WO2019177053A1

WO2019177053A1 - Prophylactic or therapeutic agent for systemic lupus erythematosus or diseases caused by systemic lupus erythematosus, and prophylactic or therapeutic agent for connective tissue diseases

Info

Publication number: WO2019177053A1
Application number: PCT/JP2019/010363
Authority: WO
Inventors: 勝実前仲; 喜美子黒木; 直良前田; 千聖山田; 愛実 ▲高▼橋
Original assignee: 国立大学法人北海道大学
Priority date: 2018-03-13
Filing date: 2019-03-13
Publication date: 2019-09-19
Also published as: JPWO2019177053A1

Abstract

This prophylactic or therapeutic agent for systemic lupus erythematosus or diseases caused by systemic lupus erythematosus contains HLA-G or an HLA-G multimer as an active ingredient.

Description

Preventive or therapeutic agent for diseases caused by systemic lupus erythematosus or systemic lupus erythematosus, and preventive or therapeutic agent for collagen disease

The present invention relates to a preventive or therapeutic agent for diseases caused by systemic lupus erythematosus or systemic lupus erythematosus and a preventive or therapeutic agent for collagen diseases.

HLA-G is one of the non-classical MHCI molecules, which bind to inhibitory receptors such as leukocyte Ig-like receptors (LILRs) to cause myeloid monocytes, T cells and It inhibits the immune response of a wide range of immune cells including NK cells and induces immune tolerance.

HLA-G protein exists in various forms in the human body and functions as a natural inhibitory molecule. The HLA-G1 isoform exists as a heterotrimer consisting of a peptide, heavy chain, and β2 microglobulin. On the other hand, the domain-deficient HLA-G2 isoform is a homodimer consisting only of a domain-deficient heavy chain. HLA-G2 was known to have an activity that complements the function of HLA-G1, but the detailed function was unknown for a long time.

In recent years, various reports have been made on HLA-G2. Non-Patent Document 1 discloses that HLA-G2 exists as a homodimer and that a signal is transmitted through the immunosuppressive receptor LILRB2 as a receptor. Non-Patent Document 2 and Patent Document 1 show that HLA-G2 was analyzed in vivo for anti-inflammatory effects, and as a result, it was found to bind tightly to mouse receptor PIR-B. It is disclosed that a long-term immunosuppressive effect was obtained by administration.

Systemic lupus erythematosus (systemic lupus erythematosus: SLE) is an autoimmune disease characterized by systemic inflammatory lesions caused by tissue deposition of immune complexes such as DNA-anti-DNA antibodies. In pharmacological treatment of systemic lupus erythematosus, non-steroidal anti-inflammatory drugs, steroids such as prednisolone, etc. are generally used.

JP 2015-140322 A

However, there has been no report on HLA-G or its multimer involved in the prevention or treatment effect of systemic lupus erythematosus.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel pharmaceutical use for HLA-G or a multimer thereof. More specifically, the present invention provides a novel pharmaceutical use of HLA-G or a multimer thereof as a preventive or therapeutic agent for diseases caused by systemic lupus erythematosus or systemic lupus erythematosus and collagen diseases. With the goal.

In order to achieve the above object, a preventive or therapeutic agent for a disease caused by systemic lupus erythematosus or systemic lupus erythematosus according to the first aspect of the present invention,
HLA-G or HLA-G multimer is used as an active ingredient.

For example, the HLA-G multimer is a protein multimer having an amino acid sequence in which an α1 domain of HLA-G and an α3 domain of HLA-G are linked.

For example, the HLA-G multimer is
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1,
Because
It is a homomultimer between (a) or (b) or a heteromultimer between (a) and (b).

For example, the HLA-G or HLA-G multimer is
The modified protein or a salt thereof, wherein at least one amino acid residue in the amino acid sequence constituting the HLA-G or HLA-G multimeric protein is PEGylated with polyethylene glycol (PEG).

For example, the HLA-G multimer is
Consisting of a multimer of proteins having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked,
The modified protein or a salt thereof, wherein at least one amino acid residue in the amino acid sequence constituting the protein is PEGylated with polyethylene glycol (PEG).

For example, the molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.

For example, the protein is a protein comprising an amino acid sequence described in (a) or (b) below,
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
The multimer is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b), and has a binding activity to leukocyte Ig-like receptor B2. .

The preventive or therapeutic agent for collagen disease according to the second aspect of the present invention,
HLA-G or HLA-G multimer as an active ingredient, used for systemic administration,
It is characterized by that.

According to the present invention, HLA-G or a multimer thereof can provide a novel medicinal use as a preventive or therapeutic agent for diseases and collagen diseases caused by systemic lupus erythematosus or systemic lupus erythematosus.

(A) is a diagram showing the results of subjecting HLA-G2 to gel filtration chromatography, (B) is the result of subjecting the fraction fractionated from the peak (arrow part) of (A) to SDS-PAGE. FIG. (A) is a view showing the binding of HLA-G2 to LILRB2 or BSA (negative control) (solid line: when LILRB2 is immobilized on the sensor chip, dotted line: when BSA is immobilized on the sensor chip), (B) shows the results of surface plasmon resonance experiments evaluating the reaction (binding and dissociation) of HLA-G2 at various concentrations (13.3 nM, 26.6 nM, 53.1 nM) to LILRB2. The result of having evaluated the reaction (binding and dissociation) of HLA-G2 at various concentrations (0.095 μM, 0.19 μM, 0.38 μM, 0.76 μM, 1.52 μM) to PIR-B by surface plasmon resonance experiments is shown. FIG. (A) is a graph showing the change in body weight of the SLE model mouse after administration of PBS, and (B) is a graph showing the change in body weight of the SLE model mouse after administration of HLA-G2. FIG. 7 is a graph showing the amount of antinuclear antibody in blood of SLE model mice after HLA-G2 administration, (A) is a graph showing the results after 72 days of administration, and (B) is a mouse with a significantly high body weight. It is a graph which shows the result except one animal. It is the graph which measured the urine protein amount of the SLE model mouse | mouth after HLA-G2 administration, (A) is a graph figure which shows the result 76 days after administration, (B) is the graph figure which shows the result 86 days after administration. It is. It is the figure which evaluated the kidney tissue of the SLE model mouse after HLA-G2 administration. It is a figure which shows the result analyzed by ELISA about the cytokine in IFN-DC after 2 days incubation with HLA-G2, (A) is a graph figure which shows IL-6 production, (B) is IL- It is a graph which shows 10 production. It is a figure showing the primary structure (left) and molecule | numerator schematic diagram (right) of each recombinant protein, (a) is HLA-G2, (b) is a schematic diagram which shows LILRB2. (A) is a diagram showing a chromatogram of SEC purification using a HiLoad26 / 60 Superdex75 pg column after unwinding inclusion bodies in the preparation of HLA-G2, and (b) is obtained by adding a reducing agent (TCEP). It is the figure which showed the result of CBB dyeing | staining of obtained PEGylated HLA-G2. It is the figure which showed the comparison of the reaction efficiency by PEG molecular weight, (a) is a figure which shows the result of CBB dyeing | staining, (b) is BaI2 dyeing | staining. It is a figure which shows the result of carrying out SEC purification using Superdex 200 10/300 GL of each molecular weight PEG using Superdex 200 10/300 GL, concentrating, and silver-staining after SDS-PAGE, (a) is PEG5-HLA-G2, (B) shows the results of PEG10-HLA-G2, (c) shows the results of PEG20-HLA-G2, and (d) shows the results of PEG40-HLA-G2. It is a schematic diagram showing the principle of biotinylated protein immobilization on a sensor chip CAP, (a) is a schematic diagram showing a state in which single-stranded DNA is immobilized on the sensor chip CAP, and (b) is a streptogram. It is a schematic diagram showing a mode that avidin was fixed on the chip | tip, (c) is a schematic diagram showing a mode that biotinylated LILRB2 and biotinylated BSA were fix | immobilized. FIG. 4 is a diagram showing sensorgrams obtained by a binding experiment with a LILRB2 receptor, where (a) is HLA-G2, (b) is PEG5-HLA-G2, (c) is PEG10-HLA-G2, (d) FIG. 4 is a view showing the results of PEG20-HLA-G2. It is a figure which shows the outline | summary of the test using a SLE model mouse. It is the graph which showed the weight transition of the SLE model mouse. (A) is the graph which measured the blood anti- dsDNA antibody titer of the SLE model mouse, (b) is a graph which shows the result 90 days after administration. It is the graph which measured the urinary albumin index of the SLE model mouse. It is the graph which measured the Blys blood concentration of a SLE model mouse. (A) is the graph which measured the blood anti- dsDNA antibody titer of the SLE model mouse, (b) is the graph which measured the urinary albumin index of the SLE model mouse.

The agent for preventing or treating a disease caused by systemic lupus erythematosus or systemic lupus erythematosus according to this embodiment will be described in detail.

The preventive or therapeutic agent for systemic lupus erythematosus or systemic lupus erythematosus according to this embodiment comprises HLA-G or HLA-G multimer as an active ingredient (HLA: Human Leukocyte Antigen (human leukocyte antigen) ).

The HLA-G or HLA-G multimer targeted by this embodiment will be described.

The HLA-G targeted by this embodiment is preferably human-derived HLA-G. The sequence information of human HLA-G molecules is already known, for example, by NCBI. Specifically, as one of the isoforms, NCBI describes the human-derived HLA-G full-length gene sequence (= HLA-G1) in NM_002127.5, of which the α1 domain of HLA-G And the base sequence corresponding to the gene region of the α3 domain are “1st to 270th base sequence” and “271st to 540th base sequence” of SEQ ID NO: 2 shown below, respectively. There are various isoforms in the HLA-G molecule, and their amino acid sequences are slightly different depending on the isoform.

The HLA-G multimer targeted by this embodiment is, for example, a multimer of a protein having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked,
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1,
Because
It is a homomultimer between (a) or (b) or a heteromultimer between (a) and (b).

In the present specification, “a protein multimer having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked” may be referred to as “HLA-G2” or “HLA-G2 multimer”. is there.

The above “protein consisting of the amino acid sequence shown in SEQ ID NO: 1” is one of dimers of proteins having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked. . Here, the HLA-G molecule is preferably human-derived HLA-G. In SEQ ID NO: 1, “1st to 90th amino acid region” corresponds to the amino acid sequence of the α1 domain, and “91st to 180th amino acid region” corresponds to the amino acid sequence of the α3 domain. Further, in SEQ ID NO: 2 representing the base sequence encoding the amino acid sequence (SEQ ID NO: 1), the “1st to 270th base sequence” region corresponds to the gene region encoding the α1 domain, and the “271st to 540th bases” The “sequence” region corresponds to the gene region encoding the α3 domain.

The amino acid sequence (sequence) of a conjugate of α1 domain and α3 domain (α1-3 conjugate) of HLA-G molecule as the “protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added”. A protein having an amino acid sequence (SEQ ID NO: 3) encoded by intron 4 of the HLA-G molecule is further exemplified on the C-terminal side of No. 1), and this may be referred to as “HLA-G2” in the present specification. . As another “protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added”, the C1 of the amino acid sequence (SEQ ID NO: 1) of a conjugate of α1 domain and α3 domain of HLA-G molecule A protein having an amino acid sequence (SEQ ID NO: 4) of the transmembrane domain and intracellular domain of the HLA-G2 molecule on the end side can be exemplified.

Of the “protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added”, the function of a multimer of a conjugate of α1 domain and α3 domain of the above HLA-G molecule (leukocyte Ig-like receptor) And the amino acid sequence of the α1-3 conjugate (SEQ ID NO: 1) is preferably 90% or more, preferably 95% or more. It is a protein formed by deletion, substitution or addition. More preferably, it is a protein having an amino acid sequence of 96% or more, more preferably 98% or more identical to the amino acid sequence (SEQ ID NO: 1) of the above conjugate.

In the present specification, among the HLA-G multimers, (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or (b) one or several amino acids are missing in the amino acid sequence shown in SEQ ID NO: 1. A protein comprising an amino acid sequence deleted, substituted, or added, which is a homomultimer between (a) or (b) or a heteromultimer between (a) and (b), “HLA-G2 Sometimes referred to as "multimer".

The HLA-G2 multimer targeted by the present embodiment is not limited to the above-mentioned ligated body of α1 domain and α3 domain of the HLA-G molecule (hereinafter also simply referred to as “α1-3 ligated product”). Any structure may be used as long as it has a linked structure of α1 domain and α3 domain of HLA-G molecule and the multimer has binding activity to leukocyte Ig-like receptor B2 (LILRB2). For example, as long as the above conditions are satisfied, one or more amino acids are missing in the amino acid sequence (SEQ ID NO: 1) of the conjugate of α1 domain and α3 domain (α1-3 conjugate) of the HLA-G molecule. It may be a protein consisting of a deleted, substituted or added amino acid sequence. Here, “binding activity to leukocyte Ig-like receptor B2 (LILRB2)” is an activity in which an HLA-G2 multimer directly binds to LILRB2, thereby transmitting a signal via LILRB2 and having an immunoregulatory effect. Means an activity capable of exerting.

Whether or not the HLA-G2 multimer has binding activity with leukocyte Ig-like receptor B2 (LILRB2) can be confirmed by, for example, a reporter assay using a T cell hybridoma. Examples of such T cell hybridomas include NFAT-GFP introduced reporter cells (mouse T cell hybridomas) expressing a chimeric molecule in which the extracellular domain of LILRB2 and the transmembrane / intracellular domain of the active receptor PILRβ are fused. Can do. In this case, when the HLA-G2 multimer binds to the LILRB2 receptor, a signal through the intracellular domain of PILRβ is transmitted, and the transcription factor NFAT is activated. The reporter assay is an assay system using the fact that the expression of GFP is induced by the activation of the NFAT. GFP expression indicates that LILRB2 and HLA-G2 multimer are bound, and the appearance of fluorescence due to such GFP expression can be confirmed by flow cytometry.

The HLA-G2 multimer is a multimerized product in which the aforementioned HLA-G2s are disulfide bonded or non-covalently bonded without using disulfide bonds. In the HLA-G2 multimer in the present embodiment, for example, in the case of a naturally formed homodimer, the HLA-G2 multimer is multimerized regardless of the disulfide bond, and the cysteine residue (Cys42) remains free on the molecular surface. In the case of a further dimer (tetramer) having a homodimer as one unit or a further multimer thereof, cysteine residues (Cys42) present on the surface of the homodimer molecule may be It may be multimerized by an intervening disulfide bond. Furthermore, in the case of a homodimer that spontaneously forms, it multimerizes regardless of disulfide bonds, and in the case of a further dimer (tetramer) having a homodimer as one unit or a further multimer thereof, It may be multimerized by binding via amino acid residues other than cysteine residues present on the surface of the homodimer molecule. In the present embodiment, in the case of a homodimer, the dimerization is preferably performed regardless of the disulfide bond. Further, at least one amino acid residue in the amino acid sequence constituting the protein of the HLA-G2 multimer may be PEGylated with polyethylene glycol (PEG). The PEGylated HLA-G2 multimer will be described later.

The HLA-G2 multimer may be a homomultimer composed of α1-3 conjugates or mutants as described above, or a heteromultimer composed of α1-3 conjugates and mutants. There is no limitation. Preferably, it is a homomultimer composed of α1-3 conjugates.

The HLA-G2 multimer has a structure in which the binding site to the leukocyte Ig-like receptor B2 (LILRB2) is exposed on the surface, and the binding to these receptors is not hindered by steric hindrance. Therefore, the HLA-G2 multimer has a structure capable of retaining the same type of function (binding activity to leukocyte Ig-like receptor) as HLA-G2.

The HLA-G or HLA-G multimer targeted by this embodiment includes, for example, HLA-G1 (NCBI, NM_002127.5) (extracellular domain: SEQ ID NO: 11), (full length (NCBI, NM_002127. 5, including the signal sequence and the Stokes region and lower): SEQ ID NO: 12) and proteins consisting of amino acid sequences in which one or several amino acids are deleted, substituted or added in HLA-G1.

An example of a method for preparing the HLA-G2 multimer will be described.

First, using a known gene recombination technique, a recombinant vector in which a gene encoding the amino acid sequence of the HLA-G2 multimer (eg, SEQ ID NO: 2) is incorporated into an expression vector or the like is constructed. The transformation can be carried out by expressing the recombinant HLA-G2 multimer and recovering it by introducing the constructed recombinant vector into a host by various transformation methods to obtain a transformant and culturing the transformant.

Here, the gene encoding the amino acid sequence of the HLA-G2 multimer includes the gene (SEQ ID NO: 2) encoding the amino acid sequence (SEQ ID NO: 1) of the conjugate of α1 domain and α3 domain of HLA-G, A gene encoding the amino acid sequence of the aforementioned HLA-G2 (hereinafter sometimes referred to as “mutant HLA-G2” for convenience) obtained by deleting, replacing or adding a part of the amino acid sequence of the conjugate. It can also be used. Further, from the viewpoint of increasing the yield, as a gene encoding the amino acid sequence of HLA-G2, for example, the first to 18th amino acids of the nucleotide sequence shown in SEQ ID NO: 2 (the 1st to 6th amino acids in the amino acid sequence of HLA-G2 are encoded) A gene obtained by substituting the base sequence of SEQ ID NO: 5 (hereinafter referred to as “modified gene”) (SEQ ID NO: 6) can also be used.

The gene encoding the mutant HLA-G2 multimer may be prepared by introducing a mutation into the DNA sequence of the α1-3 linked gene. For example, Molecular Cloning, A Laboratory Manual 2nd ed. , Cold Spring Harbor Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) and the like. Specifically, it can be prepared using a mutation introduction kit using a site-specific mutagenesis method by a known method such as the Kunkel method or the Gapped duplex method. Such kits include, for example, QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTailorTM Site-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRaSite-MustimatedMut-SensitMight-SensitMist-Smut-SensitMist-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Strain-Sensor-Mut-SensitMut-Sk (Takara Bio Inc.) and the like are preferred.

The host used for the preparation of the transformant is not particularly limited as long as it can express HLA-G2 (α1-3 conjugate, mutant) from the introduced recombinant vector or the like. Known cells that can serve as hosts, such as cells derived from various animals such as humans and mice, cells derived from various insects, prokaryotic cells such as E. coli, eukaryotic cells such as yeast, and plant cells can be used.

Specifically, the production of the recombinant HLA-G2 multimer should be carried out by a method including the step of culturing the above-mentioned transformant and the step of collecting the recombinant HLA-G2 multimer from the resulting culture. Can do. Here, “cultured product” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The transformant can be cultured according to a usual method used for host culture. The protein of interest is accumulated in the culture.

When the recombinant HLA-G2 multimer is produced extracellularly, use the culture solution as it is, or remove the cells by centrifugation, filtration or the like. Thereafter, the recombinant HLA-G2 multimer is collected from the culture by extraction with ammonium sulfate precipitation, if necessary, and further subjected to dialysis, various chromatography (gel filtration, ion exchange chromatography, affinity chromatography) as necessary. Etc.) can be isolated and purified.

When a recombinant HLA-G2 multimer is produced in a cell, the recombinant HLA-G2 multimer can be collected by disrupting the cell. If the soluble fraction contains an HLA-G2 multimer, the disrupted cell residue (including the cell extract insoluble fraction) is removed as necessary by centrifugation or filtration after disruption. The supernatant after removal of the residue is a cell extract soluble fraction and can be a crude protein solution. On the other hand, when the HLA-G2 multimer is expressed as inclusion bodies in the insoluble fraction, after crushing, the insoluble fraction is isolated by centrifugation, washed with a buffer containing a surfactant, etc., and repeatedly centrifuged. Remove cell debris. The obtained inclusion body is solubilized with a buffer containing a denaturing agent such as guanidine or urea, and then the protein is unwound using a dilution method or a dialysis method. The functionally unwound HLA-G2 multimer can be isolated and purified using various types of chromatography (gel filtration, ion exchange chromatography, affinity chromatography, etc.).

Recombinant HLA-G2 multimers can be produced not only using a protein synthesis system using a transformant, but also using a cell-free protein synthesis system that does not use any living cells. The -G2 multimer can be purified by appropriately selecting means such as chromatography.

The HLA-G2 multimer may be obtained by any method, and the acquisition method is not particularly limited.

The HLA-G or HLA-G multimer is a modification in which at least one amino acid residue in the amino acid sequence constituting the protein of the HLA-G or HLA-G multimer is PEGylated with polyethylene glycol (PEG) It may be a protein or a salt thereof.

The HLA-G multimer comprises a multimer of a protein having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked, and at least one amino acid in the amino acid sequence constituting the protein It may be a modified protein or a salt thereof whose residue is PEGylated with polyethylene glycol (PEG).

The amino acid residue to be PEGylated is preferably present at a site that does not affect the binding to the receptor in the protein. Examples of amino acid residues to be PEGylated include lysine residues and cysteine residues. For example, when a cysteine residue is PEGylated, the cysteine residue may be, for example, the 42nd free cysteine residue of HLA-G2. Alternatively, an amino acid residue present at an arbitrary site may be substituted with a cysteine residue, and the cysteine residue may be modified with PEG. For example, the 86th asparagine which is a sugar chain modification site of HLA-G And may be modified by PEGylation. Further, a cysteine residue may be added to an arbitrary site, and the cysteine residue may be modified with PEG. For example, cysteine may be added to the C-terminus of HLA-G and modified with PEG. Any amino acid residue capable of PEGylation modification can be selected without limitation to lysine residues and cysteine residues.

In this embodiment, the molecular weight of PEG used for PEGylation modification is, for example, 5 kDa to 100 kDa, and preferably 5 kDa to 40 kDa. Details of the PEGylation modification will be described later.

The protein is, for example, a protein comprising an amino acid sequence described in the following (a) or (b),
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
The multimer is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b), and has a binding activity to leukocyte Ig-like receptor B2. May be. Whether or not the multimer has binding activity with leukocyte Ig-like receptor B2 can be confirmed in the same manner as described above.

Next, the modified protein production method according to the present embodiment will be described in detail.

The method for producing a modified protein according to this embodiment includes, for example,
(A) preparing HLA-G or HLA-G multimer;
(B) a step of deaerating the protein of HLA-G or HLA-G multimer obtained in step (A), followed by a reduction treatment;
(C) PEGylation modification of the protein reduced in step (B);
including.

Details of HLA-G or HLA-G multimer are as described above. HLA-G or HLA-G multimers can be prepared by any method.

Step (B) is a step in which the HLA-G or HLA-G multimeric protein obtained in step (A) is degassed and then reduced.

In step (B), a deaeration process is performed to advance the PEGylation reaction under anaerobic conditions. The deaeration process may be performed for 1 hour using an aspirator, for example. Prior to the deaeration treatment, for example, the HLA-G or HLA-G multimeric protein obtained in the step (A) is transferred to a PEGylation buffer (eg, 1 × Phosphate-Buffer Saline (PBS), 5 mM EDTA). Replacement may be performed. Other known methods may be used as the deaeration process.

In step (B), the reducing agent is added to cleave the disulfide bond by reduction treatment to reexpose the thiol group of the residue serving as the PEGylation target and improve the reaction efficiency. As the reducing agent, any reducing agent having a reducing action can be used, and for example, tris (2-carboxyethyl) phosphine (TCEP) frequently used in the reaction using maleimide and cysteine may be used. When TCEP is used as the reducing agent, for example, TCEP may be added so as to have a final concentration of 0.1 mM or more.

Step (C) is a step of PEGylating the protein that has been reduced in step (B).

In step (C), specifically, the PEGylated modification is carried out by reacting the PEGylation reagent having a reactive functional group such as a maleimide group or a succinimide group at the end of PEG with the protein of this embodiment in a solution. Protein can be obtained. Examples of the PEGylation reagent used include linear methyl PEGn (n is the number of PEG repeats) maleimide and branched (methyl-PEGn) n-PEGn maleimide that form a thioether bond with the SH group of cysteine. It is done. The molecular weight of PEG used for PEGylation modification is, for example, 5 kDa to 100 kDa, and preferably 5 kDa to 40 kDa. Examples of the PEGylation reagent include ME-400MA (MW: 42,653 Da) (PEG40), ME-200MAOB (MW: MW), which are high-purity linear PEGs that have a maleimide group as a reactive group and recognize and react with a thiol group. 20,841 Da) (PEG20), ME-100MA (MW: 10,303 Da) (PEG10), ME-050MA (MW: 5,393 Da) (PEG5) (all are NOF Corporation), and the like.

In step (C), the PEGylation reagent is used, for example, with PEG: protein = 5: 1, 10: 1, 20: 1, 30: 1 (molar ratio) or the like so that the amount of PEG is excessive with respect to the protein May be mixed. PEGylation can be modified by adding a PEGylation reagent and reacting at 4 ° C. overnight, for example. As described above, at least one amino acid residue (for example, cysteine residue) in the amino acid sequence constituting the protein is PEGylated with PEG.

The PEG-modified protein may be purified by, for example, gel filtration chromatography (SEC).

A method for producing a modified protein according to another embodiment includes, for example,
(A ′) preparing a multimer of a protein having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked;
(B ′) a degassing treatment of the protein multimer obtained in step (A), followed by a reduction treatment;
(C ′) PEGylation modification of the multimer of the protein reduced in step (B);
including.

Details of the protein multimer (HLA-G2 or HLA-G2 multimer) having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked are as described above.

The HLA-G2 multimer targeted by the present embodiment is, for example, a protein multimer having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked, as described above.
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1,
Because
It is a homomultimer between (a) or (b) or a heteromultimer between (a) and (b).

Details of the method for preparing the HLA-G2 multimer in the step (A ′) are as described above.

As a salt of the modified protein, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or a base (eg, alkali metal salt) is used, and particularly a physiologically acceptable acid addition salt. Is preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

The preventive or therapeutic agent according to this embodiment is used for the prevention or treatment of diseases caused by systemic lupus erythematosus (SLE) or SLE.

Systemic lupus erythematosus (systemic lupus erythematosus: SLE) is an autoimmune disease characterized by systemic inflammatory lesions caused by tissue deposition of immune complexes such as DNA-anti-DNA antibodies. As diagnostic criteria, there are the following 11 items, and if 4 or more of these items are satisfied, SLE is diagnosed (refer to the intractable disease information center homepage (http://www.nanbyou.or.jp/)).
(1) Facial erythema (2) Discoid eruption (3) Photosensitivity (4) Oral ulcers (painless and appear in the oral cavity or nasopharynx)
(5) Arthritis (Non-destructive with 2 or more joints)
(6) Serositis (pleurisy or pericarditis)
(7) Renal lesions (appearance of continuous proteinuria or cellular casts of 0.5 g / day or more)
(8) Neurological lesions (convulsive seizures or mental disorders)
(9) Hematological abnormalities (hemolytic anemia, leukopenia of 4,000 / mm ³ or less, lymphocyte depletion of 1,500 / mm ³ or less, or thrombocytopenia of 100,000 / mm ³ or less)
(10) Immunological abnormality (anti-double-stranded DNA antibody positive, anti-Sm antibody positive or anti-phospholipid antibody positive (anti-cardiolipin antibody, lupus anticoagulant, syphilis false positive))
(11) Antinuclear antibody positive

The preventive or therapeutic agent according to the present embodiment has a preventive or therapeutic effect on SLE. When the prophylactic or therapeutic agent according to this embodiment is administered to a subject diagnosed with SLE, for example, the blood antinuclear antibody amount in the subject can be reduced. In addition, when the prophylactic or therapeutic agent according to the present embodiment is administered to a subject diagnosed with SLE, for example, the amount of urine protein in the subject can be reduced.

The prophylactic or therapeutic agent according to the present embodiment has a prophylactic or therapeutic effect on diseases caused by SLE. Diseases caused by SLE are concepts including so-called SLE reserve groups such as proteinuria, nephritis, lymphadenopathy, etc. For example, even if a definitive diagnosis of SLE has not been made, protein A preventive or therapeutic effect can be obtained by administering the preventive or therapeutic agent according to the present embodiment to a subject exhibiting symptoms such as urine, nephritis, and lymphadenopathy.

The administration method of the prophylactic or therapeutic agent according to the present embodiment can be appropriately selected from oral administration, intravenous administration, intraperitoneal administration, intradermal administration, sublingual administration, topical administration, etc. for humans or non-human mammals. The dosage form may be arbitrary, for example, oral solid preparations such as tablets, granules, powders and capsules, oral liquid preparations such as internal liquids and syrups, and parenteral liquid preparations such as injections. Can be appropriately prepared. An appropriate drug delivery system (DDS) may also be used.

The dose and administration interval of the prophylactic or therapeutic agent according to this embodiment can be appropriately set depending on the patient's age, body weight, indication symptoms, etc. For example, 3 to 4 administrations or 2 to 3 administrations per week Although there may be, it is not restricted to this. The prophylactic or therapeutic agent according to this embodiment has an effect on SLE or a disease caused by SLE by inducing production of cytokines IL-10 and IL-6 not locally but systemically. It is. From the viewpoint of acquiring self-tolerance described later, the dose of the prophylactic or therapeutic agent according to the present embodiment is preferably 0.35 mg / kg body weight or more, 0.4 mg / kg body weight or more, 0.45 mg / kg body weight or more, It is 0.5 mg / kg body weight or more, 1 mg / kg body weight or more, and the administration interval is preferably at least once a week. Moreover, there is a possibility that the dose and the number of administrations of the preventive or therapeutic agent according to the present embodiment can be reduced by the combined use with existing drugs having different action mechanisms. The administration of the therapeutic agent according to the present embodiment can be any of administration during meals, administration after meals, administration before meals, administration between meals, administration before going to bed, and the like.

The preventive or therapeutic agent for diseases caused by SLE or SLE according to the present embodiment contains HLA-G or HLA-G multimer as an active ingredient. HLA-G or HLA-G multimers may be used in the form of various salts, hydrates, etc., if necessary, and appropriate chemical modification in consideration of storage stability (especially maintaining activity) May be used in a state in which s. Is used, or may be used in a crystallized state or in a dissolved state.

The preventive or therapeutic agent for a disease caused by SLE or SLE according to the present embodiment may contain other components in addition to containing HLA-G or HLA-G multimer as an active ingredient. Examples of the other components include various pharmaceutical components (various pharmaceutically acceptable carriers and the like) required depending on the usage (usage form) of the prophylactic or therapeutic agent. These other components can be appropriately contained as long as the preventive or therapeutic effect on the disease caused by SLE or SLE exerted by the active ingredient of the present invention is not impaired.

Further, it is preferable that the HLA-G or HLA-G multimer as an active ingredient is purified so as to have a higher purity. Specifically, for example, a purity of 50% or more is preferable, more preferably 80% or more, and still more preferably 90% or more.

Next, the preventive or therapeutic agent for collagen disease according to this embodiment will be described in detail.

The preventive or therapeutic agent for collagen disease according to the present embodiment is characterized in that HLA-G or HLA-G multimer is used as an active ingredient and is used for systemic administration. Preferably, the HLA-G multimer is a protein multimer having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked. Preferably, the HLA-G multimer has (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, or (b) one or several amino acids deleted or substituted in the amino acid sequence represented by SEQ ID NO: 1. Alternatively, it is a protein comprising an added amino acid sequence, which is a homomultimer between (a) or (b) or a heteromultimer between (a) and (b). Details of each term are the same as described above.

Examples of the preventive or therapeutic agent for collagen disease according to this embodiment include systemic lupus erythematosus, systemic scleroderma, polymyositis / dermatomyositis, Sjogren's syndrome, mixed connective tissue disease, antiphospholipid antibody syndrome, Behcet's disease, Allergic granulomatous vasculitis (Chirge-Strauss syndrome), adult Still's disease, eosinophilic fasciitis, nodular periarteritis (nodular polyarteritis / microscopic polyangiitis), aortitis syndrome (Takayasu) Arteritis), Wegener's granulomatosis, temporal arteritis, malignant rheumatoid arthritis and other collagen diseases including systemic administration. The administration method of the prophylactic or therapeutic agent according to the present embodiment includes oral administration, intravenous administration, intraperitoneal administration, intradermal administration, sublingual administration and the like for use in systemic administration to humans or non-human mammals. Selected. The dosage form, dosage, administration interval, active ingredient and the like are the same as described above.

As described above, the preventive or therapeutic agent for a disease caused by SLE or SLE according to the present embodiment includes HLA-G or HLA-G multimer as an active ingredient, and HLA-G or HLA-G multimer. Is mainly mediated by the inhibitory receptor LILRB2 expressed in antigen-presenting cells, so that the effect on effector cell functions is reduced, and a mild and long-term immunosuppressive effect via antigen-presenting cells is expected . Moreover, it can be used as a concomitant drug with other preparations of diseases caused by SLE or SLE. As a preventive or therapeutic agent for diseases caused by SLE or SLE for which no therapeutic method has been established, development as a new preparation having a different mechanism of action from existing biological preparations is expected. Further, the preventive or therapeutic agent for collagen disease according to this embodiment is 1L-6, which is a cytokine involved in acquiring self-tolerance in peripheral blood-derived myeloid cells such as monocytes, macrophages and dendritic cells, as described later. And 1L-10 production-inducing action, and systemic administration provides a preventive or therapeutic effect against collagen disease.

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

Example 1
(Preparation of HLA-G2 protein)
(1) Expression of α1-3 conjugate as inclusion body in E. coli After cutting the pGMT7 vector with restriction enzymes NdeI and HindIII, a conjugate of α1 domain and α3 domain of HLA-G molecule (α1-3 conjugate) is obtained. A modified gene (SEQ ID NO: 6) encoding the gene was inserted using T4 DNA ligase to construct a modified HLA-G2-pGMT7.

The modified HLA-G2-pGMT7 was performed by the following method. First, using the HLA-G2-pGMT7 plasmid as a template, PCR buffer (Promega), deoxyNTP mixture (TOYOBO), 5'-side primer (atgggttagcattagtatgcgtttattttaggtaggactag-cctact cctac cc SEQ ID NO: 10) (final concentration of 0.2 μM each) and Pfu TurboDNA Polymerase (Promega) were added to carry out PCR. At that time, the reaction was performed for 25 cycles with denaturation 30 seconds (95 ° C.), annealing 1 minute (60 ° C.), and extension 8 minutes (68 ° C.). Next, DpnI (manufactured by NEB) was added to the PCR product, reacted at 37 ° C. for 1 hour, the template was removed, and agarose gel electrophoresis was performed to confirm the presence of the PCR product. Then, the base sequence was confirmed with a DNA sequencer to obtain a modified HLA-G2-pGMT7 plasmid.

Next, E. coli BL21 (DE3) pLysS strain was transformed with this modified HLA-G2-pGMT7 plasmid, and 2 × YT medium (0.5% sodium chloride, 1.6% tryptone containing 100 mg / L ampicillin) was transformed. The cells were cultured at 37 ° C. in 1% dry yeast extract (manufactured by Nacalai Tesque). Next, when the culture suspension reached OD600 = 0.4 to 0.6, IPTG was added to 1 mM, and expression was further induced at 37 ° C. for 4 to 6 hours.

(2) Rewinding of E. coli inclusion bodies Cell suspensions that had been induced to express by adding IPTG were centrifuged to collect the bacterial bodies, and a suspension buffer (50 mM Tris pH 8.0, 100 mM sodium chloride) was added and suspended. The bacterial cells were crushed by ultrasonic crushing and then centrifuged to obtain inclusion bodies. The inclusion bodies were thoroughly washed with Triton washes buffer (0.5% Triton X-100, 50 mM Tris pH 8.0, 100 mM sodium chloride) and Resuspension buffer (50 mM Tris pH 8.0, 100 mM sodium chloride), and then 6.0 M Guandine. Solubilized with solution (6.0 M guanidine, 50 mM female pH 6.5, 10 mM EDTA). At this time, when the HLA-G2 solution was measured by ultraviolet absorption, the A280 value was about 70, and the expression level of HLA-G2 is considered to be about 100 mg / L. Winding while stirring for 72 hours at 4 ° C in a general dilution method using a refolding buffer (0.1 M Tris pH 8.0, 0.4 M L-arginine, 5 mM EDTA, 3.7 mM cystamine, 6.4 mM cysteamine) Returned. And after concentrating this, it used for the gel filtration chromatography of the following conditions, and refine | purified.
<Gel filtration chromatography conditions>
Column: HiLoad 26/60, Superdex 75 (60 cm, id 26 mm)
Mobile phase: 20 mM Tris-HCl, 100 mM NaCl buffer (pH 8)
Flow rate: 2.5 ml / min

A chromatogram obtained by gel filtration chromatography is shown in FIG. The desired peak fraction (arrow part) was collected and subjected to SDS-PAGE (15% acrylamide gel) under non-reducing conditions. The result is shown in FIG. From the molecular weight of the HLA-G2 dimer (42 kDa), it was confirmed that the peak was an elution fraction of HLA-G2, and the fraction was recovered and concentrated to obtain HLA-G2 (test sample).

(3) Activity measurement The N-terminal 2 domain (D1D2) of LILRB2, in which a biotinylated sequence was added to the C terminus in Reaction buffer (50 mM D-biotin, 100 mM ATP, 15 μM BirA), was biotinylated at the C terminus. Biotinylated LILRB2 was separated from the reaction buffer and purified by gel filtration chromatography (Superdex 75).

Using BIAcore (registered trademark) 3000 (GE healthcare BIAcore), the binding property between HLA-G2 and LILRB2 was evaluated by a surface plasmon resonance experiment. First, streptavidin was covalently immobilized on a research sensor chip, and biotinylated LILRB2 and negative control BSA were immobilized via the streptavidin. Next, HLA-G2 dissolved in HBS-EP (10 mM pes pH 7.5, 150 mM sodium chloride, 3.4 mM EDTA, 0.005% Surfactant P20) as a running buffer was flowed at 10 μL / min. The binding response at each concentration was calculated by subtracting the response measured in the control flow cell from the response in the sample flow cell.

FIG. 2 is a diagram showing the response of HLA-G2 to LILRB2 or BSA which is a negative control. The solid line shows the case where LILRB2 is fixed to the sensor chip, and the dotted line shows the case where BSA is fixed to the sensor chip. According to FIG. 2 (A), LILRB2 is bound to HLA-G2 compared to BSA. FIG. 2 (B) is a diagram showing apparent Kd values of HLA-G2 dimer and LILRB2. According to FIG. 2 (B), the apparent Kd value is 1.5 nM as a result of analysis using a 1: 1 binding model by BIAevaluation software. From this result, it was confirmed that HLA-G2 binds to LILRB2.

(Confirmation of binding to mouse immunosuppressive receptor PIR-B)
The binding of HLA-G2 to PIR-B (primed-immunoglobulin-like receptor B), a mouse homolog of LILRB, which is a human immunosuppressive receptor (leukocyte immunoglobulin-like receptor B), is expressed by surface plasmon resonance sr Was used to evaluate.

(1) Preparation of PIR-B The extracellular domain of PIR-B was transfected into HEK293T cells and cultured in 1% FCS-DMEM for 72 hours. The amino acid sequence of the extracellular domain of PIR-B and the base sequence of the gene encoding it are shown in SEQ ID NOs: 7 and 8, respectively. The full-length amino acid sequence of PIR-B is published in NM_011095 of NCBI, and the PIR-B extracellular domain is a portion corresponding to domains 1-6.

Next, the supernatant was recovered from the culture, the PIR-B extracellular domain was purified by Ni affinity chromatography, and subjected to Western blotting (12.5% acrylamide gel), and the protein obtained by purification from the estimated molecular weight of 75 kDa was converted to PIR. -Confirmed to be B extracellular domain.

The purified PIR-B extracellular domain was dissolved and biotinylated in Reaction buffer (50 mM D-biotin, 100 mM ATP, 15 μM BirA) to 15 μM. The biotinylated PIR-B extracellular domain was separated from the reaction buffer by gel filtration chromatography (Superdex 200) and purified.

(2) Evaluation of HLA-G2 binding to PIR-B (extracellular domain) Using BIAcore (registered trademark) 3000 (GE Healthcare BIAcore), HLA-G2 and PIR-B extracellular prepared above The binding property with the domain was evaluated by surface plasmon resonance experiment. First, streptavidin was covalently immobilized on a research sensor chip, and biotinylated PIR-B extracellular domain and BSA as a negative control were immobilized via the streptavidin. Next, HLA-G2 dimer dissolved in HBS-EP (running buffer: HBS-EP (10 mM to pH 7.5, 150 mM sodium chloride, 3.4 mM EDTA, 0.005% Surfactant P20)) was flowed at 5 μL / min. The binding response at each concentration was calculated by subtracting the response measured in the control flow cell from the response in the sample flow cell. The apparent binding constant (Kd) was obtained by analysis with a BIAevaluation software using a 1: 1 binding model.

FIG. 3 is a diagram showing the reaction of PIR-B to each concentration of HLA-G2 (0.095 μM, 0.19 μM, 0.38 μM, 0.76 μM, 1.52 μM). From the results of FIG. 3, the apparent dissociation constant (Kd value) by analysis using the 1: 1 binding model was 142 nM. From this result, it was confirmed that HLA-G2 binds to the extracellular domain of mouse PIR-B corresponding to human LILRB2, in the same manner as it binds to human LILRB2 which is an immunosuppressive receptor (see FIG. 2). It was done.

(Example 2)
(SLE model mouse experiment)
HLA-G2 was administered to SLE model mice to verify the therapeutic effect of HLA-G2 on SLE.

As a negative control, HLA-G2 prepared in Example 1 or PBS was administered to SLE model mice as follows ("HLA-G2 administration group" or "PBS administration group"). MRL / MpJJmsSlc-lpr / lpr mice (Japan SLC Co., Ltd.) were used as SLE model mice, and the test was started from 12 mice in each administration group. As for HLA-G2, a new lot (newly unwound to re-purified) was administered about every 3 weeks.
・ Administration route: intraperitoneal administration ・ Dose: 15 μg / animal (HLA-G2 0.075 mg / mL, 200 μL administration)
・ Dosing interval: twice a week ・ Dosing period: 12 weeks in total

Sampling was performed 4 animals every 4 weeks. It was n = 10 in the HLA-G2 administration group and n = 10 in the PBS administration group until the 20th administration. In addition, n = 6 in the HLA-G2 administration group and n = 4 in the PBS administration group ended up to the 28th administration, and the administration was terminated at this point.

Measured body weight of mice as an index of occurrence of side effects. FIG. 4 shows the weight transition of the mouse. There was no significant change in body weight in both the PBS administration group (FIG. 4A) and the HLA-G2 administration group (FIG. 4B).

(Anti-dsDNA antibody ELISA)
The amount of antinuclear antibody in the blood was measured using Levis anti-dsDNA-mouse ELISA KIT, Shibayagi. A mouse plasma specimen diluted with a buffer and a standard solution for preparing a calibration curve were added to an antigen-immobilized microplate. After 2 hours of incubation and well washing, a labeled antibody (peroxidase-conjugated anti-mouse IgG antibody) was added. After further incubation for 2 hours and well washing, the mixture was reacted with the color developing solution (TMB) for 20 minutes, and the reaction was stopped by adding an acidic solution (1M H ₂ SO ₄ ). The absorbance at 450 nm (subwavelength 620 nm) was measured using a spectrophotometer, and the anti-dsDNA antibody titer of each plasma sample was calculated from the standard curve obtained by plotting the absorbance against the standard solution concentration.

FIG. 5 shows the amount of antinuclear antibody in the blood of mice 72 days after administration. In the HLA-G2 administration group, a decrease in the blood antinuclear antibody amount was confirmed as compared to the PBS administration group (FIG. 5 (A)). As a result of verification with the exception of one mouse, which was significantly heavier than other mice, a significant decrease in blood antinuclear antibody level was confirmed in the HLA-G2 administration group compared to the PBS administration group. (FIG. 5B).

(Urine protein content)
Urinary albumin was measured using an ELISA kit (Levis albumin-mouse, Shibayagi). A mouse urine specimen diluted with a buffer solution and a standard solution for preparing a calibration curve were added to an anti-albumin antibody-immobilized microplate. After 1 hour of incubation and well washing, a labeled antibody (peroxidase-conjugated anti-mouse IgG antibody) was added. After further incubation for 1 hour and well washing, the mixture was reacted with the color developing solution (TMB) for 20 minutes, and the reaction was stopped by adding an acidic solution (1M H ₂ SO ₄ ). The absorbance at 450 nm (subwavelength 620 nm) was measured using a spectrophotometer, and the urine albumin value of each urine sample was calculated from the standard curve obtained by plotting the absorbance against the standard solution concentration.

Urinary creatinine was measured using an ELISA kit (Urine creatinine measurement ELISA kit, Transgenic). A mouse urine sample diluted with a buffer solution, a standard solution for preparing a calibration curve, and an HRP-labeled anti-creatinine antibody were mixed and incubated for 30 minutes. After incubation, the mixture was added to the antigen-immobilized microplate. After incubating for 2 hours and washing the wells, the mixture was reacted with a color developing solution (orthophenylenediamine solution) for 10 minutes, and the reaction was stopped by adding an acidic solution (1M H ₂ SO ₄ ). The absorbance at 490 nm was measured using a spectrophotometer, and the urinary creatinine value of each urine sample was calculated from the standard curve obtained by plotting the absorbance against the standard solution concentration.

FIG. 6 shows the amount of urine protein (albumin (μg) / creatinine (mg)) in mice 76 days and 86 days after administration. At both 76 days and 86 days after administration, a decrease in the amount of urine protein was confirmed in the HLA-G2 administration group compared to the PBS administration group (FIGS. 6 (A) and (B)).

(Immunohistochemical staining)
After collecting kidneys from mice 79 days after administration and fixing with formalin, embedding in paraffin, slicing, hematoxylin and eosin stain (H & E) staining, mesoamine silver periodate (Periodic acid-methenamine-silver stain; PAM), A histopathological specimen was prepared by Periodic acid-Schiff stain (PAS).

FIG. 7 shows a mouse kidney tissue specimen 79 days after administration. In HLA-G2 administration group compared to PBS administration group, H & E stained specimens reduced mononuclear cell infiltration and vasculitis, and PAM / PAS stained specimens reduced glomerular cell proliferation and mesangial area expansion. Was observed.

From the above, in the SLE model mice (MRL / MpJJmsSlc-lpr / lpr mice), the intraperitoneal administration of HLA-G2 twice a week reduced the amount of blood antinuclear antibody and urine protein. It was revealed that G2 has a therapeutic effect on SLE.

(Example 3)
(Induction of cytokine production)
Next, using IFN-DC, which is a human monocyte-derived dendritic cell, it was examined whether cytokine production induction was caused by HLA-G2 binding to LILRB2.

Exp Dermatol. 2015 Jan; 24 (1): 35-41. doi: 10.1111 / exd. 12581. Epub 2014 Dec 8. IFN-DC was cultured based on Nieda M et al. After 2 days from the start of culture, medium (10% FBS RPMI-) at 37 ° C. and 5% CO ₂ together with HLA-G2 (2.3 μL) or PBS as a control. 1640 (with antibiotics + 1000 U / mL GM-CSF and IFN-α). Four days after the start of the culture, the medium was changed, and the supernatant was collected and subjected to ELISA. In ELISA, OptEIA ELISA human IL-6 and IL-10 (BD) was used as a kit.

The result of ELISA is shown in FIG. In IFN-DC, induction of production of cytokines IL-10 and IL-6 was confirmed after 2 days incubation with HLA-G2.

From the above, HLA-G2 induces the production of cytokines IL-10 and IL-6, suggesting that systemic administration of HLA-G2 has a preventive or therapeutic effect on collagen disease.

Example 4
(SLE model mouse experiment with PEGylated HLA-G2)
PEGylated HLA-G2 was administered to SLE model mice to verify the therapeutic effect on SLE.

(Preparation of various recombinant proteins)
Prior to examination, various recombinant proteins were prepared and purified as follows.

(HLA-G2 recombinant protein expression plasmid)
The extracellular region (Gly1-Trp182) of HLA-G2 (WT) in which the signal sequence was removed and a methionine residue as a translation initiation codon was added to the N-terminus was incorporated into the E. coli expression vector pGM7 to further enhance the expression level. The expression plasmid (HLA-G2-pGMT7) possessed by the inventors of the present application in which the base sequence of 5 residues near the N-terminal is synonymously substituted was used (FIG. 9 (a), base sequence: SEQ ID NO: 13, amino acid) Sequence: SEQ ID NO: 14).

(LILRB2biaA recombinant protein expression plasmid)
Two Ig-like domains (Gry1-Pro197) on the N-terminal side of the extracellular region involved in ligand binding of LILRB2 to which the signal sequence was removed and methionine as the start codon was added were incorporated into the pGM7 vector, and 17 at the C-terminus. A plasmid for expression of E. coli (LILRB2birA-pGM7) possessed by the inventors of the present application, to which a biotinylated enzyme recognition sequence consisting of amino acid residues (GSHLHILDaqKMVWWNR (SEQ ID NO: 15)) was added was used (Shiroishi, M., Tsumoto, K). Amano, K., Shirakihara, Y., Colonna, M., Braud, VM, Allan, D.S.J., Makadzange, A., Rowland-Jones, S., Willcox, B.,. Jo es, EY, van der Merwe, PA, Kumagai, I., and Maenaka, K. (2003) Human inhibitory receptors 2 (ILT2) and ILT4, ILT4, and ILT4. and bind preferred to HLA-G.Proc.Natl.Acad.Sci.U.S.A.100, 8856-61) (FIG. 9 (b), nucleotide sequence: SEQ ID NO: 16, amino acid sequence: SEQ ID NO: 17).

(Preparation of inclusion bodies of various recombinant proteins using E. coli expression system)
All recombinant proteins used in this study were expressed as inclusion bodies using E. coli. The LILRB2biirA recombinant protein was expressed as inclusion bodies by transforming E. coli BL21 (DE3) pLysS strain (Novagen) with the aforementioned plasmid. In addition, HLA-G2 protein can be obtained by using the above-mentioned plasmids from Escherichia coli ClearColi (registered trademark) BL21 (DE3) competent cell (ClearColi (registered trademark) BL21 (DE3) competitive cell (Lucigen)) by the inventors of the present application. The cells were transformed into cells) and expressed as inclusion bodies. Specifically, after transformation, seeded on an LB agar medium containing 100 μg / mL ampicillin, and then cultured at 37 ° C. overnight. The obtained colonies were inoculated into 2 × YT medium (10 mL) containing 100 μg / mL ampicillin and cultured with shaking at 37 ° C. overnight. 10 mL of the precultured bacterial solution was inoculated in 1 L of 2 × YT medium containing 100 μg / mL ampicillin, and cultured with shaking at 37 ° C. When Optical Density (OD) 600 = 0.6 in the early logarithmic growth phase was reached, Isopropyl β-D-1-thiogalactopyroside (IPTG) was added to a final concentration of 1 mM to induce expression of the recombinant protein. Thereafter, the cells were cultured with shaking in INNOVA (Eppendorf) at 150 rpm and 37 ° C. for 5 hours. The bacterial cells obtained by centrifuging the cultured bacterial solution (5000 rpm, 4 ° C., 10 minutes) are suspended in a suspension buffer (50 mM Tris hydroxylamine [Tris] -HCl pH 8.0, 150 mM NaCl) on ice. Was subjected to ultrasonic crushing. After disruption, the mixture is centrifuged at 8000 rpm and 4 ° C. for 5 minutes, and the resulting precipitate is used as an inclusion body and further suspended in a washing buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% Triton X-100). The washing operation of centrifuging at 8000 rpm and 4 ° C. for 5 minutes was repeated 4 times. Thereafter, in order to remove the surfactant Triton X-100 from the inclusion body, the same operation was repeated four times using a suspension buffer to obtain an inclusion body. The resulting inclusion bodies were overnight in a solubilization buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 6 M guanidine-HCl, 10 mM Ethylenediamine-N, N, N ′, N ′, -tetraacetic acid [EDTA]). The solution was completely dissolved by standing at 4 ° C. After solubilization, the supernatant obtained by centrifugation at 5000 × g and 4 ° C. for 5 minutes was stored as inclusion bodies at −80 ° C. All centrifugation operations after the induction of protein expression were performed using a universal cooling centrifuge (KUBOTA).

(Preparation of recombinant protein by unwinding method)
HLA-G2 and LILRB2birA recombinant proteins were prepared by unwinding E. coli inclusion bodies by a dilution method. Dithiothreitol (DTT) was added to each solubilized inclusion body of HLA-G2 (8 mg) and LILRB2biaA (4 mg) so that the final concentration at the time of dilution was about 1 to 2 μM, and the final concentration was 10 mM. Incubated for 1 hour. A refolding buffer (0.1 M Tris-HCl pH 8.0, 1 M L-argine-HCl, 2 mM EDTA, 3.73 mM cystein, which contains arginine having an effect of suppressing protein aggregation, in the inclusion body denaturing solution reduced by DTT. 6.73 mM cysteine) was added drop by drop until the guanidine concentration reached 1.5 M (concentration considered to form a secondary structure with disulfide bonds). The diluted solution was further diluted by adding dropwise to 200 mL of refolding buffer and stirred at 4 ° C. for 72 hours.

(Purification of unwound recombinant protein)
Since the volume of each recombinant protein unwound by the dilution method increases due to dilution, ultrafiltration using a VIVAFLOW system (MWCO: 10000 Da, Sartorius), and then if necessary, Amicon Ultra (MWCO: 10000 Da, Merck) After performing ultrafiltration using Millipore, fine particles such as aggregates are removed using a Millex-GV (0.22 μm, PVDF, Merk Millipore) filter, and gel filtration chromatography (Size Exclusion Chromatography): Purification by SEC) was performed. For SEC, AKTApurifier or AKTApure system (both GE Healthcare) was used, and 20 mM Tris-HCl pH 8.0, 100 mM NaCl was used as a running buffer.

Specifically, HLA-G2 was concentrated to 15 mL or less with a VIVAFLOW system, filtered, and purified using a Hiload 26/60 Superdex75 pg (GE Healthcare) column. The target peak fraction obtained by SEC was concentrated to 5 mL or less using Amicon Ultra (MWCO: 10000 Da, Merck Millipore), and then replaced with 20 mM Tris-HCl pH 8.0 using a dialysis method. After filtering, it was injected into a Resource Q 1 mL (GE Healthcare) column. IEX was performed under the conditions of 20 mM Tris-HCl pH 8.0, 0-0.5 M NaCl / 20 Column volume (CV).

The LILRB2biaA recombinant protein was concentrated to 0.5 mL or less using a VIVAFLOW system and Amicon Ultra, filtered, and purified with a Superdex 75 10/300 GL (GE Healthcare) column. As the running buffer, 20 mM Tris-HCl pH 8.0, 200 mM NaCl was used.

(Preparation of biotinylated LILRB2)
LILRB2 birA purified by the SEC method was mixed with LILRB2 birA: 5 × BiomixA buffer (0.25 M bicine buffer pH 8.3): 5 × BiomixB buffer (50 mM Adenosine triphosphat) The mixture was mixed at 1: 1, and 1 μL of BirA enzyme (prepared by the inventors of the present application) was added, and the mixture was incubated at 30 ° C. for 1 hour. Thereafter, in order to remove unreacted biotin, SEC purification was performed on a Superdex 75 10/300 GL (GE Healthcare) column. As a running buffer, 20 mM Tris-HCl pH 8.0, 400 mM NaCl was used.

As a result of SEC purification of the HLA-G2 protein prepared as described above, an elution peak appears at a position corresponding to a molecular weight (44 kDa) approximately twice the target molecular weight (HLA-G2: 22 kDa), consistent with previous findings. Obtained (FIG. 10A).

(PEGylation reaction specific to Cys42 residue of HLA-G2)
PEGylation reaction was performed using the HLA-G2 protein prepared as described above. PEGylation reagents are ME-400MA (MW: 42,653 Da) (PEG40), ME-200MAOB (MW: 20,841 Da) (PEG20), ME-100MA (MW: 10,303 Da), which are high-purity linear PEGs. (PEG10), ME-050MA (MW: 5,393 Da) (PEG5) (both NOF Corporation) were used. These reagents have a maleimide group as a reactive group and react by recognizing a thiol group.

(Reducing agent)
Prior to the PEGylation reaction, a disulfide bond was cleaved by a reduction treatment, and the thiol group of the Cys42 residue serving as a PEGylation target was exposed again, and a reducing agent was added to improve the reaction efficiency.

The purified HLA-G2 protein was concentrated by ultrafiltration using Amicon Ultra (MWCO: 10000 Da, Merck Millipore), and replaced with PEGylation buffer (1 × Phosphate-Buffer Saline (PBS), 5 mM EDTA). In order to advance the PEGylation reaction under anaerobic conditions, the solution prepared to 0.5 mL was degassed for 1 hour using an aspirator. Subsequently, the reducing agent tris (2-carboxyethyl) phosphine (TCEP) was added to a final concentration of 0.1, 0.5, 1, 5, 10 mM, PEG 20 was added, and the mixture was reacted at 4 ° C. overnight. It was. As the reducing agent, tris (2-carboxyethyl) phosphine (TCEP), which is odorless and easy to use and is frequently used in the reaction using maleimide and cysteine, was used.

The progress of the PEGylation reaction was carried out by subjecting the reaction solution to Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) (12.5% acrylamide gel, 30 mA / plate, 70 minutes) and Coomassie Brilliant (for 70 minutes). ) Staining and Barium Iodide (BaI ₂ ) staining to detect PEG molecules. BaI2 staining was performed by a procedure in which the acrylamide gel after electrophoresis was immersed in a 5% BaI ₂ solution (15 minutes), ion-exchanged water (30 minutes), and 0.1 M iodine solution (5 minutes) in this order and shaken.

PEGylated HLA-G2 was purified using Superdex 200 10/300 GL (GE Healthcare), Superdex 75 10/300 GL (GE Healthcare), or Superose 6 10/300 GL (GE HealthEC) column. The PEGylation reaction solution was replaced with SEC running buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl) using Amicon Ultra (MWCO: 10000 Da, Merck Millipore), concentrated to 0.5 mL and loaded onto the column. . For the SEC, an AKTA purifier system (GE Healthcare) was used. The degree of purification was confirmed by developing each fraction sample by SDS-PAGE under non-reducing conditions and detecting it with silver staining (2D-SILVER STAIN REAGENT II, Cosmo Bio Inc.).

When the reaction solution was developed by SDS-PAGE and stained with CBB as described above, the band of the target PEGylated protein could be confirmed (FIG. 10 (b)). From the above results, it was found that the reaction efficiency was greatly improved by adding TCEP, and that the highest reaction efficiency was obtained under the condition that the TCEP final concentration was 0.1 mM. Therefore, in the subsequent experiments, the PEGylation reaction was performed under the condition of 0.1 mM TCEP treatment.

(Examination of PEG molecular weight)
Subsequently, in order to verify whether the PEGylation reaction efficiency and the degree of purification of the PEGylated protein differ depending on the molecular weight of PEG to be bound, four types of molecular weight (5, 10, 20, 40 kDa) having maleimide as a reactive group HLA-G2 was PEGylated using molecular weight PEGylation reagents (PEG5, PEG10, PEG20, PEG40) (described above). The obtained PEGylated product is as follows.
・ PEG5-HLA-G2 (HLA-G2 PEGylated with PEG5)
・ PEG10-HLA-G2 (HLA-G2 PEGylated with PEG10)
・ PEG20-HLA-G2 (HLA-G2 PEGylated with PEG20)
・ PEG40-HLA-G2 (HLA-G2 PEGylated with PEG40)

PEGylated reaction solution was developed by SDS-PAGE, then CBB staining and BaI ₂ staining were performed to confirm the progress of the reaction. As a result, all four types of PEGylated proteins were confirmed (FIGS. 11 (a) and (b)). . Also, from the density of the PEGylated protein band, it was revealed that the PEGylation reaction rate was higher for low molecular weight PEG (PEG5, PEG10).

SEC was selected as a method for purifying these PEGylated HLA-G2 by using the difference in molecular weight before and after PEGylation. SEC purification was performed on the four types of PEGylation reaction solutions, and the obtained peak fractions were confirmed by SEC using PEG10-HLA-G2 and PEG20-HLA-G2 by SEC after silver-staining after SDS-PAGE. The target PEGylated HLA-G2 was separated from the unreacted HLA-G2 protein and purified (FIGS. 12 (b) and 12 (c)).

(Binding experiment with receptor LILRB2)
Subsequently, PEG10-HLA-G2 and PEG20-HLA-G2 that could be purified as PEGylated proteins, and SEC-purified samples of PEG5-HLA-G2 obtained as SEC peaks mainly containing PEGylated proteins, An interaction analysis with the receptor LILRB2 by Surface Plasmon Resonance (SPR) was performed, and it was confirmed whether HLA-G2 to which each molecular weight of PEG was bound maintained the receptor binding ability. As a comparative control, non-PEGylated HLA-G2 prepared by the method described above was used.

As described above, LILRB2 was prepared as biotinylated LILRB2 by SEC purification after site-specific biotinylation using a purified LILRB2biaA protein with a biotinylated tag added to the C-terminal side.

As analytes, HLA-G2 (0.2-0.7 μM), PEG5-HLA-G2 (0.4-1.6 μM), and PEG10-HLA-G2 (0.3-1. 1 μM) and PEG20-HLA-G2 (0.3 to 1.1 μM).

(Interaction analysis by Surface Plasma Resonance (SPR))
In order to confirm the influence of PEGylated HLA-G2 on LILRB2 binding, interaction analysis by SPR method was performed using Biacore 3000 (GE Healthcare). On the sensor chip, biotinylated LILRB2 and biotinylated BSA as a control were immobilized on the sensor chip CAP according to the protocol using Biotin CAPture Kit (GE Healthcare). Single-stranded DNA is immobilized on the sensor chip CAP (FIG. 13 (a)). Streptavidin is immobilized on the chip by hybridization with complementary-strand DNA added with streptavidin (FIG. 13). (B)) Furthermore, biotinylated LILRB2 and biotinylated BSA were immobilized at an immobilized amount of 200 RU to 500 RU by utilizing the interaction between streptavidin and biotin (FIG. 13 (c)).

As the analyte, HBS-EP buffer (10 mM Na-HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v) was obtained by ultrafiltration using Amicon Ultra (MWCO: 10000, Millipore). / V) Samples of HLA-G2 and PEGylated HLA-G2 protein solutions substituted with Surfactant P20: GE Healthcare) were diluted two-fold in three steps, starting with the lowest concentration, at a flow rate of 10 μL / min. Washed away. Kinetics measurements were performed at a measurement temperature of 25 ° C., a binding time and a dissociation time of 120 seconds each. BIAevaluation version: 4.1.1 (GE Healthcare) was used for the analysis.

As a result of the binding analysis, it was confirmed that all three types of PEGylated products were bound to LILRB2 (FIGS. 14B to 14D).

From the above results, since there is no significant difference in the PEGylation efficiency and the binding ability with the receptor at the three molecular weights, the degree of purification of the PEGylated protein is high, and further contributes to the stability of the PEGylated protein. The SLE model mouse experiment was conducted using “PEG20-HLA-G2” modified with 20 kDa PEG having the largest PEG molecular weight among the three.

(Preparation of each administered protein (HLA-G2, PEG20-HLA-G2))
Subsequently, each protein administered to mice was prepared. As described above, HLA-G2 and PEG20-HLA-G2 obtained by SEC purification were replaced with PBS buffer by dialysis to obtain a protein solution to be administered.

(Dosage test)
HLA-G2, PEG20-HLA-G2 prepared as described above or PBS as a negative control was administered to SLE model mice as follows ("HLA-G2 administration group", "PEG20-HLA-G2 administration group" or "PBS Administration group "). MRL / MpJJmsSlc-lpr / lpr mice (Japan SLC Co., Ltd.) were used as SLE model mice, and the test was started from 8 mice in each administration group. For HLA-G2 and PEG20-HLA-G2, a new lot (newly unwound to repurified) was administered about every 3 weeks. The outline of the test is shown in FIG.
・ Administration route: intraperitoneal administration ・ Dose:
HLA-G2 administration group: 15 μg / animal (dissolved in 200 μL of PBS)
PEG20-HLA-G2 administration group: 15 μg / animal (dissolved in 200 μL of PBS)
PBS administration group: PBS 200 μL
・ Dose interval: twice a week ・ Dose period: 95 days

From 8 days before the first administration to 23 days after the end of the administration, plasma and urine were collected once every 3 or 2 weeks on the day indicated by the arrow (FIG. 13), and used in the diagnosis and monitoring of SLE. The dsDNA antibody titer and urinary albumin index were measured.

As an indicator of the occurrence of side effects, mouse body weights were measured according to the timing of administration. FIG. 16 shows the weight transition of the mouse. In the HLA-G2 administration group, the PEG20-HLA-G2 administration group and the PBS administration group, there was no significant difference in body weight transition among the administration groups.

FIG. 17 (a) shows the anti-dsDNA antibody titers of each group. A decrease in the anti-dsDNA antibody titer was confirmed in the HLA-G2 administration group and the PEG20-HLA-G2 administration group as compared to the PBS administration group (FIG. 17 (a)). FIG. 17 (b) shows the anti-dsDNA antibody titer of mice 90 days after administration. A significant decrease in the anti-dsDNA antibody titer was confirmed in the HLA-G2 administration group and the PEG20-HLA-G2 administration group as compared to the PBS administration group (FIG. 17 (b)).

(Urine protein content)
Urinary albumin was measured using an ELISA kit (Levis albumin-mouse, Shibayagi). A mouse urine specimen diluted with a buffer solution and a standard solution for preparing a calibration curve were added to an anti-albumin antibody-immobilized microplate. After 1 hour of incubation and well washing, a labeled antibody (peroxidase-conjugated anti-mouse IgG antibody) was added. After further incubation for 1 hour and well washing, the mixture was reacted with the color developing solution (TMB) for 20 minutes, and the reaction was stopped by adding an acidic solution (1M H ₂ SO ₄ ). The absorbance at 450 nm (subwavelength 620 nm) was measured using a spectrophotometer, and the urine albumin index of each urine sample was calculated from the standard curve obtained by plotting the absorbance against the standard solution concentration. Furthermore, urinary creatinine was measured using an ELISA kit (Urine creatinine measurement ELISA kit, Transgenic). A labeled antibody (HRP-labeled anti-creatinine antibody) was added to a mouse urine sample diluted with ultrapure water and a standard solution for preparing a calibration curve, incubated for 30 minutes, and added to an antigen-immobilized microplate. After 1 hour of incubation and well washing, the reaction was stopped with a color developing solution (orthophenylenediamine) for 10 minutes and the reaction was stopped by adding an acidic solution (5.4% H ₂ SO ₄ ). The absorbance at 490 nm was measured using a spectrophotometer, and the urinary creatinine concentration of each urine sample was calculated from the standard curve obtained by plotting the absorbance against the standard solution concentration. The urinary albumin index (unit: mg albumin / g creatinine) is obtained by dividing the value of urinary albumin concentration (unit: mg / mL) obtained from the above by the value of urinary creatinine concentration (unit: g / mL). Calculated.

FIG. 18 shows the urinary albumin index of each group. Although there was no significant difference in the urinary albumin index between the groups, the PEG20-HLA-G2 administration group had a lowering effect on the urinary albumin index compared to the PBS administration group and the HLA-G2 administration group. There was a tendency to persist (FIG. 18).

(Blys blood concentration)
On the 90th day after the start of administration, the blood concentration of soluble B lymphocyte stimulating factor (Blys) was measured. More specifically, the BLys concentration in plasma was measured using a Mouse BAFF / BLyS / TNFSF13B Quantikine ELISA Kit (manufactured by R & D systems) according to the protocol. A mouse plasma specimen diluted with a buffer and a standard solution for preparing a calibration curve were added to a mouse BLys-specific monoclonal antibody-immobilized microplate. After 2 hours of incubation and well washing, a labeled antibody (peroxidase-conjugated anti-mouse BLys polyclonal antibody) was added. After further incubation for 2 hours and well washing, the mixture was reacted with the coloring solution (TMB) for 30 minutes, and the reaction was stopped by adding an acidic solution (dilute hydrochloric acid). The absorbance at 450 nm (subwavelength 540 nm) was measured using a spectrophotometer, and the BLys concentration in each plasma was calculated from the standard curve obtained by plotting the absorbance against the standard solution concentration.

FIG. 19 shows the Blys blood concentration of each group. In the PEG20-HLA-G2 administration group, a significant decrease in Blys blood concentration was observed compared to the PBS administration group and the HLA-G2 administration group (FIG. 19).

(Extended test)
Next, an extension test was performed using the PBS administration group, and the therapeutic effect of PEG20-HLA-G2 on SLE was verified.

For the mice in the PBS administration group, plasma and urine were collected on days 118 and 132 from the first administration, and the plasma anti-dsDNA antibody titer and urinary albumin index were measured in the same manner as described above. PEG20-HLA for mice with elevated plasma anti-dsDNA antibody titers and urinary albumin index values on day 132 above those on day 118 (ie, developing SLE) -G2 or PBS was administered to verify the therapeutic effect (PEG20-HLA-G2 administration: n = 1, PBS administration: n = 1). Administration was started from day 132 (administration interval: administered twice a week, administered 4 times in total for 2 weeks), and the administration route and dose were the same as described above. Plasma and urine were collected on days 138 and 145, and the plasma anti-dsDNA antibody titer and urinary albumin index were measured as described above.

FIG. 20 (a) shows the change in the anti-dsDNA antibody titer. In mice treated with PEG20-HLA-G2, a decrease in the anti-dsDNA antibody titer was observed as compared with PBS administration, confirming the therapeutic effect of PEG20-HLA-G2.

FIG. 20 (b) shows changes in the urinary albumin index. In mice treated with PEG20-HLA-G2, the urinary albumin index decreased as compared with PBS administration, confirming the therapeutic effect of PEG20-HLA-G2.

From the above, in the SLE model mouse (MRL / MpJJmsSlc-lpr / lpr mouse), the intraperitoneal administration of PEG20-HLA-G2 twice a week showed a decrease in blood antinuclear antibody amount and urinary protein. It was revealed that PEG20-HLA-G2 has a therapeutic effect on SLE. The use of PEGylated HLA-G2 can be expected to reduce the dose and the number of administrations, and is expected to reduce the financial and physical burden on the patient even when concomitant with other drugs.

The present invention is capable of various embodiments and modifications without departing from the broad spirit and scope of the present invention. The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. In other words, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.

This application is based on Japanese Patent Application No. 2018-046023 filed on Mar. 13, 2018. The specification, claims, and entire drawings of Japanese Patent Application No. 2018-046023 are incorporated herein by reference.

The present invention is applicable to preventive or therapeutic agents for diseases caused by systemic lupus erythematosus or systemic lupus erythematosus and preventive or therapeutic agents for collagen disease.

Claims

A preventive or therapeutic agent for systemic lupus erythematosus or a disease caused by systemic lupus erythematosus comprising HLA-G or an HLA-G multimer as an active ingredient.
The HLA-G multimer is a multimer of proteins having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked.
The preventive or therapeutic agent for a disease caused by systemic lupus erythematosus or systemic lupus erythematosus according to claim 1.
The HLA-G multimer is
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1,
Because
It is a homomultimer between (a) or (b) or a heteromultimer between (a) and (b).
The systemic lupus erythematosus or systemic lupus erythematosus according to claim 1 or 2, wherein the disease is caused by systemic lupus erythematosus.
The HLA-G or HLA-G multimer is
The HLA-G or HLA-G multimeric protein is an altered protein or a salt thereof in which at least one amino acid residue in the amino acid sequence is PEGylated with polyethylene glycol (PEG);
The preventive or therapeutic agent for a disease caused by systemic lupus erythematosus or systemic lupus erythematosus according to claim 1.
The HLA-G multimer is
Consisting of a multimer of proteins having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked,
At least one amino acid residue in the amino acid sequence constituting the protein is a modified protein or a salt thereof that is PEGylated with polyethylene glycol (PEG);
The preventive or therapeutic agent for a disease caused by systemic lupus erythematosus or systemic lupus erythematosus according to claim 4.
The molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.
The systemic lupus erythematosus or systemic lupus erythematosus disease-preventing or treating agent according to claim 4 or 5.
The protein is a protein having an amino acid sequence described in (a) or (b) below,
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
The multimer is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b), and has a binding activity to leukocyte Ig-like receptor B2. ,
The agent for preventing or treating systemic lupus erythematosus or a disease caused by systemic lupus erythematosus according to any one of claims 4 to 6.
HLA-G or HLA-G multimer as an active ingredient, used for systemic administration,
A preventive or therapeutic agent for collagen disease characterized by the above.
The HLA-G multimer is a multimer of proteins having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked.
The preventive or therapeutic agent for collagen disease according to claim 8.
The HLA-G multimer is
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1,
Because
It is a homomultimer between (a) or (b) or a heteromultimer between (a) and (b).
The preventive or therapeutic agent for collagen disease according to claim 8 or 9.
The HLA-G or HLA-G multimer is
The HLA-G or HLA-G multimeric protein is an altered protein or a salt thereof in which at least one amino acid residue in the amino acid sequence is PEGylated with polyethylene glycol (PEG);
The preventive or therapeutic agent for collagen disease according to claim 8.
The HLA-G multimer is
Consisting of a multimer of proteins having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked,
At least one amino acid residue in the amino acid sequence constituting the protein is a modified protein or a salt thereof that is PEGylated with polyethylene glycol (PEG);
The preventive or therapeutic agent for collagen disease according to claim 11.
The molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.
The preventive or therapeutic agent for collagen disease according to claim 11 or 12.
The protein is a protein having an amino acid sequence described in (a) or (b) below,
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
The multimer is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b), and has a binding activity to leukocyte Ig-like receptor B2. ,
The preventive or therapeutic agent for collagen disease according to any one of claims 11 to 13, wherein