WO2009101856A1

WO2009101856A1 - Remedy for rheumatoid arthritis

Info

Publication number: WO2009101856A1
Application number: PCT/JP2009/051269
Authority: WO
Inventors: Hiroshi Nakada; Yutaka Kawahito
Original assignee: University Educational Foundation Kyoto Sangyo University
Priority date: 2008-02-13
Filing date: 2009-01-27
Publication date: 2009-08-20
Also published as: JP2011093804A

Abstract

Disclosed is a novel remedy for rheumatoid arthritis which has a high joint specificity with little side effects such as immune depression. An agent for treating rheumatoid arthritis, which comprises as the active ingredient a polypeptide containing the whole scavenger receptor type A or a part of the same and having a ligand-binding activity, is provided as a remedy for rheumatoid arthritis acting specifically on joints. The above polypeptide is preferably a polypeptide containing a collagen-like domain and/or an α-helical coiled coil domain.

Description

Rheumatoid arthritis treatment

The present invention relates to a therapeutic agent for rheumatoid arthritis, and more particularly to a therapeutic agent for rheumatoid arthritis containing a polypeptide derived from scavenger receptor type A as an active ingredient. The therapeutic agent for rheumatoid arthritis of the present invention has particularly high joint specificity.

Conventionally, various drugs such as non-steroidal anti-inflammatory drugs, steroid drugs, anti-rheumatic drugs, immunosuppressive drugs have been used as therapeutic agents for rheumatoid arthritis (Patent Document 1). More recently, biological preparations (cytokine inhibitors) targeting inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) Has been developed and applied to the treatment of rheumatoid arthritis. For example, an anti-TNF-α antibody or soluble TNF-α receptor as a TNF-α inhibitor, an anti-IL-6 receptor antibody as an IL-6 inhibitor, and an IL-1 receptor antagonist as an IL-1 inhibitor Biological products, each of which is an active ingredient, have been developed. The effects of these formulations on rheumatoid arthritis are rapid and dramatic, with high therapeutic results in many patients with rheumatoid arthritis.

On the other hand, problems of cytokine inhibitors targeting these inflammatory cytokines have also been pointed out. That is, since these cytokines are not joint-specific but are related to the entire physiological function, suppression thereof may reduce the patient's immunity and increase the risk of developing infections and cancer. In addition, the proliferation of synovial cells cannot be completely suppressed, and only the progress of bone destruction is delayed. Under such circumstances, a therapeutic agent for rheumatoid arthritis that acts joint-specifically is demanded.
JP 2007-326824 A

In view of the above situation, an object of the present invention is to provide a novel therapeutic agent for rheumatoid arthritis that acts joint-specifically.

One aspect of the present invention for solving the above-described problems is a therapeutic agent for rheumatoid arthritis comprising, as an active ingredient, a polypeptide containing all or part of scavenger receptor class A and having ligand binding activity.

The therapeutic agent for rheumatoid arthritis of this aspect comprises a polypeptide containing all or part of scavenger receptor class A as an active ingredient, and the polypeptide has a ligand binding activity. Since the therapeutic agent for rheumatoid arthritis in this aspect utilizes the action of scavenger receptor class A, it has high joint specificity. Therefore, the side effect of immunity reduction is small, and the onset risk such as infectious diseases can be kept low. In addition, the therapeutic agent for rheumatoid arthritis of this aspect has a particularly high effect of suppressing the proliferation of synovial cells, and can highly inhibit the progress of bone destruction.

Examples of the “polypeptide containing all or part of scavenger receptor class A” include the following (a) to (c).
(A) a polypeptide comprising all of scavenger receptor A (full length of scavenger receptor A, scavenger receptor A itself),
(B) a polypeptide comprising a part of scavenger receptor A (a partial fragment of scavenger receptor A),
(C) The above (a) or (b) added with other polypeptides / proteins.
Furthermore, specific examples of (c) include fusion proteins of (a) or (b) with other proteins.

Preferably, the polypeptide contains a collagen-like domain.

Preferably, the polypeptide contains an α helical coiled coil domain.

Preferably, the scavenger receptor class A consists of the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence. .

Preferably, the scavenger receptor class A comprises an amino acid sequence having a homology of 80% or more with the protein comprising the amino acid sequence represented by SEQ ID NO: 2.

Since the therapeutic agent for rheumatoid arthritis of the present invention has high joint specificity, the side effect of reduced immunity is small, and the risk of developing an infection or the like can be kept low. Furthermore, the therapeutic agent for rheumatoid arthritis of the present invention has a particularly high effect of suppressing the proliferation of synovial cells, and can highly inhibit the progress of bone destruction.

It is explanatory drawing which represents typically the domain structure of SCR-AI. It is explanatory drawing which represents typically the domain structure of soluble SCR created in the Example. It is a graph showing the relationship between the number of days after subcutaneous injection of IFA emulsion and the incidence of arthritis. It is a graph showing the relationship between the days after IFA emulsion subcutaneous injection, and an arthritis score. It is a photograph showing the result of joint tissue staining, (a) and (b) are the results of the control group, and (c) and (d) are the results of the SCR administration group. It is a graph showing the relationship between the number of days after IFA emulsion subcutaneous injection and a change in body weight.

Hereinafter, the best mode for carrying out the present invention will be described in detail.

First, the scavenger receptor will be explained. The scavenger receptor (hereinafter sometimes abbreviated as “SCR”) is a general term for a group of receptors that recognize denatured low density lipoprotein (denatured LDL). SCR is purified by Kodama et al. As a receptor involved in arteriosclerosis, and its gene structure has also been elucidated (Proc. Natl. Acad. Sci. USA, 85, 9238-9242, 1988, and Nature, N353, 531). -535, 1990). SCR is classified into three classes A to C according to structural features. Among these, scavenger receptor class A (hereinafter sometimes abbreviated as “SCR-A”) is the first SCR found, and is mainly expressed in macrophage cells. Furthermore, four types of isoforms (type I, type II, type III, MACRO) generated by alternative splicing are known for SCR-A.

Scavenger receptor class A (SCR-A) is a complex protein having a structure in which three glycoproteins (subunits) having a molecular weight of about 80,000 form a homotrimer. In SCR-A, the N-terminal side is located inside the cell and the C-terminal side is located outside the cell. SCR-A type I (SCR-AI) is composed of an intracellular domain, a transmembrane domain, a spacer domain, an α helical coiled coil domain, a collagen-like domain, and a cysteine-rich domain in this order from the N-terminus. (Fig. 1). SCR-A type II (SCR-AII) has a similar structure to SCR-AI except that it lacks a cysteine-rich domain. A collagen-like domain containing a Lys / Arg cluster plays an important role in the ligand binding activity of SCR-A, and this domain is known to interact with a negatively charged ligand. That is, a wide range of molecules such as macromolecules having a negative charge, such as nucleic acids, sugar chains, modified proteins, and high molecular weight compounds, can serve as SCR-A ligands. In the present specification, the complex protein and its subunit are not particularly distinguished from each other. For example, in the present specification, the term “SCR-A” is used to mean both “trimer-formed SCR-A” and “SCR-A subunit”.

The amino acid sequence of SCR-A has high homology between humans and other mammals. For example, in the case of type I, 73% between human and cow, 72% between human and mouse, 96% between human and chimpanzee, 95% between human and monkey, 80% between human and horse, between human and rabbit Shows 80% homology. This strongly suggests that SCR-A plays a common role at least in mammals.

SCR-A classified into other types also has a structure similar to SCR-AI and SCR-AII, and is common in that it has a collagen-like domain and an α helical coiled coil domain.

The active ingredient of the therapeutic agent for rheumatoid arthritis according to one aspect of the present invention is a polypeptide containing all or part of scavenger receptor type A (SCR-A) and having ligand binding activity. First, examples of the method for obtaining “polypeptide containing all or part of SCR-A” include a genetic engineering technique for constructing a gene encoding the polypeptide and expressing the gene. The gene can be constructed based on, for example, SCR-A cDNA. As a method for obtaining SCR-A cDNA, a method generally used in the art can be applied. For example, RNA can be extracted and purified from macrophages, and single-stranded cDNA can be obtained by reverse transcription reaction, and further double-stranded cDNA can be obtained by DNA synthesis reaction. In addition, since SCR-A cDNA has already been isolated from various animals and information on the base sequences thereof is available, primers and probes can be designed with reference to the information. Here, the amino acid sequence corresponding to the nucleotide sequence of human SCR-AII cDNA is shown in SEQ ID NO: 1, and only the amino acid sequence is shown in SEQ ID NO: 2. In SEQ ID NO: 2, amino acid numbers 1 (Met) to 50 (Lys) are intracellular domains, amino acid numbers 51 (Ala) to 76 (Leu) are transmembrane domains, and amino acid numbers 77 (Lys) to 451 (Leu) are cells. Corresponds to the outer domain. Furthermore, among the extracellular domains, amino acid numbers 171 (Asn) to 255 (Arg) correspond to the α helical coiled coil domain, and amino acid numbers 273 (Gly) to 341 (Gly) correspond to the collagen-like domain.

Furthermore, the amino acid sequence corresponding to the nucleotide sequence of mouse SCR-AII cDNA is shown in SEQ ID NO: 3, and only the amino acid sequence is shown in SEQ ID NO: 4. In SEQ ID NO: 4, amino acid numbers 1 (Met) to 55 (Lys) are intracellular domains, amino acid numbers 56 (Ala) to 78 (Ala) are transmembrane domains, and amino acid numbers 79 (Gln) to 354 (Val) are cells. Corresponds to the outer domain. Furthermore, among the extracellular domains, amino acid numbers 175 (Asn) to 259 (Arg) correspond to the α helical coiled coil domain, and amino acid numbers 277 (Gly) to 348 (Val) correspond to the collagen-like domain.

As described above, examples of the “polypeptide containing all or part of SCR-A” include the following (a) to (c):
(A) a polypeptide comprising all of SCR-A (full length of SCR-A, SCR-A itself),
(B) a polypeptide comprising a part of SCR-A (partial fragment of SCR-A),
(C) The above (a) or (b) added with other polypeptides / proteins,
Is mentioned.

When the polypeptide (a) is employed, for example, cDNA encoding the desired full-length SCR-A may be obtained and expressed. On the other hand, when the polypeptide (b) is employed, for example, cDNA encoding a part of the desired SCR-A may be obtained and expressed. In a preferred embodiment, a polypeptide comprising a collagen-like domain and / or an alpha helical coiled coil domain is employed. The polypeptide of this preferred embodiment can reliably retain ligand binding activity. For example, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 contains both the collagen-like domain of mouse SCR-AII and the α helical coiled coil domain. That is, the amino acid sequence of SEQ ID NO: 5 corresponds to amino acid numbers 97 (Asp) to 354 (Val) of SEQ ID NO: 4. FIG. 2 schematically shows the domain structure of the polypeptide.

When the polypeptide (c) is employed, various functions can be imparted depending on the polypeptide / protein to be added. For example, for the purpose of extending the blood half-life, it can be a fusion protein of the polypeptide (a) or (b) and an immunoglobulin Fc region. For the purpose of imparting solubility, a fusion protein of the polypeptide (a) or (b) and a molecular chaperone can be prepared. In this embodiment as well, it is preferable to include a collagen-like domain and / or an α helical coiled coil domain.

In another embodiment, the SCR-A in the “polypeptide containing all or part of SCR-A” is the amino acid sequence represented by SEQ ID NO: 2, or one or several amino acid residues in the amino acid sequence. It consists of an amino acid sequence that is deleted, substituted or added. In other words, it is a polypeptide comprising all or part of a protein that can be regarded as substantially identical to human SCR-AII. Here, “1 or several” usually means “1 to 10”, preferably “1 to 5”, and more preferably “1 to 3”.

In another embodiment, SCR-A in “polypeptide containing all or part of SCR-A” consists of an amino acid sequence having 80% or more homology with the protein consisting of the amino acid sequence represented by SEQ ID NO: 2. Is. In a more preferred embodiment, the homology is 90% or more. In a further preferred embodiment, the homology is 95% or more. The amino acid homology can be examined using, for example, a BLAST (Basic, Local, Alignment, Search, Tool) database.

In the present invention, a polypeptide containing all or part of SCR-A needs to have ligand binding activity. As described above, a wide range of macromolecules (nucleic acids, sugar chains, modified proteins, polymer composites, etc.) having a negative charge can be ligands. Examples of ligands include acetylated LDL (AcLDL), oxidized LDL (OxLDL), fucoidan, polyinosinic acid (Poly I), dextran sulfate, lipopolysaccharide (LPS), and mucin. The presence or absence of ligand binding activity can be examined by a known method, for example, by a competitive assay using a labeled compound.

As a method for expressing cDNA, a known method can be applied as it is. For example, cDNA encoding a desired polypeptide is incorporated into an appropriate expression vector. Further, a transformant capable of producing a desired polypeptide is prepared by introducing the vector into an appropriate host cell. Then, the transformant can be cultured, and a desired polypeptide can be collected from the culture. For purification of the polypeptide, methods commonly used for protein purification can be applied as they are, and purification can be performed by appropriately combining various methods such as chromatography, salting out, concentration, membrane separation and the like.

There are no particular limitations on the route of administration of the therapeutic agent for rheumatoid arthritis of the present invention, and either oral administration or parenteral administration may be used. However, considering that the active ingredient is a polypeptide, parenteral administration, particularly administration by injection is preferred. . For example, direct administration to subcutaneous, intravenous, intramuscular, or affected area can be employed. Other parenteral administration routes, for example, transdermal administration (such as a patch) or rectal administration (such as a suppository) may be employed. On the other hand, when administered orally, dosage forms such as tablets, capsules, powders, fine granules, liquids, troches, and jelly can be employed. These administration routes and dosage forms can be used alone or in combination. In the preparation, stabilizers, preservatives, excipients, disintegrants, binders, lubricants and the like can be used as pharmaceutically acceptable carriers. For example, in the case of a freeze-dried injection, the drug solution prepared by adding a stabilizer or excipient consisting of albumin, amino acid, polyol, saccharide, etc. to the above-mentioned polypeptide that is the drug substance is aseptically filtered and sterilized. Filling and lyophilization may be performed.

The dose of the therapeutic agent for rheumatoid arthritis of the present invention may be appropriately selected according to the purpose of administration and the patient's condition (for example, sex, age, weight, etc.). For example, in the case of adults, oral administration may be performed at about 1 mg to 1000 mg per day, preferably about 15 mg to 150 mg, and parenteral administration may be performed at about 0.1 mg to 100 mg, preferably about 1.5 mg to 15 mg per day. .

The therapeutic agent for rheumatoid arthritis of the present invention can be used in combination with conventional therapeutic agents for rheumatoid arthritis. For example, a therapeutic agent for rheumatoid arthritis such as a non-steroidal anti-inflammatory drug, a steroid drug, an anti-rheumatic drug, an immunosuppressant, and an inhibitor of inflammatory cytokines (TNF-α, IL-1, IL-6, etc.) Can be used together.

The present invention can provide a method for treating rheumatoid arthritis, which comprises administering an effective amount of a scavenger receptor class A polypeptide comprising all or part of a scavenger receptor class A and having ligand binding activity. In addition, the present invention can provide a polypeptide comprising all or part of scavenger receptor class A and having ligand binding activity as a medicament for treating rheumatoid arthritis. Furthermore, the present invention provides a composition for treating rheumatoid arthritis, comprising a polypeptide having all or part of scavenger receptor class A as an active ingredient and having a ligand binding activity, and a pharmaceutically acceptable carrier. Can be done.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Preparation of polypeptide (soluble SCR) containing a part of mouse-derived macrophage scavenger receptor type A

A reverse transcription reaction was performed using mRNA prepared from mouse macrophages as a template to prepare cDNA. Using this cDNA as a template, PCR is performed using the oligonucleotides represented by SEQ ID NOs: 6 and 7 as primer pairs, and a DNA fragment containing a gene encoding a part of the mouse-derived scavenger receptor type A extracellular domain (soluble SCR) Acquired. The domain structure of the soluble SCR is shown in FIG. 2, and the amino acid sequence is shown in SEQ ID NO: 5.

The obtained amplified DNA fragment was inserted into the multicloning site of pFLAG-CMV3 vector (Sigma Aldrich) to construct a mammalian cell expression vector that expresses soluble SCR. At this time, the expression vector was designed so that the FLAG sequence as a tag was fused to the C-terminal side of the soluble SCR. This expression vector was transfected into CHO-K1 cells, and transformed cells expressing soluble SCR were cloned. The transformed cells were cultured in Opti-MEM medium (Invitrogen) containing 2% fetal bovine serum, 500 μg / mL G418, 50 units / mL penicillin, 50 μg / mL streptomycin at 37 ° C. under 5% CO ₂ . .

The condition medium of the transformed cells was collected, and ammonium sulfate (50% saturation) was added to the supernatant after centrifugation. The precipitate after centrifugation was redissolved in Tris-HCl buffer and dialyzed against the same buffer. This dialyzed sample was applied to a column packed with agarose beads cross-linked with anti-FLAG antibody (M1). After washing the column, the bound protein was eluted. This elution fraction was dialyzed against PBS to obtain a soluble SCR preparation. The purity of the soluble SCR in this fraction was confirmed by SDS-PAGE with silver staining. The endotoxin concentration of the prepared soluble SCR preparation was measured using a pyrogent single test (Cambrex Bio Science Walkersville), and it was confirmed that the endotoxin concentration was 0.05 EU / mL or less.

2. Evaluation of Rheumatoid Arthritis Treatment Effect of Soluble SCR Using Mouse Arthritis Model Collagen induced arthritis (CIA) model mice were used to evaluate the CIA inhibitory effect of soluble SCR.

Complete Freund's adjuvant (CFA; Chondrex # 7008; 1 mg / mL) 1 mL and bovine collagen (Bovine Collagen; Chondrex # 20022) 1 mL were mixed on ice for about 15 minutes to prepare a CFA emulsion. Eighteen 6-week-old DBA / 1J mice (female) were prepared, and each mouse was subcutaneously injected with 100 μL of CFA emulsion at a location about 2 cm away from the base of the tail. The animals were reared for 21 days.

1 mL of incomplete Freund's adjuvant (IFA; Chondrex # 7002; 1 mg / mL) and 1 mL of bovine collagen (Bovine Collagen; Chondrex # 20022) were mixed for about 15 minutes on ice to prepare an IFA emulsion. For each mouse on the 21st day of breeding, 100 μL of IFA emulsion was subcutaneously injected at a location about 2 cm away from the base of the tail. At this point, each mouse was divided into two groups (administration group and control group).

SCR 5 μg (0.031 μg / μL diluted solution 160 μL) was intraperitoneally administered every other day for 28 days for mice (8 mice) in the administration group (100 μg in total). On the other hand, since dialysis is performed at the time of SCR purification, PBS, which is this dialysis external solution, is used as a control. It was intraperitoneally administered every other day for 28 days. During this period, joint swelling and redness were visually observed every two days, and the incidence of arthritis and arthritis score were calculated (FIGS. 3 and 4). Regarding the incidence of arthritis, it was determined as “onset” when arthritis occurred even in one of the extremities. The arthritis score is 0 for arthritis, 1 point for complete swelling / redness on both wrists and ankles, 0.5 point for incomplete swelling / redness, 0.1 points for each finger and heel. They were evaluated and added to give an arthritis score. After completion of SCR administration on the 28th day, the onset state of arthritis was examined histochemically in the left ankle of a representative example (one individual having a median arthritis score) of each group of mice (FIG. 5).

FIG. 3 shows the relationship between the number of days after subcutaneous injection of IFA emulsion and the incidence of arthritis. The control group developed arthritis with a frequency of 100% 12 days after the subcutaneous injection of the IFA emulsion, whereas the SCR administration group developed arthritis with a frequency of 100% after 18 days. That is, it was shown that administration of SCR has an effect of delaying the onset of arthritis.

FIG. 4 shows the relationship between the number of days after subcutaneous injection of the IFA emulsion and the arthritis score. That is, in the control group, the arthritis score reached “3.6” 26 to 28 days after the subcutaneous injection of the IFA emulsion, whereas in the SCR administration group, “2.7” after 26 days and after 28 days. The value was as low as “2.3”, and arthritis was significantly suppressed.

Fig. 5 (a) to (d) show the results (photographs) of joint tissue staining. (A) and (b) are the results of the control group, and (c) and (d) are the results of the SCR administration group. That is, synovial cell proliferation, inflammatory cell infiltration, and bone destruction were observed in the control group of mice, but in the SCR-administered group, synovial cell proliferation and inflammatory cell infiltration were small, and bone destruction was suppressed. .

Furthermore, changes in body weight were graphed for each group of mice (FIG. 6). As a result, the SCR-administered group always maintained a higher body weight, and it was considered that inflammatory thinness was suppressed.

Claims

A therapeutic agent for rheumatoid arthritis comprising, as an active ingredient, a polypeptide comprising all or part of scavenger receptor class A and having ligand binding activity.
The rheumatoid arthritis therapeutic agent according to claim 1, wherein the polypeptide comprises a collagen-like domain.
The therapeutic agent for rheumatoid arthritis according to claim 1 or 2, wherein the polypeptide comprises an α helical coiled coil domain.
The scavenger receptor class A consists of the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence. The therapeutic agent for rheumatoid arthritis according to any one of 1 to 3.
The therapeutic agent for rheumatoid arthritis according to any one of claims 1 to 3, wherein the scavenger receptor class A comprises an amino acid sequence having a homology of 80% or more with a protein comprising the amino acid sequence represented by SEQ ID NO: 2. .