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WO2007011044A1 - Composition pharmaceutique comprenant un dimere hla-g lie disulfide et procede de fabrication de ce dimere hla-g lie disulfide - Google Patents

Composition pharmaceutique comprenant un dimere hla-g lie disulfide et procede de fabrication de ce dimere hla-g lie disulfide Download PDF

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WO2007011044A1
WO2007011044A1 PCT/JP2006/314537 JP2006314537W WO2007011044A1 WO 2007011044 A1 WO2007011044 A1 WO 2007011044A1 JP 2006314537 W JP2006314537 W JP 2006314537W WO 2007011044 A1 WO2007011044 A1 WO 2007011044A1
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hla
dimer
disulfide
binding
pharmaceutical composition
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Japanese (ja)
Inventor
Katsumi Maenaka
Mitsunori Shiroishi
Daisuke Kohda
Kimiko Kuroki
Linda Rasubala
Hisashi Arase
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Kyushu University NUC
University of Osaka NUC
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Kyushu University NUC
Osaka University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • 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.
  • 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.
  • HLA-G the suppressive effect of immune system cells such as natural killer (NK) cells and macrophages has been confirmed.
  • NK natural killer
  • Ig-like receptor eg, Ig-
  • Ig- leukocyte Ig-like receptor
  • 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.
  • 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.
  • the background art of the present invention will be described in more detail.
  • 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)).
  • 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.
  • LILRB1 / LILRB2 also known as LILRB1 / LILRB2, LIRlZLIR2 and ILT2 / ILT4
  • 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 (
  • 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).
  • NK natural killer
  • DC dendritic cells
  • Both LILRBs have four Ig-like domains in the extracellular region, and two N-like domains play an important role in MHC I recognition.
  • MHC I molecules bind to the domain, LILRB mediates negative signals through three or four immunoreceptor tyrosine inhibitory motifs (ITIM) in the intracellular domain.
  • ITIM immunoreceptor tyrosine inhibitory motifs
  • 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.
  • 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.).
  • HLA-G-LILRB and CD8 interactions can induce extensive immunological tolerance.
  • HLA-G can present a diverse set of peptides (Diehl, ⁇ , 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.
  • HLA'G will select a high-affinity raw peptide set limited for presentation, even if it has the classical MHC I peptide presentation mechanism.
  • 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.).
  • 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.
  • Cys67 free cysteine
  • 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.
  • HLA-G is one of the non-classical MHC I molecules and has several unique properties that differ from classical MHC I as follows.
  • the HLA-G receptor already reported is leukocyte Ig-like receptor B1 / B2 (
  • LILRB1 / LILRB2 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.
  • LILRB1 Expressed mainly in monocytes and dendritic cells (DC) (D.
  • LILRB1 is a natural killer (NK) cell and It is expressed in a wide range of leukocytes, including T cells
  • 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.
  • ITIM immunoreceptor tyrosine inhibitory motif
  • 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.
  • HLA-G has some splice variants It has a soluble form (P. Le Bouteiller, et al., (2003) Placenta, 24 Suppl A, S10-5 .; T.
  • 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.
  • 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 ⁇ 3 domain of the heavy chain and the region spanning j8 2-microglobulin opposite the free cysteine residue (Cys42) .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention is as follows.
  • a pharmaceutical composition comprising an HLA-G dimer.
  • the pharmaceutical composition of the present invention has an effect of suppressing activation of immune cells.
  • 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).
  • 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 prophylactic and / or therapeutic agent for allergic diseases comprising the pharmaceutical thread and composition according to (1) above.
  • 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.
  • An immunosuppressive therapeutic agent used for organ transplantation, bone marrow transplantation, or regenerative medicine comprising the pharmaceutical composition according to (1) above.
  • a method for producing a disulfide-linked HLA-G dimer comprising adding a reducing agent to a solution containing HLA-G.
  • the reducing agent examples include dithiothreitol (DTT).
  • DTT dithiothreitol
  • 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.
  • 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.
  • 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.
  • 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).
  • the sugar binding site (position 86) is indicated by a green dotted circle.
  • LILRB1 red and light blue
  • Mono HLA-G dimer pink and blue
  • CD8 green and yellow
  • Mono HLA-G dimer pink and blue
  • C Mono HLA-G dimer
  • 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
  • the two dotted circles red and cyan
  • the thick orange dotted line represents a schematic representation of the cell membrane.
  • 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
  • Lane 1 is dimer (15 ⁇ ) only
  • lanes 2 to 4 show binding between the dimer (15 ⁇ ) and each molar ratio of receptors
  • lane 5 is LILRB1 only (30 ⁇ ) It is.
  • 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 ⁇ L LILRB 1.
  • F Binding between HLA-G dimer and LILRB2. Lane 1 is dimer (15 ⁇ M) only, and lanes 2 and 3 show binding between HLA-G dimer (15 ⁇ M) and each molar ratio of receptors. Since LILRB2 migrated in the opposite direction, the band could not be shown using the same gel.
  • HLA-G dimer equilibrium gel filtration analysis of LILRB2 interaction. Similar to ( ⁇ ) above, a mixture of HLA-G dimer and LILRB1 was injected onto a column previously equilibrated with 15 ⁇ L LILRB2.
  • Figure 1-14 Surface plasmon resonance analysis of HLA-G dimer and monomer (Cys42Sr mutant) binding to immobilized LILRB1 and LILRB2
  • 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.
  • 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.
  • MHC I MHC class I
  • LLR leukocyte Ig-like receptor
  • CD8 CD8
  • HLA-G is expressed in disulfide-linked dimer form in solution and on the cell surface.
  • MHC I dimer organization is involved in pathogenesis and T cell activation.
  • the three-dimensional structure and receptor binding properties of MHC I dimers were not known at all.
  • 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.
  • the HLA-G dimer has a structural orientation suitable for efficient LILR-mediated signaling that produces the most potent immunosuppressive effect.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • Cys42 cysteine residue 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • kits include QuickChange TM Site-Directed Mutagenesis Kit (Stratagene), GeneTailor TM Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km Etc .: manufactured by Takara Bio Inc.).
  • 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.
  • the foreign gene was introduced by introducing plasmid DNA or the like into the host (transformation).
  • those into which foreign genes have been introduced by infecting a host with various viruses and phages (transduction) are included.
  • it is capable of expressing HLA-G from the introduced thread recombination vector, it is not limited.
  • various animal cells such as human mice, various plant cells, bacteria, fermentation
  • Known hosts such as mother and plant cells can be used.
  • 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.
  • 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.
  • recombinant HLA-G 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the disulfide-bonded HLA-G dimer can be produced more efficiently by adding a reducing agent in the presence of the HLA-G monomer.
  • the reducing agent examples include dithiothreitol (DTT), -mercaptoethanol, reduced dartathione, cysteamine, etc. Among them, DTT is particularly preferable.
  • 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.
  • 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.
  • concentration of an HLA-G solution can be mentioned.
  • a reducing agent into a small quantity with respect to a HLA-G monomer
  • final concentration is 0.1-:! OmM, Preferably it is 1-5mM.
  • 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.
  • the present invention includes an HLA-G dimerization promoter containing a reducing agent (eg, DTT).
  • a reducing agent eg, DTT
  • the pharmaceutical composition of the present invention is characterized by containing a disulfide-linked HLA-G dimer.
  • 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.
  • 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.
  • 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.
  • 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).
  • oral or parenteral eg, intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration.
  • 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.
  • Preferred oral dosage forms include, for example, tablets, powders, fine granules, granules, coated tablets, capsules, syrups, and lozenges for oral preparations.
  • 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
  • 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.
  • 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.
  • 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.
  • a binder for example, 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.
  • sugar coating can be used, and if necessary, other known methods can be used for appropriate coating.
  • syrups and injectable preparations for example, pH adjusters, solubilizers, tonicity agents, etc., and if necessary, solubilizing agents, stabilizers, etc.
  • the production method is not limited and can be produced by a conventional method.
  • the base material to be used various raw materials usually used for pharmaceuticals, quasi drugs, cosmetics, etc. can be used.
  • 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.
  • components such as blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, humectants, and keratolytic agents can be added as necessary.
  • the ratio of the HLA-G dimer to the carrier is not limited and can be appropriately set between 1 and 90% by weight.
  • the effective dose is, for example, the degree of symptoms, age, gender, body weight, dosage form, salt type, and specific disease
  • the effective dose is, for example, the degree of symptoms, age, gender, body weight, dosage form, salt type, and specific disease
  • the pharmaceutical composition 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).
  • pharmaceutical compositions such as immune system cell activation inhibitors (particularly anti-inflammatory agents).
  • 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.
  • 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.
  • 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.
  • the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
  • Crystals were obtained at 20 ° C. under the conditions of Wizard 11-39 (lOOmM CHAPS ⁇ 5, 20% (w / v) PEG8000, 200 mM NaCl).
  • Diffraction data was processed and scaled using the HKL2000 program package.
  • Native polyacrylamide gel electrophoresis 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,
  • 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.
  • BiAcore bivalent analytevaluation v3.2
  • Affinity constants K d s
  • 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).
  • 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.).
  • GFP NFAT-green fluorescent protein
  • 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.
  • HLA-G HLA-G * 0101, 1-274 residue
  • RIIPRHLQL SEQ ID NO: 3
  • 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) .
  • 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.
  • 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”) .
  • HLA-G dimer two HLA-G dimers were found in the crystals, and their structural properties suggested both the structural flexibility and structural rigidity of the dimer.
  • this dimeric interface is responsible for any electrostatic and hydrogen bond interactions (Clu61 ( ⁇ )-Lys68 ( ⁇ ), 42 (CO ) -Arg44 ( ⁇ ), Glu58 ( ⁇ 1, ⁇ 2) -Cln72 ( ⁇ 2, ⁇ )) (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.
  • 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 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). .
  • 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.
  • HLA-G dimer-LILRB1 and LILRB2 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 ⁇ L LILRB1 or 15 ⁇ L 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.
  • 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).
  • SPR Surface Plasmon Resonance
  • HLA-G binding to LILRB were further investigated by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • 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.
  • the affinity constants (K d s) of the monomers for LILRB1 and LILRB2 derived from the equilibrium analysis are 3.5 ⁇ and 15 ⁇ , respectively (Fig. 1-4E).
  • NFase NFAT-green fluorescent protein
  • LILRB1 chimeric gene consisting of extracellular domain of LILRB1 and transmembrane and intracellular domain of activated PILR ⁇ 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.
  • 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
  • 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.
  • HLA-G dimer LILRB or CD8 receptor
  • Figure 1, 1-2B, C the two receptors can assemble in the proper orientation at the cell surface for efficient signaling.
  • the biochemical test in this example showed a much higher affinity for LILRB of HLA-G dimer than monomer by increasing binding activity.
  • 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.
  • 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.
  • 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.
  • 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 (Lieri, 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.
  • 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.
  • 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.
  • refolding buffer 0.1M Tris-HCl (pH8.0), 0.4M L-arginine, 3.7mM cystamine, 6.4mM cysteamine, 5mM EDTA
  • 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.
  • a cryoprotectant solution 0.1 M CAPS (pH 10.5), 20% PEG8000, 200 mM NaCl, 20% glycerol
  • 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.
  • 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).
  • 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.
  • 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.
  • allergic diseases such as asthma, atopic dermatitis, allergic rhinitis
  • inflammatory diseases such as asthma, atopic dermatitis, allergic rhinitis
  • infertility infertility
  • Prophylactic and Z or therapeutic agents for use in organ transplantation, bone marrow transplantation or regenerative medicine
  • 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.

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Abstract

L'invention concerne une nouvelle utilisation d'un dimère HLA-G. L'invention concerne également un procédé de fabrication très efficace d'un dimère HLA-G. L'invention concerne donc une composition pharmaceutique comprenant un dimère HLA-G ; et un procédé de fabrication d'un dimère HLA-G comprenant une étape d'ajout d'un agent réducteur à une solution contenant HLA-G.
PCT/JP2006/314537 2005-07-15 2006-07-18 Composition pharmaceutique comprenant un dimere hla-g lie disulfide et procede de fabrication de ce dimere hla-g lie disulfide Ceased WO2007011044A1 (fr)

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EP2184070A1 (fr) 2008-11-07 2010-05-12 Hla-G Technologies Protéines HLA-G et leurs utilisations pharmaceutiques
JP5637981B2 (ja) * 2009-03-03 2014-12-10 国立大学法人北海道大学 関節リウマチまたはその関連疾患の予防または治療剤
WO2010101192A1 (fr) 2009-03-03 2010-09-10 国立大学法人九州大学 Agent prophylactique ou thérapeutique destiné au traitement de la polyarthrite rhumatoïde ou de maladies liées à la polyarthrite rhumatoïde
EP2264067A1 (fr) 2009-06-18 2010-12-22 Hla-G Technologies Multimères de HLA-G alpha 1 et leurs utilisations pharmaceutiques
WO2010146094A1 (fr) * 2009-06-18 2010-12-23 Hla-G Technologies Multimères alpha 1 de hla-g et leurs utilisations pharmaceutiques
JP2012530106A (ja) * 2009-06-18 2012-11-29 エイチエルエイ−ジー・テクノロジーズ ＨＬＡ−Ｇα１多量体及びその薬学的使用
JP2012531400A (ja) * 2009-06-25 2012-12-10 コミッサリア ア レネルジ アトミック エ オー エネルジ アルターネイティブス 少なくとも２つのα３ドメインを含むＨＬＡ−Ｇの多量体ポリペプチド及び医薬としてのその使用
WO2010150233A3 (fr) * 2009-06-25 2011-05-12 Commissariat A L'energie Atomique Et Aux Energies Alternatives Polypeptides multimères de hla-g comprenant au moins deux domaines alpha3 et leurs utilisations pharmaceutiques
WO2010150233A2 (fr) 2009-06-25 2010-12-29 Commissariat A L'energie Atomique Et Aux Energies Alternatives Polypeptides multimères de hla-g comprenant au moins deux domaines alpha3 et leurs utilisations pharmaceutiques
WO2010150235A1 (fr) 2009-06-25 2010-12-29 Commissariat A L'energie Atomique Et Aux Energies Alternatives Polypeptides multimères de hla-g comprenant des monomères alpha1-alpha3 et leurs utilisations pharmaceutiques
US11401318B2 (en) 2009-06-25 2022-08-02 Commissariat A L'energie Atomique Et Aux Energies Alternatives Multimeric polypeptides of HLA-G including at least two alpha3 domains and pharmaceutical uses thereof
JP2013544250A (ja) * 2010-11-23 2013-12-12 エタブリスモン フランセ ドュ サン 骨吸収に付随する疾患の治療におけるｈｌａ−ｇアイソフォームの使用
JP2015140322A (ja) * 2014-01-29 2015-08-03 国立大学法人北海道大学 関節リウマチまたはその関連疾患の予防または治療剤
JP2018512113A (ja) * 2015-02-04 2018-05-17 ウニヴェルズィテート チューリッヒ 癌治療のためのｈｌａホモ二量体の使用
US11484572B2 (en) 2015-02-04 2022-11-01 Universität Basel Use of HLA-B27 homodimers for cancer treatment
JP2019512231A (ja) * 2016-03-08 2019-05-16 ウニヴェルズィテート チューリッヒ Ｈｌａ−ｂ５７オープンコンフォーマー
US12383602B2 (en) 2016-03-08 2025-08-12 Universitat Zurich HLA-B57 open conformers

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