JPH10259198A - Connected t-cell epitope and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new peptide that comprises a peptide including a specific amino acid sequence having T-cell epitope, deactivates human T-cell that specifically reacts with the cedar pollen allergen and is useful as an active ingredient for anti-cedar pollinosis. SOLUTION: This new peptide contains the amino acid sequence of the formula (α1 -αn have different sequences comprising 11-19 amino acids and represents the amino acid sequence constituting T-cell epitope originating from human cedar allergen protein Cryj1 or Crtj2; β1 -βn-1 represent an arbitrary sequence of 0-3 amino acids; m is an integer of >=2), its conjugated product, their derivatives or their polymer. When these peptides are given to general mammalians including human, the T-cell specific to the cedar pollen allergen is deactivated with no induction of anaphylaxis and is useful as an anti-cedar pollinosis. This peptide is prepared by solid phase synthesis according to the amino acid sequence of the cedar pollen allergen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

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

It has been known to date that at least two types of allergens differing in antigenicity exist in cedar pollen. One of them is based on the literature by Yasueda et al. [J. Allergy
Clin. Immunol. 71, 77-86 (1983)] and a protein called “Cryj1”.
88)] and Sakaguchi et al. [Allergy, No. 45, 309]
-312 (1990)]. Note that Cryj1, Cry
The entire amino acid sequence of both j2 has been determined to date [WO93 / 01213 and Japanese Translation of PCT International Publication No. 8-5052.
No. 84]. In cedar pollen, usually Cryj1
And Cryj2 are present in a ratio of about 50: 1 to 5: 1, and most of the serum collected from hay fever patients is Cryj
It is said that it reacts to both 1 and Cryj2. In addition,
Sawatani et al. [Allergy, Vol. 42, pp. 738-747 (199
3 years)], in the intradermal reaction test and RAST test,
Cryj1 and Cryj2 are reported to exhibit the same degree of antigenicity.

As described above, several cedar pollen allergens have already been isolated, and their properties and properties have been elucidated to some extent. The prospect of treating / preventing is coming. Recently, several desensitizing agents for this purpose have been devised. For example, JP-A-1-156926 and JP-A-3-93730 disclose an amino acid sequence from the N-terminus of Asp-Asn-Pro-Ile-. Asp-Ser or Ala-Ile
It has been proposed that a saccharide is covalently bonded to cedar pollen allergen represented by -Asn-Ile-Phe-Asn, and the resulting complex is administered to a human as a desensitizer.

However, diagnosis of allergic disease or hyposensitization therapy usually requires a large amount of high-purity allergen, and the amount of allergen in cedar pollen is low and the stability is low. It would be very difficult to cover the agent and desensitizer with cedar pollen alone. For this reason, in the recent treatment and prevention of allergic diseases, instead of administering the entire allergen to a patient as in the past, the minimum region that T cells in the allergen specifically recognizes, Attention has been drawn to immunotherapy in which small peptides consisting essentially of T cell epitopes are administered.

[0006] Generally, when an allergen is taken up by an antigen presenting cell such as a macrophage, it is digested there,
Digested fragments are HLA (Human Leukocyt) on the surface of antigen presenting cells.
e Antigen) binds to a protein and is presented with an antigen. Fragments presented as antigens are limited to some specific regions in the allergen due to affinity for the HLA protein and the like. Among these regions, those specifically recognized by T cells are usually called "T cell epitopes". .
For immunotherapy in which a peptide consisting essentially of a T cell epitope is administered,

(I) Since the peptide lacks the B cell epitope, ie, the immunoglobulin E antibody specific to the allergen does not react, side effects such as anaphylaxis frequently occurring with conventional crude or purified allergens may occur. No; (ii) the time from starting small to reaching an effective dose can be significantly reduced compared to conventional desensitizers; (iii) induce oral tolerance and reduce allergic reactions to allergens It can be attenuated. However, not all cedar pollinosis patients respond uniformly to a common T cell epitope,
In some cases, administration of only one type of T cell epitope has no effect. Therefore, first examine which T cell epitope the patient responds to before administering it or prepare multiple T cell epitopes and prepare them. Is not expected to be effective unless administered simultaneously. However, in the former method, it is necessary to examine in advance which T cell epitope the patient responds to, and in the latter method, a plurality of T cell epitopes must be separately prepared.

[0008] In recent years, attempts have been made to use a peptide in which a plurality of different T cell epitopes are artificially linked [for example, see Japanese Patent Application Laid-Open No. 7-503362]. However, it is not known at all that a similar effect can be obtained with a human T cell epitope derived from cedar pollen allergen. Japanese Patent Application Laid-Open No. Hei 8-505284 discloses the entire amino acid sequence of cedar pollen allergen Cryj2 and Cryj
There is a description of a T cell epitope derived from No. 2, but it is shown that the 13 amino acid peptide exemplified therein has no activity as a T cell epitope. That is, it was not experimentally determined at which site in the entire amino acid sequence of Cryj2 the human T cell epitope was present.

[0009]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a peptide comprising a continuous amino acid sequence of a plurality of different human T cell epitopes derived from cedar pollen allergen. A second object of the present invention is to provide an anti-cedar pollinosis agent comprising the peptide as an active ingredient.

[0010]

The present invention relates to a compound of the formula I: α ₁ -β ₁ -α ₂ -β ₂ -α ₃ -β ₃ -...- β _n-1 -α _n (I) In the formula (I), α _{1 to} α _n each have a different amino acid sequence consisting of 11 to 19 amino acids, and in humans, the Sia allergen protein Cryj1 or Cry
represents the amino acid sequence constituting the T cell epitope derived from j2; β _{1 to} β _n-1 represent any identical or different amino acid sequences consisting of 0 to 3 amino acids; n represents an integer of 2 or more) And a peptide, a complex thereof, a derivative thereof, or a polymer thereof comprising the amino acid sequence represented by Here, "different from each other" means that the amino acid sequence composed of consecutive 5 or more amino acids does not match each other. The present invention also provides a compound of formula I
A peptide comprising an amino acid sequence represented by, a complex thereof,
An anti-cedar pollinosis agent comprising a derivative thereof or a polymer thereof as an active ingredient is also provided.

Hereinafter, the present invention will be described in detail. The peptides of the present invention preferably comprise groups A, B, C
2 to 7, more preferably 4 amino acids selected from the amino acid sequence groups of group A, group D, group E, group F and group G one by one.
From 7 to 7, most preferably 6 or 7 amino acid sequences linked in no particular order:

Group A: a group consisting of amino acid sequences consisting of 11 to 14 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing and comprising an amino acid sequence constituting a human T cell epitope; Group B: A group consisting of amino acid sequences consisting of 11 to 14 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 8 in the Sequence Listing and comprising an amino acid sequence constituting a human T cell epitope; Group C: shown in SEQ ID NO: 9 in the Sequence Listing An amino acid sequence consisting of 11 to 14 contiguous amino acids in the amino acid sequence, wherein the amino acid sequence constitutes a human T cell epitope;

Group D: an amino acid sequence consisting of 11 to 14 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing, a group consisting of an amino acid sequence constituting a human T cell epitope; Group E: A group consisting of amino acid sequences consisting of 11 to 14 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 11 of the Sequence Listing and comprising a human T cell epitope; Group F: shown in SEQ ID NO: 16 of the Sequence Listing A group consisting of 11 to 14 consecutive amino acids in the amino acid sequence of the present invention, the group consisting of amino acid sequences constituting a human T cell epitope; Group G: continuous amino acid sequence represented by SEQ ID NO: 17 in the sequence listing An amino acid sequence consisting of 11 to 19 amino acids, preferably an amino acid consisting of 18 or 19 amino acids A group consisting of amino acid sequences that constitute human T cell epitopes.

As used herein, the term "amino acid sequence constituting a T cell epitope derived from the Cryj1 or Cryj2 protein in humans" refers to a group of peripheral blood mononuclear cells derived from a cedar pollinosis patient, When cultured under the above conditions, the peripheral blood mononuclear cell group has an activity of making the DNA synthesis rate of the peripheral blood mononuclear cell group more than twice that of the peripheral blood mononuclear cell group cultured in the absence of the peptide having the amino acid sequence. Refers to the amino acid sequence. Also suitable as the amino acid sequence included in each of the above groups are, for example, the amino acid sequences described below:

Group A A: Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser
A ': Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-
Trp (amino acids 4-15 of SEQ ID NO: 7 in the sequence listing) A ″: Asp-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-
Ser (amino acid number 3-15 of SEQ ID NO: 7 in the sequence listing)

Group B B: Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met
(Amino acid number 5-15 of SEQ ID NO: 8 in Sequence Listing) B ': Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met-
Gly (amino acid number 5-16 of SEQ ID NO: 8 in the sequence listing) B ″: Leu-Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-
Met-Gly (amino acid number 4-1 of SEQ ID NO: 8 in the sequence listing)
6)

Group C: Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn
(Amino acids 4-14 of SEQ ID NO: 9 in the sequence listing); C ': Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-
Thr (amino acid No. 4-15 of SEQ ID NO: 9 in Sequence Listing) C ″: Ile-Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-
Asn (amino acids 3-14 of SEQ ID NO: 9 in the sequence listing)

Group D D: Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu
(Amino acid number 4-14 of SEQ ID NO: 10 in the sequence listing) D ': Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-
Asn (amino acids 4-15 of SEQ ID NO: 10 in the sequence listing) D ″: Leu-Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-
Leu-Asn (amino acid number 3-1 of SEQ ID NO: 10 in the sequence listing)
5)

Group E E: Met-Lys-Val-Thr-Val-Ala-Phe-Asn-Gln-Phe-Gly
(Amino acid number 4-14 of SEQ ID NO: 11 in Sequence Listing) E ': Ser-Met-Lys-Val-Thr-Val-Ala-Phe-Asn-Gln-Phe-
Gly (amino acids 3-14 of SEQ ID NO: 11 in the sequence listing) E ″: Ser-Met-Lys-Val-Thr-Val-Ala-Phe-Asn-Gln-Phe-
Gly-Pro (amino acid number 3-1 of SEQ ID NO: 11 in the sequence listing)
5);

Group F F: Tyr-Gly-Leu-Val-His-Val-Ala-Asn-Asn-Asn-Tyr-
Asp-Pro (amino acid number 6-1 of SEQ ID NO: 16 in the sequence listing)
8); F ': Gly-Leu-Val-His-Val-Ala-Asn-Asn-Asn-Tyr-Asp-
Pro-Trp-Thr (amino acid number 7 of SEQ ID NO: 16 in the sequence listing)
-20); F ″: Tyr-Gly-Leu-Val-His-Val-Ala-Asn-Asn-Asn-Tyr-
Asp-Pro-Trp-Thr-Ile (amino acids 6-21 of SEQ ID NO: 16 in the sequence listing)

Group G G: Ser-Gly-Lys-Tyr-Glu-Gly-Gly-Gly-Asn-Ile-Tyr-Thr-
G ′: Ser-Gly-Lys-Tyr-Glu-Gly-Gly-Asn-Ile -Tyr-Thr-
Lys-Lys-Glu-Ala-Phe-Asn-Val (SEQ ID NO: 17 in the sequence listing)
G ″: Ser-Ser-Gly-Lys-Tyr-Glu-Gly-Gly-Asn-Ile-Tyr-
Thr-Lys-Lys-Glu-Ala-Phe-Asn-Val (amino acids 2-20 of SEQ ID NO: 17 in the sequence listing)

In the above, groups E, F and G are groups of amino acid sequences constituting a human major T cell epitope derived from cedar pollen allergen Cryj1, and all other groups constitute a human major T cell epitope derived from Cryj2. A group of amino acid sequences. Further, examples of preferred peptides in the present invention are as described below. (1) Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-
Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-Lys-Le
u-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu (Peptide 1; SEQ ID NO: 1 in Sequence Listing) (2) Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser -Cys-Leu-
Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-Gly-Il
e-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser (Peptide 2; SEQ ID NO: 2 in Sequence Listing) (3) Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro -Ala-Ser-
Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Phe-Al
a-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn (Peptide 3; SEQ ID NO: 3 in Sequence Listing)

(4) Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-
Gln-Lys-Asn-Tyr-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pr
o-Ala-Ser-Tyr-Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-
Cys-Leu (Peptide 4; SEQ ID NO: 4 in Sequence Listing) (5) Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-
Tyr-Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-Ty
r-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser
(Peptide 5; SEQ ID NO: 5 in Sequence Listing) (6) Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-
Tyr-Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Ty
r-Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn
(Peptide 6; SEQ ID NO: 6 in Sequence Listing)

(7) Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-
Pro-Ala-Ser-Trp-Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gl
n-Lys-Asn-Thr-Met-Lys-Val-Thr-Val-Ala-Phe-Asn-Gln-
Phe-Gly-Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Le
u-Asn (peptide 7; SEQ ID NO: 12 in Sequence Listing) (8) Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-
Asn-Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-Th
r-Met-Lys-Val-Thr-Val-Ala-Phe-Asn-Gln-Phe-Gly-Gly-
Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-Trp (Peptide 8; SEQ ID NO: 13 in Sequence Listing) (9) Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn -Pro-Ala-Ser-
Trp-Met-Lys-Val-Thr-Val-Ala-Phe-Asn-Gln-Phe-Gly-Ph
e-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-Thr-Lys-
Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn (Peptide 9; SEQ ID NO: 14 in Sequence Listing)

(10) Lys-Leu-Thr-Ser-Gly-Lys-Ile-Al
a-Ser-Cys-Leu-Asn-Met-Lys-Val-Thr-Val-Ala-Phe-Asn-
Gln-Phe-Gly-Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Ly
s-Asn-Thr-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-
Ser-Trp (peptide 10; SEQ ID NO: 15 in sequence listing) (11) Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-As
n-Thr-Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-
Trp-Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-As
n-Met-Lys-Val-Thr-Val-Ala-Phe-Asn-Gln-Phe-Gly (peptide 11: SEQ ID NO: 18 in Sequence Listing) (12) Met-Lys-Val-Thr-Val-Ala- Phe-Asn-Gln-Phe-Gl
y-Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn-
Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-Trp-Ph
e-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-Thr (peptide 12: SEQ ID NO: 19 in Sequence Listing)

(13) Gly-Ile-Ile-Ala-Ala-Tyr-Gln-As
n-Pro-Ala-Ser-Trp-Ser-Met-Lys-Val-Thr-Val-Ala-Phe-
Asn-Gln-Phe-Gly-Pro-Phe-Ala-Ser-Lys-Asn-Phe-His-Le
u-Gln-Lys-Asn-Thr-Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-
Ser-Cys-Leu-Asn-Tyr-Gly-Leu-Val-His-Val-Ala-Asn-As
n-Asn-Tyr-Asp-Pro-Ser-Gly-Lys-Tyr-Glu-Gly-Gly-Asn-
Ile-Tyr-Thr-Lys-Lys-Glu-Ala-Phe-Asn-Val-Glu (peptide 13: SEQ ID NO: 20 in Sequence Listing)

In the above (1), the amino acid sequence constituting the human major T cell epitope of the cedar pollen allergen protein Cryj2 is an amino acid sequence that is continuous from the N-terminus in the order of CAD. Similarly, the above (2) corresponds to Cr
the major human T-cell epitope of yj2 is
An amino acid sequence continuous in the order of -A, and the above (3) is an amino acid sequence continuous in the order of ADC from the N-terminus.

The above (7), (8), (9), (1)
0), (11) and (12) show that the amino acid sequences constituting the human major T cell epitopes of cedar pollen allergen proteins Cryj1 and Cryj2 are A'-C'-ED 'and D'-C '-EA', A'-
E-C'-D ', D'-E-C'-A', C'-A'-
The amino acid sequence is D'-E and ED'-A'-C '. Similarly, (13) above is an amino acid sequence that is continuous in the order of A'-E "-C'-D'-FG from the N-terminus.

Furthermore, in the above (4), tyrosine (Y) is inserted one residue at a time between amino acid sequences constituting each T cell epitope, and the residue is successively arranged in the order of CYAYAD from the N-terminal. Amino acid sequence. Similarly, (5) is N
The amino acid sequence continued in the order of D-Y-C-Y-A from the terminal, and the above (6) is the amino acid sequence continued in the order of A-Y-D-Y-C from the N-terminal.

Further, T other than the above (1) to (13)
Permutation of cell epitopes, ie, ACD, CD
-A or D-A-C; A-Y-C-Y-D, C-Y-
DYA or DYACYC; or A'-
C'-D'-E, A'-D'-C'-E, A'-D'-
E-C ', A'-ED'-C', C'-A'-D'-
E, C'-A'-ED ', C'-D'-A'-E,
C'-D'-EA ', C'-EA'-D', C'-
ED'-A ', D'-A'-C'-E, D'-A'-
EC ', D'-C'-A'-E, D'-EA'-
C ', EA'-C'-D', EA'-D'-C ',
E-C'-A'-D ', E-C'-D'-A', E-
D'-A'-C 'or ED'-C'-A', A'-
E-C'-D'-FG, A'-E "-C'-D'-G
-F or A'-EC'-D'-GF is also suitable as the peptide of the present invention.

Further, as described in the above (4) to (6), the peptide of the present invention provides a human major T
Also included are those in which an amino acid sequence (β in Formula I) irrelevant to the constitution of the T cell epitope is interposed between cell epitopes. In the present invention, the length of such an intervening amino acid sequence is one to three amino acids. The amino acid sequence is not particularly limited, and examples of such an intervening amino acid sequence include tyrosine used in the above (4) to (6). Alternatively, it may be an amino acid sequence that can be specifically recognized and cleaved by a protease present in the human body. Such amino acid sequences include, for example, cathepsin B (EC
Arg that can be cleaved by 3.4.2.21); can be cleaved by cathepsin D (EC 3.4.4.23.5)
Leu-Tyr, Tyr-Leu, Phe-Phe, Phe-Tyr and Leu-Phe;
Phe-Phe, Phe-Ile, Phe-Val, Tyr-Val and Le that can be cleaved by cathepsin E (EC 3.4.23.34)
u-Phe and the like can be mentioned.

Further, the peptide of the present invention has, in addition to the above-mentioned constitution, an N-terminal and / or a C-terminal thereof.
Also includes those in which an additional amino acid sequence independent of the composition of the T cell epitope is present. Such an additional amino acid sequence is not limited as long as it does not impair the function of the peptide of the present invention as a T cell epitope donor or has any detrimental activity such as toxicity when administered to humans. Is not particularly limited.

[0033]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide described in the present invention can be easily prepared by a peptide synthesis method commonly used in the art, which is known as "solid phase method" or "liquid phase method". For example, “The New Chemistry Experiment Course” edited by The Biochemical Society of Japan, Vol. 1, “Protein VI”, pp. 3-44, 1
In 992, published by Tokyo Kagaku Dojin, details of peptide synthesis are described. In addition, the peptide was synthesized using Fmoc (9-fluorenyl methyloxycar) using a multi-peptide synthesizer (Symphony; manufactured by Protein Technology).
bonyl) It can be synthesized by the solid phase synthesis method according to the protocol of the same apparatus. That is, an Fmoc-L-amino acid Wang resin into which an amino acid corresponding to the C-terminus of each peptide to be synthesized is introduced is set in a reaction vessel of the above-mentioned peptide synthesizer, and Fmoc is removed using a deprotection solution. Further, the amino acid solution corresponding to the second amino acid from the C-terminus was reacted with the activator solution.
The target peptide can be synthesized by deprotecting the group and repeating the same operation.

The peptide of the present invention is not limited to those prepared by chemical synthesis, and may be those prepared by recombinant DNA technology. For example, the peptides (1) to (1)
3) preparing a DNA encoding the amino acid sequence described in 3), inserting the DNA into a vector capable of autonomous propagation, and introducing it into an appropriate host such as Escherichia coli, Bacillus subtilis, actinomycete, yeast or the like to obtain a transformant; The peptide of the present invention may be collected from the culture. DNA having a desired nucleotide sequence
Can be prepared by chemically synthesizing sense and antisense nucleotides, which are partial sequence nucleotides of the desired DNA and having both ends overlapping, followed by the polymerase chain reaction [Saiki, RK e
tal (1988) Science 239, 487-491], and a method in which those partial sequences are linked to each other.

By incorporating the DNA encoding the amino acid sequence of the peptide of the present invention obtained as described above into a suitable vector, prokaryotic or eukaryotic host cells can be transformed. Furthermore, by introducing an appropriate promoter and a sequence related to transformation into these vectors, the DNA can be expressed in each host cell. Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. In order to express the gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing an origin of replication and regulatory sequences. Further, it is desirable that the vector has a sequence capable of imparting phenotypic (phenotypic) selectivity to the transformed cells.

For example, Escherichia coli includes E. coli. coli K1
Two strains are commonly used, and the vector is generally pBR
Although 322 and pUC-based plasmids are often used, the present invention is not limited thereto, and any of various known strains and vectors can be used. Promoters include tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) in Escherichia coli.
Promoter, lambda (λ) from bacteriophage
PL promoter, polypeptide chain elongation factor Tu (tu
fB) promoter and the like, and any promoter can be used for production of the peptide of the present invention.

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

For example, when Escherichia coli is used as a host cell, an expression vector having a pBR322 origin of replication, capable of autonomous growth in Escherichia coli, and having a transcription promoter and a translation initiation signal is used. be able to. The expression vector was prepared by the calcium-chloride method [Mandel, M. and Higa, A. (1970) J. Mol.
Biol. 53, 154], Hanahan's method [Hanahan, D .;
and Meselson, M. (1980) Gene 10, 63] and electric pulse drilling [Neumann, E., et al. (1982) EMBO J.
1, 841-845], and the like, whereby cells transfected with the desired vector can be obtained.

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

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

As eukaryotic microorganisms, yeasts are generally used. Among them, yeasts belonging to the genus Saccharomyces,
For example, Saccharomyces cerevisiae
revisiae) is preferred. Examples of expression vectors for eukaryotes such as yeast include a promoter for an alcohol dehydrogenase gene [Bennetzen, JL and Hall, BD (198
2) J. Biol. Chem. 257, 3018-3025] and the promoter of the acid phosphatase gene [Miyanohara, A., et al.
Natl. Acad. Sci. USA 80, 1-5] can be preferably used.

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

The desired transformant obtained above can be cultured according to a conventional method, and the polypeptide of the present invention is produced intracellularly or extracellularly by the culture. The medium used for the culture can be appropriately selected from various ones commonly used depending on the host cell used. For example, in the case of Escherichia coli, a tryptone-yeast medium (1.6% bactotryptone, yeast extract 1) is used. 0.0%, NaCl
0.5% (pH 7.0)) or a peptone medium (manufactured by Difco) can be used. If the above COS cells are used, a medium such as RPMI-1640 medium or Dulbecco's modified Eagle medium (DMEM) may be used, if necessary, for fetal bovine serum (FB).
Those to which a serum component such as S) is added can be used.

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

When a foreign gene is introduced into Escherichia coli or the like and expressed in a large amount, the produced peptide may form a water-insoluble aggregate called an inclusion body. In such a case, the peptide can be solubilized by denaturing the peptide with a strong denaturing agent such as guanidine isothiocyanate. Further, the peptide of the present invention may be in the form of a complex obtained by adding a saccharide or polyethylene glycol to the peptide thus obtained, and further, the peptide may be subjected to acetylation, amidation and / or cross-linking polymerization with a polyfunctional reagent. It may be in the form of a derivative or a polymer obtained by the above method.

In producing such a conjugate, derivative or polymer of the peptide of the present invention, addition of a saccharide or the like,
Acetylation, amidation, and / or cross-linking polymerization do not impair the function of the amino acid sequence constituting the human T-cell epitope derived from the cedar pollen allergen protein in the peptide of the present invention. It is preferably carried out on an additional peptide at the C-terminal or an intervening peptide.

The conjugate of the peptide of the present invention has, for example, a repeating unit of maltotriose such as polyethylene glycol, monomethoxypolyethylene glycol, dextran, or pullulan or ercinan at the N-terminal or the amino group of Lys residue. Such as those to which polysaccharides have been added,
Those well-known to those skilled in the art in the technical field of the present invention can be mentioned, for example, "The New Chemistry Experiment Course" edited by The Biochemical Society of Japan, Vol. 1, "Protein IV", No. 23.
It can be produced according to the description on pages 6 to 252 (1991, issued by Tokyo Kagaku Dojin).

Examples of the derivatives of the peptide of the present invention include those well-known to those skilled in the technical field of the present invention, such as those having acetylated N-terminal and those having amidated C-terminal Gly residue. Can be. Such derivatives are described, for example, in “Shinsei Kagaku Kenkyusho” Vol.
V, pp. 18-20 and Vol. 9, "Hormon I", No. 2
It can be produced according to the description on pages 90 to 298 (all published by Tokyo Chemical Dojin, 1991). further,
Examples of the polymer of the peptide of the present invention include those well-known to those skilled in the art of the present invention, such as those obtained by polymerizing two molecules of the peptide of the present invention with a divalent crosslinking reagent such as disuccinimidyl suberate. Can be mentioned. The preparation of such a polymer can be carried out, for example, according to the description in “Shinsei Kagaku Jikken Koza,” Vol. 1, “Protein IV”, pp. 207-226.

The peptide of the present invention exerts the desired therapeutic and / or prophylactic effects even when administered in a relatively crude form, but is usually purified prior to use. Purification includes, for example, filtration, concentration, centrifugation, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, adsorption chromatography, high performance liquid chromatography,
Conventional methods in the art for purifying peptides or proteins, such as affinity chromatography, gel electrophoresis, and isoelectric focusing, are used, and these methods may be appropriately combined as needed. Then, depending on the final use form, the purified peptide may be concentrated and lyophilized to a liquid or solid state. The activity of the peptide of the present invention as a T cell epitope can be confirmed by measuring the incorporation of tritiated thymidine into T cells specific to cedar pollen allergen.
For this measurement, for example, the method described below can be used.

That is, a peripheral blood mononuclear cell group of a hay fever patient is separated by Ficoll-Hypaque specific gravity centrifugation or the like.
This cell group was suspended in a medium such as RPMI1640, and 9
Dispense onto a 6-well microplate. Next, a peptide as a test substance is added and cultured. Conditions such as temperature and time during the culture can be appropriately adjusted for each experiment.
7 ° C. for 3 days is preferred. Thereafter, tritiated thymidine is added to the medium, culture is further continued for a certain period of time, and the activity of the peptide of the present invention as a T cell epitope can be calculated by measuring the uptake of tritiated thymidine in the mononuclear cell group. . In the present invention,
At the same time, a system containing no peptide was provided and used as a negative control. A system in which the incorporation of tritiated thymidine reached twice or more that of the negative control was “positive”, and a system that did not reach “negative”.
And

It can be confirmed, for example, by the following experiment that the peptide of the present invention has a therapeutic effect on cedar pollinosis. Since the peptide of the present invention has an activity specific to human T cells, its effect may not be exhibited even when experiments are performed on animals other than humans.
Since the lymphocytes of the H / HeN mouse are reactive with the peptides included in the group A, it is possible to evaluate the effects of the peptides containing the amino acid sequence of the group A by the method described below. it can.

The peptide of the present invention is previously administered to C3H / HeN mice. After a certain period of time, the mouse has a human major T of Cryj2 having the sequence described below.
Cell epitope peptide: Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser (peptide 14; amino acid number 4-1 of SEQ ID NO: 7 in the sequence listing)
4) is administered together with an adjuvant such as Freund's complete adjuvant and immunized. Further, after a certain period of time, lymph node cells are extracted from the experimental animal from the popliteal region or the like to prepare a cell suspension. In addition to the above peptide 14 or Cryj
2 to continue the culture, further add tritiated thymidine, and measure the uptake of tritiated thymidine, whereby the proliferation of T cells can be measured.

In mice to which the peptide of the present invention has not been administered in advance, immunization with peptide 14 causes the proliferation of T cells in response to peptide 14 or Cryj2. On the other hand, in mice to which the peptide of the present invention has been previously administered to induce immune tolerance, T cells do not respond to peptide 14 or Cryj2 and do not proliferate even after immunization with peptide 14. By measuring the difference, the therapeutic effect of the peptide of the present invention on cedar pollinosis can be confirmed. Since the peptide of the present invention does not substantially react with the immunoglobulin E antibody specific to cedar pollen allergen, when administered to mammals including humans in general, substantially no anaphylaxis is caused, and the T specific for cedar pollen allergen is not caused. Cells can be inactivated.

The anti-cedar pollinosis agent of the present invention containing the peptide of the present invention, a complex thereof, a derivative thereof, or a polymer thereof as an active ingredient can be administered to mammals including humans in general.
It exerts a remarkable therapeutic effect on cedar pollinosis without substantially causing anaphylaxis. The anti-cedar pollinosis agent comprising the peptide of the present invention, a complex thereof, a derivative thereof, or a polymer thereof as an active ingredient, when administered to mammals including humans suffering from cedar pollinosis, has substantially side effects such as anaphylaxis. Cedar pollinosis can be treated without causing it. On the other hand, when the cedar pollinosis agent of the present invention is administered to a healthy individual or a potential cedar hay fever before the cedar pollen starts to fly, while exhibiting a remarkable preventive effect against cedar hay fever, It is extremely effective in relieving allergic symptoms at onset.

The anti-cedar pollinosis agent of the present invention will be described in more detail. Generally, the anti-cedar pollinosis agent of the present invention is one or two or more of the peptide, complex thereof, derivative thereof, or polymer thereof of the present invention. The above is 0.01 to 100% (w
/ W), preferably 0.05 to 50% (w / w),
More preferably, it comprises 0.5 to 5.0% (w / w). The anti-cedar hay fever of the present invention is not only a single form of the active ingredient such as the peptide, but also other physiologically acceptable, for example, serum albumin, gelatin, glucose, sucrose, lactose, maltose,
Carriers such as trehalose, sorbitol, maltitol, lactitol, mannitol, pullulan, excipients, immunostimulants, stabilizers, and if necessary, anti-inflammatory agents such as steroid hormones and sodium crimoglycate, antihistamines, anti-leukotrienes , In combination with one or more other drugs, including anti-tachykinin drugs. Furthermore, the anti-cedar pollinosis agent of the present invention also includes a drug in a dosage unit form, and the drug in the dosage unit form refers to the peptide of the present invention, a complex thereof, a derivative thereof, or a polymer thereof, for example, Daily dose or an integer multiple thereof (up to 4 times) or a submultiple thereof (1
/ 40) means a drug in a physically separate, unitary dosage form suitable for administration. Drugs in such dosage unit form include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, ointments, plasters, cataplasms, suppositories, eye drops ,
Nasal drops, sprays, injections and the like can be mentioned.

The method of using the anti-cedar pollinosis agent of the present invention will now be described. The anti-cedar pollinosis agent of the present invention is useful for the treatment and prevention of cedar pollinosis.
Oral, nasal, ophthalmic or injection administration. The dosage in humans varies depending on the administration method and symptoms, but is usually 0.01 to 1.0 g, preferably 0.0 to 1.0 g per day for adults while observing the symptoms and progress of the subjects after administration.
1 to 0.1 g (converted to the amount of active ingredient)
Once a day or once a month, for about one to six months,
It is usually administered repeatedly in increasing doses.

[0057]

EXAMPLES The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the technical scope of the present invention. Example 1 Synthesis of Peptide 1 A peptide was chemically synthesized by a method (solid phase synthesis method) in which amino acids were bonded one by one to the amino acid derivative fixed to the resin from the carboxyl terminal side. The amino acid used in each cycle used was a special amino acid derivative in which the α-amino group and the reactive group at the residue were blocked with a protecting group. Here, each α amino group is Fmoc (9-fluorenyl.
Amino acids blocked by methyloxycarbonyl) were used (Fmoc method). In addition, peptide synthesis was performed by sequentially repeating a reaction of deprotecting the Fmoc of the α-amino group of the amino acid bound to the resin, and then binding an amino acid derivative having an activated carboxyl group.

Each peptide of Peptides 1, 2 and 3 is
The peptide was synthesized using a peptide synthesizer (Model 430A; manufactured by Applied Biosystems) by the Fmoc solid phase synthesis method described above according to the FastMoc Chemistry method of the same apparatus. That is, Fmoc-Leu-Wa into which an amino acid (Ser) corresponding to the C-terminal residue of the peptide to be synthesized is introduced.
100 ng resin (0.69 mmol / g; Aloka)
The equivalent of μmol was set in the reaction vessel of the peptide synthesizer, and 7 ml of a deprotection solution (piperidine / N-methylpyrrolidone (hereinafter referred to as “NMP”)) was reacted twice for 3 minutes and 12 minutes to bind to the resin. The Fmoc group of the amino acid was removed, and the residue was washed 5 times with 7 ml of NMP.
1 mmol of Fm corresponding to the second amino acid from the C-terminal side
Activator solution (1 ml of 2M diisopropylethylamine (DIEA) / NMP and 0.45M 2- (1H-benzotriazol-1-yl) -1,1,3,3, -tetramethyluro) was added to oc-Cys (Trt). (HBTU) / 1-hydroxybenzotriazole (HOBt) / dimethylformamide (DMF) (2.2 ml) and NMP (1 ml) and activated (10-fold equivalent to the bound amino acid). The amino acid bound to the resin excluding the Fmoc group was reacted for 30 minutes at room temperature. Fmoc-C generated here
After washing the ys (Trt) -Leu-Wang resin seven times with 7 ml of NMP,
The Fmoc group is deprotected again (provided that the piperidine concentration is from 23% in the first cycle to 4% in the 30th cycle).
It was gradually increased according to the length of the peptide to be synthesized at 2%. ) After washing 5 times with 7 ml of NMP, activated Fmoc-Ser
(tBu) solution.

By repeating the same operation, the desired protected peptide resin (Fmoc-Phe-Ala-S
er (tBu) -Lys (Boc) -Asn (Trt)
-Phe-His (Trt) -Leu-Gln (Tr
t) -Lys (Boc) -Asn (Trt) -Gly-
Ile-Ile-Ala-Ala-Tyr (tBu)-
Gln (Trt) -Asn (Trt) -Pro-Ala
-Ser (tBu) -Lys (Boc) -Leu-Th
r (tBu) -Ser (tBu) -Gly-Lys (B
oc) -Ile-Ala-Ser (tBu) -Cys
(Trt) -Leu-Wang resin) was synthesized.

Example 1 The amino acids used in the following peptide synthesis are as follows: Fmoc-Ala, Fmoc-Arg (Pmc), Fmoc-Asn (Trt), Fm
oc-Asp (OtBu), Fmoc-Cys (Trt), Fmoc-Gln (Trt), Fmoc-G
lu (OtBu), Fmoc-Gly, Fmoc-His (Trt), Fmoc-Ile, Fmoc-
Leu, Fmoc-Lys (Boc), Fmoc-Met, Fmoc-Phe, Fmoc-Pro,
Fmoc-Ser (tBu), Fmoc-Thr (tBu), Fmoc-Trp (Boc), Fmoc-
Tyr (tBu), Fmoc-Val. (The numbers in parentheses indicate protecting groups that protect the reactive group at the residue; PerkinElmer Japan Applied Biosystems Co., Ltd.)

First, the protected peptide resin (Fmoc-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Tr
t) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Gly
-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Al
a-Ser (tBu) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys
(Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Wang resin) was reacted with 7 ml of the deprotection solution twice for 3 minutes and 12 minutes to deprotect the N-terminal Fmoc group. Then 7ml of N
After washing 6 times with MP, washing 3 times with dichloromethane,
Further drying was performed for 15 minutes by blowing argon gas.

The resin was taken out, and a clivage solution (trifluoroacetic acid (hereinafter referred to as “TFA”): phenol: water:
Thioanisole: ethanedithiol = 82.5: 5: 5: 5:
2.5) was added thereto, and the mixture was reacted at room temperature for 2 hours to cleave the peptide from the resin and remove the amino acid side chain protecting group to obtain a peptide solution [King, DS (199
0) Int. J. Peptide Protein Reg. 36, 255]. This peptide solution was filtered using a filter, and the filtrate was collected in a centrifuge tube. To this, add 40 ml of cold ether,
The peptide was precipitated. After cooling for a while, the mixture was centrifuged (3000 rpm, 10 minutes) to collect a precipitate, and the operation of dispersing by adding cold ether again and recovering was repeated 6 times to wash the peptide. The obtained peptide was dried to obtain 230 mg of a crude peptide. Hereinafter, the above operation is referred to as a “cleavage reaction”.

[0076] Of the crude peptide obtained, 76 mg was added to 0.1 mg.
After dissolving in a 20% acetonitrile aqueous solution containing 1% TFA, the solution was subjected to high performance liquid chromatography (hereinafter, referred to as “HPLC”). HPLC conditions were as follows.

Column: ODS column (TSK-gel)
ODS-120T, φ21 mm × 300 mm; manufactured by Tosoh Corporation) Mobile phase: 27-35% acetonitrile / 0.1% TF
A, 20 minutes Flow rate: 8 ml / min Detection wavelength: 230 nm. The fraction eluted at 21 to 23 minutes was collected, concentrated, and lyophilized to obtain 9 mg of the desired peptide. When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequence analyzer (PPSQ-10; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing was confirmed. Was.

Example 2 Synthesis of Peptide 2 By the same operation as in Example 1, the protected peptide resin (Fmoc
-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-A
la-Ser (tBu) -Cys (Trt) -Leu-Phe-Ala-Ser (tBu) -Lys (Boc)
-Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (T
rt) -Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt)
-Pro-Ala-Ser (tBu) -Wang resin) was synthesized and subjected to a cribage reaction to obtain 270 mg of a crude peptide. 91 mg of the obtained crude peptide was added to 20 containing 0.1% TFA.
After dissolving in a% acetonitrile aqueous solution, it was subjected to HPLC.
HPLC conditions were as follows.

Column: C4 column (YMC-Pack C4-AP, φ20 mm × 250 mm;
Mobile phase: 27-35% acetonitrile / 0.1% TF
A, 30 minutes Flow rate: 7 ml / min Detection wavelength: 230 nm. The fraction eluted between 16 and 18 minutes was collected, concentrated, and lyophilized to obtain 47 mg of the desired peptide. Amino acid sequence analysis was performed on 100 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing was confirmed.

Example 3 Synthesis of Peptide 3 A peptide (Fmoc-Gly-Ile-Ile-Al-Al) was prepared in the same manner as in Example 1.
a-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu)-
Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Al
a-Ser (tBu) -Cys (Trt) -Leu-Phe-Ala-Ser (tBu) -Lys (Boc)-
Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Tr
t) -Wang resin) and subjected to a crevage reaction to obtain 210 mg of a crude peptide. 70 mg of the obtained crude peptide was dissolved in a 20% aqueous acetonitrile solution containing 0.1% TFA, and then subjected to HPLC. HPLC conditions were as follows.

Column: Octadecyl-4PW (TSK-
gel octadecyl-4PW, φ21.5mm × 15
0 mm; manufactured by Tosoh Corporation) Mobile phase: 25-40% acetonitrile / 0.1% TF
A, 15 minutes Flow rate: 8 ml / min Detection wavelength: 230 nm. The fraction eluted in 10 to 12 minutes was collected, concentrated, and lyophilized to obtain 10 mg of the desired peptide. Amino acid sequence analysis was performed on 100 pmol of this peptide in the same manner as in Example 1, and as a result, SEQ ID NO: 3 in the sequence listing was obtained.
Was confirmed.

Example 4 Synthesis of Peptide 4 Peptides 4, 5 and 6 were prepared using the above-mentioned Fmoc solid-phase synthesis method using a multi-peptide synthesizer (Symphony; manufactured by Protein Technologies). Synthesized according to the protocol. That is, Fmoc-Leu-Wang resin (0.69 mmol) into which an amino acid (Leu) corresponding to the C-terminal residue of the peptide to be synthesized is introduced.
/ G) is set in the reaction vessel of the above peptide synthesizer, 1.25 ml of a deprotection solution (20% piperidine / dimethylformamide) is reacted twice for 5 minutes, and the Fmoc group of the amino acid bound to the resin is reacted. Was removed. The resin was washed with 1.25 ml of a dimethylformamide solution six times for 30 seconds, and 1.25 ml of a 100 mM Fmoc-Cys (Trt) dimethylformamide solution corresponding to the second amino acid from the C-terminal side was added to a 100 mM activator solution (100 mM). O-benzotriazole-N, N ',
1.25 ml (N ',-tetramethyl-uronium-hexafluorophosphate / 400 mM N-methylmorpholine / dimethylformamide) was added (5 equivalents to the bound amino acid), and the mixture was reacted for 20 minutes at room temperature. The Fmoc-Leu-Cys (Trt) -Wang resin produced here was washed with 1.25 ml of dimethylformamide six times for 30 seconds, and then Fm
The oc group was deprotected and washed with 1.25 ml of dimethylformamide six times for 30 seconds. Then, an Fmoc-Ser (tBu) solution and an activator solution were added and reacted. By repeating the same operation, the protected peptide resin (Fmoc-Phe-A
la-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln
(Trt) -Lys (Boc) -Asn (Trt) -Tyr (tBu) -Gly-Ile-Ile-Ala-A
la-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu) -Tyr
(tBu) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc)-
Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Wang resin) was synthesized.

The protected peptide resin synthesized as described above was subjected to a cribage reaction according to the method described in Example 1 to obtain 50 mg of a crude peptide. 9.5 mg of the obtained crude peptide was added to 20 containing 0.1% TFA.
After dissolving in a% acetonitrile aqueous solution, it was subjected to HPLC.
HPLC conditions were as follows.

Column: ODS column (TSK guard column ODS, φ21.5 mm × 75 mm; manufactured by Tosoh Corporation) Mobile phase: 25-40% acetonitrile / 0.1% TF
A, 20 minutes Flow rate: 8 ml / min Detection wavelength: 220 nm. The fraction eluted in 9 to 10 minutes was collected, concentrated, and lyophilized to obtain 0.7 mg of the desired peptide. Amino acid sequence analysis was performed on 100 pmol of this peptide in the same manner as in Example 1, and as a result, SEQ ID NO: 4 in the sequence listing was obtained.
Was confirmed.

Example 5 Synthesis of Peptide 5 The protected peptide resin (Fmoc-Lys (B
oc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser
(tBu) -Cys (Trt) -Leu-Tyr (tBu) -Phe-Ala-Ser (tBu) -Lys (B
oc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -As
n (Trt) -Tyr (tBu) -Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (T
rt) -Asn (Trt) -Pro-Ala-Ser (tBu) -Wang resin)
Cleavage reaction was performed to obtain 45 mg of the crude peptide.
14 mg of the obtained crude peptide was 0.1% TFA
After dissolving in 20% acetonitrile aqueous solution containing
Was served. HPLC conditions were as follows.

Column: ODS column (TSK guard column ODS, φ21.5 mm × 75 mm; manufactured by Tosoh Corporation) Mobile phase: 25-40% acetonitrile / 0.1% TF
A, 20 minutes Flow rate: 8 ml / min Detection wavelength: 220 nm. The fraction eluted at 12 to 13 minutes was collected, concentrated, and lyophilized to obtain 1.0 mg of the desired peptide. Amino acid sequence analysis was performed on 100 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing was confirmed.

Example 6 Synthesis of Peptide 6 In the same manner as in Example 4, the protected peptide resin (Fmoc-Gly-I
le-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-
Ser (tBu) -Tyr (tBu) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -G
ly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Tyr (tBu)
-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -L
eu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Wang resin)
Cleavage reaction was performed to obtain 42 mg of the crude peptide.
10 mg of the obtained crude peptide was 0.1% TFA
After dissolving in 20% acetonitrile aqueous solution containing
Was served. HPLC conditions were as follows.

Column: ODS column (TSK guard column ODS, φ21.5 mm × 75 mm; manufactured by Tosoh Corporation) Mobile phase: 25-27% acetonitrile / 0.1% TF
A, 15 minutes Flow rate: 8 ml / min Detection wavelength: 220 nm. The fraction eluted at 14 to 15 minutes was collected, concentrated, and lyophilized to obtain 0.9 mg of the desired peptide. Amino acid sequence analysis was performed on 100 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing was confirmed.

Example 7 Synthesis of Peptide 7 By the same operation as in Example 1, the protected peptide resin (Fmoc
-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pr
o-Ala-Ser (tBu) -Trp-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (T
rt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Th
r (tBu) -Met-Lys (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn (T
rt) -Gln (Trt) -Phe-Gly-Lys (Boc) -Leu-Thr (tBu) -Ser (tB
u) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn
(Trt) -Wang resin) and perform a cribage reaction,
532 mg of crude peptide were obtained. 11 mg of the obtained crude peptide was dissolved in a 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. HPLC conditions were as follows.

Column: ODS column (TSK guard column ODS, φ21.5 mm × 75 mm; manufactured by Tosoh Corporation) Mobile phase: 30-50% acetonitrile / 0.1% TF
A, 15 minutes Flow rate: 5 ml / min Detection wavelength: 220 nm. The fraction eluted at 10 to 11.5 minutes is collected, concentrated,
Lyophilization was performed to obtain 1.9 mg of the desired peptide. An amino acid sequence analysis was performed on 500 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence represented by SEQ ID NO: 12 in the sequence listing was confirmed.

Example 8 Synthesis of Peptide 8 The protected peptide resin (Fmoc
-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-A
la-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Phe-Ala-Ser (tBu)
-Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (B
oc) -Asn (Trt) -Thr (tBu) -Met-Lys (Boc) -Val-Thr (tBu) -Va
l-Ala-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-Gly-Ile-Ile-Al
a-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu)-
Trp-Wang resin) was synthesized and subjected to a cribage reaction to obtain 288 mg of a crude peptide. 12 mg of the obtained crude peptide was dissolved in a 10% aqueous acetonitrile solution containing 0.1% TFA, and then subjected to HPLC. HPLC conditions were as follows.

Column: ODS column (TSK guard column ODS, φ21.5 mm × 75 mm; manufactured by Tosoh Corporation) Mobile phase: 35-55% acetonitrile / 0.1% TF
A, 15 minutes Flow rate: 5 ml / min Detection wavelength: 220 nm. The fraction eluted between 7.5 and 8.5 minutes was collected, concentrated,
Lyophilization was performed to obtain 4.9 mg of the desired peptide. An amino acid sequence analysis was performed on 500 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence represented by SEQ ID NO: 13 in the sequence listing was confirmed.

Example 9 Synthesis of Peptide 9 By the same operation as in Example 1, the protected peptide resin (Fmoc
-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pr
o-Ala-Ser (tBu) -Trp-Met-Lys (Boc) -Val-Thr (tBu) -Val-A
la-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-Phe-Ala-Ser (tBu)-
Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Bo
c) -Asn (Trt) -Thr (tBu) -Lys (Boc) -Leu-Thr (tBu) -Ser (tB
u) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn
(Trt) -Wang resin) and perform a cribage reaction,
340 mg of the crude peptide were obtained. 16 mg of the obtained crude peptide was dissolved in a 20% aqueous acetonitrile solution containing 0.1% TFA, and then subjected to HPLC. HPLC conditions were as follows.

Column: ODS column (TSK guard column ODS, φ21.5 mm × 75 mm; manufactured by Tosoh Corporation) Mobile phase: 30-50% acetonitrile / 0.1% TF
A, 15 minutes Flow rate: 5 ml / min Detection wavelength: 220 nm. The fraction eluted at 10 to 13 minutes was collected, concentrated, and lyophilized to obtain 6.8 mg of the desired peptide. An amino acid sequence analysis was performed on 500 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence shown in SEQ ID NO: 14 in the sequence listing was confirmed.

Example 10 Synthesis of Peptide 10 By the same operation as in Example 1, the protected peptide resin (Fmoc
-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-A
la-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Met-Lys (Boc) -Val
-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-Ph
e-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-
Gln (Trt) -Lys (Boc) -Asn (Trt) -Thr (tBu) -Gly-Ile-Ile-Al
a-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu)-
Trp-Wang resin) was synthesized, and a cribage reaction was performed to obtain 444 mg of a crude peptide. 8 mg of the obtained crude peptide was dissolved in a 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. HPLC conditions were as follows.

Column: ODS column (TSK guard column ODS, φ21.5 mm × 75 mm; manufactured by Tosoh Corporation) Mobile phase: 30-45% acetonitrile / 0.1% TF
A, 15 minutes Flow rate: 5 ml / min Detection wavelength: 220 nm. The fraction eluted at 11.5-13 minutes is collected, concentrated,
Lyophilization was performed to obtain 3.0 mg of the desired peptide. An amino acid sequence analysis was performed on 500 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence represented by SEQ ID NO: 15 in the sequence listing was confirmed.

Example 11 Synthesis of Peptide 11 By the same operation as in Example 1, the protected peptide resin (Fmoc
-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -L
eu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Thr (tBu) -Gly-Ile-Ile
-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tB
u) -Trp-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc)
-Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Met-Lys (Bo
c) -Val-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt) -Phe-
Gly-Wang resin), perform a cribage reaction,
The crude peptide was obtained. 120 m of the crude peptide obtained
g was dissolved in a 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. HPLC conditions were as follows.

Column: ODS column (TSK guard column ODS, φ21.5 mm × 75 mm, manufactured by Tosoh Corporation) Mobile phase: 32-40% acetonitrile / 0.1% T
FA, 15 minutes Flow rate: 5 ml / min Detection wavelength: 230 nm. The fraction eluted at 10 to 11.5 minutes is collected, concentrated,
Lyophilization was performed to obtain 12.5 mg of the desired peptide. Amino acid sequence analysis was performed on 100 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence represented by SEQ ID NO: 18 in the sequence listing was confirmed.

Example 12 Synthesis of Peptide 12 By the same operation as in Example 1, the protected peptide resin (Fmoc
-Met-Lys (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gl
n (Trt) -Phe-Gly-Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-
Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Gl
y-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-A
la-Ser (tBu) -Trp-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt)
-Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Thr (t
Bu) -Wang resin) was synthesized and subjected to a cribage reaction to obtain a crude peptide. 42 mg of the obtained crude peptide
Was dissolved in a 20% aqueous acetonitrile solution containing 0.1% TFA, and then subjected to HPLC. HPLC conditions were as follows.

Column: ODS column (TSK guard column ODS, φ21.5 mm × 75 mm; manufactured by Tosoh Corporation) Mobile phase: 39-45% acetonitrile / 0.1% T
FA, 15 minutes Flow rate: 5 ml / min Detection wavelength: 230 nm. The fraction eluted at 7.5 to 10 minutes was collected, concentrated, and lyophilized to obtain 14.6 mg of the desired peptide.
Amino acid sequence analysis was performed on 100 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence shown in SEQ ID NO: 19 in the sequence listing was confirmed.

Example 13 Synthesis of Peptide 13 The synthesis of peptide 13 (SEQ ID NO: 20 in the sequence listing) was divided into five steps as described below to confirm the synthesis status and to add reagents. I did it. First, Fmoc which is a peptide from the C-terminal to the 16th residue was obtained by the method described in Example 1 (however, a double coupling method in which the amino acid binding reaction was repeated twice for each residue was used).
-Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu)-
Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (T
The reaction was stopped when rt) -Val-Glu (OtBu) -Wang resin was synthesized.

A part of the resin was taken out, and 0.5 ml of 20%
The Fmoc reaction was performed with piperidine / DMF for 20 minutes, and after filtration, 0.5 ml of a peptide 13 peptide solution (TFA: ethanedithiol: water: triisopropylsilane = 92.5: 2.5: 2.5: 2.5) was added to the resin. Cleavage reaction was performed for 1 hour, and ether was added to the obtained peptide solution to precipitate the peptide. The molecular weight of the obtained peptide was measured using a mass spectrometer (VG platform: manufactured by VG Biotech) by electrospray ionization (ESI) to confirm the synthesized product.

The synthesis is continued using the remaining resin,
Fmoc-Ala-A, a peptide from the C-terminal to the 26th residue
sn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-S
er (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-As
n (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu
The reaction was stopped when (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was synthesized. A part of the resin was taken out, Fmoc removal reaction and crevage reaction were performed, and the synthesized product was confirmed in the same manner as described above.

Synthesis was continued using the remaining resin, and Fmoc-A, a peptide from the C-terminal to the 46th residue, was synthesized.
sn (Trt) -Thr (tBu) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gl
y-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt)-
Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn
(Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Ser (tBu) -Gly
-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile-
Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-
The reaction was stopped when Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was synthesized. A part of the resin was taken out, a Fmoc removal reaction and a cribage reaction were performed, and the synthesized product was confirmed in the same manner as described above.

The synthesis was continued using the remaining resin, and Fmoc-T, a peptide from the C-terminal to the 65th residue, was synthesized.
hr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-Pro-
Phe-Ala-Ser (tBu) -Lys- (Boc) -Asn (Trt) -Phe-His (Trt) -L
eu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Thr (tBu) -Lys (Boc) -Le
u-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu)-
Cys (Trt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His (Tr
t) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp
(OtBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtB
u) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc)
-Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu)
The reaction was stopped when the -Wang resin was synthesized. A part of the resin was taken out, Fmoc removal reaction and crevage reaction were performed, and the synthesized product was confirmed in the same manner as described above.

At this point, the desired molecular weight (718)
4) and the defective body (5119) were found at a ratio of about 6: 4. Since the target peptide was also synthesized,
The synthesis was continued using the remaining resin, and 8
Peptides up to the first residue were synthesized. However, the Fmoc-Ser (tBu) at the 69th residue from the C-terminal and the Fmoc-Tr at the 70th residue from the C-terminal
Since the reaction that binds p (Boc) and Fmoc-Ser (tBu) at the 71st residue was predicted to have low reaction efficiency, the reaction with the deprotection solution was repeated three times more to bind to the peptide. After completely removing the Fmoc group, the reaction time for binding the amino acid was extended to 120 minutes.

According to the above method, the protected peptide resin (Fm
oc-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt)-
Pro-Ala-Ser (tBu) -Trp (Boc) -Ser (tBu) -Met-Lys (Boc) -Va
l-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-P
ro-Phe-Ala-Ser (tBu) -Lys- (Boc) -Asn (Trt) -Phe-His (Tr
t) -Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Thr (tBu) -Lys (Bo
c) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser
(tBu) -Cys (Trt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-H
is (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tB
u) -Asp (OtBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Gl
u (OtBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys
(Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu
(OtBu) -Wang resin) was synthesized.

The resulting protected peptide resin was reacted twice with 7 ml of a deprotection solution for 3 minutes and 12 minutes to deprotect the N-terminal Fmoc group. Next, the substrate was washed six times with 7 ml of NMP, then three times with dichloromethane, and dried for 15 minutes by blowing argon gas. The resin was taken out, 30 ml of a peptide 13 cleavage solution was added, and the mixture was reacted at room temperature for 2 hours to cleave the peptide from the resin and remove the amino acid side chain protecting group to obtain a peptide solution.

The peptide solution was filtered using a filter, and the filtrate was collected in a centrifuge tube. This was concentrated with an aspirator, and 80 ml of cold ether was added to precipitate the peptide. After cooling for a while, centrifuge it (3000
The operation of collecting the precipitate, adding cold ether again to disperse, and then collecting the precipitate was repeated three times to wash the peptide. The obtained peptide was dried to obtain 640 mg of crude peptide.

After dissolving the obtained crude peptide in a 20% aqueous acetonitrile solution containing 0.1% TFA, HPLC
Was served. HPLC conditions were as follows: Column: TMS column (YMC-Pak TMS)
SH-142-5, φ20mm × 150mm;
Mobile phase: 33% acetonitrile / 0.1% TFA,
After 5 minutes (isocratic), 33-36% acetonitrile / 0.1% TFA, 30 minutes (linear concentration gradient) Flow rate: 5 ml / min Detection wavelength: 230 nm.

The fraction eluted between 19 and 22 minutes was collected and
After concentration, lyophilization was performed to obtain a peptide (21 mg). The resulting peptide still contained impurities,
Further, HPLC was performed under the conditions described below. Column: ODS column (TSK-gel ODS-120)
T, φ21.5 mm × 300 mm; manufactured by Tosoh Corporation) Mobile phase: 34-35% acetonitrile / 0.1% T
FA, 30 minutes Flow rate: 5 ml / min Detection wavelength: 230 nm.

The fraction eluted between 38 and 42 minutes was collected and
After concentration, lyophilization is performed to obtain the desired peptide (9.
3 mg). The molecular weight of this peptide was confirmed by ESI using a mass spectrometer to confirm the molecular weight. Further, amino acid sequence analysis was performed on 100 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence of 10 residues from the N-terminal was found to be the amino acid sequence represented by amino acid numbers 1 to 10 of SEQ ID NO: 20 in the sequence listing. Was confirmed to match.

Example 14 Cedar Pollen Antigen T Cell Epitope Activity Using T cells derived from a cedar pollinosis patient, it was determined that peptides 1 to 13 prepared in the above Examples had cedar pollen antigen T cell epitope activity. Confirmed by the method described in 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, a buffy coat was obtained, and further, peripheral blood mononuclear cells (peripheral blood mononuclear cells) were obtained by Ficoll-pack specific gravity centrifugation.
mononuclear cells: PBMC). This PB
MC was added to the medium (R containing 5% heat-inactivated human AB serum).
PMI1640).

In a 96-well flat bottom plate, 5 × 10 ⁵ cells were dispensed per well, and 1 μg of antigen (peptides 1 to 13, Cryj1 or Cryj2) was added to 1 μg of each well.
37) in 200 μl of medium containing
The cells were cultured at 5 ° C. under 5% carbon dioxide for 72 hours. Separately, a culture group containing no peptide was provided and used as a negative control. Thereafter, 1 μCi of tritiated thymidine was added,
The culture was further performed for 16 hours. The DNA contained in the cells in each well was collected on a glass fiber filter using a cell harvester, and the amount of tritiated thymidine incorporated into the cells was measured using a liquid scintillation counter. The group in which the value (stimulation index) obtained by dividing the amount of thymidine taken up in each group by the amount taken up by the negative control group was 2 or more was defined as “positive” T cell epitope activity. The results are shown in Table 1 below.

[0102]

[Table 1]

In particular, peptide 13 showed activity higher than Cryj1 or Cryj2 in cells from one patient (Table 2).

[0104]

[Table 2]

Example 15 Induction of Oral Tolerance by Peptide 1) Peptide 14 (human major T cell epitope peptide of Cryj2): Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-
Pro-Ala-Ser (amino acid number 4- of SEQ ID NO: 7 in the sequence listing)
14) was prepared according to the method described below. First, a protected peptide resin (Fmoc-Gly-Ile
-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Se
r (tBu) -Wang resin) was synthesized and subjected to a cribage reaction to obtain 67 mg of a crude peptide. 11 mg of the obtained crude peptide was dissolved in a 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. HPLC conditions were as follows.

Column: ODS column (TSK-gel)
ODS-120T, φ7.8 mm × 300 mm; manufactured by Tosoh Corporation) Mobile phase: 21% acetonitrile / 0.1% TFA, 2
0 min (isocratic) Flow rate: 2 ml / min Detection wavelength: 220 nm The fraction eluted at 9 to 11 minutes was collected, concentrated, and lyophilized to obtain 7 mg of the desired peptide. Amino acid sequence analysis was performed on 100 pmol of this peptide in the same manner as in Example 1, and as a result, the amino acid sequence represented by amino acid numbers 4 to 14 in SEQ ID NO: 7 in the sequence listing was confirmed. In addition, cedar pollen allergen protein Cryj2
Was prepared according to the method for producing a substance described as “cedar pollen allergen A” in Japanese Patent Publication No. 1275751/1996.

C3H / HeN mouse (Charles Japan, Inc.)
(Purchased from River Co., Ltd.), 500 μg of Peptide 1 dissolved in 200 μl of Dulbecco's PBS (-) (Dainippon Pharmaceutical Co., Ltd .; hereinafter, referred to as “PBS”) was orally administered four times every two weeks. The control group received only PBS. Thereafter, 10 μg of peptide 14 was injected subcutaneously into both footpads and ridges of mice of both groups together with Freund's complete adjuvant. Eight days later, inguinal and popliteal lymph node cells of both groups of mice were removed, suspended in RPMI1640 containing 2% mouse serum, and then placed in a 96-well plate at about 5 × 10 ⁵ cells / well. Was dispensed. To each well was added an equal amount of RPMI1640 as the cell suspension, with or without antigen (800 nM peptide 14 or 800 nM Cryj2), and cultured for 3 days in the presence or absence of antigen (final concentration). :
Antigen 400 nM or 0 nM; 1% mouse serum). Thereafter, 0.5 μCi of tritiated thymidine was added per well, and the cells were further cultured for 16 hours. The thymidine uptake of lymph node cells in the presence and absence of the antigen was measured for the peptide-administered group and the control group, respectively, and the value obtained by dividing the thymidine uptake in the presence of the antigen by the thymidine uptake in the absence of the antigen was determined as the proliferation activity. It was calculated as The proliferative activity of lymph node cells derived from mice to which peptide 1 had been previously administered was 39 times higher for peptide 14 than for the control group.
% And 30% for Cryj2.

2) 50 C3H / HeN mice
0 μg of Peptide 2 dissolved in 200 μl of PBS was orally administered four times every two weeks. The control group received only PBS. Thereafter, 10 μg of peptide 14 was injected subcutaneously into both footpads and ridges of mice of both groups together with Freund's complete adjuvant. Eight days later, lymph node cells in the inguinal and popliteal regions of the mice in both groups were removed and
% Mouse serum and suspended in RPMI 1640
About 5 × 10 ⁵ cells per well were dispensed into a 96-well plate. The cells were treated with antigen (400 nM peptide 1).
The cells were cultured for 3 days in the presence or absence of 4 or 400 nM Cryj2). Then 0.5μC per well
The tritiated thymidine of i was added, and the cells were further cultured for 16 hours. The thymidine uptake of lymph node cells in the presence and absence of the antigen was measured for the peptide-administered group and the control group, respectively, and the value obtained by dividing the thymidine uptake in the presence of the antigen by the thymidine uptake in the absence of the antigen was determined as the proliferation activity. It was calculated as The proliferation activity of lymph node cells derived from mice to which peptide 2 was previously administered was 15% for peptide 14 and 2% for Cryj2 as compared to the control group.
It was down 8%.

3) 50 C3H / HeN mice
0 μg of Peptide 3 dissolved in 200 μl of PBS was orally administered four times every two weeks. The control group received only PBS. Thereafter, 10 μg of peptide 14 was injected subcutaneously into both footpads and ridges of mice of both groups together with Freund's complete adjuvant. Eight days later, lymph node cells in the inguinal and popliteal regions of the mice in both groups were removed and
% Mouse serum and suspended in RPMI 1640
About 5 × 10 ⁵ cells per well were dispensed into a 96-well plate. The cells were treated with antigen (400 nM peptide 1).
The cells were cultured for 3 days in the presence or absence of 4 or 400 nM Cryj2). Then 0.5μ per well
The tritiated thymidine of Ci was added and the cells were further cultured for 16 hours. The thymidine uptake of lymph node cells in the presence and absence of the antigen was measured for the peptide-administered group and the control group, respectively, and the value obtained by dividing the thymidine uptake in the presence of the antigen by the thymidine uptake in the absence of the antigen was determined as the proliferation activity. It was calculated as The proliferation activity of lymph node cells derived from mice to which peptide 3 was previously administered was 49% for peptide 14 and 3% for Cryj2 as compared to the control group.
It was down 7%.

Formulation Example 1 Injection The peptide obtained by the method described in Examples 1 to 13 was added to a physiological saline containing 1% (w / v) sucrose as a stabilizer at a final concentration of 0.01 or 0.1. Alternatively, after dissolving to a concentration of 1 mg / ml and sterilizing by filtration, 2 ml is dispensed into a sterilized vial, lyophilized, and sealed. Prior to administration, 1 ml of distilled water for injection etc. is first placed in a vial bottle.
In addition, the contents are then uniformly dissolved and used. The product having excellent stability and comprising the peptide of the present invention as an active ingredient is useful as a dry preparation for treating and preventing cedar pollinosis.

Formulation Example 2 Syrup A trehalose powder (Trehaose: manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) was added to 0.1 mg / ml of any of the peptides obtained by the methods described in Examples 1 to 13. % (W
/ V), each is dissolved in distilled water, and the solution is sterile-filtered by a conventional method to obtain 13 kinds of syrups. Each 2 ml of these syrups is separately dispensed into sterile vials and sealed. This product, which has excellent stability and is easy to ingest, is useful as a syrup for treating and preventing cedar pollinosis.

[Acute Toxicity Test] The preparation obtained by the method described in Preparation Example 1 or 2 is orally or intraperitoneally administered to mice 20 days after birth by a conventional method. The test sample had an LD50 of 2 (as peptide) by any route of administration.
More than 00 mg / kg mouse body weight, indicating that the peptide of the present invention can be safely used in anti-cedar pollinosis agents to be administered to mammals including humans.

[0113]

According to the present invention, the T of cedar pollen allergen
A peptide comprising a cell epitope and an anti-cedar pollinosis agent comprising them as an active ingredient could be provided. And, since the peptide of the present invention does not substantially react with the immunoglobulin E antibody specific to cedar pollen allergen, when administered to mammals including humans in general, substantially no anaphylaxis is caused, and the cedar pollen allergen specific T cells can be inactivated. Moreover, since the peptide of the present invention contains a plurality of different T cell epitopes in its molecule, an anti-cedar pollinosis agent comprising the peptide of the present invention as an active ingredient is useful for a wider range of cedar pollinosis patients. Effective.

[0114]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 33 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn Gly Ile Ile Ala Ala 1 5 10 15 Tyr Gln Asn Pro Ala Ser Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys 20 25 30 Leu

SEQ ID NO: 2 Sequence length: 33 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Leu Thr Ser Gly Lys Ile Ala Ser
Cys Leu Phe Ala Ser Lys Asn 15
10 15 Phe His Leu Gln Lys Asn Gly Ile Ile
Ala Ala Tyr Gln Asn Pro Ala 20 25
30 Ser

SEQ ID NO: 3 Sequence length: 33 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Ile Ile Ala Ala Tyr Gln Asn Pro
Ala Ser Lys Leu Thr Ser Gly 15
10 15 Lys Ile Ala Ser Cys Leu Phe Ala Ser
Lys Asn Phe His Leu Gln Lys 20 25
30 Asn

SEQ ID NO: 4 Sequence length: 35 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn Tyr Gly Ile Ile Ala 1 5 10 15 Ala Tyr Gln Asn Pro Ala Ser Tyr Lys Leu Thr Ser Gly Lys Ile Ala 20 25 30 Ser Cys Leu 35

SEQ ID NO: 5 Sequence Length: 35 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Tyr Phe Ala Ser Lys 1 5 10 15 Asn Phe His Leu Gln Lys Asn Tyr Gly Ile Ile Ala Ala Tyr Gln Asn 20 25 30 Pro Ala Ser 35

SEQ ID NO: 6 Sequence Length: 35 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Tyr Lys Leu Thr Ser 1 5 10 15 Gly Lys Ile Ala Ser Cys Leu Tyr Phe Ala Ser Lys Asn Phe His Leu 20 25 30 Gln Lys Asn 35

SEQ ID NO: 7 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Val Asp Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys 1 5 10 15 Asn

SEQ ID NO: 8 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Ile Trp Leu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly 1 5 10 15 Lys Gly

SEQ ID NO: 9 Sequence Length: 17 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Ile Asp Ile Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn Thr Ile 1 5 10 15 Gly

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

SEQ ID NO: 11 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Lys Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asn 1 5 10 15 Cys

SEQ ID NO: 12 Sequence length: 47 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Phe Ala Ser Lys 1 5 10 15 Asn Phe His Leu Gln Lys Asn Thr Met Lys Val Thr Val Ala Phe Asn 20 25 30 Gln Phe Gly Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn 35 40 45

SEQ ID NO: 13 Sequence length: 47 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn Phe Ala Ser Lys 1 5 10 15 Asn Phe His Leu Gln Lys Asn Thr Met Lys Val Thr Val Ala Phe Asn 20 25 30 Gln Phe Gly Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp 35 40 45

SEQ ID NO: 14 Sequence length: 47 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Met Lys Val Thr 1 5 10 15 Val Ala Phe Asn Gln Phe Gly Phe Ala Ser Lys Asn Phe His Leu Gln 20 25 30 Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn 35 40 45

SEQ ID NO: 15 Sequence length: 47 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn Met Lys Val Thr 1 5 10 15 Val Ala Phe Asn Gln Phe Gly Phe Ala Ser Lys Asn Phe His Leu Gln 20 25 30 Lys Asn Thr Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp 35 40 45

SEQ ID NO: 16 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asn Pro Arg Ala Arg Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr 1 5 10 15 Asp Pro Trp Thr Ile 20

SEQ ID NO: 17 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Ser Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu 1 5 10 15 Ala Phe Asn Val Glu 20

SEQ ID NO: 18 Sequence length: 47 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn Thr Gly Ile Ile Ala 1 5 10 15 Ala Tyr Gln Asn Pro Ala Ser Trp Lys Leu Thr Ser Gly Lys Ile Ala 20 25 30 Ser Cys Leu Asn Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly 35 40 45

SEQ ID NO: 19 Sequence length: 47 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Lys Leu Thr Ser Gly 1 5 10 15 Lys Ile Ala Ser Cys Leu Asn Gly Ile Ile Ala Ala Tyr Gln Asn Pro 20 25 30 Ala Ser Trp Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn Thr 35 40 45

SEQ ID NO: 20 Sequence length: 81 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Ile Ile Ala Ala Tyr Gln Asn Pro
Ala Ser Trp Ser Met Lys Val 15
10 15 Thr Val Ala Phe Asn Gln Phe Gly Pro
Phe Ala Ser Lys Asn Phe His 20 25
30 Leu Gln Lys Asn Thr Lys Leu Thr Ser
Gly Lys Ile Ala Ser Cys Leu 35 40
45 Asn Tyr Gly Leu Val His His Val Ala Asn
Asn Asn Tyr Asp Pro Ser Gly 50 55
60 Lys Tyr Glu Gly Gly Asn Ile Tyr Thr
Lys Lys Glu Ala Phe Asn Val 65 70
75 80 Glu

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1: 125) (C12P 21/02 C12R 1: 865) (C12P 21/02) (C12R 1:91) (72) Inventor Junko Kawaguchi 1-58, Hiromachi, Shinagawa-ku, Tokyo Sankyo Stock Company (72) Inventor Akio Shiraishi 1-2-58, Hiromachi, Shinagawa-ku, Tokyo Sankyo Stock Company (72) Inventor Shinki Serizawa 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Stock Company (72) Inventor Mibun Taniguchi 5-3-1-5 Hirai, Okayama City, Okayama Prefecture

Claims (13)

[Claims] (1) Formula I: α ₁ -β ₁ -α ₂ -β ₂ -α ₃ -β ₃ -...- β _n-1 -α _n (I) (wherein α _{1 to} α _n Is a different amino acid sequence consisting of 11 to 19 amino acids, and in humans, the spear allergen protein Cryj1 or Cryj1
represents the amino acid sequence constituting the T cell epitope derived from j2; β _{1 to} β _n-1 represent any identical or different amino acid sequences consisting of 0 to 3 amino acids; n represents an integer of 2 or more) A peptide, a complex thereof, a derivative thereof, or a polymer thereof comprising the amino acid sequence represented by 2. The peptide according to claim 1, wherein n is an integer of 2 to 7, a conjugate thereof, a derivative thereof, or a polymer thereof. 3. The peptide according to claim 1, wherein n is an integer of 4 to 7, a conjugate thereof, a derivative thereof, or a polymer thereof. 4. The peptide according to claim 1, wherein n is 6 or 7, a conjugate thereof, a derivative thereof, or a polymer thereof. 5. The peptide according to claim _1, wherein β _{1 to} β _n-1 are any identical or different amino acid sequences consisting of 0 or 1 amino acid, a complex thereof, and a derivative thereof. Or a polymer thereof. 6. The peptide according to claim _1, wherein β _{1 to} β _n-1 are all tyrosine, a complex thereof, a derivative thereof, or a polymer thereof. 7. An amino acid sequence in which α _{1 to} α _n are different from each other, wherein the amino acid sequence is composed of 11 to 14 consecutive amino acids in the amino acid sequences shown in SEQ ID NOs: 7 to 11 in the sequence listing, SEQ ID NO: 16 And an amino acid sequence consisting of 11 to 16 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 17 and an amino acid sequence consisting of 11 to 19 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 17. The peptide according to any one of claims 1 to 6, a conjugate thereof, a derivative thereof, or a polymer thereof. 8. An amino acid sequence in which α _{1 to} α _n are different from each other, wherein the amino acid sequence consists of 11 to 14 consecutive amino acids in the amino acid sequences shown in SEQ ID NOs: 7 to 11 in the sequence listing, SEQ ID NO: 16 And an amino acid sequence consisting of consecutive 14 to 16 amino acids in the amino acid sequence shown in SEQ ID NO: 17, and an amino acid sequence consisting of consecutive 18 or 19 amino acids in the amino acid sequence shown in SEQ ID NO: 17. The peptide according to any one of claims 1 to 6, a complex thereof, a derivative thereof, or a polymer thereof. 9. An amino acid sequence consisting of 11 to 14 consecutive amino acids in the amino acid sequence shown in SEQ ID NOs: 7 to 11 of the sequence listing, and 14 to 16 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 16 The group consisting of the amino acid sequence consisting of amino acids and the amino acid sequence consisting of consecutive 18 or 19 amino acids in the amino acid sequence shown in SEQ ID NO: 17 corresponds to amino acid number 4-1 of SEQ ID NO: 7.
Amino acid sequence of SEQ ID NO: 4, amino acid No. 4-15 of SEQ ID NO: 7
Amino acid sequence of amino acid No. 3-15 of SEQ ID No. 7, amino acid sequence of amino acid No. 5-15 of SEQ ID No. 8, amino acid sequence of amino acid No. 5-16 of SEQ ID No. 8, amino acid No. 4 of SEQ ID No. 8 -16 amino acid sequence, amino acid number 4-14 amino acid sequence of SEQ ID NO: 9, amino acid number 4-15 amino acid sequence of SEQ ID NO: 9, amino acid number 3-14 of SEQ ID NO: 9, amino acid sequence of SEQ ID NO: 10 No. 4-14 amino acid sequence, No. 10 amino acid sequence No. 4-15 amino acid sequence, No. 3 amino acid sequence No. 3-15 amino acid sequence, No. 11 amino acid sequence No. 4-14 amino acid sequence, SEQ ID No. 11 The amino acid sequence of amino acid No. 3-14, the amino acid sequence of amino acid No. 3-15 of SEQ ID No. 11, Amino acid sequence of amino acid No. 6-18, amino acid sequence of amino acid No. 7-20 of SEQ ID No. 16, amino acid sequence of amino acid No. 6-21 of SEQ ID No. 16, amino acid sequence of amino acid No. 3-21 of SEQ ID No. 17, sequence 9. The peptide according to claim 8, which has the amino acid sequence of amino acid No. 3-20 of No. 17 and the amino acid sequence of amino acid No. 2-20 of SEQ ID No. 17, a complex thereof,
A derivative thereof, or a polymer thereof. 10. The peptide according to claim 1, wherein the amino acid sequence represented by the formula I is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 6, 12 to 15, and 18 to 20 in the sequence listing. , A complex thereof, a derivative thereof, or a polymer thereof. 11. An anti-cedar pollinosis agent comprising the peptide according to any one of claims 1 to 10, a complex thereof, a derivative thereof, or a polymer thereof as an active ingredient. 12. The method for treating cedar pollinosis according to claim 11.
The anti-cedar pollinosis agent according to the above. 13. The method for preventing cedar pollinosis according to claim 11.
The anti-cedar pollinosis agent according to the above.