WO2014017599A1 - Novel t cell epitope peptide of japanese cypress pollen allergen - Google Patents

Description

Novel cypress pollen allergen T cell epitope peptide

The present invention relates to a novel allergen protein T-cell epitope peptide derived from hinoki pollen and a composition for peptide immunotherapy against hinoki hay fever, comprising the peptide as an active ingredient.

The amount of cypress pollen scattered was slight in the 1940s, but increased more than three times over the next 20 years. Cedar tree planting was intensively carried out in the Showa 20s, and in the latter half of the Showa 40s, it reached a golden age when pollen began to fly. Cypress trees were planted intensively in the Showa 30s after planting cedar trees, and have reached the middle age after cedar trees. In recent years, the number of cypress vegetation is comparable to the number of cedar vegetation, and it is expected that cypress pollinosis will increase in the future. Therefore, the need for immunotherapy using cypress pollen-specific antigens is necessary. Has been pointed out.

The major problem with immunotherapy is the anaphylactic reaction. The anaphylactic reaction is triggered by a chemical mediator that is released by binding of antigen-specific IgE antibody and antigen on mast cells. Peptide immunotherapy using a T cell epitope is an immunotherapy that does not contain a portion that binds to an IgE antibody and contains a T cell epitope related to immune tolerance as an active ingredient. To date, feline, tick, cedar pollen, ragweed pollen Peptide immunotherapeutic agents using T cell epitopes identified from such antigens have been clinically applied (Non-patent Documents 1 and 2). In addition, regarding cypress pollen, T cell epitope peptides have been identified for Cha o1 and Cha o2, which are the main antigens (Patent Document 1, Non-Patent Documents 3 and 4).

JP 2008-195718 A

Valenta R. et al., Annu. Rev. Immunol., 2010: 211-241, 2010 Casale T. et al., J. Allergy Clin. Immunol., 127: 8-15, 2011 Sone, T. et al., Clin. Exp. Allergy,: 35: 664-671,6712005 Sone, T. et al., Allergol. Int., 58: 237-245, 2009

The present invention relates to providing a novel cypress pollen allergen T cell epitope peptide different from known cypress pollen allergens in order to contribute to improving the efficacy of immunotherapy for cypress pollinosis, which is expected to increase in the future.

The present inventors have found a novel hinoki pollen allergen present in a large amount in cypress pollen and having allergen activity equal to or higher than Cha o1 and Cha o2, and has already filed a patent application (PCT / JP2012 / 052110). Further, T cell lines specific to the cypress pollen allergen were individually established from cedar pollinosis cypress pollen sensitized cases, and major T cell epitopes were determined from the T cell lines, thereby completing the present invention.
That is, the present invention relates to the following 1) to 4).
1) A peptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 3 to 16, or a peptide in which 1 to 3 amino acids are substituted and / or 1 to 10 amino acids are deleted or added in the peptide And a peptide having T cell stimulating activity.
2) The peptide according to 1) above, wherein the peptide in which 1 to 10 amino acids are deleted is a peptide in which the N-terminal side and / or C-terminal side amino acids of the peptide are deleted.
3) A peptide having the amino acid sequence represented by SEQ ID NO: 7 and lacking the amino acid at the N-terminal side and / or C-terminal side thereof and having at least “FKEKIVEYGHW”, a peptide comprising the amino acid sequence represented by SEQ ID NO: 10 N-terminal and / or C-terminal amino acids are deleted, and at least the peptide having “PYYQIIYHPAS” or the amino acid sequence shown by SEQ ID NO: 15 lacks the N-terminal and / or C-terminal amino acids. The peptide according to 2) above, which is a peptide having at least “FKVITTNKQ”.
4) A peptide immunotherapeutic agent for cypress pollinosis containing one or more of the peptides described in any one of 1) to 3) as an active ingredient.
5) Use of one or more of the aforementioned peptides 1 to 3 for producing a peptide immunotherapeutic agent for cypress pollinosis.
6) One or more of the aforementioned peptides 1 to 3 for use in peptide immunotherapy for cypress pollinosis.
7) Peptide immunotherapy for cypress pollinosis in which one or more of the above-mentioned peptides 1 to 3 are administered.

The peptide group of the present invention is useful as an immunotherapeutic agent for hinoki hay fever alone or in combination. Furthermore, it is useful for cypress pollen-sensitized cases where the effect of immunotherapy using cedar pollen extract was insufficient by combining with known cypress pollen allergens, Cha o1 and Cha o2, T cell epitope peptides. Peptide immunotherapy is possible.

The T cell stimulating activity of each overlapping peptide is shown. The binding ability of the peptide of the present invention to cypress pollen-specific IgE is shown. The T cell stimulation activity of a shortened peptide (peptide number: # 52) is shown. The T cell stimulation activity of a shortened peptide (peptide number: # 24) is shown. The T cell stimulation activity of a shortened peptide (peptide number: # 38) is shown.

Hereinafter, the present invention will be described in detail. In addition, the amino acid sequence in this specification is described by one-letter code.
The peptide consisting of the amino acid sequence shown by SEQ ID NO: 3 to 16 of the present invention is a cypress pollen allergen protein consisting of the amino acid sequence shown by SEQ ID NO: 1, or its isoform (the second amino acid in the amino acid sequence shown by SEQ ID NO: 1). Pro is replaced with Thr, 230th Val is replaced with Ile, and 271nd Val is replaced with Ile). 28 amino acids from the N-terminal which is a signal peptide from the primary structure of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 Is a partial peptide consisting of 20 amino acid residues (partially 18 amino acid residues) of a protein consisting of all 528 amino acids from N-terminal Leu to C-terminal Leu. The positions on the amino acid sequence represented by SEQ ID NO: 1 or 2 of each peptide of the present invention are as shown in Tables 1 to 3 below, that is, SEQ ID NO: 3 (peptide number: # 12): 111 of SEQ ID NO: 1 ~ 130th, SEQ ID NO: 4 (peptide number: # 13): 121-140th of SEQ ID NO: 1, SEQ ID NO: 5 (peptide number: # 20): 191st to 210th of SEQ ID NO: 1, SEQ ID NO: 6 (peptide number) : # 21): 201 to 220 of SEQ ID NO: 1, SEQ ID NO: 7 (peptide number: # 24): 231 to 250 of SEQ ID NO: 1, SEQ ID NO: 8 (peptide number: # 31): 301 of SEQ ID NO: 1 320th, SEQ ID NO: 9 (peptide number: # 37): 361 to 380th of SEQ ID NO: 1, SEQ ID NO: 10 (peptide number: # 38): 371 to 390th of SEQ ID NO: 1, SEQ ID NO: 1 (peptide number: # 39): 381-400th of SEQ ID NO: 1, SEQ ID NO: 12 (peptide number: # 41): 401-420th of SEQ ID NO: 1, SEQ ID NO: 13 (peptide number: # 42): sequence 411 to 430 of SEQ ID NO: 1, SEQ ID NO: 14 (peptide number: # 49): 481 to 500 of SEQ ID NO: 1, SEQ ID NO: 15 (peptide number: # 52): 511 to 528 of SEQ ID NO: 1, SEQ ID NO: 16 (peptide number: # 56): positions 261 to 280 of SEQ ID NO: 2.

The peptide of the present invention individually establishes a T cell line specific to the cypress pollen allergen protein consisting of the amino acid sequence represented by SEQ ID NO: 1 from the cedar pollinosis cypress pollen sensitized case, It was identified as a T cell epitope by evaluating the proliferation activity against 57 overlapping peptides of 20 residues covering the entire primary structure of the isoform (see Examples 2 to 4). Therefore, the peptide of the present invention has at least one T cell epitope, and the peptide of the present invention has T cell stimulating activity.
Here, the T cell stimulating activity refers to an activity for inducing a T cell response such as T cell proliferation or lymphokine secretion, and means an action capable of inducing immune tolerance of T cells by administration to humans or animals. Here, the T cell stimulating activity can be determined, for example, by measuring [ ³ H] thymidine incorporation into cells and calculating the stimulation coefficient. The stimulation factor (SI value) of the T cell response used here is the cpm of the radioactivity of [ ³ H] thymidine incorporated into the cell in the presence of the peptide, as the cpm in the absence of the peptide (control). Calculate as divided. The peptides of the present invention have an average S.P. I. The value is 2.0 or more.
Of these, the average S.D. I. It is preferable that the “importance index” obtained by multiplying the value by the appearance frequency (%) of a patient exhibiting a T cell response to the peptide is at least 70 or more, and as such a peptide, SEQ ID NO: 4 (peptide number: # 13), SEQ ID NO: 5 (peptide number: # 20), SEQ ID NO: 7 (peptide number: # 24), SEQ ID NO: 8 (peptide number: # 31), SEQ ID NO: 10 (peptide number: # 38), SEQ ID NO: 11 (Peptide number: # 39), the peptide which consists of an amino acid sequence shown by sequence number 12 (peptide number: # 41) and sequence number 15 (peptide number: # 52) is mentioned. Such peptides do not bind to IgE antibodies specific to cedar pollen and cypress pollen allergens and do not cause anaphylaxis, and therefore can induce immune tolerance against T cells specific to cypress pollen allergens. It is particularly suitable as an immunotherapeutic agent that can be used.

In the present invention, in addition to the peptide consisting of the amino acid sequence shown in SEQ ID NOs: 3 to 16, in the peptide, 1 to 3 amino acids are substituted and / or 1 to 10 amino acids are deleted or added And peptides having T cell stimulating activity.
Here, the peptide from which 1 to 10 amino acids have been deleted includes, for example, a peptide from which the N-terminal and / or C-terminal amino acids have been deleted. For example, the N-terminal amino acids 0 to 10 residues, C-terminal amino acids 0 to 10 residues, a total of 1 to 10 amino acids deleted.
Specifically, for example, in the case of the peptide (# 24) consisting of the amino acid sequence represented by SEQ ID NO: 7, the amino acid residues 1-3 on the N-terminal side and / or the amino acid residues 1-6 on the C-terminal side are missing. In the case of a peptide having at least “FKEKIVIEGGHW” and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 10 (# 38), 6 to 8 amino acids on the N-terminal side and / or 1 amino acid residue on the C-terminal side Is a peptide having at least “PYYQIIYHPAS” and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 15 (# 52), amino acid 1 to 2 residues on the N-terminal side and / or amino acid 1 on the C-terminal side Peptides lacking ˜7 residues and having at least “FKVITTNKQ”.

The peptide to which 1 to 10 amino acids are added includes a peptide to which 1 to 10 amino acids are added to the N-terminal side and / or the C-terminal side. In this case, the added amino acid residues include In the amino acid sequence represented by SEQ ID NO: 1 or 2, examples include 1 to 10 amino acid residues adjacent to the N-terminal side or C-terminal side of the amino acid sequence corresponding to the peptide.
In addition, “having T cell stimulating activity” herein means having T cell stimulating activity equal to or higher than that of the peptide consisting of the amino acid sequences shown in SEQ ID NOs: 3 to 16. The meaning of the T cell stimulating activity is as described above.

The T cell epitope peptide of the present invention can be synthesized by solid phase synthesis, liquid phase synthesis, or a peptide synthesis method combining these, which is used for general peptide synthesis. When the T cell epitope peptide of the present invention is synthesized by recombinant DNA technology, host cells transformed with a nucleic acid having a sequence encoding the peptide are cultured in a medium suitable for the cells, and the culture supernatant is obtained. Or the host cell can be synthesized using techniques known to those skilled in the art. As the host, Escherichia coli, yeast, mammalian cells and the like can be used.

Peptide immunotherapy since the T cell epitope peptide of the present invention can modulate an allergic response to an individual's allergen when administered to a human, preferably when administered to an individual sensitized to cypress pollen allergen Useful for. Furthermore, in addition to the T-cell epitope peptides of other cypress pollen allergens, Cha o1 and Cha o2, the T-cell epitope peptides of Cry j1 and Cry j2, which are cedar pollen allergens (JP-A-7-118295, JP-A-8- 47392, Sone, T. et al., J. Immunol., 161: 448-457, 1998), useful for peptide immunotherapy for spring tree hay fever patients represented by Sugi and Hinoki It is.

The cypress pollinosis peptide immunotherapeutic agent of the present invention can be administered by a normal administration route such as injection (subcutaneous, intravenous injection, etc.), eye drop, nasal drop, oral, inhalation, transdermal, etc. Tablets, capsules, granules, powders, troches, sublingual tablets, syrups, injections, suppositories, inhalants, transdermal absorption agents, eye drops, nasal drops, intranasal sprays, poultices, It can be prepared as preparations of various dosage forms such as cataplasms, ointments, lotions and creams.
In this case, the T cell epitope peptide of the present invention is used as it is or dried to form a powder, and a carrier such as a diluent and an additive, such as a stabilizer, an excipient, a solubilizing agent, an emulsifying agent, a buffer, It is preferably prepared as a compounding agent to which a soothing agent, a preservative, a coloring agent and the like are added by a conventional method.

The dosage of the cypress pollinosis peptide immunotherapeutic agent of the present invention can be, for example, by injection, about 1 μg to 30 mg, preferably 20 μg to 10 mg of the peptide of the present invention per dosage unit.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by these Examples.

Example 1 Purification of cypress pollen allergen protein 1000 ml of extraction buffer (25 mM Tris buffer, pH 8.0) was added to 50 g of cypress pollen and stirred for 10 minutes, followed by sonication for 15 minutes. The obtained suspension was centrifuged (3,000 rpm, 15 minutes) to obtain a supernatant, which was filtered to obtain an extract. This extract was added to a DE52 anion exchange column equilibrated with 10 mM Tris buffer (pH 8.0), and non-adsorbed fractions were collected. Subsequently, the resultant was adsorbed on a CM52 cation exchange column equilibrated with 20 mM acetate buffer (pH 5.2), and gradient-eluted with NaCl-containing 10 mM Tris buffer (pH 7.8) was divided into fractions and collected. A part of each fraction was analyzed by SDS-PAGE, and fractions containing a protein with a molecular weight of about 66 kDa were combined.

Example 2 Synthesis of Overlapping Peptides Overlapping peptides were synthesized under commission by the PepTrack Peptide Library service (JPT Peptide Technologies). Based on the primary structure of the cypress pollen allergen protein (hereinafter referred to as “Protein X”) (SEQ ID NO: 1) obtained in Example 1, starting from the N-terminal first Leu to the C-terminal Leu, 10 52 types of overlapping peptides (# 1 to # 52) containing 20 overlapping residues (18 residues for # 52 (SEQ ID NO: 52)) were synthesized (Tables 1-2). As an isoform of this protein, there is a protein in which the second Pro is replaced with Thr, the 230th Val is Ile, and the 271st Val is Ile in the amino acid sequence represented by SEQ ID NO: 1. (SEQ ID NO: 2) For these, 5 types of overlapping peptides (# 53 to # 57) were similarly prepared (Table 3).

Example 3 Establishment of a novel cypress pollen-specific T cell line Lymphocytes were isolated from the peripheral blood of 20 patients sensitized to cypress pollen due to cedar pollinosis by the commonly used Ficoll-paque specific gravity centrifugation method, A portion was stored in liquid nitrogen. Peripheral blood lymphocytes (1.2 × 10 ⁷ cells) were suspended in RPMI-1640 supplemented with 6 ml of autologous plasma 10% and cultured for 7-8 days on a 24-well culture plate with Protein X purified from cypress pollen. did. When T cells activated by antigen stimulation were confirmed under a microscope, 20 Unit / ml IL-2 was added and cultured overnight. From the next day, the culture medium was changed daily with RPMI-1640 supplemented with 20 Unit / ml IL-2, 10% autologous plasma, and cultured for 10-12 days as a T cell line for T cell epitope identification. did.

Example 4 Identification of Peptide Containing T Cell Epitope T cell lines established from 20 hay fever patients were cultured with each synthesized overlapping peptide, and Protein X specific T cell epitope peptide was identified. That is, peripheral blood mononuclear cells frozen and stocked from the same patient as the T cell line were thawed as antigen-presenting cells, X-irradiated (36 Gy), and suspended in RPMI-1640 supplemented with 10% autologous plasma. The antigen-presenting cells were seeded in a 96-well flat bottom plate (1 × 10 ⁵ cells / well), and then Protein X (final concentration 10 μg / ml) or each peptide (final concentration 2 μM) was added to each well. Next, a T cell line (2 × 10 ⁴ cells / well) suspended in RPMI-1640 supplemented with 10% autologous plasma was seeded in each well, and after culturing at 37 ° C. and 5% CO ₂ for 48 hours, 1 μCi [ ³ H] thymidine was added to the wells and incubated for another 16 hours. The cells were collected on a glass filter using a harvester, dried, and the radioactivity (cpm) of [ ³ H] thymidine incorporated into the cells was measured with a top count (manufactured by PerkinElmer). As an index of proliferative activity, the value obtained by dividing the radioactivity of the T cell line by peptide stimulation by the radioactivity without the addition of peptide (control) was obtained from S. cerevisiae. I. Calculated as a value. I. Peptides showing values of 2 or more were identified as T cell epitope peptides. FIG. 1 shows the “average SI value” (average value of 20 SI values) and “appearance frequency (%)” of all overlapping peptides (the SI values in 20 people are the same). 2) and “importance index” (a value obtained by multiplying the average SI value and the appearance frequency).

Example 5 Confirmation of binding ability to cypress pollen-specific IgE Binding ability to cypress pollen-specific IgE was confirmed using 5 cypress pollinosis patient sera. A preferred T cell epitope peptide (importance index of 100 or more) or cypress pollen allergen of the present invention is added to a Maxisorp 96-well plate (Nunk) at 1 μg / 50 μL / well, and then allowed to stand overnight at 4 ° C. To be solid-phased. On the next day, after washing the plate, blocking was performed by adding PBS containing 1% bovine serum albumin and allowing to stand at room temperature for 1 hour. After washing the plate, patient serum or negative control serum diluted 5-fold was added and allowed to stand at room temperature for 2 hours. As a negative control, health donor serum (hinoki RAST Score = 0) was used. After the plate was washed, a secondary antibody (HRP-labeled goat anti-human IgE antibody, Novus) diluted 7000 times with PBS containing 1% bovine serum albumin was added, and left at room temperature for 40 minutes. After washing the plate, TMB color development was performed at room temperature in a TMB Substrate Reagent Set (BD Biosciences) for 20 minutes in a light-shielded state. After completion, color development was stopped by adding 1N sulfuric acid, and absorbance at a wavelength of 450 nm was measured with a microplate reader. The result is shown in FIG. In FIG. 2, V012, V014, V031, V047, and V051 indicate five cypress pollinosis patient IDs, respectively.
It was confirmed that the T cell epitope peptide of the present invention does not bind to cypress pollen-specific IgE.

Example 6 Establishment of 20-mer epitope-specific T cell line Lymphocytes were isolated from the peripheral blood of a patient sensitized to Japanese cypress pollen in Japanese cedar pollinosis by a commonly used Ficoll-paque specific gravity centrifugation method Stored in liquid nitrogen. Peripheral blood lymphocytes are suspended in RPMI-1640 supplemented with 10% autologous plasma and cultured in 96-well culture plates (1.5 × 10 ⁵ cells / well) for 7-8 days with 20-mer epitope peptide at a final concentration of 1 μM. did. In parallel, antigen-presenting cells necessary for passage were prepared on the 6th to 7th days of culture as follows. That is, peripheral blood mononuclear cells frozen from the same patient were put to sleep, suspended in RPMI-1640 medium supplemented with 10% autologous plasma, and then added to a 20-mer epitope peptide with a final concentration of 10 μM on a 24-well culture plate. Incubated overnight. On the next day, the cells were subjected to X-ray irradiation (45 Gy), and the 20-mer epitope peptide in the medium was washed and removed, and used as antigen-presenting cells for passage. On day 7 to 8 of lymphocyte culture, 1/3 of each well of lymphocytes inoculated in a 96-well culture plate was treated with human IL-2 at a final concentration of 100 Unit / ml and human at a final concentration of 50 ng / ml. The cells were cultured for 7 days on a 96-well culture plate with antigen-presenting cells for passage suspended in RPMI-1640 medium supplemented with IL-4 and 10% autologous plasma. The remaining 2/3 of each lymphocyte well was used for evaluation of antigen specificity. Of the lymphocytes cultured for 7 days with IL-2, IL-4, and passage antigen-presenting cells, all the lymphocytes of wells determined to be positive for 20-mer epitope peptide-specific T cells were collected, and a final concentration of 100 Unit / ml human Suspended in RPMI-1640 medium supplemented with IL-2, human IL-4 at a final concentration of 50 ng / ml and 10% autologous plasma, together with antigen-presenting cells for passage prepared as described above, in a 24-well culture plate for 7 days The culture was expanded. Thereafter, expansion culture was continued with the antigen-presenting cells for passage every 7 days, and a cell population showing 20-mer epitope peptide-specific cell proliferation was defined as a 20-mer epitope peptide-specific T cell line.

Example 7 Estimation of Epitope Core Sequence Peptides (# 52-1 to # 52-8, # 24-1 to # 24-8, # 38-1) with one amino acid residue deleted from the C-terminus of epitope 20mer ~ # 24-3), peptides in which one amino acid residue is deleted from the N-terminal (# 52-9 to # 52-13, # 24-9 to # 24-13), and 6 to 9 from the N-terminal Peptides (# 38-4 to # 24-7) lacking amino acid residues were synthesized. The established 20-mer epitope peptide-specific T cell line was cultured together with the shortened peptide to estimate the epitope core sequence. That is, peripheral blood mononuclear cells frozen and stocked from the same patient as the T cell line were thawed as antigen-presenting cells, X-ray irradiated (45 Gy), and suspended in RPMI-1640 supplemented with 10% autologous plasma. The antigen-presenting cells were seeded on a 96-well flat bottom plate (1 × 10 ⁵ cells / well), and then 20mer peptide or each shortened peptide (final concentration 2 μM) was added to each well. Next, a T cell line (2 × 10 ⁴ cells / well) suspended in RPMI-1640 supplemented with 10% autologous plasma was seeded in each well, and after culturing at 37 ° C. and 5% CO ₂ for 48 hours, 1 μCi [3H] thymidine was added to the wells and incubated for an additional 16 hours. The cells were collected on a glass filter using a harvester, dried, and the radioactivity (cpm) of [ ³ H] thymidine incorporated into the cells was measured with a top count (manufactured by PerkinElmer). As an index of proliferative activity, the value obtained by dividing the radioactivity of the T cell line by peptide stimulation by the radioactivity without the addition of peptide (control) was obtained from the S. p. I. Calculated as value. An example of the result is shown in FIGS.
The core sequence was determined by determining both ends based on the minimum sequence that showed T cell proliferation activity with a shortened peptide lacking the N-terminus and C-terminus (SI value of 2 or more). In the # 24 epitope peptide (SEQ ID NO: 7), the core sequence in the # 24 epitope peptide, # 38 epitope peptide and # 52 epitope peptide that can be estimated by the above method is the 4th to 14th “FKEKIVYEGHW”, and the # 38 epitope peptide In the peptide (SEQ ID NO: 10), it is the 9th to 19th “PYYQIIYHPAS”, and in the # 52 epitope peptide (SEQ ID NO: 15), it is the 3rd to 11th “FKVITTNNKQ”.

Claims (7)

A peptide selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 3 to 16, or a peptide in which 1 to 3 amino acids are substituted and / or 1 to 10 amino acids are deleted or added in the peptide, And a peptide having T cell stimulating activity. The peptide according to claim 1, wherein the peptide in which 1 to 10 amino acids are deleted is a peptide in which an amino acid on the N-terminal side and / or C-terminal side of the peptide is deleted. N-terminal and / or C-terminal side amino acids of the peptide consisting of the amino acid sequence shown by SEQ ID NO: 7 and having at least “FKEKIVEYGHW”, N-terminal of the peptide consisting of the amino acid sequence shown by SEQ ID NO: 10 And / or the C-terminal side amino acid is deleted, and the peptide having at least “PYYQIIYHPAS” or the peptide consisting of the amino acid sequence represented by SEQ ID NO: 15 is deleted at the N-terminal side and / or C-terminal side. The peptide according to claim 2, which is a peptide having at least “FKVITTNKQ”. A peptide immunotherapeutic agent for Japanese cypress pollinosis containing one or more of the peptides according to any one of claims 1 to 3 as an active ingredient. Use of one or more of the peptides described in any one of claims 1 to 3 for producing a peptide immunotherapeutic agent for cypress pollinosis. The peptide according to any one of claims 1 to 3, which is used for peptide immunotherapy for cypress pollinosis. Peptide immunotherapy for hinoki hay fever, wherein one or more of the peptides according to any one of claims 1 to 3 is administered.

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