WO2000078804A1 - Proteines regulatrices de la reponse immunitaire - Google Patents

Proteines regulatrices de la reponse immunitaire Download PDF

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Publication number
WO2000078804A1
WO2000078804A1 PCT/JP2000/003970 JP0003970W WO0078804A1 WO 2000078804 A1 WO2000078804 A1 WO 2000078804A1 JP 0003970 W JP0003970 W JP 0003970W WO 0078804 A1 WO0078804 A1 WO 0078804A1
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amino acid
antigen
acid sequence
gif
seq
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PCT/JP2000/003970
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English (en)
Japanese (ja)
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Yasuyuki Ishii
Hiroshi Watarai
Ayako Tokunaga
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Kirin Beer Kabushiki Kaisha
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Priority to AU52505/00A priority Critical patent/AU5250500A/en
Publication of WO2000078804A1 publication Critical patent/WO2000078804A1/fr

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a derivative protein of a non-antigen-specific glycosylation inhibitor (hereinafter, referred to as “GIF”) which can be used to suppress a human immune response to an antigen;
  • GIF non-antigen-specific glycosylation inhibitor
  • the present invention relates to a DNA, a recombinant expression vector containing the DNA, a cell transformed with the DNA, a method for producing the GIF derivative protein, and a pharmaceutical composition containing the GIF derivative protein.
  • allergic diseases such as those found in hay fever are caused by IgE antibodies to allergens (antigens). Therefore, the control and suppression of IgE antibody production against allergens is considered to be a fundamental treatment for allergic diseases.
  • desensitization therapy in which allergic patients are repeatedly injected with a very small amount of allergen has been performed in medical practice. Although this patient may improve clinical symptoms in some patients, it may also cause anaflex shock. Therefore, desensitization therapy is only given to a limited number of patients with good surveillance.
  • IgE-binding factor enhances IgE production and the other suppresses IgE production. These two factors have the same protein portion, but different sugar chains. Factors that enhance IgE production have high-mannose N-glycans and bind to lentil lectin. On the other hand, factors that suppress IgE production have no affinity for lentil lectin (Yodoi et al., J. Immunol., 128, 289, 1982). It was also shown that the difference in the sugar chain structure resulted in different biological activities of the two IgE binding factors. Under physiological conditions, the process of glycosylation of this IgE-binding factor is controlled by two T-cell factors that promote or inhibit this process.
  • GEFs dalycosylation-promoting factors
  • GIF glycosylation inhibitor
  • T cells producing GIF are antigen-specific sub-cell T cells (Jardieu et al., J. Immunol., 133, 3266, 1984).
  • OVA ovalbumin
  • T-cell hybridomas show that these cells constitutively produce GIF, and that the GIF does not show any specificity for antigen (antigen).
  • stimulation of these cells with ovalbumin-treated antigen-presenting cells could induce the production of GIF (antigen-specific GIF) having affinity for ovalbumin.
  • antigen-specific GIF is composed of an antigen-binding polypeptide chain and a non-specific GIF.
  • Antigen-specific GIF has an antigenic determinant common to antigen-specific subcellular T-cell factor (Steele, JK et al., J. Immunol. 142, 2213-2220, 1989), and antigen (carrier) -specific Suppressed the antibody response to the virus (Jardieu, P. et al., J. Immunol. 138, 1494- 1501, 1987).
  • the produced recombinant GIF showed extremely low biological activity as compared to GIF derived from subpressor T cells.
  • the produced recombinant GIF has almost the same biological activity as GIF derived from a subpressor T cell. It was found to have Subsequently, it was shown that some post-translational modifications are required for GIF peptides to exhibit sufficiently high biological activity (Liu, Y- et al., Proc. Natl. Acad. Sci.
  • GIF derivatives were produced based on speculation of the cause of the low biological activity of recombinant GIF (International Publication, W097 / 959), but the production of GIF derivatives based on the structure of active natural GIF was Not reported.
  • An object of the present invention is to analyze the structure of a natural GIF, and to provide an active GIF derivative based on the structure and a method for producing the same. Disclosure of the invention
  • the subject of the present invention is as follows.
  • Antigen-specific non-specific glycosylation inhibitor having the amino acid sequence of SEQ ID NO: 2 A cystine residue at position 60 of the amino acid sequence of SEQ ID NO: 2 which is a cysteine derivative, and a derivative of an amino acid residue other than the cystine residue at position 60.
  • a derivative having at least one amino acid sequence which may be substituted, deleted, inserted or modified, and which has an immunosuppressive activity of an antigen non-specific dalycosylation inhibitor.
  • the antigen-nonspecific immunoglycosylation inhibitor inducer according to any one of (1) to (6), wherein the methionine residue at position 1 of the amino acid sequence of SEQ ID NO: 2 is deleted. .
  • the cysteine residue at position 57 is substituted with alanine and the cysteine residue at position 60 has a cysteine-containing amino acid sequence, and immunosuppression by a non-antigen-specific glycosylation inhibitor. Derivatives with activity.
  • At least one amino acid residue other than the cystine residue at position 60 of the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 is substituted, deleted,
  • the polypeptide is produced by culturing cells that have been transformed by a recombinant vector having a polynucleotide encoding the amino acid sequence that has been introduced or modified (12). ).
  • a pharmaceutical composition comprising, as an active ingredient, the antigen-nonspecific human glycosylation inhibitory factor derivative according to any one of (1) to (9).
  • the pharmaceutical composition according to (28), wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and type I diabetes.
  • the term “derivative” refers to any post-translational modification of the polypeptide defined by the amino acid sequence deduced from the DNA sequence of a certain gene, regardless of its origin and production method. Alternatively, it is defined as meaning a polypeptide having any amino acid sequence modification.
  • GIF found in the culture supernatant of the human subpressor T cell hybridoma was separated into the following four peaks by reversed-phase HPLC. 1) Unmodified after translation (abbreviated as WT), 2) Mass number increased by 83 daltons with respect to WT, and it is estimated that cysteine at position 9 is phosphorylated.
  • WT Unmodified after translation
  • Mass number increased by 83 daltons with respect to WT, and it is estimated that cysteine at position 9 is phosphorylated.
  • the structure of the activated GIF was identified from the culture supernatant of the human suppressor T cell hybridoma, and it was possible to produce the activated GIF in a small scale.
  • the production of GIF by human suppressor T-cell hybridomas is low, and large-scale supply is difficult. Therefore, the inventors have devised a method of producing GIF by recombinant DNA technology and then cystinizing the GIF by adding cystine. Briefly, cystine at position 60 is selectively cystinized by mixing recombinant GIF produced in E. coli with cystine, and then cystinized recombinant GIF is obtained using a cation column and gel filtration chromatography. Is a method for isolating
  • protein of the present invention a GIF derivative protein having high immunosuppressive activity (hereinafter, referred to as “protein of the present invention”) is provided.
  • the immunosuppressive activity of GIF refers to the production of a specific antibody belonging to the immunoglobulin E and immunoglobulin G1 induced by an antigen in vivo. It is an inhibitory activity. This activity is measured in vitro as the ability to convert mouse T-cell hybridoma 12H5 cells from the production of dalycosylated IgE binding factor to the production of non-dalicosylated IgE binding factor. (Iwata and Ishizaka, J. Immunol., 141, 3270 1988).
  • anti-DNP-IgE antibodies produced by stimulating ovalbumin-specific helper ⁇ cells and MP-specific ⁇ cells with DNP-ovalbumin antigen are also provided.
  • a method for measuring the ability to inhibit an anti-DNP-IgGl antibody was used.
  • the present invention provides a GIF derivative in which Cys at position 60 in the amino acid sequence of GIF has been cystinated based on the identification of the structure of the modified natural GIF.
  • the amino acid sequence of GIF may have other mutations such as substitution and deletion. Mutations may be introduced at 57 and 91 sites in the amino acid sequence of GIF. Also, the Met residue at position 1 may be deleted. The N-terminal and C-terminal amino acids may be deleted as long as the activity of the GIF derivative of the present invention is maintained. Specific examples of the mutation include substitution of the 57-position cysteine residue with an alanine residue or a serine residue.
  • Polynucleotides encoding the amino acid sequence of GIF into which these mutations have been introduced can be obtained by site-directed mutagenesis using a known GIFc DNA (Mikayama, ⁇ ⁇ et al., Supra). Alternatively, it can be produced by chemically synthesizing a partial polynucleotide containing a site to be mutated and replacing it with the corresponding region of G1 Fc DNA, or by chemically synthesizing the whole.
  • the polynucleotide encoding the amino acid sequence of GIF or a mutated GIF is a polynucleotide containing any of the nucleotide sequences that are in a degenerate relationship as a result of the genetic code. Including.
  • the expression level of the protein can be improved by employing a preferential codon suitable for the host in which the GIF derivative protein is to be expressed, for a part or all of the GIF coding region.
  • the preferential codon is used for a part of the GIF coding region, it is effective to use the N-terminal coding region.
  • polynucleotide of the present invention examples include a polynucleotide containing the nucleotide sequence of SEQ ID NO: 3 in the sequence listing.
  • the polynucleotides of the present invention can include the provision of a cleavage site with a restriction enzyme and / or the additional provision of DNA at the start and end to construct an expression vector to facilitate expression. .
  • a host cell transformed with the vector and a protein of the present invention produced by culturing the host cell and producing the same
  • a method for producing the protein of the present invention which is characterized by being modified.
  • the host cells in this case, prokaryotic (eg, bacteria, preferably E. coli) and eukaryotic (eg, yeast, insect, or mammalian) cells can be used.
  • mammalian cells include COS cells, Chinese Hamster Ovary cells, X63.6.5.3.
  • C-127 cells C-127 cells, BHK (Baby Hamster Kidney) cells, and human-derived cells ( For example, HeLa cells).
  • insect cells include silkworm (Bombyxmori) cultured cells (for example, Sf21 cells), etc.
  • yeast cells include Saccharomyces ce revisiae. And j_chia apas tor is. can give.
  • Vectors used to transform these host cells include pKC30 (Shimatake H. and M. Rosenberg, Nature. 292, 128-132, 1981) and pTrc99A (Amann E. et al., Gene6 , 69, 301-315, 1988).
  • pSV2-neo Southern and Berg; J. Mol. Appl. Genet., 1, 327-341, 1982
  • pCAGGS Newa3 ⁇ 4; Gene, 108, 193-200, 1991
  • Or pcD SRa296 Takebe et al .; Mol. Cell. Biol., 8, 466-472, 1988.
  • yeast pG-1 (Schena M.
  • vectors may optionally contain an origin of replication, a selection tool, and a promoter.
  • an eukaryotic cell vector may be added with an RNA splice site, a polyadenylation signal, and the like, as necessary.
  • a vector derived from SV40, adenovirus, or papilloma virus can be used as a vector for mammalian cells.
  • a vector for Escherichia coli those derived from ColEl, R factor and F factor can be used.
  • yeast 2 mDNA and those derived from ARS1 can be used.
  • vectors for mammalian cells include those derived from viruses, for example, those derived from retrovirus, poliovirus, adenovirus, SV40, and those derived from chromosomes. (Eg, EF1- ⁇ ) can be used.
  • Escherichia coli vectors include those derived from pacteriophage ⁇ , trp, You can use lpp, lac, and tac promoters.
  • ADH, PH05, GPD, PGK, MAF ⁇ promoter for the vector for Pichia pas toris, A0X1 promoter, etc. can be used.
  • a promoter derived from nuclear polyhedrosis virus can be used.
  • vectors for mammalian cells include neomycin (neo) resistance gene, thymidine kinase (TK) gene, dihydrofolate reductase (DHFR) gene, and Escherichia coli xanthinguanine phosphoribosyltransferase.
  • Escherichia coli a vector for Escherichia coli
  • a kanamycin resistance gene an ampicillin resistance gene, a tetracycline resistance gene and the like can be used.
  • yeast, Leu2, Trpl, Ura3 genes and the like can be used.
  • the following may be performed. After transforming a host cell with a recombinant DNA having the DNA of the present invention incorporated into an appropriate site of the above vector, the resulting transformant is cultured and further cultured in a cell or in culture.
  • the protein of the present invention may be separated and purified from the liquid. Known means and methods may be used in combination.
  • Such purification methods include the steps generally used for protein purification (ion exchange chromatography, hydrophobic mutual chromatography, gel filtration chromatography, reversed phase chromatography, isoelectric focusing, Preparative electrophoresis, isoelectric focusing, etc.).
  • affinity purification using an antibody that recognizes GIF can be mentioned (International Publication W094 / 26923).
  • a method for cysteinylation at position 60 of the amino acid sequence of GIF More specifically, a method for allowing selective cysteinylation of the cystine residue at position 60 using L (-)-cystine and the purified recombinant GIF wild type or derivative under the conditions of the mouth of the intestine. It is. Specifically, the method includes the following steps. Prepare an acidic solution of L (-)-cystine, preferably 1 M or more hydrochloric acid, with cystine to a concentration of 80 mg / ml or more, and add this solution to a salt, preferably 0.1 M or more of sodium chloride. Dilute with H6-8 buffer to prepare L (-)-cystine containing buffer.
  • this cystine-containing The buffer and the purified recombinant GIF (one having a natural amino acid sequence or one having a modified amino acid sequence) are mixed and allowed to stand.
  • the ratio of cystine to recombinant GIF can be selected as appropriate, but the amount of cystine can be adjusted to a weight ratio of cystine to recombinant GIF of at least 5: 1 (molar ratio of 250: 1) to achieve cystine. Protein can be obtained efficiently.
  • the reaction can be performed at a standing temperature in the range of 4 ° C to 37 ° C, and the standing time can be appropriately selected in the range of about 4 hours to 20 hours.
  • the recombinant GIF mixture is concentrated with an ultrafiltration membrane, preferably an ultrafiltration membrane that eliminates those having a molecular weight of 300 or less, and the solution is replaced with a buffer solution of PH 6 to 8, and the cation is removed.
  • the protein is eluted by a salt concentration gradient (linearly increasing the salt concentration from 0M, preferably from 0M to 0.5M) on an exchange column.
  • a salt concentration gradient linearly increasing the salt concentration from 0M, preferably from 0M to 0.5M
  • the earliest eluting peak is a ciscinated GIF.
  • the fraction containing the cysylated GIF can be concentrated and gel-filtrated to recover the cystine GIF.
  • Example 3 of the present specification Although detailed experimental conditions of the present method are disclosed in Example 3 of the present specification, in the present method, a person skilled in the art can appropriately adjust the experimental conditions shown in Example 3 within a range expected from the state of the art. This may involve changes in selectable concentrations, reaction times, experimental materials, etc.
  • the protein of the present invention may involve the phosphorylation of serine at position 91 of the amino acid sequence of GIF. Phosphorylation is performed using protein kinase. It can be carried out by an enzymatic reaction that transfers the phosphoric acid group to the hydroxyl group of serine, threonine or tyrosine.
  • the protein of the present invention may have a chemical modification at residues other than the 60th and 91st positions of the amino acid sequence of GIF.
  • Specific chemical modifications include phosphorylation, alkylation, acylation, EMTS modification, DTNB modification, etc.
  • select cysteine residues Specific examples include carboxymethylation and pyridylethylation for chemically modifying, acetylation and formylation for modifying the N-terminal.
  • Alkylation of the SH group can be carried out using compounds containing alkyl halides (monoacetic acid and its amides, monobromoacetic acid and its amides, iodopropionic acid, / 9-bromoethylamine, monochloroacetic acid, chloroacetophenoacetic acid). ) Can be.
  • alkyl halides monoacetic acid and its amides, monobromoacetic acid and its amides, iodopropionic acid, / 9-bromoethylamine, monochloroacetic acid, chloroacetophenoacetic acid).
  • a lipid such as a huanesyl group by using huanesyl bromide.
  • aliphatic aldehydes, ketones containing carbonyl, and the like can be used for modifying the amino group.
  • the acylation is carried out by using a carboxylic acid anhydride or a carboxylic acid chloride, or by using a carboxylic acid and a dehydrating condensing agent such as carbodiimide to convert the hydrogen of a hydroxyl group, a sulfhydryl group or an amino group into an acyl group.
  • a substitution reaction for example, N-acetylimidazole N-acetyl succinimide or the like is used.
  • Dehydration condensation of poly (ethylene glycol) having a carboxyl group at the end with N-hydroxysuccinimide using carbodiimide gives activated PEG which reacts with the amino group, and N-protein of the protein is obtained. The end can be converted to PEG.
  • glycine is added to the N-terminus, and palmitoylation, retinoylation, and riboylation of myristoylated lysine, tyrosine, threonine, and cystine can be performed.
  • Cysteine residues of the protein may be modified using a thiol reagent, such as ethyl methylcuricosalicylate (hereinafter referred to as "EMTS”), or DTNB or methyl methanethiosulfate.
  • EMTS ethyl methylcuricosalicylate
  • DTNB methyl methanethiosulfate
  • modification with a polymer compound include a method of binding soluble dextrin (Wileman, TE et al., J. Pharin. Phamaco 1.33, 85, 1982) and poly-DL- And a method using alanine (Uren, JR et al., Cancer Res., 42, 4068-4071, 1982).
  • the degree of chemical modification can be confirmed by quantifying the amount of unreacted protein-side reactive groups. For example, when the free S group of a cysteine residue is modified with monoacetic acid or 4-vinylpyridin and then subjected to S-carboxymethylation or S-pyridylethylation, respectively, the unreacted SH group becomes It can be quantified by the Ellmann method (Glazer, AN, The Proteins, 3rd., II, Academic Press, NY, 1976).
  • the iodination reagent (triiodide ion ( 1-3 ), If iodine chloride is used, the tyrosine residue will be modified, if acetyl pyrocarbonate is used, the histidine residue will be modified, and if phenyldalioxal is used, the arginine residue will be modified.
  • the amount of the corresponding amino acid residue can be confirmed by analyzing the amino acid composition or by changing the ultraviolet absorption spectrum. Alternatively, if the molecular weight changes significantly due to the modification, use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to follow changes in the mobility of the modified protein. Confirmed by. Further, which amino acid residue has been modified can be confirmed by performing peptide mapping.
  • Isolation and purification of the modified protein can also be performed by a combination of known means and methods as described in the method for producing the protein of the present invention, which is a mutant form by genetic recombination. .
  • the present invention provides a pharmaceutical composition containing the protein of the present invention as an active ingredient (hereinafter, referred to as “the pharmaceutical composition of the present invention”).
  • the pharmaceutical compositions of the present invention are used for immunosuppression and, more particularly, for alleviating or preventing an undesired immune response in the human being treated.
  • Undesirable immune responses include autoimmune diseases such as rheumatism, systemic lupus erythematosus multiple sclerosis and diabetes, and various other diseases. Allergic disease to the antigen, recipient versus graft (HVG), and graft versus recipient rejection.
  • the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a protein of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may contain diluents, preservatives, solubilizers, emulsifiers and other adjuvants.
  • therapeutically effective amount refers to an amount that provides a therapeutic effect for a specified condition and mode of administration.
  • Such pharmaceutical compositions may be in liquid or lyophilized or otherwise dried dosage forms, and may be buffered at various pHs and ionic strengths (eg, Tris-HCl, acetic acid).
  • Diluents selected from salts and phosphates; additives such as albumin or gelatin to prevent adsorption to surfaces; surfactants (eg Tween 20, Tween 80, Pluronic F68) , Bile salts); solubilizers (eg, glycerol, polyethylene glycol); antioxidants (eg, ascorbic acid, sodium metabisulfite); preservatives (eg, thimerosal, benzyl alcohol, para) Ben); in the form of a formulation containing excipients or isotonic agents (eg lactose, mannitol).
  • surfactants eg Tween 20, Tween 80, Pluronic F68
  • solubilizers eg, glycerol, polyethylene glycol
  • antioxidants eg, ascorbic acid, sodium metabisulfite
  • preservatives eg, thimerosal, benzyl alcohol, para
  • Ben in the form of a formulation containing ex
  • the protein of the present invention which is an active ingredient, is Complexed with ribosomes, polylactic acid, polydalicholic acid, hydrogels, etc. It may be incorporated into chloemaldione, micelles, unilamellar or multilamellar vesicles, red blood cells, or spheroplasts. Such a pharmaceutical composition would affect the physical state, solubility, stability, in vivo release rate and in vivo clearance of the protein of the present invention, so that the choice of the composition was effective. It depends on the physical and chemical properties of the constituent proteins of the invention.
  • the pharmaceutical composition of the present invention can be administered pulmonary, nasal, oral, intravenous, intraperitoneal, intramuscular, subcutaneous, intracavitary or transdermal, depending on the route of administration.
  • Li was granulated form, protective coating, Protea Ichize inhibitor or protein of the present invention that the absorption-promoting agent may also be incorporated, usually 0.0 0 1 17 1 8 1 ⁇ weight to 2 ⁇ (1 Depending on the patient's age, medical condition, sex and severity of the disease, and the route of administration, 1 ⁇ body weight can be administered once or several times daily or every other day.
  • FIG. 1 shows a Western plot analysis of native GIF purified with anti-GIF antibody titers.
  • Figure 2 shows the molecular weight of native GIF purified by anti-GIF antibody column on SDS-PAGE.
  • FIG. 3 shows the pattern of mass spectrometry of native GIF.
  • FIG. 4 shows the fractionation pattern of native GIF by reversed-phase chromatography.
  • Fig. 5 shows the fractionation pattern of each peptide fragment of API and AspN-digested recombinant GIF on a Super ODS column, and their amino acid sequences.
  • Figure 6 shows the fractionation of each peptide fragment of API and AspN-digested native GIF on a Super ODS column.
  • FIG. 7 shows the mass spectrometry patterns of the native GIF fragmentation peptides (X) and (Y).
  • FIG. 8 shows the pattern of mass spectrometry after carboxypeptidase digestion of native GIF fragmented peptides (X) and (Y).
  • FIG. 9 shows the fractionation pattern of the recombinant GIF C57A on the CM-5PW column after cystine treatment.
  • FIG. 10 shows the molecular species in the peak fractionated in FIG.
  • Figure 11 shows the fractionation pattern of cystinylated GIF C57A on a gel filtration column.
  • Japanese cedar pollen antigen-specific human suppressor T cell hybridoma 31E9 cells in culture [10% fetal calf serum, 10% NCTC 135 medium (Gibco), 10 mM HEPES buffer (Sigma), 0.2 U / ml Inshuri down, 50Aig / ml pyruvate, were grown up to 0.99 g / ml Okisa port high glucose containing DME basic medium added with acetic acid (Gibco)] in at IX 10 6 cells number / ml, cells were harvested And washed with PBS. The collected and washed cells were cultured in a serum-free high glucose-containing DME basic medium at a cell density of 2 ⁇ 10 e cells / ml for 24 hours, and then the culture supernatant was recovered.
  • IgG immunoglobulin G
  • the column is HPL
  • the sample was connected to C and monitored by absorption at a wavelength of 280 nm.
  • the eluate was neutralized by adding 1/100 volume of 1 M Tris-HCl buffer (pH 8.5), and then NAP25 (manufactured by Amers am Pharmacia Biotech). If the molecular extinction coefficient of IgG at 280 nm is 0.75, it is 21.7 mg / 7 ml, which is the center to exclude those with a molecular weight of less than 1000 daltons.
  • the antibody was concentrated to 2.5 ml with Replus 10 (Amicon) to bind this antibody to Hi Trap NHS-activated Sepharose (5 ml) (Amersham Pharmacia Biotech).
  • the final concentration of the antibody in the antibody column was 3.8 mg / ml.
  • the supernatant was concentrated 40 times with an ultrafiltration membrane YM3 (manufactured by Amicon), and 3 ml of the concentrated solution was adsorbed to the above antibody column at 4 ° C for 6 hours. Minute
  • the column was washed for 150 minutes at the flow rate, and eluted with 0.1 M glycine hydrochloride buffer (pH 2.8) .
  • the washing and elution procedures were performed by connecting the column to HPLC and absorbing at a wavelength of 280 nm.
  • the eluate was neutralized by adding 1 / 10,000 volume of 1 M Tris-HCl buffer (pH 8.5), and then terminated to prevent adsorption.
  • Peptide mapping was performed for the purpose of determining the position and modified structure of the modified amino acid of native GIF.
  • 400 pm 01 (5.0 g) of purified recombinant GIF The mixture was added to 6M guanidine hydrochloride and 0.3 M Tris-HCl buffer (pH 6.8) and denatured at 60 ° C for 1 hour.
  • 5 pmol of achromopacter protease (Achromobacter Protease I: API, manufactured by Wako Pure Chemical Industries) dissolved in water 401 was added and digested at 37 ° C for 6 hours.
  • Figure 5 shows the fractionation pattern and sequence of each peptide obtained by enzymatic treatment using the recombinant GIF API and AspN.
  • the purified natural GIF was concentrated to 10 pmo1 / ⁇ 1 using Ultra Free MC (manufactured by Amicon) which excludes those having a molecular weight of less than 500 daltons.
  • Ultra Free MC manufactured by Amicon
  • 301 of 8-guanidine hydrochloride and 0.4-tris-hydrochloric acid buffer (pH 6.8) were added, and denatured with 6 CTC for 1 hour.
  • API manufactured by Wako Pure Chemical Industries
  • FIG. 6 shows the fractionation pattern of each peptide by enzymatic treatment of native GIF with API and AspN.
  • Example 2 Biological activity of native GIF produced by Sablessa-1 T cell hybridoma This example relates to the in vitro biological activities of the four natural GIFs identified in Example 1.
  • an in vitro antibody production system was constructed. As the primary stimulation of T cells, anti-Stimulation of ovalbumin (OVA) -specific T-cell receptor (TCR) was carried out from whole spleen cells of transgenic mice (Sato et al., Er. J. Immunol., 24: 1512, 1994).
  • OVA ovalbumin
  • TCR T-specific T-cell receptor
  • T cells 8 x 10 5 cells
  • 10 ⁇ g of 0VA (Seikagaku Corporation) adsorbed BALB / c mice to 2 mg of Aram Thereafter, B cells (4 ⁇ 10 6 cells) purified from anti-B220 antibody microphone mouth beads (manufactured by Miltenyi Biotec) from all spleen cells excised on day 1 were cultured in 4 ml of a culture solution [10% fetal bovine embryo].
  • the cells were washed twice with a culture solution, and 200 ⁇ 1 of the culture solution was added thereto, followed by culturing for 4 days.
  • Anti MP- I g E in the culture supernatant, anti-DNP-IgGl weight was measured by enzyme immunoassay.
  • the natural GIF used in the evaluation of biological activity was prepared by concentrating the culture supernatant of 31E9 cells described in Example 1 using NAP-25 (manufactured by Pharmacia) in 20 mM sodium acetate buffer (pH 6.0). ), And apply to a TSKgel CM-5PW (7.5 mm x 7.5 cm) (manufactured by Tosoh Corporation) column equilibrated with the same buffer solution.
  • the sodium chloride concentration was adjusted to 0 to 0 at a flow rate of 1 ml / min. ⁇ Obtained by elution with a concentration gradient that increases linearly to 5M. Peaks in ascending elution order
  • composition ratios of the four natural GIFs contained in each peak were determined by separation by reversed-phase chromatography and mass spectrometry described in Example 1.
  • Table 5 The composition ratios of the four natural GIFs contained in each peak, as shown in Table 5, were determined by separation by reversed-phase chromatography and mass spectrometry described in Example 1.
  • Peak 3 3.85 ⁇ 0.63 1.81 ⁇ 0.40 Peak 3 is composed of only native GIF WT, but has no inhibitory activity on anti-DNP-IgGl production. Therefore, natural GIF WT was determined to be inactive. Peak 1 contained 25% of native GIF + 83, but since it had no inhibitory activity, natural GIF + 83 was also determined to be inactive. Peak 2 contained 26% of native GIF + 121, and its inhibitory activity was detected. Since natural GIF + 205 was present in both peak 1 and peak 2 and was in a trace amount, the biological activity could not be determined. From the above results, it became clear that cystin modification is an essential condition for the expression of biological activity of GIF.
  • the nucleotide at the corresponding site in the GIF gene of SEQ ID NO: 1 was mutated and substituted with an alanine residue.
  • the expression plasmid pTMK-hGIF (described above, W094 / 26923) in which human GIF cDNA (wild type) was inserted into the expression vector PST811 for E. coli (JP-A-63-269983) was used as the template DNA.
  • a polymerase chain reaction (PCR) (Mullis et al., Method in Enzymol., 155, 335-350, 1987) was performed using the following oligonucleotide primers.
  • the nucleotide sequence of the DNA fragment re-amplified by PCR was confirmed by ordinary DNA sequencing. These DNA fragments were ligated to the expression vector pST811, which had been cut with AflH and BamHI, by the action of DNA ligase.
  • This expression plasmid contains a human GIF cDNA (SEQ ID NO: 3) in which the nucleotide sequence encoding the cysteine residue at position 57 has been converted to a nucleotide sequence encoding an alanine residue, and contains pC57A- Named hGIF. This expression plasmid was introduced into a recombinant RR1 E. coli host cell to prepare a recombinant protein expression cell.
  • RRI E. coli containing the expression plasmid pTMK-hGIF or pC57A-hGIF was cultured in a 20 ml luria medium containing 50 mg / L ampicillin at -37 ° C. Furthermore, the cells were transferred to 1 L of M9 medium composed of 0.8% darcose, 0.4% casamino acid, 10 mg / L thiamine, and 50 mg / L ampicillin, and cultured at 37 ° C for 3 hours. Further, 40 mg of indoleacrylic acid was added, and the cells were cultured at 37 ° C for 5 hours.
  • E. coli cells with a wet weight of about 5 g are recovered by centrifugation, suspended in 200 ml of 20 mM sodium acetate buffer (PH6.0), and disrupted with a French press (8000 psi). Was. After centrifugation at 15000 g for 10 minutes, the supernatant fraction was collected, adjusted to pH 6.0 by adding acetic acid, and equilibrated with 20 mM sodium acetate buffer (pH 6.0).
  • This example describes a method for selectively cystinizing cysteine at position 60 in the amino acid sequence of SEQ ID NO: 2 by mixing purified recombinant GIF protein with L (-)-cystine in vitro.
  • the present invention relates to a method for isolating and purifying a recombinant GIF.
  • the first 80 mg / ml dissolved in 1M hydrochloric acid L (-) - cystine (manufactured by Wako Pure Chemical Industries, Ltd.) solution was diluted with 150mM chloride isocyanatomethyl Li um containing 20mM carbonate isocyanatomethyl Trim buffer (P H7.6)
  • a buffer containing 1 mg / ml L (-)-cystine was prepared.
  • the cystine-containing buffer and the purified recombinant GIF-C57A were mixed at a weight ratio of 5: 1 (molar ratio of 250: 1) and allowed to stand at room temperature for 18 hours.
  • the solution is replaced with 20 mM sodium acetate buffer (pH 6.0) using NAP- 25 (manufactured by Pharmacia).
  • TSKgel CM-5PW 7.5 mm X 7.5 cm
  • Toso Ichisha equilibrated in the above procedure.
  • cysteine-modified GIF wild type (abbreviated as wild type-Cys) and the cysteine-modified site of C57A-Cys were analyzed using the method for determining a modification site in Example 1, and as a result, SEQ ID NO: 2 It was confirmed that only the cysteine at position 60 of the amino acid sequence was modified.
  • Example 4 Biological activity of cystinylated recombinant GIF derivative
  • Example 3 The biological activities of wild type-Cys and C57A-Cys produced in Example 3 were determined using the in vitro antibody production system described in Example 2. On the day of the primary stimulation, the sample was added at a final concentration of 500 ng / ml, and the anti-DNP-IgGl and anti-DNP-IgE values produced in the culture supernatant were measured by the enzyme antibody method. As a result, it was confirmed that wild type-Cys and C57A-Cys exhibited higher inhibitory effects than C57A-N106S disclosed in W097 / 959 (Table 7). Table 7
  • Example 3 The biological activity of C57A-Cys produced in Example 3 was evaluated using an in vivo antibody production system. After intraperitoneal immunization of BDF1 mice with 0.1 ⁇ g of DNP-0VA adsorbed on 2 mg of ararum, secondary sensitization was performed at week 13 by injecting 0.1 DNP-OVA intraperitoneally. The anti-DNP-IgE value in the serum of each mouse immediately before the second immunization was between 200 and 500 ng / ml. The samples were intraperitoneally administered 10 g each on the first and second days immediately after the second sensitization.
  • Example 2 The solution containing C57A-Cys-GIF obtained in Example 3 was subjected to aseptic filtration, and then filled in a 1 Oml vial to prepare an injection.
  • Example 3 The solution containing C57A-Cys-GIF obtained in Example 3 was aseptically filtered and concentrated.This was aseptically filled into 5 ml vials of 10 ml and freeze-dried at 120 ° C. Thereafter, the freeze-dried product sealed in a rubber stopper was used as an injection.
  • Example 3 The solution containing Wildtype-Cys-GIF obtained in Example 3 was aseptically filtered, and then filled into a 10 ml vial to prepare an injection.
  • the solution containing Wildtype-Cys-GIF obtained in Example 3 was aseptically filtered, concentrated, and filled with aseptic operation into 5 ml vial of 10 ml vial and freeze-dried at 120 ° C.
  • the lyophilized product sealed in a rubber stopper was used as an injection.
  • GIF derivative proteins having high immunosuppressive activity can be stably produced in large amounts. These GIF derivative proteins are useful for allergic diseases, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosi ss (MS), etc. It can be used for the treatment and Z or prevention of autoimmune diseases, graft-versus-host disease (GvHD) and the like.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • MS multiple sclerosi ss
  • GvHD graft-versus-host disease

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Abstract

La présente invention concerne des dérivés de l'inhibiteur de glycosylation humain non-spécifique d'antigène pourvu de la séquence d'acide aminé représenté par SEQ ID NO:2 qui en sont dérivés par conversion en cystéine de la souche de cystéine en position 60 de la séquence d'acide aminé SEQ ID NO:2, accompagnée éventuellement d'une substitution, suppression, insertion ou modification d'au moins une souche d'acide aminé autre que la souche de cystéine en position 60, et présentent une activité immunorégulatrice de l'inhibiteur de glycosylation spécifique d'antigène.
PCT/JP2000/003970 1999-06-18 2000-06-16 Proteines regulatrices de la reponse immunitaire WO2000078804A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997000959A1 (fr) * 1995-06-22 1997-01-09 Kirin Beer Kabushiki Kaisha Derives de facteur d'inhibition de la glycosylation non specifique d'antigene

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997000959A1 (fr) * 1995-06-22 1997-01-09 Kirin Beer Kabushiki Kaisha Derives de facteur d'inhibition de la glycosylation non specifique d'antigene

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
T. MIKAYAMA ET AL.: "Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor", PROC. NATL. ACAD. SCI. USA,, vol. 90, 1993, pages 10056 - 10060, XP002927748 *
T. NAKANO ET AL.: "Conversion of inactive glycosylation inhibiting factor to bioactive derivatives by modification of a SH group", PROC. NATL. ACAD. SCI. USA,, vol. 94, 1997, pages 202 - 207, XP002927746 *
YUN-CAI LIU ET AL.: "Requirement of posttranslational modifications for the generation of biologic activity of glycosylation-inhibiting factor", PROC. NATL. ACAD. SCI. USA,, vol. 91, 1994, pages 11227 - 11231, XP002927747 *

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