JPH08127591A - Peptide and its use - Google Patents

Abstract

PURPOSE: To obtain a peptide not reacting with IgE specific to cedar pollen allergen, capable of activating a T cell specific to cedar pollen allergen by judging taking-in of<3> H-thymidine and useful as an immunotherapeutic agent for treating cedar pollinosis, etc. CONSTITUTION: This new peptide contains either one of amino acid sequences expressed by formulas I to VII and does not react with immunoglobulin E(IgE) antibody specific to cedar pollen allergen and significantly activates a T cell specific to cedar pollen allergen, compared with negative reference examination using a method to judge taking in of<3> H-thymidine and exhibits remarkable treating and preventing effect on cedar pollen in a short period without almost giving adverse effect due to administering to Mammalia including Human as an active ingredient for immunotherapeutic agents. The peptide is obtained by extracting pollen collected from male flower of cedar by immersing it in 0.125M aqueous solution of sodium hydrogen carbonate and purifying the extracted solution by salting out using ammonium sulfate, treatments with anion exchanger and by gel filtration, etc., or by recombinant gene technique.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide and its use, and in particular to a T specific for cedar pollen allergen.
The present invention relates to a peptide that activates cells and an immunotherapeutic agent containing the peptide as an active ingredient.

[0002]

2. Description of the Related Art In the past ten years, the number of people complaining of rhinitis and conjunctivitis due to cedar pollinosis has been increasing in Japan since early spring. Due to the large number of patients and the onset of various events during the early spring, it has been frequently picked up by the media and is now one of the issues that cannot be ignored in public health.

Cedar pollinosis is a type of allergic disease,
It is said that the main cause is the antigenic substance in cedar pollen, that is, the 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 the cedar pollen invades next in this state, the allergen in the pollen and the immunoglobulin E antibody cause an immune reaction, which causes allergic symptoms.

At present, it is known that at least two types of allergens having different antigenicities are present in cedar pollen. One of them is Yasueda's "Journal of Allergies and Clinical Immunology",
Vol. 71, No. 1, pp. 77-86 (1983), this is an allergen, and today it is "Cryj.
It is called "I". The other is Tanyai et al., "F.B.S.Letters", Vol. 239, Vol.
No. 329-332 (1988) and Sakaguchi et al., "Allergy", No. 45, 309-312 (19).
90) and is today referred to as "Cry j II". Cry j I and Cry j II are usually present in cedar pollen at a ratio of about 50: 1 to 5: 1, and most of the sera collected from pollinosis patients have Cry j I and Cry j II.
It is said that it also reacts to. Sawaya et al., "Allergy", Vol. 42, No. 6, 738-747 (199).
3 years), Cryj II was tested by intradermal test and RAS
It is reported that the T-test exhibits the same antigenicity as Cry j I.

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 are administered to humans to desensitize them, so that cedar pollinosis is caused. The prospect of being able to treat and prevent Recently, some desensitizing agents for that purpose have been devised. For example, in JP-A-1-156926 and JP-A-3-93730, the amino acid sequence from the N-terminal is Asp-Asn-Pro-Ile. -Asp
-Ser or Ala-Ile-Asn-Ile-Phe
It has been proposed that a sugar is covalently bound to a cedar pollen allergen represented by -Asn, and the resulting complex is administered to humans as a desensitizing agent. However, a large amount of high-purity allergens is usually required for the diagnosis of allergic diseases and desensitization therapy.The allergens in cedar pollen are scarce and their stability is low, and the diagnosis of cedar pollinosis is not possible. It is expected that there will be a great deal of difficulty if the cedar pollen alone is used as the agent and desensitizing agent.

From the above, in the recent treatment / prevention of allergic diseases, instead of administering the whole allergen to the patient as in the past, the minimum region specifically recognized by T cells in the allergen, That is, immunotherapy, which administers a low-molecular peptide essentially consisting of a T cell epitope, is receiving attention.

[0007] In general, when an allergen is taken up by an antigen-presenting cell such as a macrophage, it is digested there, and the digested fragment is bound to the HLA protein on the surface layer of the immuno-presenting cell to present the antigen. The antigen-presented fragment is HL
Due to the affinity for the A protein and the like, it is limited to a part of a specific region in the allergen, and a region specifically recognized by T cells is usually called a "T cell epitope". For immunotherapy in which a peptide consisting essentially of a T cell epitope is administered, (i) the peptide lacks a B cell epitope, ie, the immunoglobulin E antibody specific for the allergen does not react, and thus the conventional crude or Side effects such as anaphylaxis that frequently occur with purified allergens cannot occur. (Ii) The period from starting from a small amount to reaching an effective dose can be significantly shortened as compared with conventional desensitizing agents. There are advantages such as.

At present, the T cell epitopes of various allergens have been vigorously analyzed. However, in order to analyze T cell epitopes, the entire amino acid sequence of the allergen is usually essential, and at least as far as the cedar pollen allergen is concerned, T
The reality is that the cellular epitopes have not been elucidated substantially.

[0009]

In view of such circumstances, a first object of the present invention is to provide a peptide essentially consisting of a T cell epitope of cedar pollen allergen and a peptide homologous thereto.

A second object of the present invention is to provide an immunotherapeutic agent containing the above peptide as an active ingredient.

[0011]

The present invention provides a method for determining the above-mentioned first object by incorporating ³ H-thymidine without substantially reacting with an immunoglobulin E antibody specific to cedar pollen allergen. When tested, it is resolved by a peptide that significantly activates T cells specific for cedar pollen allergen compared to a negative control.

The present invention solves the above-mentioned second problem by an immunotherapeutic agent containing such a peptide as an active ingredient.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Since the peptide of the present invention does not substantially react with immunoglobulin E antibody specific to cedar pollen allergen, it does not cause anaphylaxis substantially when administered to mammals including humans in general. Activates T cells specific to the cedar pollen allergen.

The immunotherapeutic agent of the present invention comprising such a peptide as an active ingredient, when administered to mammals including humans in general, does not cause anaphylaxis substantially, and is markedly treated / prevented against cedar pollinosis. Be effective.

The present invention will be described below with reference to experimental examples, examples and the like. The present invention is based on the discovery of a peptide essentially consisting of a T cell epitope of cedar pollen allergen.

As a result of many years of research on cedar pollen allergen, the present inventors found last year that one main component of cedar pollen allergen has the amino acid sequence shown in SEQ ID NO: 14 in the sequence listing, Japanese Patent Application 5-3
No. 44,596. On the other hand, International Patent Publication No. 93/01213 discloses that another component of cedar pollen allergen has an amino acid sequence represented by SEQ ID NO: 15 in the sequence listing. At the "6th Annual Meeting of the Japanese Society of Allergology Spring Clinical Conference" held in Kumamoto City, Kumamoto Prefecture from April 14 to 16, 2006,
The same amino acid sequence has been published.

Therefore, in order to elucidate the T cell epitope of the cedar pollen allergen, the present inventor has set these sequence numbers 14
On the basis of the amino acid sequences shown in 15 and 15, peptides of 180 or more different amino acid sequences consisting of consecutive 11, 14, or 17 amino acid residues in the amino acid sequences were synthesized, and the peptides specific to the cedar pollen allergen were synthesized. The reactivity to immunoglobulin E antibody and the activating effect on T cells specific to the cedar pollen allergen were tested. As a result, SEQ ID NOS: 8 to 13 in the sequence listing
The peptide having the amino acid sequence shown in 1) does not substantially react with the immunoglobulin E antibody specific to the cedar pollen allergen, and when tested by the method of determining by incorporation of ³ H-thymidine, it is compared with the negative control. It was revealed that T cells specific to the cedar pollen allergen were significantly activated. This suggests that the peptides having the amino acid sequences shown in SEQ ID NOs: 8 to 13 essentially consist of T cell epitopes of cedar pollen allergen. Further, when the amino acid sequences shown in SEQ ID NOs: 8 to 13 were further analyzed, the amino acid sequences shown in SEQ ID NOs: 1 to 7 in the sequence listing were
It was found that it is an essential sequence for recognizing the peptides having the amino acid sequences shown in FIGS.

In the following Experimental Examples 1 and 2, a series of experiments for clarifying these facts will be described.

[0019]

[Experimental Example 1 Preparation of peptide and cedar pollen allergen]

[0020]

Experimental Example 1-1 Preparation of Peptide As described above, it has been known that cedar pollen has at least two kinds of allergens having different properties and properties.
The mature proteins of these cedar pollen allergens are recombinant DN
It has been revealed by the A technique that it has the amino acid sequence shown in SEQ ID NO: 14 or 15 in the sequence listing,
Actually, from cedar pollen, the 46th to 433rd positions or the 51st to 433rd positions in the amino acid sequence shown in SEQ ID NO: 14
The cedar pollen allergen having the amino acid sequence corresponding to the second (hereinafter referred to as “allergen A”), and the cedar pollen allergen having the amino acid sequences 1 to 353 in the amino acid sequence shown in SEQ ID NO: 15 (hereinafter referred to as “allergen B”). Has been isolated. In addition, allergen A
In the gene encoding, the signal peptide has not been determined yet, so in SEQ ID NO: 14,
Provisionally, the code "1" was assigned to the first amino acid residue on the N-terminal side in the amino acid sequence decoded from the cDNA base sequence.
Is attached.

In this experimental example, SEQ ID NO: 1 in the sequence listing
For the amino acid sequence shown in 4, the 46th to 43rd amino acids
Over the third region, consisting of 11 or 14 amino acid residues, with 10 overlapping amino acid residues, 9
Peptides with 5 different amino acid sequences (Sample A-1
To A-95), the amino acid sequence shown in SEQ ID NO: 15 extends over the region from the 1st to the 353rd position, and the amino acid sequence of 10 amino acid residues is overlapped by 10 amino acid residues. 86 different peptides having different amino acid sequences (Samples B-1 to B-
86) was chemically synthesized and used in Experimental Example 2 described later to search for the peptide of the present invention.

That is, 11 or 1 according to the conventional method
181 kinds of peptides consisting of 4 amino acid residues and having the amino acid sequences shown in Tables 1 to 6 below were synthesized by a solid phase method using a peptide synthesis kit “Multipin” manufactured by Cambridge Research Biochemicals, and after synthesis. Then, a part thereof was taken and analyzed by a peptide sequencer "470A type" manufactured by Perkin-Elmer, and it was confirmed that it had a desired amino acid sequence.

[0023]

[Experimental Example 1-2 Preparation of Japanese cedar pollen allergen] About 16 parts by weight of 0.1 part of 0.125 M sodium hydrogen carbonate aqueous solution (pH 8.
It was immersed in 2) and extracted at 4 ° C. for 1 hour with gentle stirring. The extract was centrifuged, the residue was re-extracted in the same manner as above, the obtained supernatant and the first supernatant were pooled, and to this was added cetablon at 0.1% (w / v), While gently stirring, the mixture was allowed to stand at 4 ° C for 1 hour to precipitate the polysaccharides, and after centrifugation, 80% ammonium sulfate was added to the supernatant.
The mixture was added so as to be saturated, and allowed to stand overnight at 4 ° C. for salting out.

The precipitation portion of the salted out product was collected and
10 against 0 mM Tris-HCl buffer (pH 7.8)
After dialysis for a period of time, filtration, and loading on a DEAE-Sephadex column that had been equilibrated with 50 mM Tris-HCl buffer (pH 7.8) in advance, the same buffer solution was passed through the column to remove protein components. The containing fraction was eluted. After collecting this fraction and adjusting the pH to 5.0 by adding acetic acid, 10
C equilibrated with mM acetate buffer (pH 5.0)
Load M-Sephadex column and load the column to 10 mM.
After washing with acetate buffer (pH 5.0), add 0.3M to the column.
0.1 M phosphate buffer containing sodium chloride (pH 7.
0) was passed through, and a fraction containing a protein component was collected.

Next, this fraction was loaded on a Mono S column which had been equilibrated with 10 mM acetate buffer (pH 5.0) in advance, and the column was loaded with 10 mM acetate buffer (pH 5.
After washing with 0), a 10 mM phosphate buffer solution (pH) was applied to the column under a concentration gradient of sodium chloride increasing from 0 M to 0.5 M.
After passing 7.0), allergen B was eluted at a sodium chloride concentration near 0.1 to 0.3 and allergen A was eluted at a sodium chloride concentration near 0.4M. Fractions containing allergens A or B were separately collected, appropriately concentrated, lyophilized, and used in the following Experimental Example 2. The yield is about 0.01% of allergen A, based on the solid content of the Japanese cedar pollen,
Allergen B was about 0.02%.

[0026]

[Experimental Example 2 Search for peptides containing T cell epitopes of cedar pollen allergen]

[0027]

[Experimental Example 2-1 Activation of T cells specific to Japanese cedar pollen allergen] By Ficoll-Hipack specific gravity centrifugation
A mononuclear cell group containing T cells specific to the cedar pollen allergen was isolated from heparinized peripheral blood of a hay fever patient. RPMI supplementing this mononuclear cell population with 5% (v / v) AB serum
Float in 1640 medium (pH 7.0), dispense 5 × 10 ⁵ cells / well on a 96-well microplate,
The peptides or cedar pollen allergens prepared in Experimental Examples 1-1 and 1-2 were added at 1 μg / well, and the volume was adjusted to 200 μl / well with the same fresh medium, and then 3% in a 5% CO ₂ incubator.
Incubated at 7 ° C for 2 days. After that, ³ H-thymidine was added at 1.0 μCi / well and incubated under the same conditions for 16 hours, and then the amount of ³ H-thymidine uptake in the mononuclear cell group was measured by a known method using a scintillation counter. did. At the same time, a system containing neither peptide nor cedar pollen allergen was set up and treated in the same manner as above to serve as a negative control.

The presence or absence of the activating effect on the T cells specific to the cedar pollen allergen is determined based on the ³ H-thymidine uptake amount (cpm) in the mononuclear cell group containing the T cells, and the uptake amount is a negative control. The system that reached about 2 times or more of the above was defined as "positive", and the system that did not reach it was defined as "negative". The results are shown in Tables 1 to 6.

[0029]

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

The results in Tables 1 to 6 show that the peptides and cedar pollen allergens tested behaved distinctly differently to T cells specific for the cedar pollen allergen. That is, samples A-19, A-20, A-
23, A-48, A-49, A-89, B-39, B-
In the system to which 80 or Japanese cedar pollen allergen A or B was added, a significantly significant ³ H-thymidine uptake promotion was observed as compared with the negative control, whereas in the system to which the other sample was added. , No significant promotion of uptake was observed. This means that samples A-19, A-20, A
-23, A-48, A-49, A-89, B-39, B
It is meant that only -80 and cedar pollen allergens A and B significantly activated the cedar pollen allergen-specific T cells in the mononuclear cell population.

Furthermore, samples A-19, A-20, A-48 and A-49 and T having overlapping amino acid sequences with each other were used.
Peptides of the amino acid sequences shown in SEQ ID NOs: 8 to 11 separately consisting of 17 amino acid residues for samples A-23 and A-89, which had a slightly lower cell activating effect than other positive samples Was chemically synthesized.

That is, peptides (samples C-1 to C-4) having the amino acid sequences shown in SEQ ID NOs: 8 to 11 in the sequence listing were prepared according to a conventional method using a peptide synthesizer "Excel" manufactured by Milligen / Bioresearch. Separately synthesized, Bio-Rad chromatography column "Hi-P"
ore RP-318 type ”and purified to 95% purity by reverse phase high performance liquid chromatography. After purification, a part of Samples C-1 to C-4 was taken and analyzed by a peptide sequencer "470A type" manufactured by Perkin-Elmer, and all four kinds of peptides involved in synthesis had the desired amino acid sequence. It was

When these samples C-1 to C-4 were tested in the same manner as described above, they were all positive, and it was found that T cells specific to the cedar pollen allergen were significantly activated.

[0034]

[Experimental example 2-2 Reactivity to immunoglobulin E antibody specific to cedar pollen allergen] Sample B which was found to significantly activate T cells specific to cedar pollen allergen in Experimental example 2-1 -39, B-80, C
-1 to C-4 and allergens A and B, Taniyai et al., "Molecular Immunology", Vol. 30, Vol.
No. pp. 183-189 (1993), the EIA method was applied to examine the reactivity with the immunoglobulin E antibody specific to the cedar pollen allergen collected from the blood of the cedar pollinosis patients.

That is, 1 g of a cross-linking agent manufactured by Pierce "(sulfosuccinimidyl) suberate (BS ₃ )" was added to 1 part of distilled water.
Dissolve in 0 ml and dispense 50 μl / well into Nunch's "Kovalink-type" 96-well microplate, 37
Incubated at ℃ for 3 hours. Sample B-3 prepared in Experimental Examples 1 and 2 by washing the microplate with distilled water
9, B-80, C-1 to C-4 or allergen A or B was dissolved in PBS at 20 μg / ml or 5 μg / ml, dispensed at 50 μl / well into a microplate, and further incubated at 37 ° C. for 3 days. Incubated for a period of time to allow covalent attachment to the microplate. And PBS containing 1% (w / v) bovine serum albumin in the microplate
50 μl / well was added and the mixture was allowed to stand overnight at 4 ° C. to block unreacted active groups, and then washed with PBS containing 0.1% (w / v) bovine serum albumin to obtain the same amount of bovine serum albumin. Serum of a Japanese cedar pollinosis patient diluted 5-fold with fresh PBS containing 50 μl / well was added and reacted at 37 ° C. for 1 hour.

Next, the microplate is set to 0.1% (w /
v) Washing with PBS containing bovine serum albumin, adding 50 μl / well of biotin-labeled anti-human ε-chain antibody manufactured by Kirkegaard and Perry diluted to 1 μg / ml with fresh PBS containing the same amount of bovine serum albumin, 37 After incubating at 0 ° C for 1 hour, it was washed again with PBS containing 0.1% (w / v) bovine serum albumin, and then diluted 5,000 times with fresh PBS containing the same amount of bovine serum albumin. 50 labeled avidin
μl / well was added, and the mixture was further incubated at 37 ° C. for 1 hour. And, the microplate is 0.1% (w /
v) After washing with PBS containing bovine serum albumin, 0.1M citric acid-phosphate buffer (pH 5.0) containing 0.03% (v / v) hydrogen peroxide and 0.5 mg / ml orthophenylenediamine. Was added at 100 μl / well and left to stand at room temperature for 5 minutes for enzyme reaction. 100 μl of 2N sulfuric acid
/ Well was added to each well to stop the reaction, and then the absorbance at a wavelength of 492 nm was measured by a known method using a spectrophotometer.

At the same time, a system was used in which the serum of a healthy individual was used in place of the serum of a Japanese cedar pollen patient, and the same treatment was performed as a negative control. The results are shown in Tables 1 to 6.

As is clear from the results shown in Tables 1 to 6, the allergens A and B strongly reacted with the immunoglobulin E antibody specific to the cedar pollen allergen derived from the cedar pollinosis patient, while the sample B- 39, B-80 and C-
1 to C-4 did not substantially react. This is
This means that these samples lack the B cell epitope of cedar pollen allergen contained in allergens A and B. Comprehensively judging from these results and the results of Experimental Example 2-1, the above-mentioned sample, that is, the peptides having the amino acid sequences shown in SEQ ID NOs: 8 to 13 in the sequence listing are essentially derived from the T cell epitope of cedar pollen allergen. It is determined that

[0039]

[Experimental example 3 Search for amino acid sequence essential for T cell to recognize T cell epitope] In this experimental example, the 6 types of T cell epitopes clarified in Experimental example 2 were further analyzed, and T cell identified them. The amino acid sequence essential for recognition was searched.

That is, by the method of Experimental Example 1-1, the amino acid sequences shown in SEQ ID NOs: 8 to 13 in the sequence listing and samples B-38, B-81 and B With respect to the 82 amino acid sequence, various peptides consisting of 14 or 17 amino acids in which one or more of the amino acids at one or both ends thereof were substituted with alanine were chemically synthesized. Then, the activation of T cells specific to the cedar pollen allergen and the reactivity to the immunoglobulin E antibody specific to the cedar pollen allergen were examined for these peptides by the method of Experimental Example 2.

As a result, as shown in Table 7, sample D containing the amino acid sequences shown in SEQ ID NOS: 1 to 7 of the sequence listing.
The peptides -1 to D-7 behave substantially the same as the peptides having the amino acid sequences shown in SEQ ID NOs: 8 to 13 against T cells specific to cedar pollen allergen and immunoglobulin E antibodies specific to cedar pollen allergen. Was found to show. This strongly suggests that the amino acid sequences shown in SEQ ID NOs: 1 to 7 in the sequence listing are indispensable sequences for T cells to recognize the peptides having the amino acid sequences shown in SEQ ID NOs: 8 to 13.

[0042]

[Table 7]

As described above, the present invention does not substantially react with the immunoglobulin E antibody specific to the cedar pollen allergen, and when tested by the method of determining by incorporation of ³ H-thymidine, it is compared with the negative control. The present invention also relates to a peptide that significantly activates T cells specific to the cedar pollen allergen. As long as the peptide has such a property, the present invention includes all peptides regardless of the structure, source / origin, and preparation method.

The peptides of this invention are usually 5 to 50
Individual, preferably 10 to 20 amino acids are peptide-bonded. Examples of the individual peptides include those having the amino acid sequences shown in SEQ ID NOS: 8 to 13 in the sequence listing and those having the amino acid sequences homologous to those amino sequences. A peptide having a homologous amino acid sequence is one in which one or more amino acids in the amino acid sequences shown in SEQ ID NOs: 8 to 13 in the sequence listing are replaced with other amino acids without substantially changing the above-mentioned immunological action. Alternatively, it can be obtained by linking one or more appropriate amino acids to one or both ends of the amino acid sequence.

Specifically, for example, SEQ ID NO: 8 in the sequence listing
In the amino acid sequences shown in 13 to 13, only the amino acid sequences essential for T cells to recognize them are unchanged, and other amino acids substantially change the immunological action of cedar pollen as a T cell epitope. Substitute with another amino acid within the range not present. Alternatively, if necessary, for example, one or two or more appropriate amino acids such as alanine are bound to one or both ends of the essential amino acid sequence, and the resulting peptide has a length that can be recognized by T cells as a whole, That is, usually, the number of amino acid residues is 1
The number should be 0 to 20. Examples of such amino acid sequences include the amino acid sequences shown in SEQ ID NOS: 1 to 7 in the sequence listing, and examples of such homologues include, for example, the amino acid sequences shown in SEQ ID NOS: 16 to 24 in the sequence listing. Mention may be made of peptides.

The peptides of this invention can be readily prepared by peptide synthesis methods commonly used in the art known as "solid phase methods" or "liquid phase methods". Since the present invention does not relate to peptide synthesis itself, detailed description thereof will be omitted. For example, “Shinsei Chemistry Experimental Course”, Vol.
I ”, pp. 3 to 44, 1992, published by Tokyo Kagaku Dojin, etc., for details of peptide synthesis. However,
The peptide of the present invention is not limited to those prepared by chemical synthesis, for example, those collected from cedar pollen or male flowers, or those obtained by degrading cedar pollen allergen prepared by recombinant DNA technology as appropriate May be Alternatively, for example, a DNA encoding a peptide having an amino acid sequence shown in SEQ ID NOs: 8 to 13 in the sequence listing or an amino acid sequence homologous thereto is prepared and inserted into an autonomously replicable vector to give a recombinant DNA. Alternatively, this may be introduced into an appropriate host such as Escherichia coli, Bacillus subtilis, actinomycete, or yeast to form a transformant, and the peptide of the present invention may be collected from the culture. DNAs encoding the peptides having the amino acid sequences shown in SEQ ID NOs: 8 to 13 in the sequence listing are, for example, the nucleotide sequences of cDNAs described in Japanese Patent Application No. 5-344596 and International Patent Publication No. 93/01213. Can be prepared based on
Furthermore, 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, the peptide is acetylated, amidated and / or crosslinked by a polyfunctional reagent. It may be in the form of a derivative or polymer obtained by polymerization.

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

As described above, the peptide of the present invention does not substantially react with the immunoglobulin E antibody specific to the cedar pollen allergen, and significantly activates the T cells specific to the cedar pollen allergen. , Has a wide range of uses as an immunotherapeutic agent for treating and preventing cedar pollinosis. An immunotherapeutic agent comprising the peptide of the present invention as an active ingredient treats cedar pollinosis without substantially causing side effects such as anaphylaxis when administered to mammals including humans suffering from cedar pollinosis in general. You can On the other hand, when the immunotherapeutic agent of the present invention is administered to healthy individuals or individuals with potential cedar pollinosis before cedar pollen begins to scatter, while exerting a significant preventive effect against cedar pollinosis, It is very effective in relieving allergic symptoms at the onset.

The immunotherapeutic agent of the present invention will be described in more detail. Usually, the immunotherapeutic agent of the present invention contains 0.01 to 10 of one or more peptides according to the present invention.
0% (w / w), preferably 0.05 to 50% (w
/ W), and more preferably 0.5 to 5.0% (w /
w) comprises. The immunotherapeutic agent of the present invention is not only in the form of the peptide alone, but is physiologically acceptable other than that, for example, a carrier such as serum albumin, gelatin, mannitol, maltose, trehalose, an excipient, an immunostimulant, It also includes a stabilizer, and optionally, a composition as a composition with one or more other drugs containing an anti-inflammatory agent such as a steroid hormone or sodium cryomoglycate or an antihistamine. Furthermore, the immunotherapeutic agent of the present invention also includes a drug in a dosage unit form, which means that the polypeptide of the present invention is, for example, a daily dose or an integral multiple (up to 4 times). ) Or a sub-multiple thereof (up to 1/40), and means a physically separate, unitary dosage form suitable for administration. Examples of the drug in such dosage unit form include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, ointments, plasters, and poultices.
Examples include suppositories, eye drops, nasal drops, sprays, injections and the like.

The method of using the immunotherapeutic agent of the present invention will be described. The immunotherapeutic agent of the present invention is generally used for the treatment / prevention of cedar pollinosis in mammals including humans in general transdermal, oral, nasal, instillation or Administered by injection. The dose in humans varies depending on the purpose and symptoms of administration, but is usually 0.01 to 1.0 g, preferably 0. 0 g per day for an adult while observing the subject's symptoms and the course after administration. The dose is usually from once to once a month to once a month for about 1 to 6 months, usually in the range of 01 to 0.1 g, and is usually repeatedly administered while increasing the dose.

The preparation and use of the peptide according to the present invention will be described below with reference to a few examples.

[0052]

Example A-1 Preparation of Peptide Using a peptide synthesizer "Excel" manufactured by Milligen / Bioresearch, peptides having amino acid sequences shown in SEQ ID NOs: 8 to 11 in the sequence listing were separately synthesized according to a conventional method, Bio-Rad chromatography column "Hi-Pore RP-"
After purification by reverse-phase high performance liquid chromatography using "Model 318" to a purity of 95%, it was freeze-dried to give a solid. When a part of the solid substance was taken and analyzed by a peptide sequencer "470A type" manufactured by Perkin-Elmer, all four kinds of peptides related to the synthesis had the desired amino acid sequences.

[0053]

Example A-2 Preparation of Peptide A peptide synthesis kit "Multipin" manufactured by Cambridge Research Biochemicals was used, and peptides having amino acid sequences shown in SEQ ID NOs: 12 and 13 in the sequence listing were separately prepared according to a conventional method. After being synthesized and purified in the same manner as in Example A-1 to a purity of 95%, respectively, it was freeze-dried to give a solid. When a portion of the solid matter was taken and analyzed in the same manner as in Example A-1, all had the desired amino acid sequence.

[0054]

Example A-3 Preparation of Peptide In the same manner as in Example A-1, a peptide having the amino acid sequence shown in SEQ ID NO: 16 in the sequence listing was chemically synthesized and purified to a purity of 95%.
After purification, a part of the peptide was taken and analyzed in the same manner as in Example A-1, and it had the desired amino acid sequence.

[0055]

Example A-4 Preparation of Peptide In the same manner as in Example A-2, a peptide having the amino acid sequence shown in SEQ ID NO: 17 in the sequence listing was chemically synthesized and purified to a purity of 95%.
After purification, a part of the peptide was taken and analyzed in the same manner as in Example A-1, and it had the desired amino acid sequence.

[0056]

Example A-5 Preparation of Peptides Examples A-1 to A
-2, peptides of amino acid sequences represented by SEQ ID NOs: 18 to 24 in the sequence listing corresponding to Samples D-1 to D-7 of Experimental Example 3 were chemically synthesized, and each had a purity of 95.
%, And then freeze-dried to give a solid. When a part of the solid substance was taken and analyzed by a peptide sequencer "470 type" manufactured by Perkin-Elmer, all seven kinds of peptides related to the synthesis had the desired amino acid sequence.

[0057]

Example B-1 Liquid formulation Any of the six peptides obtained by the method of Examples A-1 and A-2 was used at a final concentration of 0.
It was dissolved in distilled water containing 1% (w / v) purified gelatin as a stabilizer so as to be 1 g / ml, and sterile filtered by a conventional method to obtain 6 types of liquid agents.

Since the sensitivity to the peptide of the present invention generally varies from individual to individual, this product is used by appropriately mixing 6 kinds of liquid agents so that the composition is most suitable for each individual. This highly stable product is useful as a liquid for eye drops, nasal drops, and oral sprays for treating and preventing cedar pollinosis.

[0059]

Example B-2 Injection: 1% (w / v) as a stabilizer
Six kinds of peptides obtained by the method of Examples A-1 and A-2 were dissolved in physiological saline containing human serum albumin to a final concentration of 0.01, 0.1 or 1 mg / ml, respectively, and sterilized. After filtration, 2 ml each was dispensed into a sterile vial, lyophilized, and sealed.

Prior to administration, this product is used by first adding 1 ml of distilled water for injection or the like into a vial and then dissolving the contents uniformly. This product, which is excellent in stability and contains 6 kinds of polypeptides according to the present invention as an active ingredient, is useful as a dry injection for treating and preventing cedar pollinosis.

[0061]

Example B-3 Tablets 2 g of purified pullulan having an average molecular weight of about 20,000 daltons were uniformly dissolved in 100 ml of distilled water, and 2 ml of 1.7% (w / v) acetone solution of cyanuric chloride was added to the solution. % (W / v) sodium carbonate aqueous solution, while maintaining the pH at around 7, the mixture was reacted at 5 ° C. for 2 hours with stirring. Then, while keeping the pH of the reaction product around 7, in the same manner, it was dialyzed against cold water at 4 ° C. overnight to obtain 20 ml of an aqueous solution containing activated pullulan.

In this aqueous solution, the peptides having the amino acid sequences shown in SEQ ID NOS: 8, 10 and 11 in the sequence listing obtained by the method of Example A-1 and SEQ ID NO: 13 in the sequence listing obtained by the method of Example A-2 0.2 mg each of the peptide having the amino acid sequence shown in, the peptide obtained by the method of Example A-3, and the peptide obtained by the method of Example A-4.
In addition, while maintaining the pH of the solution at around 7, the reaction was carried out at 37 ° C. for 12 hours while gently stirring. After the reaction, 4g of glycine was added to the reaction product, and gently stirred at 37 ° C.
Incubated for 5 hours to block unreacted active groups.

The reaction product was concentrated and Sephadex G which had been equilibrated with 0.1 M phosphate buffer (pH 7.0) in advance.
The column was loaded on a -50 column, and the same buffer solution was passed through the column to collect a fraction containing the complex of the peptide of the present invention and pullulan. The yield is per solid content of the starting peptide,
It was about 30%.

According to a conventional method, this fraction was sterilized by filtration, concentrated, freeze-dried and pulverized, and then mannitol was uniformly mixed and the mixture was tabletted to give 2 parts of the complex per tablet (200 mg) of the product. Tablets containing 10 or 50 mg were obtained.

The product, which is excellent in ingestibility and stability, is useful as a sublingual agent for treating and preventing cedar pollinosis.

[0066]

Example B-4 Syrup Agent 1 g of purified lipopolysaccharide derived from Escherichia coli was dissolved in 100 ml of 10 mM calcium phosphate aqueous solution, 6 ml of 100 mM sodium periodate was added to the solution, and the mixture was reacted at room temperature for 20 minutes to activate lipopolysaccharide. did. The reaction product was added to a 1 M glycine-hydrochloric acid buffer solution (pH: 4 ° C)
After dialysis against 4.4) overnight to remove unreacted periodic acid, pH is adjusted with 0.1M sodium hydrogen carbonate buffer.
While adjusting to around 9.5, separately, Examples A-1 and A
6 kinds of peptides obtained by the method of No. 2 were dissolved in 100 ml of 0.1 M phosphate buffer (pH 7.0) (10 mg each), added to the reaction product containing activated lipopolysaccharide, and allowed to stand at room temperature for 12 hours. And reacted.

Thereafter, the newly obtained reaction product was used in Example B.
-3, the obtained fraction containing the complex of the peptide of the present invention and lipopolysaccharide is concentrated, lyophilized,
Crushed into a solid. The yield was about 30% based on the starting peptide solids.

The solid matter and sucrose were added to distilled water containing 1% (w / v) of purified gelatin as a stabilizer so that the final concentration of each was 0.1 or 1 mg / ml or 50% (w / w). After dissolution, the solution was sterilized by a conventional method to obtain a syrup. Each 2 ml of this syrup was dispensed into a sterilized vial bottle, which was tightly closed to obtain a product.

The product having excellent stability and containing the complex of the peptide of the present invention and the lipopolysaccharide as an active ingredient is useful as a syrup for treating and preventing cedar pollinosis.

[0070]

Example B-5 Liquid formulation Any of the seven peptides obtained by the method of Example A-5 was added to a final concentration of 0.1 g / ml.
Was dissolved in distilled water containing 1% (w / v) purified gelatin as a stabilizer and sterilized by a conventional method to obtain 7 types of liquid preparations.

Since the sensitivity to the peptide of the present invention generally varies from individual to individual, this product is used by appropriately mixing 7 kinds of liquid agents so that the composition is most suitable for each individual. This highly stable product is useful as a liquid for eye drops, nasal drops, and oral sprays for treating and preventing cedar pollinosis.

[0072]

Example B-6 Injection: 1% (w / v) as a stabilizer
Seven kinds of peptides obtained by the method of Example A-5 were dissolved in physiological saline containing human serum albumin to a final concentration of 0.01, 0.1 or 1 mg / ml, respectively, and sterilized and filtered. 2 ml each was dispensed into a sterile vial, lyophilized and sealed.

Prior to administration, this product is used by first adding 1 ml of distilled water for injection or the like into a vial and then dissolving the contents uniformly. This product, which has excellent stability and contains 7 kinds of polypeptides according to the present invention as an active ingredient, is useful as a dry injection for treating and preventing cedar pollinosis.

[0074]

[Example B-7 Syrup] 1% of purified gelatin (w
/ V) distilled water containing 0.1 mg / ml of each of the seven peptides obtained by the method of Example A-5 and 50 sucrose.
The solution was dissolved so that it had a% (w / v) content, and the solution was sterilized by a conventional method to obtain a syrup. 2 this syrup
It was dispensed into a sterile vial bottle by ml and tightly stoppered to obtain a product.

The product having excellent stability and containing the peptide of the present invention as an active ingredient is useful as a syrup for treating and preventing cedar pollinosis.

[0076]

[Experimental Example 4 Acute toxicity test] The immunotherapeutic agents obtained by the methods of Examples B-1 to B-7 were orally or intraperitoneally administered to mice on the 20th day of life by a conventional method. As a result, 200 mg of these immunotherapeutic agents were administered by any route of administration.
It was found that the LD50 was not less than / kg. This indicates that the peptide of the present invention can be safely incorporated into an immunotherapeutic agent administered to mammals including humans.

[0077]

INDUSTRIAL APPLICABILITY As described above, the present invention is based on the discovery of a peptide essentially consisting of a T cell epitope of cedar pollen allergen. The peptide of this invention is an immunoglobulin E specific to cedar pollen allergen.
Since it does not substantially react with the antibody, when administered to mammals including humans, it activates T cells specific to the cedar pollen allergen without substantially causing anaphylaxis. Therefore, the immunotherapeutic agent of the present invention containing such a peptide as an active ingredient, when administered to mammals including humans, exerts a remarkable therapeutic / preventive effect against cedar pollinosis in a short period with few side effects. Moreover, since the peptide of the present invention can be easily produced in a desired amount and the quality control is easy, it can be used very safely for the treatment / prevention of cedar pollinosis.

It can be said that the present invention, which exerts such a remarkable effect, is a significantly significant invention that contributes to the field.

[0079]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Lys Val Asp Gly Ile Ile Ala Ala Tyr Gln Asn 1 5 10

SEQ ID NO: 2 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser 1 5 10

SEQ ID NO: 3 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asn Arg Ile Trp Leu Gln Phe Ala Lys Leu 1 5 10

SEQ ID NO: 4 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Phe Ala Ser Lys Asn Phe His Leu Gln Lys 1 5 10

SEQ ID NO: 5 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Leu Lys Leu Thr Ser Gly Lys Ile Ala Ser 1 5 10

SEQ ID NO: 6 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Leu Thr Leu Arg Thr Ala Thr Asn 1 5

SEQ ID NO: 7 Sequence Length: 5 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Ala Phe Asn Val Glu 15

SEQ ID NO: 8 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Val Asp Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Asn 1 5 10 15

SEQ ID NO: 9 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asn Arg Ile Trp Leu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly 1 5 10 15

SEQ ID NO: 10 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Ile Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn Thr Ile Gly Thr 1 5 10 15

SEQ ID NO: 11 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Ile Ser Leu Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn Asp 1 5 10 15

SEQ ID NO: 12 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Thr Leu Arg Thr Ala Thr Asn Ile Trp Ile Asp His Asn 1 5 10

SEQ ID NO: 13 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu 1 5 10

SEQ ID NO: 14 Sequence length: 514 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Cryptomeria japonica Individual / isolated organism name: Sugi sequence Met Ala Met Lys Phe Ile Ala Pro Met Ala Phe Val Ala Met Gln Leu Ile 1 5 10 15 Ile Met Ala Ala Ala Glu Asp Gln Ser Ala Gln Ile Met Leu Asp Ser Asp 20 25 30 Ile Glu Gln Tyr Leu Arg Ser Asn Arg Ser Leu Arg Lys Val Glu His Ser 35 40 45 50 Arg His Asp Ala Ile Asn Ile Phe Asn Val Glu Lys Tyr Gly Ala Val Gly 55 60 65 Asp Gly Lys His Asp Cys Thr Glu Ala Phe Ser Thr Ala Trp Gln Ala Ala 70 75 80 85 Cys Lys Lys Pros Ser Ala Met Leu Leu Val Pro Gly Asn Lys Lys Phe Val 90 95 100 Val Asn Asn Leu Phe Phe Asn Gly Pro Cys Gln Pro His Phe Thr Phe Lys 105 110 115 Val Asp Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Asn Asn 120 125 130 135 Arg Ile Trp Leu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Lys 140 145 150 Gly Val Ile Asp Gly Gln Gly Lys Gln Trp Trp Ala Gly Gln Cys Lys Trp 155 160 165 170 Val Asn Gly Arg Glu Ile Cys Asn Asp Arg Asp Arg Pro Thr Ala Ile Lys 175 180 185 Phe Asp Phe Ser Thr Gly Leu Ile Ile Gln Gly Leu Lys Leu Met Asn Ser 190 195 200 Pro Glu Phe His Leu Val Phe Gly Asn Cys Glu Gly Val Lys Ile Ile Gly 205 210 215 220 Ile Ser Ile Thr Ala Pro Arg Asp Ser Pro Asn Thr Asp Gly Ile Asp Ile 225 230 235 Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn Thr Ile Gly Thr Gly Asp 240 245 250 255 Asp Cys Val Ala Ile Gly Thr Gly Ser Ser Asn Ile Val Ile Glu Asp Leu 260 265 270 Ile Cys Gly Pro Gly His Gly Ile Ser Ile Gly Ser Leu Gly Arg Glu Asn 275 280 285 Ser Arg Ala Glu Val Ser Tyr Val His Val Asn Gly Ala Lys Phe Ile Asp 290 295 300 305 Thr Gln Asn Gly Leu Arg Ile Lys Thr Trp Gln Gly Gly Ser Gly Met Ala 310 315 320 Ser His Ile Ile Tyr Glu Asn Val Glu Met Ile Asn Ser Glu Asn Pro Ile 325 330 335 340 Leu Ile Asn Gln Phe Tyr Cys Thr Ser Ala Ser Ala Cys Gln Asn Gln Arg 345 350 355 Ser Ala Val Gln Ile Gln Asp Val Thr Tyr Lys Asn Ile Arg Gly Thr Ser 360 365 370 Ala Thr Ala Ala Ala Ile Gln Leu Lys Cys Ser Asp Ser Met Pro Cys Lys 375 380 385 390 Asp Ile Lys Leu Ser Asp Ile Ser Leu Lys Leu Thr Ser Gly Lys Ile Ala 395 400 405 Ser Cys Leu Asn Asp Asn Ala Asn Gly Tyr Phe Ser Gly His Val Ile Pro 410 415 420 425 Ala Cys Lys Asn Leu Ser Pro Ser Ala Lys Arg Lys Glu Ser Lys Ser His 430 435 440 Lys His Pro Lys Thr Val Met Val Lys Asn Met Gly Ala Tyr Asp Lys Gly 445 450 455 Asn Arg Thr Arg Ile Leu Leu Gly Ser Arg Pro Pro Asn Cys Thr Asn Lys 460 465 470 475 Cys His Gly Cys Ser Pro Cys Lys Ala Lys Leu Val Ile Val His Arg Ile 480 485 490 Met Pro Gln Glu Tyr Tyr Pro Gln Arg Trp Met Cys Ser Arg His Ala Lys 495 500 505 510 Ile Tyr His Pro

SEQ ID NO: 15 Sequence length: 353 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Cryptomeria japonica Individual / isolated organism name: Sugi sequence Asp Asn Pro Ile Asp Ser Cys Trp Arg Gly Asp Ser Asn Trp Ala Gln Asn 1 5 10 15 Arg Met Lys Leu Ala Asp Cys Ala Val Gly Phe Gly Ser Ser Thr Met Gly 20 25 30 Gly Lys Gly Gly Asp Leu Tyr Thr Val Thr Asn Ser Asp Asp Asp Pro Val 35 40 45 50 Asn Pro Ala Pro Gly Thr Leu Arg Tyr Gly Ala Thr Arg Asp Arg Pro Leu 55 60 65 Trp Ile Ile Phe Ser Gly Asn Met Asn Ile Lys Leu Lys Met Pro Met Tyr 70 75 80 85 Ile Ala Gly Tyr Lys Thr Phe Asp Gly Arg Gly Ala Gln Val Tyr Ile Gly 90 95 100 Asn Gly Gly Pro Cys Val Phe Ile Lys Arg Val Ser Asn Val Ile Ile His 105 110 115 Gly Leu Tyr Leu Tyr Gly Cys Ser Thr Ser Val Leu Gly Asn Val Leu Ile 120 125 130 135 Asn Glu Ser Phe Gly Val Glu Pro Val His Pro Gln Asp Gly Asp Ala Leu 140 145 150 Thr Leu Arg Thr Ala Thr Asn Ile Trp Ile Asp His Asn Ser Phe Ser Asn 155 160 165 170 Ser Ser Asp Gly Leu Val Asp Val Thr Leu Thr Ser Thr Gly Val Thr Ile 175 180 185 Ser Asn Asn Leu Phe Phe Asn His His Lys Val Met Leu Leu Gly His Asp 190 195 200 Asp Ala Tyr Ser Asp Asp Lys Ser Met Lys Val Thr Val Ala Phe Asn Gln 205 210 215 220 Phe Gly Pro Asn Cys Gly Gln Arg Met Pro Arg Ala Arg Tyr Gly Leu Val 225 230 235 His Val Ala Asn Asn Asn Tyr Asp Pro Trp Thr Ile Tyr Ala Ile Gly Gly 240 245 250 255 Ser Ser Asn Pro Thr Ile Leu Ser Glu Gly Asn Ser Phe Thr Ala Pro Asn 260 265 270 Glu Ser Tyr Lys Lys Gln Val Thr Ile Arg Ile Gly Cys Lys Thr Ser Ser 275 280 285 Ser Cys Ser Asn Trp Val Trp Gln Ser Thr Gln Asp Val Phe Tyr Asn Gly 290 295 300 305 Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys 310 315 320 Lys Glu Ala Phe Asn Val Glu Asn Gly Asn Ala Thr Pro Gln Leu Thr Lys 325 330 335 340 Asn Ala Gly Val Leu Thr Cys Ser Leu Ser Lys Arg Cys 345 350

SEQ ID NO: 16 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asn Arg Ile Trp Leu Gln Phe Ala Lys Leu Gln Gly Phe Thr Leu Met Gly 1 5 10 15

SEQ ID NO: 17 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Leu Ala Leu Arg Thr Ala Thr Asn Ile Trp Ile Asp His Asn 1 5 10

SEQ ID NO: 18 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Val Asp Gly Ile Ile Ala Ala Tyr Gln Asn Ala Ala Ala Ala Ala Ala 1 5 10 15

SEQ ID NO: 19 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Ala Ala Ala Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Ala Ala Ala 1 5 10 15

SEQ ID NO: 20 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asn Arg Ile Trp Leu Gln Phe Ala Lys Leu Ala Ala Ala Ala Ala Ala Ala 1 5 10 15

SEQ ID NO: 21 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Ala Phe Ala Ser Lys Asn Phe His Leu Gln Lys Ala Ala Ala Ala Ala 1 5 10 15

SEQ ID NO: 22 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Ala Ser Leu Lys Leu Thr Ser Gly Lys Ile Ala Ser Ala Ala Ala Ala 1 5 10 15

SEQ ID NO: 23 Sequence Length: 14 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Leu Thr Leu Arg Thr Ala Thr Asn Ala Ala Ala Ala Ala Ala 1 5 10

SEQ ID NO: 24 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Phe Asn Val Glu 1 5 10

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 39/36 ABF 47/42 JB C07K 7/08 ZNA 8318-4H 14/415 8318-4H

Claims (13)

[Claims] 1. When tested by a method that does not substantially react with an immunoglobulin E antibody specific to a cedar pollen allergen and is determined by incorporation of ³ H-thymidine, it is specific to a cedar pollen allergen as compared with a negative control. A peptide that significantly activates T cells. 2. The peptide according to claim 1, which comprises any of the amino acid sequences shown in SEQ ID NOs: 1 to 7 in the sequence listing. 3. The peptide according to claim 1, which comprises any of the amino acid sequences shown in SEQ ID NOs: 8 to 13 in the sequence listing or an amino acid sequence homologous to the amino acid sequence. 4. The amino acid sequence according to SEQ ID NO: 1 in the sequence listing of the peptide according to claim 1. 5. The amino acid sequence according to SEQ ID NO: 2 in the sequence listing of the peptide according to claim 1. 6. The amino acid sequence of SEQ ID NO: 3 in the sequence listing of the peptide of claim 1. 7. The amino acid sequence of SEQ ID NO: 4 in the sequence listing of the peptide of claim 1. 8. The amino acid sequence of SEQ ID NO: 5 in the sequence listing of the peptide of claim 1. 9. The amino acid sequence according to SEQ ID NO: 6 in the sequence listing of the peptide according to claim 1. 10. The amino acid sequence of SEQ ID NO: 7 in the sequence listing of the peptide of claim 1. 11. An immunotherapeutic agent comprising the peptide according to claim 1 as an active ingredient. 12. The immunotherapeutic agent according to claim 11, which comprises 0.01 to 100% (w / w) of the peptide according to any one of claims 1 to 3 as an active ingredient. 13. The immunotherapeutic agent according to claim 11 or 12, which comprises serum albumin, gelatin, mannitol, maltose and / or trehalose as a stabilizer or excipient.