JP2003002897A - Peptide and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain peptides composed of a T-cell epitope of a Japanese cedar pollen allergen which does not substantially react with an immunoglobulin E antibody specific for the Japanese cedar pollen allergen, does not substantially cause an anaphylaxis when administered to the mammals in general including human, and enables the inactivation of a T-cell specific for the Japanese cedar pollen allergen, and an anti-Japanese ceder pollenosis medicine containing the peptides as an active ingredient which is effective for a Japanese ceder pollenosis patient. SOLUTION: This peptide, its complex, derivative, or polymer has the peptides composed of specific amino acid sequences or amino acid subsequences containing at least one of the amino acid subsequences, does not combine with the immunoglobulin E antibody derived from the Japanese ceder pollenosis patient, and has an activity proliferating the T-cell derived from the Japanese ceder pollenosis patient, and the anti-Japanese ceder pollenosis medicine contains them as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide that inactivates human T cells that specifically react with cedar pollen allergen, and an immunotherapeutic agent containing the peptide as an active ingredient.

[0002]

2. Description of the Related Art In recent decades, the number of people complaining of rhinitis and conjunctivitis due to cedar pollinosis has been increasing in Japan in the early spring. Cedar pollinosis is a type of allergic disease, and it is said that the main cause thereof is an antigenic substance in cedar pollen, that is, a cedar pollen allergen. When cedar pollen scattered in the air enters the human body, an immunoglobulin E antibody against the cedar pollen allergen is produced. When cedar pollen invades next in this state, the allergen in the pollen and the immunoglobulin E antibody cause an immune reaction, and allergic symptoms are exhibited.

It is known to date that there are at least two types of allergens having different antigenicities in cedar pollen. One of them is the article by Jasueda [J. Allergy.
Clin. Immunol. 71, 77-86 (1983)], which is a protein called "Cryj1", and the other is the protein of Taniyai et al. [FEBS Letters, 239, 329-332 (19).
88)] and Sakaguchi et al. [Allergy, No. 45, 309].
-312 (1990)], it is a protein called "Cryj2". Cryj1 and Cry
To date, the entire amino acid sequence of both j2 has been determined [WO93 / 01213 and JP-A-8-5052].
No. 84].

In Japanese cedar pollen, Cryj1 and Cry are usually used.
j2 is present in a ratio of about 50: 1 to 5: 1, and most of the sera collected from hay fever patients are Cryj1 and Cry.
It is said to react to j2. Sawaya et al. [Allergy, Vol. 42, pp. 738-747 (1993)] describes Cryj1 in the intradermal reaction test and RAST test.
And Cryj2 are reported to exhibit similar antigenicity.

Since several cedar pollen allergens have already been isolated and their properties and properties have been elucidated to some extent, the purified cedar pollen allergens were administered to humans to desensitize them to obtain cedar pollinosis. The prospect of being able to treat and prevent Recently, some desensitizing agents for that purpose have been devised. For example, JP-A-1-156926 and JP-A-3-93730 disclose a complex produced by covalently binding a sugar to a cedar pollen allergen. Has been proposed as a desensitizing agent for humans.

However, a large amount of high-purity allergen is usually required for the diagnosis of allergic disease and desensitization therapy, and the allergen in cedar pollen is scarce and its stability is low, so that the diagnosis of cedar pollinosis is made. There is a great deal of difficulty in trying to cover the agents and desensitizing agents with only cedar pollen.

Therefore, in the recent treatment / prevention of allergic diseases, the whole area of the allergen is not administered to the patient as in the past, but the minimum region specifically recognized by T cells in the allergen. That is, immunotherapy in which a low-molecular peptide consisting essentially of a T cell epitope is administered has attracted attention.

[0008] Generally, when an allergen is taken up by antigen-presenting cells such as macrophages, it is digested there,
The digested fragments are HLA (Human Leukocyt
e Antigen) will bind to the protein and be presented as an antigen. The fragment presented as an antigen is limited to a part of a specific region in the allergen due to affinity for the HLA protein and the like, and the region specifically recognized by T cells is generally called "T cell epitope". .

In immunotherapy in which a peptide consisting essentially of a T cell epitope is administered, (i) the peptide lacks a B cell epitope, ie, an immunoglobulin E antibody specific for the allergen does not react, Side effects such as anaphylaxis that frequently occur with conventional crude or purified allergens cannot occur; (ii) The time from starting with a small amount to reaching an effective dose is significantly greater than that with conventional desensitizing agents. (Iii) Oral tolerance can be induced, and allergic reaction to allergen can be reduced.

However, all the Japanese cedar pollinosis patients have a common T
Since it does not respond uniformly to cell epitopes, and in some cases it may not be effective to administer only one type of T cell epitope, so it is necessary to first investigate which T cell epitope the patient responds to and then administer. Alternatively, a certain effect cannot be expected unless a plurality of T cell epitopes are prepared and they are administered at the same time. However, in the former method, it is necessary to previously check which T cell epitope the patient responds to, and in the latter method, a plurality of T cell epitopes must be prepared separately.

Therefore, in recent years, attempts have been made to use peptides in which a plurality of different T cell epitopes are artificially linked [see, for example, Japanese Patent Publication No. 7-503362]. In addition, such a linked T cell epitope has also been prepared in a cedar pollen allergen [JP-A-10-259].
No. 198].

[0012]

An object of the present invention is to provide a peptide which is excellent in providing a preventive or therapeutic effect on cedar pollinosis. A second object of the present invention is to provide an anti-cedar pollinosis agent containing the above peptide as an active ingredient.

[0013]

[Means for Solving the Problems] The present inventors have newly limited the range of T cell epitopes which are particularly effective as hyposensitizing agents for Japanese cedar pollinosis, or modified some amino acid sequences. The present invention has been completed by creating an additive to provide a prophylactic or therapeutic agent for cedar pollinosis containing these peptides as an active ingredient.

That is, the present invention provides a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing (peptide 1), a complex, derivative or polymer thereof; a peptide comprising the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. (Peptide 2), a complex, derivative or polymer thereof; a peptide (peptide 3) consisting of the amino acid sequence shown by SEQ ID NO: 3 in the Sequence Listing, its complex, derivative or polymer; shown by SEQ ID NO: 4 in the Sequence Listing Peptide consisting of amino acid sequence (peptide 4), its complex, derivative or polymer; Peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 (peptide 5), its complex, derivative or polymer; Sequence listing Of the amino acid sequence represented by SEQ ID NO: 6 (peptide 6), its complex, derivative or polymer; Peptide comprising the amino acid sequence shown in issue 7 (peptide 7), the complex, derivative or polymer; peptide (peptide 8) comprising an amino acid sequence shown in SEQ ID NO: 8 in the Sequence Listing and the complex,
A derivative or polymer; and an immunoglobulin E antibody derived from a cedar pollinosis patient, which has an amino acid sequence containing any one or more of the amino acid sequences represented by SEQ ID NOS: 1 to 8 in the sequence listing, It relates to a peptide, a complex, a derivative or a polymer thereof, which is not bound and has an activity of expanding T cells derived from a Japanese cedar pollinosis patient.

Of the above peptides 1 to 8, peptide 1,
2, 3, 6 and 7 are partial amino acid sequences of Cryj1;
Peptide 8 has a partial amino acid sequence of Cryj2, and peptides 4 and 5 have an amino acid sequence in which one residue in the partial amino acid sequence of Cryj2 is substituted.

As used herein, the term "amino acid sequence constituting a human T cell epitope" refers to the presence of a peptide having the amino acid sequence of a peripheral blood mononuclear cell group (which contains a large number of T cells) derived from a cedar pollinosis patient. When cultured under the following conditions, the rate of DNA synthesis of the peripheral blood mononuclear cell group is more than twice that of the peripheral blood mononuclear cell group cultured in the absence of the peptide having the amino acid sequence, more preferably 5 It means an amino acid sequence having a doubling rate or more (in the present invention, this is referred to as "activity for growing T cells derived from cedar pollinosis patients").

Further, the peptides of the present invention include those in which an additional amino acid sequence is present at the N-terminal and / or C-terminal thereof. The structure of such an additional amino acid sequence is one that impairs the function of the peptide of the present invention as a T cell epitope donor, or one that exhibits some harmful activity such as toxicity when administered to humans. Unless otherwise, there is no particular limitation.

[0018]

The peptides described in the present invention can be prepared by peptide synthesis methods known in the art known as "solid phase method" or "liquid phase method". For example, "Renewal Chemistry Experiment Course", Volume 1, "Protein VI", edited by The Society of Biochemistry, Japan, pp. 3 to 44, 1992.
The details of peptide synthesis are described in, for example, published by Tokyo Kagaku Dojin. In addition, the peptide used was Fmoc (9-fluorenyl methyloxycarbonyl) using a multi-peptide synthesizer (Symphony, manufactured by Protein Technology).
It can be synthesized by the solid phase synthesis method according to the protocol of the same apparatus. That is, Fmoc-L-amino acid W into which an amino acid corresponding to the C-terminal of each peptide to be synthesized has been introduced
Set the ang resin (or Cl-Trt resin) in the reaction vessel of the peptide synthesizer, and use the deprotection solution.
Excluding Fmoc. Further, react an amino acid solution corresponding to the second amino acid from the C-terminal with an activator solution,
After the reaction, the Fmoc group is deprotected again, and the same procedure is repeated to synthesize the desired peptide.

The peptides of the present invention are not limited to those prepared by chemical synthesis and may be those prepared by recombinant DNA technology. For example, the peptides 1 to 8 described above.
A DNA encoding any one of the above is prepared and inserted into a vector capable of self-sustaining growth, and then introduced into a host such as Escherichia coli, Bacillus subtilis, actinomycete, or yeast to obtain a transformant, and the present invention is obtained from the culture. May be collected.

A method for preparing a DNA having a desired nucleotide sequence is, for example, chemically synthesizing sense and antisense nucleotides which are partial sequence nucleotides of the desired DNA and have overlapping ends. Chain reaction [Saiki, RK et
al (1988) Science 239, 487-491] and the like, and a method for obtaining a product in which those partial sequences are linked by utilizing a DNA polymerase reaction or a ligase reaction.

A prokaryotic or eukaryotic host cell can be transformed by incorporating the DNA encoding the amino acid sequence of the peptide of the present invention obtained as described above into a suitable vector. Furthermore, by introducing an appropriate promoter and a sequence involved in transformation into these vectors, the DNA can be expressed in each host cell.

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis.
Etc. In order to express the gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing an origin of replication and regulatory sequences.
Further, the vector preferably has a sequence capable of imparting phenotypic (phenotypic) selectivity to the transformed cell.

For example, E. coli is E. coli. coli K1
2 strains, JM109 strains and the like are often used, and pBR322 and pUC type plasmids are commonly used as the vector, but not limited to these, and various known strains and vectors can be used.

As a promoter, in E. coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan-lactose (tac) promoter, lipoprotein (lpp) promoter, bacteriophage-derived lambda (λ) P.
L promoter, polypeptide chain elongation factor Tu (tuf
B) promoter, lacUV5 promoter and the like, and any promoter can be used for production of the peptide of the present invention.

As the Bacillus subtilis, for example, strain 207-25 is preferable, and as the vector, pTUB228 [Ohmura,
K., et al. (1984) J. Biochem. 95, 87-93], etc., but are not limited thereto. As a promoter for Bacillus subtilis, a regulatory sequence of the α-amylase gene of Bacillus subtilis is often used, and if necessary, a DNA sequence encoding a signal peptide sequence of α-amylase is ligated to thereby secrete outside the cell. Will also be possible.

Taking the case of using Escherichia coli as a host cell as an example, an expression vector having a pBR322 origin of replication, capable of autonomous growth in Escherichia coli, and further provided with a transcription promoter and a translation initiation signal is used. be able to. The expression vector is a calcium-chloride method [Mandel, M. and Higa, A. (1970) J. Mol.
Biol. 53, 154], Hanahan's method [Hanahan, D. a.
nd Meselson, M. (1980) Gene 10, 63] and electric pulse perforation [Neumann, E., et al. (1982) EMBO J.
1, 841-845], etc., and incorporated into Escherichia coli, and thus cells transfected with the desired vector can be obtained.

Eukaryotic host cells include cells of vertebrates, insects, yeasts and the like, and examples of vertebrate cells include COS cells that are monkey cells [Gluzman, Y. (1981).
Cell23, 175-182] and Chinese hamster ovary cells (CHO) deficient in dihydrofolate reductase [Urlaub, G. and Chasin, LA (1980) Proc. Natl.
Acad. Sci. USA 77, 4216-4220], human Namalwa cells, hamster BHK cells and the like are often used, but not limited thereto.

As an expression vector for vertebrate cells, one having a promoter located upstream of a gene to be expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used, and this is further required. May have a replication origin. As an example of the expression vector, pSV2dhfr having a SV40 early promoter [Subramani, S., et al. (1981) Mo
l. Cell. Biol. 1, 854-864], etc.
It is not limited to this.

Yeast is commonly used as a eukaryotic microorganism. Among them, yeast of the genus Saccharomyces, for example, Saccharomyces cerevisiae.
visiae) is preferred. Examples of eukaryotic expression vectors such as yeast include promoters of alcohol dehydrogenase gene [Bennetzen, JL and Hall, BD (1982)
J. Biol. Chem. 257, 3018-3025] and the promoter of the acid phosphatase gene [Miyanohara, A., et al.
(1983) Proc. Natl. Acad. Sci. USA 80, 1-5] and the like can be preferably used.

Taking the case of using COS cells as a host cell, the expression vector has an SV40 origin of replication and is capable of autonomous growth in COS cells. Furthermore, a transcription promoter, a transcription assembly signal and an RNA splice are used. It is possible to use a device having a part. The expression vector is a DEAE-dextran method [Luth
man, H. and Magnusson, G. (1983) Nucleic Acids Re
s. 11, 1295-1308], calcium phosphate-DNA
Coprecipitation method [Graham, FL and van der Ed, AJ (197
3) Virology 52, 456-457] and electric pulse perforation [Neumann, E., et al. (1982) EMBO J. 1, 841-845.
Reference] etc., and can be incorporated into COS cells,
Thus, the desired transformed cell can be obtained. When CHO cells are used as the host cells, they function as a G418 resistance marker together with the expression vector.
A vector capable of expressing the eo gene, for example pRSVne
o [Sambrook, J., et al. (1989) "Molecular Clonin
g: A Laboratory Manual "Cold Spring Harbor Laborat
ory, NY reference] and pSV2neo [Southern, PJ and
Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341
[Reference] etc., and a G418-resistant colony is selected to obtain a transformed cell stably producing the peptide of the present invention.

The desired transformant obtained above can be cultured according to a conventional method, and the polypeptide of the present invention is produced intracellularly or extracellularly by the culture. As the medium used for the culturing, various kinds of medium commonly used can be appropriately selected depending on the adopted host cell. For example, in the case of Escherichia coli, tryptone-yeast medium (bactotryptone 1.6%, yeast extract 1 0.0%, sodium chloride 0.5% (pH 7.0)), peptone medium (manufactured by Difco) and the like can be used. As the COS cells, RPMI1640 medium and Dulbecco's modified Eagle medium (DMEM) may be used, to which serum components such as fetal bovine serum (FBS) are added, if necessary.

As described above, the peptide of the present invention produced inside or outside the transformant can be isolated or separated by various known separation operation methods utilizing the physical properties and chemical properties of the protein. It can be purified. Specific examples of such a method include treatment with an ordinary protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC). ) Etc., various chromatographies, dialysis methods, and combinations thereof.

When a foreign gene is introduced into Escherichia coli or the like for large-scale expression, the produced peptide may form a water-insoluble aggregate called inclusion body. In such a case, the peptide can be solubilized by denaturing the peptide with a strong denaturing agent such as guanidine isothiocyanate.

Further, the peptide of the present invention is in the form of a complex obtained by adding a sugar or polyethylene glycol to the peptide thus obtained, and further by acetylation, amidation and / or polyfunctional reagent of the peptide. It may be in the form of a derivative obtained by cross-linking polymerization or a homo or hetero polymer. In the production of such a complex, derivative or polymer of the peptide of the present invention, addition of sugars, etc., acetylation, amidation and / or cross-linking polymerization is carried out from the cedar pollen allergen protein in the peptide of the present invention. It is preferable, but not limited to, to be performed in the additional peptide at the N-terminal and / or C-terminal of the above-mentioned peptide of the present invention so as not to impair the function of the amino acid sequence constituting the human T cell epitope.

The complex of the peptide of the present invention comprises, for example, polyethylene glycol, monomethoxy polyethylene glycol, dextran, and maltotriose such as pullulan and erucinan as repeating units at the N-terminal or amino group of Lys residue. Those with added polysaccharides,
Examples thereof include those that can be synthesized by a method well known to those skilled in the technical field of the present invention, and these include, for example, "Newborn Chemistry Experimental Course", Vol. 1, "Protein IV", pages 236 to 252, edited by the Japan Biochemical Society. 1991, published by Tokyo Kagaku Doujin) etc.

Derivatives of the peptide of the present invention can be synthesized by methods well known to those skilled in the technical field of the present invention, such as acetylated N-terminal and amidated C-terminal Gly residue. I can list things. Such a derivative can be obtained, for example, from the above-mentioned "Newborn Chemistry Laboratory"
Volume 1, "Protein IV," pages 18-20 and 9
Volume "Hormones I" pages 290-298 (both 1991
, Tokyo Kagaku Dojin, etc.) and the like. Further, the polymer of the peptide of the present invention includes a polymer of one or more peptides of the present invention. For example, the peptide of the present invention can be prepared by using a divalent cross-linking reagent such as disuccinimidyl suberate. Examples thereof include those obtained by polymerizing two molecules and the like, which can be synthesized by a method known to those skilled in the technical field of the present invention. Such a polymer can be prepared, for example, according to the description in the above-mentioned "Shinsei Kagaku Koza", Vol. 1, "Protein IV", pages 207 to 226.

For example, a polymer in which a plurality of single or different peptides selected from SEQ ID NOS: 1 to 8 in the sequence listing are linked via a linker peptide containing an amino acid sequence that can be specifically cleaved by a protease It is also possible to obtain the peptide of the present invention by recombinantly producing Escherichia coli in a host such as Escherichia coli and then digesting the polymer with the protease. At that time, the polymer may be either a homopolymer in which one of the peptides of the present invention is polymerized or a heteropolymer in which a plurality of types of peptides of the present invention are polymerized, but it is possible to produce a large amount of a single peptide. If desired, homopolymers are selected. As the protease, any known protease can be used as long as it recognizes and cleaves a specific amino acid sequence.
Preferred are trypsin, cathepsin B, cathepsin D, cathepsin E and the like.

Further, the present invention has an amino acid sequence containing any one or more of the amino acid sequences shown by SEQ ID NOS: 1 to 8 in the sequence listing in the molecule, and is an immunoglobulin derived from a cedar pollinosis patient. Peptides characterized by having the activity of proliferating T cells derived from cedar pollinosis patients without binding to E antibody are included. Such peptides include, for example, one or more amino acid sequences selected from SEQ ID NOs: 1 to 8 in the sequence listing, and already known human T cell epitopes derived from cedar pollen allergens (Cryj1 and Cryj2). A peptide having an amino acid sequence linked to an amino acid sequence can be mentioned.
A technique for producing a peptide useful for desensitizing therapy for Japanese cedar pollinosis by linking a plurality of T cell epitopes is disclosed in JP-A-10-259198.

The peptide of the present invention exerts the desired therapeutic / prophylactic effect even when administered in a relatively crude form, but it is usually purified before use. For purification, for example, filtration, concentration, centrifugation, gel filtration chromatography, ion exchange chromatography, high performance liquid chromatography,
Methods commonly used in the art for purifying peptides or proteins such as affinity chromatography, gel electrophoresis, and isoelectric focusing are used, and these methods may be appropriately combined as necessary. Then, the purified peptide may be concentrated and lyophilized into a liquid or solid form depending on the final use form.

The activity of the peptide of the present invention as a T cell epitope can be confirmed by measuring the uptake of tritiated thymidine by T cells specific to the cedar pollen allergen. For this measurement, for example, the method described below can be used.

That is, a peripheral blood mononuclear cell group of a Japanese cedar pollinosis patient is separated by Ficoll-Hypaque gravity centrifuge, etc., and this cell group is suspended in a medium such as RPMI1640 and dispensed on a 96-well microplate. . Next, a peptide as a test substance is added and cultivated. Conditions such as temperature and time during this culture can be appropriately adjusted for each experiment, but 37 ° C. and 3 days are preferable. After that, tritiated thymidine was added to the medium, and the culture was further continued for a certain period
The activity of the peptide of the present invention as a T cell epitope can be calculated by measuring the uptake of tritiated thymidine in the mononuclear cell group. In the present invention, a system containing no peptide is provided at the same time as a negative control, and a system in which the tritiated thymidine uptake amount exceeds a value twice that of the negative control is “positive”, and a system in which the system does not reach the value is The result was "negative".

The fact that a peptide consisting of an amino acid sequence constituting a T cell epitope generally has a therapeutic effect on cedar pollinosis can be confirmed by the following experiment, for example. Since the peptide of the present invention has a human T cell-specific activity, its effect may not be exhibited even when experiments are conducted in animals other than humans. However, lymphocytes of C3H / HeN mice are amino acids derived from cedar pollen allergen. Since it has reactivity with the sequence, it is necessary to induce tolerance.
A peptide containing the amino acid sequence is previously administered.
Immunization is performed by administering the peptide again after a certain period of time, and after a certain period of time, lymph node cells are removed from the popliteal part of the mouse to prepare a cell suspension. The above-mentioned peptide is added thereto to continue culturing, and tritiated thymidine is further added to measure the uptake of tritiated thymidine to measure T cell proliferation. In mice that have not been previously induced with immune tolerance, immunization activates T cells specific for the peptide and proliferates in vitro in response to the peptide presented by the antigen presenting cells. On the other hand, in an animal into which immune tolerance has been induced in advance, the T cells specific to the peptide are not activated even after immunization, and the animal does not react with the peptide presented by the antigen-presenting cell in vitro and does not proliferate. Therefore, by measuring the difference, the therapeutic effect on the cedar pollinosis of the peptide consisting of the amino acid sequence constituting the T cell epitope can be confirmed.

Since the peptide of the present invention does not substantially react with the immunoglobulin E antibody specific to the cedar pollen allergen, it is specific to the cedar pollen allergen without substantially causing anaphylaxis when administered to mammals including human in general. T cells can be inactivated.

The anti-cedar pollinosis agent of the present invention comprising the peptide of the present invention, its complex, its derivative or its polymer as an active ingredient substantially causes side effects such as anaphylaxis when administered to a patient suffering from cedar pollinosis. Can be treated without causing it. On the other hand, when the anti-cedar pollinosis agent of the present invention is administered to healthy individuals or individuals with potential cedar pollinosis before the cedar pollen begins to disperse, while exerting a remarkable preventive effect against cedar pollinosis , Remarkably effective in ameliorating allergic symptoms at onset.

The anti-sugi pollinating agent of the present invention will be described in more detail. The anti-sugi pollinating agent of the present invention is usually one or more of the peptide of the present invention, a complex thereof, a derivative thereof or a polymer thereof. 0.01 to 100% (w /
w), preferably 0.05 to 50% (w / w), more preferably 0.5 to 5.0% (w / w). The anti-cedar pollinosis agent of the present invention is not only in the form of the peptide alone, but is physiologically acceptable other than the peptide, for example, serum albumin, gelatin, glucose, sucrose, lactose, maltose, trehalose, sorbitol, maltitol. , Carriers such as lactitol, mannitol, pullulan, excipients, immunostimulants, stabilizers, and if necessary, anti-inflammatory agents and antihistamines such as steroid hormones and sodium cromoglycate,
Includes forms as compositions in combination with one or more other agents including anti-leukotriene agents, anti-tachykinin agents. Furthermore, the anti-cedar pollinosis agent of the present invention also includes a drug in a dosage unit form, and the drug in a dosage unit form means the peptide of the present invention, a complex thereof, a derivative thereof, or a polymer thereof, for example. Means a drug in a physically separate, unitary dosage form suitable for administration containing a daily dose or an integer multiple (up to 4) or equivalent thereof (up to 1/40) . Such dosage unit forms include powders, fine granules, granules, pills, tablets, capsules, troches, oral preparations, syrups, emulsions, ointments, plasters, poultices, suppositories. , Eye drops, nasal drops, sprays, injections and the like.

The anti-cedar pollinosis agent of the present invention is administered orally, dermally, nasally, instilled or by injection for the purpose of treating or preventing cedar pollinosis. The dosage in humans varies depending on the purpose of administration, method of administration, and symptoms, but is usually 0.0 per adult per day, while observing the subject's symptoms and the course after administration.
The dosage is usually 1 to 1000 mg, preferably 1 to 10 mg, once a day or once a month for about 1 to 6 months, and is usually repeatedly administered while increasing the dose.

[0047]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited by these.

[Example 1] Peptide 1 Peptides were chemically synthesized by a method in which amino acids are bonded to a resin-immobilized amino acid derivative one by one from the carboxyl terminal side (solid phase synthesis method). The amino acid used in each cycle was a special amino acid derivative in which the reactive group in the α-amino group and the residue portion was blocked with a protecting group. For example, the α-amino group of each amino acid derivative is Fmoc
Blocked by (9-fluorenyl methyloxycarbonyl). The amino acid derivatives used for peptide synthesis in this Example and the following are as follows: Fmoc-A
la, Fmoc-Arg (Pmc), Fmoc-Asn (Trt), Fmoc-Asp (OtBu),
Fmoc-Gly, Fmoc-His (Trt), Fmoc-Ile, Fmoc-Leu, Fmoc-
Lys (Boc), Fmoc-Met, Fmoc-Phe, Fmoc-Pro, Fmoc-Ser (t
Bu), Fmoc-Thr (tBu), Fmoc-Trp (Boc), Fmoc-Val, Fmoc-
Glu (OtBu), Fmoc-Cys (Trt) (inside the parentheses represent a protecting group for protecting the reactive group at the residue portion. Perkin Elmer Japan Applied Biosystems Division).

In the peptide synthesis, the reaction of deprotecting the Fmoc of the α-amino group of the amino acid bound to the resin and then binding the amino acid derivative having the activated carboxyl group was sequentially repeated (Fmoc method). Each peptide of Peptides 1 to 6 was synthesized using a multi-peptide synthesizer (Symphony; manufactured by Protein Technology Co.) by the above Fmoc solid phase synthesis method according to the protocol of the same apparatus.

That is, Fmoc-Asn in which an amino acid (Asn) corresponding to the C-terminal residue of the peptide to be synthesized has been introduced.
25 μm of (Trt) -Wang resin (0.50 mmol / g)
ol equivalent is set in the reaction vessel of the peptide synthesizer, and 1.25 ml of the deprotection solution (20% piperidine / dimethylformamide) is reacted twice for 5 minutes,
The Fmoc group of amino acids bound to the resin was removed. The resin was washed with 1.25 ml of dimethylformamide solution for 6 times for 30 seconds to give 100 corresponding to the second amino acid from the C-terminal side.
mM Fmoc-Met dimethylformamide solution 1.25m
l, 100 mM activator solution (100 mM
2- (1H-benzotriazol-1-yl-)-1,
1,3,3-Tetramethyluronium-hexafluorophosphate / 400 mM N-methylmorpholine /
Dimethylformamide) (1.25 ml) was added (5 times the equivalent amount of the bound amino acid), and the mixture was reacted at room temperature for 20 minutes. The Fmoc-Met-Asn (Trt) -Wang resin produced here was washed with 1.25 ml of dimethylformamide 6 times for 30 seconds, and then the Fmoc group was deprotected again and washed with 1.25 ml of dimethylformamide 6 times for 30 seconds. , Fmoc-A
The sn (Trt) solution and the activator solution were added and reacted.

A protected peptide resin (Fmoc-Asp (OtBu) -Arg (Pmc) -Pro-Leu- was obtained by repeating the same operation.
Trp (Boc) -Ile-Ile-Phe-Ser (tBu) -Gly-Asn (Trt) -Met-Asn
(Trt) -Wang resin) deprotection solution 1.25m
1 was reacted twice for 5 minutes to deprotect the N-terminal Fmoc group. Next, the resin was washed 6 times with 1.25 ml of DMF, then washed 9 times with dichloromethane, and further dried by blowing nitrogen gas for 20 minutes.

The resin was taken out and the cleaved solution (trifluoroacetic acid (hereinafter referred to as "TFA"): ethanedithiol: water: triisopropylsilane = 92.5: 2.5: 2.5: 2.
5 ml of 5) was added and the reaction was carried out at room temperature for 2 hours to cleave the peptide from the resin and remove the amino acid side chain protecting group to obtain a peptide solution [1997/98 Peptide Synthesis Handbook S48; NOVABIOCHEM].

The peptide solution was filtered using a filter, and the filtrate was collected in a centrifuge tube. To this, 10 ml of cold ether was added to precipitate the peptide. After cooling for a while, this was centrifuged (3,000 rpm, 10 minutes) to collect the precipitate, and the operation of adding cold ether again to disperse and collect was repeated 4 times to wash the peptide. The obtained peptide was dried to obtain a crude peptide (hereinafter, the above operation is referred to as "cribage reaction").

5 mg of the obtained crude peptide was added to 0.
After dissolving in a 10% acetonitrile aqueous solution containing 1% TFA, it was subjected to high performance liquid chromatography (hereinafter referred to as “HPLC”). The HPLC conditions were as follows: Column: TSK Guard Column ODS, φ21.5 mm ×
75 mm (manufactured by Tosoh Corporation); mobile phase: 25-35% acetonitrile / 0.1% T
FA, 20 minutes; Flow rate: 5 ml / min; Detection wavelength: 220 nm.

Fractions eluted at 20 to 21 minutes by HPLC under the above conditions were collected, concentrated and freeze-dried to obtain 1.5 mg of the desired peptide. About 50 pmol of the obtained synthetic peptide, an amino acid sequence analyzer (PP
When amino acid sequence analysis was performed using SQ-10 type; manufactured by Shimadzu Corporation, the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing was confirmed.

Example 2 Peptide 2 By the same procedure as in Example 1, the protected peptide resin (Fmoc
-Pro-Leu-Trp (Boc) -Ile-Ile-Phe-Ser (tBu) -Gly-Asn (Tr
t) -Met-Asn (Trt) -Ile-Lys (Boc) -Wang resin)
A crude peptide was obtained by performing a cribage reaction.

Of the resulting crude peptide, 5 mg was
After dissolving in a 30% aqueous acetonitrile solution containing 1% TFA, it was purified by HPLC under the conditions described below: Column: TSK guard column ODS, φ21.5 mm ×
75 mm; mobile phase: 30-40% acetonitrile / 0.1% T
FA, 20 minutes; Flow rate: 5 ml / min; Detection wavelength: 220 nm. Fractions eluted at 17 to 20 minutes by HPLC under the above conditions are collected, concentrated, and lyophilized to give the desired peptide.
1.6 mg was obtained. Obtained synthetic peptide 50pmo
When amino acid sequence analysis was carried out for l, the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing was confirmed.

Example 3 Peptide 3 By the same procedure as in Example 1, the protected peptide resin (Fmoc
-Trp (Boc) -Ile-Ile-Phe-Ser (tBu) -Gly-Asn (Trt) -Met-As
n (Trt) -Ile-Lys (Boc) -Leu-Lys (Boc) -Wang resin) was synthesized and subjected to a cribage reaction to obtain a crude peptide.

5 mg of the obtained crude peptide was added to 0.
After dissolving in a 30% aqueous acetonitrile solution containing 1% TFA, it was purified by HPLC under the conditions described below: Column: TSK guard column ODS, φ21.5 mm ×
75 mm; mobile phase: 30-40% acetonitrile / 0.1% T
FA, 20 minutes; Flow rate: 5 ml / min; Detection wavelength: 220 nm. Fractions eluted at 10 to 13 minutes by HPLC under the above conditions are collected, concentrated, and lyophilized to obtain the desired peptide.
2.0 mg was obtained. Obtained synthetic peptide 50pmo
When amino acid sequence analysis was carried out for l, the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing was confirmed.

Example 4 Peptide 4 By the same procedure as in Example 1, the protected peptide resin (Fmoc
-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-A
La-Ser (tBu) -Ser (tBu) -Leu-Asn (Trt) -Wang resin) was synthesized and subjected to a cribage reaction to obtain a crude peptide.

4.2 mg of the obtained crude peptide was dissolved in a 10% aqueous acetonitrile solution containing 0.1% TFA, and then purified by HPLC under the following conditions: Column: TSK guard column ODS, φ21. 5 mm x
75 mm; mobile phase: 15-30% acetonitrile / 0.1% T
FA, 20 minutes; Flow rate: 5 ml / min; Detection wavelength: 220 nm.

Fractions eluted by HPLC under the above conditions at 10 to 11 minutes were collected, concentrated and lyophilized to obtain 1.2 mg of the desired peptide. When 50 pmol of the obtained synthetic peptide was subjected to amino acid sequence analysis, the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing was confirmed.

Example 5 Peptide 5 By the same procedure as in Example 1, the protected peptide resin (Fmoc
-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-A
La-Ser (tBu) -Ala-Leu-Asn (Trt) -Wang resin) was synthesized, and a cavitation reaction was performed to obtain a crude peptide.

4.6 mg of the obtained crude peptide was dissolved in a 10% aqueous acetonitrile solution containing 0.1% TFA, and then purified by HPLC under the following conditions: Column: TSK guard column ODS, φ21. 5 mm x
75 mm; mobile phase: 17-25% acetonitrile / 0.1% T
FA, 20 minutes; Flow rate: 5 ml / min; Detection wavelength: 220 nm. Fractions eluted at 11 to 12 minutes by HPLC under the above conditions are collected, concentrated, and lyophilized to give the desired peptide.
Obtained 1.7 mg. Obtained synthetic peptide 50pmo
When amino acid sequence analysis was carried out for l, the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing was confirmed.

Example 6 Peptide 6 By the same procedure as in Example 1, the protected peptide resin (Fmoc
-Leu-Thr (tBu) -Leu-Arg (Pmc) -Thr (tBu) -Ala-Thr (tBu) -A
Sn (Trt) -Ile-Trp (Boc) -Ile-Asp (OtBu) -His (Trt) -Wang resin) was synthesized and a cavitation reaction was performed to obtain a crude peptide.

Of the obtained crude peptide, 5 mg was added to
After dissolving in 10% acetonitrile aqueous solution containing 1% TFA, it was purified by HPLC under the conditions described below: Column: TSK guard column ODS, φ21.5 mm ×
75 mm; mobile phase: 25-35% acetonitrile / 0.1% T
FA, 20 minutes; Flow rate: 5 ml / min; Detection wavelength: 220 nm. Fractions eluted at 11 to 12 minutes by HPLC under the above conditions were collected, concentrated, and lyophilized to obtain 1.8 mg of the desired peptide. Obtained synthetic peptide 50pmo
When amino acid sequence analysis was carried out for l, the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing was confirmed.

Example 7 Peptide 7 For peptide 7, a peptide synthesizer (Model 433A: manufactured by Applied Biosystems) was used, and the above-mentioned Fmoc solid phase synthesis method was carried out by the “0.1 mmol fast mock method ( FastMoc Chemistry) ”.

That is, Fmoc-Tyr in which an amino acid (Tyr) corresponding to the C-terminal residue of the peptide to be synthesized has been introduced.
100μ of (tBu) -Wang-resin (0.46mmol / g)
Mol equivalent was set in a small-scale vessel (8 ml) reaction vessel of the above peptide synthesizer, and 2 ml of deprotection solution (22% piperidine / NMP) was reacted twice for 4 minutes and 7 minutes, and the amino acid bound to the resin. The Fmoc group of was removed (deprotected) and washed 4 times with 2 ml of NMP solution. Next, add 1 mmol of Fmoc-Leu corresponding to the second amino acid from the C-terminal side to the activator solution {2M
Diisopropylethylamine (DIEA) / NMP 1
ml and 0.45M 2- (1H-benzotriazole-
1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) / 1-hydroxybenzotriazole (HOBt) / dimethylformamide (DMF) 2.0 g and NMP 2.1
g) was added to activate (10-fold equivalent amount relative to the bound amino acid), and the amino acid bound to the resin excluding the Fmoc group was reacted at room temperature for 9 minutes. Fm generated here
After washing the oc-Leu-Tyr (tBu) -Wang resin 4 times with 2 ml of NMP, the Fmoc group was deprotected again, and 4 ml of 2 ml of NMP solution was used.
After washing twice, the activated Fmoc-Gly solution was reacted. Hereinafter, the same operation was repeated.

The protected peptide resin (Fmoc-Ile-Lys (Boc) -Arg (Pmc) -Val-Ser (tBu) -Asn obtained by synthesizing as described above was used.
(Trt) -Val-Ile-Ile-His (Trt) -Gly-Leu-Tyr (tBu) -Wang resin) was reacted with a deprotection solution to deprotect the N-terminal Fmoc group. Next, after washing 4 times with 2 ml of NMP, it was washed 6 times with dichloromethane, and further dried by blowing nitrogen gas.

The resin was taken out and the cleaved solution (trifluoroacetic acid (hereinafter referred to as "TFA"): phenol: water:
Thioanisole: ethanedithiol = 82.5: 5: 5:
5: 2.5) was added and reacted at room temperature for 2 hours to cleave the peptide from the resin and remove the amino acid side chain protecting group to obtain a peptide solution [King, DS
(1990) Int. J. peptide Protein Reg. 36, 255]. The peptide solution was filtered using a filter, and the filtrate was collected in a centrifuge tube. After concentrating the filtrate with an aspirator, 4
0 ml of cold ether was added to precipitate the peptide. After cooling for a while, this was centrifuged (3,000 rpm, 10 minutes) to collect the precipitate, and cold ether was added again to disperse and collect the precipitate, which was repeated 4 times to wash the peptide. The obtained peptide was dried to obtain a crude peptide.

Of the crude peptide obtained, 5 mg was
After dissolving in a 20% aqueous acetonitrile solution containing 1% TFA, it was purified by HPLC under the conditions described below: Column: TSK guard column ODS, φ21.5 mm ×
75 mm; mobile phase: 20-30% acetonitrile / 0.1% T
FA, 15 minutes; Flow rate: 5 ml / min; Detection wavelength: 220 nm. Fractions eluted at 16 to 17 minutes by HPLC under the above conditions were collected, concentrated and lyophilized to obtain 0.6 mg of the desired peptide. Obtained synthetic peptide 50 pm
When amino acid sequence analysis was performed on ol, the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing was confirmed.

Example 8 Peptide 8 By the same procedure as in Example 1, the protected peptide resin (Fmoc
-Val-Ala-Ile-Gly-Thr (tBu) -Gly-Ser (tBu) -Ser (tBu) -As
n (Trt) -Ile-Val-Ile-Glu (OtBu) -Asp (OtBu) -Leu-Ile-Cys
(Trt) -Gly-Pro-Gly-Wang resin) was synthesized and subjected to a cribage reaction to obtain a crude peptide.

Of the obtained crude peptide, 9.6 mg was
After being dissolved in a 25% aqueous acetonitrile solution containing 0.1% TFA, it was purified by HPLC under the conditions described below: Column: C22 column (docosyl-B, φ10 mm × 2)
50 mm: Senshu Scientific Co., Ltd.); Mobile phase: 35-40% acetonitrile / 0.1% T
FA, 20 minutes; Flow rate: 1.5 ml / min; Detection wavelength: 220 nm. The fraction eluted at 14 to 15.5 minutes by HPLC under the above conditions was collected, concentrated and lyophilized to obtain 0.3 mg of the desired peptide. Obtained synthetic peptide 50
When pmol was subjected to amino acid sequence analysis, the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing was confirmed.

Example 9 Cedar Pollen Antigen T Cell Epitope Activity Using cedar pollinosis patient-derived T cells, the cedar pollen antigen T cell epitope activity of peptides 1 to 8 of the present invention was confirmed by the method described below. .

Patients with cedar pollinosis symptoms (40-45
50 ml of peripheral blood was collected from Hanks 'Balanced Salt Solution (Hanks' Balanced Salt Solution, H
After diluting with BSS), peripheral blood mononuclear cells (Peripheral Blood Mononuclear C
ells, hereinafter referred to as “PBMC”) and fractionated in a medium (5%
Was suspended in RPMI 1640) containing human AB type serum.

Then, 4 × 10 ⁵ cells were dispensed per well into a 96-well flat bottom plate. At the same time, the peptide was added to each well so that the final concentration was 1 μg / ml. As a negative control, a group to which no peptide was added was prepared. The cells were cultured in 200 μl of medium at 37 ° C. under 5% carbon dioxide gas for 72 hours. Then, 0.5 μCi of tritiated thymidine was added, and the cells were further cultured for 16 hours. The cells in each well were collected on a glass fiber filter using a cell harvester, and the amount of tritiated thymidine incorporated into the cells was measured by a liquid scintillation counter. The value obtained by dividing the amount of thymidine uptake by the amount of uptake in the negative control group is defined as a T cell stimulating index, and when this T cell stimulating index reaches a value of more than 2 in at least one of the whole peripheral blood donors, T cell The epitope activity was defined as "positive". As a result, all the peptides prepared in Examples 1 to 8 were "positive" for T cell epitope activity.
showed that.

Example 10 Importance of T Cell Epitope Peptides 40-45 Sugi Pollinosis Patients Whose T Cell Responsiveness to either the Cryptomeria Allergen Cryj1 or Cryj2 Exceeds a T Cell Stimulation Index of 2 The T cell epitope activity of peptides 1 to 8 was evaluated according to the method described in Example 9 to determine the importance index. The importance index (Positivity Index) is a value obtained by multiplying the average T cell stimulation index by the positive frequency, and is described in WO94 / 01560. The average T cell stimulation index represents the geometric mean value of the T cell stimulation index for subjects who showed a positive reaction to each peptide, and the positive frequency represents the ratio (percentage) of the subjects who showed a positive reaction to the peptide. The results are shown in Table 1.

[0078]

[Table 1]

As described above, all of Peptides 1 to 8 showed a high importance index among cedar pollinosis patients, and were shown to be useful as peptides for hyposensitization therapy against cedar pollinosis.

Among the peptides prepared in Examples 1 to 8, peptides 1, 2, 3, 6 and 7 are partial amino acid sequences of Cryj1, peptide 8 is a partial amino acid sequence of Cryj2, and peptides 4 and 5 are partial Cryj2. Each of the peptides has an amino acid sequence in which one residue in the amino acid sequence is substituted, and each of these peptides has a different portion having a similar chain length in the vicinity of the region occupied by each sequence in the amino acid sequence of Cryj1 or Cryj2. Compared with the peptide or the peptide of the natural amino acid sequence without substitution, it showed an advantageous result in the positive frequency or the importance index.

[Formulation Example 1] The peptide obtained by the method described in Examples 1 to 8 was added to physiological saline containing 1% (w / v) human serum albumin as a stabilizer for the final concentration of 0.01, 0. Dissolve the solution to 1 or 1 mg / ml, sterilize by filtration, dispense in 2 ml aliquots into sterile vials, freeze-dry and seal tightly. Prior to administration, this product should first contain 1 ml of distilled water for injection in a vial.
Then, the contents are uniformly dissolved and used. The product, which has excellent stability and contains the peptide according to the present invention as an active ingredient, is useful as a dry preparation for treating and preventing cedar pollinosis.

[Formulation Example 2] A syrup was prepared by adding trehalose powder (Treha, manufactured by Hayashibara Co., Ltd.) to 0.1 mg / ml of any of the peptides obtained by the method described in Examples 1 to 8. Dissolve each in 50% (w / v) in distilled water, and sterilize and filter the solution by a conventional method to obtain 8 kinds of syrups. 2 ml of each of these syrups is separately dispensed into a sterile vial bottle and sealed. This product, which is highly stable and easy to take, is useful as a syrup for treating and preventing cedar pollinosis.

[Acute toxicity test] The mice obtained on the 20th day of life are orally or intraperitoneally administered with the preparation obtained by the method described in Preparation Example 1 or 2. LD50 (as a peptide) was 2 in the test sample regardless of the route of administration.
It is more than 00 mg / kg of mouse body weight, which means that the peptide of the present invention can be safely compounded and used as an anti-cedar pollinosis agent to be administered to mammals including human.

[0084]

According to the present invention, T of cedar pollen allergen
It was possible to provide a peptide comprising a cell epitope and an anti-cedar pollinosis agent containing them as an active ingredient. Since the peptide of the present invention does not substantially react with the immunoglobulin E antibody specific to the cedar pollen allergen, when administered to mammals including humans in general, it does not cause anaphylaxis and is specific to the cedar pollen allergen. T cells can be inactivated. Further, the anti-cedar pollinosis agent comprising the peptide of the present invention as an active ingredient is effective for a wide range of cedar pollinosis patients.

[0085]

[Sequence list]                          SEQUENCE LISTING <110> Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo       Sankyo Company, Limited <120> Peptides and The Use Thereof <130> P01-0647 <150> JP 2000-279932 <151> 2000-09-14 <160> 8 <170> PatentIn version 3.0 <210> 1 <211> 13 <212> PRT <213> Cryptomeria japonica <400> 1 Asp Arg Pro Leu Trp Ile Ile Phe Ser Gly Asn Met Asn 1 5 10 <210> 2 <211> 13 <212> PRT <213> Cryptomeria japonica <400> 2 Pro Leu Trp Ile Ile Phe Ser Gly Asn Met Asn Ile Lys 1 5 10 <210> 3 <211> 13 <212> PRT <213> Cryptomeria japonica <400> 3 Trp Ile Ile Phe Ser Gly Asn Met Asn Ile Lys Leu Lys 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Modified human T cell epitope  of Japanese ceder pollen allergen <400> 4 Lys Leu Thr Ser Gly Lys Ile Ala Ser Ser Leu Asn 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Modified human T cell epitope  of Japanese ceder pollen allergen <400> 5 Lys Leu Thr Ser Gly Lys Ile Ala Ser Ala Leu Asn 1 5 10 <210> 6 <211> 13 <212> PRT <213> Cryptomeria japonica <400> 6 Leu Thr Leu Arg Thr Ala Thr Asn Ile Trp Ile Asp His 1 5 10 <210> 7 <211> 13 <212> PRT <213> Cryptomeria japonica <400> 7 Ile Lys Arg Val Ser Asn Val Ile Ile His Gly Leu Tyr 1 5 10 <210> 8 <211> 20 <212> PRT <213> Cryptomeria japonica <400> 8 Val Ala Ile Gly Thr Gly Ser Ser Asn Ile Val Ile Glu Asp Leu Ile 1 5 10 15 Cys Gly Pro Gly             20

[0086]

[Sequence list free text]

SEQ ID NO: 4: Variant of human T cell epitope of cedar pollen allergen SEQ ID NO: 5: Variant of human T cell epitope of cedar pollen allergen

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 14/00 A61K 37/02 (72) Inventor Junko Kawaguchi 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Kazuki Hirahara 1-25-2 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Akio Shiraishi 1-2-2 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. In-company (72) Inventor Shinji Serizawa 1-258 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. In-company F-term (reference) 4C084 AA02 AA07 BA01 BA18 NA14 ZA33 ZA34 ZB13 ZB21 ZC54 4H045 AA10 AA30 BA16 BA17 EA22 FA33

Claims (10)

[Claims] 1. A peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, a complex, a derivative or a polymer thereof. 2. A peptide comprising the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its complex, derivative or polymer. 3. A peptide comprising the amino acid sequence represented by SEQ ID NO: 3 in the Sequence Listing, a complex, a derivative or a polymer thereof. 4. A peptide comprising the amino acid sequence represented by SEQ ID NO: 4 in the Sequence Listing, a complex, derivative or polymer thereof. 5. A peptide comprising the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing, a complex, a derivative or a polymer thereof. 6. A peptide comprising the amino acid sequence represented by SEQ ID NO: 6 in the sequence listing, a complex, a derivative or a polymer thereof. 7. A peptide comprising the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing, a complex, a derivative or a polymer thereof. 8. A peptide comprising the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing, a complex, a derivative or a polymer thereof. 9. A molecule having an amino acid sequence containing any one or more of the amino acid sequences represented by SEQ ID NOs: 1 to 8 in the sequence listing and binding to an immunoglobulin E antibody derived from a Japanese cedar pollinosis patient. A peptide, a complex, a derivative or a polymer thereof, which has the activity of proliferating T cells derived from a Japanese cedar pollinosis patient. 10. An anti-cedar pollinosis agent, which comprises the peptide according to any one of claims 1 to 9, a complex thereof, a derivative thereof or a polymer thereof as an active ingredient.