JP2021080171A - Modified protein, drug, prophylactic or therapeutic agent for inflammatory disease, and method for producing modified protein - Google Patents

Abstract

To provide a modified protein that is high in storage stability and in vivo stability and has high prophylactic or therapeutic effect on diseases, and a drug, a prophylactic or therapeutic agent for inflammatory diseases, and a method for producing a modified protein.SOLUTION: A 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).SELECTED DRAWING: Figure 1

Description

The present invention relates to modified proteins, pharmaceuticals, prophylactic or therapeutic agents for inflammatory diseases, and methods for producing modified proteins.

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

The 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 peptides, heavy chains and β2 microglobules. On the other hand, the domain-deficient HLA-G2 isoform is a homodimer consisting only of domain-deficient heavy chains. HLA-G2 was known to have an activity that complements the function of HLA-G1, but the detailed function was unknown for a long period of time.

In recent years, various reports have been made on HLA-G2. Non-Patent Document 1 discloses that HLA-G2 exists as a homodimer and transmits a signal via the immunosuppressive receptor LILRB2 as a receptor. Further, in Non-Patent Document 2 and Patent Document 1, as a result of analyzing the anti-inflammatory effect of HLA-G2 in vivo, it is strongly bound to the mouse receptor PIR-B, and a single dose to a rheumatoid arthritis model mouse. It is disclosed that long-term immunosuppressive effect was obtained by administration.

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 can be shielded from attack by enzymes and exposure of hydrophobic parts, and as a result, the effect of suppressing protein decomposition and aggregation can be expected. In addition, since the addition of PEG increases the molecular size and suppresses filtration in the glomerulus, the half-life of protein in blood is extended, and since PEG itself does not have antigenicity, side effects due to protein administration are reduced. Can be expected (Non-Patent Document 3). In fact, more than a dozen PEGylated drugs are currently approved by the Food and Drug Administration (FDA) (Non-Patent Documents 4 and 5) and are in clinical use.

Japanese Unexamined Patent Publication No. 2015-14022

Cutting Edge: Class II-like Structure Structures and Strong Receptor Binding of the Nonclassical HLA-G2 Isoform Homedimer. Kuroki K, Mio K, Takahashi A, Matsubara H, Kasai Y, Manaka S, Kikawa M, Hamada D, Sato C, Maenaka K. J Immunol. 2017 May 1; 198 (9): 3399-3403. doi: 10.4049 / jimmul. 1601296. The immunosuppressive effect of domain-deleted dimer of HLA-G2 isoform in collagen-induced arthritis meeting. Takahashi A, Kuroki K, Okabe Y, Kasai Y, Matsumoto N, Yamada C, Takai T, Ose T, Kon S, Masada T, Maenaka. Hum Immunol. 2016 Sep; 77 (9): 754-9. doi: 10.1016 / j. humimm. 2016.01.010. Harris, J.M. M. , And Chess, R.M. B. (2003) Effect of PEGylation on pharmaceuticals. Nat. Rev. Drag Discov. 2, 214-221 Dozier, J. et al. K. , And Distefano, M.D. D. (2015) Site-specific PEGylation of therapeutic proteins. Int. J. Mol. Sci. 16, 25831-25864 Turek, P.M. L. , Bossard, M.D. J. , Schoetens, F. , And Events, I. A. (2016) PEGylation of Biopharmacyticals: A Review of Chemistry and Nonclinical Safety Information of Applied Drugs. J. Pharm. Sci. 105, 460-475

However, although HLA-G2 is expected to be used as a pharmaceutical product, it has a problem that its stability and homogeneity as a recombinant protein decrease with time, which leaves a problem in terms of long-term storage. Was there.

The present invention has been made in view of the above circumstances, and is a modified protein, a drug, a prophylactic or therapeutic agent for an inflammatory disease, which has high storage stability and in vivo stability and is highly effective in preventing or treating a disease. It is an object of the present invention to provide a method for producing a modified protein.

In order to achieve the above object, the modified protein according to the first aspect of the present invention is
It consists of a multimeric protein 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 cysteine residue in the amino acid sequence constituting the protein is PEGylated and modified with polyethylene glycol (PEG).
It is characterized by that.

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

For example, the protein is a protein consisting of the amino acid sequence described in (a) or (b) below.
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
(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.
The multimer is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b), and has a binding activity to the leukocyte Ig-like receptor B2. ..

The pharmaceutical product according to the second aspect of the present invention is
The modified protein or a salt thereof according to the first aspect of the present invention is contained as an active ingredient.

The prophylactic or therapeutic agent for an inflammatory disease according to the third aspect of the present invention is
The modified protein or a salt thereof according to the first aspect of the present invention is contained as an active ingredient.

The method for producing a modified protein according to the fourth aspect of the present invention is
(A) A step of preparing 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
(B) A step of degassing and then reducing the multimer of the protein obtained in the step (A), and a step of reducing the protein.
(C) A step of PEGylating and modifying the multimer of the protein subjected to the reduction treatment in the step (B), and
including.

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

For example, the protein is a protein consisting of the amino acid sequence described in (a) or (b) below.
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
(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.
The multimer is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b), and has a binding activity to the leukocyte Ig-like receptor B2. ..

According to the present invention, there is provided a method for producing a modified protein, a drug, a prophylactic or therapeutic agent for an inflammatory disease, and a modified protein, which are highly stable in storage and in vivo and have a high preventive or therapeutic effect on a disease. Can be done.

It is a figure which shows typically the PEGylated HLA-G2. (A) is a diagram showing a chromatogram of SEC purification using a HiLoad 26/60 Superdex75 pg column after rewinding the inclusion body in the preparation of HLA-G2, and (b) is obtained by adding a reducing agent (TCEP). It is a figure which showed the result of CBB staining of the PEGylated HLA-G2. It is a figure which showed the comparison of the reaction efficiency by the PEG molecular weight, (a) is the figure which shows the result of _{CBB staining, (b) is the figure which shows the result of BaI 2 staining.} It is a figure which shows the result of SEC purification using Superdex 200 10/300 GL of the PEGylation reaction solution by PEG of each molecular weight, concentration, SDS-PAGE, and silver staining, and (a) is PEG5-HLA-G2. (B) is a diagram showing the results of PEG10-HLA-G2, (c) is a diagram showing the results of PEG20-HLA-G2, and (d) is a diagram showing the results of PEG40-HLA-G2. It is a figure which shows the sensorgram obtained by the binding experiment with LILRB2 receptor, (a) is HLA-G2, (b) is PEG5-HLA-G2, (c) is PEG10-HLA-G2, (d). Is a diagram showing the results of PEG20-HLA-G2. It is a figure which compared the PEGylation reaction efficiency by PEG20 of HLA-G2 mutants (HLA-G2N86C, HLA-G2CTER) having different PEGylation sites, (a) is the result of CBB staining, (b) is the result of _{BaI 2 staining.} It is a figure which shows. (A) is a figure showing the result of SDS-PAGE and CBB staining of HLA-G2 and PEG20-HLA-G2 with and without freeze-drying treatment, and (b) is the amount of HLA-G2 (LILRB2 immobilized amount) before and after freeze-drying treatment. It is a figure which shows the sensorgram obtained by the binding experiment of: 512RU), and (c) is the sensorgram obtained by the binding experiment of PEG20-HLA-G2 (LILRB2 immobilization amount: 272RU) before and after the freeze-drying treatment. It is a figure which shows. It is a figure which shows the comparison result (CBB staining) of the protein aggregation and decomposition degree by heat treatment, (a) is HLA-G2, (b) is PEG20-HLA-G2. It is a figure which shows the serum stability comparison of HLA-G2, PEG20-HLA-G2 protein, (a) is a figure which shows the serum stability of HLA-G2, PEG20-HLA-G2, (b) is a figure which shows. It is a figure which shows the change of the survival rate over time in the serum of HLA-G2, PEG20-HLA-G2. (A) is a diagram showing the induction of dermatitis by atopic dermatitis-inducing ointment, the protein administration schedule, and the recording date of the auricle thickness, and (b) is a diagram showing the degree of swelling of the mouse auricle. , (C) are photographic views showing the mouse pinna on the 18th day of administration. It is a figure showing the primary structure (left) and molecular schematic diagram (right) of each recombinant protein, (a) is HLA-G2, (b) is HLA-G2C42S, (c) is HLA-G2N86C, (d). Is HLA-G2CTER, (e) is HLA-G1C42S heavy chain, (f) is β2m, and (g) is LILRB2. It is a schematic diagram which shows the principle of biotinylated protein immobilization on a sensor chip CAP.

First, the modified protein according to this embodiment will be described in detail.

The modified protein according to the present embodiment consists of a multimeric protein having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked, and at least one cysteine residue in the amino acid sequence constituting the protein. The group is PEGylated and modified with polyethylene glycol (PEG) (Fig. 1).

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

The HLA-G multimer targeted by the present 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, and preferably.
(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.
And
It is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b).

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

The above-mentioned "protein consisting of the amino acid sequence shown in SEQ ID NO: 1" is one of the dimers of the protein having the 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, the "1-90th amino acid region" corresponds to the amino acid sequence of the α1 domain, and the "91-180th 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 "1-270th base sequence" region corresponds to the gene region encoding the α1 domain, and the "271-540th base". The "sequence" region corresponds to the gene region encoding the α3 domain.

As the above-mentioned "protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added", the amino acid sequence (sequence) of the link (α1-3 link) between the α1 domain and the α3 domain of the HLA-G molecule. A protein having an amino acid sequence (SEQ ID NO: 3) encoded by the 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. .. Further, as another "protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added", C of the amino acid sequence (SEQ ID NO: 1) of the link between the α1 domain and the α3 domain of the HLA-G molecule. Examples of proteins having the amino acid sequences (SEQ ID NO: 4) of the transmembrane domain and the intracellular domain of the HLA-G2 molecule on the terminal side can be exemplified.

Among "proteins consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added", preferably, the function of the multimer of the link between the α1 domain and the α3 domain of the HLA-G molecule (leukocyte Ig-like acceptance). The amino acid has the same amino acid sequence as that of the α1-3 conjugate (SEQ ID NO: 1), preferably 90% or more, preferably 95% or more. A protein that is deleted, substituted or added. A protein having an amino acid sequence that is 96% or more identical to the amino acid sequence (SEQ ID NO: 1) of the above conjugate, and more preferably 98% or more the same.

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

The HLA-G2 multimer targeted by the present embodiment is not limited to the above-mentioned conjugate itself of the α1 domain and the α3 domain of the HLA-G molecule (hereinafter, this is also simply referred to as “α1-3 conjugate”). , The dimer has a linking structure between the α1 domain and the α3 domain of the HLA-G molecule, and the dimer has binding activity to the leukocyte Ig-like receptor B2 (LILRB2). For example, as long as it has the above conditions, one or more amino acids are missing from the amino acid sequence (SEQ ID NO: 1) of the link (α1-3 link) between the α1 domain and the α3 domain of the HLA-G molecule. It may be a protein consisting of a lost, substituted or added amino acid sequence. Here, the "binding activity to leukocyte Ig-like receptor B2 (LILRB2)" is an activity in which the HLA-G2 multimer directly binds to LILRB2, thereby transmitting a signal via LILRB2 and having an immunoregulatory effect. It means the activity that can exert.

Whether or not the HLA-G2 multimer has a binding activity to the 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 active receptor PILRβ are fused. Can be done. In this case, when the HLA-G2 multimer binds to the LILRB2 receptor, a signal is transmitted via the intracellular domain of PILRβ, and the transcription factor NFAT is activated. The reporter assay is an assay system utilizing the fact that the expression of GFP is induced by the activation of the NFAT. GFP expression indicates that LILRB2 and the HLA-G2 multimer were bound, and the appearance of fluorescence due to such GFP expression can be confirmed by flow cytometry.

The HLA-G2 multimer is a multimerized product of the above-mentioned HLA-G2 by disulfide bond or non-covalent bond without disulfide bond. In the HLA-G2 multimer of the present embodiment, which cysteine residue in the amino acid sequence constituting the protein is PEGylated and modified, for example, in the case of a naturally formed homodimer, It is multimerized regardless of the disulfide bond, and the cysteine residue (Cys42) may remain free on the surface of the molecule, or a further dimer (tetramer) having a homodimer as one unit or In the case of these additional multimers, they may be multimerized by disulfide bonds via cysteine residues (Cys42) existing on the surface of the homodimer molecule.

The HLA-G2 multimer may be a homomultimer composed of the above-mentioned α1-3 conjugates or mutants, or may be a heteromultimer composed of the α1-3 conjugate and the mutant. , Not limited. It is preferably a homomultimer composed of α1-3 conjugates.

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

The method for preparing the HLA-G2 multimer will be described later.

As described above, the modified protein according to the present embodiment comprises a multimeric protein having an amino acid sequence in which the α1 domain of HLA-G and the α3 domain of HLA-G are linked, and at least in the amino acid sequence constituting the protein. One cysteine residue is PEGylated and modified with polyethylene glycol (PEG).

The PEGylated modified cysteine residue is preferably present at a site in the protein that does not affect receptor binding. The PEGylated modified cysteine residue may be, for example, one cysteine residue in the amino acid sequence constituting the protein, for example, the 42nd free cysteine residue of HLA-G2. Further, the amino acid residue existing at an arbitrary site may be replaced with a cysteine residue, and the cysteine residue may be PEGylated and modified. For example, the 86th asparagine, which is the sugar chain modification site of HLA-G, may be cysteine. It may be substituted with PEGylation modification. Further, a cysteine residue may be added to an arbitrary site and the cysteine residue may be PEGylated and modified. For example, cysteine may be added to the C-terminal of HLA-G and PEGylated and modified. The methods of these substitutions and additions will be described later.

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

As a method for confirming the storage stability of the PEGylated modified protein according to the present embodiment, for example, the modified protein is lyophilized and then dissolved in sterilized water, and SDS-PAGE is performed to band the degree of aggregation / degradation. Method of confirming as a pattern; Method of confirming binding activity to LILRB2 by the above-mentioned method after freeze-drying of the modified protein; SDS-PAGE after incubating the modified protein under high temperature conditions to determine the degree of aggregation / degradation. Examples include a method of confirming as a band pattern. Further, as a method for confirming the stability of the PEGylated modified protein according to the present embodiment in vivo, for example, the modified protein is incubated in serum (for example, FBS) and then degraded by Western blotting. There is a method of confirming the degree of.

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

The method for producing a modified protein according to this embodiment is
(A) A step of preparing 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
(B) A step of degassing and then reducing the multimer of the protein obtained in the step (A), and a step of reducing the protein.
(C) A step of PEGylating and modifying the multimer of the protein subjected to the reduction treatment in the step (B), and
including.

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

The HLA-G2 multimer targeted by the present embodiment is, for example, a 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, as described above, and is preferably preferred.
(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.
And
It is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b).

In the step (A), a method for preparing an HLA-G2 multimer will be described as an example.

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

Here, as the gene encoding the amino acid sequence of the HLA-G2 multimer, in addition to the gene (SEQ ID NO: 2) encoding the amino acid sequence (SEQ ID NO: 1) of the link between the α1 domain and the α3 domain of HLA-G, It is also possible to use a gene encoding the amino acid sequence of the above-mentioned HLA-G2 (hereinafter, this is referred to as “mutant HLA-G2” for convenience) obtained by deleting, substituting or adding a part of the amino acid sequence of the conjugate. it can. Further, from the viewpoint of increasing yield, as a gene encoding the amino acid sequence of HLA-G2, for example, the 1st to 18th amino acids of the base 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 (hereinafter referred to as "modified gene") (SEQ ID NO: 6) obtained by substituting the base sequence of (corresponding to the base sequence) with the base sequence shown in SEQ ID NO: 5 can also be used.

The gene encoding the mutant HLA-G2 multimer may be prepared by introducing a mutation into the DNA sequence of the α1-3 conjugate gene. For example, Molecular Cloning, A Laboratory Manual 2nd ed. , Cold Spring Harbor Laboratory Press (1989), Cold Spring Harbor Laboratory, John Wiley & Sons (1987-1997), and the like. Specifically, it can be prepared using a mutation introduction kit utilizing a site-specific mutation introduction method by a known method such as the Kunkel method or the Gapped duplex method. Examples of the kit include QuickChangeTM Site-Directed Mutagesis Kit (manufactured by Stratagene), GeneTailorTM Site-Directed Mutagesys System (manufactured by Invitrogen), TakaRageSiteMuteMute-Mut (Manufactured by Takara Bio Inc.) and the like are preferably mentioned.

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

The production of the recombinant HLA-G2 multimer is specifically carried out by a method including a step of culturing the above-mentioned transformant and a step of collecting the recombinant HLA-G2 multimer from the obtained culture. Can be done. Here, the "culture" means any of a culture supernatant, cultured cells, cultured cells, or crushed cells or cells. Culturing of the transformant can be carried out according to the usual method used for culturing the host. The protein of interest is accumulated in the culture.

When the recombinant HLA-G2 multimer is produced extracellularly, the culture solution is used as it is, or the cells are removed by centrifugation, filtration, or the like. Then, if necessary, the recombinant HLA-G2 multimer is collected from the culture by extraction with ammonium sulfate precipitation or the like, and if necessary, dialysis and various chromatographys (gel filtration, ion exchange chromatography, affinity chromatography) are performed. Etc.) can be used for isolation and purification.

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

In addition, the recombinant HLA-G2 multimer can be produced using a protein synthesis system using a transformant or a cell-free protein synthesis system that does not use live cells at all, and the produced recombinant HLA can be produced. The -G2 multimer can be purified by appropriately selecting a means such as chromatography.

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

The step (B) is a step of degassing and then reducing the multimer of the protein obtained in the step (A).

In step (B), degassing is performed to allow the PEGylation reaction to proceed under anaerobic conditions. The degassing treatment may be performed for 1 hour using, for example, an aspirator. Prior to the degassing treatment, for example, the multimer of the protein obtained in step (A) may be replaced with a PEGylated buffer (for example, 1 × Phosphate-Buffer Saline (PBS), 5 mM EDTA). .. Other known methods may be used as the degassing treatment method.

In step (B), the reducing agent is added in order to cleave the disulfide bond by the reduction treatment, re-expose the thiol group of the residue to be PEGylated, and improve the reaction efficiency. As the reducing agent, any reducing agent having a reducing action can be used, and for example, tris (2-carboxythyl) phosphorine (TCEP), which is often used in a reaction using maleimide and cysteine, may be used. When TCEP is used as the reducing agent, for example, TCEP may be added so that the final concentration is 0.1 mM or more.

The step (C) is a step of PEGylating and modifying the multimer of the protein reduced in the step (B).

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

In step (C), for example, PEG: protein multimer = 5: 1, 10: 1, 20: 1, 30: 1 (molar ratio) so that PEG is in excess of the protein multimer. ) Etc. may be mixed with the PEGylation reagent. PEGylation modification can be performed by adding a PEGylation reagent and reacting at 4 ° C. overnight, for example. As described above, at least one cysteine residue in the amino acid sequence constituting the protein is PEGylated and modified with PEG.

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

Next, a drug and a preventive or therapeutic agent for an inflammatory disease according to the present embodiment will be described in detail.

The pharmaceutical product and the prophylactic or therapeutic agent for an inflammatory disease according to the present embodiment include the above-mentioned modified protein or a salt thereof.

As the salt of the modified protein, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal salt) is used, and in particular, a physiologically acceptable acid addition salt is used. Is preferable. 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). Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.

The prophylactic or therapeutic agent for inflammatory diseases according to the present embodiment contains the above-mentioned modified protein or a salt thereof, and is used for inflammatory diseases such as atopic dermatitis, contact dermatitis, rash, psoriasis, and pemphigus vulgaris. On the other hand, it has a preventive or therapeutic effect.

The drug according to the present embodiment contains the above-mentioned modified protein or a salt thereof, and can be used for a desired pharmaceutical use, for example, as a preventive or therapeutic agent for the above-mentioned inflammatory disease. The drug according to this embodiment is, for example, an ophthalmic disease such as rheumatoid arthritis, Sjogren's syndrome, keratoconjunctivitis sicca and the resulting dry eye, rheumatoid nodule, amyloidosis perforation, amyloidosis and amyloidosis; interstitial. Respiratory disorders such as pneumonia, obstructive bronchitis, pleural inflammation, pneumococcal, pyorrhea, airway lesions, pleural lesions, rheumatoid nodules, vascular lesions and sleep amyloidosis (jaw joint lesions, cricoid arthritis lesions); Cardiac diseases such as epidermitis, symptomatic epicarditis, chronic contractile peritonitis, valve dysfunction, embolism, conduction disorders, myocardial disorders, aortitis, aortic valve insufficiency and aneurysm rupture; Gastrointestinal diseases such as AA amyloidosis due to inflammation and ischemic enteritis due to rheumatoid vasculitis; interstitial nephritis due to Sjogren's syndrome associated with rheumatoid arthritis, interstitial renal lesions, proteinuria, secondary amyloidosis, and rheumatoid arthritis Kidney diseases such as glomerular lesions (membranous nephropathy) due to AA amyloidosis; spinal cord disorders due to cervical spinal deformity, compressive neuropathy due to rheumatoid arthritis, and multiple mononeuritis due to vasculitis associated with rheumatoid arthritis System diseases; dermatological diseases such as rheumatoid nodules, cutaneous vasculitis (leukocyte crushing vasculitis) and ischemic skin ulcers; anemia (microcytic hypopigmentation anemia), splenoma, leukocyte (neutrophil only) decrease It can be used as a prophylactic or therapeutic agent for blood system diseases such as Felty's syndrome. Further, the drug according to the present embodiment can be used, for example, as a preventive or therapeutic agent for collagen disease, and the collagen disease includes, for example, systemic lupus erythematosus, systemic granulomatosis, polyarteritis nodosa / dermatomyositis, Schegren's syndrome, etc. Mixed binding tissue disease, antiphospholipid antibody syndrome, Bechet's disease, allergic granulomatous vasculitis (Charg-Strauss syndrome), adult Still's disease, eosinophilic myositis, polyarteritis nodosa (polyarteritis nodosa) Includes arteritis / microscopic polyarteritis), aortitis syndrome (Takayasu's arteritis), Wegener's granulomatosis, temporal arteritis, malignant rheumatoid arthritis, etc.

The method for administering the drug and the prophylactic or therapeutic agent for the inflammatory disease according to the present 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 oral liquids and syrups, parenteral liquid preparations such as injections and the like. Can be appropriately prepared. Alternatively, a suitable drug delivery system (DDS) may be used.

As described above, to provide a modified protein, a drug, a prophylactic or therapeutic agent for an inflammatory disease, and a method for producing a modified protein, which are highly stable in storage and in vivo and have a high preventive or therapeutic effect on diseases. Can be done.

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

(Example 1)
(Examination of conditions for HLA-G2 site-specific PEGylation)
The HLA-G2 protein, which is the target protein for PEGylation, and the mutant protein (HLA-G2C42S), in which the 42nd free cysteine residue (Cys42), which is the modification site of the PEG reagent used this time, is replaced with a serine residue. And by comparing the PEGylation progress of these proteins, it was confirmed whether the PEGylation progressed site-specifically with respect to the Cys42 residue. In order to evaluate the binding ability of the PEGylated HLA-G2 protein to the receptor and the mutant protein for PEGylation site examination, a C-terminal virA tag-specific biotinylated labelable LILRB2 virA protein was prepared and binding analysis was performed. went.

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

(HLA-G2 recombinant protein expression plasmid)
The extracellular region (Gly1-Trp182) of HLA-G2 (WT) with the signal sequence removed and the methionine residue, which is the translation initiation codon, added to the N-terminal was incorporated into the Escherichia coli expression vector pGM7 to further enhance the expression level. An expression plasmid (HLA-G2-pGMT7) possessed by our laboratory in which the base sequences of 5 residues near the N-terminal were synonymously substituted was used (FIG. 11 (a), base sequence: SEQ ID NO: 7, amino acid sequence: SEQ ID NO: 8).

(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 replaced with serine. -G2C42S-pGMT7 plasmid was used (FIG. 11 (b), base sequence: SEQ ID NO: 9, amino acid sequence: SEQ ID NO: 10). It has been confirmed in our laboratory that the HLA-G2C42S recombinant protein maintains the same molecular structure and receptor binding ability as HLA-G2.

In addition, a cysteine variant was also prepared for studying PEG modification sites targeting cysteine residues. A new cysteine mutation was introduced into the HLA-G2C42S-pGMT7 plasmid so that the HLA-G2 had one free cysteine. The prepared 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), base sequence: SEQ ID NO: 11, amino acid sequence: SEQ ID NO: 12). )
(2) A construct (HLA-G2CTER-pGMT7) in which cysteine is added to the C-terminal (FIG. 11 (d), base sequence: SEQ ID NO: 13, amino acid sequence: SEQ ID NO: 14).

The above two types of plasmids were prepared by a mutation introduction method using a restriction enzyme DpnI. Specifically, each mutation was introduced by PCR reaction using DNA synthase KOD Plus (TOYOBO) and the following primers using HLA-G2C42S-pGMT7 as a template.
-Primers used for mutant production (1) HLA-G2-N86C
N86C_F: 5'-CGCGGCTACTACTGCCAGAGCGGCC-3'(SEQ ID NO: 21)
N86C_R: 5'-GCCGCCGATAGATGACGGTCCGCTCCGG-3'(SEQ ID NO: 22)
(2) HLA-G2-CTER
CTER_F: 5'-TGGAAGCAGTGCGGGTTAAAAGCTTGAA-3'(SEQ ID NO: 23)
CTER_R: 5'-ACCTTCCGTCACGCCAAATTTCGAACTT-3'(SEQ ID NO: 24)

The PCR reaction was performed using a Veriti® thermal cycler (Applied Biosystems) at 94 ° C for 2 minutes, then heat denaturation at 98 ° C for 10 seconds, 60 ° C for 30 seconds annealing, and 68 ° C for 4 minutes. The cycle of the extension reaction was repeated 20 times. After the reaction, 1 μL of DpnI enzyme (TOYOBO) was added, and the mixture was incubated at 37 ° C. for 1 hour.

The sequences of the obtained mutant plasmids were confirmed using the sequencing method. Specifically, 0.5 μL of each plasmid was added to 100 μL of Escherichia 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 100 μg / mL ampicillin-containing Luria-Bertani (LB) agar medium and then cultured overnight at 37 ° C. The obtained single colony was inoculated into 2 × Yeast-tryptone (YT) medium (5 mL) containing 100 μg / mL ampicillin, and cultured with shaking at 37 ° C. and 150 rpm overnight. Centrifugate the entire amount of the cultured bacterial solution (13,200 rpm, 4 ° C., 1 minute), and use a plasmid purification kit (QIAprep® Spin Miniprep kit (250), QIAGEN) from the obtained E. coli pellets to obtain plasmid DNA. Was purified. Purified plasmids were used on a Big Dye® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), T7 promotor or T7 Terminator universal primer, 1 min at Veriti® thermal cycler, and 96% at Veriti® thermal cycler. After heat denaturation, a sequence reaction was carried out by repeating heat denaturation at 96 ° C. for 10 seconds, annealing at 50 ° C. for 5 seconds, and extension reaction at 60 ° C. for 4 minutes 30 times. The sequence reaction solution was precipitated with ethanol, 20 μL of Hi-DiTM formamide (Applied Biosystems) was added, and the mixture was incubated at 95 ° C. for 2 minutes and then analyzed with a sequencer 3130 Genetic Analyzer (Applied Biosystems).

(HLA-G1 recombinant protein expression plasmid)
To prepare the HLA-G1 monomer, the signal sequence was removed, a methionine residue, which is a translation initiation codon, was added to the N-terminal, and the 42nd cysteine of HLA-G1 was replaced with serine in an extracellular expression region (Gly1). An Escherichia coli expression plasmid (HLA-G1C42S-pGMT7) possessed by our laboratory in which −Gln276) was incorporated into a pGM7 vector was used (FIG. 11 (e), nucleotide sequence: SEQ ID NO: 15, amino acid sequence: SEQ ID NO: 16).

(Β2m recombinant protein expression plasmid)
An Escherichia coli expression plasmid (β2m-pGM7) possessed by our laboratory in which the Ig-like domain moiety (Ile1-Met99) having a methionine residue, which is a translation initiation codon, added to the N-terminal, excluding the signal sequence, was incorporated into the pGMT7 vector was used. It was used (FIG. 11 (f), base sequence: SEQ ID NO: 17, amino acid sequence: SEQ ID NO: 18).

(LILRB2virA 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 with the start codon methionine added by removing the signal sequence were integrated into the pGM7 vector, and 17 at the C-terminal. A plasmid for expressing Escherichia coli (LILRB2virA-pGM7) possessed by our laboratory to which a biotinylated enzyme recognition sequence consisting of amino acid residues (GSLHHILDAQKMVWNHR (SEQ ID NO: 25)) was added was used (Shiroishi, M., Tsumoto, K., Amano, K., Shirakihara, Y., Colonna, M., Brand, VM, Allan, DSJ, Makadzage, A., Rowland-Jones, S., Willcox, B., Jones, EY, van der Merwe, PA, Kumagai, I., and Maenaka, K. (2003) Human inhibitory acceptor's Ig-like transcript 2 (ILT2) and ILT4 index Premierly to HLA-G. Proc. Natl. Acad. Sci. USA 100, 8856-61) (FIG. 11 (g), base sequence: SEQ ID NO: 19, amino acid sequence: SEQ ID NO: 20).

(Preparation of inclusions of various recombinant proteins using E. coli expression system)
All recombinant proteins used in this study were expressed as inclusion bodies using Escherichia coli. The HLA-G1, β2m, and LILRB2virA recombinant proteins were expressed as inclusion bodies by transforming Escherichia coli BL21 (DE3) pLysS strain (Novagen) with the various plasmids described above. In addition, HLA-G2 and HLA-G2 mutants (HLA-G2C42S, HLA-G2N86C, HLA-G2CTER) recombinant proteins were prepared using their respective plasmids for Escherichia coli ClearColi® BL21 (DE3) competent cell (ClearColi). The registered trademark) BL21 (DE3) competent cell (Lucien) was transformed into a chemic competent cell in our laboratory) and expressed as an enclosure. Specifically, after transformation, the cells were seeded on LB agar medium containing 100 μg / mL ampicillin and then cultured overnight at 37 ° C. 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 pre-cultured bacterial solution was inoculated into 1 L of 2 × YT medium containing 100 μg / mL ampicillin, and cultured with shaking at 37 ° C. When the Exponential Density (OD) 600 = 0.6, which is the early stage of logarithmic growth, was reached, Isopropanol β-D-1-thiogalactopylanoside (IPTG) was added to a final concentration of 1 mM to induce the expression of the recombinant protein. Then, it was cultured with shaking in INNOVA (Eppendorf) at 150 rpm and 37 ° C. for 5 hours. The bacterial solution obtained by centrifuging the cultured bacterial solution (5000 rpm, 4 ° C., 10 minutes) was suspended in a suspension buffer (50 mM Tris hydroxymethyl aminomethane [Tris] -HCl pH 8.0, 150 mM NaCl) and placed on ice. Ultrasonic crushing was performed in. After crushing, centrifuge at 8000 rpm at 4 ° C. for 5 minutes, and suspend the obtained precipitate as an inclusion body 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. Then, in order to remove the surfactant Triton X-100 from the inclusion body, the same operation was repeated 4 times using a suspension buffer to obtain an inclusion body. The resulting inclusions were placed overnight in solubilization buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 6 M guanidine-HCl, 10 mM Ethylenediamine-N, N, N', N', -tellaacetic acid [EDTA]). It was completely dissolved by allowing it to stand at 4 ° C. After solubilization, the supernatant obtained by centrifuging at 5000 × g at 4 ° C. for 5 minutes was stored as an inclusion body at −80 ° C. All centrifugation operations after protein expression induction were performed using a universal cooling centrifuge (KUBOTA).

(Preparation of recombinant protein by rewinding method)
HLA-G2, HLA-G2 mutants (HLA-G2C42S, HLA-G2N86C, HLA-G2CTER), HLA-G1 and LILRB2virA recombinant proteins were prepared by rewinding by dilution of E. coli inclusion bodies. The final concentration of Dithiothreitol (DTT) in each solubilized inclusion body of HLA-G2 (8 mg), HLA-G2 mutant (8 mg), and LILRB2virA (4 mg) so that the final concentration at the time of dilution is about 1 to 2 μM. It was added to 10 mM and incubated for 1 hour at room temperature. Refolding buffer (0.1M Tris-HCl pH 8.0, 1M L-arginine-HCl, 2 mM EDTA, 3.73 mM cystamine,) containing arginine, which has an inhibitory effect on protein aggregation, in the inclusion denaturing solution reduced by DTT. 6.73 mM cystamine) was added drop by drop until the guanidine concentration reached 1.5 M (a concentration believed to form a disulfide bond and form a secondary structure). Further, the diluted solution was further diluted by adding drop by drop to 200 mL of refolding buffer, and the mixture was stirred at 4 ° C. for 72 hours.

The HLA-G1 recombinant protein needs to be rewound as a heterotrimer of the HLA-G1 heavy chain, β2m, peptide. The β2m inclusion body (4 mg) was diluted in the same manner as in the above method and stirred at 4 ° C. for 4 to 6 hours in order to rewind in order from a unit that is easy to rewind and is stable as a simple substance to obtain a complex. Subsequently, 0.2 mg / 0.2 mL DMSO of completely dissolved HLA-G1 binding synthetic peptide (RIIPRHLQL) was slowly added dropwise to the β2 m refolding solution, and the mixture was further stirred at 4 ° C. for 1 to 2 hours. Finally, the HLA-G1 inclusion body (4 mg) was diluted with a refolding solution containing β2 m and a peptide in the same manner as described above, and stirred at 4 ° C. for 72 hours.

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

Specifically, HLA-G2, HLA-G2 mutants (HLA-G2C42S, HLA-G2N86C, HLA-G2CTER), and HLA-G1 recombinant protein are concentrated to 15 mL or less in the VIVAFLOW system and then filtered. , Hiload 26/60 Superdex75 pg (GE Healthcare) column was used for purification. Furthermore, for the HLA-G1 protein, ion exchange chromatography (IEX) was performed as a secondary purification. The desired 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 dialysis. After filtering, it was injected into a Resource Q 1 mL (GE Healthcare) column. IEX was performed under the condition of 20 mM Tris-HCl pH 8.0, 0-0.5 M NaCl / 20 Volume volume (CV).

The LILRB2virA recombinant protein was concentrated to 0.5 mL or less using the VIVAFLOW system and Amicon Ultra, filtered, and purified on a Superdex75 10/300 GL (GE Healthcare) column. As the running buffer, 20 mM Tris-HCl pH 8.0, 200 mM NaCl was used.

(Preparation of biotinylated LILRB2)
LILRB2virA purified by the SEC method, by volume ratio, LILRB2virA: 5 × BiomixA buffer (0.25M bicine buffer pH 8.3): 5 × BiomixB buffer (50 mM Biotin Atembito The mixture was mixed at a ratio of 1: 1, and 1 μL of BirA enzyme (prepared in our laboratory) was added, and the mixture was incubated at 30 ° C. for 1 hour. Then, in order to remove unreacted biotin, SEC purification was performed on a Superdex 75 10/300 GL (GE Healthcare) column. As the running buffer, 20 mM Tris-HCl pH 8.0, 400 mM NaCl was used.

As a result of SEC purification of the HLA-G2 protein prepared as described above, an elution peak was found at a position corresponding to a molecular weight (44 kDa) approximately twice the target molecular weight (HLA-G2: 22 kDa), in agreement with the findings so far. It was obtained (Fig. 2 (a)).

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

(Reducing agent)
Prior to the PEGylation reaction, the disulfide bond was cleaved by a reduction treatment to re-expose the thiol group of the Cys42 residue to be the PEGylation target, and a reducing agent was added to improve the reaction efficiency.

The purified HLA-G2 protein was concentrated by ultrafiltration using an Amazon Ultra (MWCO: 10000 Da, Merck Millipore) and replaced with a PEGylated buffer (1 x Phosphate-Buffer Saline (PBS), 5 mM EDTA). The solution prepared to 0.5 mL was degassed using an aspirator for 1 hour in order to allow the PEGylation reaction to proceed under anaerobic conditions. Subsequently, the reducing agent tris (2-carboxythyl) phophine (TCEP) was added to a final concentration of 0.1, 0.5, 1, 5, and 10 mM, then PEG20 was added, and the mixture was reacted overnight at 4 ° C. It was. As the reducing agent, tris (2-carboxythyl) phosphorine (TCEP), which is odorless and easy to use and is often used in the reaction using maleimide and cysteine, was used.

To proceed with the PEGylation reaction, the reaction solution was subjected to Sodium Dodecyl Sulfate-Poly Stain Gel Electrophoresis (SDS-PAGE) (12.5% acrylamide gel, 30 mA / sheet, 70 minutes electrophoresis) under non-reducing conditions, and Coomassie Brilli (Coomasie Brilli). ) Staining and Barium Iodide (BaI ₂ ) staining to detect PEG molecules. BaI2 staining, 5% BaI ₂ solution (15 min) acrylamide gel after electrophoresis, ion-exchanged water (30 minutes) and shaken immersed in the order of 0.1M iodine solution (5 min), was carried out in the procedure of.

Purification of PEGylated HLA-G2 was performed using Superdex 200 10/300 GL (GE Healthcare), Superdex 75 10/300 GL (GE Healthcare), or Superose6 10/300 GL (GE Healthcare) columns. 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 driven into a column. .. For 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 by silver staining (2D-SILVER STAIN REAGENT II, Cosmo Bio Co., Ltd.).

When the reaction solution was developed by SDS-PAGE as described above and stained with CBB, 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 of a final TCEP concentration of 0.1 mM. Therefore, in the subsequent experiments, the PEGylation reaction was performed under the condition of 0.1 mM TCEP treatment.

In order to confirm whether PEG specifically binds to the Cys42 residue of the target HLA-G2 protein, a variant in which the cysteine residue is replaced with a serine residue (HLA-G2C42S, FIG. 11B). ) Recombinant protein was prepared and compared with HLA-G2 protein. The HLA-G2C42S protein was prepared in the same manner as HLA-G2, and the desired peak fraction was recovered and used in the PEGylation reaction. Since HLA-G2C42S lost a free cysteine residue, multimer formation did not occur, and no band was observed at the high molecular weight position suggesting multimer formation even in non-reducing SDS-PAGE (not shown).

From the results of examining 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 ejector degassing (1 hour, on ice).
(2) Addition of reducing agent during PEGylation reaction: After degassing the aspirator, TCEP is added to the protein solution to a final concentration of 0.1 mM before adding the PEG reagent.
(3) 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: PEG: HLA-G2 = 10: 1 (molar ratio).

(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 the PEG to be bound, four kinds of molecular weights (5, 10, 20, 40 kDa) having maleimide as a reactive group are used. PEGylation of HLA-G2 was performed 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)

After developing the PEGylated reaction solution with SDS-PAGE, CBB staining and BaI ₂ staining were performed to confirm the progress of the reaction, and all four types of PEGylated proteins were confirmed (FIGS. 3 (a) and 3 (b)). .. Further, from the density of the band of the PEGylated protein, it was clarified that the low molecular weight PEG (PEG5, PEG10) had a higher PEGylation reaction rate.

As a method for purifying these PEGylated HLA-G2, SEC for separation using the difference in molecular weight before and after PEGylation was selected. SEC purification was performed on four types of PEG-forming reaction solutions, and the obtained peak fraction was confirmed by silver staining after SDS-PAGE. As a result, PEG10-HLA-G2 and PEG20-HLA-G2 were confirmed by SEC. The target PEGylated HLA-G2 could be separated from the unreacted HLA-G2 protein and purified (FIGS. 4 (b) and 4 (c)).

(Experiment with binding to receptor LILRB2)
Subsequently, PEG10-HLA-G2 and PEG20-HLA-G2 that could be purified as PEGylated proteins, and PEG5-HLA-G2 SEC-purified samples obtained as SEC peaks mainly containing PEGylated proteins were used. The interaction with the receptor LILRB2 was analyzed by Surface Plasmon Resonance (SPR), and it was confirmed whether HLA-G2 to which the PEG of each molecular weight was bound maintained the receptor binding ability. HLA-G2 that was not PEGylated was used as a comparative control.

As described above, LILRB2 was prepared as biotinylated LILRB2 by SEC purification after site-specific biotinlation with an enzyme using a purified LILRB2virA protein having a biotinylated tag added to the C-terminal side.

As analysts, HLA-G2 (0.2 to 0.7 μM), PEG5-HLA-G2 (0.4 to 1.6 μM), and PEG10-HLA-G2 (0.3 to 1.), which were continuously diluted 2-fold each. 1 μM), PEG20-HLA-G2 (0.3 to 1.1 μM) was flowed.

(Interaction analysis by Surface Plasmon Resonance (SPR))
In order to confirm the effect of PEGylated HLA-G2 on LILRB2 binding, an interaction analysis by the SPR method was performed using Biacore3000 (GE Healthcare). Biotinylated LILRB2 on the sensor chip 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)), and streptavidin is immobilized on the chip by hybridizing the complementary strand DNA to which streptavidin is added (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)).

As an analyzer, HBS-EP buffer (10 mM Na-HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA), 0.005% (v), which is a running buffer by ultrafiltration using Amicon Ultra (MWCO: 10000, Millipore). / V) For each HLA-G2 and PEGylated HLA-G2 protein solution substituted with Buffertant P20: GE Healthcare), samples diluted 2-fold in 3 steps were serially diluted in order from the lowest concentration at a flow rate of 10 μL / min. Shed. Kinetics were measured at a measurement temperature of 25 ° C. and a binding time and a dissociation time of 120 seconds each. BIA evolution version: 4.1.1 (GE Healthcare) was used for the analysis.

As a result of the binding analysis, it was possible to confirm the binding to LILRB2 for all three types of PEGylated products (FIGS. 5 (b)-(d)).

From the above results, since there is no significant difference in PEGylation efficiency and binding ability to the receptor between the three molecular weights, it is considered that the degree of purification of the PEGylated protein is high and further contributes to the stability of the PEGylated protein. It was decided to carry out the future PEGylation reaction and analysis using 20 kDa PEG having the largest PEG molecular weight among the three.

(Examination of PEG introduction site)
So far, PEG has been introduced by targeting the Cys42 residue originally possessed by the HLA-G2 protein, but the site to be PEGylated is expected to improve reactivity and functionality by changing the introduction site of PEG. Was examined.

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-terminal of the HLA-G2 protein located on the cell membrane side in the membrane-bound HLA-G2, And a construct (HLA-G2N86C) in which the 86th asparagine residue, which is predicted to have a sugar chain added to HLA-G2, is replaced with a cysteine residue because it is a glycosylated site of HLA-G1. It was prepared and the protein was prepared. Introducing PEG into these locations was not considered to affect receptor binding.

In these mutant proteins, the free cysteine residue (Cys42 residue) originally possessed by the HLA-G2 protein is replaced with a serine residue, and the PEG molecule is introduced into only one of these mutation sites. Is expected.

Each HLA-G2 mutant protein was expressed as an inclusion body using Escherichia coli as described above. Further, as described above, after rewinding each protein, SEC purification was performed, and the eluted fraction of each HLA-G2 mutant was collected.

Subsequently, each purified mutant protein was PEGylated with PEG20 in the same manner as described above. The progress of the PEGylation reaction was confirmed from the results _{of SDS-PAGE, CBB and BaI 2} staining of the PEGylation reaction solution for all HLA-G2 mutant proteins in which the PEG introduction site was changed (FIG. 6 (a)). ), (B)).

(Example 2)
(In vitro stability evaluation of PEGylated HLA-G2)
The PEG20-HLA-G2 obtained in Example 1 was used to evaluate in vitro stability.

(Stability evaluation against freeze-drying)
Biopharmacy is generally stored lyophilized from the time it is manufactured until it is actually used, in order to avoid the effects of external factors such as oxygen, sunlight and pH. Therefore, whether or not the lyophilized drug does not decompose or aggregate even after re-dissolution and maintains its biological activity is a very important index for commercializing a biopharmacy. Therefore, in this study, we verified whether PEGylation improves stability against freeze-drying.

SEC-purified HLA-G2 and PEG20-HLA-G2 were freeze-dried, redissolved in sterilized water, and then SDS-PAGE was performed. 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 proteins were redissolved in an equivalent amount (50 μL) of sterilized water, subjected to SDS-PAGE under non-reducing conditions, and detected by CBB staining to compare the protein states before and after lyophilization.

The degree of aggregation / decomposition, which is an index of stability as an HLA-G2 molecule, was compared as a band pattern with or without freeze-drying treatment. As a result, no aggregation or decomposition of the HLA-G2 molecule by freeze-drying occurred regardless of the presence or absence of PEGylation, and no clear difference was observed (FIG. 7 (a)).

Subsequently, in order to verify whether the receptor binding activity of HLA-G2 and PEG20-HLA-G2 is changed by the lyophilization treatment and whether there is a difference in the degree due to PEGylation, the lyophilization treatment is carried out in the same manner as described above. HLA-G2 and PEG20-HLA-G2 were subjected to a binding experiment with the receptor LILRB2 using the SPR method. In the same manner as described above, biotinylated LILRB2 was immobilized on the sensor chip via streptavidin, and there was 2-fold serial dilution (HLA-G2: 0.3 to 1.1 μM, PEG20-HLA-G2: 0.3 to 1. 1 μM) freeze-dried HLA-G2 and PEG20-HLA-G2 were flowed as an analyzer.

First, in order to compare the proportion of molecules that lost their ability to bind to the receptor due to the freeze-drying treatment between HLA-G2 and PEG20-HLA-G2, the sensorgram obtained in this binding experiment was freeze-dried. It was compared with the sensorgrams (FIGS. 5 (a) and 5 (d)) of the results of a binding experiment with LILRB2 using untreated HLA-G2 and PEG20-HLA-G2. In both binding experiments, HLA-G2 and PEG20-HLA-G2 used samples from the same lot. The amount of LILRB2 immobilization was matched between the two.

Focusing on the sensorgram when flowing at a sample concentration of around 0.6 μM, the binding response value for LILRB2 decreased from about 250 RU to about 150 RU before and after freeze-drying in HLA-G2 (FIG. 7 (b)). On the other hand, PEG20-HLA-G2 did not show a significant decrease in response (Fig. 7 (c)).

From this result, it was found that the proportion of molecules in PEG20-HLA-G2 that retain the receptor-binding activity even after lyophilization is higher than that in HLA-G2. From the above, it was suggested that the HLA-G2 protein is improved in stability against freeze-drying by being PEGylated.

(Evaluation of thermal stability)
Purified HLA-G2 and PEG20-HLA-G2 were incubated under 50 ° C. and 60 ° C. conditions to verify whether PEGylation improved the thermal stability of the HLA-G2 protein. 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 collecting all the samples, SDS-PAGE was performed under reducing or non-reducing conditions, and the degree of proteolysis / aggregation was compared.

More specifically, SEC-purified HLA-G2 and PEG20-HLA-G2 (0.08 mg / mL, 20 mM Tris-HCl pH 8.0, 100 mM NaCl) were incubated at 50, 60, and 70 ° C., respectively, at 7 ° C. , 5 × SDS-PAGE Sample buffer (25 mM Tris-HCl pH 6.5, 5% glycerol, 1% SDS, 0.05% chloride blue) was added to the sample collected after 24 and 48 hours, and the sample was stored at 4 ° C. After collecting all samples, SDS-PAGE was performed under non-reducing conditions (12.5% or 15% acrylamide gel, 30 mA / sheet, 70 minutes electrophoresis), and detection by CBB staining was performed to determine the degree of protein degradation and the degree of protein degradation. The degree of multimer / aggregate formation was compared.

From the results of electrophoresis under non-reducing conditions, the band of the target substance disappeared in HLA-G2 under high temperature conditions, and the number of aggregates of 40 kDa or more and the band considered to be a decomposition product around 12 kDa increased (FIG. 8 (a)). On the other hand, in PEG20-HLA-G2, the disappearance of the target band and the appearance of non-target bands were small even under high temperature conditions (FIG. 8 (b)).

From the above results, it was found that PEGylation suppresses protein aggregation and degradation caused under high temperature conditions.

(Evaluation of serum stability)
Regarding the prepared PEG20-HLA-G2, it was investigated whether the degradation or aggregation of HLA-G2 in serum (FBS) was suppressed by PEGylation in an in vitro system.

Purified HLA-G2 and PEG20-HLA-G2 were incubated under 5% FBS, 37 ° C. conditions. Samples were collected every hour from 24 hours after the start of incubation as 0 hours, SDS-PAGE Sample buffer was added, and the mixture was stored at 4 ° C. After collecting all the samples, the residual amounts of HLA-G2 and PEG20-HLA-G2 were quantified using SDS-PAGE and Western blotting under non-reducing conditions.

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%. Incubation was carried out under 37 ° C. conditions for 22 to 30 hours, 5 × SDS-PAGE Sample buffer was added to the sample collected every 2 hours, and the sample was stored at 4 ° C. After collecting all the samples, the degree of protein degradation was compared by Western blotting. Western blotting involves developing a sample on SDS-PAGE (12.5% or 15% acrylamide gel, 30 mA / sheet, 70 minutes electrophoresis) under non-reducing conditions and then transferring it to a Poly Vinylidene Di Fluoride (PVDF) membrane (Bio Rad). did. Membranes were blocked with 5% skim milk (Skim Milk) / Phosphate-buffered saline (PBS) -T by shaking at room temperature for 1 hour or overnight. Then, 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. As a secondary antibody, it was reacted with an anti-mouse IgG (Fc) -Horserdic peroxidase (HRP) -labeled antibody (Thremo Fisher Scientific) (× 1/10000) by shaking at room temperature for 1 hour. After that, the membrane was washed with PBS-T several times, emitted with ECL Prime (GE Healthcare), and detected using Image Quant LAS4000mini (GE Healthcare).

The MEM-G1 antibody used for the Western blotting method this time is an HLA-G specific antibody, and although the epitope site is unknown, it has been confirmed in our laboratory that the denatured HLA-G2 can be detected. (Unpublished data). In addition, in a preliminary experiment, it was confirmed that PEG20-HLA-G2 can be detected by Western blotting using the MEM-G1 antibody, and this experiment was performed.

Comparing the changes over time in the amounts of HLA-G2 and PEG20-HLA-G2 in the presence of 5% FBS, the rate of protein degradation and disappearance was higher in HLA-G2 than in PEG20-HLA-G2. Especially, it was high in 26 to 30 hours (Fig. 9 (a)). This band was quantified using ImageQuant LAS 4000, and the residual amount of the band for each incubation time was shown in the graph. As a result, the protein disappearance rate of PEG20-HLA-G2 was slower than that of HLA-G2. Was found (Fig. 9 (b)).

From the above results, it was found that the stability of the HLA-G2 protein in serum tends to be improved by PEGylation.

(Example 3)
(Evaluation of HLA-G2, PEGylated HLA-G2 anti-inflammatory effect in vivo)
The in vivo anti-inflammatory effects of HLA-G2 and PEG20-HLA-G2 proteins were examined using atopic dermatitis disease model mice.

Specifically, HLA-G2, PEG20-HLA-G2 in the auricle of a dermatitis onset model mouse, and HLA-G1 whose therapeutic effect was confirmed in a mite antigen-induced dermatitis model mouse as a positive control in our laboratory. Was administered, and the degree of inflammation was observed. PBS was used as a negative control. Atopic dermatitis disease model mice were prepared by applying an atopic dermatitis-inducing ointment (Biosta AD) containing a Kona leopard mite component to the surface of the auricle of a mouse.

(Preparation of atopic dermatitis model mouse)
Prior to protein administration, 100 mg of Biosuta AD was applied to NC / Nga Slc mice every 3 days for a total of 6 times in order to prepare atopic dermatitis model mice. More specifically, 16 NC / Nga Slc mice (10 weeks old, male) purchased from Nippon SLC Co., Ltd. were used with an electric clipper and a hair removal cream to remove hair from the back of the ear. Atopic dermatitis was caused by applying 100 mg of atopic dermatitis-inducing ointment (Biosta AD (registered trademark)) containing a Kona leopard mite component to the surface of the auricle from which hair was removed every 3 days for a total of 6 times. (Fig. 10 (a)). However, in the second and subsequent induction operations, the operation of applying 4% SDS to the auricle was performed before applying Biosuta AD. The group (1 animal) not treated with the atopic dermatitis-inducing ointment was used as a control group for the onset of dermatitis.

In the group of mice to which Biosuta AD was applied, symptoms such as pruritus, erythema, and bleeding characteristic of atopic dermatitis were observed as compared with the group of control mice to which Biosuta AD was not applied, and atopic dermatitis was observed. I was able to confirm the induction of.

(Preparation of each administered protein (HLA-G2, PEG20-HLA-G2, HLA-G1))
Subsequently, each administered 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 a dialysis method to prepare a protein solution to be administered.

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 an endotoxin-containing BL21 (DE3) pLysS strain, a solution that had been purified by IEX and replaced with PBS buffer and then LPS-removed was used as the administered protein.

(Protein administration to the prepared dermatitis model mouse)
The prepared HLA-G2, PEG20-HLA-G2, and HLA-G1 were transdermally administered to the auricle surface of mice 10 times every other day at 5 μg / ear (4 animals in each group) to determine the degree of ear inflammation. , The thickness of the auricle was measured using a dial thickness gauge (Ozaki Seisakusho) on the 0th, 6th, 10th, 14th, 18th, and 22nd days after the start of administration (FIG. 10 (a)).

Statistical analysis was performed using JMP® 11 (SAS Institute Inc., Cary, NC, USA) software. A t-test was performed on the degree of auricular inflammation 22 days after the start of protein administration, and HLA-G2, PEG20-HLA-G2 administration group and PBS administration group, and HLA-G2 administration group and PEG20-HLA-G2 administration. We examined whether there was a statistically significant difference between the groups.

The anti-inflammatory effect was confirmed by administration of HLA-G2, PEG20-HLA-G2, and HLA-G1 from the 0th day to the 22nd day after the start of administration. Furthermore, when comparing the HLA-G2 administration group and the PEG20-HLA-G2 administration group, a stronger anti-inflammatory effect was observed in the PEG20-HLA-G2 administration group as compared with the HLA-G2 administration group (FIG. 10 (b), FIG. (C)). Regarding the degree of swelling of both auricles on the 22nd day, 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)). In addition, no weight loss was observed by administration of the test substance (not shown).

From the results of the above protein administration experiments, it was found that PEG20-HLA-G2 exhibits a sufficient anti-inflammatory effect and therapeutic effect as an anti-inflammatory agent on atopic dermatitis mice without any side effects. It was also revealed that the effect was higher with PEG20-HLA-G2 than with HLA-G2.

From the above, it was confirmed that the addition of PEG to the free cysteine residue (Cys42) exposed on the surface of the HLA-G2 molecule improved the temperature stability, freeze-drying resistance, and blood stability in vitro. .. Furthermore, it was clarified that the anti-inflammatory effect in atopic dermatitis mice was enhanced by PEGylation in vivo, and the PEGylated HLA-G2 had a greater anti-inflammatory effect than HLA-G2 in vivo in mice. It was suggested that the stability was improved. By using PEGylated HLA-G2, it is expected that the dose and the number of administrations can be reduced, and it is expected that the financial and physical burden on the patient can be reduced even when it is assumed that the drug is used in combination with other drugs.

Claims (8)

It consists of a multimeric protein 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 cysteine residue in the amino acid sequence constituting the protein is PEGylated and modified with polyethylene glycol (PEG).
A modified protein characterized by that. The molecular weight of PEG used for PEGylation modification is 5 kDa to 100 kDa.
The modified protein according to claim 1. The protein is a protein consisting of the amino acid sequence described in (a) or (b) below.
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
(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.
The multimer is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b), and has a binding activity to the leukocyte Ig-like receptor B2. ,
The modified protein according to claim 1 or 2. A pharmaceutical product containing the modified protein according to any one of claims 1 to 3 or a salt thereof as an active ingredient. A prophylactic or therapeutic agent for an inflammatory disease containing the modified protein according to any one of claims 1 to 3 or a salt thereof as an active ingredient. (A) A step of preparing 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
(B) A step of degassing and then reducing the multimer of the protein obtained in the step (A), and a step of reducing the protein.
(C) A step of PEGylating and modifying the multimer of the protein subjected to the reduction treatment in the step (B), and
A method for producing a modified protein containing. 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, wherein the modified protein is produced. The protein is a protein consisting of the amino acid sequence described in (a) or (b) below.
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
(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.
The multimer is a homomultimer of (a) or (b) or a heteromultimer of (a) and (b), and has a binding activity to the leukocyte Ig-like receptor B2. ,
The method for producing a modified protein according to claim 6 or 7.

Images