WO2019177056A1 - Modified protein, drug, prophylactic or therapeutic agent for inflammatory disease, and method for producing modified protein - Google Patents

Modified protein, drug, prophylactic or therapeutic agent for inflammatory disease, and method for producing modified protein Download PDF

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WO2019177056A1
WO2019177056A1 PCT/JP2019/010366 JP2019010366W WO2019177056A1 WO 2019177056 A1 WO2019177056 A1 WO 2019177056A1 JP 2019010366 W JP2019010366 W JP 2019010366W WO 2019177056 A1 WO2019177056 A1 WO 2019177056A1
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hla
protein
amino acid
acid sequence
multimer
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PCT/JP2019/010366
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French (fr)
Japanese (ja)
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勝実 前仲
喜美子 黒木
直良 前田
千聖 山田
愛実 ▲高▼橋
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国立大学法人北海道大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a modified protein, a pharmaceutical, a preventive or therapeutic agent for inflammatory diseases, and a method for producing the modified protein.
  • 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.
  • 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.
  • LILRs leukocyte Ig-like receptors
  • 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 ⁇ 2 microglobulin.
  • 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.
  • 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.
  • Polyethylene glycol is a polymer compound having a structure in which ethylene glycol is polymerized.
  • a polymer such as protein (PEGylation)
  • PEGylation By adding hydrophilic PEG to a polymer such as protein (PEGylation), the protein is shielded from attack by an enzyme and exposure of a hydrophobic part, and as a result, an effect of suppressing protein degradation and aggregation can be expected.
  • the increase in the molecular size due to the addition of PEG suppresses filtration in the glomerulus, so the protein half-life is prolonged, but PEG itself has no antigenicity, reducing the side effects of protein administration Can be expected (Non-patent Document 3).
  • FDA Food and Drug Administration
  • HLA-G2 is expected to be used as a pharmaceutical, it has a problem that the stability and homogeneity as a recombinant protein deteriorates with time, leaving a problem in terms of long-term storage. It was.
  • the present invention has been made in view of the above circumstances, and is a modified protein, a pharmaceutical, a prophylactic or therapeutic agent for inflammatory diseases, which has high storage stability and in vivo stability, and is highly effective in preventing or treating diseases.
  • An object is to provide a method for producing a modified protein.
  • the modified protein according to the first aspect of the present invention comprises: Consisting of a multimer of proteins having an amino acid sequence in which the ⁇ 1 domain of HLA-G and the ⁇ 3 domain of HLA-G are linked, At least one amino acid residue in the amino acid sequence constituting the protein is PEGylated with polyethylene glycol (PEG), It is characterized by that.
  • PEG polyethylene glycol
  • the molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.
  • 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 pharmaceutical product according to the second aspect of the present invention is: According to the first aspect of the present invention, a modified protein or a salt thereof is included as an active ingredient.
  • a modified protein or a salt thereof is included as an active ingredient.
  • the method for producing a modified protein according to the fourth aspect of the present invention comprises: (A) preparing a multimer of a protein having an amino acid sequence in which the ⁇ 1 domain of HLA-G and the ⁇ 3 domain of HLA-G are linked; (B) a step of degassing the protein multimer obtained in step (A), followed by a reduction treatment; (C) PEGylating and modifying the protein multimer reduced in step (B); including.
  • the molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.
  • 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. .
  • a modified protein a pharmaceutical, a prophylactic or therapeutic agent for inflammatory diseases, and a method for producing the modified protein, which have high storage stability and in vivo stability and are highly effective in preventing or treating diseases. Can do.
  • FIG. 1 shows PEGylated HLA-G2 typically.
  • 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
  • Is a diagram showing a comparison of the reaction efficiency with PEG molecular weight is a diagram showing (a) shows CBB staining, (b) the results of BaI 2 staining.
  • TCEP reducing agent
  • FIG. 2 is a diagram showing the results of SEC purification using Superdex 200 10/300 GL of a PEGylation reaction solution with PEG of each molecular weight, concentrating, SDS-PAGE, and silver staining, (a) is PEG5-HLA- G2, (b) are the results of PEG10-HLA-G2, (c) are the results of PEG20-HLA-G2, and (d) are the results of PEG40-HLA-G2.
  • 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.
  • FIG. 4 is a view showing the results of PEG20-HLA-G2. It is the figure which compared the PEGylation reaction efficiency by PEG20 of the HLA-G2 variant (HLA-G2N86C, HLA-G2CTER) from which a PEGylation site differs, (a) is the result of CBB staining, (b) is the result of BaI 2 staining.
  • FIG. (A) shows the results of SDS-PAGE and CBB staining of HLA-G2 and PEG20-HLA-G2 with and without lyophilization treatment, and (b) shows the amount of HLA-G2 (LILRB2 immobilized amount before and after lyophilization treatment).
  • FIG. It is a figure which shows the comparison result (CBB dyeing
  • FIG. 1 It is a figure which shows the stability in serum of HLA-G2, PEG20-HLA-G2 protein
  • (a) is a figure which shows the stability in serum of HLA-G2, PEG20-HLA-G2
  • (b) It is a figure which shows the change of the survival rate in the serum of HLA-G2 and PEG20-HLA-G2 over time.
  • (A) is a figure which shows the dermatitis induction by an atopic dermatitis induction ointment, the administration schedule of protein, and the recording date of the thickness of an auricle
  • (b) is a figure which shows the swelling degree of a mouse
  • (C) is a photograph showing the mouse auricle on the 18th day after administration.
  • FIG. 2 is a diagram showing the primary structure (left) and molecular schematic diagram (right) of each recombinant protein, where (a) is HLA-G2, (b) is HLA-G2C42S, (c) is HLA-G2N86C, (d) FIG. 4 is a schematic diagram showing HLA-G2CTER, (e) HLA-G1C42S heavy chain, (f) ⁇ 2m, and (g) LILRB2. 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.
  • (c) is a schematic diagram showing a mode that avidin was fixed on the chip
  • (c) is a schematic diagram showing a mode that biotinylated LILRB2 and biotinylated BSA were fix
  • (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 modified protein according to this embodiment comprises a multimer of proteins having an amino acid sequence in which the ⁇ 1 domain of HLA-G and the ⁇ 3 domain of HLA-G are linked, and at least one amino acid residue in the amino acid sequence constituting the protein is present.
  • the group is PEGylated with polyethylene glycol (PEG) (FIG. 1).
  • the HLA-G targeted by this embodiment is preferably human-derived HLA-G.
  • the HLA-G multimer targeted by this embodiment is, for example, a multimer of a protein having an amino acid sequence in which the ⁇ 1 domain of HLA-G and the ⁇ 3 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).
  • HLA-G2 a protein multimer having an amino acid sequence in which the ⁇ 1 domain of HLA-G and the ⁇ 3 domain of HLA-G are linked
  • HLA-G2 a protein multimer having an amino acid sequence in which the ⁇ 1 domain of HLA-G and the ⁇ 3 domain of HLA-G are linked
  • 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 ⁇ 1 domain of HLA-G and the ⁇ 3 domain of HLA-G are linked.
  • the HLA-G molecule is preferably human-derived HLA-G.
  • “1st to 90th amino acid region” corresponds to the amino acid sequence of the ⁇ 1 domain
  • “91st to 180th amino acid region” corresponds to the amino acid sequence of the ⁇ 3 domain.
  • 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 ⁇ 1 domain
  • the “sequence” region corresponds to the gene region encoding the ⁇ 3 domain.
  • amino acid sequence of a conjugate of ⁇ 1 domain and ⁇ 3 domain ( ⁇ 1-3 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. .
  • the C1 of the amino acid sequence (SEQ ID NO: 1) of a conjugate of ⁇ 1 domain and ⁇ 3 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.
  • the function of a multimer of a conjugate of ⁇ 1 domain and ⁇ 3 domain of the above HLA-G molecule (leukocyte Ig-like receptor)
  • the amino acid sequence of the ⁇ 1-3 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.
  • 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.
  • the HLA-G2 multimer targeted by the present embodiment is not limited to the above-mentioned ligated body of ⁇ 1 domain and ⁇ 3 domain of the HLA-G molecule (hereinafter also simply referred to as “ ⁇ 1-3 ligated product”). Any structure may be used as long as it has a linking structure of ⁇ 1 domain and ⁇ 3 domain of HLA-G molecule and the dimer 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 ⁇ 1 domain and ⁇ 3 domain ( ⁇ 1-3 conjugate) of the HLA-G molecule.
  • binding activity to leukocyte Ig-like receptor B2 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 can be confirmed by, for example, a reporter assay using a T cell hybridoma.
  • 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 PILR ⁇ are fused. Can do.
  • NFAT-GFP introduced reporter cells mouse T cell hybridomas
  • a signal through the intracellular domain of PILR ⁇ 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 form in which the above-mentioned HLA-G2s are disulfide bonded or non-covalently bonded without using disulfide bonds.
  • cysteine residue in the amino acid sequence constituting the protein is PEGylated, for example, in the case of a naturally formed homodimer, It multimerizes regardless of the disulfide bond, and the cysteine residue (Cys42) may be left free on the surface of the molecule, or a further dimer (tetramer) having a homodimer as one unit or In the case of those further multimers, they may be multimerized by disulfide bonds via cysteine residues (Cys42) existing on the surface of the homodimer molecule.
  • a homodimer that spontaneously forms it multimerizes regardless of disulfide bonds
  • 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.
  • the HLA-G2 multimer may be a homomultimer composed of the above-mentioned ⁇ 1-3 conjugates or variants, or a heteromultimer composed of an ⁇ 1-3 conjugate and a variant. There is no limitation. Preferably, it is a homomultimer composed of ⁇ 1-3 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.
  • LILRB2 leukocyte Ig-like receptor B2
  • the modified protein according to the present embodiment comprises a multimer of proteins having an amino acid sequence in which the ⁇ 1 domain of HLA-G and the ⁇ 3 domain of HLA-G are linked, and at least in the amino acid sequence constituting the protein.
  • One cysteine 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.
  • amino acid residues to be PEGylated include lysine residues and cysteine residues.
  • the cysteine residue may be, for example, the 42nd free cysteine residue of HLA-G2.
  • 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.
  • the 86th asparagine which is a sugar chain modification site of HLA-G And may be modified by PEGylation.
  • cysteine residue may be added to an arbitrary site, and the cysteine residue may be modified with PEG.
  • 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.
  • 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 modified protein is dissolved in sterilized water after lyophilization, and the degree of aggregation / degradation is determined by SDS-PAGE.
  • a method for confirming as a pattern, a method for confirming the binding activity to LILRB2 by the above-mentioned method after lyophilizing the modified protein, and a degree of aggregation / degradation by performing SDS-PAGE after incubating the modified protein under high temperature conditions The method of confirming as a band pattern etc. are mentioned.
  • the modified protein is decomposed by incubating in serum (for example, FBS) and then performing Western blotting.
  • serum for example, FBS
  • Western blotting The method of confirming the grade of etc. is mentioned.
  • the method for producing a modified protein according to the present embodiment includes: (A) preparing a multimer of a protein having an amino acid sequence in which the ⁇ 1 domain of HLA-G and the ⁇ 3 domain of HLA-G are linked; (B) a step of degassing the protein multimer obtained in step (A), followed by a reduction treatment; (C) PEGylating and modifying the protein multimer reduced in step (B); including.
  • HLA-G2 or HLA-G2 multimer having an amino acid sequence in which the ⁇ 1 domain of HLA-G and the ⁇ 3 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 ⁇ 1 domain of HLA-G and the ⁇ 3 domain of HLA-G are linked, as described above.
  • step (A) as an example, a method for preparing an HLA-G2 multimer will be described.
  • 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.
  • 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 ⁇ 1 domain and ⁇ 3 domain of HLA-G,
  • a gene encoding the amino acid sequence of the aforementioned HLA-G2 (hereinafter referred to as “mutant HLA-G2” for convenience) obtained by deleting, substituting or adding a part of the amino acid sequence of the conjugate may be used. it can.
  • 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 ⁇ 1-3 ligated gene. See, 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 site-directed mutagenesis by a known technique such as Kunkel method or Gapped duplex method.
  • kits include QuickChange TM Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTailor TM ite-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRa Site-Mit-Sensitized-Might-Sensitive-Mid-Sensitive-Mid-Sensitive-Mit-Sensitive-Mid-Sequential Etc .: manufactured by Takara Bio Inc.).
  • the host used for the preparation of the transformant is not particularly limited as long as it can express HLA-G2 ( ⁇ 1-3 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.
  • 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.
  • 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.
  • the recombinant HLA-G2 multimer 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.
  • various chromatography gel filtration, ion exchange chromatography, affinity chromatography
  • the recombinant HLA-G2 multimer 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.
  • Step (B) is a step in which the protein multimer obtained in step (A) is degassed and then reduced.
  • 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.
  • the protein multimer obtained in the step (A) may be replaced with a PEGylation buffer (for example, 1 ⁇ Phosphate-Buffer Saline (PBS), 5 mM EDTA).
  • PBS Phosphate-Buffer Saline
  • 5 mM EDTA 5 mM EDTA
  • 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.
  • 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.
  • TCEP tris (2-carboxyethyl) phosphine
  • 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 multimer of the protein reduced in step (B).
  • 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.
  • 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.
  • 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.
  • PEGylation reagent for example, PEGylation can be modified by adding a PEGylation reagent and reacting at 4 ° C. overnight, for example.
  • at least one amino acid residue for example, cysteine residue
  • the PEG-modified protein may be purified by, for example, gel filtration chromatography (SEC).
  • SEC gel filtration chromatography
  • the pharmaceutical agent and the preventive or therapeutic agent for inflammatory diseases according to this embodiment include the above-described modified protein or a salt thereof.
  • a salt with a physiologically acceptable acid eg, inorganic acid, organic acid
  • a base eg, alkali metal salt
  • a physiologically acceptable acid addition salt e.g., sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium tartaric acid, sodium tartaric acid, sodium tartaric acid, sodium tartaric acid, sodium tartaric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid.
  • the preventive or therapeutic agent for inflammatory diseases contains the aforementioned modified protein or a salt thereof, for example, for inflammatory diseases such as atopic dermatitis, contact dermatitis, rash, psoriasis, pemphigus vulgaris, etc. It has a preventive or therapeutic effect.
  • the pharmaceutical product according to the present embodiment includes the above-described modified protein or a salt thereof and is used for a desired pharmaceutical use, and can be used, for example, as a prophylactic or therapeutic agent for the above-mentioned inflammatory disease.
  • the pharmaceutical product according to the present embodiment includes, for example, ophthalmic diseases such as rheumatoid arthritis, Sjogren's syndrome, dry keratoconjunctivitis and dry eye, rheumatoid nodule, perforated scleral softening, superior sclera and scleritis; Respiratory diseases such as pneumonia, obstructive bronchiolitis, pleurisy, pneumothorax, empyema, respiratory tract lesions, pleural lesions, rheumatoid nodules, vascular lesions and sleep apnea syndrome (temporomandibular joint lesions, ring-shaped hip joint lesions); Cardiovascular diseases such as epidermitis, symptom
  • the pharmaceutical product according to the present embodiment can be used, for example, as a preventive or therapeutic agent for collagen disease.
  • collagen disease include systemic lupus erythematosus, systemic scleroderma, polymyositis / dermatomyositis, Sjogren's syndrome, Mixed connective tissue disease, antiphospholipid syndrome, Behcet's disease, allergic granulomatous vasculitis (Chirge-Strauss syndrome), adult Still's disease, eosinophilic fasciitis, nodular periarteritis (nodular polyposis) Arteritis / microscopic polyangiitis), aortitis syndrome (Takanian arteritis), Wegener's granulomatosis, temporal arteritis, malignant rheumatoid arthritis and the like.
  • the administration method of the pharmaceutical agent and the preventive or therapeutic agent for inflammatory diseases can be appropriately selected from oral administration, local administration, intravenous administration, intraperitoneal administration, intradermal administration, sublingual administration, and the like.
  • 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.
  • a modified protein As described above, to provide a modified protein, a pharmaceutical, a prophylactic or therapeutic agent for inflammatory diseases, and a method for producing the modified protein, which have high storage stability and in vivo stability and are highly effective in preventing or treating diseases. Can do.
  • Example 1 (Examination of conditions for HLA-G2 site-specific PEGylation) An HLA-G2 protein as a target protein for PEGylation, and a mutant protein (HLA-G2C42S) in which the 42nd free cysteine residue (Cys42) as a modification site of the PEG reagent used this time is substituted with a serine residue; And the progress of PEGylation of these proteins was compared to confirm whether PEGylation proceeded site-specifically to Cys42 residue.
  • 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.
  • HLA-G2 recombinant protein mutant expression plasmid In order to confirm that the PEGylation reagent reacts site-specifically with the free cysteine residue (Cys42 residue) possessed by HLA-G2, the 42nd cysteine was substituted with serine. -The G2C42S-pGMT7 plasmid was used (FIG. 11 (b), base sequence: SEQ ID NO: 9, amino acid sequence: SEQ ID NO: 10). The inventors of the present application have confirmed that the HLA-G2C42S recombinant protein maintains the same molecular structure and receptor binding ability as HLA-G2.
  • cysteine mutants for PEG modification site targeting cysteine residues were also prepared.
  • a new cysteine mutation was introduced into the HLA-G2C42S-pGMT7 plasmid so that the free cysteine of HLA-G2 was at one place.
  • the produced mutant expression plasmids are the following two. (1) A construct (HLA-G2N86C-pGMT7) in which the 86th asparagine which is the sugar chain modification site of HLA-G is replaced with cysteine (FIG. 11 (c), nucleotide sequence: SEQ ID NO: 11, amino acid sequence: SEQ ID NO: 12) ) (2) Construct added with cysteine at the C-terminus (HLA-G2CTER-pGMT7) (FIG. 11 (d), base sequence: SEQ ID NO: 13, amino acid sequence: SEQ ID NO: 14)
  • each mutation was introduced by PCR reaction using HLA-G2C42S-pGMT7 as a template and DNA synthase KOD Plus (Toyobo Co., Ltd.) and the following primers.
  • the PCR reaction was performed using a Veriti (registered trademark) thermal cycler (Applied Biosystems), heat-denatured at 94 ° C. for 2 minutes, heat-denatured at 98 ° C. for 10 seconds, annealed at 60 ° C. for 30 seconds, and heated at 68 ° C. for 4 minutes.
  • the extension reaction cycle was repeated 20 times.
  • 1 ⁇ L of DpnI enzyme (TOYOBO) was added and incubated at 37 ° C. for 1 hour.
  • the sequence of the obtained mutant plasmid was confirmed using a sequencing method. Specifically, 0.5 ⁇ L of each plasmid was added to 100 ⁇ L of E. coli DH5 ⁇ competent cells, allowed to stand on ice for about 30 minutes, and then incubated at 42 ° C. for 45 seconds for transformation. The transformed Escherichia coli was inoculated on Luria-Bertani (LB) agar medium containing 100 ⁇ g / mL ampicillin and cultured at 37 ° C. overnight. The obtained single colony was inoculated into 2 ⁇ Yeast-trytone (YT) medium (5 mL) containing 100 ⁇ g / mL ampicillin and cultured overnight at 37 ° C. and 150 rpm.
  • LB Luria-Bertani
  • LB Luria-Bertani
  • the obtained single colony was inoculated into 2 ⁇ Yeast-trytone (YT) medium (5 mL) containing 100 ⁇ g
  • the whole culture broth is centrifuged (13,200 rpm, 4 ° C., 1 minute), and plasmid DNA is obtained from the resulting E. coli pellet using a plasmid purification kit (QIAprep (registered trademark) Spin Miniprep kit (250), QIAGEN).
  • plasmid purification kit QIAprep (registered trademark) Spin Miniprep kit (250), QIAGEN).
  • a plasmid purification kit QIAprep (registered trademark) Spin Miniprep kit (250), QIAGEN).
  • a plasmid purification kit QIAprep (registered trademark) Spin Miniprep kit (250), QIAGEN).
  • a plasmid purification kit QIAprep (registered trademark) Spin Miniprep kit (250), QIAGEN).
  • Applied Biosystems Using the Big Dye (registered trademark) Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), T7 promote
  • sequence reaction solution was ethanol precipitated, 20 ⁇ L of Hi-DiTM formamide (Applied Biosystems) was added, incubated at 95 ° C. for 2 minutes, and then analyzed with a sequencer 3130 Genetic Analyzer (Applied Biosystems).
  • HLA-G1 recombinant protein expression plasmid For preparation of HLA-G1 monomer, an extracellular expression region (Gly1) in which the signal sequence was removed, a methionine residue as a translation initiation codon was added to the N-terminus, and the 42nd cysteine of HLA-G1 was replaced with serine.
  • the plasmid for expression of E. coli (HLA-G1C42S-pGMT7) possessed by the inventors of the present application in which -Gln276) was incorporated into the pGM7 vector was used (FIG. 11 (e), nucleotide sequence: SEQ ID NO: 15, amino acid sequence: SEQ ID NO: 16). ).
  • 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 Escherichia 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: 25)) 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.,.
  • HLA-G1, ⁇ 2m, and LILRB2biaA recombinant proteins were expressed as inclusion bodies by transforming E. coli BL21 (DE3) pLysS strain (Novagen) with the aforementioned various plasmids.
  • HLA-G2, HLA-G2 mutant HLA-G2C42S, HLA-G2N86C, HLA-G2CTER
  • HLA-G2CTER HLA-G2C42S, HLA-G2N86C, HLA-G2CTER
  • coli ClearColi (registered trademark) BL21 (DE3) competent cell (ClearColi (ClearColi) (Registered trademark) BL21 (DE3) competent cell (Lucigen) was transformed into a chemical competent cell by the inventors of the present application) and transformed into an inclusion body. Specifically, after transformation, seeded on an LB agar medium containing 100 ⁇ g / mL ampicillin, and then cultured at 37 ° C. overnight. The obtained colonies were inoculated into 2 ⁇ YT medium (10 mL) containing 100 ⁇ g / mL ampicillin and cultured with shaking at 37 ° C. overnight.
  • 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.
  • a suspension buffer 50 mM Tris hydroxylamine [Tris] -HCl pH 8.0, 150 mM NaCl
  • the mixture was centrifuged at 8000 rpm and 4 ° C. for 5 minutes, and the resulting precipitate was 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.
  • HLA-G2 HLA-G2 mutants (HLA-G2C42S, HLA-G2N86C, HLA-G2CTER), HLA-G1 and LILRB2biaA recombinant proteins were prepared by unwinding by E. coli inclusion body dilution method.
  • the final concentration of Dithiothreitol (DTT) is added to each of the solubilized inclusion bodies of HLA-G2 (8 mg), HLA-G2 mutant (8 mg), and LILRB2biaA (4 mg) so that the final concentration upon dilution is about 1 to 2 ⁇ M.
  • the solution was added to 10 mM and incubated at room temperature 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.
  • the HLA-G1 recombinant protein needs to be unwound as a heterotrimer of HLA-G1 heavy chain, ⁇ 2m, and peptide.
  • the ⁇ 2m inclusion body (4 mg) was diluted in the same manner as described above, and stirred at 4 ° C. for 4 to 6 hours in order to obtain a composite by rewinding from a stable unit as a simple substance.
  • completely dissolved HLA-G1 binding synthetic peptide (RIIPRHLQL) 0.2 mg / 0.2 mL DMSO was slowly added dropwise to the ⁇ 2m refolding solution, and the mixture was further stirred at 4 ° C. for 1 to 2 hours.
  • HLA-G1 inclusion bodies (4 mg) were diluted in the same manner as described above using a refolding solution containing ⁇ 2m and peptide, and stirred at 4 ° C. for 72 hours.
  • HLA-G2, HLA-G2 mutants (HLA-G2C42S, HLA-G2N86C, HLA-G2CTER) and HLA-G1 recombinant protein were concentrated to 15 mL or less with a VIVAFLOW system and then filtered. It was purified using a Hiload 26/60 Superdex75 pg (GE Healthcare) column. Furthermore, the HLA-G1 protein was subjected to ion exchange chromatography (IEX) as secondary purification. 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).
  • IEX was
  • 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.
  • LILRB2 birA (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 ⁇ L 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.
  • PEGylation reaction specific to Cys42 residue of HLA-G2 A PEGylation reaction was attempted 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.
  • 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).
  • PBS Phosphate-Buffer Saline
  • TCEP reducing agent tris (2-carboxyethyl) phosphine
  • TCEP tris (2-carboxyethyl) phosphine
  • 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.
  • BaI 2 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. .
  • 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.).
  • HLA-G2C42S a mutant in which a cysteine residue is substituted with a serine residue
  • Recombinant protein was prepared and compared with HLA-G2 protein.
  • HLA-G2C42S protein was prepared in the same manner as HLA-G2, and the target peak fraction was collected and used for the PEGylation reaction. Since HLA-G2C42S lost the free cysteine residue, multimer formation did not occur, and no band was observed at a high molecular weight position suggesting multimer formation even in non-reduced SDS-PAGE (not shown).
  • the PEGylation reaction conditions were determined as follows. All subsequent PEGylation reactions were carried out under these reaction conditions.
  • 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 could be confirmed (FIGS. 3A and 3B). . 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. 4B and 4C).
  • 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.
  • HLA-G2 (0.2-0.7 ⁇ M), PEG5-HLA-G2 (0.4-1.6 ⁇ M), and PEG10-HLA-G2 (0.3-1. 1 ⁇ M) and PEG20-HLA-G2 (0.3 to 1.1 ⁇ M).
  • 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. 12 (c)).
  • 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).
  • Amicon Ultra MWCO: 10000, Millipore.
  • 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 ⁇ L / 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.
  • HLA-G2CTER a construct in which a cysteine residue is added to the C-terminus of the HLA-G2 protein located on the cell membrane side in membrane-bound HLA-G2.
  • HLA-G1 glycosylation site a construct in which the 86th asparagine residue, which is predicted to have a sugar chain added to HLA-G2, is substituted with a cysteine residue.
  • Each HLA-G2 mutant protein was expressed as inclusion bodies using E. coli as described above. Furthermore, as described above, after unwinding each protein, SEC purification was performed, and the eluted fraction of each HLA-G2 mutant was collected.
  • each of the purified mutant proteins was PEGylated with PEG20 as described above.
  • the progress of the PEGylation reaction could be confirmed from the results of SDS-PAGE, CBB and BaI 2 staining of the PEGylation reaction solution (FIG. 6 (a ), (B)).
  • Example 2 In vitro stability evaluation of PEGylated HLA-G2 Using PEG20-HLA-G2 obtained in Example 1, in vitro stability was evaluated.
  • HLA-G2 and PEG20-HLA-G2 were lyophilized, redissolved in sterile water, and then subjected to SDS-PAGE. More specifically, 50 ⁇ L each of HLA-G2 and PEG20-HLA-G2 (0.15 mg / mL, HBS-EP buffer) were lyophilized using a lyophilizer. Immediately after lyophilization, the protein state before and after lyophilization was compared by re-dissolving in an equivalent amount (50 ⁇ L) of sterile water, performing SDS-PAGE under non-reducing conditions, and detecting by CBB staining.
  • the degree of aggregation / decomposition which is an indicator of stability as an HLA-G2 molecule, with and without lyophilization treatment was compared as a band pattern. As a result, regardless of the presence or absence of PEGylation, no aggregation or degradation of HLA-G2 molecules occurred by freeze-drying treatment, and no clear difference was observed (FIG. 7 (a)).
  • lyophilization treatment is performed in the same manner as described above.
  • the HLA-G2 and PEG20-HLA-G2 were subjected to binding experiments with the receptor LILRB2 using the SPR method.
  • biotinylated LILRB2 was immobilized on the sensor chip via streptavidin and serially diluted 2-fold (HLA-G2: 0.3 to 1.1 ⁇ M, PEG20-HLA-G2: 0.3 to 1.. 1 ⁇ M) lyophilized HLA-G2 and PEG20-HLA-G2 were run as analytes.
  • PEG20-HLA-G2 has a higher proportion of molecules that retain the binding activity to the receptor even after lyophilization compared to HLA-G2. From the above, it was suggested that the stability to lyophilization is improved by PEGylating the HLA-G2 protein.
  • HLA-G2 and PEG20-HLA-G2 (0.08 mg / mL, 20 mM Tris-HCl pH 8.0, 100 mM NaCl) after SEC purification were incubated at 50, 60, and 70 ° C., respectively.
  • the target band of HLA-G2 disappears under high temperature conditions, and the bands that are considered to be aggregates of 40 kDa or more and degradation products around 12 kDa increase (FIG. 8 (a)).
  • the disappearance of the target band and the appearance of bands other than the target were small even under high temperature conditions (FIG. 8 (b)).
  • HLA-G2 and PEG20-HLA-G2 were incubated under conditions of 5% FBS and 37 ° C. Samples were collected every hour from 24 hours after the start of incubation as 0 hours, and SDS-PAGE Sample buffer was added, followed by storage at 4 ° C. After collecting all the samples, the remaining amounts of HLA-G2 and PEG20-HLA-G2 were quantified using SDS-PAGE and Western blotting under non-reducing conditions.
  • HLA-G2 and PEG20-HLA-G2 HLA-G2: 0.3 mg / mL, PEG20-HLA-G2: 0.15 mg / mL, 20 mM Tris-HCl pH 8.0 after SEC purification, 100 mM NaCl
  • FBS Fetal Bovine Serum
  • 5 ⁇ SDS-PAGE Sample buffer was added to the sample collected every 2 hours and stored at 4 ° C. After all samples were collected, the degree of protein degradation was compared by performing Western blotting.
  • a secondary antibody was reacted with anti-mouse IgG (Fc) -horserdisse peroxide (HRP) -labeled antibody (Thremo Fisher Scientific) ( ⁇ 1/10000) by shaking at room temperature for 1 hour. Thereafter, the membrane was washed several times with PBS-T, and light was emitted with ECL Prime (GE Healthcare), and detection was performed using Image Quant LAS4000mini (GE Healthcare).
  • the MEM-G1 antibody used in this Western blotting method is an HLA-G-specific antibody, and the epitope site is unknown, but the inventors of the present application confirmed that it can detect denatured HLA-G2. (Unpublished data). In a preliminary experiment, it was confirmed that PEG20-HLA-G2 could be detected by Western blotting using the MEM-G1 antibody, and this experiment was conducted.
  • Example 3 Evaluation of anti-inflammatory effects of HLA-G2 and PEGylated HLA-G2 in vivo
  • the anti-inflammatory effect of HLA-G2 and PEG20-HLA-G2 proteins in vivo was verified using atopic dermatitis disease model mice.
  • HLA-G2 and PEG20-HLA-G2 were applied to the pinna of the dermatitis onset model mouse, and HLA- which the inventors of the present application confirmed the therapeutic effect in the mite antigen-induced dermatitis model mouse as a positive control.
  • G1 was administered and the degree of inflammation was observed.
  • PBS was used for the negative control.
  • An atopic dermatitis disease model mouse was prepared by applying an atopic dermatitis-inducing ointment (Biosta AD) containing a mite beetle component to the auricle surface of the mouse.
  • atopic dermatitis model mouse Prior to protein administration, 100 mg of Biosta AD was applied to NC / Nga Slc mice a total of 6 times every 3 days in order to prepare atopic dermatitis model mice. More specifically, body hair at the back of the ear was removed from 16 NC / Nga Slc mice (Japan SLC Inc., 10 weeks old, male) using an electric clipper and a hair removal cream.
  • Atopic dermatitis was developed by applying 100 mg of atopic dermatitis-inducing ointment (Biosta AD (registered trademark)) containing the mite mite insect component to the surface of the auricle from which body hair had been removed a total of 6 times every 3 days.
  • Biosta AD registered trademark
  • FIG. 10 (a) An operation of applying 4% SDS to the auricle was performed before applying Biosta AD.
  • a group not treated with the atopic dermatitis-induced ointment one animal was used as a control group for developing dermatitis.
  • each administration protein (HLA-G2, PEG20-HLA-G2, HLA-G1)) Subsequently, each administration protein to the prepared atopic dermatitis disease model mouse 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.
  • HLA-G1 molecular weight 44 kDa
  • IEX IEX was performed as secondary purification.
  • HLA-G1 was expressed using the endotoxin-containing BL21 (DE3) pLysS strain, the solution treated with LPS removal after IEX purification and substitution with PBS buffer was used as the administered protein.
  • the prepared HLA-G2, PEG20-HLA-G2, and HLA-G1 were transdermally administered to the auricular surface of the mouse 10 times every other day at a rate of 5 ⁇ g / ear (4 mice in each group), and the degree of inflammation of the auricle was determined.
  • the thickness of the auricle was recorded by measuring on the 0th, 6th, 10th, 14th, 18th, and 22nd days after the start of administration using a dial thickness gauge (Ozaki Seisakusho) (FIG. 10 (a)).
  • PEG20-HLA-G2 exhibits sufficient anti-inflammatory and therapeutic effects as an anti-inflammatory agent to atopic dermatitis mice without side effects. It was also revealed that the effect was higher with PEG20-HLA-G2 compared with HLA-G2.
  • Example 4 (SLE model mouse experiment) PEG20-HLA-G2 was administered to SLE (systemic lupus erythematosus) model mice to verify the therapeutic effect on SLE.
  • HLA-G2 PEG20-HLA-G2 prepared in Example 3 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.
  • a new lot newly unwound to repurified was administered about every 3 weeks. An outline of the test is shown in FIG.
  • FIG. 14 shows the weight transition of the mouse.
  • the PEG20-HLA-G2 administration group and the PBS administration group there was no significant difference in body weight transition among the administration groups.
  • 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 ).
  • TMB color developing solution
  • 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. 15 (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. 15 (a)).
  • FIG. 15 (b) shows the anti-dsDNA antibody titer of mice 90 days after administration. In the HLA-G2 administration group and the PEG20-HLA-G2 administration group, a significant decrease in the anti-dsDNA antibody titer was confirmed as compared to the PBS administration group (FIG. 15 (b)).
  • 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 ).
  • TMB color developing solution
  • the absorbance at 450 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.
  • 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. 16 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. Tended to last.
  • 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.
  • a labeled antibody peroxidase-conjugated anti-mouse BLys polyclonal antibody
  • 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).
  • TMB coloring solution
  • 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. 17 shows the Blys blood concentration of each group.
  • 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.
  • 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.
  • 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. 18 (a) shows the change in the anti-dsDNA antibody titer.
  • 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. 18 (b) shows changes in the urinary albumin index.
  • the urinary albumin index decreased as compared with PBS administration, confirming the therapeutic effect of PEG20-HLA-G2.

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Abstract

This modified protein is characterized by: comprising 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; and at least one amino acid residue in the amino acid sequence constituting the protein being PEGylated by polyethylene glycol (PEG).

Description

改変タンパク質、医薬品、炎症性疾患の予防又は治療剤及び改変タンパク質の製造方法Modified protein, pharmaceutical, preventive or therapeutic agent for inflammatory disease, and method for producing modified protein
　本発明は、改変タンパク質、医薬品、炎症性疾患の予防又は治療剤及び改変タンパク質の製造方法に関する。 The present invention relates to a modified protein, a pharmaceutical, a preventive or therapeutic agent for inflammatory diseases, and a method for producing the modified protein.
　ＨＬＡ－Ｇは、非古典的ＭＨＣＩ分子の１つであり、ＨＬＡ－Ｇ分子は、白血球Ｉｇ様受容体（ＬＩＬＲ）などの抑制性受容体への結合により、骨髄系単球細胞、Ｔ細胞及びＮＫ細胞をはじめとする広範な免疫細胞の免疫応答を阻害し、免疫寛容を誘導する。 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タンパク質は、ヒト生体内で多様な形態で存在し、天然の抑制性分子として機能している。ＨＬＡ－Ｇ１アイソフォームは、ペプチド、重鎖、β２ミクログロブリンからなるヘテロ三量体として存在する。一方、ドメイン欠損型のＨＬＡ－Ｇ２アイソフォームは、ドメイン欠損重鎖のみからなるホモ二量体である。ＨＬＡ－Ｇ２については、ＨＬＡ－Ｇ１の機能を補完する活性を持つことが知られていたが、詳細な機能については長期間にわたって不明であった。 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.
　近年、ＨＬＡ－Ｇ２について、種々の報告がなされている。非特許文献１には、ＨＬＡ－Ｇ２がホモ二量体として存在すること、受容体として免疫抑制性受容体ＬＩＬＲＢ２を介してシグナルを伝達することが開示されている。また、非特許文献２及び特許文献１には、ＨＬＡ－Ｇ２について、ｉｎ　ｖｉｖｏで抗炎症効果を解析した結果、マウス受容体ＰＩＲ－Ｂに強固に結合すること、関節リウマチモデルマウスへの単回投与により長期間の免疫抑制効果が得られたことが開示されている。 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.
　ポリエチレングリコール（ＰＥＧ）は、エチレングリコールが重合した構造を持つ高分子化合物である。親水性のＰＥＧをタンパク質などの高分子に付加すること（ＰＥＧ化）により、酵素による攻撃や疎水部の露出からタンパク質を遮蔽し、結果としてタンパク質の分解や凝集を抑制する効果が期待できる。また、ＰＥＧの付加により分子サイズが大きくなることで糸球体での濾過が抑制されるため、タンパク質血中半減期の延長が、ＰＥＧ自身は抗原性を持たないことから、タンパク質投与による副作用の軽減が期待できる（非特許文献３）。実際に、現在十数種のＰＥＧ化医薬品がＦｏｏｄ　ａｎｄ　Ｄｒｕｇ　Ａｄｍｉｎｉｓｔｒａｔｉｏｎ　（ＦＤＡ）により承認されており（非特許文献４、５）、臨床的に使用されている。 Polyethylene glycol (PEG) is a polymer compound having a structure in which ethylene glycol is polymerized. By adding hydrophilic PEG to a polymer such as protein (PEGylation), the protein is shielded from attack by an enzyme and exposure of a hydrophobic part, and as a result, an effect of suppressing protein degradation and aggregation can be expected. In addition, the increase in the molecular size due to the addition of PEG suppresses filtration in the glomerulus, so the protein half-life is prolonged, but PEG itself has no antigenicity, reducing the side effects of protein administration Can be expected (Non-patent Document 3). In fact, more than a dozen types of PEGylated drugs are currently approved by Food and Drug Administration (FDA) (Non-Patent Documents 4 and 5) and are used clinically.
特開２０１５－１４０３２２号公報JP 2015-140322 A
　しかしながら、ＨＬＡ－Ｇ２は、医薬品としての使用が期待されるものの、組換え蛋白質としての安定性及び均一性が経時的に低下するという問題を有しており、長期的保存の点で課題を残していた。 However, although HLA-G2 is expected to be used as a pharmaceutical, it has a problem that the stability and homogeneity as a recombinant protein deteriorates with time, leaving a problem in terms of long-term storage. It was.
　本発明は、上記事情に鑑みてなされたものであり、保存安定性及び生体内での安定性が高く、疾患に対する予防又は治療効果の高い改変タンパク質、医薬品、炎症性疾患の予防又は治療剤及び改変タンパク質の製造方法を提供することを目的とする。 The present invention has been made in view of the above circumstances, and is a modified protein, a pharmaceutical, a prophylactic or therapeutic agent for inflammatory diseases, which has high storage stability and in vivo stability, and is highly effective in preventing or treating diseases. An object is to provide a method for producing a modified protein.
　上記目的を達成するため、本発明の第１の観点に係る改変タンパク質は、
　ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体からなり、
　前記タンパク質を構成するアミノ酸配列中の少なくとも１つのアミノ酸残基は、ポリエチレングリコール（ＰＥＧ）でＰＥＧ化修飾されている、
　ことを特徴とする。 In order to achieve the above object, the modified protein according to the first aspect of the present invention comprises:
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 PEGylated with polyethylene glycol (PEG),
It is characterized by that.
　例えば、ＰＥＧ化修飾に用いるＰＥＧの分子量は、５ｋＤａ～１００ｋＤａである。 For example, the molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.
　例えば、前記タンパク質は、下記（ａ）または（ｂ）に記載するアミノ酸配列からなるタンパク質であって、
　（ａ）配列番号１に示されるアミノ酸配列からなるタンパク質、
　（ｂ）配列番号１に示されるアミノ酸配列において、１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質、
　前記多量体が、前記（ａ）同士若しくは（ｂ）同士のホモ多量体又は（ａ）と（ｂ）とのヘテロ多量体であり、白血球Ｉｇ様受容体Ｂ２との結合活性を有するものである。 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. .
　本発明の第２の観点に係る医薬品は、
　本発明の第１の観点に係るに改変タンパク質又はその塩を有効成分として含む。 The pharmaceutical product according to the second aspect of the present invention is:
According to the first aspect of the present invention, a modified protein or a salt thereof is included as an active ingredient.
　本発明の第３の観点に係る炎症性疾患の予防又は治療剤は、
　本発明の第１の観点に係るに改変タンパク質又はその塩を有効成分として含む。 The preventive or therapeutic agent for inflammatory diseases according to the third aspect of the present invention,
According to the first aspect of the present invention, a modified protein or a salt thereof is included as an active ingredient.
　本発明の第４の観点に係る改変タンパク質の製造方法は、
　（Ａ）ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体を調製する工程と、
　（Ｂ）工程（Ａ）で得られた前記タンパク質の多量体を脱気処理した後、還元処理する工程と、
　（Ｃ）工程（Ｂ）で還元処理された前記タンパク質の多量体をＰＥＧ化修飾する工程と、
　を含む。 The method for producing a modified protein according to the fourth aspect of the present invention comprises:
(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 step of degassing the protein multimer obtained in step (A), followed by a reduction treatment;
(C) PEGylating and modifying the protein multimer reduced in step (B);
including.
　例えば、ＰＥＧ化修飾に用いるＰＥＧの分子量は、５ｋＤａ～１００ｋＤａである。 For example, the molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.
　例えば、前記タンパク質は、下記（ａ）または（ｂ）に記載するアミノ酸配列からなるタンパク質であって、
　（ａ）配列番号１に示されるアミノ酸配列からなるタンパク質、
　（ｂ）配列番号１に示されるアミノ酸配列において、１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質、
　前記多量体が、前記（ａ）同士若しくは（ｂ）同士のホモ多量体又は（ａ）と（ｂ）とのヘテロ多量体であり、白血球Ｉｇ様受容体Ｂ２との結合活性を有するものである。 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. .
　本発明によれば、保存安定性及び生体内での安定性が高く、疾患に対する予防又は治療効果の高い改変タンパク質、医薬品、炎症性疾患の予防又は治療剤及び改変タンパク質の製造方法を提供することができる。 According to the present invention, there are provided a modified protein, a pharmaceutical, a prophylactic or therapeutic agent for inflammatory diseases, and a method for producing the modified protein, which have high storage stability and in vivo stability and are highly effective in preventing or treating diseases. Can do.
ＰＥＧ化ＨＬＡ－Ｇ２を模式的に示す図である。It is a figure which shows PEGylated HLA-G2 typically. （ａ）はＨＬＡ－Ｇ２の調製において封入体巻き戻し後、ＨｉＬｏａｄ２６／６０　Ｓｕｐｅｒｄｅｘ７５　ｐｇカラムを用いたＳＥＣ精製のクロマトグラムを示した図であり、（ｂ）は還元剤（ＴＣＥＰ）の添加によって得られたＰＥＧ化ＨＬＡ－Ｇ２のＣＢＢ染色の結果を示した図である。(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. ＰＥＧ分子量による反応効率の比較を示した図であり、（ａ）はＣＢＢ染色、（ｂ）はＢａＩ２染色の結果を示す図である。Is a diagram showing a comparison of the reaction efficiency with PEG molecular weight is a diagram showing (a) shows CBB staining, (b) the results of BaI 2 staining. 各分子量のＰＥＧによるＰＥＧ化反応液のＳｕｐｅｒｄｅｘ　２００　１０／３００　ＧＬを用いたＳＥＣ精製を行い、濃縮し、ＳＤＳ－ＰＡＧＥ後、銀染色した結果を示す図であり、（ａ）はＰＥＧ５－ＨＬＡ－Ｇ２、（ｂ）はＰＥＧ１０－ＨＬＡ－Ｇ２、（ｃ）はＰＥＧ２０－ＨＬＡ－Ｇ２、（ｄ）はＰＥＧ４０－ＨＬＡ－Ｇ２の結果を示す図である。FIG. 2 is a diagram showing the results of SEC purification using Superdex 200 10/300 GL of a PEGylation reaction solution with PEG of each molecular weight, concentrating, SDS-PAGE, and silver staining, (a) is PEG5-HLA- G2, (b) are the results of PEG10-HLA-G2, (c) are the results of PEG20-HLA-G2, and (d) are the results of PEG40-HLA-G2. ＬＩＬＲＢ２受容体との結合実験により得られたセンサグラムを示す図であり、（ａ）はＨＬＡ－Ｇ２、（ｂ）はＰＥＧ５－ＨＬＡ－Ｇ２、（ｃ）はＰＥＧ１０－ＨＬＡ－Ｇ２、（ｄ）はＰＥＧ２０－ＨＬＡ－Ｇ２の結果を示す図である。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. ＰＥＧ化部位の異なるＨＬＡ－Ｇ２変異体（ＨＬＡ－Ｇ２Ｎ８６Ｃ、ＨＬＡ－Ｇ２ＣＴＥＲ）のＰＥＧ２０によるＰＥＧ化反応効率を比較した図であり、（ａ）はＣＢＢ染色、（ｂ）はＢａＩ２染色の結果を示す図である。It is the figure which compared the PEGylation reaction efficiency by PEG20 of the HLA-G2 variant (HLA-G2N86C, HLA-G2CTER) from which a PEGylation site differs, (a) is the result of CBB staining, (b) is the result of BaI 2 staining. FIG. （ａ）は凍結乾燥処理有無によるＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２のＳＤＳ－ＰＡＧＥ、ＣＢＢ染色の結果を示す図であり、（ｂ）は凍結乾燥処理前後のＨＬＡ－Ｇ２（ＬＩＬＲＢ２固定化量：５１２ＲＵ）の結合実験によって得られたセンサグラムを示す図であり、（ｃ）は凍結乾燥処理前後のＰＥＧ２０－ＨＬＡ－Ｇ２（ＬＩＬＲＢ２固定化量：２７２ＲＵ）の結合実験によって得られたセンサグラムを示す図である。(A) shows the results of SDS-PAGE and CBB staining of HLA-G2 and PEG20-HLA-G2 with and without lyophilization treatment, and (b) shows the amount of HLA-G2 (LILRB2 immobilized amount before and after lyophilization treatment). : 512RU) shows a sensorgram obtained by a binding experiment, and (c) shows a sensorgram obtained by a binding experiment of PEG20-HLA-G2 (LILRB2 immobilization amount: 272RU) before and after lyophilization treatment. FIG. 加熱処理によるタンパク質凝集及び分解度の比較結果（ＣＢＢ染色）を示す図であり、（ａ）はＨＬＡ－Ｇ２、（ｂ）はＰＥＧ２０－ＨＬＡ－Ｇ２である。It is a figure which shows the comparison result (CBB dyeing | staining) of the protein aggregation and decomposition degree by heat processing, (a) is HLA-G2, (b) is PEG20-HLA-G2. ＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２タンパク質の血清中安定性比較を示す図であり、（ａ）はＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２の血清中安定性を示す図であり、（ｂ）はＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２の血清中の継時的残存率の変化を示す図である。It is a figure which shows the stability in serum of HLA-G2, PEG20-HLA-G2 protein, (a) is a figure which shows the stability in serum of HLA-G2, PEG20-HLA-G2, (b) It is a figure which shows the change of the survival rate in the serum of HLA-G2 and PEG20-HLA-G2 over time. （ａ）はアトピー性皮膚炎誘発軟膏による皮膚炎の誘導及びタンパク質の投与スケジュール並びに耳介の厚さの記録日を示す図であり、（ｂ）はマウス耳介の腫脹度を示す図であり、（ｃ）は投与１８日目のマウス耳介を示す写真図である。(A) is a figure which shows the dermatitis induction by an atopic dermatitis induction ointment, the administration schedule of protein, and the recording date of the thickness of an auricle, (b) is a figure which shows the swelling degree of a mouse | mouth ear. (C) is a photograph showing the mouse auricle on the 18th day after administration. 各組換えタンパク質の一次構造（左）及び分子模式図（右）を表す図であり、（ａ）はＨＬＡ－Ｇ２、（ｂ）はＨＬＡ－Ｇ２Ｃ４２Ｓ、（ｃ）はＨＬＡ－Ｇ２Ｎ８６Ｃ、（ｄ）はＨＬＡ－Ｇ２ＣＴＥＲ、（ｅ）はＨＬＡ－Ｇ１Ｃ４２Ｓ重鎖、（ｆ）はβ２ｍ、（ｇ）はＬＩＬＲＢ２を示す模式図である。FIG. 2 is a diagram showing the primary structure (left) and molecular schematic diagram (right) of each recombinant protein, where (a) is HLA-G2, (b) is HLA-G2C42S, (c) is HLA-G2N86C, (d) FIG. 4 is a schematic diagram showing HLA-G2CTER, (e) HLA-G1C42S heavy chain, (f) β2m, and (g) LILRB2. センサーチップＣＡＰ上へのビオチン化タンパク質固定化の原理を表す模式図であり、（ａ）はセンサーチップＣＡＰ上に一本鎖ＤＮＡを固定化した様子を表す模式図であり、（ｂ）はストレプトアビジンをチップ上に固定した様子を表す模式図であり、（ｃ）はビオチン化ＬＩＬＲＢ２及びビオチン化ＢＳＡを固定化した様子を表す模式図である。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. ＳＬＥモデルマウスを用いた試験の概要を示す図である。It is a figure which shows the outline | summary of the test using a SLE model mouse. ＳＬＥモデルマウスの体重推移を示したグラフ図である。It is the graph which showed the weight transition of the SLE model mouse. （ａ）はＳＬＥモデルマウスの血中抗ｄｓＤＮＡ抗体価を測定したグラフ図であり、（ｂ）は投与９０日後の結果を示すグラフ図である。(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. ＳＬＥモデルマウスの尿中アルブミン指数を測定したグラフ図である。It is the graph which measured the urinary albumin index of the SLE model mouse. ＳＬＥモデルマウスのＢＬｙｓ血中濃度を測定したグラフ図である。It is the graph which measured the BLys blood concentration of a SLE model mouse. （ａ）はＳＬＥモデルマウスの血中抗ｄｓＤＮＡ抗体価を測定したグラフ図であり、（ｂ）はＳＬＥモデルマウスの尿中アルブミン指数を測定したグラフ図である。(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.
　まず、本実施形態による改変タンパク質について詳細に説明する。 First, the modified protein according to this embodiment will be described in detail.
　本実施形態による改変タンパク質は、ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体からなり、該タンパク質を構成するアミノ酸配列中の少なくとも１つのアミノ酸残基は、ポリエチレングリコール（ＰＥＧ）でＰＥＧ化修飾されている（図１）。 The modified protein according to this embodiment comprises 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, and at least one amino acid residue in the amino acid sequence constituting the protein is present. The group is PEGylated with polyethylene glycol (PEG) (FIG. 1).
　本実施形態が対象とするＨＬＡ－Ｇは、好ましくはヒト由来のＨＬＡ－Ｇである。なお、ヒトのＨＬＡ－Ｇ分子の配列情報は、例えばＮＣＢＩ等により既に公知である。具体的には、アイソフォームの一つとして、ＮＣＢＩには、ＮＭ＿００２１２７．５にヒト由来のＨＬＡ－Ｇ全長（＝ＨＬＡ－Ｇ１）の遺伝子配列が記載されており、このうちＨＬＡ－Ｇのα１ドメイン及びα３ドメインの遺伝子領域に相当する塩基配列が、それぞれ下記に示す配列番号２の「１～２７０番目の塩基配列」及び「２７１～５４０番目の塩基配列」である。なお、ＨＬＡ－Ｇ分子には種々のアイソフォームが存在しており、アイソフォームによってそれらのアミノ酸配列は若干相違する。 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.
　本実施形態が対象とするＨＬＡ－Ｇ多量体は、例えば、ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体であり、好ましくは、
　（ａ）配列番号１に示されるアミノ酸配列からなるタンパク質、又は
　（ｂ）配列番号１に示されるアミノ酸配列において、１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質、
　であって、
　前記（ａ）同士若しくは（ｂ）同士のホモ多量体又は（ａ）と（ｂ）とのヘテロ多量体である。 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).
　本明細書において、「ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体」を“ＨＬＡ－Ｇ２”又は“ＨＬＡ－Ｇ２多量体”と称する場合がある。 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.
　上記の「配列番号１に示されるアミノ酸配列からなるタンパク質」は、ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の二量体のうちの一つである。ここでＨＬＡ－Ｇ分子は、好ましくはヒト由来のＨＬＡ－Ｇである。当該配列番号１において「１～９０番目のアミノ酸領域」がα１ドメインのアミノ酸配列に相当し、「９１～１８０番目のアミノ酸領域」がα３ドメインのアミノ酸配列に相当する。また、当該アミノ酸配列（配列番号１）をコードする塩基配列を表す配列番号２において「１～２７０番目の塩基配列」領域がα１ドメインをコードする遺伝子領域に相当し、「２７１～５４０番目の塩基配列」領域がα３ドメインをコードする遺伝子領域に相当する。 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.
　上記の「１以上のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質」として、ＨＬＡ－Ｇ分子のα１ドメインとα３ドメインとの連結体（α１－３連結体）のアミノ酸配列（配列番号１）のＣ端側にさらにＨＬＡ－Ｇ分子のイントロン４によってコードされるアミノ酸配列（配列番号３）を有するタンパク質が例示され、本明細書においてこれを“ＨＬＡ－Ｇ２”と称する場合がある。また、他の「１以上のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質」として、ＨＬＡ－Ｇ分子のα１ドメインとα３ドメインとの連結体のアミノ酸配列（配列番号１）のＣ端側にさらにＨＬＡ－Ｇ２分子の膜貫通ドメイン及び細胞内ドメインのアミノ酸配列（配列番号４）を有するタンパク質を例示することができる。 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.
　「１以上のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質」のうち、好ましくは上記ＨＬＡ－Ｇ分子のα１ドメインとα３ドメインとの連結体の多量体の機能（白血球Ｉｇ様受容体Ｂ２との結合活性）を備え、かつ当該α１－３連結体のアミノ酸配列（配列番号１）と好ましくは９０％以上、好ましくは９５％以上同一のアミノ酸配列を有することを限度として、アミノ酸が欠失、置換若しくは付加してなるタンパク質である。より好ましくは上記連結体のアミノ酸配列（配列番号１）と９６％以上同一、さらに好ましくは９８％以上同一のアミノ酸配列を有するタンパク質である。 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.
　本明細書において、ＨＬＡ－Ｇ多量体のうち、（ａ）配列番号１に示されるアミノ酸配列からなるタンパク質、又は（ｂ）配列番号１に示されるアミノ酸配列において、１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質、であって、前記（ａ）同士若しくは（ｂ）同士のホモ多量体又は（ａ）と（ｂ）とのヘテロ多量体を、“ＨＬＡ－Ｇ２多量体”と称する場合がある。 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".
　本実施形態が対象とするＨＬＡ－Ｇ２多量体は、前述するＨＬＡ－Ｇ分子のα１ドメインとα３ドメインとの連結体そのもの（以下これを単に「α１－３連結体」ともいう）に限定されず、ＨＬＡ－Ｇ分子のα１ドメインとα３ドメインとの連結構造を有し、かつ当該二量体が白血球Ｉｇ様受容体Ｂ２（ＬＩＬＲＢ２）に対して結合活性を有するものであればよい。例えば、上記条件を有するものである限り、当該ＨＬＡ－Ｇ分子のα１ドメインとα３ドメインとの連結体（α１－３連結体）のアミノ酸配列（配列番号１）のうち、１以上のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質であってもよい。ここで、「白血球Ｉｇ様受容体Ｂ２（ＬＩＬＲＢ２）に対する結合活性」とは、ＨＬＡ－Ｇ２多量体がＬＩＬＲＢ２と直接結合する活性であって、これによりＬＩＬＲＢ２を介してシグナルを伝達し、免疫制御効果を発揮することができる活性を意味する。 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 linking structure of α1 domain and α3 domain of HLA-G molecule and the dimer 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.
　なお、ＨＬＡ－Ｇ２多量体が、白血球Ｉｇ様受容体Ｂ２（ＬＩＬＲＢ２）との結合活性を有するか否かの確認は、例えば、Ｔ細胞ハイブリドーマを用いたレポーターアッセイなどにより行うことができる。かかるＴ細胞ハイブリドーマとしては、ＬＩＬＲＢ２の細胞外ドメインと活性型受容体ＰＩＬＲβの膜貫通・細胞内ドメインを融合させたキメラ分子を発現したＮＦＡＴ－ＧＦＰ導入レポーター細胞（マウスＴ細胞ハイブリドーマ）を例示することができる。この場合、ＨＬＡ－Ｇ２多量体がＬＩＬＲＢ２受容体に結合すると、ＰＩＬＲβの細胞内ドメインを介したシグナルが伝達され、転写因子ＮＦＡＴが活性化される。レポーターアッセイは、当該ＮＦＡＴの活性化によりＧＦＰの発現が誘導されることを利用したアッセイ系である。ＧＦＰ発現は、ＬＩＬＲＢ２とＨＬＡ－Ｇ２多量体とが結合したことを示すが、かかるＧＦＰ発現による蛍光の出現はフローサイトメトリーで確認することができる。 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.
　ＨＬＡ－Ｇ２多量体は、前述するＨＬＡ－Ｇ２同士が、ジスルフィド結合するか、又はジスルフィド結合によらないで非共有結合的に結合することにより、多量体化したものである。なお、本実施形態におけるＨＬＡ－Ｇ２多量体において、該タンパク質を構成するアミノ酸配列中のどのシステイン残基がＰＥＧ化修飾されているかにもよるが、例えば、自然形成するホモ二量体の場合、ジスルフィド結合によらず多量体化し、システイン残基（Ｃｙｓ４２）は分子表面にフリーのまま存在していてもよく、また、ホモ二量体を１単位とするさらなる二量体（四量体）又はそれらのさらなる多量体の場合、ホモ二量体分子表面に存在するシステイン残基（Ｃｙｓ４２）同士を介したジスルフィド結合により多量体化していてもよい。さらに、自然形成するホモ二量体の場合、ジスルフィド結合によらず多量体化し、また、ホモ二量体を１単位とするさらなる二量体（四量体）又はそれらのさらなる多量体の場合、ホモ二量体分子表面に存在するシステイン残基以外のアミノ酸残基同士を介した結合により多量体化していてもよい。 The HLA-G2 multimer is a multimerized form in which the above-mentioned HLA-G2s are disulfide bonded or non-covalently bonded without using disulfide bonds. In the HLA-G2 multimer in the present embodiment, depending on which cysteine residue in the amino acid sequence constituting the protein is PEGylated, for example, in the case of a naturally formed homodimer, It multimerizes regardless of the disulfide bond, and the cysteine residue (Cys42) may be left free on the surface of the molecule, or a further dimer (tetramer) having a homodimer as one unit or In the case of those further multimers, they may be multimerized by disulfide bonds via cysteine residues (Cys42) existing on the surface of the homodimer molecule. 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.
　ＨＬＡ－Ｇ２多量体は、前述するα１－３連結体同士又は変異型同士からなるホモ多量体であってもよいし、又はα１－３連結体と変異型からなるヘテロ多量体であってもよく、限定はされない。好ましくはα１－３連結体同士からなるホモ多量体である。 The HLA-G2 multimer may be a homomultimer composed of the above-mentioned α1-3 conjugates or variants, or a heteromultimer composed of an α1-3 conjugate and a variant. There is no limitation. Preferably, it is a homomultimer composed of α1-3 conjugates.
　また、ＨＬＡ－Ｇ２多量体は、白血球Ｉｇ様受容体Ｂ２（ＬＩＬＲＢ２）との結合部位を表面に露出した構造を有し、これらの受容体との結合が立体障害により妨げられないものである。そのため、ＨＬＡ－Ｇ２多量体は、ＨＬＡ－Ｇ２と同種の機能（白血球Ｉｇ様受容体への結合活性）を保持しうる構造を有する。 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.
　ＨＬＡ－Ｇ２多量体の調製方法については後述する。 The method for preparing the HLA-G2 multimer will be described later.
　本実施形態による改変タンパク質は、上述の通り、ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体からなり、該タンパク質を構成するアミノ酸配列中の少なくとも１つのシステイン残基は、ポリエチレングリコール（ＰＥＧ）でＰＥＧ化修飾されている。 As described above, the modified protein according to the present embodiment comprises 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, and at least in the amino acid sequence constituting the protein. One cysteine residue is PEGylated with polyethylene glycol (PEG).
　ＰＥＧ化修飾されるアミノ酸残基は、該タンパク質において受容体との結合に影響しない部位に存在することが好ましい。ＰＥＧ化修飾されるアミノ酸残基として、例えば、リジン残基、システイン残基等を挙げることができる。例えば、システイン残基がＰＥＧ化修飾される場合、該システイン残基は、例えば、ＨＬＡ－Ｇ２の４２番目のフリーのシステイン残基であってもよい。また、任意の部位に存在するアミノ酸残基をシステイン残基に置換し、該システイン残基をＰＥＧ化修飾してもよく、例えば、ＨＬＡ－Ｇの糖鎖修飾部位である８６番目のアスパラギンをシステインに置換し、ＰＥＧ化修飾してもよい。また、任意の部位にシステイン残基を付加し、該システイン残基をＰＥＧ化修飾してもよく、例えば、ＨＬＡ－ＧのＣ末端にシステインを付加し、ＰＥＧ化修飾してもよい。なお、ＰＥＧ化修飾が可能なアミノ酸残基であれば、リジン残基、システイン残基に限らず選択することができる。 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.
　本実施形態において、ＰＥＧ化修飾に用いるＰＥＧの分子量は、例えば、５ｋＤａ～１００ｋＤａであり、好ましくは５ｋＤａ～４０ｋＤａである。ＰＥＧ化修飾の詳細については、後述する。 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.
　本実施形態によるＰＥＧ化修飾された改変タンパク質の保存安定性を確認する方法として、例えば、改変タンパク質を凍結乾燥処理後に滅菌水に溶解させ、ＳＤＳ－ＰＡＧＥを行うことで凝集・分解の程度をバンドパターンとして確認する方法、改変タンパク質を凍結乾燥処理後に前述の方法によりＬＩＬＲＢ２への結合活性を確認する方法、改変タンパク質を高温条件下でインキュベートした後にＳＤＳ－ＰＡＧＥを行うことで凝集・分解の程度をバンドパターンとして確認する方法等が挙げられる。また、本実施形態によるＰＥＧ化修飾された改変タンパク質の生体内での安定性を確認する方法として、例えば、改変タンパク質を血清（例えば、ＦＢＳ）中でインキュベートした後に、ウエスタンブロッティングを行うことで分解の程度を確認する方法等が挙げられる。 As a method for confirming the storage stability of the PEGylated modified protein according to this embodiment, for example, the modified protein is dissolved in sterilized water after lyophilization, and the degree of aggregation / degradation is determined by SDS-PAGE. A method for confirming as a pattern, a method for confirming the binding activity to LILRB2 by the above-mentioned method after lyophilizing the modified protein, and a degree of aggregation / degradation by performing SDS-PAGE after incubating the modified protein under high temperature conditions The method of confirming as a band pattern etc. are mentioned. In addition, as a method for confirming the in vivo stability of the modified protein modified with PEG according to the present embodiment, for example, the modified protein is decomposed by incubating in serum (for example, FBS) and then performing Western blotting. The method of confirming the grade of etc. is mentioned.
　次に、本実施形態による改変タンパク質の製造方法について詳細に説明する。 Next, the modified protein production method according to the present embodiment will be described in detail.
　本実施形態による改変タンパク質の製造方法は、
　（Ａ）ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体を調製する工程と、
　（Ｂ）工程（Ａ）で得られた前記タンパク質の多量体を脱気処理した後、還元処理する工程と、
　（Ｃ）工程（Ｂ）で還元処理された前記タンパク質の多量体をＰＥＧ化修飾する工程と、
　を含む。 The method for producing a modified protein according to the present embodiment includes:
(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 step of degassing the protein multimer obtained in step (A), followed by a reduction treatment;
(C) PEGylating and modifying the protein multimer reduced in step (B);
including.
　ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体（ＨＬＡ－Ｇ２又はＨＬＡ－Ｇ２多量体）の詳細については、前述の通りである。 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.
　本実施形態が対象とするＨＬＡ－Ｇ２多量体は、前述同様、例えば、ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体であり、好ましくは、
　（ａ）配列番号１に示されるアミノ酸配列からなるタンパク質、又は
　（ｂ）配列番号１に示されるアミノ酸配列において、１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質、
　であって、
　前記（ａ）同士若しくは（ｂ）同士のホモ多量体又は（ａ）と（ｂ）とのヘテロ多量体である。 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).
　工程（Ａ）において、一例として、ＨＬＡ－Ｇ２多量体の調製方法について説明する。 In the step (A), as an example, a method for preparing an HLA-G2 multimer will be described.
　まず、公知の遺伝子組換え技術を用いて、ＨＬＡ－Ｇ２多量体のアミノ酸配列をコードする遺伝子（例えば、配列番号２）を発現ベクター等に組込んだ組換えベクターを構築し、次いで、公知の各種形質転換法により、構築した組換えベクターを宿主に導入して形質転換体を得、これを培養することにより、組換えＨＬＡ－Ｇ２多量体を発現させ、回収することにより行うことができる。 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.
　ここでＨＬＡ－Ｇ２多量体のアミノ酸配列をコードする遺伝子としては、ＨＬＡ－Ｇのα１ドメインとα３ドメインとの連結体のアミノ酸配列（配列番号１）をコードする遺伝子（配列番号２）のほか、当該連結体のアミノ酸配列の一部を欠失、置換又は付加してなる前述のＨＬＡ－Ｇ２（以下、これを便宜上「変異型ＨＬＡ－Ｇ２」という）のアミノ酸配列をコードする遺伝子を用いることもできる。また、収量増加の観点から、ＨＬＡ－Ｇ２のアミノ酸配列をコードする遺伝子として、例えば配列番号２に示す塩基配列の第１～１８番目（ＨＬＡ－Ｇ２のアミノ酸配列において１～６番目のアミノ酸をコードする塩基配列に相当する）の塩基配列を、配列番号５に示す塩基配列で置換して得られる遺伝子（以下、「改変遺伝子」という）（配列番号６）を用いることもできる。 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 referred to as “mutant HLA-G2” for convenience) obtained by deleting, substituting or adding a part of the amino acid sequence of the conjugate may be used. it can. 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.
　変異型ＨＬＡ－Ｇ２多量体をコードする遺伝子は、α１－３連結体の遺伝子のＤＮＡ配列に変異を導入して調製すればよく、例えば、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　２ｎｄ　ｅｄ．，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ　（１９８９），Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｊｏｈｎ　Ｗｉｌｅｙ　＆　Ｓｏｎｓ（１９８７－１９９７）等に記載の部位特異的変異導入法に準じて調製することができる。具体的には、Ｋｕｎｋｅｌ法やＧａｐｐｅｄ　ｄｕｐｌｅｘ法等の公知手法により、部位特異的突然変異導入法を利用した変異導入用キットを用いて調製することができる。当該キットとしては、例えば、ＱｕｉｃｋＣｈａｎｇｅＴＭ　Ｓｉｔｅ－Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（ストラタジーン社製）、ＧｅｎｅＴａｉｌｏｒＴＭ　ｉｔｅ－Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｓｙｓｔｅｍ（インビトロジェン社製）、ＴａＫａＲａ　Ｓｉｔｅ－Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｓｙｓｔｅｍ（Ｍｕｔａｎ－Ｋ、Ｍｕｔａｎ－Ｓｕｐｅｒ　Ｅｘｐｒｅｓｓ　Ｋｍ等：タカラバイオ社製）等が好ましく挙げられる。 The gene encoding the mutant HLA-G2 multimer may be prepared by introducing a mutation into the DNA sequence of the α1-3 ligated gene. See, 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 site-directed mutagenesis by a known technique such as Kunkel method or Gapped duplex method. Examples of such kits include QuickChange ™ Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTailor ™ ite-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRa Site-Mit-Sensitized-Might-Sensitive-Mid-Sensitive-Mid-Sensitive-Mit-Sensitive-Mid-Sequential Etc .: manufactured by Takara Bio Inc.).
　形質転換体の作成に使用される宿主は、導入された組換えベクター等からＨＬＡ－Ｇ２（α１－３連結体、変異型）を発現し得るものであれば、特に限定はされず、例えば、ヒトやマウス等の各種動物に由来する細胞、各種昆虫に由来する細胞、大腸菌などの原核細胞、酵母などの真核細胞、植物細胞等、宿主となりえる公知の細胞が使用できる。 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.
　組換えＨＬＡ－Ｇ２多量体の製造は、具体的には、上述の形質転換体を培養する工程と、得られる培養物から組換えＨＬＡ－Ｇ２多量体を採取する工程とを含む方法により行うことができる。ここで、「培養物」とは、培養上清、培養細胞、培養菌体、又は細胞若しくは菌体の破砕物のいずれをも意味するものである。上記形質転換体の培養は、宿主の培養に用いられる通常の方法に従って行うことができる。目的のタンパク質は、上記培養物中に蓄積される。 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.
　組換えＨＬＡ－Ｇ２多量体が細胞外に生産される場合は、培養液をそのまま使用するか、遠心分離やろ過等により細胞を除去する。その後、必要に応じて硫安沈澱による抽出等により、培養物中から組換えＨＬＡ－Ｇ２多量体を採取し、さらに必要に応じて透析、各種クロマトグラフィー（ゲルろ過、イオン交換クロマトグラフィー、アフィニティクロマトグラフィー等）を用いて単離精製することができる。 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.
　組換えＨＬＡ－Ｇ２多量体が細胞内に生産される場合は、細胞を破砕することにより組換えＨＬＡ－Ｇ２多量体を採取することができる。可溶性画分にＨＬＡ－Ｇ２多量体が含まれる場合は、破砕後、遠心分離やろ過などにより、必要に応じて細胞の破砕残渣（細胞抽出液不溶性画分を含む）を除く。残渣除去後の上清は、細胞抽出液可溶性画分であり、粗精製したタンパク質溶液とすることができる。一方、不溶性画分に封入体としてＨＬＡ－Ｇ２多量体が発現する場合は、破砕後、遠心分離により不溶性画分を単離し、界面活性剤等を含んだバッファーで洗浄、遠心を繰り返すことにより、細胞の破砕残渣を取り除く。得られた封入体はグアニジンや尿素などの変性剤を含むバッファーで可溶化した後、希釈法や透析法を利用した蛋白質の巻き戻しを行う。機能的に巻き戻ったＨＬＡ－Ｇ２多量体の精製は各種クロマトグラフィー（ゲルろ過、イオン交換クロマトグラフィー、アフィニティクロマトグラフィー等）を用いて単離精製することができる。 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.).
　また、組換えＨＬＡ－Ｇ２多量体の産生は、形質転換体を用いたタンパク質合成系のほか、生細胞を全く使用しない無細胞タンパク質合成系を用いて行うこともでき、産生された組換えＨＬＡ－Ｇ２多量体は、クロマトグラフィー等の手段を適宜選択して精製することができる。 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.
　ＨＬＡ－Ｇ２多量体は、どのような方法で得られるものであってもよく、その取得方法に特に限定はされない。 The HLA-G2 multimer may be obtained by any method, and the acquisition method is not particularly limited.
　工程（Ｂ）は、工程（Ａ）で得られた前記タンパク質の多量体を脱気処理した後、還元処理する工程である。 Step (B) is a step in which the protein multimer obtained in step (A) is degassed and then reduced.
　工程（Ｂ）において、嫌気条件下でＰＥＧ化反応を進行させるために、脱気処理を行う。脱気処理は、例えば、アスピレーターを用いて１時間行ってもよい。脱気処理の前に、例えば、工程（Ａ）で得られた前記タンパク質の多量体をＰＥＧ化バッファー（例えば、１×Ｐｈｏｓｐｈａｔｅ－Ｂｕｆｆｅｒｄ　Ｓａｌｉｎｅ（ＰＢＳ），５ｍＭ　ＥＤＴＡ）への置換を行ってもよい。脱気処理の方法として、他の公知の方法を用いてもよい。 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 protein multimer obtained in the step (A) may be replaced with a PEGylation buffer (for example, 1 × Phosphate-Buffer Saline (PBS), 5 mM EDTA). . Other known methods may be used as the deaeration process.
　工程（Ｂ）において、還元剤は、還元処理によってジスルフィド結合を切断し、ＰＥＧ化ターゲットとなる残基のチオール基を再度露出させ、反応効率を向上させるために、添加される。還元剤は、還元作用を有する任意の還元剤を用いることができ、例えば、マレイミドとシステインを用いた反応時に多用されるｔｒｉｓ（２－ｃａｒｂｏｘｙｅｔｈｙｌ）ｐｈｏｓｐｈｉｎｅ（ＴＣＥＰ）を用いてもよい。還元剤としてＴＣＥＰを用いる場合、例えば、ＴＣＥＰを終濃度０．１ｍＭ以上となるように加えてもよい。 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.
　工程（Ｃ）は、工程（Ｂ）で還元処理された該タンパク質の多量体をＰＥＧ化修飾する工程である。 Step (C) is a step of PEGylating the multimer of the protein reduced in step (B).
　工程（Ｃ）において、具体的には、ＰＥＧの末端にマレイミド基、スクシンイミド基などの反応性官能基を有するＰＥＧ化試薬と本実施形態のタンパク質を溶液中で反応させることによりＰＥＧ化された改変タンパク質を得ることができる。使用されるＰＥＧ化試薬としては、例えば、システインのＳＨ基とチオエーテル結合を形成する直鎖型メチルＰＥＧｎ（ｎはＰＥＧのリピート数）マレイミド、分岐型（メチル－ＰＥＧｎ）ｎ－ＰＥＧｎマレイミド等が挙げられる。ＰＥＧ化修飾に用いるＰＥＧの分子量は、例えば、５ｋＤａ～１００ｋＤａであり、好ましくは５ｋＤａ～４０ｋＤａである。ＰＥＧ化試薬として、例えば、反応基としてマレイミド基を持ち、チオール基を認識して反応する高純度直鎖ＰＥＧであるＭＥ－４００ＭＡ（ＭＷ：４２，６５３Ｄａ）（ＰＥＧ４０）、ＭＥ－２００ＭＡＯＢ（ＭＷ：２０，８４１Ｄａ）（ＰＥＧ２０）、ＭＥ－１００ＭＡ（ＭＷ：１０，３０３Ｄａ）（ＰＥＧ１０）、ＭＥ－０５０ＭＡ（ＭＷ：５，３９３Ｄａ）（ＰＥＧ５）（いずれも日油株式会社）等を用いることができる。 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.
　工程（Ｃ）において、ＰＥＧが該タンパク質の多量体に対して過剰量となるように、例えば、ＰＥＧ：タンパク質の多量体＝５：１、１０：１、２０：１、３０：１（モル比）等でＰＥＧ化試薬を混合してもよい。ＰＥＧ化試薬を加え、例えば、４℃で一晩反応させることでＰＥＧ化修飾することができる。前述の通り、該タンパク質を構成するアミノ酸配列中の少なくとも１つのアミノ酸残基（例えば、システイン残基）が、ＰＥＧでＰＥＧ化修飾される。 In step (C), for example, PEG: protein multimer = 5: 1, 10: 1, 20: 1, 30: 1 (molar ratio) so that PEG is in an excess amount relative to the protein multimer. ) Or the like may be mixed with the PEGylation reagent. 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.
　ＰＥＧ化修飾されたタンパク質は、例えば、ゲルろ過クロマトグラフィー（Ｓｉｚｅ　Ｅｘｃｌｕｓｉｏｎ　Ｃｈｒｏｍａｔｏｇｒａｐｈｙ：ＳＥＣ）による精製を行ってもよい。 The PEG-modified protein may be purified by, for example, gel filtration chromatography (SEC).
　次に、本実施形態による医薬品及び炎症性疾患の予防又は治療剤について詳細に説明する。 Next, the drug and the preventive or therapeutic agent for inflammatory diseases according to this embodiment will be described in detail.
　本実施形態による医薬品及び炎症性疾患の予防又は治療剤は、上述した改変タンパク質又はその塩を含む。 The pharmaceutical agent and the preventive or therapeutic agent for inflammatory diseases according to this embodiment include the above-described modified protein or a salt thereof.
　改変タンパク質の塩としては、生理学的に許容される酸（例、無機酸、有機酸）や塩基（例、アルカリ金属塩）などとの塩が用いられ、とりわけ生理学的に許容される酸付加塩が好ましい。このような塩としては、例えば、無機酸（例えば、塩酸、リン酸、臭化水素酸、硫酸）との塩、あるいは有機酸（例えば、酢酸、ギ酸、プロピオン酸、フマル酸、マレイン酸、コハク酸、酒石酸、クエン酸、リンゴ酸、蓚酸、安息香酸、メタンスルホン酸、ベンゼンスルホン酸）との塩などが用いられる。 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.
　本実施形態による炎症性疾患の予防又は治療剤は、前述の改変タンパク質又はその塩を含み、例えば、アトピー性皮膚炎、接触性皮膚炎、かぶれ、乾癬、尋常性天疱瘡等の炎症性疾患に対して予防又は治療効果を奏する。 The preventive or therapeutic agent for inflammatory diseases according to the present embodiment contains the aforementioned modified protein or a salt thereof, for example, for inflammatory diseases such as atopic dermatitis, contact dermatitis, rash, psoriasis, pemphigus vulgaris, etc. It has a preventive or therapeutic effect.
　本実施形態による医薬品は、前述の改変タンパク質又はその塩を含み、所望の医薬用途に用いられ、例えば、前述の炎症性疾患の予防又は治療剤等として用いられ得る。本実施形態による医薬品は、例えば、関節リウマチ、シェーグレン症候群、乾燥性角結膜炎及びそれによるドライアイ、リウマトイド結節、穿孔性強膜軟化症、上強膜及び強膜炎等の眼科疾患；間質性肺炎、閉塞性細気管支炎、胸膜炎、気胸、膿胸、気道病変、胸膜病変、リウマチ結節、血管病変及び睡眠時無呼吸症候群（顎関節病変、輪状披裂関節病変）等の呼吸器系の疾患；心外膜炎、症候性心外膜炎、慢性収縮性心膜炎、弁機能障害、塞栓症、伝導障害、心筋障害、大動脈炎、大動脈弁閉鎖不全及び動脈瘤破裂等の心臓系の疾患；慢性炎症によるＡＡアミロイドーシス、リウマトイド血管炎による虚血性腸炎等の消化管系疾患；関節リウマチに合併するシェーグレン症候群による間質性腎炎、間質性腎病変、蛋白尿、続発性アミロイドーシス、関節リウマチに合併するＡＡアミロイドーシスによる糸球体病変（膜性腎症）等の腎臓系疾患；頚椎変形による脊髄障害、腱滑膜炎による圧迫性神経障害、関節リウマチに合併する血管炎による多発性単神経炎などの神経系疾患；リウマトイド結節、皮膚血管炎（白血球破砕性血管炎）及び虚血性皮膚潰瘍等の皮膚科系の疾患；貧血（小球性低色素性貧血）、脾腫、白血球（好中球のみ）減少をきたしたフェルティー症候群等の血液系の疾患の予防又は治療剤として用いられ得る。また、本実施形態による医薬品は、例えば、膠原病の予防又は治療剤として用いられ得、膠原病には、例えば、全身性エリテマトーデス、全身性強皮症、多発性筋炎・皮膚筋炎、シェーグレン症候群、混合性結合組織病、抗リン脂質抗体症候群、ベーチェット病、アレルギー性肉芽腫性血管炎（チャーグ・ストラウス症候群）、成人スティル病、好酸球性筋膜炎、結節性動脈周囲炎（結節性多発動脈炎・顕微鏡的多発血管炎）、大動脈炎症候群（高安動脈炎）、ウェゲナー肉芽腫症、側頭動脈炎、悪性関節リウマチ等が含まれる。 The pharmaceutical product according to the present embodiment includes the above-described modified protein or a salt thereof and is used for a desired pharmaceutical use, and can be used, for example, as a prophylactic or therapeutic agent for the above-mentioned inflammatory disease. The pharmaceutical product according to the present embodiment includes, for example, ophthalmic diseases such as rheumatoid arthritis, Sjogren's syndrome, dry keratoconjunctivitis and dry eye, rheumatoid nodule, perforated scleral softening, superior sclera and scleritis; Respiratory diseases such as pneumonia, obstructive bronchiolitis, pleurisy, pneumothorax, empyema, respiratory tract lesions, pleural lesions, rheumatoid nodules, vascular lesions and sleep apnea syndrome (temporomandibular joint lesions, ring-shaped hip joint lesions); Cardiovascular diseases such as epidermitis, symptomatic pericarditis, chronic constrictive pericarditis, valve dysfunction, embolism, conduction disorder, myocardial disorder, aortitis, aortic regurgitation and aneurysm rupture; chronic Gastrointestinal diseases such as AA amyloidosis due to inflammation, ischemic enteritis due to rheumatoid vasculitis; interstitial nephritis due to Sjogren's syndrome associated with rheumatoid arthritis, interstitial renal lesions, proteinuria, secondary amyloid Renal diseases such as glomerular lesions (membrane nephropathy) caused by AA amyloidosis associated with cis and rheumatoid arthritis; spinal cord injury due to cervical spine deformity, compression neuropathy due to tendon synovitis, frequent occurrence due to vasculitis associated with rheumatoid arthritis Dermatological diseases such as rheumatoid nodules, dermatovascular inflammation (leukocyte vasculitis) and ischemic skin ulcers; anemia (microcytic hypochromic anemia), splenomegaly, leukocytes (Only for neutrophils) It can be used as a preventive or therapeutic agent for blood system diseases such as Feltie syndrome, which has decreased. The pharmaceutical product according to the present embodiment can be used, for example, as a preventive or therapeutic agent for collagen disease. Examples of collagen disease include systemic lupus erythematosus, systemic scleroderma, polymyositis / dermatomyositis, Sjogren's syndrome, Mixed connective tissue disease, antiphospholipid syndrome, Behcet's disease, allergic granulomatous vasculitis (Chirge-Strauss syndrome), adult Still's disease, eosinophilic fasciitis, nodular periarteritis (nodular polyposis) Arteritis / microscopic polyangiitis), aortitis syndrome (Takanian arteritis), Wegener's granulomatosis, temporal arteritis, malignant rheumatoid arthritis and the like.
　本実施形態による医薬品及び炎症性疾患の予防又は治療剤の投与方法は、経口投与、局所投与、静脈内投与、腹腔内投与、皮内投与、舌下投与等、適宜選択され得る。投与剤型も任意であってよく、例えば、錠剤、顆粒剤、散剤、カプセル剤等の経口用固形製剤、内服液剤、シロップ剤等の経口用液体製剤、注射剤などの非経口用液体製剤等に適宜調製することができる。また、適切なドラッグデリバリーシステム（ＤＤＳ）を用いてもよい。 The administration method of the pharmaceutical agent and the preventive or therapeutic agent for inflammatory diseases according to this embodiment can be appropriately selected from oral administration, local administration, intravenous administration, intraperitoneal administration, intradermal administration, sublingual administration, and the like. 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.
　以上説明したように、保存安定性及び生体内での安定性が高く、疾患に対する予防又は治療効果の高い改変タンパク質、医薬品、炎症性疾患の予防又は治療剤及び改変タンパク質の製造方法を提供することができる。 As described above, to provide a modified protein, a pharmaceutical, a prophylactic or therapeutic agent for inflammatory diseases, and a method for producing the modified protein, which have high storage stability and in vivo stability and are highly effective in preventing or treating diseases. Can do.
　以下、実施例を挙げて本発明を具体的に説明する。ただし、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.
（実施例１）
（ＨＬＡ－Ｇ２部位特異的ＰＥＧ化の条件検討）
　ＰＥＧ化のターゲットタンパク質となるＨＬＡ－Ｇ２タンパク質と、今回用いるＰＥＧ試薬の修飾部位となる４２番目のフリーのシステイン残基（Ｃｙｓ４２）をセリン残基に置換した変異体タンパク質（ＨＬＡ－Ｇ２Ｃ４２Ｓ）と、を作製し、これらのタンパク質のＰＥＧ化進行を比較することで、Ｃｙｓ４２残基に対して部位特異的にＰＥＧ化が進行するかを確認した。ＰＥＧ化部位検討用の変異体タンパク質及び、ＰＥＧ化ＨＬＡ－Ｇ２タンパク質の受容体に対する結合能を評価するために、Ｃ末端のｂｉｒＡタグ特異的ビオチン化標識可能なＬＩＬＲＢ２ｂｉｒＡタンパク質を調製し、結合解析を行った。 Example 1
(Examination of conditions for HLA-G2 site-specific PEGylation)
An HLA-G2 protein as a target protein for PEGylation, and a mutant protein (HLA-G2C42S) in which the 42nd free cysteine residue (Cys42) as a modification site of the PEG reagent used this time is substituted with a serine residue; And the progress of PEGylation of these proteins was compared to confirm whether PEGylation proceeded site-specifically to Cys42 residue. In order to evaluate the binding ability of the mutant protein for PEGylation site investigation and the receptor of the PEGylated HLA-G2 protein to the receptor, a C-terminal birA tag specific biotinylated labelable LILRB2birA protein was prepared and the binding analysis was performed. went.
（各種組換えタンパク質の調製）
　検討に先立ち、以下の通り、各種組換えタンパク質を以下の通り調製及び精製した。 (Preparation of various recombinant proteins)
Prior to examination, various recombinant proteins were prepared and purified as follows.
（ＨＬＡ－Ｇ２組換えタンパク質発現プラスミド）
　シグナル配列を除去し、Ｎ末端に翻訳開始コドンであるメチオニン残基を付加したＨＬＡ－Ｇ２（ＷＴ）の細胞外領域（Ｇｌｙ１－Ｔｒｐ１８２）を大腸菌発現用ベクターｐＧＭ７に組み込み、さらに発現量増強のためＮ末端近傍５残基分の塩基配列を同義置換した、本願の発明者ら保有の発現用プラスミド（ＨＬＡ－Ｇ２－ｐＧＭＴ７）を用いた（図１１（ａ）、塩基配列：配列番号７、アミノ酸配列：配列番号８）。 (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 nucleotide sequence of 5 residues near the N-terminal is synonymously substituted was used (FIG. 11 (a), nucleotide sequence: SEQ ID NO: 7, amino acid) Sequence: SEQ ID NO: 8).
（ＨＬＡ－Ｇ２組換えタンパク質変異体発現プラスミド）
　ＰＥＧ化試薬がＨＬＡ－Ｇ２が保有するフリーのシステイン残基（Ｃｙｓ４２残基）に対して部位特異的に反応することを確認するため、４２番目のシステインをセリンに置換した、当研究保有のＨＬＡ－Ｇ２Ｃ４２Ｓ－ｐＧＭＴ７プラスミドを用いた（図１１（ｂ）、塩基配列：配列番号９、アミノ酸配列：配列番号１０）。ＨＬＡ－Ｇ２Ｃ４２Ｓ組換えタンパク質がＨＬＡ－Ｇ２と同様の分子構造、受容体結合能を維持していることは本願の発明者らが確認している。 (HLA-G2 recombinant protein mutant expression plasmid)
In order to confirm that the PEGylation reagent reacts site-specifically with the free cysteine residue (Cys42 residue) possessed by HLA-G2, the 42nd cysteine was substituted with serine. -The G2C42S-pGMT7 plasmid was used (FIG. 11 (b), base sequence: SEQ ID NO: 9, amino acid sequence: SEQ ID NO: 10). The inventors of the present application have confirmed that the HLA-G2C42S recombinant protein maintains the same molecular structure and receptor binding ability as HLA-G2.
　さらに、システイン残基を標的としたＰＥＧ修飾部位検討のためのシステイン変異体も作製した。ＨＬＡ－Ｇ２が有するフリーのシステインが１か所になるように、ＨＬＡ－Ｇ２Ｃ４２Ｓ－ｐＧＭＴ７プラスミドに新たなシステイン変異を導入した。作製した変異体発現プラスミドは、以下の２つである。
　（１）ＨＬＡ－Ｇの糖鎖修飾部位である８６番目のアスパラギンをシステインに置換したコンストラクト（ＨＬＡ－Ｇ２Ｎ８６Ｃ－ｐＧＭＴ７）（図１１（ｃ）、塩基配列：配列番号１１、アミノ酸配列：配列番号１２）
　（２）Ｃ末端にシステインを付加したコンストラクト（ＨＬＡ－Ｇ２ＣＴＥＲ－ｐＧＭＴ７）（図１１（ｄ）、塩基配列：配列番号１３、アミノ酸配列：配列番号１４） Furthermore, cysteine mutants for PEG modification site targeting cysteine residues were also prepared. A new cysteine mutation was introduced into the HLA-G2C42S-pGMT7 plasmid so that the free cysteine of HLA-G2 was at one place. The produced mutant expression plasmids are the following two.
(1) A construct (HLA-G2N86C-pGMT7) in which the 86th asparagine which is the sugar chain modification site of HLA-G is replaced with cysteine (FIG. 11 (c), nucleotide sequence: SEQ ID NO: 11, amino acid sequence: SEQ ID NO: 12) )
(2) Construct added with cysteine at the C-terminus (HLA-G2CTER-pGMT7) (FIG. 11 (d), base sequence: SEQ ID NO: 13, amino acid sequence: SEQ ID NO: 14)
　上記２種のプラスミドは、制限酵素ＤｐｎＩを用いた変異導入法により作製した。具体的には、ＨＬＡ－Ｇ２Ｃ４２Ｓ－ｐＧＭＴ７をテンプレートとしてＤＮＡ合成酵素ＫＯＤ　Ｐｌｕｓ（東洋紡株式会社）と、以下のプライマーを用いたＰＣＲ反応により、それぞれの変異を導入した。
・変異体作製に用いたプライマー
　（１）ＨＬＡ－Ｇ２－Ｎ８６Ｃ
　Ｎ８６Ｃ＿Ｆ：５’－ＣＧＣＧＧＣＴＡＣＴＡＣＴＧＣＣＡＧＡＧＣＧＡＧＧＣＣ－３’　（配列番号２１）
　Ｎ８６Ｃ＿Ｒ：５’－ＧＣＧＣＣＧＡＴＧＡＴＧＡＣＧＧＴＣＴＣＧＣＴＣＣＧＧ－３’　（配列番号２２）
　（２）ＨＬＡ－Ｇ２－ＣＴＥＲ
　ＣＴＥＲ＿Ｆ：５’－ＴＧＧＡＡＧＣＡＧＴＧＣＧＧＴＴＡＡＡＡＧＣＴＴＧＡＡ－３’　（配列番号２３）
　ＣＴＥＲ＿Ｒ：５’－ＡＣＣＴＴＣＧＴＣＡＣＧＣＣＡＡＴＴＴＴＣＧＡＡＣＴＴ－３’　（配列番号２４） The above two types of plasmids were prepared by a mutagenesis method using the restriction enzyme DpnI. Specifically, each mutation was introduced by PCR reaction using HLA-G2C42S-pGMT7 as a template and DNA synthase KOD Plus (Toyobo Co., Ltd.) and the following primers.
・ Primers used for mutant preparation (1) HLA-G2-N86C
N86C_F: 5′-CGCGGCCTACTACTCCCAGACGGAGGCC-3 ′ (SEQ ID NO: 21)
N86C_R: 5′-GCCGCCGATGATGACGGTCTCGCTCCGG-3 ′ (SEQ ID NO: 22)
(2) HLA-G2-CTER
CTER_F: 5′-TGGAAGCAGTGCGGTTAAAAGCTTGAA-3 ′ (SEQ ID NO: 23)
CTER_R: 5′-ACCCTCGTCACGCCCAATTTCGAACTT-3 ′ (SEQ ID NO: 24)
　ＰＣＲ反応は、Ｖｅｒｉｔｉ（登録商標）サーマルサイクラー（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）を使用し、９４℃で２分熱変性させた後、９８℃で１０秒熱変性、６０℃で３０秒アニーリング、６８℃で４分の伸長反応のサイクルを２０回繰り返した。反応後、ＤｐｎＩ酵素（ＴＯＹＯＢＯ）１μＬを加え、３７℃で１時間インキュベートした。 The PCR reaction was performed using a Veriti (registered trademark) thermal cycler (Applied Biosystems), heat-denatured at 94 ° C. for 2 minutes, heat-denatured at 98 ° C. for 10 seconds, annealed at 60 ° C. for 30 seconds, and heated at 68 ° C. for 4 minutes. The extension reaction cycle was repeated 20 times. After the reaction, 1 μL of DpnI enzyme (TOYOBO) was added and incubated at 37 ° C. for 1 hour.
　得られた変異プラスミドについて、シークエンス法を用いて配列を確認した。具体的には、各プラスミド０．５μＬを大腸菌ＤＨ５αコンピテントセル１００μＬに加え、氷上に約３０分静置した後、４２℃で４５秒間インキュベートして形質転換した。形質転換後の大腸菌を、１００μｇ／ｍＬのアンピシリン含有Ｌｕｒｉａ－Ｂｅｒｔａｎｉ（ＬＢ）寒天培地に播種後、３７℃で一晩培養した。得られたシングルコロニーを、１００μｇ／ｍＬアンピシリン含有２×Ｙｅａｓｔ－ｔｒｙｐｔｏｎｅ（ＹＴ）培地（５ｍＬ）に植菌し、３７℃、１５０ｒｐｍの条件で一晩振盪培養した。培養菌液全量を遠心分離し（１３，２００ｒｐｍ、４℃、１分間）、得られた大腸菌のペレットからプラスミド精製キット（ＱＩＡｐｒｅｐ（登録商標）Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（２５０）、ＱＩＡＧＥＮ）を用いてプラスミドＤＮＡを精製した。精製したプラスミドを、Ｂｉｇ　Ｄｙｅ（登録商標）Ｔｅｒｍｉｎａｔｏｒ　ｖ３．１　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ｋｉｔ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）、Ｔ７　ｐｒｏｍｏｔｏｒ又はＴ７　ｔｅｒｍｉｎａｔｏｒユニバーサルプライマーを用いて、Ｖｅｒｉｔｉ（登録商標）サーマルサイクラーを使用し、９６℃で１分熱変性させた後、９６℃で１０秒熱変性、５０℃で５秒アニーリング、６０℃で４分の伸長反応を３０回繰り返すことによってシークエンス反応を行った。シークエンス反応液をエタノール沈殿し、Ｈｉ－ＤｉＴＭ　ｆｏｒｍａｍｉｄｅ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）を２０μＬ加え、９５℃で２分間インキュベートした後、シークエンサー３１３０　Ｇｅｎｅｔｉｃ　Ａｎａｌｙｚｅｒ　（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）で解析した。 The sequence of the obtained mutant plasmid was confirmed using a sequencing method. Specifically, 0.5 μL of each plasmid was added to 100 μL of E. coli DH5α competent cells, allowed to stand on ice for about 30 minutes, and then incubated at 42 ° C. for 45 seconds for transformation. The transformed Escherichia coli was inoculated on Luria-Bertani (LB) agar medium containing 100 μg / mL ampicillin and cultured at 37 ° C. overnight. The obtained single colony was inoculated into 2 × Yeast-trytone (YT) medium (5 mL) containing 100 μg / mL ampicillin and cultured overnight at 37 ° C. and 150 rpm. The whole culture broth is centrifuged (13,200 rpm, 4 ° C., 1 minute), and plasmid DNA is obtained from the resulting E. coli pellet using a plasmid purification kit (QIAprep (registered trademark) Spin Miniprep kit (250), QIAGEN). Was purified. Using the Big Dye (registered trademark) Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), T7 promoter or T7 terminator universal primer, using the Veriti (registered trademark) thermal cycler at 96 ° C for 1 minute. After heat denaturation, a sequence reaction was performed by repeating 30 times of heat denaturation at 96 ° C. for 10 seconds, annealing at 50 ° C. for 5 seconds, and extension at 60 ° C. for 4 minutes 30 times. The sequence reaction solution was ethanol precipitated, 20 μL of Hi-DiTM formamide (Applied Biosystems) was added, incubated at 95 ° C. for 2 minutes, and then analyzed with a sequencer 3130 Genetic Analyzer (Applied Biosystems).
（ＨＬＡ－Ｇ１組換えタンパク質発現プラスミド）
　ＨＬＡ－Ｇ１単量体調製には、シグナル配列を除去し、Ｎ末端に翻訳開始コドンであるメチオニン残基を付加し、ＨＬＡ－Ｇ１の４２番目のシステインをセリンに置換した細胞外発現領域（Ｇｌｙ１－Ｇｌｎ２７６）をｐＧＭ７ベクターに組み込んだ本願の発明者ら保有の大腸菌発現用プラスミド（ＨＬＡ－Ｇ１Ｃ４２Ｓ－ｐＧＭＴ７）を用いた（図１１（ｅ）、塩基配列：配列番号１５、アミノ酸配列：配列番号１６）。 (HLA-G1 recombinant protein expression plasmid)
For preparation of HLA-G1 monomer, an extracellular expression region (Gly1) in which the signal sequence was removed, a methionine residue as a translation initiation codon was added to the N-terminus, and the 42nd cysteine of HLA-G1 was replaced with serine. The plasmid for expression of E. coli (HLA-G1C42S-pGMT7) possessed by the inventors of the present application in which -Gln276) was incorporated into the pGM7 vector was used (FIG. 11 (e), nucleotide sequence: SEQ ID NO: 15, amino acid sequence: SEQ ID NO: 16). ).
（β２ｍ組換えタンパク質発現プラスミド）
　シグナル配列を除き、Ｎ末端に翻訳開始コドンであるメチオニン残基を付加したＩｇ－ｌｉｋｅドメイン部分（Ｉｌｅ１－Ｍｅｔ９９）をｐＧＭＴ７ベクターに組み込んだ本願の発明者ら保有の大腸菌発現用プラスミド（β２ｍ－ｐＧＭ７）を使用した（図１１（ｆ）、塩基配列：配列番号１７、アミノ酸配列：配列番号１８）。 (Β2m recombinant protein expression plasmid)
The Escherichia coli expression plasmid (β2m-pGM7) possessed by the inventors of the present application, in which the Ig-like domain portion (Ile1-Met99), in which a methionine residue as a translation initiation codon is added to the N terminus except for the signal sequence, is incorporated into the pGMT7 vector. (FIG. 11 (f), base sequence: SEQ ID NO: 17, amino acid sequence: SEQ ID NO: 18).
（ＬＩＬＲＢ２ｂｉｒＡ組換えタンパク質発現プラスミド）
　シグナル配列を除去し、開始コドンであるメチオニンを付加したＬＩＬＲＢ２のリガンド結合に関与する細胞外領域Ｎ末端側の２つのＩｇ－ｌｉｋｅドメイン（Ｇｒｙ１－Ｐｒｏ１９７）をｐＧＭ７ベクターに組み込み、さらにＣ末端に１７アミノ酸残基からなるビオチン化酵素認識配列（ＧＳＬＨＨＩＬＤＡＱＫＭＶＷＮＨＲ（配列番号２５））を付加した、本願の発明者ら保有の大腸菌発現用プラスミド（ＬＩＬＲＢ２ｂｉｒＡ－ｐＧＭ７）を使用した（Ｓｈｉｒｏｉｓｈｉ，Ｍ．，Ｔｓｕｍｏｔｏ，Ｋ．，Ａｍａｎｏ，Ｋ．，Ｓｈｉｒａｋｉｈａｒａ，Ｙ．，Ｃｏｌｏｎｎａ，Ｍ．，Ｂｒａｕｄ，Ｖ．Ｍ．，Ａｌｌａｎ，Ｄ．Ｓ．Ｊ．，Ｍａｋａｄｚａｎｇｅ，Ａ．，Ｒｏｗｌａｎｄ－Ｊｏｎｅｓ，Ｓ．，Ｗｉｌｌｃｏｘ，Ｂ．，Ｊｏｎｅｓ，Ｅ．Ｙ．，ｖａｎ　ｄｅｒ　Ｍｅｒｗｅ，Ｐ．Ａ．，Ｋｕｍａｇａｉ，Ｉ．，ａｎｄ　Ｍａｅｎａｋａ，Ｋ．（２００３）Ｈｕｍａｎ　ｉｎｈｉｂｉｔｏｒｙ　ｒｅｃｅｐｔｏｒｓ　Ｉｇ－ｌｉｋｅ　ｔｒａｎｓｃｒｉｐｔ　２（ＩＬＴ２）ａｎｄ　ＩＬＴ４　ｃｏｍｐｅｔｅ　ｗｉｔｈ　ＣＤ８　ｆｏｒ　ＭＨＣ　ｃｌａｓｓ　Ｉ　ｂｉｎｄｉｎｇ　ａｎｄ　ｂｉｎｄ　ｐｒｅｆｅｒｅｎｔｉａｌｌｙ　ｔｏ　ＨＬＡ－Ｇ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．１００，８８５６－６１）（図１１（ｇ）、塩基配列：配列番号１９、アミノ酸配列：配列番号２０）。 (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 Escherichia 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: 25)) 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. 11 (g), nucleotide sequence: SEQ ID NO: 19, amino acid sequence: SEQ ID NO: 20).
（大腸菌発現系を用いた各種組換えタンパク質の封入体調製）
　本研究で用いた組換えタンパク質はすべて大腸菌を用いて封入体として発現させた。ＨＬＡ－Ｇ１、β２ｍ、ＬＩＬＲＢ２ｂｉｒＡ組換えタンパク質は、前述の各種プラスミドを用いて、大腸菌ＢＬ２１（ＤＥ３）ｐＬｙｓＳ株（Ｎｏｖａｇｅｎ）を形質転換することにより封入体として発現させた。また、ＨＬＡ－Ｇ２、ＨＬＡ－Ｇ２変異体（ＨＬＡ－Ｇ２Ｃ４２Ｓ、ＨＬＡ－Ｇ２Ｎ８６Ｃ、ＨＬＡ－Ｇ２ＣＴＥＲ）組換えタンパク質は、それぞれのプラスミドを用いて大腸菌ＣｌｅａｒＣｏｌｉ（登録商標）ＢＬ２１（ＤＥ３）ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（ＣｌｅａｒＣｏｌｉ（登録商標）ＢＬ２１（ＤＥ３）ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ（Ｌｕｃｉｇｅｎ）を本願の発明者らによりｃｈｅｍｉｃａｌ　ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌに作り替えたもの）を形質転換することにより封入体として発現させた。具体的には、形質転換後、１００μｇ／ｍＬアンピシリン含有ＬＢ寒天培地に播種後、３７℃で一晩培養した。得られたコロニーを１００μｇ／ｍＬアンピシリン含有２×ＹＴ培地（１０ｍＬ）に植菌し、３７℃一晩振盪培養した。１００μｇ／ｍＬ　アンピシリンを含む２×ＹＴ培地１Ｌに前培養した１０ｍＬの菌液を植菌し、３７℃で振盪培養した。対数増殖前期であるＯｐｔｉｃａｌ　Ｄｅｎｓｉｔｙ（ＯＤ）６００＝０．６に達したところで、Ｉｓｏｐｒｏｐｙｌ　β－Ｄ－１－ｔｈｉｏｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ（ＩＰＴＧ）を終濃度１ｍＭとなるように加えて組換えタンパク質の発現を誘導し、その後ＩＮＮＯＶＡ（エッペンドルフ）で１５０ｒｐｍ、３７℃で５時間振盪培養した。培養後の菌液を遠心分離（５０００ｒｐｍ、４℃、１０分間）して得た菌体を懸濁バッファー（５０　ｍＭ　Ｔｒｉｓ　ｈｙｄｒｏｘｙｍｅｔｈｙｌ　ａｍｉｎｏｍｅｔｈａｎｅ［Ｔｒｉｓ］－ＨＣｌ　ｐＨ８．０，１５０ｍＭ　ＮａＣｌ）で懸濁し、氷上で超音波破砕を行った。破砕後、８０００ｒｐｍ、４℃で５分間遠心し、得られた沈殿を封入体として、さらに洗浄バッファー（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０，１５０ｍＭ　ＮａＣｌ，０．５％　Ｔｒｉｔｏｎ　Ｘ－１００）で懸濁、８０００ｒｐｍ、４℃で５分間遠心する洗浄操作を４回繰り返した。その後、封入体から界面活性剤であるＴｒｉｔｏｎ　Ｘ－１００を除去するために、懸濁バッファーを用いて同様の操作を４回繰り返し、封入体を得た。得られた封入体は可溶化バッファー（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０，１００　ｍＭ　ＮａＣｌ，６Ｍ　ｇｕａｎｉｄｉｎｅ－ＨＣｌ，１０ｍＭ　Ｅｔｙｌｅｎｅｄｉａｍｉｎｅ－Ｎ，Ｎ，Ｎ’，Ｎ’，－ｔｅｔｒａａｃｅｔｉｃ　ａｃｉｄ［ＥＤＴＡ］）で一晩４℃静置することによって、完全に溶解させた。可溶化後、５０００×ｇ、４℃で５分間遠心して得られた上清を封入体として－８０℃で保存した。タンパク質発現誘導以降の遠心操作はすべてユニバーサル冷却遠心機を用いて行った（ＫＵＢＯＴＡ）。 (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. HLA-G1, β2m, and LILRB2biaA recombinant proteins were expressed as inclusion bodies by transforming E. coli BL21 (DE3) pLysS strain (Novagen) with the aforementioned various plasmids. In addition, HLA-G2, HLA-G2 mutant (HLA-G2C42S, HLA-G2N86C, HLA-G2CTER) recombinant proteins can be obtained using E. coli ClearColi (registered trademark) BL21 (DE3) competent cell (ClearColi (ClearColi) (Registered trademark) BL21 (DE3) competent cell (Lucigen) was transformed into a chemical competent cell by the inventors of the present application) and transformed into an inclusion body. 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 was centrifuged at 8000 rpm and 4 ° C. for 5 minutes, and the resulting precipitate was 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).
（巻き戻し法による組換えタンパク質の調製）
　ＨＬＡ－Ｇ２、ＨＬＡ－Ｇ２変異体（ＨＬＡ－Ｇ２Ｃ４２Ｓ、ＨＬＡ－Ｇ２Ｎ８６Ｃ、ＨＬＡ－Ｇ２ＣＴＥＲ）、ＨＬＡ－Ｇ１及びＬＩＬＲＢ２ｂｉｒＡ組換えタンパク質は、大腸菌封入体の希釈法による巻き戻しにより調製した。希釈時の最終濃度が１～２μＭ程度になるように、ＨＬＡ－Ｇ２（８ｍｇ）、ＨＬＡ－Ｇ２変異体（８ｍｇ）、ＬＩＬＲＢ２ｂｉｒＡ（４ｍｇ）の各可溶化済み封入体にＤｉｔｈｉｏｔｈｒｅｉｔｏｌ（ＤＴＴ）を終濃度１０ｍＭとなるように加え、室温で１時間インキュベートした。ＤＴＴによる還元処理した封入体変性溶液に、タンパク質の凝集抑制効果を持つアルギニンを含むリフォールディングバッファー（０．１Ｍ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０，１Ｍ　Ｌ－ａｒｇｉｎｉｎｅ－ＨＣｌ，２ｍＭ　ＥＤＴＡ，３．７３ｍＭ　ｃｙｓｔａｍｉｎｅ，６．７３ｍＭ　ｃｙｓｔｅａｍｉｎｅ）を１滴ずつグアニジン濃度が１．５Ｍ（ジスルフィド結合を組んで、２次構造を取ると考えられている濃度）になるまで加えた。さらに、その希釈溶液を２００ｍＬのリフォールディングバッファーに１滴ずつ加えることによってさらに希釈し、４℃で７２時間攪拌した。 (Preparation of recombinant protein by unwinding method)
HLA-G2, HLA-G2 mutants (HLA-G2C42S, HLA-G2N86C, HLA-G2CTER), HLA-G1 and LILRB2biaA recombinant proteins were prepared by unwinding by E. coli inclusion body dilution method. The final concentration of Dithiothreitol (DTT) is added to each of the solubilized inclusion bodies of HLA-G2 (8 mg), HLA-G2 mutant (8 mg), and LILRB2biaA (4 mg) so that the final concentration upon dilution is about 1 to 2 μM. The solution was added to 10 mM and incubated at room temperature 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.
　ＨＬＡ－Ｇ１組換えタンパク質は、ＨＬＡ－Ｇ１重鎖、β２ｍ、ペプチドのヘテロ三量体として巻き戻す必要がある。巻き戻りやすく、単体として安定なユニットから順に巻き戻して複合体として得るために、β２ｍ封入体（４ｍｇ）を上記方法と同様に希釈し、４℃で４～６時間攪拌した。続いて、完全に溶解したＨＬＡ－Ｇ１結合合成ペプチド（ＲＩＩＰＲＨＬＱＬ）０．２　ｍｇ／０．２ｍＬ　ＤＭＳＯをβ２ｍリフォールディング溶液にゆっくり滴下し、さらに４℃で１～２時間攪拌した。最後に、ＨＬＡ－Ｇ１封入体（４ｍｇ）を、β２ｍとペプチドを含むリフォールディング溶液を用いて上記と同様の方法で希釈し、４℃で７２時間攪拌した。 The HLA-G1 recombinant protein needs to be unwound as a heterotrimer of HLA-G1 heavy chain, β2m, and peptide. The β2m inclusion body (4 mg) was diluted in the same manner as described above, and stirred at 4 ° C. for 4 to 6 hours in order to obtain a composite by rewinding from a stable unit as a simple substance. Subsequently, completely dissolved HLA-G1 binding synthetic peptide (RIIPRHLQL) 0.2 mg / 0.2 mL DMSO was slowly added dropwise to the β2m refolding solution, and the mixture was further stirred at 4 ° C. for 1 to 2 hours. Finally, HLA-G1 inclusion bodies (4 mg) were diluted in the same manner as described above using a refolding solution containing β2m and peptide, and stirred at 4 ° C. for 72 hours.
（巻き戻した組換えタンパク質の精製）
　希釈法により巻き戻した各組換えタンパク質は、希釈により容量が増えるため、ＶＩＶＡＦＬＯＷ　ｓｙｓｔｅｍ（ＭＷＣＯ：１００００Ｄａ、Ｓａｒｔｏｒｉｕｓ）を用いた限外ろ過法、必要に応じてその後Ａｍｉｃｏｎ　Ｕｌｔｒａ（ＭＷＣＯ：１００００　Ｄａ、Ｍｅｒｋ　Ｍｉｌｌｉｐｏｒｅ）を用いた限外ろ過法を行ったのちに、Ｍｉｌｌｅｘ－ＧＶ（０．２２μｍ、ＰＶＤＦ、Ｍｅｒｋ　Ｍｉｌｌｉｐｏｒｅ）フィルタを用いて、凝集体などの微粒子を取り除き、ゲルろ過クロマトグラフィー（Ｓｉｚｅ　Ｅｘｃｌｕｓｉｏｎ　Ｃｈｒｏｍａｔｏｇｒａｐｈｙ：ＳＥＣ）による精製を行った。ＳＥＣは、ＡＫＴＡｐｕｒｉｆｉｅｒもしくはＡＫＴＡｐｕｒｅシステム（いずれもＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用い、ランニングバッファーとして２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０，１００ｍＭ　ＮａＣｌを使用した。 (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.
　具体的には、ＨＬＡ－Ｇ２、ＨＬＡ－Ｇ２変異体（ＨＬＡ－Ｇ２Ｃ４２Ｓ、ＨＬＡ－Ｇ２Ｎ８６Ｃ、ＨＬＡ－Ｇ２ＣＴＥＲ）、ＨＬＡ－Ｇ１組換えタンパク質は、ＶＩＶＡＦＬＯＷ　ｓｙｓｔｅｍで１５ｍＬ以下まで濃縮した後、フィルタ処理を行い、Ｈｉｌｏａｄ　２６／６０　Ｓｕｐｅｒｄｅｘ７５　ｐｇ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）カラムを用いて精製した。さらに、ＨＬＡ－Ｇ１タンパク質については、二次精製としてイオン交換クロマトグラフィー（Ｉｏｎ　Ｅｘｃｈａｎｇｅ　Ｃｈｒｏｍａｔｏｇｒａｐｈｙ：　ＩＥＸ）を行った。ＳＥＣで得られた目的のピークフラクションをＡｍｉｃｏｎ　Ｕｌｔｒａ（ＭＷＣＯ：１００００　Ｄａ、Ｍｅｒｋ　Ｍｉｌｌｉｐｏｒｅ）を使用して５ｍＬ以下まで濃縮した後、透析法を用いて２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０に置換した。フィルタ処理後、Ｒｅｓｏｕｒｃｅ　Ｑ　１ｍＬ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）カラムに注入した。ＩＥＸは、２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０，０－０．５Ｍ　ＮａＣｌ／２０　Ｃｏｌｕｍｎ　ｖｏｌｕｍｅ（ＣＶ）の条件で行った。 Specifically, HLA-G2, HLA-G2 mutants (HLA-G2C42S, HLA-G2N86C, HLA-G2CTER) and HLA-G1 recombinant protein were concentrated to 15 mL or less with a VIVAFLOW system and then filtered. It was purified using a Hiload 26/60 Superdex75 pg (GE Healthcare) column. Furthermore, the HLA-G1 protein was subjected to ion exchange chromatography (IEX) as secondary purification. 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).
　ＬＩＬＲＢ２ｂｉｒＡ組換えタンパク質はＶＩＶＡＦＬＯＷ　ｓｙｓｔｅｍ及びＡｍｉｃｏｎ　Ｕｌｔｒａを用いて０．５ｍＬ以下まで濃縮した後、フィルタ処理を行ない、Ｓｕｐｅｒｄｅｘ７５　１０／３００　ＧＬ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）カラムで精製した。ランニングバッファーは２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０，２００ｍＭ　ＮａＣｌを使用した。 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.
（ビオチン化ＬＩＬＲＢ２の調製）
　ＳＥＣ法により精製したＬＩＬＲＢ２ｂｉｒＡを、体積比でＬＩＬＲＢ２ｂｉｒＡ：５×ＢｉｏｍｉｘＡ　ｂｕｆｆｅｒ（０．２５Ｍ　ｂｉｃｉｎｅ　ｂｕｆｆｅｒ　ｐＨ８．３）：５×ＢｉｏｍｉｘＢ　ｂｕｆｆｅｒ（５０ｍＭ　Ａｄｅｎｏｓｉｎｅ　ｔｒｉｐｈｏｓｐｈａｔｅ［ＡＴＰ］，５０ｍＭ　ＭｇＯＡｃ，２５０μＭ　ｄ－ｂｉｏｔｉｎ）＝３：１：１になるように混合して、ＢｉｒＡ酵素１μＬ（本願の発明者らが調製したもの）を加え、３０℃で１時間インキュベートした。その後未反応のビオチンを取り除くために、Ｓｕｐｅｒｄｅｘ　７５　１０／３００　ＧＬ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）カラムでＳＥＣ精製を行った。ランニングバッファーは２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０，４００ｍＭ　ＮａＣｌを用いた。 (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.
　上記の通り調製したＨＬＡ－Ｇ２タンパク質をＳＥＣ精製した結果、これまでの知見と一致して、目的の分子量（ＨＬＡ－Ｇ２：２２ｋＤａ）の約２倍の分子量（４４ｋＤａ）相当の位置に溶出ピークが得られた（図２（ａ））。 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. 2 (a)).
（ＨＬＡ－Ｇ２のＣｙｓ４２残基特異的なＰＥＧ化反応）
　上記の通り調製したＨＬＡ－Ｇ２タンパク質を用いて、ＰＥＧ化反応を試みた。ＰＥＧ化試薬は、高純度直鎖ＰＥＧであるＭＥ－４００ＭＡ（ＭＷ：４２，６５３Ｄａ）（ＰＥＧ４０）、ＭＥ－２００ＭＡＯＢ（ＭＷ：２０，８４１Ｄａ）（ＰＥＧ２０）、ＭＥ－１００ＭＡ（ＭＷ：１０，３０３Ｄａ）（ＰＥＧ１０）、ＭＥ－０５０ＭＡ（ＭＷ：５，３９３Ｄａ）（ＰＥＧ５）（いずれも日油株式会社）を用いた。これらの試薬は反応基としてマレイミド基を持ち、チオール基を認識して反応する。 (PEGylation reaction specific to Cys42 residue of HLA-G2)
A PEGylation reaction was attempted 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.
（還元剤）
　ＰＥＧ化反応前に、還元処理によってジスルフィド結合を切断し、ＰＥＧ化ターゲットとなるＣｙｓ４２残基のチオール基を再度露出させ、反応効率を向上させるために、還元剤を添加した。 (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.
　精製したＨＬＡ－Ｇ２タンパク質について、Ａｍｉｃｏｎ　Ｕｌｔｒａ（ＭＷＣＯ：１００００Ｄａ、Ｍｅｒｋ　Ｍｉｌｌｉｐｏｒｅ）を用いた限外ろ過による濃縮、及びＰＥＧ化バッファー（１×Ｐｈｏｓｐｈａｔｅ－Ｂｕｆｆｅｒｄ　Ｓａｌｉｎｅ（ＰＢＳ），　５ｍＭ　ＥＤＴＡ）への置換を行い０．５ｍＬに調製した溶液を、嫌気条件下でＰＥＧ化反応を進行させるために、アスピレーターを用いて１時間脱気処理を行った。続いて、還元剤ｔｒｉｓ（２－ｃａｒｂｏｘｙｅｔｈｙｌ）ｐｈｏｓｐｈｉｎｅ（ＴＣＥＰ）を終濃度０．１、０．５、１、５、１０ｍＭとなるように加えたあと、ＰＥＧ２０を加え、４℃で一晩反応させた。還元剤として、無臭で使いやすく、マレイミドとシステインを用いた反応時に多用されるｔｒｉｓ（２－ｃａｒｂｏｘｙｅｔｈｙｌ）ｐｈｏｓｐｈｉｎｅ（ＴＣＥＰ）を用いた。 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.
　ＰＥＧ化反応の進行は、反応液を非還元条件でＳｏｄｉｕｍ　Ｄｏｄｅｃｙｌ　Ｓｕｌｆａｔｅ－Ｐｏｌｙ　Ａｃｒｙｌａｍｉｄｅ　Ｇｅｌ　Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ（ＳＤＳ－ＰＡＧＥ）（１２．５％　アクリルアミドゲル、３０ｍＡ／枚、７０分間泳動）し、Ｃｏｏｍａｓｓｉｅ　Ｂｒｉｌｌｉａｎｔ　Ｂｌｕｅ（ＣＢＢ）染色、及び、ＰＥＧ分子を検出するＢａｒｉｕｍ　Ｉｏｄｉｄｅ（ＢａＩ２）染色により確認した。ＢａＩ２染色は、泳動後のアクリルアミドゲルを５％　ＢａＩ２溶液（１５分間）、イオン交換水（３０分間）、０．１Ｍヨウ素溶液（５分間）の順に浸して振盪する、という手順で行った。 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. BaI 2 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. .
　ＰＥＧ化ＨＬＡ－Ｇ２の精製はＳｕｐｅｒｄｅｘ　２００　１０／３００　ＧＬ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）、Ｓｕｐｅｒｄｅｘ　７５　１０／３００　ＧＬ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）、あるいはＳｕｐｅｒｏｓｅ６　１０／３００　ＧＬ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）カラムを用いたＳＥＣにより行った。ＰＥＧ化反応液を、Ａｍｉｃｏｎ　Ｕｌｔｒａ（ＭＷＣＯ：１００００Ｄａ、Ｍｅｒｋ　Ｍｉｌｌｉｐｏｒｅ）を使用してＳＥＣランニングバッファー（２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０，１００ｍＭ　ＮａＣｌ）へ置換し、０．５ｍＬまで濃縮してカラムへ打ち込んだ。ＳＥＣには、ＡＫＴＡｐｕｒｉｆｉｅｒシステム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用いた。精製度は、各フラクションサンプルを非還元条件でＳＤＳ－ＰＡＧＥにより展開し、銀染色（２Ｄ－ＳＩＬＶＥＲ　ＳＴＡＩＮ　ＲＥＡＧＥＮＴ　ＩＩ、コスモ・バイオ株式会社）で検出して確認した。 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.).
　反応液を上記の通りＳＤＳ－ＰＡＧＥで展開し、ＣＢＢ染色したところ、目的のＰＥＧ化タンパク質のバンドを確認することができた（図２（ｂ））。以上の結果からＴＣＥＰを加えることにより反応効率は大きく向上すること、さらに、ＴＣＥＰ終濃度が０．１ｍＭの条件で最も高い反応効率が得られることがわかった。そのため、ＰＥＧ化反応は０．１ｍＭ　ＴＣＥＰ処理をした条件下で行うこととした。 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. 2 (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, the PEGylation reaction was carried out under the condition of 0.1 mM TCEP treatment.
　なお、ＰＥＧがターゲットとしているＨＬＡ－Ｇ２タンパク質のＣｙｓ４２残基特異的に結合しているかを確認するために、システイン残基をセリン残基に置換した変異体（ＨＬＡ－Ｇ２Ｃ４２Ｓ、図１１（ｂ））組換えタンパク質を調製し、ＨＬＡ－Ｇ２タンパク質と比較した。ＨＬＡ－Ｇ２Ｃ４２Ｓタンパク質はＨＬＡ－Ｇ２と同様に調製し、目的のピークフラクションを回収してＰＥＧ化反応に用いた。ＨＬＡ－Ｇ２Ｃ４２Ｓはフリーのシステイン残基を失ったため、多量体形成は起こらず、非還元ＳＤＳ－ＰＡＧＥでも多量体形成を示唆する高分子量の位置にバンドは認められなかった（図示せず）。 In order to confirm whether PEG is specifically bound to the Cys42 residue of the target HLA-G2 protein, a mutant in which a cysteine residue is substituted with a serine residue (HLA-G2C42S, FIG. 11 (b) ) Recombinant protein was prepared and compared with HLA-G2 protein. HLA-G2C42S protein was prepared in the same manner as HLA-G2, and the target peak fraction was collected and used for the PEGylation reaction. Since HLA-G2C42S lost the free cysteine residue, multimer formation did not occur, and no band was observed at a high molecular weight position suggesting multimer formation even in non-reduced SDS-PAGE (not shown).
　以上、ＰＥＧ化反応時の種々の条件検討を行った結果から、ＰＥＧ化反応条件を以下のように確定した。以後のＰＥＧ化反応はすべてこの反応条件で行った。
　（１）ＰＥＧ化反応前の脱気処理：調製したＨＬＡ－Ｇ２溶液についてアスピレーター脱気（１時間、氷上）を行う。
　（２）ＰＥＧ化反応時の還元剤の添加：アスピレーター脱気処理後、ＰＥＧ試薬を加える前にＴＣＥＰを終濃度０．１ｍＭとなるようにタンパク質溶液に加える。
　（３）ＰＥＧ試薬量：ＰＥＧ：ＨＬＡ－Ｇ２＝１０：１（モル比）となるように、脱気処理及び還元剤処理後のＨＬＡ－Ｇ２溶液にＰＥＧ試薬を加える。 As described above, from the results of examination of various conditions during the PEGylation reaction, the PEGylation reaction conditions were determined as follows. All subsequent PEGylation reactions were carried out under these reaction conditions.
(1) Degassing treatment before PEGylation reaction: The prepared HLA-G2 solution is subjected to aspirator degassing (1 hour on ice).
(2) Addition of reducing agent during PEGylation reaction: After aspirator deaeration treatment, TCEP is added to the protein solution to a final concentration of 0.1 mM before adding the PEG reagent.
(3) The PEG reagent is added to the HLA-G2 solution after the degassing treatment and the reducing agent treatment so that the amount of PEG reagent is PEG: HLA-G2 = 10: 1 (molar ratio).
（ＰＥＧ分子量の検討）
　続いて、結合させるＰＥＧの分子量によって、ＰＥＧ化反応効率、ＰＥＧ化タンパク質の精製度が異なるかどうかを検証するため、反応基としてマレイミドを持つ４種類の分子量（５、１０、２０、４０ｋＤａ）の分子量のＰＥＧ化試薬（ＰＥＧ５、ＰＥＧ１０、ＰＥＧ２０、ＰＥＧ４０）（前述）を用いてＨＬＡ－Ｇ２のＰＥＧ化を行った。得られたＰＥＧ化体は以下の通りである。
　・ＰＥＧ５－ＨＬＡ－Ｇ２（ＨＬＡ－Ｇ２をＰＥＧ５でＰＥＧ化したもの）
　・ＰＥＧ１０－ＨＬＡ－Ｇ２（ＨＬＡ－Ｇ２をＰＥＧ１０でＰＥＧ化したもの）
　・ＰＥＧ２０－ＨＬＡ－Ｇ２（ＨＬＡ－Ｇ２をＰＥＧ２０でＰＥＧ化したもの）
　・ＰＥＧ４０－ＨＬＡ－Ｇ２（ＨＬＡ－Ｇ２をＰＥＧ４０でＰＥＧ化したもの） (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)
　ＰＥＧ化した反応液についてＳＤＳ－ＰＡＧＥで展開後、ＣＢＢ染色及びＢａＩ２染色し反応の進行を確認したところ、４種類のＰＥＧ化タンパク質すべてを確認できた（図３（ａ）、（ｂ））。また、ＰＥＧ化タンパク質のバンドの濃さから、ＰＥＧ化反応率については低分子量のＰＥＧ（ＰＥＧ５、ＰＥＧ１０）のほうが高いことが明らかとなった。 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 could be confirmed (FIGS. 3A and 3B). . 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).
　これらのＰＥＧ化ＨＬＡ－Ｇ２を精製する方法として、ＰＥＧ化前後の分子量差を利用して分離するＳＥＣを選択した。４種類のＰＥＧ化反応液についてＳＥＣ精製を行ない、得られたピークフラクションについてＳＤＳ－ＰＡＧＥ後、銀染色によって精製度の確認を行ったところ、ＳＥＣによって、ＰＥＧ１０－ＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２については目的のＰＥＧ化ＨＬＡ－Ｇ２を未反応のＨＬＡ－Ｇ２タンパク質と分離して精製することができた（図４（ｂ）、（ｃ））。 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. 4B and 4C).
（受容体ＬＩＬＲＢ２との結合実験）
　続いて、ＰＥＧ化タンパク質として精製できたＰＥＧ１０－ＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２、並びにＰＥＧ化タンパク質をメインに含むＳＥＣピークとして得られたＰＥＧ５－ＨＬＡ－Ｇ２のＳＥＣ精製後サンプルを用いて、Ｓｕｒｆａｃｅ　Ｐｌａｓｍｏｎ　Ｒｅｓｏｎａｎｃｅ（ＳＰＲ）による受容体ＬＩＬＲＢ２との相互作用解析を行い、各分子量のＰＥＧが結合したＨＬＡ－Ｇ２が受容体結合能を維持しているかを確認した。比較対照としてＰＥＧ化していないＨＬＡ－Ｇ２を用いた。 (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. Non-PEGylated HLA-G2 was used as a comparative control.
　ＬＩＬＲＢ２は、上述の通り、Ｃ末端側にビオチン化タグが付加されている精製ＬＩＬＲＢ２ｂｉｒＡタンパク質を用いて、酵素による部位特異的ビオチン化後、ＳＥＣ精製によりビオチン化ＬＩＬＲＢ２として調製した。 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.
　アナライトとして、２倍ずつ連続希釈したＨＬＡ－Ｇ２（０．２～０．７μＭ）、ＰＥＧ５－ＨＬＡ－Ｇ２（０．４～１．６μＭ）、ＰＥＧ１０－ＨＬＡ－Ｇ２（０．３～１．１μＭ）、ＰＥＧ２０－ＨＬＡ－Ｇ２（０．３～１．１μＭ）を流した。 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).
（Ｓｕｒｆａｃｅ　Ｐｌａｓｍｏｎ　Ｒｅｓｏｎａｎｃｅ（ＳＰＲ）による相互作用解析）
　ＰＥＧ化ＨＬＡ－Ｇ２のＬＩＬＲＢ２結合への影響を確認するために、ＳＰＲ法による相互作用解析をＢｉａｃｏｒｅ３０００（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用いて行った。センサーチップ上にはビオチン化ＬＩＬＲＢ２及びコントロールとしてビオチン化ＢＳＡをＢｉｏｔｉｎ　ＣＡＰｔｕｒｅ　Ｋｉｔ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を使用して、プロトコル通りにセンサーチップＣＡＰに固定化した。センサーチップＣＡＰ上には一本鎖ＤＮＡが固定化されており（図１２（ａ））、そこへストレプトアビジンが付加した相補鎖ＤＮＡをハイブリダイゼーションさせることによってストレプトアビジンをチップ上に固定（図１２（ｂ））、さらにストレプトアビジンとビオチンの相互作用を利用してビオチン化ＬＩＬＲＢ２及びビオチン化ＢＳＡを２００ＲＵ～５００ＲＵの固定化量で固定化した（図１２（ｃ））。 (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. 12 (a)). Streptavidin is immobilized on the chip by hybridization with complementary strand DNA added with streptavidin (FIG. 12). (B)) Further, 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. 12 (c)).
　アナライトとしては、Ａｍｉｃｏｎ　Ｕｌｔｒａ（ＭＷＣＯ：１００００、Ｍｉｌｌｉｐｏｒｅ）を用いた限外ろ過によりランニングバッファーであるＨＢＳ－ＥＰバッファー（１０ｍＭ　Ｎａ－ＨＥＰＥＳ　ｐＨ７．４、１５０ｍＭ　ＮａＣｌ、３ｍＭ　ＥＤＴＡ、０．００５％（ｖ／ｖ）Ｓｕｒｆａｃｔａｎｔ　Ｐ２０：ＧＥ　Ｈｅａｌｔｈｃａｒｅ）に置換したＨＬＡ－Ｇ２、ＰＥＧ化ＨＬＡ－Ｇ２各タンパク質溶液について、３段階に２倍ずつ段階希釈したサンプルを、低濃度のものから順番に流速１０μＬ／ｍｉｎで流した。測定温度は２５℃、結合時間及び解離時間はそれぞれ１２０秒でカイネティクス測定を行った。解析にはＢＩＡｅｖａｌｕａｔｉｏｎ　ｖｅｒｓｉｏｎ：４．１．１（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用いた。 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.
　結合解析の結果、３種類すべてのＰＥＧ化体についてＬＩＬＲＢ２との結合を確認することができた（図５（ｂ）－（ｄ））。 As a result of the binding analysis, it was confirmed that all three types of PEGylated products were bound to LILRB2 (FIGS. 5B to 5D).
　以上の結果から、３種類の分子量でＰＥＧ化効率及び受容体との結合能に大きな差は見られないことから、ＰＥＧ化タンパク質の精製度が高く、さらにＰＥＧ化タンパク質の安定性に寄与すると考えられるＰＥＧ分子量が３つのうちで最も大きい２０ｋＤａのＰＥＧを用いて今後のＰＥＧ化反応、及び解析を行うこととした。 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 future PEGylation reaction and analysis were performed using 20 kDa PEG having the largest PEG molecular weight.
（ＰＥＧ導入部位の検討）
　これまで、ＨＬＡ－Ｇ２タンパク質が本来保有するＣｙｓ４２残基をターゲットとしてＰＥＧを導入してきたが、ＰＥＧの導入箇所を変えることにより反応性や機能性が向上することを期待して、ＰＥＧ化する部位の検討を行った。 (Examination of PEG introduction site)
So far, PEG has been introduced targeting the Cys42 residue originally possessed by the HLA-G2 protein, but the site to be PEGylated is expected to improve the reactivity and functionality by changing the PEG introduction site. Was examined.
　ＨＬＡ－Ｇ２分子上のＬＩＬＲＢ２結合領域が明らかになっていないため、膜結合型ＨＬＡ－Ｇ２で細胞膜側に位置するＨＬＡ－Ｇ２タンパク質のＣ末端にシステイン残基を付加したコンストラクト（ＨＬＡ－Ｇ２ＣＴＥＲ）、及びＨＬＡ－Ｇ１の糖鎖付加サイトであるために、ＨＬＡ－Ｇ２においても糖鎖が付加していると予測される８６番目のアスパラギン残基をシステイン残基に置換したコンストラクト（ＨＬＡ－Ｇ２Ｎ８６Ｃ）を作製し、タンパク質を調製した。これらの場所へのＰＥＧ導入は、受容体への結合に影響しないと考えた。 Since the LILRB2 binding region on the HLA-G2 molecule has not been clarified, a construct (HLA-G2CTER) in which a cysteine residue is added to the C-terminus of the HLA-G2 protein located on the cell membrane side in membrane-bound HLA-G2. And the HLA-G1 glycosylation site, a construct (HLA-G2N86C) in which the 86th asparagine residue, which is predicted to have a sugar chain added to HLA-G2, is substituted with a cysteine residue. Prepared and prepared protein. It was considered that introduction of PEG at these locations did not affect binding to the receptor.
　これらの変異体タンパク質において、ＨＬＡ－Ｇ２タンパク質が本来保有するフリーのシステイン残基（Ｃｙｓ４２残基）はセリン残基に置換させており、ＰＥＧ分子は、これらの変異部位１か所のみに導入されると予想される。 In these mutant proteins, the free cysteine residue (Cys42 residue) originally possessed by the HLA-G2 protein is substituted with a serine residue, and the PEG molecule is introduced only at one of these mutation sites. It is expected.
　各ＨＬＡ－Ｇ２変異体タンパク質を、前述の通り、大腸菌を用いて封入体として発現させた。さらに、前述の通り、各タンパク質を巻き戻し後、ＳＥＣ精製を行い、各ＨＬＡ－Ｇ２変異体の溶出画分を回収した。 Each HLA-G2 mutant protein was expressed as inclusion bodies using E. coli as described above. Furthermore, as described above, after unwinding each protein, SEC purification was performed, and the eluted fraction of each HLA-G2 mutant was collected.
　続いて、精製した各変異体タンパク質について、前述同様にＰＥＧ２０によってＰＥＧ化を行った。ＰＥＧの導入箇所を変えたいずれのＨＬＡ－Ｇ２変異体タンパク質についても、ＰＥＧ化反応溶液のＳＤＳ－ＰＡＧＥ、ＣＢＢ及びＢａＩ２染色の結果から、ＰＥＧ化反応の進行が確認できた（図６（ａ）、（ｂ））。 Subsequently, each of the purified mutant proteins was PEGylated with PEG20 as described above. For any HLA-G2 mutant protein in which the PEG introduction site was changed, the progress of the PEGylation reaction could be confirmed from the results of SDS-PAGE, CBB and BaI 2 staining of the PEGylation reaction solution (FIG. 6 (a ), (B)).
（実施例２）
（ＰＥＧ化ＨＬＡ－Ｇ２のｉｎ　ｖｉｔｒｏでの安定性評価）
　実施例１で得られたＰＥＧ２０－ＨＬＡ－Ｇ２を用いて、ｉｎ　ｖｉｔｒｏでの安定性を評価した。 (Example 2)
(In vitro stability evaluation of PEGylated HLA-G2)
Using PEG20-HLA-G2 obtained in Example 1, in vitro stability was evaluated.
（凍結乾燥に対する安定性評価）
　バイオ医薬品は、酸素、日光、ｐＨなどの外的要因による影響を避けるために、製造されてから実際に使用されるまで、凍結乾燥した状態で保存されるのが一般的である。そのため凍結乾燥した医薬品が再溶解後も分解、凝集せず、生物活性を維持しているかどうかは、バイオ医薬品を製品化する上で非常に重要な指標となる。そこで本研究では、ＰＥＧ化により凍結乾燥に対する安定性が向上するかどうかの検証を行った。 (Stability evaluation against freeze-drying)
In order to avoid the influence of external factors such as oxygen, sunlight, pH, etc., biopharmaceuticals are generally stored in a lyophilized state from the time they are manufactured until they are actually used. Therefore, whether or not a freeze-dried drug product does not decompose or aggregate after re-dissolution and maintains biological activity is a very important index for commercializing a biopharmaceutical product. In this study, we examined whether PEGylation improves the stability against freeze-drying.
　ＳＥＣ精製したＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２を凍結乾燥し、滅菌水に再溶解した後、ＳＤＳ－ＰＡＧＥを行った。より具体的には、ＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２（０．１５ｍｇ／ｍＬ、ＨＢＳ－ＥＰバッファー）各５０μＬを、凍結乾燥器を用いて凍結乾燥した。凍結乾燥直後に、当量（５０μＬ）の滅菌水に再溶解し、非還元条件でＳＤＳ－ＰＡＧＥを行い、ＣＢＢ染色で検出することにより凍結乾燥前後のタンパク質の状態を比較した。 SEC-purified HLA-G2 and PEG20-HLA-G2 were lyophilized, redissolved in sterile water, and then subjected to SDS-PAGE. More specifically, 50 μL each of HLA-G2 and PEG20-HLA-G2 (0.15 mg / mL, HBS-EP buffer) were lyophilized using a lyophilizer. Immediately after lyophilization, the protein state before and after lyophilization was compared by re-dissolving in an equivalent amount (50 μL) of sterile water, performing SDS-PAGE under non-reducing conditions, and detecting by CBB staining.
　凍結乾燥処理有無で、ＨＬＡ－Ｇ２分子としての安定性の指標となる凝集・分解の程度をバンドパターンとして比較した。その結果、ＰＥＧ化の有無に関わらず、凍結乾燥処理によるＨＬＡ－Ｇ２分子の凝集や分解は起きておらず、明らかな差は認められなかった（図７（ａ））。 The degree of aggregation / decomposition, which is an indicator of stability as an HLA-G2 molecule, with and without lyophilization treatment was compared as a band pattern. As a result, regardless of the presence or absence of PEGylation, no aggregation or degradation of HLA-G2 molecules occurred by freeze-drying treatment, and no clear difference was observed (FIG. 7 (a)).
　続いて、凍結乾燥処理により、ＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２の受容体結合活性が変化するか、またＰＥＧ化による程度の差があるかどうかを検証するため、上記と同様に凍結乾燥処理したＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２について、ＳＰＲ法を用いて受容体ＬＩＬＲＢ２との結合実験を行った。前述同様に、ビオチン化ＬＩＬＲＢ２をセンサーチップにストレプトアビジンを介して固定化し、そこへ２倍段階希釈（ＨＬＡ－Ｇ２：０．３～１．１μＭ、ＰＥＧ２０－ＨＬＡ－Ｇ２：０．３～１．１μＭ）した凍結乾燥処理済みＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２をアナライトとして流した。 Subsequently, in order to verify whether the receptor binding activities of HLA-G2 and PEG20-HLA-G2 are changed by lyophilization treatment, and whether there is a difference in the degree due to PEGylation, lyophilization treatment is performed in the same manner as described above. The HLA-G2 and PEG20-HLA-G2 were subjected to binding experiments with the receptor LILRB2 using the SPR method. As described above, biotinylated LILRB2 was immobilized on the sensor chip via streptavidin and serially diluted 2-fold (HLA-G2: 0.3 to 1.1 μM, PEG20-HLA-G2: 0.3 to 1.. 1 μM) lyophilized HLA-G2 and PEG20-HLA-G2 were run as analytes.
　まず、凍結乾燥処理により受容体との結合能を失った分子の割合をＨＬＡ－Ｇ２とＰＥＧ２０－ＨＬＡ－Ｇ２との間で比較するため、今回の結合実験で得られたセンサグラムを、凍結乾燥処理していないＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２を用いてＬＩＬＲＢ２との結合実験を行った結果のセンサグラム（図５（ａ）、（ｄ））と比較した。両結合実験において、ＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２は同じロットのサンプルを使用した。ＬＩＬＲＢ２固定化量を両者で一致させた。 First, in order to compare the proportion of molecules that have lost the receptor-binding ability by lyophilization treatment between HLA-G2 and PEG20-HLA-G2, the sensorgrams obtained in this binding experiment were lyophilized. The results were compared with the sensorgrams (FIGS. 5 (a) and (d)) of the results of binding experiments with LILRB2 using untreated HLA-G2 and PEG20-HLA-G2. In both binding experiments, HLA-G2 and PEG20-HLA-G2 used the same lot of sample. The amount of LILRB2 immobilized was matched between the two.
　サンプル濃度０．６μＭ付近で流した時のセンサグラムに着目すると、ＨＬＡ－Ｇ２ではＬＩＬＲＢ２に対する結合レスポンス値が凍結乾燥前後で約２５０ＲＵから約１５０　ＲＵに低下している（図７（ｂ））のに対し、ＰＥＧ２０－ＨＬＡ－Ｇ２では大きなレスポンスの低下は見られなかった（図７（ｃ））。 Paying attention to the sensorgram when the sample concentration is about 0.6 μM, the binding response value for LILRB2 in HLA-G2 decreases from about 250 RU to about 150 RU before and after lyophilization (FIG. 7 (b)). In contrast, PEG20-HLA-G2 did not show a significant decrease in response (FIG. 7 (c)).
　この結果から、ＰＥＧ２０－ＨＬＡ－Ｇ２ではＨＬＡ－Ｇ２と比較して、凍結乾燥後も受容体との結合活性を保持している分子の割合が多いことがわかった。以上より、ＨＬＡ－Ｇ２タンパク質は、ＰＥＧ化することにより凍結乾燥に対する安定性は向上することが示唆された。 From this result, it was found that PEG20-HLA-G2 has a higher proportion of molecules that retain the binding activity to the receptor even after lyophilization compared to HLA-G2. From the above, it was suggested that the stability to lyophilization is improved by PEGylating the HLA-G2 protein.
（熱安定性の評価）
　ＰＥＧ化によりＨＬＡ－Ｇ２タンパク質の熱安定性が向上するかどうかを検証するため、精製したＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２を、５０℃、６０℃条件下でインキュベートした。インキュベーション開始時を０時間として、７、２４、４８時間後に回収し、ＳＤＳ－ＰＡＧＥ　Ｓａｍｐｌｅ　ｂｕｆｆｅｒを加えたのち、４℃で保存した。全てのサンプルを回収後、還元条件下あるいは非還元条件下でＳＤＳ－ＰＡＧＥを行ない、タンパク質分解・凝集の程度を比較した。 (Evaluation of thermal stability)
In order to verify whether PEGylation improves the thermal stability of the HLA-G2 protein, purified HLA-G2 and PEG20-HLA-G2 were incubated at 50 ° C. and 60 ° C. conditions. It was collected after 7, 24, and 48 hours with the start of incubation as 0 hour, and after adding SDS-PAGE Sample buffer, it was stored at 4 ° C. After all samples were collected, SDS-PAGE was performed under reducing or non-reducing conditions to compare the degree of proteolysis and aggregation.
　より具体的には、ＳＥＣ精製後のＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２（０．０８ｍｇ／ｍＬ、２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０、１００ｍＭ　ＮａＣｌ）を５０、６０、７０℃でインキュベートし、それぞれ７、２４、４８時間後に回収したサンプルについて５×ＳＤＳ－ＰＡＧＥ　Ｓａｍｐｌｅ　ｂｕｆｆｅｒ（２５ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ６．５、５％　ｇｌｙｃｅｒｏｌ、１％　ＳＤＳ、０．０５％　ｂｒｏｍｏｐｈｅｎｏｌ　ｂｌｕｅ）を加え、４℃で保存した。全てのサンプルを回収した後、非還元条件でＳＤＳ－ＰＡＧＥを行い（１２．５％又は１５％アクリルアミドゲル、３０ｍＡ／枚、７０分間泳動）、ＣＢＢ染色で検出することによって、タンパク質の分解度及び多量体・凝集体形成度を比較した。 More specifically, HLA-G2 and PEG20-HLA-G2 (0.08 mg / mL, 20 mM Tris-HCl pH 8.0, 100 mM NaCl) after SEC purification were incubated at 50, 60, and 70 ° C., respectively. Samples collected after 24 and 48 hours were added with 5 × SDS-PAGE Sample buffer (25 mM Tris-HCl pH 6.5, 5% glycerol, 1% SDS, 0.05% bromophenol blue) and stored at 4 ° C. After all samples are collected, SDS-PAGE is performed under non-reducing conditions (12.5% or 15% acrylamide gel, 30 mA / plate, run for 70 minutes) and detected by CBB staining. The degree of multimer / aggregate formation was compared.
　非還元条件で泳動した結果から、ＨＬＡ－Ｇ２は高温条件下において目的物のバンドが消失し、４０ｋＤａ以上の凝集体及び１２ｋＤａ周辺の分解産物とみられるバンドが増える（図８（ａ））のに対し、ＰＥＧ２０－ＨＬＡ－Ｇ２では高温条件下でも目的物のバンドの消失及び目的物以外のバンドの出現は少なかった（図８（ｂ））。 From the results of electrophoresis under non-reducing conditions, the target band of HLA-G2 disappears under high temperature conditions, and the bands that are considered to be aggregates of 40 kDa or more and degradation products around 12 kDa increase (FIG. 8 (a)). On the other hand, with PEG20-HLA-G2, the disappearance of the target band and the appearance of bands other than the target were small even under high temperature conditions (FIG. 8 (b)).
　以上の結果から、ＰＥＧ化は、高温条件下で引き起こされるタンパク質凝集及び分解を抑制することを見出した。 From the above results, it was found that PEGylation suppresses protein aggregation and degradation caused under high temperature conditions.
（血清中安定性評価）
　調製したＰＥＧ２０－ＨＬＡ－Ｇ２について、ｉｎ　ｖｉｔｒｏの系において、血清（ＦＢＳ）中でのＨＬＡ－Ｇ２の分解又は凝集がＰＥＧ化により抑制されるかどうかを調べた。 (Evaluation of serum stability)
Regarding the prepared PEG20-HLA-G2, whether or not the degradation or aggregation of HLA-G2 in serum (FBS) is suppressed by PEGylation in an in vitro system was examined.
　精製したＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２を、５％ＦＢＳ、３７℃条件下でインキュベートした。インキュベーション開始時を０時間として、２４時間後より１時間ごとにサンプルを回収し、ＳＤＳ－ＰＡＧＥ　Ｓａｍｐｌｅ　ｂｕｆｆｅｒを加えたのち、４℃で保存した。全てのサンプルを回収後、非還元条件下でＳＤＳ－ＰＡＧＥ、ウエスタンブロッティング法を用いてＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２残存量を定量した。 Purified HLA-G2 and PEG20-HLA-G2 were incubated under conditions of 5% FBS and 37 ° C. Samples were collected every hour from 24 hours after the start of incubation as 0 hours, and SDS-PAGE Sample buffer was added, followed by storage at 4 ° C. After collecting all the samples, the remaining amounts of HLA-G2 and PEG20-HLA-G2 were quantified using SDS-PAGE and Western blotting under non-reducing conditions.
　より具体的には、ＳＥＣ精製後のＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２（ＨＬＡ－Ｇ２：０．３ｍｇ／ｍＬ、ＰＥＧ２０－ＨＬＡ－Ｇ２：０．１５ｍｇ／ｍＬ、２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０、１００ｍＭ　ＮａＣｌ）を１０％のＦｅｔａｌ　Ｂｏｖｉｎｅ　Ｓｅｒｕｍ（ＦＢＳ）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）と、ＦＢＳの終濃度が５％となるように混合した。３７℃条件下で２２～３０時間インキュベートし、２時間おきに回収したサンプルに５×ＳＤＳ－ＰＡＧＥ　Ｓａｍｐｌｅ　ｂｕｆｆｅｒを加え、４℃で保存した。全てのサンプルを回収した後、ウエスタンブロッティングを行うことによりタンパク質の分解度を比較した。ウエスタンブロッティングは、サンプルを非還元条件でＳＤＳ－ＰＡＧＥ（１２．５％あるいは１５％　アクリルアミドゲル、３０ｍＡ／枚、７０分間泳動）により展開後、Ｐｏｌｙ　Ｖｉｎｙｌｉｄｅｎｅ　Ｄｉ　Ｆｌｕｏｒｉｄｅ（ＰＶＤＦ）メンブレン（Ｂｉｏ　Ｒａｄ）に転写した。５％スキムミルク（雪印メグミルク）／Ｐｈｏｓｐｈａｔｅ－ｂｕｆｆｅｒｄ　ｓａｌｉｎｅ（ＰＢＳ）－Ｔでメンブレンを、１時間もしくは一晩室温で振盪してブロッキングした。その後メンブレンをＰＢＳＴ溶液約１０ｍＬに浸し、一次抗体として抗ＨＬＡ－Ｇ抗体（ＭＥＭ－Ｇ１、Ａｂｃａｍ）（×１／５０００）と１時間室温で振盪して反応させた。二次抗体として抗マウスＩｇＧ（Ｆｃ）－Ｈｏｒｓｅｒｄｉｓｈ　ｐｅｒｏｘｉｄａｓｅ（ＨＲＰ）標識抗体（Ｔｈｒｅｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）（×１／１００００）と１時間室温で振盪して反応させた。その後メンブレンをＰＢＳ－Ｔで数回洗浄して、ＥＣＬ　Ｐｒｉｍｅ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）で発光させてＩｍａｇｅ　Ｑｕａｎｔ　ＬＡＳ４０００ｍｉｎｉ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用いて検出を行った。 More specifically, HLA-G2 and PEG20-HLA-G2 (HLA-G2: 0.3 mg / mL, PEG20-HLA-G2: 0.15 mg / mL, 20 mM Tris-HCl pH 8.0 after SEC purification, 100 mM NaCl) was mixed with 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific) so that the final concentration of FBS was 5%. After incubating at 37 ° C. for 22 to 30 hours, 5 × SDS-PAGE Sample buffer was added to the sample collected every 2 hours and stored at 4 ° C. After all samples were collected, the degree of protein degradation was compared by performing Western blotting. In Western blotting, samples were developed by SDS-PAGE (12.5% or 15% acrylamide gel, 30 mA / sheet, 70 minutes electrophoresis) under non-reducing conditions, and then transferred to a Poly Vinylidene Fluoride (PVDF) membrane (Bio Rad). did. The membrane was blocked by shaking with 5% skim milk (Snow Brand Megmilk) / Phosphate-buffer saline (PBS) -T for 1 hour or overnight at room temperature. Thereafter, the membrane was immersed in about 10 mL of PBST solution, and reacted with anti-HLA-G antibody (MEM-G1, Abcam) (× 1/5000) as a primary antibody by shaking at room temperature for 1 hour. A secondary antibody was reacted with anti-mouse IgG (Fc) -horserdisse peroxide (HRP) -labeled antibody (Thremo Fisher Scientific) (× 1/10000) by shaking at room temperature for 1 hour. Thereafter, the membrane was washed several times with PBS-T, and light was emitted with ECL Prime (GE Healthcare), and detection was performed using Image Quant LAS4000mini (GE Healthcare).
　今回ウエスタンブロッティング法に用いたＭＥＭ－Ｇ１抗体は、ＨＬＡ－Ｇ特異的抗体であり、エピトープ部位は不明であるが、本願の発明者らが変性状態のＨＬＡ－Ｇ２を検出可能なことを確認している（未発表データ）。また、予備実験にて、ＭＥＭ－Ｇ１抗体を用いたウエスタンブロッティングにおいてＰＥＧ２０－ＨＬＡ－Ｇ２の検出も可能であることを確認し、今回の実験を行った。 The MEM-G1 antibody used in this Western blotting method is an HLA-G-specific antibody, and the epitope site is unknown, but the inventors of the present application confirmed that it can detect denatured HLA-G2. (Unpublished data). In a preliminary experiment, it was confirmed that PEG20-HLA-G2 could be detected by Western blotting using the MEM-G1 antibody, and this experiment was conducted.
　５％ＦＢＳ存在下でのＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２量の経時的変化を比較したところ、タンパク質が分解し、消失する割合が、ＨＬＡ－Ｇ２ではＰＥＧ２０－ＨＬＡ－Ｇ２と比較して、特に２６～３０時間で多かった（図９（ａ））。このバンドをＩｍａｇｅＱｕａｎｔ　ＬＡＳ　４０００を用いて定量し、インキュベート時間ごとのバンドの残存量をグラフに示したところ、ＰＥＧ２０－ＨＬＡ－Ｇ２ではＨＬＡ－Ｇ２と比較して、タンパク質の消失速度が緩やかであることがわかった（図９（ｂ））。 When the changes over time in the amounts of HLA-G2 and PEG20-HLA-G2 in the presence of 5% FBS were compared, the rate of protein degradation and disappearance was higher in HLA-G2 than in PEG20-HLA-G2. In particular, it was high in 26 to 30 hours (FIG. 9 (a)). When this band was quantified using ImageQuant LAS 4000 and the remaining amount of the band for each incubation time was shown in the graph, the disappearance rate of the protein in PEG20-HLA-G2 was slower than that in HLA-G2. Was found (FIG. 9B).
　以上の結果から、ＨＬＡ－Ｇ２タンパク質の血清中におけるタンパク質の安定性は、ＰＥＧ化により向上する傾向にあることがわかった。 From the above results, it was found that the stability of HLA-G2 protein in serum tends to be improved by PEGylation.
（実施例３）
（ｉｎ　ｖｉｖｏでのＨＬＡ－Ｇ２、ＰＥＧ化ＨＬＡ－Ｇ２抗炎症効果の評価）
　アトピー性皮膚炎疾患モデルマウスを用いて、ＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２タンパク質のｉｎ　ｖｉｖｏでの抗炎症効果を検証した。 (Example 3)
(Evaluation of anti-inflammatory effects of HLA-G2 and PEGylated HLA-G2 in vivo)
The anti-inflammatory effect of HLA-G2 and PEG20-HLA-G2 proteins in vivo was verified using atopic dermatitis disease model mice.
　具体的には、皮膚炎発症モデルマウスの耳介にＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２及び、ポジティブコントロールとして、本願の発明者らがダニ抗原誘発皮膚炎モデルマウスで治療効果を確認したＨＬＡ－Ｇ１を投与し、炎症度の観察を行った。ネガティブコントロールにはＰＢＳを用いた。アトピー性皮膚炎疾患モデルマウスは、コナヒョウヒダニ虫成分を含有したアトピー性皮膚炎誘発軟膏（ビオスタＡＤ）をマウスの耳介表面に塗布することにより作製した。 Specifically, HLA-G2 and PEG20-HLA-G2 were applied to the pinna of the dermatitis onset model mouse, and HLA- which the inventors of the present application confirmed the therapeutic effect in the mite antigen-induced dermatitis model mouse as a positive control. G1 was administered and the degree of inflammation was observed. PBS was used for the negative control. An atopic dermatitis disease model mouse was prepared by applying an atopic dermatitis-inducing ointment (Biosta AD) containing a mite beetle component to the auricle surface of the mouse.
（アトピー性皮膚炎モデルマウスの作製）
　タンパク質の投与に先立ち、アトピー性皮膚炎モデルマウスを作製するために、ビオスタＡＤ　１００ｍｇを３日おきに計６回ＮＣ／Ｎｇａ　Ｓｌｃマウスへ塗布した。より具体的には、ＮＣ／Ｎｇａ　Ｓｌｃマウス（日本エスエルシー株式会社、１０週齢、雄）１６匹について、エレクトリッククリッパーと除毛クリームを用いて耳後部の体毛を除去した。体毛を除去した耳介表面に、コナヒョウヒダニ虫成分を含有したアトピー性皮膚炎誘発軟膏（ビオスタＡＤ（登録商標））１００ｍｇを３日おきに計６回塗布することによりアトピー性皮膚炎を発症させた（図１０（ａ））。ただし、２回目以降の誘発操作の際には、ビオスタＡＤを塗布する前に、耳介に４％ＳＤＳを塗布する操作を行った。アトピー性皮膚炎誘発軟膏を処置しない群（１匹）を皮膚炎発症の対照群とした。 (Preparation of atopic dermatitis model mouse)
Prior to protein administration, 100 mg of Biosta AD was applied to NC / Nga Slc mice a total of 6 times every 3 days in order to prepare atopic dermatitis model mice. More specifically, body hair at the back of the ear was removed from 16 NC / Nga Slc mice (Japan SLC Inc., 10 weeks old, male) using an electric clipper and a hair removal cream. Atopic dermatitis was developed by applying 100 mg of atopic dermatitis-inducing ointment (Biosta AD (registered trademark)) containing the mite mite insect component to the surface of the auricle from which body hair had been removed a total of 6 times every 3 days. (FIG. 10 (a)). However, in the second and subsequent induction operations, an operation of applying 4% SDS to the auricle was performed before applying Biosta AD. A group not treated with the atopic dermatitis-induced ointment (one animal) was used as a control group for developing dermatitis.
　ビオスタＡＤを塗布したマウス群においては、ビオスタＡＤを塗布していない対照マウス群と比較して、アトピー性皮膚炎に特徴的な掻痒行動、紅斑、出血などの症状が観察され、アトピー性皮膚炎の誘発を確認できた。 In the group of mice to which Biosta AD was applied, symptoms such as pruritus behavior, erythema and hemorrhage characteristic of atopic dermatitis were observed compared to the control mouse group to which Biosta AD was not applied. I was able to confirm the induction.
（各投与タンパク質（ＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２、ＨＬＡ－Ｇ１）の調製）
　続いて、作製したアトピー性皮膚炎疾患モデルマウスへの各投与タンパク質を調製した。前述の通りＳＥＣ精製により得られたＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２を、透析法によりＰＢＳバッファーに置換し、投与タンパク質溶液とした。 (Preparation of each administered protein (HLA-G2, PEG20-HLA-G2, HLA-G1))
Subsequently, each administration protein to the prepared atopic dermatitis disease model mouse 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.
　ＨＬＡ－Ｇ１（分子量４４ｋＤａ）は、ＳＥＣ精製後、目的タンパク質を含むピークを回収し、限外濾過により塩を含まないバッファー（Ｔｒｉｓ　ｐＨ８．０）に置換し、二次精製としてＩＥＸを行った。さらに、ＨＬＡ－Ｇ１についてはエンドトキシン含有ＢＬ２１（ＤＥ３）ｐＬｙｓＳ株を用いて発現させたため、ＩＥＸ精製、及びＰＢＳバッファーへ置換後、ＬＰＳ除去処理した溶液を投与タンパク質とした。 For HLA-G1 (molecular weight 44 kDa), after SEC purification, the peak containing the target protein was recovered, replaced with a salt-free buffer (Tris pH 8.0) by ultrafiltration, and IEX was performed as secondary purification. Furthermore, since HLA-G1 was expressed using the endotoxin-containing BL21 (DE3) pLysS strain, the solution treated with LPS removal after IEX purification and substitution with PBS buffer was used as the administered protein.
（作製した皮膚炎モデルマウスへのタンパク質投与）
　調製したＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２、ＨＬＡ－Ｇ１を１日おきに計１０回マウスの耳介表面に５μｇ／ｅａｒで経皮投与し（各群４匹）、耳介の炎症度を、ダイヤルシックネスゲージ（尾崎製作所）を用いて耳介の厚さを投与開始後０、６、１０、１４、１８、２２日目に測定することにより記録した（図１０（ａ））。 (Protein administration to the prepared dermatitis model mouse)
The prepared HLA-G2, PEG20-HLA-G2, and HLA-G1 were transdermally administered to the auricular surface of the mouse 10 times every other day at a rate of 5 μg / ear (4 mice in each group), and the degree of inflammation of the auricle was determined. The thickness of the auricle was recorded by measuring on the 0th, 6th, 10th, 14th, 18th, and 22nd days after the start of administration using a dial thickness gauge (Ozaki Seisakusho) (FIG. 10 (a)).
　統計解析は、ＪＭＰ（登録商標）１１（ＳＡＳ　Ｉｎｓｔｉｔｕｔｅ　Ｉｎｃ．，Ｃａｒｙ，ＮＣ，ＵＳＡ）ソフトウェアを用いて行った。タンパク質投与開始後２２日目の耳介の炎症度についてｔ検定を行い、ＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２投与群とＰＢＳ投与群間で、及びＨＬＡ－Ｇ２投与群とＰＥＧ２０－ＨＬＡ－Ｇ２投与群間で統計的有意差が認められるかどうかを検証した。 Statistical analysis was performed using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA) software. T-test was performed on the degree of inflammation of the pinna on the 22nd day after the start of protein administration. It was verified whether there was a statistically significant difference between groups.
　投与開始０日目から２２日目について、ＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２、ＨＬＡ－Ｇ１の投与により、抗炎症効果が確認できた。さらに、ＨＬＡ－Ｇ２投与群とＰＥＧ２０－ＨＬＡ－Ｇ２投与群で比較すると、ＨＬＡ－Ｇ２投与群に比べＰＥＧ２０－ＨＬＡ－Ｇ２投与群ではより強い炎症抑制効果が観察された（図１０（ｂ）、（ｃ））。２２日目の両耳介の腫脹度について、ＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２タンパク質投与群とＰＢＳ投与群間、又はＨＬＡ－Ｇ２タンパク質投与群間とＰＥＧ２０－ＨＬＡ－Ｇ２タンパク質投与群間でｔ検定を行ったところ、すべての２群間比較において統計的有意差が認められた（図１０（ｂ））。また、被験物質の投与により、体重減少は見られなかった（図示せず）。 From day 0 to day 22 after the start of administration, anti-inflammatory effects were confirmed by administration of HLA-G2, PEG20-HLA-G2, and HLA-G1. Furthermore, when the HLA-G2 administration group and the PEG20-HLA-G2 administration group were compared, a stronger inflammation suppression effect was observed in the PEG20-HLA-G2 administration group than in the HLA-G2 administration group (FIG. 10 (b), (C)). About the swelling degree of both ears on the 22nd day, it was t between the HLA-G2 and PEG20-HLA-G2 protein administration group and the PBS administration group, or between the HLA-G2 protein administration group and the PEG20-HLA-G2 protein administration group. When the test was performed, a statistically significant difference was observed in all comparisons between the two groups (FIG. 10 (b)). Moreover, weight loss was not seen by administration of the test substance (not shown).
　以上のタンパク質投与実験の結果から、ＰＥＧ２０－ＨＬＡ－Ｇ２では抗炎症剤として、アトピー性皮膚炎マウスに対し、副作用を伴うことなく、十分な抗炎症効果及び治療効果を示すことを見出した。また、その効果はＨＬＡ－Ｇ２と比較してＰＥＧ２０－ＨＬＡ－Ｇ２でより高いことが明らかとなった。 From the results of the above protein administration experiments, it was found that PEG20-HLA-G2 exhibits sufficient anti-inflammatory and therapeutic effects as an anti-inflammatory agent to atopic dermatitis mice without side effects. It was also revealed that the effect was higher with PEG20-HLA-G2 compared with HLA-G2.
（実施例４）
（ＳＬＥモデルマウス実験）
　ＰＥＧ２０－ＨＬＡ－Ｇ２をＳＬＥ（全身性エリテマトーデス）モデルマウスに投与して、ＳＬＥに対する治療効果を検証した。 Example 4
(SLE model mouse experiment)
PEG20-HLA-G2 was administered to SLE (systemic lupus erythematosus) model mice to verify the therapeutic effect on SLE.
　実施例３で調製したＨＬＡ－Ｇ２、ＰＥＧ２０－ＨＬＡ－Ｇ２又はネガティブコントロールとしてＰＢＳをＳＬＥモデルマウスに下記の通り投与した（“ＨＬＡ－Ｇ２投与群”、“ＰＥＧ２０－ＨＬＡ－Ｇ２投与群”又は“ＰＢＳ投与群”）。ＳＬＥモデルマウスとして、ＭＲＬ／ＭｐＪＪｍｓＳｌｃ－ｌｐｒ／ｌｐｒマウス（日本エスエルシー株式会社）を用い、各投与群８匹ずつから試験を開始した。なお、ＨＬＡ－Ｇ２及びＰＥＧ２０－ＨＬＡ－Ｇ２については、約３週間ごとに新しいロット（新たに巻き戻し～精製し直したもの）を投与した。試験の概要を図１３に示す。
　・投与経路：腹腔内投与
　・投与量：
　　　ＨＬＡ－Ｇ２投与群：１５μｇ／匹（２００μＬのＰＢＳに溶解）
　　　ＰＥＧ２０－ＨＬＡ－Ｇ２投与群：１５μｇ／匹（２００μＬのＰＢＳに溶解）
　　　ＰＢＳ投与群：ＰＢＳ　２００μＬ
　・投与間隔：週２回投与
　・投与期間：９５日間 HLA-G2, PEG20-HLA-G2 prepared in Example 3 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. An 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
　初回投与の８日前から、投与終了から２３日後まで、３週間又は２週間に一度、矢印（図１３）で示した日に血漿及び尿を採取し、ＳＬＥの診断及びモニタリングに用いられる血漿中抗ｄｓＤＮＡ抗体価及び尿中アルブミン指数を測定した。 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.
　副作用発生の指標として、投与のタイミングにあわせてマウスの体重測定を行った。図１４に、マウスの体重推移を示す。ＨＬＡ－Ｇ２投与群、ＰＥＧ２０－ＨＬＡ－Ｇ２投与群及びＰＢＳ投与群において、各投与群間で体重推移に有意な差は見られなかった。 As an indicator of the occurrence of side effects, mouse body weights were measured according to the timing of administration. FIG. 14 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.
（抗ｄｓＤＮＡ抗体ＥＬＩＳＡ）
　レビス　抗ｄｓＤＮＡ－マウスＥＬＩＳＡ　ＫＩＴ、シバヤギ社を用いて、血中抗核抗体量を測定した。緩衝液を用いて希釈したマウス血漿検体、および検量線作成用の標準溶液を抗原固相化マイクロプレートに添加した。２時間のインキュベートおよびウェル洗浄後、標識抗体（ペルオキシダーゼ結合抗マウスＩｇＧ抗体）を添加した。さらに２時間のインキュベート及びウェル洗浄後、発色液（ＴＭＢ）と２０分間反応させ、酸性溶液（１Ｍ　Ｈ２ＳＯ４）を添加することで反応を停止させた。分光光度計を用いて４５０ｎｍ（副波長６２０ｎｍ）の吸光度を測定し、標準液濃度に対する吸光度をプロットすることにより得られた標準曲線から各血漿サンプルの抗ｄｓＤＮＡ抗体価を算出した。 (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.
　図１５（ａ）に、各群の抗ｄｓＤＮＡ抗体価を示す。ＰＢＳ投与群に比してＨＬＡ－Ｇ２投与群及びＰＥＧ２０－ＨＬＡ－Ｇ２投与群では、抗ｄｓＤＮＡ抗体価の低下が確認された（図１５（ａ））。また、図１５（ｂ）に、投与９０日後のマウスの抗ｄｓＤＮＡ抗体価を示す。ＰＢＳ投与群に比してＨＬＡ－Ｇ２投与群及びＰＥＧ２０－ＨＬＡ－Ｇ２投与群では、抗ｄｓＤＮＡ抗体価の有意な低下が確認された（図１５（ｂ））。 FIG. 15 (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. 15 (a)). FIG. 15 (b) shows the anti-dsDNA antibody titer of mice 90 days after administration. In the HLA-G2 administration group and the PEG20-HLA-G2 administration group, a significant decrease in the anti-dsDNA antibody titer was confirmed as compared to the PBS administration group (FIG. 15 (b)).
（尿蛋白量）
　尿中アルブミンを、ＥＬＩＳＡキット（レビス　アルブミン－マウス、シバヤギ社）を用いて測定した。緩衝液を用いて希釈したマウス尿検体、および検量線作成用の標準溶液を抗アルブミン抗体固相化マイクロプレートに添加した。１時間のインキュベートおよびウェル洗浄後、標識抗体（ペルオキシダーゼ結合抗マウスＩｇＧ抗体）を添加した。さらに１時間のインキュベートおよびウェル洗浄後、発色液（ＴＭＢ）と２０分間反応させ、酸性溶液（１Ｍ　Ｈ２ＳＯ４）を添加することで反応を停止させた。分光光度計を用いて４５０ｎｍ（副波長６２０ｎｍ）の吸光度を測定し、標準液濃度に対する吸光度をプロットすることにより得られた標準曲線から各尿サンプルの尿中アルブミン指数を算出した。さらに尿中クレアチニンをＥＬＩＳＡキット（尿中クレアチニン測定用ＥＬＩＳＡキット、トランスジェニック社）を用いて測定した。超純水を用いて希釈したマウス尿検体、及び検量線作成用の標準溶液に標識抗体（ＨＲＰ標識－抗クレアチニン抗体）を添加し３０分インキュベートさせ、抗原固相化マイクロプレートに添加した。１時間のインキュベート及びウェル洗浄後、発色液（オルトフェニレンジアミン）と１０分間反応させ、酸性溶液（５．４％　Ｈ２ＳＯ４）を添加することで反応を停止させた。分光光度計を用いて４９０ｎｍの吸光度を測定し、標準液濃度に対する吸光度をプロットすることにより得られた標準曲線から各尿サンプルの尿中クレアチニン濃度を算出した。以上から得られた尿中アルブミン濃度（単位：ｍｇ／ｍＬ）の値を尿中クレアチニン濃度（単位：ｇ／ｍＬ）の値で割ることで尿中アルブミン指数（単位：ｍｇアルブミン／ｇクレアチニン）を算出した。 (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.
　図１６に、各群の尿中アルブミン指数を示す。各群間で尿中アルブミン指数の有意な差は見られなかったが、ＰＥＧ２０－ＨＬＡ－Ｇ２投与群では、ＰＢＳ投与群及びＨＬＡ－Ｇ２投与群に比して、尿中アルブミン指数の低下効果が持続する傾向にあった。 FIG. 16 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. Tended to last.
（ＢＬｙｓ血中濃度）
　また、投与開始後９０日目に、可溶型Ｂリンパ球刺激因子（Ｂｌｙｓ）血中濃度を測定した。より具体的には、Ｍｏｕｓｅ　ＢＡＦＦ／ＢＬｙＳ／ＴＮＦＳＦ１３Ｂ　Ｑｕａｎｔｉｋｉｎｅ　ＥＬＩＳＡ　Ｋｉｔ（Ｒ＆Ｄ　ｓｙｓｔｅｍｓ社製）を用いて、プロトコルに従い血漿中ＢＬｙｓ濃度を測定した。緩衝液を用いて希釈したマウス血漿検体、及び検量線作成用の標準溶液をマウスＢＬｙｓ特異的モノクローナル抗体固相化マイクロプレートに添加した。２時間のインキュベート及びウェル洗浄後、標識抗体（ペルオキシダーゼ結合抗マウスＢＬｙｓポリクローナル抗体）を添加した。さらに２時間のインキュベート及びウェル洗浄後、発色液（ＴＭＢ）と３０分間反応させ、酸性溶液（希塩酸）を添加することで反応を停止させた。分光光度計を用いて４５０ｎｍ（副波長５４０ｎｍ）の吸光度を測定し、標準液濃度に対する吸光度をプロットすることにより得られた標準曲線から各血漿中ＢＬｙｓ濃度を算出した。 (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.
　図１７に、各群のＢｌｙｓ血中濃度を示す。ＰＥＧ２０－ＨＬＡ－Ｇ２投与群では、ＰＢＳ投与群及びＨＬＡ－Ｇ２投与群に比して、Ｂｌｙｓ血中濃度の有意な低下が認められた。 FIG. 17 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.
（延長試験）
　次に、ＰＢＳ投与群を用いて延長試験を行い、ＰＥＧ２０－ＨＬＡ－Ｇ２のＳＬＥに対する治療効果を検証した。 (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.
　上記のＰＢＳ投与群のマウスについて、初回投与から１１８日及び１３２日目に血漿及び尿を採取し、前述同様に、血漿中抗ｄｓＤＮＡ抗体価及び尿中アルブミン指数を測定した。１３２日目の血漿中抗ｄｓＤＮＡ抗体価及び尿中アルブミン指数の値が、１１８日目のそれらの値よりも上昇している（すなわち、ＳＬＥを発症している）マウスに対して、ＰＥＧ２０－ＨＬＡ－Ｇ２又はＰＢＳを投与し、治療効果を検証した（ＰＥＧ２０－ＨＬＡ－Ｇ２投与：ｎ＝ １、ＰＢＳ投与：ｎ＝１）。１３２日目から投与を開始し（投与間隔：週２回投与、２週間で計４回投与）、投与経路及び投与量については、前述同様とした。１３８日目及び１４５日目に血漿及び尿を採取し、前述同様に、血漿中抗ｄｓＤＮＡ抗体価及び尿中アルブミン指数を測定した。 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.
　図１８（ａ）に、抗ｄｓＤＮＡ抗体価の変化を示す。ＰＥＧ２０－ＨＬＡ－Ｇ２投与のマウスでは、ＰＢＳ投与のそれに比して抗ｄｓＤＮＡ抗体価の低下が見られ、ＰＥＧ２０－ＨＬＡ－Ｇ２による治療効果が確認された。 FIG. 18 (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.
　図１８（ｂ）に、尿中アルブミン指数の変化を示す。ＰＥＧ２０－ＨＬＡ－Ｇ２投与のマウスでは、ＰＢＳ投与のそれに比して尿中アルブミン指数の低下が見られ、ＰＥＧ２０－ＨＬＡ－Ｇ２による治療効果が確認された。 FIG. 18 (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.
　以上より、ＳＬＥモデルマウス（ＭＲＬ／ＭｐＪＪｍｓＳｌｃ－ｌｐｒ／ｌｐｒマウス）において、週２回のＰＥＧ２０－ＨＬＡ－Ｇ２の腹腔内投与によって血中抗核抗体量及び尿蛋白質の低下が認められたことから、ＰＥＧ２０－ＨＬＡ－Ｇ２はＳＬＥに対して治療効果を奏することが明らかとなった。 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.
　以上より、ＰＥＧを、ＨＬＡ－Ｇ２分子表面に露出したフリーのシステイン残基（Ｃｙｓ４２）に付加することで、ｉｎ　ｖｉｔｒｏでの温度安定性、凍結乾燥耐性、血中安定性の向上が認められた。さらに、ｉｎ　ｖｉｖｏでアトピー性皮膚炎マウスにおける抗炎症効果がＰＥＧ化することによって増強することが明らかとなり、ＰＥＧ化ＨＬＡ－Ｇ２ではＨＬＡ－Ｇ２に比べ、炎症抑制効果が大きく、マウス生体内での安定性が向上していることが示唆された。ＰＥＧ化ＨＬＡ－Ｇ２を用いることで、投与量や投与回数の低減が期待でき、他剤との併用を想定する上でも、患者への金銭的、身体的負担を軽減できるものと期待される。 From the above, by adding PEG to the free cysteine residue (Cys42) exposed on the surface of the HLA-G2 molecule, in vitro temperature stability, freeze-drying resistance, and blood stability were improved. . Furthermore, it became clear that the anti-inflammatory effect in atopic dermatitis mice is enhanced in vivo by PEGylation, and PEGylated HLA-G2 has a greater inflammation-inhibiting effect than HLA-G2, and thus in vivo in mice. It was suggested that the stability was improved. 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.
　本発明は、本発明の広義の精神と範囲を逸脱することなく、様々な実施の形態及び変形が可能とされるものである。また、上述した実施の形態は、この発明を説明するためのものであり、本発明の範囲を限定するものではない。すなわち、本発明の範囲は、実施の形態ではなく、特許請求の範囲によって示される。そして、特許請求の範囲内及びそれと同等の発明の意義の範囲内で施される様々な変形が、この発明の範囲内とみなされる。 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.
　本出願は、２０１８年３月１３日に出願された、日本国特許出願２０１８－０４６０２５号に基づく。本明細書中に日本国特許出願２０１８－０４６０２５号の明細書、特許請求の範囲、図面全体を参照として取り込むものと
する。 This application is based on Japanese Patent Application No. 2018-046025 filed on Mar. 13, 2018. The specification, claims, and entire drawings of Japanese Patent Application No. 2018-046025 are incorporated herein by reference.

Claims (8)

  1. 　ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体からなり、
    　前記タンパク質を構成するアミノ酸配列中の少なくとも１つのアミノ酸残基は、ポリエチレングリコール（ＰＥＧ）でＰＥＧ化修飾されている、
    　ことを特徴とする改変タンパク質。     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 PEGylated with polyethylene glycol (PEG),
    A modified protein characterized by the above.
  2. 　ＰＥＧ化修飾に用いるＰＥＧの分子量は、５ｋＤａ～１００ｋＤａである、
    　ことを特徴とする請求項１に記載の改変タンパク質。     The molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.
    The modified protein according to claim 1.
  3. 　前記タンパク質は、下記（ａ）または（ｂ）に記載するアミノ酸配列からなるタンパク質であって、
    　（ａ）配列番号１に示されるアミノ酸配列からなるタンパク質、
    　（ｂ）配列番号１に示されるアミノ酸配列において、１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質、
    　前記多量体が、前記（ａ）同士若しくは（ｂ）同士のホモ多量体又は（ａ）と（ｂ）とのヘテロ多量体であり、白血球Ｉｇ様受容体Ｂ２との結合活性を有するものである、
    　ことを特徴とする請求項１又は２に記載の改変タンパク質。     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 modified protein according to claim 1 or 2, wherein
  4. 　請求項１乃至３のいずれか１項に記載の改変タンパク質又はその塩を有効成分として含む医薬品。 A pharmaceutical comprising the modified protein according to any one of claims 1 to 3 or a salt thereof as an active ingredient.
  5. 　請求項１乃至３のいずれか１項に記載の改変タンパク質又はその塩を有効成分として含む炎症性疾患の予防又は治療剤。 A preventive or therapeutic agent for inflammatory diseases comprising the modified protein or salt thereof according to any one of claims 1 to 3 as an active ingredient.
  6. 　（Ａ）ＨＬＡ－Ｇのα１ドメインとＨＬＡ－Ｇのα３ドメインとが連結したアミノ酸配列を有するタンパク質の多量体を調製する工程と、
    　（Ｂ）工程（Ａ）で得られた前記タンパク質の多量体を脱気処理した後、還元処理する工程と、
    　（Ｃ）工程（Ｂ）で還元処理された前記タンパク質の多量体をＰＥＧ化修飾する工程と、
    　を含む改変タンパク質の製造方法。     (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 step of degassing the protein multimer obtained in step (A), followed by a reduction treatment;
    (C) PEGylating and modifying the protein multimer reduced in step (B);
    A method for producing a modified protein comprising:
  7. 　ＰＥＧ化修飾に用いるＰＥＧの分子量は、５ｋＤａ～１００ｋＤａである、
    　ことを特徴とする請求項６に記載の改変タンパク質の製造方法。     The molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.
    The method for producing a modified protein according to claim 6.
  8. 　前記タンパク質は、下記（ａ）または（ｂ）に記載するアミノ酸配列からなるタンパク質であって、
    　（ａ）配列番号１に示されるアミノ酸配列からなるタンパク質、
    　（ｂ）配列番号１に示されるアミノ酸配列において、１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質、
    　前記多量体が、前記（ａ）同士若しくは（ｂ）同士のホモ多量体又は（ａ）と（ｂ）とのヘテロ多量体であり、白血球Ｉｇ様受容体Ｂ２との結合活性を有するものである、
    　ことを特徴とする請求項６又は７に記載の改変タンパク質の製造方法。     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 method for producing a modified protein according to claim 6 or 7, wherein:
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