WO2007011044A1

WO2007011044A1 - Pharmaceutical composition comprising disulfide-linked hla-g dimer and process for production of disulfide-linked hla-g dimer

Info

Publication number: WO2007011044A1
Application number: PCT/JP2006/314537
Authority: WO
Inventors: Katsumi Maenaka; Mitsunori Shiroishi; Daisuke Kohda; Kimiko Kuroki; Linda Rasubala; Hisashi Arase
Original assignee: Kyushu University, National University Corporation; Osaka University
Priority date: 2005-07-15
Filing date: 2006-07-18
Publication date: 2007-01-25

Abstract

Disclosed is novel use of an HLA-G dimer. Also disclosed is a process for producing an HLA-G dimer with high efficiency. A pharmaceutical composition comprising an HLA-G dimer; and a process for producing an HLA-G dimer comprising the step of adding a reducing agent to a solution containing HLA-G.

Description

Specification

TECHNICAL FIELD The present invention relates to a pharmaceutical composition containing a disulfide-linked HLA-G dimer and a method for producing a disulfide-linked HLA-G dimer

The present invention relates to a pharmaceutical composition containing a disulfide-bonded HLA-G dimer, a method for producing the HLA-G dimer, and suppression of activation of immune system cells using the HLA-G dimer. The present invention relates to pharmaceuticals and inflammatory disease prevention and treatment methods that are required to have an effect, particularly an inflammation-suppressing effect. Background art

Unlike classical MHC class I molecules, HLA-G, one of the major non-classical major histocompatibility antigens (MHC), is expressed in a tissue-specific manner. HLA-G is expressed particularly in the fetal trophoblast on the placenta, which is important during pregnancy, and is involved in suppressing immunity so that the maternal immune system recognizes the fetus as a foreign body and does not attack it. It has been known. Based on this knowledge, HLA-G has been applied in various ways. For example, HLA-G molecules with improved genetic engineering can be used for infertility treatment, and rejection of organ transplantation can be suppressed. Attempts have been made to use HLA-G molecules.

Conventionally, there are many analysis examples regarding the role of HLA-G at the cellular level, and the suppressive effect of immune system cells such as natural killer (NK) cells and macrophages has been confirmed. However, what has been confirmed at the molecular level as a receptor for HLA-G is mainly CD8 molecule expressed in T cells and leukocyte Ig-like receptor (LILR) (eg, Ig-) expressed in a wide range of immune system cells. like transcript (ILT), CD85d, LIR) There are only a few members in the receptor group. Therefore, the inhibitory effect of HLA-G is probably carried out through CD8 and LILR. As a detailed analysis example of the binding between HLA-G and the receptor, the present inventor has reported the interaction between LILR group (LILRB1 and B2) and HLA-G (Shiroishi et al. al "Pro Natl. Acad. Sci. USA" 2003, Vol.100, No.15, p.8856-8861). However, other similar analysis examples are not well known.

By the way, it is also known that HLA-G molecules can exist as dimers by natural oxidation for 2 to 3 months. This dimer formation is thought to be due to the free cysteine residues of the HLA-G molecules forming disulfide bonds between the molecules. However, studies at the cellular and molecular levels have not been sufficiently conducted to determine whether HLA-G dimers have the same functions as HLA-G monomers, and useful knowledge has not been obtained. Absent. Hereinafter, the background art of the present invention will be described in more detail.

(1) Knowledge of the molecular mechanism of maternal-fetal tolerance is still limited. Unlike normal cells, the fetal extravillous trophoblast cell layer at the maternal-fetal interface expresses the majority of classical MHC I (major histocompatibility antigen class I molecules), HLA-A or HLA-B But not a few classical MHC I, HLA-C and non-classical MHC I, HLA-E and HLA-G (LeMaoult, I, et al., (2003) Tissue Antigens, 62, 273- 284.) Impaired expression of HLA-A and HLA-B significantly inhibits maternal T cell responses. HLA-C and HLA-E are expressed in normal cells, but HLA-G expression is limited to a few tissues: extravillous trophoblast, thymic epithelial cells and some tumors (LeMaoult et al ., 2003 (supra)). In contrast to the polymorphic classical MHC I, HLA-G exhibits a limited polymorphism, which indicates that HLA-G can accept genetic immunosuppression when protecting fetal cells from maternal immune cells. Suggesting that it plays a role as a common ligand to the body. Recently, several immunologically related cell surface receptors have been found to mediate negative regulation of immune cells by binding to classical and nonclassical MHC I. The receptors for HLA-G reported to date are CD8, leukocyte Ig-like receptor B1ZB2 (also known as LILRB1 / LILRB2, LIRlZLIR2 and ILT2 / ILT4) and KIR2DL4. Although the molecular rationale for KIR2DL4-HLA-G interaction is still a subject of debate (LeMaoult et al., 2003 (supra)), the recognition of LILRB1 / 2- and CD8- HLA-G has been studied. Is underway (

Chapman, TL, et al., (1999) Immunity, 11, 603-613 .; Gao, GF, et al., (2000) J Biol Chem, 275, 15232-15238 .; Shiroishi, M "et al., (2003) Proc Natl Acad Sci USA, 100, 8856-8861.).

LILRB1 is expressed on a wide range of white blood cells, including natural killer (NK) cells and T cells, while LILRB2 is expressed on limited immune cells, including monocytes and dendritic cells (DC). (Brown, D., et al., (2004) Tissue Antigens, 64, 215-225.). Both LILRBs have four Ig-like domains in the extracellular region, and two N-like domains play an important role in MHC I recognition. When MHC I molecules bind to the domain, LILRB mediates negative signals through three or four immunoreceptor tyrosine inhibitory motifs (ITIM) in the intracellular domain. HLA-G molecules at the maternal-fetal interface recognize LILRB and inhibit the immune response of a wide range of maternal immune cells, including bone marrow monocytic cells, T cells, and NK cells. On the other hand, HLA-G has several soluble forms such as splice variants (Fujii, T., et al., (1994) J Immunol, 153, 5516-5524 .; Ishitani, A. et al. (1992) Proc Natl Acad Sci USA, 89, 3947-3951.; Le Bouteiller, P., et al., (2003) Placenta, 24 Suppl A, S10-15.). Soluble form of HLA-G induced CD8 + T cell dysfunction that could promote semi-allogenic pregnancy (Contini, P., et al., (2003) Eur J Immunol, 33, 125- Fournel, S., et al., (2000) J Immunol, 164, 6100-6104 .; Morales, PJ, et al., (2003) J Immunol, 171, 6215-6224.). Thus, HLA-G-LILRB and CD8 interactions can induce extensive immunological tolerance.

Elution peptides and sequencing experiments have shown that HLA-G can present a diverse set of peptides (Diehl, Μ, et al., (1996) Curr Biol, 6, 305-314 .; Lee, N., et al., (1995) Immunity, 3, 591-600.). This peptide display is more classical MHC than non-classical MHC I, HLA-E, which can present heat shock and viral proteins as well as a very limited repertoire of peptides containing the MHC I signal sequence. Similar to I's. HLA-G has a truncated cytoplasmic domain that lacks the endocytosis motif, so it is slow to transport to the cell surface and has a long lifetime (Park, B. et al., (2003) J Biol Chem, 278 , 14337-14345 .; Park, B., et al., (2001) Immunity, 15, 213-224.). As a result, HLA'G will select a high-affinity raw peptide set limited for presentation, even if it has the classical MHC I peptide presentation mechanism. Recently reported The crystal structure of the HLA-G Cys42Ser mutant monomer (Clements, CS, et al., (2005) Proc Natl Acad Sci US A.) also shows that peptide recognition of HLA-G includes an extensive network. This supports a constrained peptide binding mode.

HLA-G, unlike most other MHC I, has two free cysteine residues (Cys42 and Cysl47). Boyson et al. Reported that bacterial recombinant soluble HLA-G can form disulfide-linked dimers with intermolecular Cys42-Cys42 disulfide bonds (Boyson, IE., Et al "( 2002) Proc Natl Acad Sci USA, 99, 16180-16185.) In addition, soluble HLA-Gl expressed by human fetal kidney 293 cells can reduce the CD8 expression level in CTL, a monomeric form (Morales et al., 2003 (supra)) However, it is not clear how much effect each form has on the other hand. Bound HLA-G can also form disulfide-linked dimers on the cell surface of HLA-G transfectants (Boyson et al., 2002 (supra); Gonen-Gross, T., et al., (2003a) J Immunol, 171, 1343-1351 .; Gonen-Gross, T., et al., (2003b) Hum Immunol, 64: 1011-1016.). Studies have suggested that HLA-G dimers play an important role in efficient LILRB1-mediated inhibition of NK cell killing activity (Gonen-Gross et al., 2003a (supra); Gonen-Gross et al ., 2003b (supra)).

A disulfide-linked homodimer also has a free cysteine (Cys67)] 3 2m free form of HLA-B27. Previous reports (Allen, R 丄., Et al., (2001) J Immunol, 167, 5543-5547 .; Kollnberger, S., et al., (2002) Arthritis Rheum, 46, 2972- 2982.) Indicate that this homodimer is directly related to the development of ankylosing spondylitis. In contrast, other reports have argued that the dimer organization may be part of a multimer that exhibits significant function in signal transduction (Colbert, RA (2004) Curr Mol Med, 4, 21-30). Damjanovich, S., et al., (2002) Immunol Lett, 82, 93-99.). Furthermore, significant levels of expression of the 32m free disulfide-bound MHC I dimer were observed on the cell surface of activated T cells rather than resting normal T cells. This suggests that MHC I dimers are important in the regulation of T cell activation (Santos, S.G., et al., (2004) J Biol Chem, 279, 53062-53070.).

In our previous report (Shiroishi, M., et al., (2003) Proc Natl Acad Sci USA, 100, 8856-8861.), LILRBl and LILRB2 bind preferentially to monomeric HLA-G compared to other classical MHC I, and the binding site overlaps with that of CD8. Proved. However, the three-dimensional structure and receptor binding properties of disulfide-linked wild-type HLA-G dimers are not explained at all.

(2) HLA-G is one of the non-classical MHC I molecules and has several unique properties that differ from classical MHC I as follows.

ω Few tissues: very limited expression in extravillous trophoblasts, thymic epithelial cells, and some tumors (J. LeMaoult, et al., (2003) Tissue Antigens, 62, 273-84.)

(ii) Limited polymorphism

Gii) Slow transportation

(iv) relatively limited peptide presentation

The HLA-G receptor already reported is leukocyte Ig-like receptor B1 / B2 (

Also known as LILRB1 / LILRB2, LIR1 / LIR2 and ILT2 / ILT4), CD8 and KIR2DL4. Reports on KIR2DL4-HLA-G recognition are still controversial (J. LeMaoult, et al., (2003) (supra)), 1 ^ 11 ^ 181 / 2- and 008-111 ^-& Interactions are well studied (TL Chapman, et al., (1999) Immunity, 11, 603-13 .; M. Shiroishi, et al "(2003) Proc Natl Acad Sci USA, 100, 8856-61 GF Gao, et al., (2000) J Biol Chem, 275, 15232-8.) Within the LILR family, LILRB1, LILRB2 and LIR6 were reported to bind to MHC I. LILRB2 Expressed mainly in monocytes and dendritic cells (DC) (D. Brown, et al., (2004) Tissue Antigens, 64, 215-25.), Whereas LILRB1 is a natural killer (NK) cell and It is expressed in a wide range of leukocytes, including T cells According to our previous studies, LILRB1 and LILRB2 preferentially become monomeric HLA-G compared to other classical MHC I. (M. Shiroishi, et al., (2 003) (supra)).

LILRB has an immunoreceptor tyrosine inhibitory motif (ITIM) in its intracellular domain and mediates a negative signal when MHC I binds. HLA-G molecules bind to LILRB and inhibit the immune response of a wide range of immune cells, including bone marrow monocytic cells, T cells and NK cells. On the other hand, HLA-G has some splice variants It has a soluble form (P. Le Bouteiller, et al., (2003) Placenta, 24 Suppl A, S10-5 .; T. Fujii, et al., (1994) J Immunol, 153, 5516-24 .; A Ishitani et al., (1992) Proc Natl Acad Sci USA, 89, 3947-51.). Soluble HLA-G induced CD8 + T cell dysfunction that could promote semi-allogenic pregnancy (PJ Morales, et al., (2003) J Immunol, 171, 6215-24 .; S. Fournel , et al., (2000) J Immunol, 164, 6100-4 .; P. Contini, et al.,

(2003) Eur J Immunol, 33, 125-34.). Thus, HLA-G'LILRB and CD8 interactions can induce a wide range of immunological tolerance.

HLA-G has two free cysteine residues (Cys42 and Cysl47). The unwound HLA-G molecule formed a disulfide-linked dimer with an intermolecular Cys42-Cys42 disulfide bond (JE Boyson, et al., (2002) Proc Natl Acad Sci USA, 99, 16180. -Five.) . Soluble HLA-G molecules expressed by human 293 cells also showed disulfide-linked dimer and further oligomeric forms. These were able to reduce the level of CD8 expression in CTL (P.J. Morales, et al., (2003) (supra)) Force It remains unclear how much effect each form has. On the other hand, membrane-bound HLA-G also showed disulfide-linked dimer in the case of HLA-G transfectant (JE Boyson, et al., (2002) (supra); T. Gonen- Gross, et al., (2003) Hum Immunol, 64, 1011-6 .; T. Gonen-Gross, et al., (2003) J Immunol, 171, 1343-51.). Furthermore, mutagenesis studies suggested that HLA-G dimers efficiently inhibit the killing activity of NK cells (T. Gonen-Gross, et al., (2003) (supra); Gonen-Gross, et al., (2003) (supra)).

LILRB 1 / HLA-A11 complex structure reveals that LILRB1 recognizes the _α 3 domain of the heavy chain and the region spanning j8 2-microglobulin opposite the free cysteine residue (Cys42) . In addition, a recent report on the monomer structure of the HLA-G Cys42Ser mutant proposed a dimer model with a Cys42-Cys42 disulfide bond, which indicates that the LILRB1 binding site is completely involved in LILRB binding. It suggests that it can be used. However, the three-dimensional structure of disulfide-linked wild-type HLA-G dimer is still unknown. Disclosure of the invention The problem to be solved by the present invention is to provide a novel use of disulfide-linked HLA-G dimer. Another object of the present invention is to provide an efficient method for producing a disulfide-linked HLA-G dimer.

The present inventor has intensively studied to solve the above problems.

Specifically, the present inventor first confirmed whether the HLA-G dimer has the ability to bind to LILR. As a result, it was confirmed by native gel shift assembly, equilibrium gel filtration method, surface plasmon resonance analysis, and the like that the HLA-G dimer has a considerably stronger binding ability than the HLA-G monomer. In particular, in surface plasmon resonance analysis, about 1000 times stronger binding was observed when HLA-G dimer was run against immobilized LILRB. As a result of X-ray crystallographic analysis of the HLA-G dimer, the structure of the HLA-G homodimer was stabilized by interactions such as the intermolecular disulfide bond and salt bridge of Cys42-Cys42. It was confirmed that The HLA-G dimer has a structure in which the binding sites of LILR and CD8 are directed toward the solvent side by dimerization, and two receptors can recognize one HLA-G dimer. Met. Furthermore, from modeling, the two HLA-G molecules in the HLA-G dimer are dimerized with their C-terminals oriented in the same direction and the binding sites for LILR and CD8 exposed. Therefore, it was also confirmed that the binding to these receptors was not hindered by steric hindrance.

Based on the above findings, the present inventor has shown that the HLA-G dimer enhances the effect of suppressing signal transduction at the cellular level, that is, the effect of suppressing activation of immune system cells, like the HLA-G monomer. I thought it was possible. Therefore, as a result of reporter assembly using H-cell hybridoma for HLA-G dimer, HLA-G dimer was mediated by LILRB1 with about 100 times higher efficiency than monomer. It was confirmed to transmit a signal. Thus, since the HLA-G dimer can effectively suppress the activation of immune system cells, it is used for pharmaceuticals and treatments for inflammatory diseases that require an anti-inflammatory effect. It was found that the above problems can be solved.

The present inventor also examined a method for producing a disulfide-bonded HLA-G dimer more efficiently. As a result, in the presence of the HLA-G monomer, As a result, it was confirmed that a sufficient amount of the HLA-G dimer can be obtained with good reproducibility, and that the above-mentioned problems could be solved.

That is, the present invention is as follows.

(1) A pharmaceutical composition comprising an HLA-G dimer.

The pharmaceutical composition of the present invention has an effect of suppressing activation of immune cells. In the pharmaceutical composition of the present invention, examples of the HLA-G dimer include those in which HLA-Gs are linked by an intermolecular disulfide bond. Examples of HLA-G include the following protein (a) or (b). In this case, the intermolecular disulfide bond can be exemplified by the one formed between the 4th and 2nd cysteine residues in the amino acid sequence shown in SEQ ID NO: 2.

(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2;

(b) comprising an amino acid sequence in which one or several amino acids except the fourth amino acid in the amino acid sequence shown in SEQ ID NO: 2 have been deleted, substituted or added, and leukocyte Ig-like receptor (LILR) and Protein with binding activity to Z or CD8

(2) A prophylactic and / or therapeutic agent for allergic diseases, comprising the pharmaceutical thread and composition according to (1) above.

(3) A drug for the prevention and treatment of atopic dermatitis, asthma, allergic rhinitis, inflammatory disease or infertility, which comprises the pharmaceutical composition described in (1) above. ,

(4) An immunosuppressive therapeutic agent used for organ transplantation, bone marrow transplantation, or regenerative medicine, comprising the pharmaceutical composition according to (1) above.

(5) A method for producing a disulfide-linked HLA-G dimer, comprising adding a reducing agent to a solution containing HLA-G.

Examples of the reducing agent include dithiothreitol (DTT). In the method of the present invention, the concentration of the HLA-G monomer in the HLA-G solution is, for example, 0.1 to 10 mg / mL, and the concentration of the reducing agent added to the solution is, for example, 0 :! The concentration of the HLA-G monomer in the HLA-G solution is, for example, 0.1 to 10 mg / mL, and the concentration of the reducing agent added to the solution is, for example, 0.1. ~: Preferably it is lOmM.

(6) A method for controlling and / or suppressing inflammation, comprising administering an effective amount of a disulfide-binding HLA-G dimer to a human or non-human mammal.

(7) A method for treating and / or preventing an inflammatory disease, which comprises administering an effective amount of a disulfide-bonded HLA-G dimer to a patient. Brief Description of Drawings

Figure 1-1: Structure of disulfide-bonded HLA-G dimer

(A) Crystal structure of disulfide bond dimer of HLA-G-peptide (RIPRHLQL) complex. HLA-G H chain and i3 2m are represented by a ribbon model and covered with a translucent surface (blue and pink; H chain, light blue and light pink: i3 2m, yellow bar model: pen Petit RIPRHLQL).

(B) Peptides are represented by bars. The HLA-G crystal packing shown in the ribbon model (one is shown in blue and the other is shown in green).

(C) Three-dimensional view of intermolecular interaction at the dimer interface. The pseudo-annealing omit map around the Cys42-Cys42 bond (blue shading, Fo-Fc (contour at 3σ)) is shown.

(D) Monomer structure (chain Α) (blue: HLA-G strand, pale blue:] 3 2m, yellow stick model: peptide). The binding sites of MHC class I specific receptors (TCR, CD8 and LILRB) are indicated by arrows. Two cysteines, Cys42 (red) and Cysl47 (magenta) are shown in the bar model.

(E) View of (D) from above. The position 67, the cysteine residue in HLA-B27, is shown in green.

(F) Structural comparison between two dimers of the crystal. The color of one dimer is the same as that in (A) above, and the overlapping dimer is green. Figure 1-2: Complex of disulfide-bonded HLA-G dimer with LILRB1 and CD8

(A) The sugar binding site (position 86) is indicated by a green dotted circle.

(B, C) LILRB1 (red and light blue) Mono HLA-G dimer (pink and blue) ) Complex (B) and CD8 (green and yellow) Mono HLA-G dimer (pink and blue) Complex (C). A side view (left) and a plan view (right) are shown. These models show the crystal structure of the previous LILRB1-HLA-A2 complex (Willcox, BE, et al., (2003) Nat Immunol, 4, 913-919.) And the CD8 aa -HLA-A2 complex. It is constructed by superimposing the HLA-G dimer structure on the body crystal structure (Gao, GF, et al., (1997) Nature, 387, 630-634.). The black dotted line represents the stalk region of each molecule, and the two dotted circles (red and cyan) represent the two C-terminal two Ig-like domains of LILRB1. The thick orange dotted line represents a schematic representation of the cell membrane.

(D) Proximity map of putative LIKB1 / 2 binding sites on HLA-G molecules. First (B)

LILRB 1, HLA-A2 and HLA-G bar models are colored magenta, blue and cyan, respectively. Higher affinity of HLA_G for LILRB 1/2 based on our previous biochemical studies (Shiroishi, M., et al., (2003) Proc Natl Acad Sci USA, 100, 8856-8861.) The key amino acids (Phel95 and Tyrl97) that we propose to be important are shown in a bar model. Figure 1-3: Purification of soluble disulfide-bonded HLA-G dimer by Native-PAGE and equilibrium gel filtration and binding of 111 ^-& dimer to 1 ^ 11 ^ 81/2

(A) Gel filtration matrix of HLA-G molecules after incubation for 20 days using a Superdex-200 10/300 column.

(B) HLA-G under non-reducing conditions (lane 1) and reducing conditions (lane 2)

SDS-PAGE ₀

(C) Native_PAGE of dimer (lane 1), dimer with 2 mM dithiothreitol (DTT) (lane 2), and monomer (Cys42Ser mutant, lane 3).

(D) Binding between dimer and LILRB1. Lane 1 is dimer (15 μΜ) only, lanes 2 to 4 show binding between the dimer (15 μΜ) and each molar ratio of receptors, and lane 5 is LILRB1 only (30 μΜ) It is.

(E) HLA-G dimer: Equilibrium gel filtration analysis of LILRB eye interactions. A mixture of HLA-G dimer and LILRB 1 at the indicated concentrations was injected onto a column previously equilibrated with 10 μL LILRB 1. (F) Binding between HLA-G dimer and LILRB2. Lane 1 is dimer (15 μM) only, and lanes 2 and 3 show binding between HLA-G dimer (15 μM) and each molar ratio of receptors. Since LILRB2 migrated in the opposite direction, the band could not be shown using the same gel.

(G) HLA-G dimer: equilibrium gel filtration analysis of LILRB2 interaction. Similar to (と) above, a mixture of HLA-G dimer and LILRB1 was injected onto a column previously equilibrated with 15 μL LILRB2. Figure 1-14: Surface plasmon resonance analysis of HLA-G dimer and monomer (Cys42Sr mutant) binding to immobilized LILRB1 and LILRB2

(A) Kinetic analysis of HLA-G dimer against immobilized LILRB1. The indicated concentration of dimer was injected into a flow cell (200RU) with LILRB1 immobilized. The response curve generally fits the bivalent analyte model (left panel, black line), and for lower concentrations (0.8-3.9 nM), the curve is a 1: 1 binding model (right panel, (Black line).

(B) Kinetic analysis of HLA-G injected into a flow cell fixed with LILRB1.

(C) Kinetic analysis of HLA-G dimer against immobilized LILRB2 (270RU). Response curves generally applied to the 1: 1 model for all concentrations (left panel, black line) and for low concentrations (63-250 nM, right panel, black line).

(D) Reaction rate analysis of HLA-G monomer injected into the flow cell with LILRB2 immobilized.

(E) Equilibrium binding analysis of HLA-G monomers to immobilized LILRB1 (black squares) and LILRB2 (red circles). The solid line represents the non-linear fit of the 1: 1 Langmuir coupled isoform. Small box; Scatchyard plot of the same data. The solid line is a linear fit.

5: LILRB1-mediated signal induced by immobilized HLA-G dimer (A) NFAT-GFP receptor cells expressing LILRBl-PILR chimeras were stimulated with the indicated concentration of fixed HLA-G monomer or dimer for 12 hours, and GFP in the receptor cells Expression was analyzed.

(B) Average fluorescence intensity (MFI) of GFP (left) and GFP positive rate of LILRB1-PILR receptor cells (right) were shown. White and black circles indicate dimer and monomer, respectively. Figure 2-1:

(A) HLA-G dimer crystals and SDS-PAGE with inverted vapor diffusion using 0.2 L protein and 0.2 pL reservoir solution.

(B) Detected using ADSC Quantumn 4R CCD force mer: Diffraction image of ELA-G dimer crystal. Circular lines represent the 8.0 A and 3.0 A resolution limits. The small square line is an image near the 3.0 A resolution limit. Figure 2-2: HLA-G dimer formation at low concentrations of DTT. Gel filtration matrix using Superdex 200 10/300

(A) Incubate for 10 days without DTT.

(B) 3 days incubation with 2 mM DTT. Each sample was incubated at 4 ° C. D: dimer peak, M: monomer peak. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. However, the scope of the present invention is not limited to these explanations, and the examples other than the following examples can be appropriately modified and implemented without departing from the spirit of the present invention.

This specification includes the entire US Provisional Patent Application No. 60 / 699,783, which is the basis of the priority claim of the present application. In addition, all prior art documents cited in this specification, as well as publications, patent publications and other patent documents are incorporated herein by reference. 1. Summary of the present invention

(1) HLA-G is a non-classical MHC class I molecule (MHC I) that induces a wide range of immunological tolerance by binding to inhibitory receptors such as leukocyte Ig-like receptor (LILR) and CD8. To do. HLA-G is expressed in disulfide-linked dimer form in solution and on the cell surface. Interestingly, MHC I dimer organization is involved in pathogenesis and T cell activation. However, the three-dimensional structure and receptor binding properties of MHC I dimers were not known at all.

To understand the potential role of HLA-G dimers for LILR signaling, we determined the crystal structure of a wild-type dimer of HLA-G with intermolecular Cys42-Cys42 disulfide bonds. did. This dimer configuration allowed two receptors to bind by exposing two LILR / CD binding sites to the surface. As expected from this result, the binding test conducted by the present inventor shows that the HLA-G dimer shows a higher overall affinity for LILRB1 / 2 than the monomer due to the significant avidity effect. Prove that. Furthermore, the cell receptor assembly demonstrated that dimer organization significantly improved LILRB1-mediated signaling at the cellular level. These results suggest that the HLA-G dimer has a structural orientation suitable for efficient LILR-mediated signaling that produces the most potent immunosuppressive effect. Furthermore, the structural and functional roles of MHC I dimers including virulence genes, HL and B27, suggest that the receptor binding is also strong. Accordingly, the present invention relates to efficient receptor signal transmission of disulfide-bound HLA-G dimers, and use for the suppression of inflammation, including the use of the soluble proteins in inflammatory diseases of animals and humans. I will provide a.

(2) The present inventors also crystallized HLA-G as a disulfide-linked dimer by adding dithiothreitol (DTT), and collected the 3.2A data set. Furthermore, the present inventor has confirmed that DTT promotes the exchange of unfolded HLA-G disulfide bonds in which free cysteine is protected, thereby promoting the dimer formation. This was a technique applicable when a protein having free cysteine was unwound to form a disulfide-linked dimer / multimer. The present invention provides an efficient method for producing a disulfide-bound HLA-G dimer, which comprises mixing an HLA-G monomer and DTT. This procedure involves an improved method for producing dimeric HLA-G protein. 2. Disulfide-linked HLA-G dimer

(1) HLA-G (monomer)

In the present invention, HLA-G (monomer), which is a constitutional unit of disulfide-binding HLA-G dimer, is not limited to wild-type HLA-G, and 1 of amino acid sequences of wild-type HLA-G. Alternatively, a protein having an amino acid sequence in which several amino acids have been deleted, substituted or added, and a protein having a binding activity to leukocyte Ig-like receptor (LILR) and / or CD8 (mutant type) ) Can also be used. Information on the wild-type HLA-G amino acid sequence (SEQ ID NO: 2) and the nucleotide sequence encoding the wild-type HLA-G amino acid sequence (SEQ ID NO: 1) was published, for example, as “Accession number: M90684J” by NCBI. ing.

Here, the “amino acid sequence in which one or several amino acids have been deleted, substituted or added” is, for example, about 1 to 15, preferably about 1 to 10, more preferably about 1 It is preferred that the amino acid sequence has about ˜5 amino acids deleted, substituted or added.

In addition, since it is important that the above-mentioned “protein consisting of a deleted, substituted or added amino acid sequence” is a protein that can stably exhibit binding activity to LILR and CD8, for example, LILR and CD8 The amino acid residues considered to be important for the binding reactivity of are preferably those that have not been mutated (deleted, substituted or added) from the amino acid sequence of wild-type HLA-G. Furthermore, since the presence of a cysteine residue (Cys42) that contributes to the disulfide bond between HLA-G molecules is important for the formation of HLA-G dimers, the 42nd amino acid residue is the wild type. Those that have not been altered (deleted, substituted or added) from the amino acid sequence of HLA-G are preferred.

Here, “binding activity with LILR and Z or CD8” is an activity in which HLA-G directly binds to LILR and / or CD8, thereby transmitting a signal via LILR or CD8, and controlling the immune system. It means an activity that can exert an effect. As long as it has an activity capable of exerting an immunoregulatory effect, the present invention is not limited to the above-mentioned binding activity with LILR and / or CD8, but includes binding activity with KIR2DL4, CD160, etc. It is done.

In addition, the above-mentioned “protein consisting of a deleted, substituted or added amino acid sequence”, whether or not it has binding activity to LILR and CD8 should be confirmed by, for example, a reporter assay using a T cell hybridoma. Can do.

HLA-G can be prepared by expressing and recovering recombinant HLA-G using the transformant. In order to express recombinant HLA-G, first, using a known gene recombination technique, construct a recombinant vector that incorporates the gene encoding the amino acid sequence of HLA-G into an expression vector, etc. It is necessary to.

As a gene encoding the amino acid sequence of HLA-G, in addition to the wild type HLA-G encoding gene, the aforementioned mutant HLA-G encoding gene can also be used. Mutant HLA-G coding genes can be prepared by introducing mutations into the DNA sequence of the wild type gene.For example, Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (1989), Current It can be prepared according to the site-specific displacement induction method described in Protocols in Molecular Biology, John Wiley & Sons (1987-1997) and the like. Specifically, it can be prepared using a mutagenesis kit using site-directed mutagenesis by a known method such as Kunkel method or Gapped duplex method. Examples of such kits include QuickChange ™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor ™ Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km Etc .: manufactured by Takara Bio Inc.).

Subsequently, the recombinant HLA-G can be expressed by introducing a recombinant vector into a host by various known transformation methods to obtain a transformant and culturing it. The term “transformant” as used in the present invention means a foreign gene introduced into the host. For example, the foreign gene was introduced by introducing plasmid DNA or the like into the host (transformation). And those into which foreign genes have been introduced by infecting a host with various viruses and phages (transduction) are included. Inn As long as it is capable of expressing HLA-G from the introduced thread recombination vector, it is not limited. For example, various animal cells such as human mice, various plant cells, bacteria, fermentation Known hosts such as mother and plant cells can be used.

Specifically, the recombinant HLA-G can be produced by a method comprising a step of culturing the above-mentioned transformant and a step of collecting the recombinant HLA-G from the obtained culture. Here, “cultured product” means any of culture supernatant, cultured cells, cultured cells, or cells or destructed cells of cells. The transformant can be cultured according to a usual method used for host culture. The protein of interest is accumulated in the culture.

When recombinant HLA-G is produced extracellularly, remove the cells by force centrifugation using a culture solution or by filtration. Then, if necessary, extract recombinant HLA-G from the culture by extraction with ammonium sulfate precipitation, and if necessary, dialysis, various chromatography (gel filtration, ion exchange chromatography, affinity chromatography). Etc.) can be isolated and purified.

When recombinant HLA-G is produced intracellularly, recombinant HLA-G can be collected by disrupting the cells. After disruption, remove cell debris (including cell extract insoluble fraction) as necessary by centrifugation or filtration. The supernatant after removal of the residue is a cell extract soluble fraction and can be a crude protein solution.

Recombinant HLA-G can be produced not only using a protein synthesis system using transformants, but also using a cell-free protein synthesis system that does not use live cells at all. Can be purified by appropriately selecting means such as chromatography. (2) HLA-G dimer

The disulfide-linked HLA-G dimer has a cross-linked structure formed between the 42nd cysteine residues in the amino acid sequence constituting the HLA-G monomer. The thiol group (SH group) is oxidized to form an intermolecular disulfide bond, resulting in dimerization (see Figure 1-11A). The HLA-G dimer may be a homodimer consisting of wild-type HLA-G and Z or mutant HLA-G, or may be a heterodimer, and is not limited. In addition, when the HLA-G monomer that is the structural unit of the HLA-G dimer is a mutant HLA-G, the cysteine residue that contributes to the intermolecular disulfide bond constitutes the wild-type HLA-G. It means the cysteine residue corresponding to the 42nd residue in the amino acid sequence. The disulfide-binding HLA-G dimer has a structure in which the binding sites for LILR and CD8 are exposed to the outside when the disulfide binding moiety is on the inside, and the binding to these receptors is sterically hindered. (See Fig. 1-2 B and C). Therefore, the HLA-G dimer has a structure capable of retaining the same function as the HLA-G monomer.

The disulfide-bonded HLA-G dimer preferably has a binding efficiency with LILR and Z or CD8 that is, for example, twice or more that of the HLA-G monomer. The binding efficiency can be measured, for example, by reporter assay using a T cell hybridoma, surface plasmon resonance analysis, native gel shift assay, and the like. Thus, HLA-G dimers are extremely useful for suppressing activation of immune system cells because of their very high binding efficiency with LILR and Z or CD8.

The disulfide bond-type HLA-G dimer may be obtained by any method and is not limited. For example, the disulfide bond-type HLA-G dimer may be obtained by a known production method that naturally forms a disulfide bond. Further, it may be a dimer obtained by a production method described later that can promote the formation of a disulfide bond.

3. Preparation of disulfide-linked HLA-G dimer

The disulfide-bonded HLA-G dimer can be produced more efficiently by adding a reducing agent in the presence of the HLA-G monomer.

Examples of the reducing agent include dithiothreitol (DTT), -mercaptoethanol, reduced dartathione, cysteamine, etc. Among them, DTT is particularly preferable. In the dimerization reaction of HLA-G, the reducing agent can attack the intermediate thiol group of the cysteine residue that contributes to disulfide bond and promote intermolecular disulfide bond formation between thiol groups by oxidation reaction. . The reaction solvent in the dimerization reaction of HLA-G is not limited, but an aqueous solvent containing, for example, Tris-HCl (pH 8.0) and NaCl at appropriate concentrations is preferable. Further, in the production method of HLA-G dimer, HLA-G monomer is preferably present at a high concentration, for example, 0.1 to: 10 mg / mL, preferably 5 to 10 mg / mL, particularly preferably. Is about 10 mg / mL. As a method for increasing the concentration of HLA-G, for example, concentration of an HLA-G solution can be mentioned.

Furthermore, in the said manufacturing method, it is preferable to make the addition amount of a reducing agent into a small quantity with respect to a HLA-G monomer, for example, final concentration is 0.1-:! OmM, Preferably it is 1-5mM. When DTT is used as the reducing agent, about 2 mM is particularly preferable. It is particularly preferable to add a small amount of such a reducing agent in a state in which the HLA-G monomer is present at a high concentration as described above, and the HLA-G dimer production efficiency is most enhanced. Can do.

Note that the present invention includes an HLA-G dimerization promoter containing a reducing agent (eg, DTT).

4. Use of disulfide-linked HLA-G dimer

(1) Pharmaceutical composition

The pharmaceutical composition of the present invention is characterized by containing a disulfide-linked HLA-G dimer. As described above, the HLA-G dimer is excellent in suppressing the activation of immune system cells. Therefore, the pharmaceutical composition is used as a prophylactic and therapeutic agent for, for example, allergic diseases (such as asthma, atopic dermatitis, allergic rhinitis), inflammatory diseases, or infertility (infertility), and organs. It can be preferably used as an immunosuppressive therapeutic agent or immunosuppressive drug used for transplantation, bone marrow transplantation or regenerative medicine.

The HLA-G dimer, which is an active ingredient in the pharmaceutical composition of the present invention, may be used in the form of various salts or hydrates as necessary, and also has storage stability (particularly activity maintenance). It may be used with appropriate chemical modifications in consideration, and is not limited. The HLA-G dimer may be used in a crystallized state or in a dissolved state. The pharmaceutical composition of the present invention can contain other components in addition to the protein of the present invention. Examples of the other components include various pharmaceutical components (various pharmaceutically acceptable carriers and the like) required depending on the usage (form of use) of the pharmaceutical composition. Other components can be appropriately contained as long as the effects exhibited by the protein of the present invention are not impaired.

In the pharmaceutical composition of the present invention, the blending ratio of the active ingredient HLA-G dimer is not limited, but for example, it is preferably 0.01% by weight or more based on the composition, more preferably 1% by weight or more, more preferably 10% by weight or more. When the content ratio of the active ingredient satisfies the above range, a sufficient immune system cell activation suppressing action, particularly an anti-inflammatory action can be exhibited. Further, the HLA-G dimer as an active ingredient is preferably purified to have a higher purity, for example, a purity of 50% or more is preferable, more preferably 80% or more, More preferably, it is 90% or more.

The pharmaceutical composition of the present invention can be administered to human or non-human mammals in various administration routes, specifically, oral or parenteral (eg, intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration). Can be administered. Therefore, the HLA-G dimer, which is the active ingredient, can be used alone, but can be formulated into an appropriate dosage form using a pharmaceutically acceptable carrier by a method commonly used depending on the administration route. Can be

Preferred oral dosage forms include, for example, tablets, powders, fine granules, granules, coated tablets, capsules, syrups, and lozenges for oral preparations. For parenteral preparations, for example, inhalants Preferred examples include suppositories, injections (including drops), ointments, eye drops, eye ointments, nasal drops, ear drops, poultices, lotions, and ribosomes.

Carriers that can be used to formulate these preparations include, for example, commonly used excipients, binders, disintegrating agents, lubricants, coloring agents, and flavoring agents, and if necessary, stabilizing agents. , Emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, extenders, wetting agents, surfactants, dispersants, buffers, preservatives, solubilizers, and soothing agents It is possible to formulate by a conventional method by blending known ingredients that can be used as raw materials for pharmaceutical preparations. Non-toxic components that can be used in the pharmaceutical composition of the present invention include, for example, animal and vegetable oils such as soybean oil, cow moonlight, and synthetic glyceride; hydrocarbons such as liquid paraffin, squalene, and solid paraffin; myristic acid Ester oils such as octyldodecyl and isopropyl myristate; higher alcohols such as cetostearyl alcohol and behenyl alcohol; silicone resin; silicone oil; polyoxyethylene fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene Surfactants such as sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, and polyoxyethylene monopolyoxypropylene block copolymer; hydroxyxetyl cellulose, polyacrylic acid, force noboxybul polymer Water-soluble polymers such as polyethylene glycolenole, polyvinylidene / levidone lydone, and methylsenololose; lower alcohols such as ethanol and isopropanol; glycerin, propylene glycol, dipropylene glycol, sorbitol, and polyethylene Polyhydric alcohols such as glycols (polyols); sugars such as gnolecose and sucrose; inorganic powders such as anhydrous caic acid, aluminum magnesium silicate and aluminum silicate; inorganic such as sodium chloride and sodium phosphate Salt; purified water and the like, and any of them may be a salt thereof or a hydrate thereof.

Excipients include, for example, lactose, fructose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, and silicon dioxide.Binders include, for example, polyvinyl alcohol and polyvinyl alcohol. Methylcellulose. Ethenorescenellose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylidene chloride, polypropylene dariconole polyoxyethylene block polymer. Medamine and the like are disintegrating agents such as starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, and carboxymethylcellulose. Examples of lubricants that can be added to pharmaceuticals include, for example, magnesium stearate, tanolec, polyethylene glycol, silica, and hydrogenated vegetable oil. Preferred examples of the flavoring agent include cocoa powder, heart beat brain, fragrance powder, heart force oil, dragon brain, and cinnamon powder. Or a hydrate thereof.

When the pharmaceutical composition of the present invention is an oral preparation, the active ingredient HLA-G dimer is mixed with an excipient, and if necessary, for example, a binder, a disintegrant, a lubricant, a coloring agent, and After adding a flavoring agent etc., it can be made into a powder, a fine granule, a granule, a tablet, a covered tablet, a capsule, etc. by a conventional method. In the case of tablets and granules, for example, sugar coating can be used, and if necessary, other known methods can be used for appropriate coating. In the case of syrups and injectable preparations, for example, pH adjusters, solubilizers, tonicity agents, etc., and if necessary, solubilizing agents, stabilizers, etc. Can be In the case of an external preparation, the production method is not limited and can be produced by a conventional method. As the base material to be used, various raw materials usually used for pharmaceuticals, quasi drugs, cosmetics, etc. can be used. For example, animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols Raw materials such as fatty acids, silicone oils, surfactants, phospholipids, alcohols, polyhydric alcohols, water-soluble polymers, clay minerals, and purified water, and pH adjusters as necessary Antioxidants, chelating agents, antiseptic / antifungal agents, coloring agents, fragrances, and the like can be added. Furthermore, components such as blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, humectants, and keratolytic agents can be added as necessary. In this case, the ratio of the HLA-G dimer to the carrier is not limited and can be appropriately set between 1 and 90% by weight.

When the pharmaceutical composition of the present invention is administered parenterally (for example, by injection), the effective dose is, for example, the degree of symptoms, age, gender, body weight, dosage form, salt type, and specific disease For example, in the case of an adult, a person with a weight of 60 kg, 10 pg ~: 1 mg per day, preferably 100 g ~ 10 mg, one or more at a time. Can be administered in divided doses.

When the pharmaceutical composition is orally administered, its dosage form and effective dosage depend on the subject of administration, administration route, formulation properties, patient condition, doctor's judgment, etc., and are not limited. However, for example, in the case of an adult, a body weight of 60 kg may be administered at a dose of 10 pg to: lmg, preferably lOOpg to 10 mg per day, in a single dose or divided into multiple doses. Given that the efficiency of the route of administration is different, The dosage can be appropriately set in a wide range.

The method for confirming the immune system cell activation inhibitory effect (particularly the anti-inflammatory effect) of the pharmaceutical composition of the present invention is not particularly limited.

The present invention also includes the use of disulfide-bonded HLA-G dimers for the production of pharmaceutical compositions such as immune system cell activation inhibitors (particularly anti-inflammatory agents). (2) Treatment methods for inflammatory diseases

The present invention includes a method for treating and / or preventing an inflammatory disease, which comprises administering an effective amount of a disulfide-conjugated HLA-G dimer to a patient. The present invention also includes the use of disulfide-conjugated HLA-G dimers for the manufacture of therapeutic and Z or prophylactic agents for inflammatory diseases in patients.

Furthermore, the present invention includes a method for controlling and / or suppressing inflammation, which comprises administering an effective amount of a disulfide-bound HLA-G dimer to a human or non-human mammal. Furthermore, the present invention includes the use of disulfide-linked HLA-G dimers for the control of inflammation and / or the production of inhibitors in human or non-human mammals.

These disulfide-bound HLA-G dimers to be administered or used in the present invention can be used as they are, but are preferably used as the aforementioned pharmaceutical composition. The preferred administration method and dosage of the pharmaceutical composition are as described above, and can be appropriately selected and set in consideration of the patient's medical condition, the presence or absence of side effects, the administration effect, and the like. Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1] Disulfide-bonded HLA-G dimer bond formation and efficient receptor signaling

(1) Materials and methods

(Production of recombinant protein and HLA-G crystallization

Our previous report (Shiroishi, M., et al., (2003) Proc Natl Acad Sci USA, 100, 8856-8861.) Recombinant extracellular domains of HLA'G monomer, LILRB1 and LILRB2 were prepared. The preparation of the piotinylated HLA-G monomer, LILRB1 and LILRB2 was essentially the same except for the absence of the tag. Specific site mutagenesis of Cys42Ser mutant HLA-G monomer was performed by a two-step PCR method. Unwound wild type HLA-G monomer spontaneously formed disulfide-linked dimers (10-30% of total protein) at 4 ° C for 20 days. For binding studies, the HLA-G dimer was purified by genomic filtration (Superdex 200 10/300, Amersham Biosciences). The crystallization of the disulfide-bonded HLA-G dimer is briefly described below.

0.2 pL of protein solution and crystallization reservoir solution were mixed at a ratio of 1: 1.

Crystals were obtained at 20 ° C. under the conditions of Wizard 11-39 (lOOmM CHAPS ρΗΙΟ5, 20% (w / v) PEG8000, 200 mM NaCl).

(ii) Data collection, structure determination and refinement

A 3.2-A diffraction data set was collected at 100K with a beamline BL38B1 from Spring 8 (Harima, Japan) (λ = 1 · 0000Α).

Diffraction data was processed and scaled using the HKL2000 program package.

Detailed crystallographic statistics are shown in Table 1-11. Use the Molrep, HLA-E-peptide complex (PDB code: 1MHE) from the CCP4 package with a probe (Collaborative Computational Project Number 4. (1994) Acta Cryst., D, 50, 760-763.) The HLA-G structure was faded by the molecular replacement procedure (O'Callaghan, CA, et al., (1998) Mol Cell, 1, 531-541.). By using E in the range 20-3.5A, two HLA-G monomers (chain A and chain B) were found. After refinement of rigid body correction, position of one cyclore and total B-factor in the program Refmac5 (Collaborative Computational Project Number 4., 1994), Fo-Fc map is crystallographically 2 for the disulfide bond between Cys42. The electron density of double monomer was clearly shown. This indicates that the two disulfide-bonded dimers present in the crystal and the monomers in each dimer complex are arranged along the crystal double axis. Grouped (2 groups per residue) For further refinement with B factor refinement Refmac5 and CNS (Brunger, AT, et al., (1998) Acta Crystallogr D Biol Crystallogr, 54 (Pt 5) , 905-921.) Manual in interactive graphics program o done in 'Rebuilding

(Jones, TA, et al "(1991) Acta Crystallogr A, 47 (Pt 2), 110-119.) The final model consists of two HLA-G molecules (H chain, 2m and peptide) 11 _∞ 23.5% between 50A and 3.2 (R _fr ee = 29.8%).

Detailed crystal statistics are shown in Table 1-1. The ramachandran plot of skeletal angle shows 85.3%, 13.4% and 0.9% in the most preferred additionally widely accepted regions, and 0.3% in the unacceptable regions, respectively. Ramachandran plot was calculated by PROCHECK (Laskowski, RA, et al "(1993) J. Appl. Crystallogr., 26, 283-291.) PyMOL (http://pymol.sourceforge.net) , BOBSCRIPT (Esnouf, RM (1997) J Mol Graph Model, 15, 132-134, 112-133.) And Raster 3D (Merritt, EA et al., (1997) Methods in Enzymology, 277, 505-524. The atomic coordinates are deposited in the Protein Data Bank under the accession number 2D31.

Data collection and refinement statistics

(The highest shell value is shown in parentheses) (iii) Native gel electrophoresis (Native-PAGE)

Native polyacrylamide gel electrophoresis (Native-PAGE) was performed with the Phast system (Amersham Biosciences) to analyze the properties of protein samples and their binding. Electrophoresis was performed at 15 ° C. using a commercially available Native-PAGE buffer strip (0.25M Tris, 0.88M L-alanine, pH 8.8, Amersham Biosciences). For complex formation experiments, the sample mixture was incubated for 1 hour at 20 ° C and then applied to a homogeneous 12.5% polyacrylamide gel (Amersham Bioscinces). A sample of less than 4 pL was applied to each lane,

(iv) Equilibrium gel filtration test

Equilibrium gel filtration was performed at a flow rate of 60 L / min using a SMART system (Amersham Biosciences) equipped with a Superdex-200 PC 3.2 / 30 column. The column was equilibrated with 10 mM Hees pH 7.4, 150 mM NaCl, 3.4 mM EDTA and 0.005% detergent P20 (HIBS-EP buffer) with 10 μΜ LILRB 1 or 15 μΜ LILRB2. Prior to injection, HLA-G dimer (10 μΜ for LILRB 1 and 15 μΜ for LILRB2) and LILRB 1/2, 1: 0 (dimer only), 1: 1, 1: 2. And 1: 3 molar ratio and the mixture was incubated at room temperature for 1 hour.

(V) Surface plasmon resonance test

Using BIAcore 2000 ™ (BIAcore AB, St. Albans, UK), surface plasmon resonance experiments were performed according to a well-known standard protocol (Shiroishi et al., 2003 (supra)). went. Piotinylated LILRB was immobilized on a research Sensor Chip CM5 (BIAcore AB) where streptavidin is covalently immobilized. HLA-G dimer or Cys42Ser mutant monomer was run at 50 pL / min.

Reaction rate constants were derived by bivalent analyte model with BIAevaluation v3.2 (BIAcore) or curve fitting to fit a simple 1: 1 binding model. For equilibrium binding analysis, the equilibrium binding response at each concentration of analyte was calculated by subtracting the response measured in the control flow cell from the response in the sample flow cell. Affinity constants (K _d s) were calculated by nonlinear curve fitting or by Scatchyard analysis with a simple 1: 1 Langmuir binding model using the program Origin ver.5.0 (MicroCal). In the reverse orientation experiment, biotinylated HLA-G Fixed. LILRB was flowed on the fixed HLA-G.

(vi) LILRB 1 NFAT-GFP reporter cell assay

A chimeric molecule (Shiratori, I., et al., (2004) J Exp Med, 199, 525-533.) Consisting of an extracellular domain of LILRB1 and a transmembrane and cytoplasmic domain of activated PILR iS By using the vector, it was transformed into mouse T-cell hybridoma with NFAT-green fluorescent protein (GFP) reporter gene and DAP12 (Arase, H., et al., (2002) Science, 296, 1323- 1326 .; Ohtsuka, M., et al., (2004) Proc Natl Acad Sci USA, 101, 8126-8131.). Various concentrations (0 to: 60 ng / mL) of HLA-G dimers and monomers were immobilized on 48-well culture plates (BD falcon) for 2 hours at 37 ° C. Reporter cells (5 × 10 ⁴ / well) expressing LILRB1-PILR chimera molecules were stimulated with immobilized HLA-G monomer or dimer for 12 hours, and GFP expression was measured using FACScalibur. analyzed.

(2) Results

(i) HLA-G dimer overall structure and peptide recognition

The HLA-G (HLA-G * 0101, 1-274 residue) -peptide (RIIPRHLQL (SEQ ID NO: 3)) complex was refolded, purified as a monomeric form, and crystallized. These crystals were diffracted to 3.2A resolution. The structure was determined by molecular replacement using the HLA-E structure (O'Callaghan, CA, et al., (1998) Mol Cell, 1, 531-541.) As a research model (see Table 1-11) .

Two wild-type HLA-G molecules exist within the asymmetric unit. Surprisingly, even though the monomeric form was used for crystallization, each monomer was covalently bound to its asymmetric partner by a Cys42-Cys42 disulfide bond along the crystal axis twice (Figure 1-1). A) A clear electron density related to this intermolecular disulfide bond was clearly observed (Fig. 11 B). The two HLA-G molecules of the asymmetric unit showed a slight difference in peptide protein interaction in the central region of the peptide, but with a root mean square distance (rmsd) of 0.29A for the 38lC a position. It was almost the same. The overall structure of this HLA-G-peptide complex was very similar to other previously reported HMC class I structures (Figure 1-1 C, D). Cys42-Cys42 disulfi No significant structural changes due to bond formation were observed.

The structural features of the main anchor residues, P2, P3 and PC, were similar to other known classical MHC I (Clements, CS, et al, (2005) Proc Natl Acad Sci US A.) Figures 1-2 and 1_3, Table 1-1). The backbone conformation of the 9-mer peptide bound to HLA-G was similar to that of the fully elongated, deeply buried HLA-E-9-mer complex (O'Callaghan et al., 1998 (Above)). However, HLA-G had fewer hydrophobic residues in the central region of its groove than HLA-E. This allows HLA-G to display a limited but still diverse liver tree peptide similar to classical MHC I (which showed very stringent peptide specificity) than HLA-E. Can be explained.

(ii) Dimer formation and LILR / CD8 complex model

Wild-type HLA-G formed disulfide-linked dimers with intermolecular Cys42-Cys42 disulfide bonds in crystals (hereinafter referred to as “HLA-G dimer” or “dimer”) . As mentioned earlier, two HLA-G dimers were found in the crystals, and their structural properties suggested both the structural flexibility and structural rigidity of the dimer. With the exception of the Cys42-Cys42 disulfide bond, this dimeric interface is responsible for any electrostatic and hydrogen bond interactions (Clu61 (Οεΐ)-Lys68 (Νζ), 42 (CO ) -Arg44 (Νηΐ), Glu58 (Οε1, Οε2) -Cln72 (Νε2, Οεΐ)) (Fig. 1- l B). Binding area is relatively small compared to the average protein-protein interaction 1600A ± 400A R ² (LO Conte, L., et al., (1999) J Mol Biol, 285, 2177-2198.) 676 A ² (chain A) and 568A ² (chain B). This suggests that these dimers have some structural flexibility. However, the angle between the monomers of the two dimers is surprisingly conserved (Fig. 1-1E), and the intermolecular interaction at the dimer interface is almost without structural adjustment. It was maintained. With the maintenance of the Cys42-Cys42 disulfide bond, a limited angle and direction is assigned for structural failure and interface area loss. In addition, the N-sugar addition site at position 86 (Rudd, PM, et al., (1999) J Mol Biol, 293, 351-366.) Is located outside the dimer interface and is attached at the close position. There was no structural interference due to (Figure 1-2 A, green dotted circle). Furthermore, in membrane-bound HLA-G, the transmembrane domain Both c-terminal sites of the dimer connecting the ins must be close to the membrane. These support the idea that this dimer conformation is possible and predominant in both membrane-bound and soluble HLA-G dimers. The structural properties of the LILRB and CD8 binding sites were maintained in the dimeric form of HLA-G and were similar to other MHC I molecules (Figure 1-2D). This orientation, therefore, allowed two functional LILR / CD8 binding sites to be sufficiently accessible to the receptor, resulting in a 1: 2 complex (HLA-G dimer: receptor) ( Figure 1 1-2 B, C). These complex models suggested that the two bound receptors are correctly oriented and close enough to assemble on the cell surface and properly signal. These were confirmed by the following biochemical and cell tests.

(iii) 1: 2 bond (HLA-G dimer: LILRB) Stoichiometry

In order to characterize the biochemical properties of the HLA-G dimer, this dimer and the Cys42Ser mutant monomer were prepared as described in the section “(1) Materials and Methods”. Intermolecular disulfide binding in the wild-type HLA-G dimer was confirmed by gel filtration, SDS-PAGE, and native polyatrylamide gel electrophoresis (Native PAGE) (Fig. 1_ 3 A to C). . As previously known (Boyson, IE., Et al., (2002) Proc Natl Acad Sci USA, 99, 16180-16185.), HLA-G due to Cys42Ser mutation is a monomeric form (Referred to as “mer” or “monomer”). This clearly shows that the HLA-G dimer was formed mainly by the Cys42-Cys42 disulfide bond.

Although binding of LILRB1 to HLA-G monomer was observed by Native PAGE, as previously known (Shiroishi et al., 2003 (supra)), due to its low affinity (Kd 2-5 μΜ), Not all of the monomers formed the LILRB1 complex. To determine the stoichiometric ratio of the complex between LILRB1 and HLA-G dimer, a series of mixtures (1: 1, 1: 2 and 1: 4 (HLA-G dimer: LILRB1) ) Was analyzed by Native PAGE (Fig. 1, 3D). The complete complex consisted of one HLA-G dimer and two LILRB1 (1: 2 complex). On the other hand, the LIRB2 binding test by Native PAGE (Figure 1-3F) shows that LIRB2 binds to the dimer, but its affinity for binding cannot be determined due to its very low affinity. It was. Equilibrium gel filtration was performed to clearly determine the binding stoichiometry (Figures 1-31E, G). If the protein concentration in the running buffer is higher than the dissociation constant, the elution profile depends on the stoichiometric ratio of the injected mixture and the true equilibrium stoichiometric ratio of the complex. The stoichiometric ratio of the HLA-G dimer-LILRB1 and LILRB2 complex is calculated as follows: 1 0, 1: 1, 1: 2, or 1: 3 on a column equilibrated with 10 μL LILRB1 or 15 μL LILRB2. (HLA-G dimer: LILRB1) Determination was made by applying a sample containing one of any molar ratio. Injections of 1: 0 and 1: 1 (HLA-G dimer: LILRB 1) showed a peak corresponding to the dimer-LILRB complex and a valley peak where free LILRB migrated. This indicates that LILRB in the running buffer was consumed for complex formation. On the other hand, a 1: 3 molar ratio sample for both LILRBs produced two peaks corresponding to the HLA-G dimer-LILRB complex and free LILRB. This indicates the presence of excess free LILRB. The sample with a molar ratio of 1: 2 represented a true equilibrium complex stoichiometry because it showed only one complex peak. A 1: 2 binding stoichiometry for LILRB1 / 2 resulted in a modest binding activity. This effect has been observed in the Surface Plasmon Resonance (SPR) analysis section below, and can be explained by the LILRB 1 complex model structure of the HLA-G dimer (Figure 1-2B).

Gv) HLA-G dimer high affinity LIRB binding

The characteristics of HLA-G binding to LILRB were further investigated by surface plasmon resonance (SPR). A HLA-G dimer and a monomer were injected on the surface of the sensor on which the biotinylated LIRBS was immobilized. Representative data on the binding of HLA-G monomer and dimer to LILRB is shown in Figure 14-14. Monomer, one at a very high dissociation rate (3.5 to 5 seconds one ^1.): Showed 1 binding (Fig. 1-4 B and D). The affinity constants (K _d s) of the monomers for LILRB1 and LILRB2 derived from the equilibrium analysis are 3.5μΜ and 15μΜ, respectively (Fig. 1-4E). It was similar to that from the reverse orientation test (Table 1 1-2) (Shiroishi et al., 2003 (supra)). In contrast, kinetic analysis of dimer binding to immobilized LILRB revealed that K _d s fits well in the bivalent analyte model (Figures 14 A and C, left). Thus, the dimer showed divalent binding. This result is based on the HLA-G dimer Consistent with the constructed 1: 2 (dimer: LILRB) complex model (Fig. 1-1-2B). The above-mentioned binding activity effect contributed significantly to the high-affinity binding clearly seen in FIGS. At low dimer concentrations where dimers can bind to two immobilized receptors simultaneously, their responses can be fitted to a simple 1: 1 binding model, resulting in high affinity constants. . That is, it was about 6.7 nM for Kd LILRB1, and about 750 nM for LILRB2 (Figures 1-4 A and C, right). On the other hand, the dissociation rate of LILRB binding to the HLA-G dimer was much slower than that of the monomer. That is, K in the bivalent model. ffl = 0.28 si (LILRB 1) or 1.0 s " ¹ (LILRB2), and in the simple 1: 1 bond model, _ff l = 0.11 s- ¹ (LILRB 1) or 0.96 s ¹ (LILRB2) These results clearly showed that the binding activity effect produced a much higher affinity at slower release rates.

(V) Efficient LILRB 1-mediated signaling of HLA-G dimers

In order to examine the above-mentioned binding activity of the dimer of the present invention on receptor-mediated signal transduction at the cellular level, a mouse T cell hybridoma (NFase, et al.) With an NFAT-green fluorescent protein (GFP) reporter system was used. al., (2002) Science, 296, 1323-1326 .; Ohtsuka, M., et al., (2004) Proc Natl Acad Sci USA, 101, 8126-8131.). LILRB1 chimeric gene consisting of extracellular domain of LILRB1 and transmembrane and intracellular domain of activated PILR β that can induce GFP expression when an appropriate reporter is bound (Shiratori et al., 2004 (supra)) And transformed those hybridoma cells. Therefore, LILRB1-mediated signal transmission could be easily detected by its GFP expression.

Various amounts of HLA-G monomer or dimer were immobilized on the wells and the LILRB1 reporter cells on these tools were incubated. After 12 hours, GJ expression in one LILRB1 reporter cell was analyzed by flow cytometry. Figure 1-15 clearly shows that the monomers can only stimulate their reporter cells even at the highest concentration of 160 ng / mL (mean fluorescence intensity (MFI) = 35.9, and GFP positive cells Only 13% of the total). Surprisingly, the dimer significantly activated cells at a much lower concentration of 1.6 ng / mL. GFP positive cells are 61% (MFI = 266) of the total at a concentration of 160 ng / mL. Therefore, HLA-G dimer Markedly improved LILRB1-mediated signaling at the cell level

(3) Discussion

Our structural analysis shows the first example of disulfide-linked dimeric MHC I, but many previous reports have shown that some disulfide-linked dimers are MHC I alleles. For example, HLA-B27 (McMichael, A. et al "(2002) Arthritis Res, 4 Suppl 3, S153-158.) Is expressed on activated T cells but expressed on resting cells The HLA-G dimer structure showed that the dimer's interfacial force was not so large and included some electrostatic and hydrogen bonding interactions, indicating that the relative orientation was The rotation of the Cys42-Cys42 bond shows a certain degree of mobility, but the two dimer conformations in the crystal were very similar, and also bound to Asn86. The glycan moiety did not show structural impairment in this dimer conformation, but controlled the relative movement of this dimer. The C-terminal sites of the two monomers in the membrane-bound dimer must be close enough to the membrane, so that the dimer conformation exists primarily on the cell surface. The soluble form is probably more flexible, but a similar conformation is likely to predominate.

Dimer formation resulted in a 1: 2 (HLA-G dimer: LILRB or CD8 receptor) complex (Figure 1, 1-2B, C). This indicates that the two receptors can assemble in the proper orientation at the cell surface for efficient signaling. To dictate this, the biochemical test in this example showed a much higher affinity for LILRB of HLA-G dimer than monomer by increasing binding activity. This result shows that the F _Ca -F _{C a} I interaction (Herr, AB, et al., (2003a) Nature, 423, 614-620 .; Herr, AB, et al., (2003b) J Mol Biol, 327 , 645-657.) And other cell surface receptor interactions (Appel, H., et al., (2000) J Biol Chem, 275, 312-321 .; De Crescenzo, G., et al., ( 2003) J Mol Biol, 328, 1173-1183.). Furthermore, the NFAT-GFP reporter assay with an active chimeric protein of LILRB1 revealed that the HLA-G dimer significantly induced LILRB1-mediated signaling at the cellular level. These results indicate that cell surface HLA-G dimers promote inhibitory signaling in natural killer cells through LILRB1 binding (Gonen- It is also in good agreement with Gross, T., et al., (2003 a) J Immunol, 171, 1343-1351.). On the other hand, it has been conventionally known that soluble HLA-G impairs the functions of CD8 + T cells, NK cells, and dendritic cells by binding to CD8, LILRB1 and LILRB2, respectively. (Contini, P., et al., (2003) Eur J Immunol, 33, 125-134 .; Fournel, S., et al., (2000) J Immunol, 164, 6100-6104 .; Liang, S et al., (2003) Hum Immunol, 64, 1025-1032 .; Morales, PJ, et al., (2003) J Immunol, 171, 6215-6224.). Soluble HLA-G protein was found to have monomeric, dimeric and oligomeric forms. Based on the significant binding affinity demonstrated by the inventor's SPR binding test, soluble HLA-G dimers are believed to have the most potent effect on LILRB signaling. Furthermore, preliminary data obtained by the present inventors showed that CD8 also showed a similar binding affinity effect and could improve CD8-mediated signaling.

From the above inventor's experiments, it is clear that both membrane-bound and soluble HLA-G dimers have the appropriate structural properties to play a major role in the broad immunosuppressive effect, particularly at the maternal-fetal interface. It has been shown to have extended functional properties. In addition, HLA-G dimer is a powerful immunosuppressive reagent in a wide range of inflammatory diseases, autoimmune diseases, infertility problems and medical treatment of organ Z bone marrow transplantation, and is naturally formed in vivo. Less likely to have side effects.

HLA-G, like classical MHC I, can theoretically present a diverse repertoire of peptides that can be recognized by T cell receptors. Already, HLA-G showed peptides derived from human megalovirus UL83 protein in HLA-G transgenic mice in vivo, and their specific T cells were induced but not efficient (Lenfant, R, et al., (2003) J Gen Virol, 84, 307-317.). The HLA_G monomer and dimer form may exist on the cell surface, but in the case of the HLA-G dimer, the peptide binding sites of each monomer of the HLA-G dimer are too close. Too much that the T cell receptor is not close enough. Thus, HLA-G dimers do not function as antigen-providing molecules for T cell responses, but are molecules that regulate immune by binding to: LILR or CD8. These properties can explain in part the inefficient cytotoxic T cell induction. However, these properties are also important for receptor recognition of the 32m free MHC I dimer that regulates T cell function.

[Example 2] Efficient production of disulfide-linked HLA-G dimer; Promotion of intermolecular disulfide bond formation by DTT

In this example, an efficient method for producing a disulfide-bonded HLA-G dimer is provided. We also show that low dithiothreitol concentrations in the crystallization drop promote dimer formation of unwound HLA-G.

HLA-G monomer was refolded with refolding buffer (0.1M Tris-HCl (pH8.0), 0.4M L-arginine, 3.7mM cystamine, 6.4mM cysteamine, 5mM EDTA) (M. Shiroishi, et al., (2003) Proc Natl Acad Sci USA, 100, 8856-61.). Thereafter, it was purified by gel filtration (Superdex 75) and ion exchange chromatography (Resource Q). The purified HLA-G monomer was concentrated to 10 mg / mL, and after 3-4 hours, dithiothreol for crystallization setting (DTT) was added to 5 mM and incubated on ice. The initial crystallization test was carried out using Crystal Scrren 1 & 2 (Hampton Research) and Wizard I & U (Art Robbins) using an inteplate using the crystallization robot Hydra plus one. Under the conditions of Wizard II No.39 (0.1M CAPS (pH10.5), 20% PEG8000, 0.2M NaCl), rod crystals suitable for data collection were obtained at 20 ° C for 6 days (Fig. 2 _ 1 A).

X-ray diffraction data was collected at the Springline (Harima, Japan) beamline BL38B1. Prior to data collection, the crystals were immersed in a cryoprotectant solution (0.1 M CAPS (pH 10.5), 20% PEG8000, 200 mM NaCl, 20% glycerol) and cooled by flash. Using an ADSC Qauntum 4R CCD system with an X-ray wavelength of 1.0000A, 3.2A diffraction data was obtained at 100K (Figure 2—IB). Diffraction data was processed and scaled with the HKL2000 program package. These crystals have unit cell dimensions a = 94.70A, b = 127.85 A and c = 72.60A, and belong to the orthorhombic space group PS ^^. Data collection statistics are shown in Table 2 _1. The crystal structure was elucidated by molecular replacement. Intermolecular Cys42-Cys42 disulfide bond Since the electron density map for was clearly observed, the disulfide-linked dimer of wild-type HLA-G was crystallized under these conditions.

Table 2

Data collection statistics for HLA-G dimers

Crystallization conditions were examined to identify the factors that promote the formation of HLA-G disulfide-linked dimers in the crystallization drop. HLA-G monomer concentrated to 10 mg / mL (buffer; 20 mM Tris-HCl (pH 8.0), lOOmM NaCl) at 4 ° C for 3 days with 2 mM DTT or 10 days without DTT Incubated. These mixtures were analyzed by gel filtration and, as a result, it was found that the addition of DTT significantly promotes HLA-G dimer formation (Figures 2-2 A and B).

Next, is the free cysteine of the refolded HLA-G molecule protected? In order to clarify whether or not, the amount of free thiol group of HLA-G monomer was measured by using 5,5'-dithiopis 2-nitrobenzoate. The recent crystal structure of the HLA-G Cys42Ser mutant shows that Cys42 is completely exposed to the solvent (CS Clements, et al., (2005) Proc Natl Acad Sci USA, 102, 3360-5.), Only less than 10% unpaired thiol groups were detected. This indicated that the majority of the unrolled HLA-G free thiol groups were protected by a reducing agent, such as cysteamine, contained in the refolding solution. High pH (CAPS pH 10.5) was only marginally effective for dimer formation.

These results indicated that low concentrations of DTT promote disulfide bond formation by attacking free thiol groups protected by reducing agents. This technique is applicable to disulfide-linked dimer formation or multimer formation of proteins in solution and in crystallization drops. Industrial applicability

According to the present invention, it is possible to provide a pharmaceutical composition (particularly, a pharmaceutical composition having an excellent immune cell activation-inhibiting action), which is a novel use of HLA-G dimer, and disulfide-binding HLA An efficient method for producing a -G dimer can be provided.

Further, according to the present invention, various therapeutic agents and preventive agents containing the above pharmaceutical composition, for example, allergic diseases (such as asthma, atopic dermatitis, allergic rhinitis), inflammatory diseases, or infertility (infertility) Prophylactic and Z or therapeutic agents, and immunosuppressive therapeutic agents for use in organ transplantation, bone marrow transplantation or regenerative medicine can be provided.

Furthermore, according to the present invention, there is provided a method for treating and / or preventing an inflammatory disease characterized by administering an effective amount of a disulfide-bonded HLA-G dimer to a patient, and disulfide in a human or non-human mammal. It is possible to provide a method for controlling and / or suppressing inflammation, which comprises administering an effective amount of a conjugated HLA-G dimer. Sequence listing free text

Sequence number 3: Synthetic peptide

Claims

The scope of the claims

1. A pharmaceutical composition comprising an HLA-G dimer.

2. The composition according to claim 1, which has an activity of suppressing activation of immune cells.

3. The composition according to claim 1, wherein the HLA-G dimer is one in which HLA-Gs are linked by an intermolecular disulfide bond.

4. HLA-G is the following protein (a) or (b), and an intermolecular disulfide bond is formed between the 4th and 2nd cysteine residues in the amino acid sequence shown in SEQ ID NO: 2. The composition according to claim 3, wherein

(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2;

5. A preventive and Z or therapeutic agent for allergic diseases, comprising the pharmaceutical composition according to claim 1.

6. A preventive and / or therapeutic agent for atopic dermatitis, asthma, allergic rhinitis, inflammatory disease or infertility, comprising the pharmaceutical composition according to claim 1.

7. An immunosuppressive therapeutic agent for organ transplantation, bone marrow transplantation or regenerative medicine, comprising the pharmaceutical composition according to claim 1.

8. A method for producing a disulfide-linked HLA-G dimer, which comprises adding a reducing agent to a solution containing HLA-G.

9. The process according to claim 8, wherein the reducing agent is dithiothreitol (DTT).

10. In HLA-G solution: concentration power of BLA-G monomer S0.1 ~: lOmg / mL, and addition concentration of reducing agent to the solution is 0.1 ~: lOmM. The method described.

1 1. A method for controlling and / or inhibiting inflammation, comprising administering an effective amount of a disulfide-binding HLA-G dimer to a human or non-human mammal.

1 2. A method for treating and / or preventing an inflammatory disease, which comprises administering an effective amount of a disulfide-bonded HLA-G dimer to a patient.