WO1997043411A1 - T-cell receptor alpha-chain constant-region peptides, processes for producing the peptides, and use thereof - Google Patents

Abstract

Polypeptides that contain substantially part or the whole of the constant region of a T-cell receptor α-chain, have immunosuppressive effects, but do not substantially cause any production of antibodies against themselves even when administered; DNAs coding for the polypeptides; a DNA having a base sequence coding for fused polypeptides represented by the general formula R1-X-R2 (wherein R1 is a carrier polypeptide; X is a protease recognition site; and R2 is a polypeptide that contains substantially part or the whole of the constant region of a T-cell receptor α-chain, has immunosuppressive activities, but does not substantially cause any production of antibodies against itself even when administered); an expression vector having the above DNA carried thereon; host cells transformed by the above vector; processes for producing the above polypeptides; and pharmaceutical compositions containing the above polypeptides as the active ingredient. These polypeptides have non-antigen-specific immunosuppressive effects and can suppress not only humoral but also cell-mediated immunoreactions. Furthermore, the administration, if ever, of them does not substantially cause any production of antibodies against them.

Description

 Specification

T cell receptor α-chain constant region peptide, method for producing the peptide and use thereof

 The present invention relates to a constant region polypeptide of a Τ-cell receptor α-chain having an antigen-specific immunosuppressive action. Background art

 ΤThe cell receptor chain (hereinafter, レ セ プ タ ー cell receptor is called “TCR” and T cell receptor chain is called “TCR”) is composed of a variable region having a specific amino acid sequence that recognizes antigen, and a unique region for each species. Consists of a constant region having an amino acid sequence. Until now, the biological function of TCR is that TC cells and the variable region of the TCR chain (hereinafter referred to as “TCR; S”) recognize antigens presented on MHC molecules, and T cells It has been thought that responding and modulating the immune response. In addition, the constant region of the TCR chain (hereinafter, referred to as “C”), which is uniform in all T cells having a TCR, ^, associates with the constant region of the TCR / S (hereinafter, referred to as “C / 3”). Its role has been to maintain the structure of the TCR and to transmit signals into cells.

 On the other hand, immunoglobulins (antibodies), which have a structure that specifically reacts with antigens like the TCR, consist of a variable region and a constant region, similar to the TCR, and the antibody molecule on the surface of the B cell is an organism similar to the TCR. It has a biological function. However, for antibodies, there are more soluble antibodies that are secreted by B cells. In this case, in addition to the variable region that binds to the antigen, the constant region also has important biological functions. That is, many cells have receptors for this constant region, and antibodies bind to these cells via the constant region to induce various antibody-dependent immune responses.

For TCRa, no clear evidence has been found to date on the presence of soluble TCR. Furthermore, the existence of a receptor for the TCR constant region has not been reported at all. That is, the constant region of the soluble TCR There were no phenomena to foresee having ability.

 There are reports suggesting that soluble TCR may be secreted or excreted from cells (Guy et al., Science, 244: 1477-1480, 1989, and Fairchild et al., J. Immunol., 145: 2001-2009, 1990). However, it is not clear what structure it has. On the other hand, there are many negative opinions that the TCR protein is released from cells while maintaining its function. That is, TCRa is degraded in the endoplasmic reticulum unless it forms a complex with the CD3 complex protein (Bonifacino et al., Science, 247: 79-82, 1990), or as a TCR-CD3 complex on the cell surface. It is shown to be degraded by lysosomes unless it is transported to other countries (Minami et al., Proc. Natl. Acad. Sci. USA, 84: 2688-2692, 1987).

In studies of antigen-specific sub-esser factors, there are reports suggesting that TCRα is involved in its activity in a soluble form. Such factors exhibit the property of reacting with anti-TCR antibody (Bissonnette et al., J. Immunol., 146: 2898-290 7, 1991; Collins et al., J. Immunol., 145: 2809- 2812, 1990, and Taka da et al., J. Immunol., 145: 2846-2853, 1990) ₀ Recently, when the TCR gene was introduced into subpressor T cells, antigen-specific subrepressor activity was reduced. Being able to be detected in culture supernatants (Kuchroo et al., J. 〖-Oki unol., 154: 5030-5038, 1995), and introducing the histidine-tagged TCR α gene into suppressor-I T cells As a result, it was found that proteins retaining the tag and having a TCR antigenic determinant were secreted (Ishii et al., J. Immunol., 156: 1735-1742, 1996). That is, TCRa is highly likely to be secreted by sublesser T cells, and it is thought that factors including this TCR may be involved in antigen-specific immunosuppressive reactions.

In addition, a DNA encoding the extracellular region of TCRa cDNA cloned from a PLA2-specific sublesser T cell hybridoma expressing the antigen-specific TCRa and 3 chains was prepared and expressed in an E. coli host. It has been reported that a specific immunosuppressive activity was confirmed (International Publication WO95-166462; June 22, 1995). These results indicate that, even if soluble TCRs do exist, their function is involved in antigen-specific immunomodulation, and in this case, naturally, the structure of the variable region plays a role. It is considered that. Onda et al. (Proc. Natl. Acad. Sci. USA, 92: 3004-3008, 1995) describe that the region of TCRa required for antigen-specific immunosuppressive activity consists of a variable region and a constant region of 25 amino acids or less. It is shown that it is.

 The involvement of the TCR variable region in regulating immune responses has also been shown for TCR0. For example, in an experimental mouse model of autoimmune encephalomyelitis, immunization with the TCR / 3 variable region peptide specific for the antigen myelin basic protein suppresses the development of this disease. (Vandenbark et al., Nature, 341: 541-544, 1989, and Howell et al., Science, 246: 668-670, 1989), or sometimes enhance (Desquenne- Clark et al., Proc. Natl. Acad. Sci. USA, 88: 7219-7223, 1991).

 The only case suggesting that the TCR may suppress immune responses that are not particularly related to its recognition antigen is the only one that has a variable region of Va 14 J 28 1 obtained from KLH-specific sub-lesser T cells. This is only for the case of TCRa (Japanese Patent Laid-Open No. 6-2988662). Here, it has been shown that TCRa containing this special variable region suppresses the onset of diabetes mellitus in autoimmune NOD (Nonobese diabetogenic) mice in addition to the response to KLH. However, this does not suggest that other TCRs also have such an antigen-specific immunosuppressive effect. The TCRa of 14Jα28 1 is completely different from the TCR on normal T cells, and is thought to be involved in the regulation of autoimmune diseases, etc.ΝΚΤExpressed in cells The TCR was found to be In other words, the function of this special TCR essentially depends on the structure of the variable region.

Conventional attempts to use TCRa for immunosuppressive applications are expected to have antigen-specific immunity involving the variable region of TCRa, but none have been clinically put to practical use. Due to its specific action, when it is used for immunosuppression, it is necessary to prepare and use a specific TCRa for each antigen involved in each immune response. It is considered that there is a therapeutic constraint that this should not be done. Further, as shown in the present specification, there is also a problem that administration of TCRa containing a variable region easily induces an antibody against the administered TCRa.

 Therefore, there has been a demand for the development of TCRα having an immunosuppressive effect without considering the histocompatible antigen of the patient to be administered and without easily inducing antibody production. An object of the present invention is to solve the above problems. Disclosure of the invention

 The present invention is based on the finding, for the first time, that the constant region, but not the variable region of TCRα, exhibits an antigen-nonspecific immunosuppressive effect. Completely overturns the idea.

 The present invention provides a polypeptide that substantially contains a part or all of the constant region of a T cell receptor chain, has an immunosuppressive effect, but does not substantially induce the production of an antibody against itself upon administration. Things.

 The present invention also includes the amino acid sequence of SEQ ID NO: 1 in which one or more amino acid residues in the amino acid sequence may be deleted, inserted, or substituted, and has an immunosuppressive effect. A polypeptide that does not substantially elicit the production of antibodies against

 Furthermore, the present invention includes an amino acid sequence of SEQ ID NO: 2 in which one or more amino acid residues of the amino acid sequence may be deleted, inserted, or substituted, or has an immunosuppressive effect. A polypeptide that does not substantially elicit the production of antibodies against

 Furthermore, the present invention provides a DNA encoding the above-mentioned polypeptide. The present invention also provides the following formula:

 R 1-X-R 2

 (Wherein, R 1 is a carrier polypeptide, X is a protease recognition site, and R 2 is the polypeptide according to any one of claims 1 to 3.)

And a DNA having a base sequence encoding the fusion polypeptide represented by R 1 may be calmodulin. X is the site recognized by thrombin The site recognized by thrombin may be represented by the amino acid sequence of SEQ ID NO: 3 below.

 Lys-Va1-Pro-Arg-Gly (SEQ ID NO: 3)

 The present invention also provides an expression vector carrying the above-mentioned DNA and a host cell transformed with the expression vector. The host cell may be a prokaryotic cell, and the prokaryotic cell may be E. coli.

 The present invention also relates to the above-mentioned method for producing a polypeptide, wherein the host cell transformed with an expression vector carrying a DNA having a nucleotide sequence encoding the polypeptide is cultured, The above-mentioned production method is also provided, wherein the peptide is isolated. The host cell may be a prokaryotic cell, and the prokaryotic cell may be E. coli.

 The present invention provides a method for producing the above-mentioned polypeptide, comprising the following formula:

 R 1 -X-R 2

 Where R 1 is the carrier polypeptide, X is the protease recognition site, and R 2 is the polypeptide described above.

A host cell transformed with an expression vector carrying DNA having a nucleotide sequence encoding the fusion polypeptide represented by is cultured, the fusion polypeptide is expressed, and the fusion polypeptide is subjected to proteolysis. The above-described production method is also provided, wherein the method is cleaved with an enzyme to isolate a polypeptide represented by R2. R 1 may be calmodulin. X is a site recognized by thrombin, and thrombin may be used as a protease. The site recognized by thrombin may be represented by the amino acid sequence of SEQ ID NO: 3 below.

 Lys-Va1-Pro-Arg-Gly (SEQ ID NO: 3)

Further, the present invention provides a pharmaceutical composition comprising the above polypeptide as an active ingredient. The pharmaceutical composition of the present invention comprises an immunosuppressant, a delayed-type hypersensitivity reaction inhibitor, an antibody production inhibitor, a prophylactic and / or therapeutic agent for an allergic disease, a prophylactic and / or therapeutic agent for an autoimmune disease, or an agent for organ transplantation. And the like. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the DNA sequence of 3B3-derived TCRa c DNA.

 FIG. 2 shows a restriction map of pCF1—3B3TCRa VJ.

 FIG. 3 shows a restriction map of pCF1 — 3B3 TCR Hi VJC25. FIG. 4 shows a restriction map of pCF1—TCRa—Ca.

 FIG. 5 shows the inhibitory effect of recombinant TCRa-C on allergic reactions induced by the 0VA antigen sensitization.

 FIG. 6 shows the DNA sequence of the TCR or cDNA derived from B4-9.5.2.

 FIG. 7 shows a restriction map of pCF1—TCRa—humanC.

 FIG. 8 shows the inhibitory effect of recombinant TCRa-Cα on transplant rejection. BEST MODE FOR CARRYING OUT THE INVENTION

 The polypeptide of the present invention may include at least a region essential for immunosuppressive action or an analog thereof. For example, Yanagi et al., Proc. Natl. Acad. Sci. USA, 82: 3430-3434, 1985 and Morris et al., Immunogenetics, 27: 174-179, 1988, all the constant regions of the T-cell receptor-α chain of various organisms, including humans and mice, which are essential for at least immunosuppressive action. As long as one or more amino acid residues of these amino acid sequences, including a region thereof, have an immunosuppressive action, deletion, insertion and / or substitution can be mentioned. The polypeptides of the present invention include polypeptides comprising the amino acid sequence of SEQ ID NO: 1 or 2, wherein one or more amino acid residues of the amino acid sequence may be deleted, inserted and / or substituted. The polypeptide of the present invention can be produced by a gene recombination technique as described below, a chemical synthesis method based on a known amino acid sequence, or the like.

The immunosuppressive action of the polypeptide of the present invention is an antigen-nonspecific action. It also suppresses not only the humoral immune response of antibody production but also the cellular immune response. Another preferred property of the polypeptides of the present invention is that they do not substantially elicit the production of antibodies to themselves as seen with administration of TCRa containing the variable region. Here, "substantially does not induce antibody production" means that an amount of the antibody detectable by the western blot is detected by at least 14 days after the administration, as described in Examples 9 and 14 described below. It is not found in the blood. The antigen-nonspecific immunosuppressive activity of the polypeptide can be easily determined by the presence or absence of an immunosuppressive effect when administered to animals in vivo as described in the Examples of the present application.

 Further, the polypeptide of the present invention may have a sugar chain. The glycosylated polypeptide of the present invention can be produced as a recombinant product by secretory expression using a host cell capable of producing a glycoprotein such as a mammalian cell or a yeast cell, as described below. .

 The present invention also provides a polypeptide which substantially comprises part or all of the constant region of a T cell receptor chain, has an immunosuppressive effect, but does not substantially induce the production of an antibody against itself upon administration. Provides the coding DNA. The DNA of the present invention can be used for producing the above-described polypeptide of the present invention by gene recombination technology. Here, the term "encode" means that the amino acid sequence information of the polypeptide of the present invention is encoded on the base sequence of the DNA, and is directly expressed in various host vector systems or in the form of a fusion protein. This means that the polypeptide of the present invention can be produced by appropriately adding a base sequence encoding another amino acid sequence suitable for intracellular or secretory production. When the polypeptide of the present invention is to be secreted and expressed by the direct expression method, a nucleotide sequence encoding another known secretory protein signal peptide depending on the host is used. This can be done by adding a peptide upstream of the nucleotide sequence to be coded. In addition, expression in a host cell can be carried out by adding a translation initiation codon upstream of a nucleotide sequence encoding the polypeptide of the present invention. Any of these techniques is a technique well known to those skilled in the gene recombination technical field, and can be appropriately implemented. Further, specific embodiments of the DNA of the present invention in expressing a fusion protein include:

The following formula:

 R 1-X-R 2

 (Where R 1 is a carrier polypeptide, X is a protease recognition site, and R 2 contains part or all of the constant region of the T cell receptor chain and has an immunosuppressive effect. Is a polybeptide that does not substantially elicit the production of antibodies against itself.)

In shown are fused Poribe peptide co - sul having the nucleotide sequence _c which include DNA In a preferred embodiment of the DNA of the present invention that can be used in the fusion protein expression method, R1 is a calmodulinka, and X is a thrombin recognition site sequence (particularly preferably Lys-Va1-Pro-Arg-G). (sequence of ly) (see Ishii et al., J. Immunol. Methods, 186: 27-36, 1995). Alternatively, other well-known techniques for recombinant production by a fusion protein method (see, for example, JP-A-54-145289) can be used. The DNA of the present invention is prepared by cloning from the cDNA library or synthesizing the DNA, or by subjecting the obtained DNA to site-directed mutagenesis or cassette mutagenesis. This can be achieved by modification using a genetic mutation technique.

 The TCR α gene and its structure are already known for various organisms including humans and mice (for example, Yanagi et al., Pro Natl. Acad. Sci. USA, 82: 3430-3434). , 1985 and Morris et al., Immunogenetics, 27: 174-1 79, 1988), based on these known base sequence or amino acid sequence information, the PCR method and the DNA The DNA encoding the TCR constant region can be appropriately obtained using synthetic techniques or the like.

 For the method of obtaining from the cDNA library, for example, in the case of the mouse Cα region, as described in the present example, the cDNA library is prepared from a known mouse Τ cell line by a conventional method. The TCRa cDNA can be obtained by the PCR method using a primer prepared based on the known nucleotide sequence of the C region of mouse TCRa. Perform PCR again using primers that amplify the DNA fragment corresponding to the C region (132 to 241 of the amino acid sequence in Fig. 1) to obtain the desired DNA fragment. be able to. Alternatively, the PCR method can be performed using the latter primer directly in the cDNA library to obtain a desired DNA fragment. In the case of the human Ca region, cDNA was also prepared from a known human T cell line, and a primer prepared based on the known nucleotide sequence of the Ca region of the human TCR was used for the mouse mouse. The desired DNA fragment can be obtained in the same manner as in the above.

When the DNA is obtained by chemical synthesis, for example, the method of Alton et al. Based on the known amino acid sequence of Cα, if necessary, considering the use of preferential codons, the nucleotide sequence is designed and DN Α Fragments can be obtained.

 In addition, a polypeptide having an amino acid sequence in which one or more amino acid residues have been deleted, inserted, Z or substituted from a known constant region amino acid sequence also includes a DNA encoding the above-mentioned TCR constant region. Using site-directed mutagenesis techniques such as oligonucleotide site-directed mutagenesis and cassette mutation (for example, DF Mark et al., Pro Natl. Acad. Sci. USA, Vol. 81, p5662). -5666, 1984, S. Inouye et al., Pro Natl. Acad. Sci. USA, Vol. 79, p3438-3441, 1982, PCT W085 / 00817, published February 28, 1985, RP Wharton et al., Nature, Vol. 316, p601-605, Aug. 15, 1985, etc.), and DNA encoding such a mutant polypeptide can be produced by chemical synthesis of DNA.

 Further, according to the present invention, an expression vector incorporating the above-described DNA of the present invention, a host cell transformed with the vector, and culturing the host cell to isolate and purify the polypeptide of the present invention. A manufacturing method is provided.

 In this case, prokaryotic (eg, bacteria, preferably E. coli) and eukaryotic (eg, yeast, insect, or mammalian) cells can be used as host cells. Examples of mammalian cells include COS cells, Chinese hamster ovary (Chines e Hamster Ovary) cells, X63.6.5.3. Cells, C-127 cells, BHK (Baby Hamster Kidney) cells, and human-derived cells (eg, , HeLa cells) and the like. Examples of yeast include baker's yeast (Saccharomyces cerevisiae) and methanol-assimilating yeast (Picia pastoris). Examples of insect cells include silkworm cultured cells (eg, Sf21 cells).

Vectors used to transform these host cells include pKC30 (Shimatake H. and M. Rosenberg, Nature ^ 292, 128-132, 1981), pTrc99A (Amann B. et al., Gene 6, 69, 301-315, 1988). For mammalian cells, PSV2-neo (Southern and Berg; J. Mol. Appl. Genet., 1, 327-341, 1982), pCAGGS (Niwa et al .; Gene, 108, 193-200, 1991), and the like are available. Is PCD SR 296 (Takebe et al .: Mol. Cell. Biol., 8, 466-472, 1988), etc. There is. For yeast, pG-1 (Scena M. and Yamamo to KR; Science, 241, 965-967, 1988) and the like are exemplified. For silkworm cells, transfer vector pAc373 for recombinant virus production (Luckow et al., Bio / Technology, 6, 47-55, 1988) and the like are listed.

 These vectors contain an origin of replication, a selection marker, and a promoter, if necessary, and an eukaryotic cell vector may contain an RNA splice site, a polyadenylation signal, etc., as necessary. .

 As the origin of replication, a vector derived from SV40, adenovirus, or espapilloma virus can be used as a vector for mammalian cells. As a vector for Escherichia coli, those derived from CollEl, R factor, F factor and the like can be used. For yeast, those derived from 2 / mDNA, ARS1, etc. can be used.

 As a promoter for gene expression, a vector derived from a virus, such as retrovirus, poliovirus, adenovirus, SV40, or a chromosome (eg, EF1-α), is used as a vector for mammalian cells. be able to. As a vector for Escherichia coli, a vector derived from bacteriophage λ, a trp, lpp, lac, or tac promoter can be used. ADH, PH05, GPD, PGK, and MAFα promoter can be used for baker's yeast, and A0X1 promoter can be used for methanol-assimilating yeast. As a vector for silkworm cells, a vector derived from nucleopolyhedrovirus can be used.

 As selection markers, vectors for mammalian cells include neomycin (neo) resistance gene, thymidine kinase (TK) gene, dihydrofolate reductase (DHFR) gene, and Escherichia coli xanthinganine phospho- Ribbon transferase (Ecogpt) gene or the like can be used. As a vector for Escherichia coli, a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene and the like can be used. For yeast, Leu2, TrpK Ura3 genes and the like can be used.

In order to obtain the polypeptide of the present invention using the host-one vector system as described above, after transforming a host cell with a recombinant DNA in which the DNA of the present invention is incorporated into an appropriate site of the above vector, The obtained transformant may be cultured, and the polypeptide may be separated and purified from the cells or the culture solution. The specific embodiment of the method for producing the polypeptide of the present invention includes the following embodiments.

 Using the DNA of the present invention in an embodiment in which a nucleotide sequence encoding a signal peptide of another known secretory protein depending on the host is added upstream of the nucleotide sequence encoding the polypeptide of the present invention. When the production is performed by genetic recombination, the polypeptide is separated and purified from the culture solution.

 In addition, when production is performed by genetic recombination using the DNA of the present invention in which a translation initiation codon is added upstream of the nucleotide sequence encoding the polypeptide of the present invention, the polypeptide may be introduced into host cells. Since the peptide is produced, the polypeptide is separated and purified from the cells.

 In the case of the polypeptide production method of the present invention using the fusion protein expression method,

The following formula:

 R 1-X-R 2

 (Wherein R 1 is a carrier polypeptide, X is a protease recognition site, and R 2 substantially or partially contains the constant region of one T cell receptor chain, and has an immunosuppressive effect. It is a polypeptide that does not substantially elicit the production of antibodies against itself upon administration.)

Culturing a host cell transformed with an expression vector carrying a DNA having a nucleotide sequence encoding the fusion polypeptide represented by, expressing the fusion polypeptide, and converting the fusion polypeptide with a protease. After cleavage, the polypeptide represented by R2 is isolated. The carrier polypeptide of R1 may be any that functions to transport the fusion polypeptide to the inclusion body, extracellular membrane, outer membrane, or, preferably, to the external environment, and may be a prokaryotic or true non-T cell receptor chain. It can be any polypeptide derived from a nuclear cell. Examples of R 1 include calmodulin, and examples of X include a thrombin recognition site sequence (particularly preferably a sequence of Lys—Va 1—Pro—Arg—G 1 y). However, it goes without saying that the method for producing the polypeptide of the present invention relating to fusion expression is not limited to this, as already described for the DNA of the present invention.

In the production method of the present invention, the separation and purification of an expression product (in the case of a fusion protein expression method, before and / or after cleavage treatment) is generally used for protein purification. Process (ion exchange chromatography, lectin affinity chromatography, dye adsorption chromatography, hydrophobic mutual chromatography, gel filtration chromatography, reverse phase chromatography, heparin affinity chromatography, Conventional purification methods, such as sulfated gel chromatography, hydroquinone patite chromatography, metal chelating chromatography, isoelectric focusing, preparative electrophoresis, and isoelectric focusing. Can be used in combination as appropriate, but it is preferable to add glycerol in the purification step for stabilizing the expression product.

 Further, the present invention provides a pharmaceutical composition comprising the boreptide of the present invention as an active ingredient. One embodiment thereof is an immunosuppressant. The immunosuppressive agent of the present invention is very useful for treating many immune diseases. One reason is that when variable regions are used, a type that perfectly matches the histocompatibility antigen must be selected for each patient, whereas the constant regions are common to all. It can be used without the need for selection.

 As shown in the examples, the polypeptide of the present invention clearly suppresses IgE and IgG1, which trigger the onset of allergic disease. More importantly, even when administered to mice that have been primed with an antigen, the increase in antibody production due to the next antigen challenge is suppressed, and those that are in the on-going antibody production state are also suppressed. That is what is shown. No drug showing these effects has been found so far, except for steroid drugs, for which strong side effects are a problem. In addition to suppressing humoral immune response, which is the suppression of antibody production, it also suppresses cell-mediated immunity such as delayed hypersensitivity.

Today, the regulation of humoral immunity and cellular immunity is thought to be controlled by the balance between two types of helper T cells (Thl and Th2). It also has an inhibitory effect on, and is very unique, indicating that it may be effective in treating many immune diseases. Therefore, since the immunosuppressant containing the polypeptide of the present invention exhibits a potent anti-antigen non-specific antibody production inhibitory effect or a delayed type hypersensitivity reaction inhibitory effect, various allergic diseases include autoimmune diseases and organ transplantation. Can be used as an inhibitor of rejection it can.

 The immunosuppressive agent of the present invention may contain a useful and suitable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier together with a therapeutically effective amount of the polypeptide of the present invention. As used herein, the term "therapeutically effective amount" refers to an amount that provides a therapeutic effect for a specified condition and mode of administration. Such formulations may be in liquid or lyophilized or otherwise dried dosage forms, having buffers of varying pH and ionic strength (eg, tris-HCl, acetate, phosphate, etc.). ) Yes, diluents of choice, additives such as albumin or gelatin to prevent adsorption on surfaces, surfactants (eg Tween 20, Tween 80, Pluronic F68, bile salts), yes Solubilizers (eg, glycerol, polyethylene glycol), antioxidants (eg, ascorbic acid, sodium metabisulfite), preservatives (eg, thimerosal, benzyl alcohol, paraben), excipients or tonicity agents (eg, lactate) And mannitol). In addition, the covalent bond of a polypeptide to a polymer such as polyethylene glycol, complexation with a metal ion, or in or on the surface of a granular preparation of a polymer compound such as polylactic acid, polyglycolic acid, or hydrogel. Includes the incorporation of the substance onto or into ribosomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or sup X-mouth plasts. Such a composition is considered to affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance of the polypeptide of the present invention. Depends on physical and chemical properties. Other dosage forms of the immunosuppressant of the present invention include a granular form, a form coated with a protective film, a form containing a protease inhibitor or an absorption enhancer, and the like. The immunosuppressive agent of the present invention can be administered by various administration routes including parenteral, pulmonary, nasal, and oral.

 The immunosuppressant containing the polypeptide of the present invention as an active ingredient is usually 1 g Z kg body weight to 2 mg Z kg body weight as an active ingredient, depending on the disease state, sex, administration route, etc.-once or several times a day. The degree can be administered.

The present invention will be more specifically described by the following examples. These examples are illustrative and do not limit the scope of the invention. Example 1 cDNA Cloning and Expression of TCR Gene of Sublesser T Cell Line Specific to Bee Venom Phospholipase A2

A. cDNA cloning of TCR gene

A PLA2-specific subcellular T cell bipridoma strain 3B3 cell expressing TCRa and ^ chain specific to bee venom phospholipase A2 (hereinafter referred to as "PLA2") has been established ( Mori et al., Int. Immunol., 5: 833-842, 1993). Acquisition of TCR acDNA from 33 cells has already been reported (International Publication WO95-164642 mentioned above). Specifically, the TCR was cloned by PCR according to the method described in Murus et al., Nucl. Acid. Res., 8: 3895-3950, 1980. Use Fast preparative track ®mRNA isolation kit (Invitrogen (Ιπν roge n)), the mRNA A was isolated from 5 X 1 0 ⁷ of 3 B 3 cells (described above). cDNA was generated using a cDNA synthesis system (Pharmacia). After its generation, the cDNA was ligated at the 5 'and 3' ends using T4 ligase (Takara) to construct a circular DNA. Oligonucleotide primers encoding mouse CDN A were synthesized using the phosphoramidite method with a DNA / RNA synthesizer (Applied Biosystems) (Beaucage et al., Tetrahedron Lett., 22). : 1859-1862, 1981). The sequences of these primers are as follows.

 5'-GTGGTCCAGTTGAGGTCTGCAAGA- 3 '(SEQ ID NO: 6)

 5 '-TTGAAAGTTTAGGTTCATATC-3' (SEQ ID NO: 7)

 PCR was performed in a Thermo 'cycler' in the presence of type I cDNA, primers and dNTPs with TaqI DNA polymerase (Even Color). The PCR conditions were as follows: denaturation step at 94 ° C. for 1 minute; annealing step at 54 ° C. for 1 minute; and elongation step at 72 ° C. for 2 minutes. The amplified cDNA was subcloned into the pCR100 vector of TA Cloning System® (Invitrogen). The DNA sequence of the input was determined by didequin sequencing.

(Sanger et al., Proc. Nalt. Acad. Sci. USA 74: 5463-5367, 1977.

). Three different TCRa cDNAs were cloned and sequenced. Two of them are fusion partner cells of the 3B3 hybridoma, BW5147 (Chien et al., Nature, 312: 31-35, 1984; Kumar et al., J. Exp. Med., 170: 2183-2188, 1989) Was identified. Other TCR c DNA may be not expressed in BW5 1 4 within 7, this TCRa heritage _c was confirmed using code sul several PCR primers scratch different portions of gene This means that this TCR ac This indicates that the DNA originated from the PLA2-specific T cells. Two independent clones encoding this TCRa cDNA were isolated and confirmed that their DNA sequences were identical. The DNA sequence of the 3B3-derived TCR cDNA is shown in FIG. 1 and SEQ ID NO: 4.

 This TCRa encoded an open reading frame of 268 amino acids, and the first 20 amino acids were identified as signal peptides.

[Example 2] Construction of expression system for recombinant 3B3-TCR derivative

 It is already known that recombinant 3B3-TCRα polypeptide (extracellular region of TCRα) has immunosuppressive activity (the above-mentioned international publication WO95-164642). . It describes how three delaminated recombinant 3B3-TCR derivatives were constructed to determine which regions of TCRa play an important role in immunosuppressive activity.

 A. Construction of recombinant 3B3 TCR-like VJ expression system in E. coli

 From pCR1000 plasmid (pCR100-0-3B3) carrying 3B3TCRcDNA described in Example 1, amino acids 21-132 (FIG. 3 DNA fragment encoding the B region-derived T CRa variable region (V region) to the joining region (J region)), and a BamHI site for the 5 'end. Amplified by PCR using two primers containing a stop codon for the 3 'end and an XbaI site. The sequences of these primers are as follows.

 5 '-AATTTAGGATCCGGACAGCAAGTGCAGCAG-3' (SEQ ID NO: 8)

5'-GACTCTAGATTACTAGTTTGGATGGACCCTAAG-3 '(SEQ ID NO: 9) The widened DNA fragment was recovered by agarose gel electrophoresis and expressed in the expression plasmid pCF1 vector (J. Immunological Methods 186: 27-36, 1995). The clone was cloned at its unique BamHI and XbaI sites, and the new plasmid was named pCF1-3B3TCRa-VJ (Figure 2). The pCF1 vector has a trp promoter and a trp terminator, and has rat calmodulin followed by a thrombin recognition (cleavage) site between the promoter and the terminator, and BamHI, XbaI, N A base sequence corresponding to the otI restriction enzyme site is arranged, and a fusion protein can be expressed by inserting a gene of a desired protein into the restriction enzyme site. Competent DH5 E. coli cells (ATCC No. 53868) were transformed with pCFl-3B3 TCR HiVJ and the DNA sequence was confirmed. In addition, the transformant W3110 E. coli cells (ATCC No. 27325) were transformed.

 B. Construction of recombinant 3B3TCRa—VJC25 expression system in E. coli

 DNA fragments encoding the amino acids 21 to 157 (the V and J regions of the 3B3-derived TCR and up to the 25th amino acid of the constant region (C region)) in Fig. 1 PCR using two primers, each containing a BamHI site for the 5 'end and a stop codon and an XbaI site for the 3' end, yielded pCR100-3. Amplified from B3 TCR plasmid. The sequences of these primers are as follows.

 5'-AATTTAGGATCCGGACAGCAAGTGCAGCAG- 3 '(SEQ ID NO: 10) 5'-TCCTCTAGATTACTAGGTGAACAGGCAGAGGGT- 3' (SEQ ID NO: 11) It was cloned at its unique BamHI and XbaI sites (FIG. 3). Competent DH5 E. coli cells were transformed with this novel plasmid, designated pCF1- 3B3TCRa-VJC25, and its DNA sequence was confirmed. In addition, transformant W3110 E. coli cells were transformed.

 C. Construction of recombinant TCRa-C expression system in E. coli

The DNA fragments encoding amino acids 1332 to 2441 in FIG. 1 (amino acids of the mouse-derived TCR. Region) were each contained an XbaI site for the 5 ′ end, and the 3 ′ end PCR using two primers containing a stop codon and a NotI site for PCR, pCR100—3B3 TCRa plasmid Was amplified. The sequences of these primers are as follows.

 5 '-CTTTCTAGAGACATCCAGAACCCAGAACCT-3' (SEQ ID NO: 12)

 5'-AAGCGGCCGCTTAGTTTTGAAAGTTTAGGTT-3 '(SEQ ID NO: 13) The amplified DNA fragment was cloned in the unique XbaI site and NotI site within one pCFl vector by the same operation as described in A. above. (Fig. 4). Combinable DH5 E. coli cells were transformed with this novel plasmid, called pCF1-TCR-C, to confirm its DNA sequence. In addition, transformant W3110 E. coli cells were transformed.

Example 3 Culture of E. coli producing TCR-VJ, VJ C25 and Ca

 W3110 E. coli carrying the respective plasmids shown in A, B and C of Example 2 were cultured in 50 ml of Luria medium containing 100 g / ml of ampicillin. The inoculum culture was transferred to 1 L of M9 medium consisting of 0.8% glucose, 0.4% casamino acid, 100/1 ampicillin so that other bacteria did not enter, and 3 hours 37 ° C. At the end of the first culture, indoleacrylic acid was added to a final concentration of 20〃M, and the culture was further cultured at 37 ° C for 5 hours. In this expression system, fusion proteins of the calmodulin TCRs VJ, VJC25 and C were expressed in a soluble form, which was about 10% of the total protein.

[Example 4] Purification of Escherichia coli recombinant 3B3TCR-VJ, VJC25 and Ca

1. About 0.5 g of each cell cultured in Example 3 was suspended in 100 ml of water, and the cells were disrupted with a French press (800 psi, repeated 4 times). The disrupted cell pellet was centrifuged at 15,000 xg for 10 minutes at 4 ° C, and the supernatant was collected. Further, calmodulin-3B3TCRa-Ca was heat-treated at 80 at 10 minutes, centrifuged at 1500 xg for 15 minutes, and the supernatant was collected.

2. The supernatant fraction was diluted with 100 volumes of 2 mM glutathione (reduced form) and 0.2 mM glutathione (oxidized form) in 5 OmM Tris HC1 buffer (pH 8.0). . C overnight. This sample solution was added to an appropriate mixture so that the final concentration in the mixture was _2.5 mM CaCl ₂ and 5 mM MgCl ₂ . 3. Transfer the mixture to 50 mM Tris HC1 buffer (pH 8.0), _2.5 mM CaCl ₂ and 5 mM MgC and equilibrate with a phenylsepharose 6 subcolumn (Falmana, 3 x 6 cm). C. and flowed at a flow rate of 0.5 ml / min. After washing the column with the same buffer, calmodulin-13B3TCR-1 VJ, VJC25 and Cα fusion protein were added to 50 mM Tris HC1 buffer (pH 8.0) and 5 mM EDTA (pH 8. 0). SDS-PAGE confirmed the expression of calmodulin-1 3B3TCRa-VJ. VJ C25 and Ca protein.

 4. The eluted fraction was reduced 10 times with a YM10 ultrafiltration membrane, and applied to a DEAE Toyopearl (T0S0H, 2 x 10 cm) column equilibrated with 5 OmM Tris HC1 buffer (pH 8.0). Run at room temperature. After washing the column with 5 OmM Tris HC1 buffer (pH 8.0) at a flow rate of 2.0 ml / min, calmodulin-1 3B3 TCR α was added using a concentration gradient of 0 to 0.5 M NaCl. -VJ, VJC25 and Ca fusion proteins were eluted. As a result, calmodulin-13B3TCR and other VJ.VJC25 and Ca fusion proteins were eluted at a concentration of about 30 OmM NaCl.

 5. The eluted fraction was dialyzed against 100 volumes of 50 mM Tris HC1 buffer (pH 8.0) at 4 ° C. Glycerol was added to a final concentration of 10%, dithiothreitol (hereinafter referred to as “DTT”) to a final concentration of 2 niM, and NaCl to a final concentration of 100 mM, to the 50 ml dialyzed fraction. The fusion protein was digested by adding thrombin (Sigma) and incubating at 25 ° C for 6 hours.

 6. The mixture is concentrated by centrifugation using MACR0SEP3K (FILTRON), and TCR-1Ca contains 10% glycerol and 2 mM DTT on a NAP (Pharmacia) column.

It was changed to Tris HC1 buffer (pH 8.0). DEAE-5PW (Toso-I., 0.1%) equilibrated with 5 OmM Tris HCi buffer (pH 8.0) containing 10% glycerol and 2 mM TTT.

(75 x 7.5 cm) column. Use this column at a flow rate of 1.0 ml / min.

After washing with 5 OmM Tris HC1 buffer (pH 8.0) containing 0% glycerol and 2 mM TTT, the TCR-Cα protein was eluted using a concentration gradient of 0 to 0.5 M NaCl. As a result, TCRa-C was eluted at a concentration of about 30 OmM NaCl. The amount of purified TCRa-Cα protein was about 1 mg / L. TCR α-protein was added to PBS containing 10% glycerol for stabilization. And stored at 20 ° C. The recombinant TCR protein in Escherichia coli was named recCa.

 On the other hand, 3B 3 TCR α-VJ and VJC 25 were replaced with 50 mM Tris HC1 buffer (ρΗ8.0) and equilibrated with 5 OmM Tris HC1 buffer (pH 8.0). The column was loaded at room temperature. The column was washed with 5 OmM Tris HC1 buffer (pH 8.0) at a flow rate of 0 ml / min, and then 3 B 3 TCR "—VJ and VJC 25 using a concentration gradient of 0 to 0.5 M NaCl. As a result of protein binding, these proteins were eluted in the DEAE-5 PW non-adsorbed fraction.The amount of purified 3B3TCR-VJ and VJC25 proteins was about 0.5 mg / L. 3B3 TCR-VJ and VJC25 proteins were stored in PBS at -2 (stored in TC. These recombinant in E. coli 3B3-TCR-HIVJ and VJC25 proteins Were named rec VJ and rec VJC 25, respectively.

 7. If necessary, add 1/10 of the amount of PyoSep C (manufactured by Daicel Chemical) to the purified sample to remove endotoxin from E. coli, and stir for 1-2 hours if necessary. Was recovered. The endotoxin level was measured using Limulus ES-II Single Test (Wako Pure Chemical Industries) or Endosushiichi ES6 (Seikagaku Corporation).

[Example 5] In vivo immunosuppressive activity of recombinant 3B3-TCRa-VJ

 1. To evaluate whether rec VJ suppresses the immune response in vivo, rec VJ protein was administered to mice immunized with bee venom PLA2. A dinitrophenyl (hereinafter referred to as “DNP”) derivative of the bee venom PLA2 was prepared as an antigen by standard procedures, and Balb / c mice were immunized with 1 mg of aluminum hydroxide (hereinafter “A1 um”). Immunization was performed by intraperitoneal injection of 1 g of DNP-PLA2 adsorbed to the DNA. recVJ was injected intraperitoneally at a dose of 5 g per day on days −5, −3, −1, 0, and control mice received only PBS. Two weeks after immunization, serum was collected from each mouse, and anti-DNP-IgGl was assayed by ELISA (Iwa evening, J. I recitation unol., 141: 3270-3277,

1988). As shown below, recVJ protein did not suppress anti-DNP-IgG1 production.

Suppression of immune response by rec VJ (Sample N anti-DNP-lgGK g / ml)

 PB S 7 1.201 ± 0.163

r e c V J 7 0.982 ± 0.230

[Example 6] In vivo immunosuppressive activity of recombinant 3B3-TCRa-VJC25 1. rec VJC25 To evaluate whether or not to suppress the immune response in vivo, rec VJC25 protein was administered to mice immunized with the bee venom PLA2. Balb / c mice were immunized by intraperitoneal injection of 1 g of DNP-PLA2 adsorbed to 1 Alum using PLA2 subjected to DNP as an antigen in the same manner as in Example 5. rec VJC25 was injected intraperitoneally at a dose of 4 μg / time on days −3, −1, 0, and 1; control mice received only PBS. Two weeks after immunization, serum was collected from each mouse, and anti-DNP-IgGl was measured by EUSA (Iwa-Yu et al., Immunol., 141: 3270-3277, 1988). As shown below, the recVJC25 protein did not suppress anti-DNP-Iggl1 production.

 Inhibition of immune response by r e c V J C 25

Sample N anti-DNP-lgGl (/ zg / ml)

 PB S 7 1.240 ± 0.555

 r e c V J C 2 5 7 1.810 ± 0.362

Example 7 Biological Activity of Recombinant TCR HiC

A. In vivo immunosuppressive activity of recombinant TCRa-Ca

1. To evaluate whether recCα suppresses the immune response in vivo, recCa protein was administered to mice immunized with bee venom PLA2. Balb / c mice were immunized by intraperitoneal injection of DNP-PLA21 adsorbed on 1 mg of Alum, using DNP-bound PLA2 as an antigen in the same manner as in Example 5. recCα was injected intraperitoneally at a dose of 5 g per day on days −1, 0, 1, and 3, and control mice received only PBS containing 10% glycerol. Two weeks after immunization, serum was collected from each mouse, and anti-DNP-IgGl was measured by EUSA (Iwa-Yu et al., J. Immunol., 141: 3270-3277, 1988). As shown below, rec C a The protein significantly suppressed anti-DNP-igGl production as compared to rec VJ and rec VJC25.

 Inhibition of anti-hapten antibody response in Balb / c mice by r e C C α Sample N Anti-DNP-lgGl (g / ml)

 PBSI10¾ Glycerol 6 1.451 ± 1.010

 r e c C α 6 0.068 ± 0.016

B. In vivo antigen-nonspecific immunosuppressive activity of recombinant TCRa-C

 1. To evaluate whether r c Ca is powerful and suppresses an immune response other than the bee venom PLA2 antigen in vivo, the r c Ca protein is converted to ovalbumin (hereinafter, referred to as ovalbumin).

The mice were immunized with “OVAj”. Using DNP-epitched OVA prepared according to the procedure of Example 5, Balb / c mice were adsorbed to 1 mg of Alum with DNP-0VA0. Immunization was performed by intraperitoneal injection of 1 zg.6. Injected intraperitoneally at a dose of 5 g per dose on days -1, 0, 1, and 3, and 10% for control mice Two weeks after immunization, serum was collected from each mouse, and anti-DNP-IgGl was assayed by ELISA (Iwa-Yu et al., J.I. 141: 3270-3277,

1988). As shown below, the production of anti-DNP-IgGl was suppressed, and it was revealed that recCa also exhibited immunosuppressive activity against other antigens.

 Inhibition of immune response to OVA antigen by r e c C α

Sample N Anti-DNP-lgGl (g / ml)

 PBS + 10¾glycerol 6 31.52 ± 6.220

 r e c C 6 8.370 ± 4.390

C. In vivo inhibitory activity of recombinant TCRa-Cα on secondary immune response

1. It was evaluated whether recCa suppresses the immune response also in the secondary response. Balb / c mice were immunized by intraperitoneal injection of 1 / zg of denatured carboxymethylated PLA 2 (hereinafter referred to as “CM-P LA 2”) adsorbed on 1 mg of Alum. On day 14 after immunization, it was induced by intraperitoneal injection of 2 g of DNP-PLA. rec Ca was sensitized in group A on days -1, 0, 1, and 3 at sensitization, and in group B at 13, On days 14, 15, 15 and 17, injection was performed intraperitoneally at a dose of 5 g each time, and control mice were administered only PBS containing 10% glycerol. Two weeks after the induction, serum was collected from each mouse, and anti-DNP-IgGl was assayed by ELISA (Iwa-Yu et al.

Immunol., 141: 3270-3277, 1988). Anti-DNP-lg as shown below

G1 production was suppressed in both groups A and B, and recCa showed that the immune response was also suppressed in the secondary immune response.

 Suppression of primary and secondary immune response by r e c Ca

 (Sample N anti-DNP- IgGK g / ml)

 Group A—PBS + 10¾iglycerol 6 499.7 ± 352.1

 Group A — r e c C α 6 221.8 ± 56.89

Group ——glycerol 6 595.1 ± 77.47

 Group B r e c C α 6 196.5 ± 105.8

D. In vivo inhibitory activity of recombinant TCR-C on on-going antibody production

1. The ability to suppress on-going antibody production with rec C was evaluated. Balb / c mice were immunized by intraperitoneal injection of 0.1 g of OVA adsorbed on 1 mg of Alum. On day 14 after immunization, IgG specific to the OVA antigen was measured by ELISA (Iwa evening, J. Immunol., 141: 3270-3277, 1988), and after confirming an increase in antibody titer, rec C was measured. Intraperitoneal injections were made at a dose of 5 g per dose on days 16, 17, and 18, and control mice were administered only PBS containing 10% glycerol. On day 28, serum was collected from each mouse, and OVA-specific IgG was measured by EUSA (Iwa evening, J. 1 unol., 141: 3270-3277, 1988). The standard at this time was obtained by pooling and diluting the sera of mice immunized with the OVA antigen and having an elevated OVA antibody titer, and was expressed as 1 000-fold dilution as 1 unit. As shown below, 0 VA specific IgG production was suppressed, and rec C also suppressed on-going antibody production.Suppression of on-going antibody production by _c rec Ca

Sample N Anti-OVA- IgG (Unit)

 PBS + 10¾; glycerol 8 8.300 ± 11.21

rec C a 6 3.510 ± 5.410 E. In vivo inhibitory activity of recombinant TCR a-C α in other mouse strains

 In order to evaluate whether recCa suppresses the immune response of other strains of mice in vivo as well, DNP-PLA adsorbed on 1 mg of Alum using BDF1 1 was immunized by intraperitoneal injection. recCa was injected intraperitoneally at a dose of 5 g per day on days 1, 0, 1, and 12, and control mice received only PBS containing 10% glycerol. On day 13 after immunization, serum was collected from each mouse, and anti-DNP-IgGl was measured by EL1SA (Iwa-Yu et al., J. 1 fraction unol., 141: 3270-3277, 1988). As shown below, anti-DNP-Iggl1 production was also remarkably suppressed in BDF1 mice, and recCa showed high immunosuppressive activity also in mice of other strains.

 Inhibition of immune response in BDF1 mice by r e c C

Sample N Anti-DNP- IgGl (g / ml)

 PBSU0¾glycerol 6 495.2 ± 82.57

 r e c C a 6 62.32 ± 38.86

Example 8 Activity of Recombinant TCRa-Ca in Delayed Type Hypersensitivity Reaction

 In this example, r e c C force 'delayed-type hypersensitivity;

DTH). Using recC, the inhibitory effect on allergic reactions induced by antigen sensitization was examined. Balb / c mice were immunized by intraperitoneal injection of 10 g of OVA adsorbed on 1 mg of Alum. recC was injected intraperitoneally at a dose of 5 / zg per day on days -1 and 0, and control mice were administered PBS and saline containing 10% glycerol. On the other hand, dexamethasone was injected intraperitoneally as a control drug at a dose of 0.1 μg / dose on days 6 and 7. Seven days after immunization, the challenge was induced by injection of 50 g of AVA adsorbed on 50 ig of Alum into the feet. At 24 hours after the induction, edema of the foot was measured.

As a result, mice to which recCα was administered showed almost the same inhibitory activity as dexamethasone (Fig. 5). [Example 9] Production of antibody against recombinant TCR α-C α in mouse serum after administration of recombinant TCRa-Ca

 In this example, the production of antibodies to the recombinant TCR-C in the serum of each mouse was examined by estanblotting on the mice to which the TCR-I-Ca was administered as shown in ① of Example 7 It is. SDS-PAGE was performed on a 10 to 20% gradient gel under reducing conditions containing 5 mM DTT at a concentration of 1 g / ml of recCα161. The protein was transferred to a nitrocellulose membrane at 150 mA per gel for 30 minutes. 20 m TrisHC1 (pH 7.5) and 500 mM NaCl (hereinafter, referred to as

Wash the membrane at room temperature for 5 minutes with “Ding BS” and then add 0.1% Tvveen-20 (hereinafter called “TTB S”) twice at room temperature for 5 minutes. did. After that, the cells were shake-cultured for 1 hour at room temperature in TTB S containing gelatin (hereinafter, referred to as “GZTTB S” (manufactured by Boehringer Mannheim)) to immobilize the membrane. Next, each mouse serum was diluted 50-fold with GZTTBS and cultured with shaking at room temperature for 1 hour. Wash the membrane with TTB S for 5 minutes at room temperature.

Using an IgG (H + L) alkaline phosphatase-labeled antibody (manufactured by Bio-Rat), a 200-fold dilution of this was shake-cultured at room temperature for 1 hour. The membrane was washed twice with TTB S at room temperature for 5 minutes, and further washed twice with TBS at room temperature for 5 minutes. Finally, color development was performed using an alkaline phosphatase color development kit (manufactured by Biorat).

 As a result, unlike the recombinant B4-9.52-TCR and recombinant 3B3-TCRa having the VCR region of the TCR shown in the Reference Example below, the recombinant TCRa-Ca No antibody to -Ca was detected.

[Reference Example 1] cDNA cloning and expression of the TCRa gene of a helper T cell line specific for bee venom phospholipase A2

A. cDNA cloning of TCR gene

1. A PLA2-specific helper T cell line expressing PLA2-specific TCRa and chains was established according to the method of Kimoto et al. (Kimoto et al., J. Exp. Med., 15 2: 759-767, 1980, J. Exp. Med., 153: 375-383, 1981). 100 g of PLA2 antigen was dissolved in PBS, and an emulsion mixed in complete 'Freund' adjuvant (CFA) and 1: 1 was immunized subcutaneously at the base of the tail of Balb / c mice. Ten days after the immunization, the lymph nodes at the radii and para-aorta, which were the injected lymph nodes, were taken out and made into a single cell suspension in the culture medium. The cells were made up to 4 xiO ⁶ / ml with Click's Medium-10% FCS and PLA2 was added to a final concentration of 200 μg / ml. Seven days after the initial culture, the cells were collected, and 2 × 10 ⁶ viable cells and spleen cells of syngeneic mice were used as antigen-presenting cells (X-irradiated at 200 OR). X 1 0 ⁶ the Click 's Medium- 1 0% were cultured in FCS, the final concentration 2 0 0 g / ml to PLA 2 at that time,

1 L 2 was added to a final concentration of 1 Ounit / ml. Furthermore, after the same antigen stimulation was performed twice, culture was performed using the ultradilution method, and a helper-I T cell line specific for the PLA2 antigen was obtained. This helper T cell line was named B4-9.52 cells.

2. The TCR cDNA of B4-9.52 cells was cloned by PCR according to the method described in Murus et al., Nucl. Acid. Res., 8: 3895-3950, 1980. Use fur strike track ®mRNA isolation kit Bok the (Invitrogen (Inv rogen)), mRNA was isolated from B4-9.52 cells 5 X 1 0 ^7. The cDNA synthesis system (Pharmacia) was used to generate cDNA. After its generation, the cDNA was ligated at the 5 'and 3' ends using T4 ligase (Takara) to construct a circular DNA. Oligonucleotide primers encoding mouse C DNA were synthesized by a DNA / RNA synthesizer (Applied Biosystems) using the phosphoramidite method (Beaucage et al., Tetra hedron Lett., 22: 1859-1862, 1981). The sequences of these primers are as follows.

 5 '1 GTGGTCCAGTTGAGGTCTGCAAGA-3' (SEQ ID NO: 6)

 5 * -TTGAAAGTTTAGGTTCATATC- 3 '(SEQ ID NO: 7)

PCR was performed in a Thermo 'cycler' with Taq I DNΑ polymerase (evening color) in the presence of type I cDNA, primers and dNTPs. The PCR conditions were as follows: denaturation step at 94 ° C. for 1 minute; annealing step at 54 ° C. for 1 minute; and elongation step at 72 ° C. for 2 minutes. Amplified c DN A was subcloned into the pCRII vector of TA Cloning System (Invitrogen). The DNA sequence of the input was confirmed by dideoxy sequencing (Sanger et al., Proc. Nalt. Acad. Sci. USA 74: 5463-5367, 1977). The DNA sequence of the T4-acR derived from B4-9.52 is shown in FIG. 6 and SEQ ID NO: 5 (Yague et al., Nucleic Acids Research 16: 11355-11364, 1988).

 B. Construction of recombinant B4-9.52-TCRa expression system in E. coli

 The DNA fragments encoding amino acids 21 to 24 (TCR extracellular region derived from B4-9.52) shown in Fig. 6 were ligated with BamHI sites for the 5 'end and for the 3' end, respectively. Amplified from pCRII-B4-9.52-TCR plasmid by PCR using two primers containing a stop codon and an XbaI site. The PCR conditions were as follows: denaturation step at 94 ° C for 1 minute; annealing step at 55 ° C for 1 minute; and elongation step at 72 ° C for 1 minute. . The sequences of these primers are as follows.

 5 '-AATTTAGGATCCGATTCCGTGACTCAAACA-3' (SEQ ID NO: 14)

 5'-GCCTCTAGATTACTATTGAAAGTTTAGGTT-3 '(SEQ ID NO: 15) The amplified DNA fragment was recovered by agarose gel electrophoresis, digested with BamHI and XbaI, and used as the expression plasmid pCF1 vector used in Example 2. In cloning at its unique BamHI and XbaI sites. Competent DH5 E. coli cells were transformed with this novel plasmid, designated pCF1-B4-9.52-TCRa, and its DNA sequence was confirmed. In addition, competent W3110 E. coli cells were transformed.

 C. Β4-9.52- Culture of TCRa-producing E. coli

W3110 E. coli harboring plasmid pCF1-B4-9.52-TCR was cultured overnight in 5 Oml of Luria medium containing 100 g / ml of ampicillin. The inoculum culture was transferred to 1 L of M9 medium consisting of 0.8% glucose, 0.4% casamino acid, 100 mg / 1 ampinline so that other bacteria did not enter the cells, and 3 hours 3 The cells were cultured at 7 ° C. At the end of the first culture, indoleacrylic acid was added to a final concentration of 20 2, and the culture was further cultured at 37 ° C for 5 hours. In this expression system, The fusion protein of rumodulin-B4-9.52-TCRa was expressed in a soluble form, about 10% of the total protein.

[Reference Example 2] Purification of Escherichia coli recombinant B4-9.52-TCR strain

 1. About 0.5 g of the cells cultured in Reference Example 1 were suspended in 100 ml of water, and the cells were disrupted with a French press (repeated 800 000 psu 4 times). The disrupted cell pellet was centrifuged at 15,000 Xg for 10 minutes at 4 ° C, and the supernatant was collected. The expression level of this fusion protein was about 5 mg / L.

 2. The supernatant fraction was added with 100 volumes of 2 mM glutathione (reduced form) and 0.2 mM guanolethione (oxidized form) in 5 OmM Tris HC1 buffer (pH 8.0) at 4 ° C. C was used for the whole analysis. Add this sample solution to the appropriate mixture to make the final concentration in the mixture

2. It was adjusted to 5 mM CaCl ₂ and 5 mM MgCl ₂ .

3. The mixture 5 Omm Bok squirrel HC1 buffer (pH8.0), 2. 5 mM CaCl 2 and 5 mM MgC 1 ₂ flat銜化the phenylene Rusefarosu of 6 sub-column (Pharmacia, 3 x 6c m ) At 4 ° C. and a flow rate of 0.5 ml / min. After washing the column with the same buffer, the Calmodulin B4-9.52-TCR fusion protein was eluted with 50 mM Tris HC1 buffer (PH8.0) and 5 mM EDTA (pH 8.0), followed by SDS-PAGE. As a result, the expression of the calmodulin-B4-9.52-TCRα fusion protein was confirmed.

 4. Concentrate the eluted fractions 10-fold on a YM10 ultrafiltration membrane and equilibrate with 5 OmM Tris HC1 buffer (pH 8.0) on a DEAE Toyopearl (TOS0H, 2 x 10 cm) column at room temperature. went out. After washing the column with 50 mM Tris HC1 buffer (pH 8.0) at a flow rate of 2.0 ml / min, calmodulin-B4-9.52 was purified using a gradient of NaCl from 0 to 0.5 M. -TCR alpha fusion protein was eluted. As a result, calmodulin-Δ4-9.52-TCRa was eluted at a concentration of about 300 mM NaCl.

5. The eluted fraction was dialyzed against 100 volumes of 50 mM Tris HC1 buffer (pH 8.0) at 4 ° C. Glycerol was added to a final concentration of 10%, dithiothreitol (hereinafter referred to as DTT) to a final concentration of 2 mM, and NaCl to a final concentration of 10 OmM to 50 ml of the dialyzed fraction, and 1% thrombin (Sigma) was added. ) Was added and the mixture was incubated at 25 ° C. for 6 hours to digest the fusion protein. 6. The mixture was concentrated by centrifugation using MACR0SEP3K (FILTR0N) and exchanged with a 50 mM Tris HC1 buffer (pH 8.0) containing 10% glycerol and 2 mM DTT using a NAP (Pharmacia) column. The column was loaded at room temperature on a DEAE-5PW (0.75 × 7.5 cm) column equilibrated with 50 mM Tris HC1 buffer (pH 8.0) containing 10% glycerol and 2 mM DTT. Run the column at a flow rate of 1.0 ml / min and a 10%

- After washing with 5 0 mM Tris HC1 buffer containing Le and 2 mM DTT (pH 8.0), using a gradient of NaCl of 0 to 0. 5M, it was eluted B4-9.52-TCR α protein _c As a result, B4-9.52-TCR was eluted at a concentration of about 300 mM NaCl. The amount of the purified B 4-9.52-TCRα protein was about 1 mg. The B4-9.52-TCRα protein was stored at 120 ° C. in PBS containing 10% glycerol for stabilization. This recombinant B4-9.52-T CRa in Escherichia coli was named rec VJ C (Va4.4).

 7. If necessary, add 1/10 of the amount of PyoSep C (manufactured by Daicel Chemical) to the purified sample to remove endotoxin derived from Escherichia coli, and stir for 1-2 hours if necessary. The supernatant was collected. The amount of endotoxin was measured by Limulus ES-II single test (manufactured by Wako Pure Chemical Industries, Ltd.) or Endosushi ES6 (manufactured by Seikagaku Corporation).

[Reference Example 3] Antibody production against the polypeptide in mouse serum treated with recombinant TCRaV JC region polypeptide

 The rec VJC (V aAA) protein was administered to mice immunized with the bee venom PLA2. As an antigen, the bee venom PLA2 dinitrophenyl (hereinafter referred to as “DNP

Derivatives were prepared by standard procedures. Balb / c mice were immunized by intraperitoneal injection of 1 g of DNP-PLA2 adsorbed to 1 mg / um. r e c V J C

(Va4.4) was injected intraperitoneally at a dose of 5 g / time on days -1, 0, and 3 and control mice were administered only PBS containing 10% glycerol. Two weeks after immunization, serum was collected from each mouse, and anti-DNP-IgGl was measured by ELISA (Iwata et al., J. 1 Oki unol., 141: 3270-3277, 1988). As a result, the production of anti-DNP-Iggl1 was strongly suppressed as in the case of C. Therefore, the antibody production against rec VJC (Vα4.4) in the serum of each of these mice was examined by eastern blotting.

 SDS-PAGE was performed on a 10% to 20% gradient gel under reducing conditions containing 1 mM / g of rec VJC (V α4.4) 16〃1 at a concentration of 5 mM DΤΤ. . This protein was transferred to a nitrocellulose membrane at 150 mA per gel for 30 minutes. The membrane was washed with TBS at room temperature for 5 minutes, and further washed twice with TTBS at room temperature for 5 minutes. Then, use G / TTB S (Boehringer's Mannheim) at room temperature

After shaking culture for 1 hour, the membrane was immobilized. Next, each mouse serum was subjected to 5 G / TTB S

The 0-fold dilution was shake-cultured at room temperature for 1 hour. Wash the membrane with TTB S for 5 minutes at room temperature, and use a goat anti-mouse IgG (H + -alkaline phosphatase-labeled antibody (manufactured by Bio-Lat)) as a secondary antibody, and dilute it 200-fold. At room temperature

The cells were cultured with shaking for 1 hour. The membrane was washed twice with TTB S at room temperature for 5 minutes, and further washed twice with TBS at room temperature for 5 minutes. Finally, coloring was performed using an alkaline phosphatase coloring kit (manufactured by Biorat).

 As a result, an antibody against recVJC (Va4.4) was detected in the serum of the mice to which recVJC (Va4.4) was administered.

Rec VJ C (V α4.4) is derived from TCR α of helper Τ cells, but is a recombinant polypeptide of the VJC region (extracellular region of TCR) of TCRa derived from suppressor Τ cells 3 33 (see above). A similar test was also conducted for the international publication WO95-166462, which also confirmed production of an antibody against the administered recombinant polypeptide.

(Example 10)

 A. Expression of human TCR gene

A PLA2-specific GIF-producing human T cell expressing PLA2-specific TCRa and / 3 chain T cell hybridoma strain AC5 cells have been established (Thomas et al., The Journa 1 of Immunology, 92: 729-737, 1992). The TCR cDNA of this cell was cloned by PCR according to the method described in Murus et al., Nucl. Acid. Res., 8: 3895-3950, 1980. Use Fast preparative track (TM) mRNA isolation kit Bok the (i Nbito androgenic (Invitrogen)), mRNA was isolated from 5 X 1 0 ⁷ AC5 cells. cDNA was generated using a cDNA synthesis system (Falmana). After its generation, the cDNA was ligated at the 5 'and 3' ends using T4 ligase (Takara) to construct a circular DNA. Based on a known TCRa chain DNA sequence (Yanagi et al., Proc. Nalt. Acad. Sci. USA, 82: 3430-3434, 1985), an oligonucleotide primer encoding human CaDNA was converted to a DNA / RNA synthesizer (Applied. It was synthesized by the phosphoramidite method using the 'biosystem' (Beaucage et al., Tetrahedron Lett., 22: 1859-1862, 1981). The sequences of these primers are as follows.

 5 '-CTAGGATCCATCCAGAACCCTGACCCT-3' (SEQ ID NO: 16)

 5 '-CTAGAATTCTCAGCTGGACCACAGCCG- 3' (SEQ ID NO: 17)

 PCR was performed in a Thermo 'cycler' with Taql DNA polymerase (Takara) in the presence of type I cDNA, primers and dNTPs. The PCR conditions were as follows: denaturation step: 94 ° C. for 1 minute; annealing step: 54 ° C. for 1 minute; and elongation step: 72 ° C. for 2 minutes. The amplified cDNA was subcloned into the pCR1000 vector of the TA Cloning System ™ (Invitrogen). This was named pCRIOOO-human TCR.

 B. Construction of recombinant TCRa- expression system in E. coli

Use two primers, each containing a Bam HI site for the 5 'end and a stop codon and an Xba I site for the 3' end, for the DNA fragment of the C region of the human-derived TCR. Amplified from pCRlOO-human TCRa plasmid by PCR ₍ The sequences of these primers are as follows. 5'-GACGGATCCATCCAGAACCCTGACCCTGCCGTG-3 '(SEQ ID NO: 18) 5'-TCCTCTAGATTACTATTGAAAGTTTAGGTTCGTATC- 3' (SEQ ID NO: 19)

 The amplified DNA fragment was cloned into the pCFl vector at its unique Bam HI and Xba I sites, as in Example 1: A (Figure 7). The novel plasmid called pCFl-TCRa-human Ca was used to transform competent X-Blue E. coli cells (Stratagene), and its DNA sequence was confirmed (shown in SEQ ID NO: 20). In addition, the transformant was transformed into W3110 E. coli cells.

[Example 11] Expression of E. coli recombinant TCRa-human Ca

 The w3U0 colon orchid holding the plasmid pCF1-TCRa-human Ca shown in Example 10 was cultured in 50 ml of Luria medium containing 100 wg / nil of ampicillin. The inoculum culture was transferred to 1 L of M9 medium consisting of 0.8% glucose, 0.4% casamino acid, 100 mg / 1 ampicillin in a manner that no other bacteria entered, and 3 hours 3 7 Cultured at ° C. At the end of the first culture, indoleacrylic acid was added to a final concentration of 20, and the culture was cultured for a further 5 hours at 37. In this expression system, the calmodulin-TCRa-human C fusion protein was expressed in a soluble form, about 10% of the total protein.

[Example 12] Purification of E. coli recombinant TCR-human Ca

 1. About 0.5 g of the cells cultured in Example 11 were disrupted in the same manner as in Example 4, and the supernatant was recovered. The expression level of these fusion proteins was about 5 mg / L. After heat treatment at 80 ° C for 10 minutes, the mixture was centrifuged at 1500 xg for 15 minutes, and the supernatant was recovered.

 2. The supernatant fraction was diluted with 50 mM Tris HC1 buffer (PH8.0) containing 100 volumes of 2 mM glutathione (reduced form) and 0.2 m glutathione (oxidized form).

Dialysis at ° C overnight. This sample solution was added to an appropriate mixture so that the final concentration in the mixture was 2.5 mM CaC and 5 mM MgCl ₂ .

3. Apply the mixture to a Phenyl Sepharose 6 subcolumn under the same conditions as in Example 4, and add calmodulin-TCRa-human C fusion protein to 50 mM Tris HC1. Elution was performed with buffer (pH 8.0) and 5mBDTA (pH 8.0). The expression of calmodulin-TCRa-human Ca protein was confirmed by SDS-PAGE.

4. The eluted fraction was concentrated 10-fold with an ultrafiltration membrane, applied to a DEAE Toyopearl column under the same conditions as in Example 4, and subjected to a concentration gradient of 0.5 M NaCl at 0. Calmodulin-TCR-human C fusion protein was eluted. As a result, the fusion protein TCRa-human Ca fusion protein was eluted at a concentration of about 500 mM NaCl.

 5. The eluted fraction was dialyzed against 100 volumes of 50 mM Tris HC1 buffer (pH 8.0) at 4 ° C overnight. Under the same conditions as in Example 4, the fusion protein was digested with thrombin (Sigma).

 6. Centrifuge the mixture with MACR0SEP 3 K (FILTR0N), and convert TCRa-human C to 50 mM Tris HC1 buffer containing 10% glycerol and 2 mM DTT on a NAP (Pharmacia) column. (PH 8.0). The protein was applied to a DEAE-5PW column under the same conditions as in Example 4 and TCRa-human Cα was eluted with a concentration gradient of 0 to 0.5 M NaCl. As a result, TCRa-human Ca was eluted at a concentration of about 200 mM NaCl. The amount of purified TCRa-human Ca protein was about 1 mg / L. TCRa-human Ca protein was stored at 120 ° C in PBS containing 10% glycerol for stabilization. The recombinant TCR-human C protein in Escherichia coli was named rechC.

 7. Endotoxin derived from Escherichia coli was removed using PyoSep C (manufactured by Daicel Chemical Industries) as necessary in the same manner as in Example 4.

[Example 13] In vivo immunosuppressive activity of recombinant TCRa-human Ca

To evaluate whether rechCa suppresses the immune response in vivo, the rechCa protein was administered to mice immunized with the bee venom PLA2. As in Example 5, DNP-modified PLA2 was used as an antigen, and Balb / c mice were immunized by intraperitoneal injection of DNP-PLA2 1 fig adsorbed to 1 mg of Alum. recCa was injected intraperitoneally at a dose of 5 per day on days -1, 0 and 1, and control mice received only PBS containing 10% glycerol. Each mouse 2 weeks after immunization Serum was collected from the sample, and anti-DNP-IgGl was measured by EL1SA (Iwa-Yu, 1 Cell unol., 141: 3270-3277, 1988). As shown below, rechC protein inhibited anti-DNP-Iggl1 production.

 Inhibition of anti-hapten antibody response in Balb / c mice by rechC

 Sample N anti-DNP- IgGl (/ z / ml)

 PBS + 10 ¾ glycerol 6 42.81 ± i9.72

 rechCa 6 14.36 ± 11.03

[Example 14] Antibody production against recombinant TCR-human Ca in mouse serum from recombinant TCR-human Cα-administered mice

 In this example, the production of antibodies to the recombinant TCR-human Cα in the serum of each mouse shown in Example 13 was examined by estanbuling. SDS-PAGE was performed on a 10 to 20% gradient gel of TCR-human C161 at a concentration of 1 g / ml under reducing conditions containing 5 mDTT. The protein was transferred to a nitrocellulose membrane at 150 mA per gel for 30 minutes. The membrane was washed with TBS at room temperature for 5 minutes, and further washed twice with TTBS at room temperature for 5 minutes. Thereafter, the cells were shake-cultured in G / TTBS for 1 hour at room temperature to fix the membrane. Next, each mouse serum diluted 50-fold with G / TTBS was cultured with shaking at room temperature for 1 hour. The membrane was washed with TTBS at room temperature for 5 minutes, and a goat anti-mouse lgG (H +) alkaline phosphatase-labeled antibody (manufactured by Bio-Rat) was used as a secondary antibody, and diluted 200-fold. The culture was shake-cultured at room temperature for 1 hour. The membrane was washed twice with TTBS at room temperature for 5 minutes, and further washed twice with TBS at room temperature for 5 minutes. Finally, coloring was performed using an alkaline phosphatase coloring kit (manufactured by Biorat).

 As a result, no antibody against the recombinant TCR human Ca was detected in the serum of the mouse administered with the recombinant TCRa-human.

[Example 15] Inhibitory activity of recombinant TCR-C cells in transplant rejection

This example shows that recC inhibits transplant rejection. The method of BiUingham et al. (Blingham et al., J. Exp. Med. 28, 285-402, 1951), and skin transplantation was performed. The tail skin of a 10-week-old DBA / 2NCrj female mouse (having an approximately 7 nun angle) was transplanted into the same-week Balb / cAnNCrj female mouse (N = 9) under ether anesthesia. The surface to be transplanted was covered with a bandage with gauze. After transplantation6, he removed the bond descendants and observed the graft every day to determine if the entire _c- graft was necrotic or indurated and fell off. 5 g of rec C was intraperitoneally administered every day for 5 days from the day before transplantation. Control mice were similarly administered only PBS containing 10% glycerol.

 As a result, the control group was rejected in all cases by day 11 after transplantation, whereas the recC-treated group was delayed in rejection up to day 19 after transplantation (Fig. 8). Industrial applicability

The polypeptide of the present invention has an antigen-nonspecific immunosuppressive action, and can suppress not only humoral immune reaction but also cell-mediated immune reaction. Furthermore, administration of the polypeptide of the present invention does not substantially induce the production of antibodies against TCRα.

Arrangement table

SEQ ID NO: 1

Array length: 1 0 9

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Origin

 Organism name: Mus musculus (Mus)

Array:

 Asn lie Gin Asn Pro Glu Pro Ala Val Tyr Gin Leu Lys Asp Pro Arg

1 5 10 15

Ser Gin Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gin lie

 20 25 30

 Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe lie Thr Asp Lys Thr

 35 40 45

 Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala He Ala

50 55 60

 Trp Ser Asn Gin Thr Ser Phe Thr Cys Gin Asp He Phe Lys Glu Thr 65 70 75 80

Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr

 85 90 95

Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gin

 100 105 SEQ ID NO: 2

Array length: 1 1 3

Sequence type: amino acid

 Topology: linear

Sequence type: peptide 9 ε: violent

 A ^: mno m ■-^ ci ^ ^ ^ ■ ourn

9: $ ¥ 0) ε: View επ on 50ΐ ΟΟΐ

 3Md us naq usy J¾ dsv J¾ "10 3Md sAq nio s sAq

S6 06 S8

dsv SAQ J9S J3S nio OJJ jas OJJ aqd aqd jqi dsy nio OJd an 311

08 S2, 01 99

J3S usv usv 3Md ^ IV usv ¾I sAg ¾jy 9Md dsy J3S s usy J9S c 丄

 09 95 09

¾IV 1¾A ^ IV ^J 3S usv J3S sAi aqj dsy) 9 ^ J9S 3JV} 9W dsv "31 eight

 Of 'SS

 JiU sA dsy JM1 311 J ΙΒΛ dsy J3S dsy sAq JSS UIQ J9S Ι¾Λ USV

 OC 02

 丄 ui3 J3S dsy 3Md dsy J41 9Md "31 sAo Ι¾Λ J3S sA dsy J3S J3S

SI 01 S ΐ s J9g dsy 3 ^J V ⁿ³ l uio J ¾ V1V OJJ dsy OJJ USV "10 311 usy

(suaid-es ouiOH) ^: dDT / XDd Array length: 8 0 7

Sequence type: nucleic acid

Number of chains: double strand

 Topology: linear

Sequence type: cDNA to mRNA

Origin

 Organism name: Mus musculus (Mus)

 Strain name: Sublesser T cell Hypri-Doma 3 B 3 strain

Array:

 9 18 27 36 45 54

ATG AAG AGC CTG CTG AGC TCT CTC CTG GGG CTT CTG TGC ACC CAG GTT TGC TGG

 Met Lys Ser Leu Leu Ser Ser Leu Leu Gly Leu Leu Cys Thr Gin Val Cys Trp

 63 72 81 90 99 108

GTG AAA GGA CAG CAA GTG CAG CAG AGT CCT GCA TCC TTG GTT CTG CAG GAG GGG

 Val Lys Gly Gin Gin Val Gin Gin Ser Pro Ala Ser Leu Val Leu Gin Glu Gly

 117 126 135 144 153 162

GAG AAC GCA GAG CTG CAG TGT AAC TTT TCC TCC ACA GCA ACC CAG CTG CAG TGG

 Glu Asn Ala Glu Leu Gin Cys Asn Phe Ser Ser Thr Ala Thr Gin Leu Gin Trp

 171 180 189 198 207 216

TTT TAC CAA CGT CCT GGG GGA AGC CTC GTC AGC CTG TTG TAC AAT CCT TCT GGG

 Phe Tyr Gin Arg Pro Gly Gly Ser Leu Val Ser Leu Leu Tyr Asn Pro Ser Gly

 225 234 243 252 261 270

ACA AAG CAC ACT GGA AGA CTG ACA TCC ACC ACA GTC ACT AAA GAA CGT CGC AGC

 Thr Lys His Thr Gly Arg Leu Thr Ser Thr Thr Val Thr Lys Glu Arg Arg Ser

 279 288 297 306 315 324

TCT TTG CAC ATT TCC TCC TCC CAG ATC ACA GAC TCA GGC ACT TAT TTC TGT GCT

 Ser Leu His lie Ser Ser Ser Gin lie Thr Asp Ser Gly Thr Tyr Phe Cys Ala

 333 342 351 360 369 378

ATG GAA GAC ACT GGA GCT AAC ACT GGA AAG CTC ACG TTT GGA CAC GGC ACC ATC Met Glu Asp Thr Gly Ala Asn Thr Gly Lys Leu Thr Phe Gly His Gly Thr He

387 396 405 414 423 432

CTT AGG GTC CAT CCA AAC ATC CAG AAC CCA GAA CCT GCT CTG TAC CAG TTA AAA

 Leu Arg Val His Pro Asn lie Gin Asn Pro Glu Pro Ala Val Tyr Gin Leu Lys

 441 450 459 468 477 486

GAT CCT CGG TCT CAG GAC AGC ACC CTC TGC CTG TTC ACC GAC TTT GAC TCC CAA

 Asp Pro Arg Ser Gin Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gin

 495 504 513 522 531 540

ATC AAT GTG CCG AAA ACC ATG GAA TCT GGA ACG TTC ATC ACT GAC AAA ACT GTG

lie Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe lie Thr Asp Lys Thr Val

 549 558 567 576 585 594

CTG GAC ATG AAA GCT ATG GAT TCC AAG AGC AAT GGG GCC ATT GCC TGG AGC AAC

 Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala He Ala Trp Ser Asn

 603 612 621 630 639 648

CAG ACA AGC TTC ACC TGC CAA GAT ATC TTC AAA GAG ACC AAC GCC ACC TAC CCC

 Gin Thr Ser Phe Thr Cys Gin Asp lie Phe Lys Glu Thr Asn Ala Thr Tyr Pro

 657 666 675 684 693 702

AGT TCA GAC GTT CCC TGT GAT GCC ACG TTG ACC GAG AAA AGC TTT GAA ACA GAT

 Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp

 711 720 729 738 747 756

ATG AAC CTA AAC TTT CAA AAC CTG TCA GTT ATG GGA CTC CGA ATC CTC CTG CTG

 Met Asn Leu Asn Phe Gin Asn Leu Ser Val Met Gly Leu Arg lie Leu Leu Leu

 765 774 783 792 801

 AAA GTA GCG GGA TTT AAC CTG CTC ATG ACG CTG AGG CTG TGG TCC AGT TGA

 Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser SEQ ID NO: 5

Array length: 8 1 9

Sequence type: nucleic acid 6 ε ζζ ^

 ^ 10 3iJd ΙΒΛ naq ^ uio ^ IO J3S J9S ^ IO O- ^ d o ^ d s ¾iv SA3 J JA IOO 111110 013 OVV OVO 30010V VDl 100 003 300 IOV 013 1D0101 OVI 3V

8ζε 69c 09ε Tss 2 εεε

 ΙΒΛ Βΐν J3S dsv J3S nio "10 Ι¾Λ J3S ¾IV δΑ naq S! H 3Md as JMI JMI

010130 031 OVO VOX OVO VVO 010 VOX 300 VVV 0V3 Oil OVO 311 DDI 03V D3V

 SIS 90S LQZ 882 6LZ

 "ID sXi usv ¾IV niO ^ IO 3J J3S J3S IO sA UJO ΑϊΟ ¾IV 丄

VVO VVV XVV 3V1 VDV 330 VVO 1X1000 VOV OOV OOV VOO OVV OVO 900 130 93V

OLZ 19Z SSS £ H ΠΖ 522

 311 ΙΒΛ s q na ngq ngq naq λιο nio ^ io OJJ JAI 3JV 1¾Λ 丄 djx aqd

LLV 310 VVV 013 DID 310 VVO VIO 100 VVO VOO 133 IVI VOO 110 IVi OOX 311

912 102 861 681 081 T

nai usy OJJ J / ί 丄 ¾i 911 J3S J¾l ¾IV J3S J jqi SAQ USV 3Π 9Π nai J3S

113 IVV 133 3V1 130 VIV OOV VOV 330 V31 IVI OOV 301 IVV VIV VIV 010 DDI

Z9i esT SCT i \\

sA ^ i J9S nio J3S 1¾Λ JMI 1¾Λ UIO Aio nio J¾ J¾ 1¾Λ J3g dsv AIO S! H OVV OOV VVO VOX 310 33V 010 VVO 300 VVO VOV VVO 13V 010 331 331 IVO V09 OVO 801 66 06 18 ZL S9

JiJ 丄 3JV Aio naq an naq na na ¾ ¾IV Ι¾Λ 9Md ^ IO o ^j d J3S J3S dsv} 9W 03V OOV V0911D VIV 310 UO VIO 010100 010 311300 V331311310V9 01V SS 9C LZ 81 6

 :

¾ z s · 6— g¾B 睐 丄 ichi: ¾ ^

 (s n ^) ^ x C K, ^: ^ ^

VN oi VN (P: ii¾ © ½a @ ^ S: ー-α

 鹩 本 二 ： 綠 0 鹩

S9SI0 / £ 6df / XDd UPZP / L6 OAS. 0 t '

9: 2 ri Honmoto J9S J3S

V9110V 031 618

dj 丄 naq Sjy na

naq naq usy 9] d AjQ ¾iv sq naq naq naq an JL 3 丄 3 00V OID 93V 01V 3X3 013 OVV 丄 丄 V09 000 VXO VVV 910 OID DID 31V

018 108 261 281 ll S9L

 3J nai ^ io 19W 1¾Λ J3S "31 usy uio aild" sy naq usy} dn dsy Jill nig aqd

VOO 313 VOO OIV 110 VOX 013 OVV VV3 111 OVV VIO DVV OIV IVO VDV VVO 丄 丄 丄

 J9S s nio JM1 naq jqi ¾iv dsy SAQ OJd \ dsy J3S J3S OJd JA 丄 jq ¾iv

30V VVV OVO ODV 911 OOV 330 IVO 191 330 1X0 DVO VOX 10V 330 3V1 33V DOO

ZL m 219 999 丄 S9 usv JMi nio sAq 9i an ds \ UIQ SAQ jqi aj J3S JMl uio ^US V ^ dJi ¾iv

3VV OOV OVO VVV 311 31V IVO VVO DDI ODV Oil 30V V3V OVD OVV 30V 091000

8 ^ 9 6 £ 9 089 129 219 809

 311 ¾iv O usv J3S sAn J3S dsy} 3W ¾1V sA dsy naq octal jqi sAq dsv 丄 丄 V 300 900 IVV OOV OVV 331 IVO OIV 130 VVV OIV OVO 019 010 13V VVV OVO

^ 6S S8S 9iS 丄 9S 8SS 6 S jiU an J 41 i9 J3S nio ^ Vi sAq OJd Ι¾Λ usv 311 "10 s dsv aqd

13V OIV 311 03V VOO 1D1 VVO OXV 33V VVV 03D OIO IVV OIV VVO 301 3V0 丄 丄 丄

0 ^ 9 185 ZZ eig 0S S6 dsv Jm sqd nai sA ^ naq Jm J3S dsy uj J3S 3JV OJd dsv sAi na ^ Ιΰ "^ 丄

3V9 33V 311013 001 313 33V OOV OVO DVO X31000 100 IVO VVV Vll 0V3 OVI

98 LL 89 ^ 6 09 ^ i

 ΙΒΛ BIV OJd nio OJd usy uio 911 S IH naq JA 丄 Ι¾Λ sA naq d \\ jqi AIQ uio

019130133 VV9 V33 DVV OVD OIV OVO 010 3V1 D10 9VV Vll VIV ODV 900 9V3

S9SI0 / Z.6dLT / XDd Ϊ t fr € 6 ΟΛ \ Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 GTGGTCCAGT TGAGGTCTGC AAGA SEQ ID NO: 7

Array length: 2 1

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 TTGAAAGTTT AGGTTCATAT C SEQ ID NO: 8

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 AATTTAGGAT CCGGACAGCA AGTGCAGCAG SEQ ID NO: 9

Array length: 2 3

Sequence type: nucleic acid

Number of chains: single strand Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 GACTCTAGAT TACTAGTTTG GATGGACCCT AAG SEQ ID NO: 10

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 AATTTAGGAT CCGGACAGCA AGTGCAGCAG SEQ ID NO: 1 1

Array length: 3 3

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 TCCTCTAGAT TACTAGGTGA ACAGGCAGAG GGT SEQ ID NO: 1 2

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: other nucleic acid synthetic DNA Array:

 CTTTCTAGAG CAATCCAGAA CCCAGAACCT SEQ ID NO: 1 3

Array length: 3 1

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 AAGCGGCCGC TTAGTTTTGA AAGTTTAGGT T SEQ ID NO: 14

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 AATTTAGGAT CCGATTCCGT GACTCAAACA SEQ ID NO: 15

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

GCCTCTAGAT TACTATTGAA AGTTTAGGTT SEQ ID NO: 1 6

Array length: 2 7

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 CTAGGATCCA TCCAGAACCC TGACCCT SEQ ID NO: 17

Array length: 2 7

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

 CTAGAATTCT CAGCTGGACC ACAGCCG SEQ ID NO: 18

Array length: 3 3

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence:

GACGGATCCA TCCAGAACCC TGACCCTGCC GTG SEQ ID NO: 19 丄 3 丄 111 OVO 1D1 VVV OVV OOV 9D100901013010V OVV DOV 3VV Dll DVO

9T2 LOZ 861 68ΐ 081 \ L \

 ^ Yi J3S 3JV law dsv Π91 \ s \ jqi sAq dsv ail J [ΈΛ dsv J9S dsy sA OIV 131 OOV OIV 3V0 V1301013V VVV 3V9 VOV OIV 1V1010 IVO 1D1 IVO OVV

Z9i mm ssi 921 LU

J9S uiO J3S Ι¾Λ usv JM1 "Ϊ9 s dsy 9qj dsv JM1 9Md na SAQ Ι¾Λ J3S sAq 10V VVO V31010 IVV VOV VVO 131 IVO 111 IVO 33V 311 V133013101019VV 801 66 06 18 ZL 89 dsy J 3S J 9S sA J3S dsy 3JV na uig丄I¾A ¾1V 0Jd dsv 0Jd usv UIQ an 3V010V 001 VVV JLOI OVO VOV 013 OVO DVI 0100301333V0100 OVV OVD OXV S 9 9C LZ 8ΐ 6

:

I-one

mm 丽 ko: thigh o 鹩 本 Z '

 9 ε ε:

 0 Z ■

31V1931 100V1110V VVOllVlOVl 1V0VI31D31

:

Suimoto: Leak: fSG? ½2S

9 ε:

S9SI0 / .6dT / 13d OAV 9 ί '

UIO 3Md us "31

VVO 丄 丄 丄 DVV VIO usv JM1 dsy J l nio 3iid J3S sXq nig naq sAq Ι¾Λ dsy sAo jas J9S nio

DVV OOV IVO V3V VVO 11 130V VVV OVO DIO 013 OVV DIO IVO 10X 33 110V VVO m sie 9οε LQZ 882 QLZ

OJd J3S OJd 3i] d 9Md J ll dsv "10 o j d 311 ail J3S US V usv ¾IV usv ¾IV

V33 OOV 333311 Oil 33V OVO VVO V3D LLV 11V 30V 3VV OVV 311 300 DVV V30

OLZ 292 ΠΖ ^ ZZ

 SAQ ¾IV aqd dsv J3S sA usy J9S dj 丄 ΈΙ / eight BIV J3S usy J8S s q 9qj dsy

Claims

The scope of the claims

1. A polypeptide that contains substantially all or part of the constant region of a T cell receptor chain, has immunosuppressive properties, but does not substantially induce the production of antibodies against itself upon administration.

 2. It contains an amino acid sequence of SEQ ID NO: 1 in which one or more amino acid residues in the amino acid sequence may be deleted, inserted and / or substituted, and has an immunosuppressive effect. A polypeptide that does not substantially elicit.

 3. Contains an amino acid sequence of SEQ ID NO: 2 in which one or more amino acid residues in the amino acid sequence may be deleted, inserted and / or substituted, and has an immunosuppressive effect. A polypeptide that does not substantially elicit.

 4. A DNA encoding the polypeptide according to any one of claims 1 to 3.

 5. The following formula:

 R 1 -X-R 2

 (Wherein, R 1 is a carrier polypeptide, X is a protease recognition site, and R 2 is a polypeptide according to any one of claims 1 to 3)

DNA having a nucleotide sequence encoding the fusion polypeptide represented by

 6. The DNA according to claim 5, wherein R 1 is calmodulin.

 7. The DN according to claim 5, wherein X is a site recognized by thrombin.

8. The DNA of claim 7, wherein the site recognized by thrombin is represented by the following amino acid sequence of SEQ ID NO: 3.

 Lys-Val-Pro-Arg-Gly (SEQ ID NO: 3)

 9. An expression vector carrying the DNA according to any one of claims 4 to 8

10. A host cell transformed with the expression vector according to claim 9.

 11. The host cell according to claim 10, wherein the host cell is a prokaryotic cell.

12. The host cell according to claim 11, wherein the prokaryotic cell is Escherichia coli.

13. A method for producing a polypeptide according to any one of claims 1 to 3, wherein the expression vector carries a DNA having a nucleotide sequence encoding the polypeptide. The above-mentioned production method, wherein the transformed host cell is cultured, and the polypeptide is isolated.

 14. The production method according to claim 13, wherein the host cell is a prokaryotic cell.

 15. The production method according to claim 13, wherein the prokaryotic cell is Escherichia coli.

 16. A method for producing a polypeptide according to any one of claims 1 to 3, which comprises the following formula:

 R 1-X-R 2

 (Wherein, R 1 is a carrier polypeptide, X is a protease recognition site, and R 2 is a polypeptide according to any one of claims 1 to 3)

Culturing a host cell transformed with an expression vector carrying a DNA having a nucleotide sequence encoding the fusion polypeptide represented by The above-mentioned production method, which comprises cleaving with a degrading enzyme and isolating the polypeptide represented by R2.

 17. The production method according to claim 16, wherein R 1 is calmodulin.

 18. The production method according to claim 16, wherein X is a site recognized by thrombin, and thrombin is used as a protease.

 19. The production method according to claim 18, wherein the site recognized by thrombin is represented by the following amino acid sequence of SEQ ID NO: 3.

 Lys-Va1Pro—Arg—Gly (SEQ ID NO: 3)

 20. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 3 as an active ingredient.

 21. The pharmaceutical composition according to claim 20, which is an immunosuppressant.

 22. The pharmaceutical composition according to claim 20, which is a delayed type hypersensitivity reaction inhibitor.

 23. The pharmaceutical composition according to claim 20, which is an antibody production inhibitor.

 24. The pharmaceutical composition according to claim 20, which is an agent for preventing and / or treating allergic diseases.

25. The preventive and / or therapeutic agent for autoimmune diseases according to claim 20 Pharmaceutical composition.

26. The pharmaceutical composition _c according to claim 20, which is an inhibitor of rejection during organ transplantation.