown in FIG. 4A, the time course of the ankle width of CIA mice is shown in FIG. 4B, and the time course of the hind limb thickness of CIA mice is shown in FIG. Of C. As can be seen from the results in FIGS. 4A to 4C, when 1 × 10 ⁹ pfu of AxCA-sTREM-1-Ig was intravenously administered, the progression of arthritis was compared to when AxCA-LacZ (control) was administered. Was significantly suppressed. In addition, when 1 × 10 ⁸ pfu of AxCA-sTREM-1-Ig was administered intravenously, arthritis was significantly suppressed compared to the control, although not as much as when 1 × 10 ⁹ pfu was administered. That is, it was revealed that AxCA-sTREM-1-Ig inhibits arthritis in a dose-dependent manner. These findings indicate that TREM-1 plays a major role in the progression of CIA, and it has been found that inhibiting TREM-1 is very likely to be useful in the treatment of arthritis.

(2) Results of Histological Evaluation of CIA in Mice Treated with sTREM-1-Ig The lesions in the joints of mice introduced with CIA and AxCA-sTREM-1-Ig in Example 1 (1) For histological evaluation, a section of a knee joint of a mouse into which AxCA-sTREM-1-Ig or AxCA-LacZ (control) was introduced 16 days after the second immunization was prepared, and hematoxylin and eosin (H & E) were used. Stained. The observation results are shown in FIG. 5A (AxCA-LacZ-introduced mouse) and B (AxCA-sTREM-1-Ig-introduced mouse). As can be seen from FIGS. 5A and 5B, the joints of AxCA-LacZ-introduced mice are unique to collagen arthritis in all four parameters: prominent infiltration of inflammatory cells, degeneration of synovial intima, cartilage destruction, and pannus formation. The characteristics were shown. In contrast, in the joints of AxCA-sTREM-1-Ig-introduced mice, the infiltration of inflammatory cells was remarkably reduced, and the degree of intimal hyperplasia and bone and cartilage destruction were low.

The above four parameters in the joints of AxCA-LacZ-introduced mice and AxCA-sTREM-1-Ig-introduced mice are classified into 4 levels from 0 to 3 (0, normal; 1, slight; 2, mild; 3, severe). Each was evaluated. The observation results are shown in FIG. As can be seen from the results in FIG. 5C, the score of each parameter of the AxCA-sTREM-1-Ig-introduced mouse was significantly lower than that of the AxCA-LacZ-introduced mouse. These results suggested that TREM-1 signaling is involved in promoting joint inflammation and destruction in collagen arthritis.

[Effect of sTREM-1-Ig gene transfer on T cell response]
(1) Results of proliferation of bovine type II collagen (CII) -specific T cells In order to examine whether in vivo introduction of AxCA-sTREM-1-Ig affects T cell immunity against CII, the above example In the method of “Proliferation of CII-specific T cells” described in Example 1 above, the proliferation of T cells in the spleen cells of mice introduced with CIA induction and AxCA-sTREM-1-Ig in (1) of 5 Therefore, it was measured in vitro. That is, various adenoviruses (AxCA-sTREM-1-Ig or AxCA-LacZ) were introduced 2 days and 9 days after the second immunization of CIA-induced mice, and 4 spleens per group were introduced on the 16th day. Removed and co-cultured with type II collagen. The observation result is shown in FIG. As can be seen from the results in FIG. 6A, each group was introduced with 10 ⁸ pfu of AxCA-sTREM-1-Ig, 10 ⁹ pfu of AxCA-sTREM-1-Ig, or AxCA-LacZ (control). No significant difference in response was observed. This result indicated that inhibition of TREM-1 did not impair the antigen-specific T cell response.

[Effect of sTREM-1-Ig gene transfer on B cell response]
(1) Results of Detection of Bovine Type II Collagen (CII) Specific Antibody In order to examine the effect of AxCA-sTREM-1-Ig on the humoral immune response against type II collagen, “Bovine” described in Example 1 above was used. 10 ⁸ pfu or 10 ⁹ pfu of AxCA-sTREM-1-Ig-introduced mouse or AxCA-LacZ-introduced mouse on the 16th day from the second immunization according to the method of “Detection of type II collagen (CII) -specific antibody” The blood samples were collected and analyzed for anti-CII antibodies (IgG1, IgG2a and IgG2b) in the serum. The result is shown in FIG. As can be seen from the results in FIG. 6B, no significant difference was observed in the blood antibody titers of the IgG1, IgG2a, and IgG2b anti-CII antibodies among the above three groups of mice. Furthermore, the antibody titers of these anti-CII antibodies were not significantly different from the second immunization to day 0 (data not shown). From these results, it was shown that treatment with AxCA-sTREM-1-Ig did not impair the production of anti-CII antibodies in collagen arthritis.

By inhibiting the function of TREM-1, it is possible to exert a preventive / therapeutic effect on collagen disease. Therefore, a TREM-1 inhibitor is effective for preventing or treating a collagen disease. In addition, a prophylactic / therapeutic agent for collagen disease can be screened by searching for a TREM-1 inhibitor.

Claims (12)

1. A preventive or therapeutic agent for collagen disease comprising a TREM-1 inhibitor.
2. The preventive / therapeutic agent for collagen disease according to claim 1, wherein the TREM-1 inhibitor is soluble TREM-1 or a soluble TREM-1 expression vector.
3. Soluble TREM-1 is a polypeptide comprising the amino acid sequence represented by amino acid numbers 1 to 194 in SEQ ID NO: 1, a polypeptide comprising the amino acid sequence represented by amino acid numbers 1 to 200 in SEQ ID NO: 5, SEQ ID NO: 3 or 7 The preventive / therapeutic agent for collagen disease according to claim 2, which is a polypeptide having the amino acid sequence shown or a polypeptide containing the amino acid sequence shown by SEQ ID NO: 3 or 7.
4. Soluble TREM-1 is selected from the amino acid sequence represented by amino acid numbers 1 to 194 in SEQ ID NO: 1, the amino acid sequence represented by amino acid numbers 1 to 200 in SEQ ID NO: 5, and the amino acid sequence represented by SEQ ID NO: 3 or 7. The amino acid sequence according to claim 2, which is a polypeptide having a TREM-1 inhibitory activity, comprising an amino acid sequence substituted, deleted, inserted, or added by one or several amino acids. Prophylactic / therapeutic agent.
5. The preventive / therapeutic agent for collagen disease according to claim 1, wherein the TREM-1 inhibitor is immunoglobulin binding-soluble TREM-1 (sTREM-1-Ig).
6. 6. The preventive / therapeutic agent for collagen disease according to claim 5, wherein the immunoglobulin binding-soluble TREM-1 (sTREM-1-Ig) is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 5.
7. A soluble TREM-1 expression vector is a polynucleotide consisting of the nucleotide sequence shown in nucleotide numbers 1 to 582 in SEQ ID NO: 2, a polynucleotide consisting of the nucleotide sequence shown in nucleotide numbers 1 to 600 in SEQ ID NO: 6, SEQ ID NO: 4 or The prevention of collagen disease according to claim 2, which is a recombinant vector comprising a polynucleotide comprising the nucleotide sequence represented by 8 and a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4 or 8. Therapeutic agent.
8. A soluble TREM-1 expression vector comprises a nucleotide sequence represented by nucleotide numbers 1 to 582 in SEQ ID NO: 2, a nucleotide sequence represented by nucleotide numbers 1 to 600 in SEQ ID NO: 6, and a nucleotide sequence represented by SEQ ID NO: 4 or 8. A recombinant vector comprising a polynucleotide encoding a polypeptide having a TREM-1 inhibitory activity, which hybridizes with a polynucleotide comprising a sequence complementary to a nucleotide sequence selected from 2. A preventive / therapeutic agent for collagen disease according to 2.
9. The preventive / therapeutic agent for collagen disease according to claim 7 or 8, wherein the soluble TREM-1 expression vector is a recombinant viral vector expressing soluble TREM-1.
10. The preventive / therapeutic agent for collagen disease according to any one of claims 1 to 9, wherein the collagen disease is rheumatoid arthritis.
11. A screening method for a prophylactic / therapeutic agent for collagen disease, comprising a step of searching for a TREM-1 inhibitor.
12. The step of searching for a TREM-1 inhibitor comprises culturing cells containing TREM-1 in the presence or absence of a test substance, measuring the TNFα concentration in the culture supernatant, and comparing the TNFα concentrations The screening method for a prophylactic / therapeutic agent for collagen disease according to claim 11, wherein

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