JP4171794B2 - Peptides and their uses - Google Patents

Description

  The present invention relates to a peptide that activates a T cell that specifically reacts with a cedar pollen allergen, and an immunotherapeutic agent comprising the peptide as an active ingredient.

  In recent decades, in Japan, the number of people complaining of rhinitis and conjunctivitis due to cedar pollinosis continues to increase in early spring. Cedar pollinosis is a type of arlegiosis, and the main cause is said to be an antigenic substance in cedar pollen, that is, cedar pollinosis allergen. When cedar pollen scattered in the atmosphere enters the human body, immunoglobulin E antibodies against cedar pollen allergen are produced. Next, when cedar pollen invades in this state, the allergen in the pollen and the immunoglobulin E antibody cause an immune reaction, and allergic symptoms are exhibited.

  It has been known to date that at least two types of allergens having different antigenicity are present in cedar pollen. One of them is the allergen reported by Yasuda et al. In “Journal of Allergy and Clinical Immunology”, Vol. 71, No. 1, pp. 77-86 (1983). This is called “Cryj1” (Non-Patent Document 1). Cryj1 has its full-length amino acid sequence determined and has been filed internationally (Patent Document 1: WO 93/01213).

  The other is Taniyai et al. “EF SB Letters”, Vol. 239, No. 2, pp. 329-332 (1988) and Sakaguchi et al., “Allergy”, No. 45, No. 309- It is an allergen reported on page 312 (1990), and today it is called “Cry j 2” (Non-patent Documents 2 and 3). Cry j 2 has been determined for its full-length amino acid sequence and has been internationally filed (Patent Document 2: WO 94/11512). Komiyama et al. Also separately determined the full-length amino acid sequence of Cryj2, but Biochem. Biophys. Res, Comm., Vol. 201, No. 2, 1021-1028, (1994) (non-patent document 4), WO The amino acid sequence described in 94/11512 differs in 4 amino acid residues.

  In cedar pollen, Cryj1 and Cryj2 are usually present in a ratio of about 50: 1 to 5: 1, and it is said that most of the serum collected from hay fever responds to both Cryj1 and Cryj2. Sawatani et al., Allergy, Vol. 42, No. 6, pp. 738-747 (1993), reported that Cryj2 exhibits the same level of antigenicity as Cryj1 in intradermal reaction tests and RAST tests. (Non-Patent Document 5).

  In this way, several cedar pollen allergens have already been isolated and their properties and properties have been elucidated to some extent. The prospects that can be prevented have come. Recently, several desensitizers have been devised for this purpose. For example, Patent Document 3: JP-A-1-156926 and Patent Document 4: JP-A-3-93730 disclose amino acid sequences from the N-terminus. Is covalently bound to a cedar pollen allergen represented by Asp-Asn-Pro-Ile-Asp-Ser or Ala-Ile-Asn-Ile-Phe-Asn, and the resulting complex is used as a desensitizing agent in humans. Proposal to administer has been made.

  However, diagnosis and desensitization of allergies usually require large amounts of high-purity allergens, and allergens in cedar pollen are scarce and have low stability. It is very difficult to cover the desensitizer with cedar pollen alone. For this reason, in the recent treatment and prevention of allergic diseases, instead of administering the whole allergen to the patient as in the past, the minimum region that T cells in the allergen specifically recognize, that is, Attention has been focused on immunotherapy in which a small peptide consisting essentially of a T cell epitope is administered.

In general, when allergens are taken into antigen-presenting cells such as macrophages, they are digested there, and the digested fragments bind to HLA (Human Leucocyte Antigen) protein on the surface of immune-presenting cells and presented as antigens. The antigen-presented fragment is limited to a specific region of the allergen due to its affinity for the HLA protein. Among these regions, the region specifically recognized by T cells is usually a “T cell epitope”. It is called. For immunotherapy in which a peptide consisting essentially of only a T cell eptop is administered,
(i) Since the peptide lacks the B cell epitope, that is, the immunoglobulin E antibody specific to the allergen does not react, side effects such as anaphylaxis that frequently occur with conventional crude or purified allergens cannot occur.
(ii) The period from starting with a small amount to reaching an effective dose can be significantly shortened compared to conventional desensitizers.
(iii) It can induce oral tolerance and attenuate allergic reactions to allergens. There are advantages such as.

WO 93/01213 WO 94/11512 JP-A-1-156926 Japanese Patent Laid-Open No. 3-93730 Yasuda et al., "Journal of Allergy and Clinical Immunology", Volume 71, No. 1, pp. 77-86 (1983) Taniai et al., FB Letters, Vol. 239, No. 2, pp. 329-332 (1988) Sakaguchi et al., “Allergy”, No. 45, pp. 309-312 (1990) Komiyama et al., “Biochemical Biophysical Research Communication” Vol. 201, No. 2, pages 1021-1028 (1994) Sawatani et al., “Allergy”, Vol. 42, No. 6, pp. 738-747 (1993)

  The present inventors have found the amino acid sequence of the minimum unit constituting the T cell epitope and completed the present invention. The first object of the present invention is to provide a peptide consisting only of a T cell epitope of an essential cedar pollen allergen. The second object of the present invention is to provide an anti-cedar pollinosis agent comprising the above peptide as an active ingredient.

The present invention
(1) A peptide consisting of the amino acid sequence of SEQ ID NO: 2.
(2) A peptide consisting of the amino acid sequence of SEQ ID NO: 3.
(3) A peptide consisting of the amino acid sequence of SEQ ID NO: 9.
(4) A peptide consisting of the amino acid sequence of SEQ ID NO: 10.
(5) A peptide consisting of the amino acid sequence of SEQ ID NO: 17.
(6) A peptide consisting of the amino acid sequence of SEQ ID NO: 18.
(7) A peptide consisting of the amino acid sequence of SEQ ID NO: 19.

(8) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
(9) An anti-cedar pollinosis agent comprising a peptide having the amino acid sequence of SEQ ID NO: 3 as an active ingredient.
(10) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 4 as an active ingredient.
(11) An anti-cedar pollinosis agent comprising a peptide having the amino acid sequence of SEQ ID NO: 5 as an active ingredient.
(12) An anti-cedar pollinosis agent comprising a peptide having the amino acid sequence of SEQ ID NO: 6 as an active ingredient.
(13) An anti-cedar pollinosis agent comprising a peptide having the amino acid sequence of SEQ ID NO: 9 as an active ingredient.

(14) An anti-cedar pollinosis agent comprising a peptide having the amino acid sequence of SEQ ID NO: 10 as an active ingredient.
(15) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 11 as an active ingredient.
(16) An anti-cedar pollinosis agent comprising a peptide having the amino acid sequence of SEQ ID NO: 12 as an active ingredient.
(17) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 13 as an active ingredient.
(18) An anti-cedar pollinosis agent comprising a peptide having the amino acid sequence of SEQ ID NO: 14 as an active ingredient.
(19) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 15 as an active ingredient.
(20) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 16 as an active ingredient.
(21) An anti-cedar pollinosis agent comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 17 as an active ingredient.

(22) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 18 as an active ingredient.
(23) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 19 as an active ingredient.
(24) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 20 as an active ingredient.
(25) An anti-cedar pollinosis agent comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 21 as an active ingredient.
(26) An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 22 as an active ingredient.
(27) An anti-cedar pollinosis agent comprising a peptide having the amino acid sequence of SEQ ID NO: 23 as an active ingredient.
(28) An anti-cedar pollinosis agent containing a peptide consisting of the amino acid sequence of SEQ ID NO: 24 as an active ingredient.

  INDUSTRIAL APPLICABILITY According to the present invention, it was possible to provide a peptide comprising only a T cell epitope of a cedar pollen allergen and an anti-cedar pollinosis agent comprising them as an active ingredient. The peptide of the present invention does not substantially react with the immunoglobulin E antibody specific for the cedar pollen allergen. Therefore, when administered to mammals including humans, it does not cause anaphylaxis substantially and is specific for the cedar pollen allergen. T cells can be activated.

  The present invention will be described in detail below. Examples of preferred peptides in the present invention are shown in Table 1.

The above peptide 1 is a peptide represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing,
Peptide 2 is a peptide represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing,
Peptide 3 is a peptide represented by the amino acid sequence of SEQ ID NO: 3 in the sequence listing,
Peptide 4 is a peptide represented by the amino acid sequence of SEQ ID NO: 4 in the sequence listing,
The peptide 5 is a peptide represented by the amino acid sequence of SEQ ID NO: 5 in the sequence listing,
The peptide 6 is a peptide represented by the amino acid sequence of SEQ ID NO: 6 in the sequence listing,
The peptide 7 is a peptide represented by the amino acid sequence of SEQ ID NO: 7 in the sequence listing,
Peptide 8 is a peptide represented by the amino acid sequence of SEQ ID NO: 8 in the sequence listing,
The peptide 9 is a peptide represented by the amino acid sequence of SEQ ID NO: 9 in the sequence listing,
The peptide 10 is a peptide represented by the amino acid sequence of SEQ ID NO: 10 in the sequence listing,
The peptide 11 is a peptide represented by the amino acid sequence of SEQ ID NO: 11 in the sequence listing,
The peptide 12 is a peptide represented by the amino acid sequence of SEQ ID NO: 12 in the sequence listing,
The peptide 13 is a peptide represented by the amino acid sequence of SEQ ID NO: 13 in the sequence listing,
The peptide 14 is a peptide represented by the amino acid sequence of SEQ ID NO: 14 in the sequence listing,
The peptide 15 is a peptide represented by the amino acid sequence of SEQ ID NO: 15 in the sequence listing,
The peptide 16 is a peptide represented by the amino acid sequence of SEQ ID NO: 16 in the sequence listing,
The peptide 17 is a peptide represented by the amino acid sequence of SEQ ID NO: 17 in the sequence listing,
The peptide 18 is a peptide represented by the amino acid sequence of SEQ ID NO: 18 in the sequence listing,
The peptide 19 is a peptide represented by the amino acid sequence of SEQ ID NO: 19 in the sequence listing,
The peptide 20 is a peptide represented by the amino acid sequence of SEQ ID NO: 20 in the Sequence Listing,
The peptide 21 is a peptide represented by the amino acid sequence of SEQ ID NO: 21 in the sequence listing,
The peptide 22 is a peptide represented by the amino acid sequence of SEQ ID NO: 22 in the sequence listing,
The peptide 23 is a peptide represented by the amino acid sequence of SEQ ID NO: 23 in the sequence listing,
Each of the peptides 24 represents a peptide represented by the amino acid sequence of SEQ ID NO: 24 in the sequence listing.

  The peptides described in the above (1) to (7) can be easily prepared by a peptide synthesis method commonly used in this field known as “solid phase method” or “liquid phase method”. For example, the details of peptide synthesis are described in the Japan Biochemical Society, “New Chemistry Laboratory”, Volume 1, “Protein VI”, pp. 3-44, 1992, published by Tokyo Kagaku Dojin. Further, the peptide can be synthesized according to the protocol of the same apparatus by Fmoc (9-fluorenyl methyloxycarbonyl) solid phase synthesis method using a multi-peptide synthesizer SYMPHONY (manufactured by Protein Technology). That is, Fmoc-L-amino acid Wang resin into which an amino acid corresponding to the C-terminal of each peptide to be synthesized is introduced is set in the reaction vessel of the peptide synthesizer, and Fmoc is removed using a deprotection solution. Furthermore, the target peptide can be synthesized by reacting an amino acid solution corresponding to the second amino acid from the C-terminus with an activator solution, deprotecting the Fmoc group again after the reaction, and repeating the same operation. .

  The peptides of the present invention are not limited to those prepared by chemical synthesis, for example, those collected from cedar pollen or male flowers, or from cedar pollen allergens prepared by recombinant DNA technology, and collected from degradation products For example, a DNA encoding the peptide described in (1) to (7) above is prepared, and this is inserted into a vector capable of autonomous replication to obtain a recombinant DNA. The peptide of the present invention may be collected from the culture by introducing it into a suitable host such as a fungus, actinomycetes, or yeast.

  Furthermore, the peptide of the present invention is in the form of a complex obtained by adding a carbohydrate or polyethylene glycol to the peptide thus obtained, and further, the peptide is crosslinked by acetylation, amidation and / or multifunctional test. It may be in the form of a derivative or polymer obtained by polymerization.

  The peptide of the present invention exerts the intended therapeutic / preventive effect even when administered in a relatively crude form, but is usually purified prior to use. For purification, for example, to purify peptides or proteins such as filtration, concentration, centrifugation, gel filtration chromatography, ion exchange chromatography, high performance liquid chromatography, affinity chromatography, gel electrophoresis, isoelectric focusing etc. Conventional methods in this field are used, and these methods may be appropriately combined as necessary. Then, the purified peptide may be concentrated and lyophilized to make it liquid or solid depending on the final use form.

It can be confirmed that the peptide of the present invention has activity as a T cell epitope by measuring the uptake of ³ H-thymidine of T cells specific for the cedar pollen allergen. For example, the following method can be used for this measurement. That is, a mononuclear cell group containing T cells specific for Cryj2 is isolated from peripheral blood of hay fever patients or experimental animals such as mice immunized with Cryj2 by Ficoll-Hypaque specific gravity centrifugation, etc., and this cell group is separated from RPMI. Suspend in a medium such as 1640 and dispense onto a 96-well microplate. Next, the test substance peptide is added and incubated. The incubation temperature and time can be appropriately adjusted for each experiment, but 37 ° C. and 2 days are preferable. Thereafter, ³ H-thymidine is added to the medium, and further incubation is continued for a certain period of time. By measuring the amount of ³ H-thymidine incorporation in the mononuclear cell group, the activity of the peptide of the present invention as a T cell epitope is calculated. Can do. In the present invention, a system that does not contain a peptide at the same time is set as a negative control. A system in which the amount of ³ H-thymidine incorporation reaches twice or more that of the negative control is “positive”, and a system that has not reached “ Negative.

Measurement of ³ H-thymidine incorporation of T cells specific for cedar pollen allergen can also be performed by the following method. An experimental animal such as a mouse is immunized with Cryj2 in advance, and then lymphocytes are collected from a submandibular lymph node or the like. Then, the activity as a T cell epitope of the peptide of the present invention can be calculated by stimulating with the subject peptide by the same method as described above and measuring the amount of ³ H-thymidine incorporation. The determination of peptide “positive” and “negative” was performed based on the same criteria as described above.

  It can be confirmed, for example, by the following experiment that the peptide of the present invention has a preventive effect on hay fever patients. In advance, the peptide of the present invention is administered to an experimental animal such as a mouse to induce immune tolerance against the peptide. After a certain period of time, the experimental animal is immunized by administering Cryj2 together with an adjuvant such as cholera toxin. Furthermore, after a certain period of time, submandibular lymph node cells are removed from the experimental animal to prepare a cell suspension.

In addition, a spleen is extracted from another untreated experimental animal to prepare a spleen cell suspension, which is irradiated with X-rays to lose cell proliferation activity. To do. This was mixed with the previous submandibular lymph node cell suspension, Cryj2 was added thereto, and the culture was continued. Further, ³ H-thymidine was added to measure the uptake of this, and T cell proliferation was observed. Can be measured.

  In animals in which immune tolerance has not been previously induced with the peptide of the present invention, the T cells respond to Cryj2 bound to antigen-presenting cells and proliferate by immunization with Cryj2. On the other hand, in animals whose immune tolerance was previously induced with the peptide of the present invention, T cells do not react with Cryj2 bound to antigen-presenting cells and do not proliferate even when immunization with Cryj2 is subsequently performed. By measuring the difference, the preventive effect against hay fever of the peptide of the present invention can be confirmed.

  Furthermore, when Cryj2 is added to the mixed solution of the above-mentioned immunized animal submandibular lymph node cell suspension and the antigen-presenting cell-containing suspension and the culture is continued, cytokines such as interleukin 4 are secreted in the culture solution. However, the hay fever of the peptide of the present invention can also be obtained by comparing the amount of secretion of this cytokine between an experimental animal that has been pre-administered with the peptide of the present invention to induce immune tolerance and an experimental animal that has not been pre-administered. The preventive effect can be confirmed.

  It can be confirmed, for example, by the following experiment that the peptide of the present invention has a therapeutic effect on pollinosis patients. An experimental animal such as a mouse is immunized by administering Cryj2 together with an adjuvant of cholera toxin. After a certain period of time, Cryj2 is administered to the experimental animal together with adjuvant of cholera toxin and boosted. Furthermore, after a certain period of time, submandibular lymph node cells are removed from the experimental animal to prepare a cell suspension, and then T cell proliferation is measured by the same method as described above.

  In animals not treated with the peptide of the present invention, the T cells respond to Cryj2 bound to antigen-presenting cells and proliferate by immunization with Cryj2. On the other hand, in the animal treated with the peptide of the present invention, T cells do not react with Cryj2 bound to antigen-presenting cells and do not proliferate even if immunization with Cryj2 is subsequently performed. By measuring the difference, the therapeutic effect on hay fever of the peptide of the present invention can be confirmed.

[Operation]
Since the peptide of the present invention does not substantially react with an immunoglobulin E antibody specific for a cedar pollen allergen, it is specific to a cedar pollen allergen without substantially causing anaphylaxis when administered to mammals including humans. T cells can be activated. The anti-cedar pollinosis agent of the present invention comprising such a peptide as an active ingredient exerts a remarkable therapeutic / preventive effect on cedar pollinosis without substantially causing anaphylaxis when administered to mammals including humans in general. To do.

  An anti-cedar pollinosis agent comprising the peptide of the present invention as an active ingredient is effective in causing cedar pollinosis without substantially causing side effects such as anaphylaxis when administered to mammals including humans suffering from cedar pollinosis. Can be treated. On the other hand, when the anti-cedar pollen case of this invention is administered to a healthy individual or an individual with potential cedar pollinosis before the cedar pollen begins to scatter, it exhibits a remarkable preventive effect against cedar pollinosis Effective in relieving allergic symptoms at the time of onset.

  The anti-cedar pollinosis agent of the present invention will be described in more detail. The anti-cedar pollinosis agent of the present invention usually contains 0.01 to 100% (w / w) of one or more of the peptides according to the present invention. Preferably, it comprises 0.05 to 50% (w / v), 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 also physiologically acceptable, for example, carriers such as serum albumin, gelatin, mannitol, excipients, immune aids, stabilizers Furthermore, it includes a form as a composition with one or two or more other drugs including anti-inflammatory drugs such as steroid hormones and sodium crimoglycate and antihistamines as required. Further, the anti-cedar pollinosis agent of the present invention includes a drug in a dosage unit form, and the drug in the dosage unit form includes, for example, a dose per day or an integral multiple thereof (4 Means an agent in a physically separate, unitary dosage form that contains an amount equivalent to (up to twice) or a fraction thereof (up to 1/40) and is suitable for administration. Such dosage unit forms include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, mild steels, plasters, poultices, suppositories, eye drops. Nasal drops, sprays, injections and the like.

  The method of using the anti-cedar pollinosis agent of the present invention will be described. The anti-cedar pollinosis agent of the present invention is generally transdermally, orally, nasally, and instilled for mammals including humans for the purpose of treating and preventing cedar pollinosis. Or it is administered by injection. The dose in humans varies depending on the purpose and symptom of administration, but is usually 0.01 to 1.0 g per day for adults, preferably 0.0 while observing the subject's symptoms and the course after administration. As a guideline, the dose is usually repeated once a week to once a month for about 1 to 6 months with increasing doses.

According to the conventional acute toxicity of the polypeptide of the present invention, the immunotherapeutic agent obtained by the method of Preparation Examples 1 to 4 described later was orally or intraperitoneally administered to a 20-day-old mouse. As a result, these immunotherapeutic agents were found to have an LD ₅₀ of 200 mg / kg or more by any administration route. This indicates that the peptide of the present invention can be safely used in an immunotherapeutic agent for mammals including humans.

Test Example 1
Using T cells isolated from a cedar pollinosis patient, it was confirmed that peptides 1 to 6 and peptides 9 to 24 of the present invention have cedar pollen antigen T cell epitope activity. In a skin test, 20 ml of peripheral blood was collected from a patient who was positive for cedar pollen allergen and positive for anti-cedar pollen allergen IgE reaction. After centrifugation, buffy coat was obtained, and peripheral blood mononuclear cells (PBMC) were collected by Ficoll-Pak specific gravity centrifugation. This PBMC was suspended in a medium (RPMI-1640, containing 5% heat-inactivated human AB serum) at 7.5 × 10 ⁵ cells / ml.

In 96-well round bottom plates, 1.5 × 10 ⁵ cells were cultured in 200 μl of each well for 48 hours in the presence of 20 ng peptide and 37 ° C. 5% CO ₂ . Thereafter, 1 μCi of tritiated thymidine was added and further cultured for 16 hours. In order to measure the count taken up by the cells, the cells were collected on a glass fiber filter using a cell harvester and measured with a liquid scintillation counter. The results are shown in Table 2 below.

  From the above results, these peptides were shown to contain a T cell epitope of Cryj2 allergen.

Test Example 2
Cryj2 was purified by the method described in the literature (Allergy, 1990, 45, 309-312). An antigen solution prepared by dissolving 1 μg of purified Cryj2 and 1 μg of cholera toxin B subunit (containing 0.5% cholera toxin) in 0.01 M phosphate buffer (pH 7.4) was added to Balb / c mice (5 to 5) under an abatin anesthesia. 6 weeks of age: Charles River Japan) was administered nasally and immunized. Two weeks later, the mice were boosted again by the same method. One week later, mouse submandibular lymph node cells were removed. This was passed through a nylon mesh and further suspended in a medium (containing RPMI 1640 10% calf fetal serum) to prepare a suspension.

In addition, spleen cells were excised from mice not immunized with Cryj2, and a lymph node cell suspension was prepared by the same method as described above. This suspension was irradiated with 3000 Rad X-rays to eliminate cell growth activity, and used as an antigen-presenting cell suspension. A flat bottom 96-well plate (Corning) was dispensed at 3 × 10 ⁶ lymph node cells and 6 × 10 ⁵ antigen-presenting cells per well, and in the presence of peptide 7 or peptide 8 (0.5 μg / ml). Or in the absence of these peptides, the cells were cultured for 3 days at 37 ° C. and 5% CO ₂ .

The last 16 hours were cultured in the presence of ³ H-Thymidine, and during this time, the amount of ³ H-Thymidine incorporated into the intracellular DNA was measured by measuring the radiation dose of DNA adsorbed on the glass filter by the liquid scintillation method. Calculated. A value obtained by dividing the amount of ³ H-Thymidine incorporation in the presence of the peptide by the amount of incorporation in the absence of the peptide was used as a reaction fold, and this was used as an index of cell proliferation activity.

  The proliferation rate of lymph node cells increased about 3 times for peptide 7 and about 5 times for peptide 8. Thus, these peptides were shown to contain the T cell epitope of the Cryj2 allergen.

Test Example 3
For peptide 7 or 8, immune tolerance was induced in Balb / c mice. That is, each peptide solution dissolved in a phosphate buffer (0.01 M (pH 7.4)) was intravenously administered to the mouse tail vein so that the amount of peptide was 20 μg per mouse. Alternatively, the peptide solution was orally administered so that the amount of the peptide was 1 mg per animal, and this oral administration was repeated 4 times in 2 weeks. Thereafter, the mouse was immunized with Cryj2 in the same manner as in Test Example 2.

Submandibular lymph node cells were removed from the mouse by the same method as in Test Example 2 to form a submandibular lymph node cell suspension, and the spleen was obtained from a separate mouse that had not been tolerized with peptide and immunized with Cryj2. And the proliferative activity is eliminated by X-rays to obtain antigen-presenting cell suspensions, which are co-cultured in the presence of Cryj2 (1 μg / ml) and incorporated with ³ H-Thymidine in the same manner as in Test Example 2. The amount was measured and the cell proliferation activity was calculated.

In addition, a suspension of lymph node cells and antigen-presenting cells was prepared using a preparation medium. Distribute the cells into 24-well plates (Corning) so that there are 1.5 × 10 ⁶ lymph node cells and 3 × 10 ⁶ antigen-presenting cells per well, and these cells are combined with Cryj2 (1 μg / ml) at 37 ° C. The cells were cultured for 3 days under 5% CO ₂ conditions. After completion of the culture, the culture supernatant was collected and stored frozen at 20 ° C. until used for measurement. The amount of interleukin 4 contained in the culture solution was measured with a commercially available measurement kit (Endogen).

(1) Induction of immune tolerance by intravenous administration of peptide 7 A solution of peptide 7 was administered to the tail vein of mice. Only the phosphate buffer (0.01 M (pH 7.4)) was intravenously administered to the control group of mice. Thereafter, both groups of mice were immunized intranasally with Cryj2 according to the method described above. Thereafter, when submandibular lymph node cells excised from the mouse and antigen-presenting cells excised from other mice were cultured with Cryj2, the proliferation activity of the lymph node cells from the mice pre-administered with peptide 7 was compared with that of the control group. 29.5%. As a result, it was revealed that peptide 7 has an activity of suppressing an immune response against a spear allergen.

(2) Induction of immune tolerance by oral administration of Peptide 7 A T cell peptide 7 solution was orally administered to mice 4 times in 2 weeks according to the above method. Only the phosphate buffer (0.01 M (pH 7.4)) was orally administered to the control group of mice. Thereafter, both groups of mice were immunized intranasally with Cryj2. Thereafter, submandibular lymph node cells excised from the mice and antigen-presenting cells excised from other mice were cultured with Cryj2 for 3 days, and the amount of cytokine in the culture supernatant was measured. As a result, the amount of interleukin 4 produced from lymph node cells from mice pre-administered with peptide 7 was 49.8% lower than that in the control group. Thus, it was shown that the peptide 7 was orally administered to suppress the immune response to squirrel allergen.

(3) Induction of immune tolerance by intravenous administration of peptide 8 A solution of peptide 8 was administered to the tail vein of mice. Only the phosphate buffer (0.01 M (pH 7.4)) was intravenously administered to the control group of mice. Thereafter, both groups of mice were immunized intranasally with Cryj2 according to the method described above. Thereafter, when submandibular lymph node cells excised from the mouse and antigen-presenting cells excised from other mice were cultured together with Cryj2, the proliferation activity of the lymph node cells from mice pre-administered with peptide 8 was compared with the control group. 30.9%. As a result, it was revealed that peptide 8 has an activity of suppressing an immune response to a spear allergen.

(4) Induction of immune tolerance by oral administration of Peptide 8 A T cell peptide 8 solution was orally administered to mice 4 times in 2 weeks according to the above method. Only the phosphate buffer (0.01 M (pH 7.4)) was orally administered to the control group of mice. Thereafter, both groups of mice were immunized intranasally with Cryj2. Thereafter, when submandibular lymph node cells excised from the mouse and antigen-presenting cells excised from other mice were cultured together with Cryj2, the proliferation activity of the lymph node cells from mice pre-administered with peptide 8 was compared with the control group. 73.1%. As a result, it was revealed that peptide 8 has an activity of suppressing an immune response to a spear allergen.

Test example 4
For peptide 8, Balb / c mice were treated. That is, an antigen solution prepared by dissolving 1 μg of purified Cryj2 and 1 μg of cholera toxin B subunit (containing 0.5% of cholera toxin) in 0.01 M phosphate buffer (pH 7.4) was added to two groups of Balb under abatin anesthesia. / c mice (5-6 weeks old: Charles River Japan) were nasally administered and immunized. From one week later, the peptide 8 solution dissolved in 0.01 M phosphate buffer (pH 7.4) was orally administered to the mice in the experimental group so that the amount of the peptide was 200 μg per mouse. And this oral administration was repeated 4 times over 2 weeks. Only 0.01M phosphate buffer (pH 7.4) was similarly administered to the control group of mice. Four days after the fourth oral administration, both groups of mice were again immunized intranasally with Cryj2. One week later, when submandibular lymph node cells excised from the mouse by the same method as in Test Example 3 and antigen-presenting cells excised from other mice were cultured with Cryj2, the proliferation of the lymph node cells derived from the experimental group mice was It was 46.0% lower than the control group. From this result, it was revealed that peptide 8 has an activity of suppressing an immune response to a skier allergen even when administered to a mouse immunized with a skier allergen.

  As described above, the peptide of the present invention can activate T cells specific for the cedar pollen allergen without substantially causing anaphylaxis when administered to mammals including humans in general. The anti-cedar pollinosis agent of the present invention comprising such a peptide as an active ingredient has a remarkable therapeutic / preventive effect on cedar pollinosis substantially without causing anaphylaxis when administered to mammals including humans. Demonstrate.

  An anti-cedar pollinosis agent comprising the peptide of the present invention as an active ingredient is effective in causing cedar pollinosis without substantially causing side effects such as anaphylaxis when administered to mammals including humans suffering from cedar pollinosis. Can be treated. On the other hand, when the anti-cedar pollinosis agent of this invention is administered to healthy individuals or individuals with potential cedar pollinosis before the cedar pollen begins to scatter, it exerts a remarkable preventive effect against cedar pollinosis. At the same time, it is effective in relieving allergic symptoms at the onset.

  EXAMPLES Hereinafter, although an Example and a formulation example demonstrate this invention further in detail, this invention does not limit the technical scope by these.

Example 1
Peptide 1:
Lys-Val-Asp-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser
Peptides were chemically synthesized by a method (solid phase synthesis method) in which amino acids were bound to the amino acid derivatives fixed on the resin one by one from the carboxyl terminal side. The amino acid used in each cycle was a special amino acid derivative in which the reactive group of the α-amino group and the residue part was blocked with a protecting group. Here, amino acids in which each α-amino group was blocked with Fmoc (9-fluorenyl methyloxycarbonyl) were used (Fmoc method). In addition, peptide synthesis was performed by sequentially repeating the reaction of deprotecting the Fmoc of the α-amino group of the amino acid bonded to the resin and then binding the amino acid derivative activated with the carboxyl group.

  Each peptide used in the experiment was synthesized by the above Fmoc solid phase synthesis method using a multi-peptide synthesizer SYMPHONY (Protein Technologies, Inc.) according to the protocol of the apparatus. That is, 25 μmol equivalent of Fmoc-Ser (tBu) -Wang-resin (0.52 mmol / g) into which the amino acid (Ser) corresponding to the C-terminal residue of the peptide to be synthesized is introduced into the reaction vessel of the peptide synthesizer. Then, 1.25 ml of a deprotection solution (20% piperidine / dimethylformamide (DMF)) was reacted twice for 5 minutes to remove the Fmoc group of the amino acid bound to the resin. After washing 6 times with 1.25 ml of DMF solution for 30 seconds, 1.25 ml of 200 mM Fmoc-Ala / DMF solution corresponding to the second amino acid from the C-terminal side and 200 mM activator solution (200 mM O-Benzotriazole-N, N, ', 1.25 ml of N ′,-Tetramethyl-Uronium-Hexafluorophosphate / 400 mM N-methylmorpholine / DMF) was added (each 10 times the theoretical equivalent: equivalent to 250 μmol) and reacted at room temperature for 20 minutes. The Fmoc-Ala-Ser (tBu) -Wang-resin produced here was washed 6 times for 30 seconds with 1.25 ml of DMF, and then again washed 6 times for 30 seconds with 1.25 ml of DMF using Fmoc group deprotection. Fmoc-Pro solution and activator solution were added and reacted. By repeating the same procedure, the target peptide (Fmoc-Lys (Boc) -Val-Asp (OtBu) -Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt ) -Pro-Ala-Ser (tBu) -Wang-resin).

The amino acids used in the synthesis are as follows (manufactured by Nisshinbo Co., Ltd.). The inside of () represents the protecting group which protects the reactive group of a residue part.
Fmoc-Ala, Fmoc-Pro,
Fmoc-Asn (Trt), Fmoc-Gln (Trt), Fmoc-Tyr (tBu),
Fmoc-Ile, Fmoc-Gly, Fmoc-Asp (OtBu),
Fmoc-Val, Fmoc-Lys (Boc),
A cleave reaction was performed in the apparatus using a peptide synthesizer SYMPHONY.

First, the protected peptide resin synthesized as described above (Fmoc-Lys (Boc) -Val-Asp (OtBu) -Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn ( Trt) -Pro-Ala-Ser (tBu) -Wang-resin) was reacted with 1.25 ml of deprotection solution twice for 5 minutes to deprotect the N-terminal Fmoc group. Then after DMF at 30 seconds 6 washes 1.25 ml, washed as in CH ₂ Cl _2, dried for 10 minutes blowing N _2, Kuribe chromatography di solution (Trifluoroacetic acid: Phenol: Water: Tioanisole: Ethanedithiol = 82.5: 5: 5: 5: 2.5) was added and reacted at room temperature for 2 hours (DSKing, Int. J. Peptide Protein Reg., 36, 255 (1990)). The side chain protecting group was removed to obtain a peptide (Lys-Val-Asp-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser).

  After completion of the reaction, the peptide solution was filtered using a filter and separated into a resin and a filtrate. Further, 2.5 ml of the washed resin was combined and collected in a centrifuge tube. The collected peptide solution was removed from the apparatus and 5 ml of cold ether was added to precipitate the peptide. After cooling for a while, this was centrifuged (3000 rpm for 10 minutes), the precipitate was collected, and cold ether was added again to disperse and recover, and the peptide was washed 5 to 6 times repeatedly.

  The obtained peptide was dried to obtain a crude peptide (50.5 mg). The crude peptide was dissolved in 10% acetonitrile aqueous solution containing 0.1% TFA, then supplied to an ODS column (TSKgel ODS-12OT, 21.5 mm x 30 cm: manufactured by Tosoh Corporation), and developed with 21% acetonitrile containing 0.1% TFA. (The flow rate was 9 ml / min, the detection wavelength was 220 nm). The fraction eluted at 31 to 35 minutes was collected, concentrated, and lyophilized to obtain the desired peptide (15.9 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 2
Peptide 2:
Val-Asp-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser
In the same manner as in Example 1, the peptide (Fmoc-Val-Asp (OtBu) -Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser ( tBu) -Wang-resin) and cleaved to obtain a peptide (Val-Asp-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser) The peptide solution was collected in a centrifuge tube. Thereafter, the peptide was precipitated to obtain a crude peptide (55.5 mg).

  The crude peptide was dissolved in 10% acetonitrile aqueous solution containing 0.1% TFA, then supplied to an ODS column (TSKgel ODS-120T, 21.5 mm x 30 cm: manufactured by Tosoh Corporation), and developed with 22% acetonitrile containing 0.1% TFA. The fraction eluted at 26 to 29 minutes was collected, concentrated and freeze-dried to obtain the desired peptide (7.1 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 3
Peptide 3:
Asp-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser
In the same manner as in Example 1, the peptide (Fmoc-Asp (OtBu) -Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu) -Wang-resin) and cleaved to obtain a peptide (Asp-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser). Collected in a tube. Thereafter, the peptide was precipitated to obtain a crude peptide (47.9 mg).

  The crude peptide was dissolved in 10% acetonitrile aqueous solution containing 0.1% TFA, then supplied to an ODS column (TSKgel ODS-120T, 21.5 mm x 30 cm: manufactured by Tosoh Corporation) and developed with 21% acetonitrile containing 0.1% TFA. (Flow rate 9 ml / min, detection wavelength 220 nm) The fraction eluted at 25 to 28 minutes was collected, concentrated, and lyophilized to obtain the desired peptide (13.8 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 4
Peptide 4:
Trp-Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met-Gly
The peptide (Fmoc-Trp-Leu-Gln- (Trt) -Phe-Ala-Lys (Boe) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Met) was prepared in the same manner as in Example 1. -Gly-Wang-resin). However, Fmoc-Gly-Wang-resin (0.50 mol page / g) was used in an amount equivalent to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are as follows.
Fmoc-Met, Fmoc-Leu, Fmoc-Thr (tBu),
Fmoc-Phe, Fmoc-Gly, Fmoc-Lys (Boc),
Fmoc-Ala, Fmoc-Gln (Trt), Fmoc-Trp,

  A peptide (Trp-Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met-Gly) was obtained by carrying out a cleave reaction in the same manner as in Example 1. The solution was collected in a centrifuge tube, and then the peptide was precipitated to obtain a crude peptide (63.3 mg).

  The crude peptide is dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA, then supplied to an ODS column (TSKgel ODS-120T, 21.5 mm x 30 cm: manufactured by Tosoh Corporation) and developed with 38% acetonitrile containing 0.1% TFA. (Flow rate: 9 ml / min, detection wavelength: 220 nm) The fraction eluted at 25-31 min was collected, concentrated, and lyophilized to obtain the desired peptide (2.0 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 5
Peptide 5:
Trp-Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met
The peptide (Fmoc-Trp-Leu-Gln- (Trt) -Phe-Ala-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Met) was prepared in the same manner as in Example 1. -Wang-resin) was synthesized. However, Fmoc-Met-Wang-resin (0.75 mmol / g) was used in an amount equivalent to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are the same as in Example 4. A peptide (Trp-Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met-) was obtained by carrying out a cleave reaction in the same manner as in Example 1, and this peptide solution Was collected in a centrifuge tube, and then the peptide was precipitated to obtain a crude peptide (29 mg).

  The crude peptide was dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA, then supplied to an ODS column (TSKgel ODS-120T, 21.5 mm x 30 cm: manufactured by Tosoh Corporation), and developed with 36% acetonitrile containing 0.1% TFA. (The flow rate was 9 ml / min, the detection wavelength was 220 nm), and the fraction eluted at 32 to 34 minutes was concentrated and then lyophilized to obtain the desired peptide (1.1 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 6
Peptide 6:
Trp-Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu
In the same manner as in Example 1, the peptide (Fmoc-Trp-Leu-Gln (Trt) -Phe-Ala-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Wang- Resin) was synthesized. However, Fmoc-Leu-Wang-resin (0.69 mmol / g) was used in an amount equivalent to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are the same as in Example 4.

  A peptide (Trp-Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu) was obtained by carrying out a cleave reaction in the same manner as in Example 1, and this peptide solution was centrifuged. After that, the peptide was precipitated to obtain a crude peptide (35.6 mg). The crude peptide is dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA, then supplied to an ODS column (TSKgel ODS-120T, 21.5 mm x 30 cm: manufactured by Tosoh Corporation) and developed with 38% acetonitrile containing 0.1% TFA. The fraction eluted at 26-30 minutes was concentrated and lyophilized to obtain the desired peptide (6.3 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 7
Peptide 7:
His-Phe-Thr-Phe-Lys-Val-Asp-Gly-Ile-Ile-Ala-Ala-Tyr-Gln
By the Fmoc method described in Example 1, using a 9050 peptide synthesizer manufactured by Milligen / Biosearch, 400 mg of crude peptide was obtained. The crude peptide was dissolved in a 0.1% TFA aqueous solution, then applied to a μBONDASPHERE 5μ C18C120 A column (19 × 150 mm), developed with a 90% acetonitrile solution containing 0.1% TFA (flow rate 5 ml / min, detection wavelength 214 nm), 28 The fraction eluted at ˜29 minutes was evaporated and then lyophilized to obtain the desired peptide (36 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 8
Peptide 8:
Arg-Ala-Glu-Val-Ser-Tyr-Val-His-Val-Asn-Gly-Ala-Lys-Phe
Using the 9050 peptide synthesizer manufactured by Milligen / Biosearch, 550 mg of crude peptide was obtained by the Fmoc method described in Example 1. The crude peptide was dissolved in a 0.1% TFA aqueous solution, then applied to a μBONDASPHERE 5μ C18C120 A column (19 × 150 mm), developed with a 90% acetonitrile solution containing 0.1% TFA (flow rate 5 ml / min, detection wavelength 214 nm), 26 The fraction eluted at ˜27 minutes was evaporated and lyophilized to obtain the desired peptide (60 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 9
Peptide 9:
Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser
Peptides were chemically synthesized by a method (solid phase synthesis method) in which amino acids were bound to the amino acid derivatives fixed on the resin one by one from the carboxyl terminal side. The amino acid used in each cycle was a special amino acid derivative in which the reactive group of the α-amino group and the residue part was blocked with a protecting group. Here, amino acids in which each α-amino group was blocked with Fmoc (9-fluorenyl methyloxycarbonyl) were used (Fmoc method). In addition, peptide synthesis was performed by sequentially repeating the reaction of deprotecting the Fmoc of the α-amino group of the amino acid bonded to the resin and then binding the amino acid derivative activated with the carboxyl group.

  Each peptide used in the experiment was synthesized by the above Fmoc solid phase synthesis method using a multi-peptide synthesizer SYMPHONY (Protein Technologies, Inc.) according to the protocol of the apparatus. That is, 25 μmol equivalent of Fmoc-Ser (tBu) -Wang-resin (0.52 mmol / g) into which the amino acid (Ser) corresponding to the C-terminal residue of the peptide to be synthesized is introduced into the reaction vessel of the peptide synthesizer. Then, 1.25 ml of a deprotection solution (20% piperidine / dimethylformamide (DMF)) was reacted twice for 5 minutes to remove the Fmoc group of the amino acid bound to the resin. After washing 6 times with 1.25 ml of DMF solution for 30 seconds, 1.25 ml of 200 mM Fmoc-Ala / DMF solution corresponding to the second amino acid from the C-terminal side and 200 mM activator solution (200 mM O-Benzotriazole-N, N, ', 1.25 ml of N ′,-Tetramethyl-Uronium-Hexafluorophosphate / 400 mM N-methylmorpholine / DMF) was added (each 10 times the theoretical equivalent: equivalent to 250 μmol) and reacted at room temperature for 20 minutes. The Fmoc-Ala-Ser (tBu) -Wang-resin produced here was washed 6 times for 30 seconds with 1.25 ml of DMF, and then again washed 6 times for 30 seconds with 1.25 ml of DMF using Fmoc group deprotection. Fmoc-Pro solution and activator solution were added and reacted. By repeating the same procedure, the target peptide (Fmoc-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu) -Wang -Resin) was synthesized.

The amino acids used in the synthesis are as follows (manufactured by Nisshinbo Co., Ltd.). The inside of () represents the protecting group which protects the reactive group of a residue part.
Fmoc-Ala, Fmoc-Pro,
Fmoc-Asn (Trt), Fmoc-Gln (Trt), Fmoc-Tyr (tBu),
Fmoc-Ile, Fmoc-Gly,
A cleave reaction was performed in the apparatus using a peptide synthesizer SYMPHONY.

First, the protected peptide resin (Fmoc-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu)- Wang-resin) was reacted with 1.25 ml of deprotection solution twice for 5 minutes to deprotect the N-terminal Fmoc group. Then after DMF at 30 seconds 6 washes 1.25 ml, washed as in CH ₂ Cl _2, dried for 10 minutes blowing N _2, Kuribe chromatography di solution (Trifluoroacetic acid: Phenol: Water: Tioanisole: Ethanedithiol = 82.5: 5: 5: 5: 2.5) was added and reacted at room temperature for 2 hours (DSKing, Int. J. Peptide Protein Reg., 36, 255 (1990)). The side chain protecting group was removed to obtain a peptide (Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser).

  After completion of the reaction, the peptide solution was filtered using a filter and separated into a resin and a filtrate. Further, 2.5 ml of the washed resin was combined and collected in a centrifuge tube. The collected peptide solution was removed from the apparatus and 5 ml of cold ether was added to precipitate the peptide. After cooling for a while, this was centrifuged (3000 rpm for 10 minutes), the precipitate was collected, and cold ether was added again to disperse and recover, and the peptide was washed 5 to 6 times repeatedly.

  The obtained peptide was dried to obtain a crude peptide. 11 mg of the obtained crude peptide was dissolved in 10% acetonitrile aqueous solution containing 2 ml of 0.1% TFA, and then divided into three times and supplied to an ODS column (TSKgel ODS-12OT, 7.8 mm × 30 cm: manufactured by Tosoh Corporation) Development with 21% acetonitrile containing 0.1% TFA (flow rate 2 ml / min, detection wavelength 220 nm), fractions eluted at 9.2-11 min were collected, concentrated, and lyophilized for the purpose Was obtained (5 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 10
Peptide 10:
Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-Trp
In the same manner as in Example 9, the peptide (Fmoc-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu) -Trp-Wang- Resin) was synthesized. However, 25 μmol of Fmoc-Trp-Wang-resin (0.66 mmol / g) was used as the C-terminal amino acid resin. The amino acids used in the synthesis are as follows.
Fmoc-Ala, Fmoc-Pro, Fmoc-Asn (Trt),
Fmoc-Gln (Trt), Fmoc-Tyr (tBu), Fmoc-Ile,
Fmoc-Gly, Fmoc-Ser (tBu)

  A peptide (Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-Trp) was obtained by the same procedure as in Example 9, and this peptide solution was centrifuged. After that, the peptide was precipitated to obtain a crude peptide.

  9 mg of the obtained crude peptide was dissolved in 10% aqueous acetonitrile containing 4 ml of 0.1% TFA, and then divided into two portions and supplied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). Then, the resultant was developed with 23% acetonitrile containing 0.1% TFA, and the fraction eluted at 32 to 38 minutes was concentrated and freeze-dried to obtain the desired peptide (2.5 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 11
Peptide 11:
Ile-Trp-Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu
In the same manner as in Example 9, the peptide (Fmoc-Ile-Trp-Leu-Gln (Trt) -Phe-Ala-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu- Wang-resin) was synthesized. However, Fmoc-Leu-Wang-resin (0.69 mmol / g) was used in an amount equivalent to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are as follows.
Fmoc-Leu, Fmoc-Thr (tBu),
Fmoc-Phe, Fmoc-Gly, Fmoc-Lys (Boc),
Fmoc-Ala, Fmoc-Gln (Trt), Fmoc-Trp,
Fmoc-Ile,

  The peptide (Ile-Trp-Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu) was obtained by carrying out a cleave reaction in the same manner as in Example 9. After collecting in a centrifuge tube, the peptide was precipitated to obtain a crude peptide.

  7 mg of the obtained crude peptide was dissolved in 4 ml of 20% acetonitrile aqueous solution containing 0.1% TFA, and then divided into three times and supplied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). Development with 37% acetonitrile containing 0.1% TFA (flow rate 2 ml / min, detection wavelength 220 nm), fractions eluted at 17-20 min are collected, concentrated, and lyophilized for the purpose. The peptide was obtained (0.7 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 12
Peptide 12:
Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu
Peptide (Fmoc-Leu-Gln (Trt) -Phe-Ala-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Wang-resin) in the same manner as in Example 9 And a cleave reaction was performed to obtain a peptide (Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu), and this peptide solution was collected in a centrifuge tube. Thereafter, the peptide was precipitated to obtain a crude peptide. The amino acids used in the synthesis are as follows.
Fmoc-Leu, Fmoc-Thr (tBu), Fmoc-Phe,
Fmoc-Gly, Fmoc-Lys (Boc), Fmoc-Ala,
Fmoc-Gln (Trt),

  Of the obtained crude peptide, 9.6 mg was dissolved in 2 ml of 20% acetonitrile aqueous solution containing 0.1% TFA, and then divided into two times and applied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). Developed with 32% acetonitrile containing 0.1% TFA (flow rate 2 ml / min, detection wavelength 220 nm), fractions eluted at 11-16 min were collected, concentrated and freeze-dried Peptide (6.4 mg) was obtained. When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 13
Peptide 13
Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met
In the same manner as in Example 9, the peptide (Fmoc-Leu-Gln (Trt) -Phe-Ala-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Met-Wang- Resin) was synthesized. However, Fmoc-Met-Wang-resin (0.75 mmol / g) was used in an amount equivalent to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are the same as in Example 12. A peptide (Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met) was obtained by carrying out a cleave reaction in the same manner as in Example 9, and this peptide solution was centrifuged. After that, the peptide was precipitated to obtain a crude peptide.

  8 mg of the obtained crude peptide was dissolved in 2 ml of 20% acetonitrile aqueous solution containing 0.1% TFA, and then divided into two portions and supplied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). Then, the resultant was developed with 30% acetonitrile containing 0.1% TFA, and the fraction eluted at 25 to 32 minutes was concentrated and then lyophilized to obtain the desired peptide (1.1 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 14
Peptide 14:
Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu
Peptide (Fmoc-Gln (Trt) -Phe-Ala-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Wang-resin) was synthesized in the same manner as in Example 9. did. However, Fmoc-Leu-Wang-resin (0.69 mmol / g) was used in an amount equivalent to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are the same as in Example 12. The peptide (Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu) was obtained by carrying out a cleave reaction in the same manner as in Example 9, and this peptide solution was recovered in a centrifuge tube. Thereafter, the peptide was precipitated to obtain a crude peptide.

  2.5 mg of the obtained crude peptide was dissolved in 1 ml of a 20% acetonitrile aqueous solution containing 0.1% TFA, and then divided into two portions on an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). The resulting peptide was developed with 30% acetonitrile containing 0.1% TFA, and the fraction eluted at 10 to 12 minutes was concentrated and then lyophilized to obtain the desired peptide (0.6 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 15
Peptide 15:
Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met
Peptide (Fmoc-Gln (Trt) -Phe-Ala-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Met-Wang-resin) in the same manner as in Example 9 Was synthesized. However, Fmoc-Met-Wang-resin (0.75 mmol / g) was used in an amount equivalent to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are the same as in Example 12. The peptide (Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met) was obtained by the same procedure as in Example 9, and this peptide solution was collected in a centrifuge tube. Thereafter, the peptide was precipitated to obtain a crude peptide.

  7 mg of the obtained crude peptide was dissolved in 4 ml of 0.1% TFA in 20% acetonitrile aqueous solution and then divided into two portions and supplied to an ODS column (TSKgel ODS-120T, 7.8 mm x 30 cm: manufactured by Tosoh Corporation). Then, it was developed with 30% acetonitrile containing 0.1% TFA, and the fraction eluted at 15 to 20 minutes was concentrated and then lyophilized to obtain the desired peptide (1.9 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 16
Peptide 16:
Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met-Gly
In the same manner as in Example 9, the peptide (Fmoc-Gln (Trt) -Phe-Ala-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Met-Gly-Wang- Resin) was synthesized. However, Fmoc-Gly-Wang-resin (0.50 mmol / g) was used in an amount equivalent to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are as follows.
Fmoc-Leu, Fmoc-Thr (tBu), Fmoc-Phe,
Fmoc-Gly, Fmoc-Lys (Boc), Fmoc-Ala,
Fmoc-Gln (Trt), Fmoc-Met

  The peptide (Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met-Gly) was obtained by the same procedure as in Example 9, and this peptide solution was centrifuged. After that, the peptide was precipitated to obtain a crude peptide.

  13 mg of the obtained crude peptide was dissolved in 6 ml of 20% acetonitrile aqueous solution containing 0.1% TFA, and then divided into three times and supplied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). Then, the resultant was developed with 29% acetonitrile containing 0.1% TFA, and the fraction eluted at 17 to 20 minutes was concentrated and then lyophilized to obtain the desired peptide (0.9 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 17
Peptide 17:
Ile-Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn
In the same manner as in Example 9, the peptide (Fmoc-Ile-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc ) -Asn (Trt) -Wang-resin). However, 25 μmol of Fmoc-Asn (Trt) -Wang-resin (0.60 mmol / g) was used as the C-terminal amino acid resin. The amino acids used in the synthesis are as follows.
Fmoc-Leu, Fmoc-Asn (Trt), Fmoc-Ile,
Fmoc-Phe, Fmoc-Lys (Boc), Fmoc-His (Trt),
Fmoc-Ala, Fmoc-Gln (Trt), Fmoc-Ser (tBu),

  A cleave reaction was carried out in the same manner as in Example 9 to obtain a peptide (Ile-Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn), and this peptide solution was centrifuged. After that, the peptide was precipitated to obtain a crude peptide.

  3.8 mg of the obtained crude peptide was dissolved in 10 ml of 10% acetonitrile solution containing 4 ml of 0.1% TFA, and then divided into two times on an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). The sample was developed with 18% acetonitrile containing 0.1% TFA (flow rate 2 ml / min, detection wavelength 220 nm), the fraction eluted at 12 to 15 minutes was collected, concentrated, and lyophilized for the purpose. Was obtained (1.9 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 18
Peptide 18:
Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-Thr
In the same manner as in Example 9, the peptide (Fmoc-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc)- Asn (Trt) -Thr (tBu) -Wang-resin) was synthesized. However, Fmoc-Thr (tBu) -Wang-resin (0.50 mmol / g) was used in an amount corresponding to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are as follows.
Fmoc-Leu, Fmoc-Asn (Trt), Fmoc-Phe,
Fmoc-Lys (Boc), Fmoc-His (Trt), Fmoc-Ala,
Fmoc-Gln (Trt), Fmoc-Ser (tBu),

  A peptide (Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-Thr) was obtained by carrying out a cleave reaction in the same manner as in Example 9, and this peptide solution was centrifuged. After that, the peptide was precipitated to obtain a crude peptide.

  5 mg of the obtained crude peptide was dissolved in 4 ml of 10% acetonitrile aqueous solution containing 0.1% TFA, and then divided into two portions and supplied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). Then, the resultant was developed with 15% acetonitrile containing 0.1% TFA, and the fraction eluted at 22 to 30 minutes was concentrated and freeze-dried to obtain the desired peptide (3.5 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 19
Peptide 19:
Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn
In the same manner as in Example 9, the peptide (Fmoc-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc)- Asn (Trt) -Wang-resin) and cleaved to obtain a peptide (Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn) Was collected in a centrifuge tube. Thereafter, the peptide was precipitated to obtain a crude peptide. The amino acids used in the synthesis are the same as in Example 18.

  6 mg of the obtained crude peptide was dissolved in 10% aqueous acetonitrile containing 4 ml of 0.1% TFA, and then divided into two portions and supplied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). The mixture was developed with 15% acetonitrile containing 0.1% TFA, and the fraction eluted at 20 to 28 minutes was concentrated and freeze-dried to obtain the desired peptide (3.8 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 20
Peptide 20:
Leu-Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu
In the same manner as in Example 9, the peptide (Fmoc-Leu-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) ) -Leu-Wang-resin). However, Fmoc-Leu-Wang-resin (0.69 mmol / g) was used in an amount equivalent to 25 μmol for the C-terminal amino acid resin. The amino acids used in the synthesis are as follows.
Fmoc-Leu, Fmoc-Thr (tBu), Fmoc-Asn (Trt),
Fmoc-Gly, Fmoc-Lys (Boc), Fmoc-Cys (Trt),
Fmoc-Ala, Fmoc-Ser (tBu), Fmoc-Ile

  The peptide (Leu-Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu) was obtained by carrying out a cleave reaction in the same manner as in Example 9, and this peptide solution was centrifuged. After that, the peptide was precipitated to obtain a crude peptide.

  10 mg of the resulting crude peptide was dissolved in 10% acetonitrile aqueous solution containing 4 ml of 0.1% TFA, and then divided into three times and supplied to an ODS column (TSKgel ODS-120T, 7.8 mm x 30 cm: manufactured by Tosoh Corporation) Development with 23% acetonitrile containing 0.1% TFA (flow rate 2 ml / min, detection wavelength 220 nm), fractions eluted at 18-22 min are collected, concentrated and freeze-dried for the purpose The peptide was obtained (0.9 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 21
Peptide 21:
Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu
In the same manner as in Example 9, the peptide (Fmoc-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt)- Leu-Wang-resin) and cleaved to obtain a peptide (Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu). Recovered. Thereafter, the peptide was precipitated to obtain a crude peptide. The amino acids used in the synthesis are the same as in Example 20.

  Of the obtained crude peptide, 6.6 mg was dissolved in 2 ml of 10% acetonitrile aqueous solution containing 0.1% TFA, and then divided into two times and applied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). Developed with 19% acetonitrile containing 0.1% TFA (flow rate 2 ml / min, detection wavelength 220 nm), fractions eluted at 17-22 min were collected, concentrated and freeze-dried Was obtained (1.5 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 22
Peptide 22:
Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn
In the same manner as in Example 1, the peptide (Fmoc-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt)- Leu-Asn (Trt) -Wang-resin) was synthesized. However, 25 μmol of Fmoc-Asn (Trt) -Wang-resin (0.60 mmol / g) was used as the C-terminal amino acid resin. The amino acids used in the synthesis are the same as in Example 20. The peptide (Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu) was obtained by carrying out a cleave reaction in the same manner as in Example 9, and this peptide solution was recovered in a centrifuge tube. Thereafter, the peptide was precipitated to obtain a crude peptide.

  Of the obtained crude peptide, 6.9 mg was dissolved in 1 ml of 10% acetonitrile aqueous solution containing 0.1% TFA, and then divided into two portions and placed on an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). The resulting peptide was developed with 22% acetonitrile containing 0.1% TFA, and the fraction eluted at 9 to 12 minutes was concentrated and then lyophilized to obtain the desired peptide (1.6 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 23
Peptide 23:
Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn
The peptide (Fmoc-Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) was prepared in the same manner as in Example 9. ) -Wang-resin) and a cleave reaction to obtain a peptide (Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn). Recovered. Thereafter, the peptide was precipitated to obtain a crude peptide. The amino acids used in the synthesis are as follows.
Fmoc-Leu, Fmoc-Thr (tBu), Fmoc-Gly,
Fmoc-Cys (Trt), Fmoc-Ala, Fmoc-Ser (tBu),
Fmoc-Ile

  6 mg of the resulting crude peptide was dissolved in 1 ml of 20% acetonitrile aqueous solution containing 0.1% TFA, and then divided into three times and supplied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). And developed with 19% acetonitrile containing 0.1% TFA (flow rate 2 ml / min, detection wavelength 220 nm), fractions eluted at 15-17 min are collected, concentrated and freeze-dried for the purpose. The peptide was obtained (0.9 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Example 24
Peptide 24:
Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn-Asp
The peptide (Fmoc-Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) was prepared in the same manner as in Example 9. ) -Asp (OtBu) -Wang-resin). However, 25 μmol of Fmoc-Asp (OtBu) -Wang-resin (0.42 mmol / g) was used as the C-terminal amino acid resin. The amino acids used in the synthesis are as follows.
Fmoc-Leu, Fmoc-Thr (tBu), Fmoc-Gly,
Fmoc-Cys (Trt), Fmoc-Ala, Fmoc-Ser (tBu),
Fmoc-Ile Fmoc-Asn (Trt)

  A peptide (Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn-Asp) was obtained by carrying out a cleave reaction in the same manner as in Example 9, and this peptide solution was centrifuged. After that, the peptide was precipitated to obtain a crude peptide.

  7.5 mg of the resulting crude peptide was dissolved in 10% acetonitrile aqueous solution containing 1 ml of 0.1% TFA, and then divided into three times and applied to an ODS column (TSKgel ODS-120T, 7.8 mm × 30 cm: manufactured by Tosoh Corporation). The resulting peptide was developed with 18% acetonitrile containing 0.1% TFA, and the fraction eluted at 17 to 19 minutes was concentrated and then lyophilized to obtain the desired peptide (0.6 mg). When 50 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer PPSQ-10 type (manufactured by Shimadzu Corporation), the amino acid sequence shown above was confirmed.

Formulation Example 1
Solution One of the 24 peptides obtained by the method described in Examples 1 to 24 was dissolved in distilled water containing 1% (w / v) purified gelatin as a stabilizer to a final concentration of 0.1 g / ml, By sterilizing filtration by a conventional method, 24 types of solutions were obtained.

  Since susceptibility to the peptide of the present invention usually varies from individual to individual, this product is used by appropriately blending 24 types of liquid preparations so that the composition is most suitable for each individual. Since this product is excellent in stability, it is useful as a solution for eye drops, nasal drops, and oral sprays for treating and preventing cedar pollinosis.

Formulation Example 2
24 kinds of peptides obtained by the method described in Examples 1 to 24 in physiological saline containing 1% (w / v) human serum albumin as an injection stabilizer to a final concentration of 0.01, 0.1 or 1 mg / ml, respectively. After dissolution in sterilization and sterile filtration, 2 ml each was dispensed into sterile vials, lyophilized and sealed.

  Prior to administration, first add 1 ml of distilled water for injection into a vial, and then dissolve the contents uniformly before use. The product having excellent stability and comprising 24 kinds of polypeptides according to the present invention as an active ingredient is useful as a dry preparation for treating and preventing cedar pollinosis.

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

  0.2 mg of each peptide obtained by the method described in Examples 1 to 24 was added, and the mixture was allowed to react at 37 ° C. for 12 hours with gentle stirring while keeping the pH of the solution at around 7. After the reaction, 4 g of glycine was added to the reaction product, and incubated at 37 ° C. for 5 hours with gentle stirring to block unreacted active groups. The reaction was concentrated and applied to a Sephadex G-50 column that had been equilibrated with 0.1 M phosphate buffer (pH 7.0), and the same buffer was passed through the column. Fractions containing the peptide and pullulan complex were collected. The yield was about 30% per raw peptide solid content.

  This fraction is sterile filtered, concentrated, lyophilized, pulverized and mixed uniformly with mannitol, and the mixture is compressed to give 2, 10, or 50 mg of complex per tablet (200 mg) according to conventional methods. A tablet containing was obtained. This product with excellent ingestion and stability is useful as a sublingual agent for treating and preventing cedar pollinosis.

Formulation Example 4
1 g of purified ribopolysaccharide derived from syrup E. coli was dissolved in 100 ml of 10 mM calcium phosphate 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 the ribopolysaccharide. The reaction product was dialyzed overnight against 1 M glycine-hydrochloric acid buffer (pH 4.4) at 4 ° C to remove unreacted periodic acid, and then adjusted to about pH 9.5 with 0.1 M sodium bicarbonate buffer. Separately, 24 types of peptides obtained by the methods described in Examples 1 to 24 were dissolved in 10 mg each in 100 ml of 0.1 M phosphate buffer (pH 7.0), added to the reaction product containing activated ribopolysaccharide, The reaction was allowed to stand for 12 hours underneath.

  Thereafter, the newly obtained reaction product is purified by the method of Formulation Example 3, and the resulting fraction containing the complex of the peptide of the present invention and ribopolysaccharide is concentrated, lyophilized, pulverized and solidified. It was. The yield was about 30% per raw peptide solid content. This solid is dissolved in distilled water containing 1% (w / w) purified gelatin as a stabilizer so that the final concentration of sucrose is 0.1 or 1 mg / ml or 50% (w / w), respectively. The solution was sterilized by filtration to obtain a syrup. Each 2 ml of this syrup was dispensed into sterile vials and sealed to give a product.

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

SEQ ID NO: 1-description of artificial sequence: synthetic peptide SEQ ID NO: 2-description of artificial sequence: synthetic peptide SEQ ID NO: 3-description of artificial sequence: synthetic peptide SEQ ID NO: 4-description of artificial sequence: synthetic peptide SEQ ID NO: 5-artificial sequence Description: Synthetic Peptide Sequence No. 6-Description of Artificial Sequence: Synthetic Peptide Sequence Number 7-Description of Artificial Sequence: Synthetic Peptide Sequence Number 8-Description of Artificial Sequence: Synthetic Peptide Sequence Number 9-Description of Artificial Sequence: Synthetic Peptide Sequence No. 10-description of artificial sequence: synthetic peptide SEQ ID NO: 11-description of artificial sequence: synthetic peptide SEQ ID NO: 12-description of artificial sequence: synthetic peptide SEQ ID NO: 13-description of artificial sequence: synthetic peptide SEQ ID NO: 14-of artificial sequence Description: Synthetic peptide SEQ ID NO: 15-description of artificial sequence: Synthetic peptide SEQ ID NO: 16-description of artificial sequence: synthetic peptide SEQ ID NO: 17 Description of Artificial Sequence: Synthetic Peptide SEQ ID NO: 18-Description of Artificial Sequence: Synthetic Peptide SEQ ID NO: 19-Description of Artificial Sequence: Synthetic Peptide SEQ ID NO: 20-Description of Artificial Sequence: Synthetic Peptide SEQ ID NO: 21-Description of Artificial Sequence: Synthesis Peptide SEQ ID NO: 22-description of artificial sequence: synthetic peptide SEQ ID NO: 23-description of artificial sequence: synthetic peptide SEQ ID NO: 24-description of artificial sequence: synthetic peptide

Claims (28)

  A peptide consisting of the amino acid sequence of SEQ ID NO: 2.   A peptide consisting of the amino acid sequence of SEQ ID NO: 3.   A peptide consisting of the amino acid sequence of SEQ ID NO: 9.   A peptide consisting of the amino acid sequence of SEQ ID NO: 10.   A peptide consisting of the amino acid sequence of SEQ ID NO: 17.   A peptide consisting of the amino acid sequence of SEQ ID NO: 18.   A peptide consisting of the amino acid sequence of SEQ ID NO: 19.   An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 2 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 3 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 4 as an active ingredient.   An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 5 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 6 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 9 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 10 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 11 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 12 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 13 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 14 as an active ingredient.   An anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 15 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 16 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 17 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 18 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 19 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 20 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 21 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 22 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 23 as an active ingredient.   The anti-cedar pollinosis agent which uses the peptide which consists of an amino acid sequence of sequence number 24 as an active ingredient.