JP2005336204A - Peptide-based immunotherapeutic agent for treating allergic disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide-based immunotherapeutic agent effective for treating allergic patients having sensitivity to two or more different allergens as well. <P>SOLUTION: A monomolecular multi-epitope peptide is obtained by binding T-cell epitope regions derived from different allergen molecules with each other. The peptide-based immunotherapeutic agent contains an effective amount of this multi-epitope peptide and is effective for the prophylaxis and treatment of a wide range of allergic diseases. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a multi-epitope peptide effective for peptide immunotherapy for allergic diseases.

  Allergic diseases are defined as type I hypersensitivity immune reactions, that is, dysfunctions based on type I immune reactions mediated by IgE antibodies, or disease groups due to disorders. The pathological conditions include hay fever, bronchial asthma, allergic rhinitis, atopic dermatitis, anaphylactic shock and the like. Hay fever is a typical allergic disease. In Japan, about 10% of people suffer from Japanese cedar pollinosis, but the number is constantly increasing. In the United States, it is estimated that there are 5-15% of patients with ragweed pollinosis. In this way, hay fever is a big social and economic problem because it has a large number of patients, it is accompanied by painful symptoms such as itchy eyes, runny nose, sneezing and nasal congestion, and it repeats every year once it occurs. There is a strong need for the development of fundamental therapies.

  Research on the establishment of type I allergic reactions is important in understanding and treating allergic diseases. At present, the focus is on the elucidation of the initial reaction in the allergen-specific immune reaction, in particular, the mechanism of allergic reaction control by T cells. Initiation of an immune response against foreign antigens, including allergens, depends on antigen presenting cells of the immune system. Antigen-presenting cells, including B cells, macrophages, and dendritic cells, take up foreign antigens and fragment them into antigenic peptides (T cell epitope peptides) that are α and β chains of MHC class II molecules (HLA class II in humans). And is expressed on the cell surface and presented to antigen-specific CD4 positive helper T cells (Th cells). HLA class II molecules consist of DR, DQ and DP molecules, the α chain of the DR molecule is encoded by HLA-DRA, the β chain is encoded by the HLA-DRB1, -DRB3, -DRB4 or -DRB5 gene, and the α chain of the DQ molecule is HLA-DQA1 and β chain are encoded by HLA-DQB1 gene, α chain of DP molecule is encoded by HLA-DPA1, and β chain is encoded by HLA-DPB1 gene. Each gene except HLA-DRA contains many alleles, and the pockets containing antigenic peptides show a high degree of polymorphism and their structures are slightly different. As a result, the type of antigenic peptide that binds to the pocket and is presented to the T cell is naturally limited to its structure.

  Th cells that have received HLA class II-restricted antigen information via the T cell receptor (TCR) are activated and secrete various cytokines to proliferate and differentiate B cells into plasma cells. Induces production. Th cells activated by antigen stimulation are Th1 cells that produce interleukin 2 (IL-2), interferon γ (IFN-γ), tumor necrosis factor β (TNF-β) due to differences in cytokine production patterns, They are classified into Th2 cells that produce IL-4, IL-5, IL-6, IL-10, and IL-13, and Th0 cells that produce both cytokines. The production of IgE antibodies that cause allergies is promoted by IL-4 and IL-13, but is suppressed by IFN-γ. That is, Th1 cells suppress IgE production and Th2 cells promote it. It can be said that whether or not allergic sensitization occurs is determined by whether Th1 cells or Th2 cells work upon antigen invasion. In fact, it is known that Th2 cells are dominant in allergic patients. Allergen-specific IgE antibodies adhere to basophils and tissue mast cells in peripheral blood, and subsequent allergen invasion causes IgE antibodies to cross-link on basophils and mast cells via the allergens. Inflammatory mediators including histamine, prostaglandins and leukotrienes are released, causing an immediate allergic reaction. In response to these inflammatory mediators, locally accumulated lymphocytes, monocytes, basophils, and eosinophils are activated and delayed by releasing mediators that cause a variety of reactions, including damage to tissues. Causes an allergic reaction.

  One of the attempts to treat specific allergies by suppressing IgE antibody production in an antigen-specific manner is desensitization therapy using allergen protein molecules. Although desensitization therapy has a long-lasting effect that cannot be obtained by drug therapy, it is not always recognized as a general therapy, although it is close to the only fundamental treatment. The reason for this is that, besides the risk of side effects (such as local swelling and anaphylactic shock) associated with this therapy, the mechanism of action is still unclear why this therapy is effective.

  Therefore, the idea of desensitization using a peptide antigen having a T cell epitope has appeared. The peptide fragment containing the T cell epitope on the allergen molecule used in this treatment method does not contain the B cell epitope or is monovalent even if it contains it, and cannot crosslink the high affinity IgE receptor of the mast cell, etc. For these reasons, side effects such as anaphylaxis do not occur even when administered to patients. Furthermore, it is known that when a T cell epitope is administered to a living body, the T cell is inactivated in an antigen-specific manner (La Salle JM, et al .: J. Exp. Med. 176). : 177-186, 1992). Under such a theoretical background, animal experiments of desensitization using a peptide containing the major T cell epitope of the feline hair allergen Fel d 1 were conducted, and T cell anergy was induced in vitro. (Briner, TJ et al .: Proc. Natl. Acad. Sci. USA, 90: 7608-7612, 1994) and clinical trials of desensitization with this peptide are currently underway (Norman , PS et al .: Am. J. Respir. Crit. Care Med. 154: 1623-1628, 1996; Simons, FE et al .: Int. Immunol. 8: 1937-1945, 1996). Such desensitization therapy using a peptide containing a major T cell epitope on the allergen molecule is called “Peptide-based Immunotherapy” (peptide immunotherapy or peptide desensitization therapy).

  As a selection criterion for T cell epitope peptides used in peptide immunotherapy, the importance index (mean T cell stimulation coefficient × frequency of appearance) has been devised (WO 94/01560). There is also a report that the peptide design should cover the diversity of HLA haplotypes in the patient population (Wallrer, B. P. & Gefter M. L .: Allergy, 49: 302-308, 1994).

  The “T cell epitope or T cell epitope peptide” means a T cell epitope having allergen-specific T cell activation ability (eg, taken as cytokine production or DNA synthesis), and an antigenic peptide containing the epitope. .

  Many allergic patients have specific IgE antibodies in each of two or more different allergen molecules. It is necessary for the fundamental treatment of allergies to develop a peptide immunotherapeutic agent effective for such patients. However, no such immunotherapeutic agent has been developed so far, and no such idea is shown in the above literature. Therefore, an object of the present invention is to provide a peptide immunotherapeutic agent that is also effective for allergic patients sensitive to two or more different allergens.

  Major allergens of cedar pollen include Cry j 1 (Yasueda, H. et al .: J. Allergy Clin. Immunol. 71: 77-86, 1983) and Cry j 2 (Taniai, M. et al .: FEBS Letter 239 : 329-332, 1988; Sakaguchi, M. et al .: Allergy. 45: 309-312, 1990), but more than 90% of Japanese cedar pollinosis patients are specific for Cry j 1 and Cry j 2 respectively. Patients with IgE antibody and the remaining less than 10% have specific IgE antibodies against either Cry j 1 or Cry j 2 (Hashimoto, M. et al .: Clin. Exp. Allergy 25: 848-852 , 1995). Therefore, the present inventors have shown that when Cry j 1 only or Cry j 2 only T cell epitopes are used for peptide immunotherapy for cedar pollinosis, the efficacy is sufficient for 90% of patients. Were not expected, and multiple epitopes were prepared that contained Cry j 1 T cell epitope and Cry j 2 T cell epitope in the same molecule. The multi-epitope peptide activates T cells of hay fever patients in vitro and does not react with IgE antibodies of the patients, and induces an immune response in vivo using mice. From the new findings, it was found that the multi-epitope peptide is effective as a peptide immunotherapy for cedar pollinosis patients.

  Furthermore, the present inventors have developed this idea, and in many cases of cedar pollinosis, there are many cases of clinical manifestations of cypress pollen. Therefore, the T-cell epitope of cypress pollen allergen Cha o 1 (particularly) Japanese cedar pollinosis patients who make multiple epitopes containing the T-cell epitope of Kohei No. 8-15527) and the cedar pollen allergen Cry j 1 in the same molecule, and in which the multi-epitope peptide does not react to each T-cell epitope And found to activate T cells of cypress pollinosis patients. Based on these novel findings, it has been found that such multi-epitope designs can be applied to T cell epitopes derived from various other allergens, not limited to cedar pollen allergens and cypress pollen allergens.

  Furthermore, as a selection criterion for T-cell epitopes when designing multiple epitopes, the HLA haplotypes in patient populations (including ethnic groups) were investigated, and HLA in populations were expected to be effective for more patients. Care should be taken to select epitopes that bind to HLA with high haplotype frequency as much as possible, and each epitope should not be presented by the same HLA class II molecule. It was clarified that the effective target patients can be further expanded by selecting the presented T cell epitope peptides.

  That is, this invention consists of an invention as described in each claim of a claim.

  The following describes the design of a multi-epitope peptide that is effective for patients sensitive to cedar pollen, cypress pollen, or both, but the invention is not limited to patients sensitive to these allergens. . For example, other allergens whose primary structure has already been clarified, such as ragweed (Amba1, Amba2, Amba5, Ambt5, Ambp5), camphor (Dacg2), barley (Lolp1, Lolp2, Lolp3), The technical idea of the present invention can also be applied to tree pollen such as Alng1), hippopotamus (Betv1, Betv2), mountain seda (Juns1), empitzabikine (Junv1), and various other allergens not described here.

  In this specification, “multi-epitope peptide” refers to a peptide in which peptides containing T cell epitopes derived from different allergen molecules (also referred to as antigenic peptides or simply peptides) are linearly linked to form one molecule. means. Moreover, in order to reduce the possibility that a newly recognized epitope site is generated between peptide regions containing T cell epitopes, it is preferable to interpose a region that is cleaved within an antigen-presenting cell. As a result, since the multi-epitope peptide is cleaved into individual antigen peptides in the cleavage region, an effect equivalent to that obtained when individual antigen peptides are administered as a mixture is expected. The cleavage region may have any structure as long as it is cleaved in vivo, but arginine dimer or lysine dimer which is a recognition sequence of cathepsin B which is an enzyme contained in lysosome can be used.

  The multi-epitope peptide design of the present invention will be described using cedar pollen allergens Cry j 1 and Cry j 2 as examples.

  Peripheral blood lymphocytes of cedar pollinosis patients are stimulated with Cry j 1 or Cry j 2, and a T cell line for each patient is prepared. About 15 amino acids covering the entire primary structure of Cry j 1 (WO 94/01560) or Cry j 2 (Komiyama, N. et al .: Biochem. Biophys. Res. Commun. 201: 1201, 1994) By stimulating a T cell line established from a patient with an overlapping peptide, the amino acid sequence recognized as a T cell epitope on the Cry j 1 or Cry j 2 molecule is identified (FIGS. 1 and 2).

  Next, HLA class II molecules that bind to these antigenic peptides are typed.

  In humans, it is known that there are DR, DQ and DP molecules at the locus of HLA class II molecules. This means that the differentiation of T cells is regulated by antigen-presenting molecules DR, DQ and DP that present antigen. Therefore, the T cells that have received antigen peptide information via which DR, DQ, or DP molecule the antigen-presenting molecule from which the Cry j 1 or Cry j 2 antigen peptide is presented. Alternatively, it is determined by using T cell clones established for each patient whether they tend to differentiate into Th2 cells (FIGS. 3 and 4).

  3 and 4, it is clear that differentiation into Th1, Th2 or Th0 after stimulation with an antigen peptide is not defined by a specific epitope or a specific combination of HLA molecules. That is, when selecting a peptide for the design of the multi-epitope peptide of the present invention, a peptide containing at least a T cell epitope site can stimulate T cells, and thus is a candidate for antigen peptide selection. obtain.

  Criteria for selecting peptides for multi-epitope peptide design are as follows: (1) First, select peptides in descending order of importance index (International Publication No. 94/01560). Select), (2) select peptides with HLA class II molecules with high frequency of appearance as antigen presenting molecules, (3) if there is not much difference in importance index, different types to increase efficacy It is to select a peptide that is presented as a constrained molecule. In other words, when selecting the T cell epitope of the allergen for a certain allergic disease, the HLA haplotype of the allergic patient of a certain group is analyzed, and the T cell epitope having a high gene frequency of the HLA haplotype of the population to which the patient group belongs is analyzed. This is the choice that is expected to be most effective. In other words, it means that the T cell epitope selected in this way may not be effective at all in other populations.

For example, in the case of DPB1 ^* 0501 of HLA haplotype, it is assumed that a Japanese patient has a high frequency of this HLA haplotype in a certain allergic disease and selects this HLA haplotype-restricted T cell epitope. On the other hand, the peptides selected in this way are hardly expected to be effective in North Americans and patients with the same allergic diseases. This is because the HLA haplotype has a very high gene frequency of 39.0% in the Japanese population, but 1.3% in the white population in North America and 0.8% in the black population. DPB1 ^* 0401 (North America; whites 30.2%, blacks 11.1%, Japaneses; 4.8%) should be selected if HLA-DP restricted T cell epitopes are selected from North Americans. In addition, it is important to select peptides that are presented at different loci levels, such as DR, DQ, DP, or different haplotype antigen-presenting molecules, even if the loci are the same.

  In this case, it is preferable that the epitope site to be selected does not contain a cysteine residue. If the cysteine residue is contained in the epitope site, it may bind nonspecifically to the HLA class II molecule, and when immunized with an antigen peptide containing a cysteine residue, a site that is not originally an antigen is It may be recognized as an epitope. When it is recognized as an epitope, it is predicted that the second and third peptide administration will recognize an epitope containing cysteine and increase the risk of side effects.

Hereinafter, specific examples of the multiple epitope design will be shown. Using the importance index of Cry j 1 and Cry j 2 shown in FIG. 1 and FIG. 2, the importance index of the T cell epitope in Cry j 1 is amino acid number 211-225 (hereinafter referred to as p211-) of peptide number 43. 225) (restrained molecule DPA1 ^* 0101-DPB1 ^* 0501) is the highest, and peptide number 22 p106-120 (restrained molecule DRB5 ^* 0101) is the second. These two can be selected as antigen peptides to be used for multi-epitope peptides. Moreover, the importance index in Cry j 2 is high in peptide number 14 p66-80 (restrained molecule DRB5 ^* 0101) and 38th p186-200 (DRB4 ^* 0101), and can be selected as an antigen peptide in the same manner. The peptide number 37 p181-195 located in front of peptide number 38 of Cry j 2 has an importance index of 280 but the constrained molecules are DPA1 ^* 0101-DPB1 ^* 0201, and the 38th constrained molecule is Different. Peptide No. 37 p181-195 overlaps with peptide No. 38 p186-200 by 10 residues, and can be selected as an HLA-DP molecule-restricted peptide by adding No. 37 4 residues before No. 38 . There is no antigen peptide showing DQ restriction among peptides selected so far. Cry j 1 peptide number 4 p16-30 is a restricted molecule of DQA1 ^* 0102-DQB1 ^* 0602, but cannot be selected because it contains a cysteine residue in the center of the epitope. P341-360 corresponding to peptide number 69 to 70 of Cry j 2 is a peptide represented by DQA1 ^* 0102-DQB1 ^* 0602, and this peptide also contains cysteine. However, since only the 69th peptide not containing cysteine can activate T cells, only 12 residues, that is, p344-355 (ISLKLTSGKIAS) can be selected. In addition, peptide number 22 p106-120 of Cry j 1 contains cysteine at position 107, but the minimum sequence required by determining the core sequence of the T cell epitope using a T cell clone is p109-117 (FIKRVSNVI) (sequence Number: 4) 9 residues. That is, it can be used even if the Pro-Cys residue at p106-107th position is removed.

  The antigen taken up into the antigen-presenting cell is degraded by lysosomes. How foreign proteins are incorporated into antigen-presenting molecules, how they are processed, and how they bind to HLA class II molecules remains unresolved. However, at present, it is pointed out that cathepsin B may be involved in antigen cleavage in this complex mechanism (Nobuhiko Katsunuma, Japanese Society for Immunology (1995) 25:75).

  For some HLA class II types, the HLA binding amino acid motif of the antigenic peptide has been determined. Although binding to HLA class II molecules has specificity, any kind of antigenic peptide can be bound by a peptide that satisfies certain rules for a particular HLA class II type (Rammensee, H.-G. et al Immunogenetics. (1995) 41: 178-228). For this reason, there is a possibility that a newly recognized epitope site is generated at the site where the antigen peptide is connected. In order to avoid this, it is preferable to design the multi-epitope peptide so that each antigen peptide is cleaved in the antigen-presenting cell. Since the peptide sequence recognized by cathepsin B is the hydrophobic amino acid-Arg-Arg or Lys-Lys, Arg-Arg or Lys-Lys is added to the latter half of the peptide containing the epitope, and the subsequent epitope sequence is Arg-Arg Or it arrange | positions so that a hydrophobic amino acid sequence may be located after Lys-Lys.

Regarding the sequence of antigen peptides in this specific example, Arg-Arg was interposed between the antigen peptides, so it is not necessary to ask the order, but peptide number 14 of Cry j 2 (FIG. 2) For Arg, when Arg is connected to the latter half of this peptide, Tyr at position 73 becomes the first anchor, and the added Arg residue can be the 9th amino acid of the peptide binding motif of DRB5 ^* 0101, becoming the second anchor There is sex. As a result, it may be recognized as a new epitope. For this reason, this sequence is preferably located at the end of the multi-epitope peptide.

The multi-epitope peptide thus obtained is shown in SEQ ID NO: 1. The multi-epitope constrained molecules are DRB4 ^* 0101, DRB5 ^* 0101, DPA1 ^* 0101-DPB1 ^* 0201, DPA1 ^* 0101-DPB1 ^* 0501, and DQA1 ^* 0102-DQB1 ^* 0602. These gene frequencies in the Japanese population were calculated at the 11th International Histocompatibility Antigen Conference (Tsuji, K. et al. HLA 1991 vol. 1 (1992) Oxford University Press). It is calculated that DRB4 ^* 0101 is 0.291, DRB5 ^* 0101 is 0.056 (DRB5 ^* 0102 is 0.070), DPB1 ^* 0201 is 0.208, DPB1 ^* 0501 is 0.399, and DQB1 ^* 0602 is 0.053 (DQB1 ^* 0601 is 0.204). From this value, the antigen frequency is calculated as DRB4 ^* 0101 = 0.50, DRB5 ^* 0101 = 0.11 (DRB5 ^* 0102 = 0.14), DPB1 ^* 0201 = 0.37, DPB1 ^* 0501 = 0.64 (0.79 in the observation by Hori et al.), DQB1 ^* Calculated as 0602 = 0.10 (DQB1 ^* 0601 = 0.37). Since DRB5 ^* 0101 and DQB1 ^* 0602 are regarded as the same because of linkage disequilibrium, the value DRB5 ^* 0101 can be used. The probability of possessing both or one of DPB1 ^* 0201 and DPB1 ^* 0501 in the Japanese population is calculated as 0.85. Also, the probability of possessing both or one of DRB4 ^* 0101 and DRB5 ^* 0101 is calculated as 0.56. From this value, it is estimated that approximately 90% of patients can recognize one or more T cell epitopes contained in the multi-epitope peptide of SEQ ID NO: 1. However, it is unclear whether patients with these HLA-types have a T cell repertoire capable of recognizing these epitope peptides even if antigen information is presented by these restriction molecules on the T cell side. In addition, since the number of epitopes for causing T cell proliferation is unknown (more than one may be necessary), the effectiveness of this multi-epitope peptide is considered to decrease. Actually, the result of proliferative response of 17 peripheral blood lymphocytes, that is, around 77% is expected to be a reasonable value.

  Furthermore, a multi-epitope peptide containing more T cell epitopes can be designed to expand the effective target personnel. For example, Cry j 1 p213-225, p108-120, Cry j 2 p182-200, p79-98, Cry j 1 p80-95, Cry j 1 p66-80 were connected in this order ( SEQ ID NO: 2) or Cry j 1 p213-225, p108-120, Cry j 2 p182-200, p79-98, Cry j 1 p67-95, Cry j 2 p238-251, p66- It is a multi-epitope peptide (SEQ ID NO: 3) in which 80s are connected in this order. These multi-epitope peptides are effective as peptide immunotherapy agents because they stimulate the peripheral blood lymphocytes of all 21 cedar pollinosis patients studied and do not react with patient IgE antibodies. By further advancing this concept, T cell epitopes of different allergens such as cypress pollen allergen and cedar pollen allergen can be prepared by the method shown in Example 13 to further increase the effectiveness.

  It is also included in the present invention to modify the antigenic peptide portion used in the multi-epitope peptide to modulate T cell activity. Modification means performing amino acid substitution, deletion, or insertion of one or more residues. The qualitative change given to T cells by amino acid substitution of the antigen peptide can be performed by a known method. For example, by replacing specific amino acids in the multi-epitope peptide of the present invention with 1) a similar amino acid, Asp is Glu, Asn is Gln, Lys is Arg, Phe is Tyr, and Ile is Leu. , Synthesizing an analog peptide with Gly replaced by Ala and Thr replaced by Ser, and comparing T cell proliferation ability or lymphokine production ability with the original peptide. 2) Substitution with a dissimilar amino acid Then, polar amino acid and hydrophilic amino acid are substituted with hydrophobic amino acid Ala, and hydrophobic amino acid is substituted with hydrophilic amino acid Ser, and compared with the original peptide. A multi-epitope peptide that is immunologically equivalent to the multi-epitope peptide of the present invention (importance index, T cell activation ability, etc.) is also included in the present invention.

Many T cells that react with antigen peptides derived from Cry j 1 or Cry j 2 have Th2 and Th0 properties (FIGS. 3 and 4). By the way, BCG vaccine prevents infection from Mycobacterium tuberculosis by activating cellular immunity. Th1 type T cells must be induced in order to stimulate cellular immunity. However, when the properties of human T cell clones inoculated with BCG are examined, it is reported that there are many Th1 type T cells (Matsushita). Sho, 45th Japanese Allergy Society, page 836, 1995). According to Matsushita's report, there is a Th1 clone that recognizes the 84-100 amino acid sequence (EEYLILSARDVLAVVSK) of Mycobacterium tuberculosis BCGa protein in an HLA-DR14 (DRB1 ^* 1405) -restricted manner. Therefore, select a DPA1 ^* 0101-DPB1 ^* 0501-restricted T cell epitope which is an HLA haplotype possessed by more than 60% of Japanese (for example, Cry j 1 43 peptide (p211-225) / KSMKVTVAFNQFGPN in FIG. 1) ), The multi-epitope peptide EEYLILSARDVLAVVSKRRMKVTVAFNQFGPN, which connects this peptide with the 84-100 T cell epitope of DRB1 ^* 1405-restricted Mycobacterium tuberculosis BCGa protein, is considered to have a much higher probability of hitting cedar pollinosis patients with a haplotype of DRB1 ^* 1405 . If such a multi-epitope peptide is used, a BC1 antigen-derived peptide can be expected to produce Th1 lymphokines, particularly IL-12. It is known in many human and mouse cases that IL-12 has a reciprocal effect on IL-4 and acts on T cells to induce Th cell differentiation to Th1 (Manetti, R., et al. al .: J. Exp. Med., 177, 1199-1204, 1993; Wu, C., et al .: J. Immunol., 151, 1938-1949, 1993; Hsieh, C., et al .: Science 260, 547-549, 1993). In particular, Manetti et al. Show that Der p 1 antigen-specific T cell clones, which are one of the mite allergens, usually induce Th2, but Th1 or Th0 is induced in the presence of IL-12. Yes. Therefore, by using a multi-epitope peptide combining a T cell epitope capable of inducing Th1 and an allergen-reactive T cell epitope, an originally Th2-inducible T cell can be induced into Th1 or Th0 type T cells. Be expected.

  Subcutaneous administration of a peptide containing at least one Cry j 1 and / or Cry j 2 T cell epitope of the present invention to mice results in T cell anergy when exposed to subsequent cedar pollen allergens (FIGS. 13 and 14). , IL-2 production is also significantly reduced compared to the control group. There is a report that IL-2 decreases during human desensitization therapy (J. Allergy Clin. Immunol. 76: 188, 1985). Furthermore, the multi-epitope peptide of the present invention activates each T cell clone for each T cell epitope peptide constituting the peptide (FIG. 10) and does not react with the patient IgE antibody (FIG. 8). These results indicate that the multi-epitope peptide of the present invention induces immune tolerance against allergens and is useful as a peptide immunotherapeutic agent for allergic diseases. The multi-epitope peptide of the invention can be administered with a pharmaceutically acceptable carrier or diluent. The effective amount will vary according to factors such as the degree of sensitivity to the cedar pollen allergen, age, sex and patient weight, and the ability of the peptide to elicit an immune response in the patient.

  The administration route can be administered by a simple method such as injection (subcutaneous, intravenous), nasal drop, eye drop, oral, inhalation, or transdermal.

  In the present specification and sequence listing, the one-letter code of amino acids is a notation established by the IUPAC Biochemical Nomenclature Committee (see Biochemical Dictionary (2nd edition), page 1468, Table 1.1).

Identification of Cry j 1 and Cry j 2 T cell epitopes using T cell lines
The peripheral blood lymphocytes of 18 cedar pollinosis patients were stimulated with cedar pollen allergens Cry j 1 or Cry j 2, and T cell lines that specifically recognize each allergen were established for each patient.

5 × 10 ⁴ autologous B cell lines treated with mitomycin C, 2 μM overlapping peptide, 2 × 10 ⁴ T cell lines on a 96-well plate, containing 0.2 ml of 15% serum The cells were cultured in RPMI-1640 medium for 2 days, and further cultured for 18 hours after adding 0.5 μCi of [ ³ H] thymidine. The cells were collected on a glass filter with a cell harvester, and the amount of [ ³ H] thymidine incorporated into the cells was measured with a liquid scintillation counter. The value of the cellular uptake of ^{[3 H]} thymidine after addition of the peptide, obtained by dividing the value of the cellular uptake of control ^{[3 H]} thymidine without added peptide value (stimulation index / Stimulation Index) Is defined as having been recognized as an antigenic peptide.

  In the case of Cry j 1, the average number of T cell epitope sites on the Cry j 1 molecule recognized by each patient was 9.8, and the range was 4 ≦ number of epitopes ≦ 15. On the other hand, in the case of Cry j 2, the average was 8.7, and the range was 2 ≦ number of epitopes ≦ 13. Since Cry j 1 is composed of 353 amino acids and Cry j 2 is composed of 379 amino acids, there are approximately 2.3 to 2.8 T cell epitope sites per 100 amino acid residues.

  Since HLA-class II types are thought to vary from patient to patient, the recognized T cell epitopes are expected to be different for each HLA-class II type. Therefore, the antigen peptide recognized by each patient was mapped for each patient. As a result, on the Cry j 1 and Cry j 2 molecules, the epitope sites that can be recognized by each patient were different. On the allergen molecule, there are sites that are easily recognized as T cell epitopes by individuals and sites that are difficult to recognize. In addition, since the proliferation rate of T cells differs for each T cell epitope, it is impossible to determine which antigen peptide may be selected for the design of multiple epitopes only with this epitope map. Therefore, for 18 patients, the average stimulation coefficient is calculated for the antigenic peptide when the stimulation coefficient is 2 or more, and the ratio (frequency of appearance) of patients holding the antigenic peptide is multiplied by this value for each epitope. An “importance index” was calculated, which indicates the superiority of each other (see International Publication No. 94/01560).

  The results are shown in FIG. 1 and FIG. In Cry j 1, the peptide number 43 (p211-225) has the highest importance index of 679, the peptide number 22 index is 578, and the peptide number 4 index is 373. In Cry j 2, the index of peptide number 14 shows the highest value at 709, the index of peptide number 38 continues to 680, and the index of peptide number 48 continues to 370. When considering peptide immunotherapy, there is a method of selecting one antigenic peptide with a high importance index and using it as peptide immunotherapy, but Cry j 1 No.22, the most frequently occurring peptide, or Even in the case of No. 43, the effect can be expected only in 72% of patients, and the actual effective rate will be further lowered. There is a need to combine several T cell epitopes to increase the efficiency. In this case, candidates for T cell epitopes with high importance index are candidates, but no matter how high the importance index is selected, HLA class II molecules presenting these epitopes as antigens If they are the same, the effectiveness rate cannot be increased. Therefore, it is necessary to identify the type of HLA class II molecule that presents the T cell epitope peptide.

Identification of T cell epitope peptides recognized by T cell clones
Among 18 cedar pollinosis patients, 2 patients [Patient B (hereinafter abbreviated as PB), Patient J (PJ)] recognizing peptide numbers 43 and 22 exhibiting a high importance index in Cry j 1. Three patients [PB, patient C (PC), patient R (PR)] who recognize peptide numbers 14, 38, 48, and 69 showing high importance index in Cry j 2 T cell clones that recognize Cry j 1 or Cry j 2 were established by stimulating peripheral blood lymphocytes of hay fever patients with Cry j 1 or Cry j 2. The HLA-class I and class II types of the 4 patients are shown below.
PB: A2 / 24 - B39 / 55 - Cw7 / w3 - DRB1 * 1501/0901 - DRB4 * 0101 - DRB5 * 0101, DQA1 * 0102/0301 - DQB1 * 0602/0303 - DPA1 * 0101/0101 - DPB1 * 0501 / 0201,
PJ: A24 /--B61 / 51-Cw3 /--DRB1 ^* 1501/0802-DRB5 ^* 0101, DQA1 ^* 0102/0401-DQB1 ^* 0602/0402-DPA1 ^* -/--DPB1 ^* 0501/0402,
PC: A-2 / 2-B54 / 51-Cw1 /-, DRB1 ^* 0405/1501-DRB4 ^* 0101-DRB5 ^* 0101-DQA1 ^* 0301/0102-DQB1 ^* 0401/0602-DPA1 ^* 0202/0202-DPB1 ^* 0201/0501,
PR: A-11 /--B60 / 35-Cw7 / w3-DRB1 ^* 0901/1501-DRB4 ^* 0101-DRB5 ^* 0101-DQA1 ^* 0301/0102-DQB1 ^* 0303/0602-DPA1 ^* 01/0202-DPB1 ^* 0201/0201).

A total of 35 T cell clones that specifically recognize Cry j 1 were established from PB-derived peripheral blood lymphocytes and 14 from PJ-derived peripheral blood lymphocytes. Similarly, a total of 31 T cell clones specifically recognizing Cry j 2 were established from PB-derived peripheral blood lymphocytes, 10 from PC-derived peripheral blood lymphocytes, and 17 from PR-derived peripheral blood lymphocytes. Since these T cell clones are all CD3 ⁺ , CD4 ⁺ , CD8 ⁻ , TCRαβ ⁺ , TCRγδ ⁻ , it was found that the restricted molecules are HLA-class II molecules. RPMI- containing 5 × 10 ⁴ autologous B cell lines treated with mitomycin C on 96-well microculture plates, 2 μM overlapping peptide and 2 × 10 ⁴ T cell clones with 0.2 ml of 15% serum The cells were cultured in 1640 culture for 2 days, and 0.5 μCi of [ ³ H] thymidine was added, followed by further incubation for 18 hours. The cells were collected on a glass filter with a cell harvester, and then [ ³ H] thymidine uptake was measured with a liquid scintillation counter. By this operation, the T cell epitope recognized by each T cell clone was identified.

  Among the prepared T cell clones that recognize Cry j 1, 69% (34/49) showed proliferative responses to peptide stimuli containing the antigen and were able to identify the antigenic peptide. Similarly, antigen peptides could be identified in 69% (40/58) of T cell clones that recognize Cry j2. T cell clones that specifically recognize Cry j 1 are peptide numbers 4, 13, 19, 22, 30, 31, 39, 43, 51, 66, and T cell clones that specifically recognize Cry j 2 , Peptide Nos. 4, 8, 14, 17, 31, 37, 38, 48, 65, 66, 68, 69, 70 were recognized. The results are summarized in FIG. 3 and FIG.

Identification of HLA class II-restricted molecules at the locus level Monoclonal antibodies that react specifically with HLA-class II DR, DQ, or DP are added to the T cell clone growth response system established in Example 2 Thus, we identified HLA class II restricted molecules at the locus level by blocking the proliferative response of T cells.

2 × 10 ⁴ autologous B cell lines treated with mitomycin C, 2 μM overlapping peptide, 3 μg / ml anti-DR, DQ or DP monoclonal antibody (Becton / Dickinson) on 96-well microculture plates 2 × 10 ⁴ T cell clones were cultured in RPMI-1640 medium containing 0.2 ml of 15% serum for 2 days, and 0.5 μCi of [ ³ H] thymidine was added for another 18 hours. Cultured. The cells were collected on a glass filter with a cell harvester, and then [ ³ H] thymidine uptake was measured with a liquid scintillation counter. The results are shown in FIG. From this figure, Cry j 1 p106-120, Cry j 2 p66-80, Cry j 2 p186-200 peptide restricted molecule is DR, Cry j 2 p341-355 peptide restricted molecule is DQ, Cry j 1 p211-225 It can be seen that the constrained molecule of Cry j 2 p181-195 is DP. The restriction molecules of other T cell clones were similarly analyzed (see FIGS. 3 and 4).

Identification of constrained molecules in individual types of HLA class II molecules
T cell clones that have been able to identify restricted molecules at the HLA class II locus level, with regard to DR, mouse L-cells into which individual types have been introduced, and with respect to DQ or DP, haplotype-matching B cell lines Can be used as an antigen-presenting cell to identify restricted molecules in individual types.

5 × 10 ⁴ mouse L-cells treated with mitomycin C on 96-well microculture plates, or haplotype-matched B cell line, 2 μM overlapping peptide, 3 μg / ml anti-DR, DQ, or DP alone Cloned antibody (Becton / Dickinson), 2 × 10 ⁴ T cell clones are cultured in RPMI-1640 medium containing 0.2 ml of 15% serum for 2 days, and 0.5 μCi of [ ³ H] thymidine is added Thereafter, the cells were further cultured for 18 hours. The cells were collected on a glass filter with a cell harvester, and then [ ³ H] thymidine uptake was measured with a liquid scintillation counter.

A restricted molecule can be identified when a proliferative response of a T cell clone is observed. Restrained molecule presenting Cry j 1 p106-120 peptide is DRB5 ^* 0101, Restrained molecule presenting Cry j 1 p211-225 peptide is DPA1 ^* 0101-DPB1 ^* 0501, Restrained molecule presenting Cry j 2 p66-80 peptide DRB5 ^* 0101, restraint molecule presenting Cry j 2 p181-195 peptide is DPA1 ^* 0101-PDB1 ^* 0201, restraint molecule presenting Cry j2 p186-200 peptide is DRB4 ^* 0101, Cry j 2 p341-355 peptide The constrained molecule presenting was DQA1 ^* 0102-DQB1 ^* 0602 (FIG. 6). The analysis results for other epitope sites are shown in FIGS.

Identification of Th type of T cell clones Th2 cells are assumed to be involved in the development of allergy. At the current research level, there are still many unresolved parts of whether the differentiation of T cells into Th1 or Th2 cells is regulated at a specific epitope peptide or HLA-class II locus level after antigen stimulation. However, when Th2 cells are predominantly induced after stimulation with peptides, there is a high possibility that cedar pollinosis will be worsened by peptide administration. The T cell clone prepared in Example 2 was stimulated with an epitope peptide recognized by T cells, and the Th type was determined by measuring the production amounts of IL-2, IL-4, and IFNγ.

RPMI-1640 containing 1 × 10 ⁵ autologous B cell lines treated with mitomycin C on 24-well microculture plates, 2 μM epitope peptide, 5 × 10 ⁵ T cell clones with 1 ml of 10% human serum The cells were cultured for 24 hours in the culture solution. Cells were precipitated by centrifugation to obtain a culture supernatant. IL-2, IL-4, and IFNγ in the culture supernatant can be obtained using commercially available ELISA kits [IL-2 (R & D)], IL-4 (Medogenics), and IFNγ (Otsuka Assay Laboratory). It was measured.

  The amounts of IL-2, IL-4, and IFNγ produced by each T cell clone are shown in FIGS. T cell clones that recognize Cry j 1 had 12 Th2, 2 Th1 cells, and 16 Th0 cells, and had more Th2 than Th1, but T cell clones that recognized Cry j 2 had Th2 cells. 10, Th1 cells were 8, Th0 cells were 8, and Th2 and Th1 were comparable. Comparing T cell epitopes, restriction molecules, and Th types recognized by individual T cell clones, Th2, Th1, and Th0 types differ depending on the individual T cell clone, and several recognize the same epitope and the same antigen-presenting molecule. Th2 cells and Th1 cells have been found in this T cell clone. These results imply that differentiation of T cells after Cry j 1 or Cry j 2 stimulation into Th2, Th1, or Th0 cells is not regulated by specific T cell epitopes or specific binding molecule combinations doing. That is, it has been found that all peptides containing a T cell epitope site can be candidates for the multi-epitope peptide of the present invention.

Generation of multi-epitope peptides
As a result of identifying IgE antibody epitope sites present in Cry j 1 and Cry j 2 molecules, Cry j 1 has no IgE epitope that recognizes this primary structure, and Cry j 2 has at least 4 IgE antibody epitopes. These IgE antibody epitope sites were found to be different from the T cell epitope sites. Based on this finding, peptides shown in FIG. 7 were selected from Cry j 1 and Cry j 2 T cell epitope sites.

  Peptides a and b in FIG. 7 correspond to peptides No. 43 and 22 of Cry j 1 in FIG. 1, respectively, peptide c corresponds to No. 14 in Cry j 2 in FIG. 2, and d and e correspond to FIG. Cry j 2 of 37-38 and a part of 69-71 amino acids.

When these five peptides are connected in series to create a multi-epitope peptide, the two peptides and a and b are fixed in the order of ab, and the remaining three peptides (c, d, and e) are randomly connected. There are the following six types of multi-epitope peptides in which Arg-Arg sequences are inserted between the peptides.
CA # 1. a-Arg-Arg-b-Arg-Arg-c-Arg-Arg-d-Arg-Arg-e
CA # 2. a-Arg-Arg-b-Arg-Arg-c-Arg-Arg-e-Arg-Arg-d
CA # 3. a-Arg-Arg-b-Arg-Arg-d-Arg-Arg-c-Arg-Arg-e
CA # 4. a-Arg-Arg-b-Arg-Arg-d-Arg-Arg-e-Arg-Arg-c
CA # 5. a-Arg-Arg-b-Arg-Arg-e-Arg-Arg-c-Arg-Arg-d
CA # 6. a-Arg-Arg-b-Arg-Arg-e-Arg-Arg-d-Arg-Arg-c

Reactivity of multi-epitope peptide against human IgE antibody Six multi-epitope peptides (CA # 1 to # 6) obtained in Example 6 were dissolved in 0.2 M acetate buffer (pH 4.5) and 0.1 ml / well. In addition to a black plate (Dainippon Pharmaceutical Co., Ltd.), it was left overnight at 4 ° C. After removing the antigen solution, the plate was washed three times with a washing solution, and 29 cedar pollen patient and healthy subject serum (4-fold dilution) was added and reacted at 37 ° C. for 4 hours. After removing the serum, the plate was washed 3 times with a washing solution and reacted with β-D-galactosidase-labeled anti-human IgE antibody (Pharmacia) overnight at room temperature. After washing with the washing solution three times, 0.1 mM 4-methylumbelliferyl-β-D-galactopyranoside / 0.01 M phosphate buffer (pH 7.0), 0.1 M NaCl, 1 mM MgCl ₂ , 0.1% NaN ₃ , 0.1 A substrate solution of% BSA was added and reacted at 37 ° C. for 2 hours. A 0.1 M glycine / NaOH, pH 10.3 solution was added thereto to stop the reaction, and the fluorescence intensity was measured with a fluorescence spectrophotometer (Labsystems). In addition, biotin-labeled rabbit anti-d-epitope IgG and galactosidase-labeled streptavidin (Pierce) were reacted as a positive control for each multi-epitope peptide.

  As a result, in all 29 human sera, the fluorescence intensity was 3 to 5 for all 6 multi-epitope peptides (C.A. # 1 to # 6) (blank value was 3 or 4). In contrast, Cry j 1, an antigen extracted and purified from cedar pollen, had a fluorescence intensity of 1,000 or more in 6 people, 100 or more in 14 people, 10 or more in 4 people, and 9 or less in 5 people. On the other hand, rabbit anti-d epitope peptide IgG showed 3,000 or more against 6 types of consensus allergens (blank value was 112, 230 for Cry j 1 allergen). Based on the above, it was found that the multi-epitope peptide does not substantially bind to the allergen-specific IgE antibody of Japanese cedar pollinosis patients, and the connection order of each epitope does not affect the reactivity with the human IgE antibody. (FIG. 8).

Presence / absence of recognition of T cell epitope of multi-epitope peptide It was examined whether the antigenic peptide constituting CA # 4 among the multi-epitope peptides obtained in Example 6 actually functions as a T cell epitope.

5 × 10 ⁴ autologous B cell lines, 2 × 10 ⁴ T cell clones treated with mitomycin C on 96-well microculture plates in RPMI-1640 medium containing 0.2 ml of 15% serum 50 μg / ml Cry j 1 as an antigen, 2 μg / ml Cry j 2, individual antigen peptides constituting multi-epitope peptide CA # 4, or 10 μg / ml CA # 4 multi-epitope peptide prepared by gene expression The cells were cultured with either of them for 2 days, and 0.5 μCi of [ ³ H] thymidine was added and further cultured for 16 hours. The cells were collected on a glass filter with a cell harvester, and then [ ³ H] thymidine uptake was measured with a liquid scintillation counter. The results are shown in FIG.

  T cell clone PB8-3 recognizing Cry j 1 p106-120, T cell clone PB8-34 recognizing Cry j 1 p211-225, T cell clone PB4-22 recognizing Cry j 2 p66-80, Cry j 2 T cell clone PB14-5 that recognizes p181-195 and T cell clone PB14-34 that recognizes Cry j 2 p186-200 both react well with antigenic peptides. On the other hand, in the case of multi-epitope peptides, T cell clones respond to proliferation with the same strength as individual peptides. A slightly weak proliferative response to multi-epitope peptide stimulation was observed for the T cell clone PB14-19 that recognizes Cry j 2 p341-355.

  The above results indicate that each of the antigen peptides contained in the multi-epitope peptide functions well as an epitope and retains the ability to activate T cells.

Multi-epitope peptide proliferative response of cedar pollinosis patients' peripheral blood lymphocytes Since multi-epitope peptides contain T-cell epitope sites, it is necessary to elicit proliferative responses in peripheral blood lymphocytes when attempting peptide immunotherapy. is there. Multi-epitope peptides stimulated peripheral blood lymphocytes to investigate whether a proliferative response was observed.

Suspended peripheral blood lymphocytes from Japanese cedar pollinosis patients or healthy individuals in RPMI-1640 culture medium containing 10% human serum, then 2.5 × 10 ⁵ cells / 200 μl in each well of a 96-well round bottom culture plate Sowing. The multi-epitope peptide of SEQ ID NO: 1, either Cry j 1 or Cry j 2, with a multi-epitope peptide at a final concentration of 0.001-20 μg / ml, Cry j 1 at 50 μg / ml, and Cry j 2 at 2 μg / ml And cultured for 6 days. 0.5 μCi of [ ³ H] thymidine was added, and the cells were further cultured for 16 hours. The cells were collected on a glass filter with a cell harvester, and then [ ³ H] thymidine uptake was measured with a liquid scintillation counter.

  Of the 6 patients, 5 peripheral blood lymphocytes showed a proliferative response to the multi-epitope peptide. The peripheral blood lymphocytes of one patient and two healthy subjects did not show a proliferative response (FIG. 10).

  Peripheral blood lymphocyte proliferative responses began to occur with 0.1 μg / ml multi-epitope peptide stimulation and proliferative responses increased in proportion to dose. From this result, it was determined that the concentration of the multi-epitope peptide that induces a sufficient T cell proliferation response in vitro was 10 μg / ml or more.

T cell responses were calculated by stimulating 17 cedar pollinosis patients and 2 healthy peripheral blood lymphocytes with 10 μg / ml of the multi-epitope peptide of SEQ ID NO: 1. T cell proliferation response ability was not observed in the peripheral blood lymphocytes of healthy individuals. Up to 9,652 cpm of [ ³ H] thymidine uptake was observed in 17 patients. The [ ³ H] thymidine uptake of peripheral blood lymphocytes without antigen stimulation was calculated as 1, and the [ ³ H] thymidine uptake value of peripheral blood lymphocytes in the presence of antigen was expressed as a stimulation coefficient (SI). Is shown in FIG. When identifying T cell epitopes, SI> 2 or higher is considered positive, and similarly, if SI> 2 or higher is considered to be a proliferative response to the peptide, 13 of 17 patients (76.5%) had a proliferative response. From this result, when a peptide is administered to a cedar pollinosis patient, it is determined that 76.5% of patients have an effect of peptide immunotherapy.

  Before attempting peptide immunotherapy with the multi-epitope peptide of the present invention for cedar pollinosis patients, investigate the proliferative response ability of the patient-derived peripheral blood lymphocytes to the multi-epitope peptide, and select patients with proliferative responses. Can do. This test can determine whether peptide immunotherapy using a multi-epitope peptide can be applied to the patient, and it can be predicted to some extent about the therapeutic effect from the high growth response ability.

Induction of immune tolerance by administration of cedar pollen allergens in mice The details of the mechanism of so-called desensitization treatment, in which treatment is carried out by administration of cedar allergen, are not known. Therefore, animal experiments using mice were conducted. Sugi pollen allergen, Cry j 1, 300 μg per mouse, CB6F1 mice (female, 5 animals) were subcutaneously administered twice at 5-day intervals. As a control, the same volume of PBS was administered subcutaneously (female, 5 animals). After 5 days, Cry j 1 100 μg was administered subcutaneously together with Alum adjuvant to immunize, and 10 days later, lymph node cells were isolated, and lymph node cells from control group mice and lymph node cells from Cry j 1 treated mice Were pooled in each group. Cry j 1 was added to the pooled lymphocytes at 0, 50, 150 μg / ml and further cultured for 3 days. The culture supernatant was collected and IL-2 contained therein was measured (manufactured by Endogen). The result is shown in FIG. In the control group, PBS-administered mice, the Cry j 1 concentration increased to 0, 50, and 150 μg / ml, and IL-2 production increased. On the other hand, Cry j 1 treated mice clearly decreased IL-2 production compared to these control mice, and cedar pollen allergen administration resulted in immune tolerance. This result reproduces an effective example of desensitization therapy using the cedar pollen allergen currently used.

Identification of T cell epitopes in CB6F1 mice
Eight-week-old male CB6F1 mice were immunized three times every two weeks with 10 μg of recombinant Cry j 2 (rCry j 2) together with an adjuvant (Imject Alum: Pierce) (ip). One week after the final immunization, splenocytes were prepared from 3 mice and combined into one. One well of 96-well plate (Falcon) contains 0.2 ml of spleen cells (5 × 10 ⁶ ) with 74 types of Cry j 2 overwrapping peptides (0.115 μM) each consisting of 15 residues. The cells were cultured in RPMI medium (10% FCS, 2 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin). As controls, responses to PBS, 50 μg / ml Cry j 1 and 0.3 μg / ml rCry j 2 were also examined. Each test reagent was seeded in 3 wells and cultured at 37 ° C. under 5% CO 2 for 3 days. After 6 hours of pulse labeling with [ ³ H] -thymidine at 0.5 μCi / well for the last 6 hours, cells were collected on a glass filter with a cell harvester (Inoteck, manufactured by Bertold Japan), and dried. [ ³ H] -thymidine intracellular uptake was measured with a liquid scintillation counter (TRI-CARB 4530, Packard Japan).

  CB6F1 mice immunized with rCry j 2 showed strong reactivity to the antigen rCry j 2, but did not react with Cry j 1, another major cedar pollen allergen. It was confirmed that there was. And CB6F1 mouse immunized with rCry j 2 showed remarkable responsiveness to No. 14 peptide and No. 48 peptide shown in FIG. 2 among 74 kinds of overwrapping peptides examined. This indicates that the peptides No. 14 and No. 48 are involved in antigen presentation as major T cell epitopes in CB6F1 mice. The CB6F1 mouse is a useful model for evaluating the efficacy of peptides used for peptide immunotherapy against cedar pollen because the peptides No. 14 and No. 48 are major T cell epitope peptides even in humans. It was determined that it could be an animal.

In vivo immune response of antigen peptide No. 14 3 mg No. 14 peptide dissolved in physiological saline per group of 8 male CB6F1 (8 weeks old, male) mice was subcutaneously administered twice at 5-day intervals . As a control group, an equal volume (200 μl) of physiological saline was similarly administered. On day 5 after the second peptide administration, all mice were immunized subcutaneously with rCry j 2 (50 μg / mouse) mixed with Imject Alum. Spleen cells were prepared from each mouse one week after immunization. Splenocytes (5 × 10 ⁶ ) per well of 96 well plate (Falcon) with 0.2 ml RPMI medium (10% FCS, 2 mM L-glutamine, 50 U / ml penicillin, 50 μg) with rCry j 2 (3 μg / ml) / ml streptomycin). As a control, the cells were cultured under conditions without rCry j2. Measurement of T cell proliferation with ³ H-thymidine was performed according to the method described in Example 1. Cytokine measurement was performed using the culture supernatant when stimulated in vitro with 0.3 μg / ml of Cry j 2 for three types of peptide administration groups (0.3, 1.3, 10 μg / ml) including controls.

  When the No.14 peptide was administered subcutaneously to CB6F1 mice in advance, the immune response of T cells was significantly suppressed (p <0.01) compared to the physiological saline administration group in response to the subsequent antigen stimulation by rCry j 2 ( FIG. 13). Regarding IL-2 production, each of the three types of peptide administration groups significantly decreased compared to the control group. From this, it was shown that the No. 14 peptide has a preventive effect of peptide immunotherapy against cedar pollen allergy in a mouse model system.

In vivo immune response of antigenic peptide No.48
3 mg No. 48 peptide dissolved in physiological saline per 6-week-old male CB6F1 mouse was subcutaneously administered twice at 5-day intervals. As a control group, an equal volume (200 μl) of physiological saline was similarly administered. The number of animals in the peptide administration group and the control group was 8 each, and all mice were immunized subcutaneously with rCry j 2 (50 μg) mixed with adjuvant (Imject Alum) on the 5th day after the second peptide administration. Spleen cells were prepared from each mouse one week after immunization. Splenocytes (5 × 10 ⁶ ) per well of 96-well plate (Falcon) with rCry j 2 (3 μg / ml) in 0.2 ml RPMI medium (10% FCS, 2 mM L-glutamine, 50 U / ml penicillin, 50 μg) / ml streptomycin). As a control, the cells were cultured under conditions without rCry j2. Measurement of T cell proliferation with ³ H-thymidine was performed according to the method described in Example 10.

  When the No. 48 peptide was subcutaneously administered to CB6F1 mice in advance, the immune response of T cells was significantly suppressed compared with the physiological saline administration group in response to the subsequent antigen stimulation by rCry j 2 (p <0.05). From this, it was shown that No. 48 peptide has a preventive effect by peptide immunotherapy against cedar pollen allergy in a mouse model system (FIG. 14).

  From the above experimental results, it has been clarified that the conventional desensitization therapy with cedar pollen extract in humans is a mechanism of action through T cell epitopes.

Determination of core sequence
In order to determine the amino acid sequence (core) necessary for the T cell line and T cell clone growth response of Cry j 1 peptide No. 22 (p106-120), as shown in FIG. P107-120 (p22-2), p108-120 (p22-3), p109-120 (p22-4), p110-120 (p22-5), p111-120 (p22-6), p106-119 (p22-7), p106-118 (p22-8), p106-117 (p22-9), p106-116 (p22-10), p106-115 (p22-11) 11 peptides were synthesized with a peptide synthesizer (PSSM-8, manufactured by Shimadzu Corporation). Three cedar pollinosis patients T cell lines (PJ, PR, PB) reacting with Cry j 1 peptide # 22 p106-120, and one patient T cell clone (PB 8-3, PB 8- 2, PB 9-39) were examined for reactivity to these 11 kinds of peptides using the methods of Examples 1 and 2. Two types of T cell lines (PJ, PB) and two types of T cell clones (PB 8-2, PB 9-39) proliferated recognizing p106-120 (p22-1). Cell lines and T cell clones did not show a proliferative response (FIG. 15). As a result, the p106-120 core sequence was found to be 9 residues of “FIKRVSNVI” (SEQ ID NO: 4) (the 9 residues are denoted as Cry j 1 # 22 core).

Peptide No. 8 (p71-90; IFSKNLNIKLNMPLYIAGNK), which is a T-cell epitope of Japanese cypress pollen allergen Cha o 1 (Japanese Patent Application No. 8-15527), or peptide number, including T cell epitopes derived from cedar pollen and cypress pollen allergen 32 (P311-330; SSGKNEGTNIYNNNEAFKVE) and Cry j 1 # 22 core sequence “FIKRVSNVI” obtained in Example 14 (Cha o 1 # 8-Cry ji # 22 core, Cha o 1 # 32 -Cry j 1 # 22 core) was synthesized with a peptide synthesizer (PSSM-8; manufactured by Shimadzu Corporation). RR sequences were inserted between Cha o 1 # 8 and Cry j 1 # 22 core, and between Cha o 1 # 32 and Cry j 1 # 22 core. That is, Cha o 1 # 8-Cry j 1 # 22 core (sequence number: 5) and Cha o 1 # 32-Cry j 1 # 22 core (sequence number: 6).

  A Cry j 1-specific T cell line and a Cha o 1-specific T cell line were prepared from a cedar pollinosis patient and a cypress pollinosis patient, respectively. Cry j 1 specific and Cha o 1 specific T cell lines do not react with M. tuberculosis antigen (PPD) and streptococcal cell wall (SCW) antigens, and Cry j 1 specific T cell lines are Cry j 1 Reacts with # 22 or Cry j 1 # 22 core, but does not react with Cha o 1 # 8 and # 32, Chao 1 specific T cell line reacts with Cha o 1 # 8 and # 32 However, it did not react with Cry j 1 # 22 or Cry j 1 # 22 core (FIG. 16). On the other hand, these T cell lines all responded to both the multiple epitope peptide of SEQ ID NO: 5 and the multiple epitope peptide of SEQ ID NO: 6. From these results, it was revealed that the multi-epitope peptide connecting T cell epitopes derived from cedar pollen and cypress pollen allergen is effective for peptide immunotherapy in cedar pollinosis patients and cypress pollinosis patients.

Growth response and cytokine production of analog peptides
It was investigated using two clones PJ7-9 and PB10-18 whether or not it was possible to regulate the activity of T cells by substituting amino acids of Cry j 1 # 22 core T cell epitope peptides. T cell clones PJ 7-9 and PB10-12 that react with Cry j 1 peptide No. 22 p106-120 recognize DRB5 ^* 0101 as a restriction molecule and recognize 9 residues of Cry j 1 # 22core. An analog peptide was synthesized in which each amino acid of 9 residues in the 13-residue peptide p108-120 (VFIKRVSNVIIHG) containing 9 residues was substituted with two types of amino acids, similar amino acids and dissimilar amino acids (FIG. 17, 18). Then, the reactivity of T cell clones PJ7-9 and PB10-18 to these analog peptides was examined by the amount of [ ³ H] thymidine incorporated. The cytokine concentration in the reaction solution was measured with a cytokine measurement kit manufactured by R & D Systems. The results are shown in FIG. 17 and FIG. Here, the amount of IFNγ, IL-4, IL-2, and IL-5 produced in the supernatant obtained by reacting the 13-residue peptide without amino acid substitution and the amount of [ ³ H] thymidine taken up by the cells were respectively determined. 100%. In the case of PJ7-9 clone, Cry j 1 # 22core “FIKRVSNVI” 3rd, 4th and 6th amino acid parts “K”, “R” and “S” are both similar and dissimilar amino acid substitutions or dissimilarity Amino acid substitution significantly suppressed [ ³ H] thymidine incorporation and cytokine production (FIG. 17). Therefore, the amino acids in these parts are considered to be important parts for complex formation of HLA molecules and T cell receptor molecules via peptides. Substituting the first amino acid (F) with a similar amino acid, Y, shows no change in [ ³ H] thymidine incorporation and IL-4, IL-5 production, but it is a dissimilar amino acid Substitution with “S” markedly increased the production of IFNγ and IL-2 despite no change in [ ³ H] thymidine incorporation. In the case of the PB10-18 clone, the incorporation of [ ³ H] thymidine is suppressed by the 1, 2, 3, 4, 6, 7, 8th amino acid substitution of Cry j 1 # 22core, and the amino acids in these parts are peptide It is considered to be an important part for the complex formation of HLA molecule and T cell receptor molecule via. Furthermore, IL-2 production suppression was recognized by the 6th, 7th, and 8th amino acid substitutions as compared with the production amount of IL-5 (FIG. 18). From these results, it is clear that “SIKRVSNVI” in which the first amino acid F of Cry j 1 # 22core is replaced with S is useful as a therapeutic agent for allergy by increasing the production of IFN-γ. became.

  The multi-epitope peptide of the present invention includes a T cell epitope peptide derived from different allergen molecules, and a peptide presented as an HLA class II molecule having a high gene frequency in the allergic patient population. Since several peptides presented with different molecules between loci (DR, DQ, DP) are included, peptide immunotherapy with an expanded number of effective target patients can be expected with the minimum length of multiple epitope peptides.

  In addition, when peptide immunotherapy is attempted for allergic patients using the multi-epitope peptide of the present invention, the proliferative response ability of peripheral blood lymphocytes derived from the patient to the peptide is investigated in advance, and patients with proliferative responses are induced. Can be selected. By this investigation, it is possible to determine whether or not peptide immunotherapy with a multi-epitope peptide can be applied to the patient, and the therapeutic effect can be predicted to some extent from the high proliferation response ability.

FIG. 1 is a diagram showing an average stimulation coefficient, appearance frequency, and importance index (average stimulation coefficient × appearance frequency) for Cry j 1 overlapping peptides of a cell line derived from a cedar pollinosis patient. FIG. 2 is a diagram showing an average stimulation coefficient, appearance frequency, and importance index (average stimulation coefficient × appearance frequency) for Cry j 2 overlapping peptides of a cell line derived from a cedar pollinosis patient. FIG. 3 is a diagram showing an HLA class II type that binds the antigen peptide of Cry j 1 and a Th type of a T cell clone that recognizes a complex of the antigen peptide and an HLA class II restricted molecule. FIG. 4 is a diagram showing an HLA class II type that binds the antigen peptide of Cry j 2 and a Th type of a T cell clone that recognizes a complex of the antigen peptide and an HLA class II restricted molecule. FIG. 5 is a diagram showing the identification results at the locus level (DR, DQ, DP) of HLA class II molecules that bind to antigenic peptides. FIG. 6 is a diagram showing the identification results at the allele level of each locus of HLA class II molecules that bind to an antigenic peptide. FIG. 7 is a view showing an antigen peptide binding sequence used for a multi-epitope peptide. In the figure, a and b correspond to peptides No. 43 and 22 of Cry j 1, c corresponds to No. 14 of Cry j 2, d and e are No. 37-38 (p181-200), Corresponds to No.69-71 (p346-365). FIG. 8 shows the reactivity of multiple epitope peptides C.A. # 1, C.A. # 2, C.A. # 3, C.A. # 4, C.A. # 5, and C.A. # 6 with human IgE. FIG. 9 is a diagram showing the recognition results of T cell epitopes contained in a multi-epitope peptide, C.A. # 4, by T cell clones. FIG. 10 is a graph showing the lymphocyte proliferation response ability by stimulation with multiple epitope peptides (SEQ ID NO: 1) at various concentrations against peripheral blood lymphocytes of Japanese cedar pollinosis patients and healthy individuals. FIG. 11 is a diagram showing the proliferation response ability by stimulation with the multi-epitope peptide of SEQ ID NO: 1 against peripheral blood lymphocytes of 2 healthy subjects and 17 cedar pollinosis patients. FIG. 12 shows the induction of immune tolerance by administration of the cedar pollen allergen Cry j 1 to CB6F1 mice. FIG. 13 is a diagram showing immune tolerance by administration of Cry j 2 No. 14 peptide (p66-80) to CB6F1 mice. FIG. 14 is a view showing immune tolerance by administration of Cry j 2 No. 48 peptide (p236-250) to CB6F1 mice. FIG. 15 shows the determination of the core amino acid sequence of Cry j 1 No. 22 peptide (p106-120). FIG. 16 is a diagram showing the reactivity of lymphocytes of a cedar pollinosis patient and a cypress pollinosis patient with respect to a multi-epitope peptide composed of a cedar pollen-specific T cell epitope peptide and a cypress pollen-specific T cell epitope peptide. FIG. 17 is a graph showing the proliferation responsiveness of the T cell clone PJ7-9 to the amino acid-substituted analog peptide of the Cry j 1 # 22core peptide and the amount of cytokine produced at that time. FIG. 18 is a graph showing the proliferation responsiveness of the T cell clone PB10-18 to the analog peptide and the subsequent cytokine production.

Claims (9)

A molecule of a linear polypeptide linking different T cell epitope regions,
(1) The importance index measured in the patient population in which each of the T cell epitope regions is sensitive to the allergen is about 100 or more,
(2) reacts with peripheral blood lymphocytes of at least 70% or more of the patient population sensitive to the allergen;
(3) A peptide immunotherapeutic agent comprising an effective amount of a multi-epitope peptide that does not substantially react with an IgE antibody of a patient population susceptible to the allergen.   The peptide immunotherapeutic agent according to claim 1, wherein different T cell epitope regions are derived from two or more different allergen molecules.   The peptide immunotherapeutic agent according to claim 2, wherein the different allergen molecules are cedar pollen allergens Cry j 1 and Cry j 2.   The peptide immunotherapeutic agent according to claim 1, wherein a site to be processed in the antigen-presenting cell is interposed between each T cell epitope region.   The peptide immunotherapeutic agent according to claim 4, wherein the site subjected to antigen-presenting intracellular processing is arginine dimer or lysine dimer.   The peptide immunotherapeutic agent of Claim 3 which is a peptide containing the amino acid sequence in any one of sequence number: 1, sequence number: 2, or sequence number: 3. An epitope comprising at least one of the HLA class II molecules DRB5 ^* 0101, DRB4 ^* 0101, DQA1 ^* 0102-DQB1 ^* 0602, DPA1 ^* 0101-DPB1 ^* 0501, or DPA1 ^* 0101-DPB1 ^* 0201 as a binding molecule 3. The peptide immunotherapy agent according to 3.   The peptide immunotherapeutic agent according to claim 2, wherein the different allergen molecules are cedar pollen allergen Cry j 1 and cypress pollen allergen Cha O 1. The peptide immunotherapeutic agent according to claim 8, which is a peptide comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5.

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