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WO1997043411A1 - Peptides a regions constantes de chaine alpha de recepteur des cellules t, leurs procedes de production et leur utilisation - Google Patents

Peptides a regions constantes de chaine alpha de recepteur des cellules t, leurs procedes de production et leur utilisation Download PDF

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Publication number
WO1997043411A1
WO1997043411A1 PCT/JP1997/001565 JP9701565W WO9743411A1 WO 1997043411 A1 WO1997043411 A1 WO 1997043411A1 JP 9701565 W JP9701565 W JP 9701565W WO 9743411 A1 WO9743411 A1 WO 9743411A1
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Prior art keywords
polypeptide
dna
tcr
seq
amino acid
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PCT/JP1997/001565
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English (en)
Japanese (ja)
Inventor
Noriko YUYAMA
Nakayuki Honma
Toshifumi Mikayama
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Kirin Beer Kabushiki Kaisha
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Priority to AU26519/97A priority Critical patent/AU2651997A/en
Publication of WO1997043411A1 publication Critical patent/WO1997043411A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • T cell receptor ⁇ -chain constant region peptide T cell receptor ⁇ -chain constant region peptide, method for producing the peptide and use thereof
  • the present invention relates to a constant region polypeptide of a ⁇ -cell receptor ⁇ -chain having an antigen-specific immunosuppressive action.
  • TCR The cell receptor chain
  • TCR T cell receptor chain
  • TCR T cell receptor chain
  • C the constant region of the TCR chain
  • ⁇ which is uniform in all T cells having a TCR
  • C / 3 the constant region of the TCR / S
  • immunoglobulins 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.
  • 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).
  • 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.
  • TCR variable region The involvement of the TCR variable region in regulating immune responses has also been shown for TCR0.
  • immunization with the TCR / 3 variable region peptide specific for the antigen myelin basic protein suppresses the development of this disease.
  • 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
  • Dosquenne- Clark et al. Proc. Natl. Acad. Sci. USA, 88: 7219-7223, 1991.
  • TCRa Japanese Patent Laid-Open No. 6-2988662
  • 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.
  • autoimmune NOD Nonobese diabetogenic mice
  • the TCRa of 14J ⁇ 28 1 is completely different from the TCR on normal T cells, and is thought to be involved in the regulation of autoimmune diseases, etc. ⁇ Expressed 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.
  • 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.
  • 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 TCR ⁇ , 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.
  • 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.
  • the present invention provides a DNA encoding the above-mentioned polypeptide.
  • the present invention also provides the following formula:
  • R 1 is a carrier polypeptide
  • X is a protease recognition site
  • R 2 is the polypeptide according to any one of claims 1 to 3.
  • 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.
  • 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 is the carrier polypeptide
  • X is the protease recognition site
  • 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.
  • 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.
  • 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-C ⁇ on transplant rejection.
  • the polypeptide of the present invention may include at least a region essential for immunosuppressive action or an analog thereof.
  • 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- ⁇ chain of various organisms, including humans and mice, which are essential for at least immunosuppressive action.
  • 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.
  • 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.
  • 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.
  • 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.
  • the DNA of the present invention can be used for producing the above-described polypeptide of the present invention by gene recombination technology.
  • 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.
  • 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.
  • 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:
  • R 1 is a carrier polypeptide
  • X is a protease recognition site
  • 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.
  • R1 is a calmodulinka
  • 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).
  • 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 ⁇ 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.
  • the cDNA library is prepared from a known mouse ⁇ 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.
  • the PCR method can be performed using the latter primer directly in the cDNA library to obtain a desired DNA fragment.
  • 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.
  • the DNA is obtained by chemical synthesis, for example, the method of Alton et al. Based on the known amino acid sequence of C ⁇ , if necessary, considering the use of preferential codons, the nucleotide sequence is designed and DN ⁇ Fragments can be obtained.
  • 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.
  • 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.
  • 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.
  • prokaryotic eg, bacteria, preferably E. coli
  • eukaryotic eg, yeast, insect, or mammalian cells
  • 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.
  • yeast include baker's yeast (Saccharomyces cerevisiae) and methanol-assimilating yeast (Picia pastoris).
  • 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).
  • pKC30 Shiatake H. and M. Rosenberg, Nature ⁇ 292, 128-132, 1981
  • pTrc99A Amann B. et al., Gene 6, 69, 301-315, 1988.
  • PSV2-neo Southern and Berg; J. Mol. Appl. Genet., 1, 327-341, 1982
  • pCAGGS Newa et al .; Gene, 108, 193-200, 1991
  • Is PCD SR 296 Takebe et al .: Mol. Cell. Biol., 8, 466-472, 1988
  • yeast pG-1 (Scena M.
  • transfer vector pAc373 for recombinant virus production (Luckow et al., Bio / Technology, 6, 47-55, 1988) and the like are listed.
  • 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. .
  • a vector derived from SV40, adenovirus, or espapilloma virus can be used as a vector for mammalian cells.
  • a vector for Escherichia coli those derived from CollEl, R factor, F factor and the like can be used.
  • yeast those derived from 2 / mDNA, ARS1, etc. can be used.
  • a vector derived from a virus such as retrovirus, poliovirus, adenovirus, SV40, or a chromosome (eg, EF1- ⁇ )
  • a vector for mammalian cells is used as a vector for mammalian cells.
  • a vector for Escherichia coli a vector derived from bacteriophage ⁇ , a trp, lpp, lac, or tac promoter can be used.
  • ADH, PH05, GPD, PGK, and MAF ⁇ promoter can be used for baker's yeast, and A0X1 promoter can be used for methanol-assimilating yeast.
  • a vector for silkworm cells a vector derived from nucleopolyhedrovirus can be used.
  • 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.
  • neomycin (neo) resistance gene thymidine kinase (TK) gene, dihydrofolate reductase (DHFR) gene
  • Escherichia coli a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene and the like can be used.
  • yeast Leu2, TrpK Ura3 genes and the like can be used.
  • the polypeptide of the present invention 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.
  • 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.
  • the polypeptide is separated and purified from the culture solution.
  • the polypeptide 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.
  • R 1 is a carrier polypeptide
  • X is a protease recognition site
  • 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.
  • 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.
  • R 1 examples include calmodulin
  • examples of X include a thrombin recognition site sequence (particularly preferably a sequence of Lys—Va 1—Pro—Arg—G 1 y).
  • 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.
  • the separation and purification of an expression product 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.
  • 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.
  • 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.
  • 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.
  • 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.).
  • 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).
  • 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, paraben
  • excipients or tonicity agents eg, lactate
  • mannitol
  • 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.
  • compositions 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.
  • Example 1 cDNA Cloning and Expression of TCR Gene of Sublesser T Cell Line Specific to Bee Venom Phospholipase A2
  • PHA2 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.
  • 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.
  • 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.
  • TCRa cDNA 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.
  • 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.
  • the transformant W3110 E. coli cells (ATCC No. 27325) were transformed.
  • 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.
  • Competent DH5 E. coli cells were transformed with this novel plasmid, designated pCF1- 3B3TCRa-VJC25, and its DNA sequence was confirmed.
  • transformant W3110 E. coli cells were transformed.
  • the DNA fragments encoding amino acids 1332 to 2441 in FIG. 1 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.
  • 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.
  • M9 medium consisting of 0.8% glucose, 0.4% casamino acid, 100/1 ampicillin so that other bacteria did not enter
  • indoleacrylic acid was added to a final concentration of 20 ⁇ M, and the culture was further cultured at 37 ° C for 5 hours.
  • 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 3 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.
  • 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.
  • calmodulin-13B3TCR-1 VJ, VJC25 and C ⁇ 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.
  • 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 ⁇ 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.
  • 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.
  • DTT dithiothreitol
  • NaCl sodium chloride
  • TCR-1Ca contains 10% glycerol and 2 mM DTT on a NAP (Pharmacia) column.
  • TCR-C ⁇ protein After washing with 5 OmM Tris HC1 buffer (pH 8.0) containing 0% glycerol and 2 mM TTT, the TCR-C ⁇ 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-C ⁇ protein was about 1 mg / L. TCR ⁇ -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.
  • 3B 3 TCR ⁇ -VJ and VJC 25 were replaced with 50 mM Tris HC1 buffer ( ⁇ 8.0) 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.
  • 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.
  • 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,
  • recVJ protein did not suppress anti-DNP-IgG1 production.
  • 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 ⁇ g / time on days ⁇ 3, ⁇ 1, 0, and 1; control mice received only PBS.
  • 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.
  • recC ⁇ 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.
  • serum was collected from each mouse, and anti-DNP-IgGl was measured by EUSA (Iwa-Yu et al., J. Immunol., 141: 3270-3277, 1988).
  • rec C a The protein significantly suppressed anti-DNP-igGl production as compared to rec VJ and rec VJC25.
  • ovalbumin ovalbumin
  • 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,
  • CM-P LA 2 denatured carboxymethylated PLA 2
  • mice were immunized by intraperitoneal injection of 0.1 g of OVA adsorbed on 1 mg of Alum.
  • 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.
  • 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.
  • dexamethasone was injected intraperitoneally as a control drug at a dose of 0.1 ⁇ g / dose on days 6 and 7.
  • 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.
  • mice to which recC ⁇ was administered showed almost the same inhibitory activity as dexamethasone (Fig. 5).
  • Example 9 Production of antibody against recombinant TCR ⁇ -C ⁇ in mouse serum after administration of recombinant TCRa-Ca
  • TTB S 0.1% Tvveen-20
  • GZTTB S manufactured by Boehringer Mannheim
  • 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.
  • CFA complete 'Freund' adjuvant
  • 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 ⁇ g / ml.
  • 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,
  • helper-I T cell line specific for the PLA2 antigen was obtained. This helper T cell line was named B4-9.52 cells.
  • 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.
  • PCR was performed in a Thermo 'cycler' with Taq I DN ⁇ 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).
  • 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.
  • 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.
  • 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.
  • indoleacrylic acid was added to a final concentration of 20 2, and the culture was further cultured at 37 ° C for 5 hours.
  • the fusion protein of rumodulin-B4-9.52-TCRa was expressed in a soluble form, about 10% of the total protein.
  • 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.
  • DTT dithiothreitol
  • 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%
  • B4-9.52-TCR ⁇ protein c 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 ⁇ 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-TCR ⁇ protein was about 1 mg. The B4-9.52-TCR ⁇ 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).
  • the rec VJC (V aAA) protein was administered to mice immunized with the bee venom PLA2.
  • the bee venom PLA2 dinitrophenyl hereinafter referred to as “DNP
  • 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 (V ⁇ 4.4) in the serum of each of these mice was examined by eastern blotting.
  • 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.
  • a goat anti-mouse IgG H + -alkaline phosphatase-labeled antibody (manufactured by Bio-Lat)
  • 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).
  • Rec VJ C (V ⁇ 4.4) is derived from TCR ⁇ of helper ⁇ cells, but is a recombinant polypeptide of the VJC region (extracellular region of TCR) of TCRa derived from suppressor ⁇ cells 3 33 (see above).
  • VJC region extracellular region of TCR
  • WO95-166462 A similar test was also conducted for the international publication WO95-166462, which also confirmed production of an antibody against the administered recombinant polypeptide.
  • 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.
  • mRNA was isolated from 5 X 1 0 7 AC5 cells.
  • cDNA was generated using a cDNA synthesis system (Falmana).
  • the cDNA was ligated at the 5 'and 3' ends using T4 ligase (Takara) to construct a circular DNA.
  • T4 ligase T4 ligase
  • 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.
  • 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 TM (Invitrogen). This was named pCRIOOO-human TCR.
  • 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).
  • the transformant was transformed into W3110 E. coli cells.
  • 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.
  • indoleacrylic acid was added to a final concentration of 20, and the culture was cultured for a further 5 hours at 37.
  • the calmodulin-TCRa-human C fusion protein was expressed in a soluble form, about 10% of the total protein.
  • Example 11 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.
  • 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).
  • 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.
  • the fusion protein TCRa-human Ca fusion protein was eluted at a concentration of about 500 mM NaCl.
  • 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).
  • 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.
  • the rechCa protein was administered to mice immunized with the bee venom PLA2.
  • DNP-modified PLA2 was used as an antigen
  • 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.
  • the production of antibodies to the recombinant TCR-human C ⁇ 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.
  • 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.
  • coloring was performed using an alkaline phosphatase coloring kit (manufactured by Biorat).
  • 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 TCR ⁇ .
  • Organism name Mus musculus (Mus)
  • Sequence type nucleic acid
  • Organism name Mus musculus (Mus)
  • Sequence type nucleic acid 6 ⁇ ⁇ ⁇
  • VN oi VN (P: ii3 ⁇ 4 ⁇ 1 ⁇ 2a @ ⁇ S: ⁇ - ⁇
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

L'invention concerne des polypeptides qui contiennent sensiblement tout ou partie de la région constante d'une chaîne-α de récepteur des cellules T, possèdent des effets immunosuppresseurs, mais n'engendrent pas sensiblement de production d'anticorps contre eux-mêmes, même en cas d'administration. L'invention concerne également de l'ADN codant pour ces polypeptides, ainsi qu'un ADN présentant une séquence de bases codant pour des polypeptides condensés représentés par la formule générale R1-X-R2, dans laquelle R1 est un polypeptide porteur; X est un site de reconnaissance de protéase; et R2 est un polypeptide qui contient sensiblement tout ou partie de la région constante d'une chaîne-α de récepteur des cellules T, présente des activités immunosuppressives, mais n'engendre pas sensiblement de production d'anticorps contre lui-même, même en cas d'administration. L'invention concerne d'autre part un vecteur d'expression portant l'ADN précité, des cellules hôtes transformées par le vecteur précité, des procédés pour produire les polypeptides précités, et des compositions pharmaceutiques les contenant comme principe actif. Ces polypeptides possèdent des effets immunosuppresseurs non spécifiques de l'antigène et peuvent supprimer des immunoréactions non seulement humorales mais également à médiation cellulaire. Par ailleurs, leur administration éventuelle n'engendre pas sensiblement de production d'anticorps contre eux-mêmes.
PCT/JP1997/001565 1996-05-10 1997-05-09 Peptides a regions constantes de chaine alpha de recepteur des cellules t, leurs procedes de production et leur utilisation WO1997043411A1 (fr)

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JP2009508517A (ja) * 2005-09-22 2009-03-05 コーエン，イルン，アール Ｔ細胞受容体定常ドメインの免疫原性断片及びそれに由来するペプチド
US7795223B2 (en) 2004-05-27 2010-09-14 Novozymes Biopharma Au Ltd. Treatment of inflammatory airway disease
WO2020138256A1 (fr) * 2018-12-27 2020-07-02 国立大学法人京都大学 Objet modifié par un récepteur de lymphocytes t

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PROC. NATL. ACAD. SCI. U.S.A., Vol. 82, (1985), Y. YANAGI et al., "Analysis of cDNA Clones Specific for Human T Cells and the alpha and beta Chains of the T-Cell Receptor Heterodimer from a Human T-Cell Line", pages 3430-3434. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7795223B2 (en) 2004-05-27 2010-09-14 Novozymes Biopharma Au Ltd. Treatment of inflammatory airway disease
JP2009508517A (ja) * 2005-09-22 2009-03-05 コーエン，イルン，アール Ｔ細胞受容体定常ドメインの免疫原性断片及びそれに由来するペプチド
US9078843B2 (en) 2005-09-22 2015-07-14 Irun R. Cohen Immunogenic fragments of T-cell receptor constant domains and peptides derived therefrom
WO2020138256A1 (fr) * 2018-12-27 2020-07-02 国立大学法人京都大学 Objet modifié par un récepteur de lymphocytes t
JPWO2020138256A1 (ja) * 2018-12-27 2021-11-04 国立大学法人京都大学 Ｔ細胞受容体の改変体

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