JP4176750B2 - T cell epitope peptide - Google Patents

Description

  The present invention relates to a pollen allergen T-cell epitope peptide and a peptide immunotherapy composition for spring hay fever comprising the peptide as an active ingredient.

  Spring hay fever, represented by cedar pollen, which is afflicted by about 10% of the entire population, is on the rise and is one of the diseases that attracts social attention.

  In general, the onset and duration of hay fever coincides with the pollen dispersal time, but there are quite a few examples in which the symptoms persist even after the cedar pollen dispersal time has passed. This is because the majority of cedar pollinosis patients are sensitized to cypress pollen at the same time, and the symptom lasts for a long time in response to cypress pollen that scatters late in the cedar pollen period. .

  That is, Japanese cedar and Japanese cypress pollen have a common antigenicity (Takeshi Ide et al., Allergy Clinical 11, 174-178, 1991), and cross-reactivity between Japanese cedar and Japanese cypress has been shown against IgE antibodies (Taniai M. et al. al .: Mol. Immunol. 30, 183-189, 1993), and the allergen-specific IgE antibody positive rate in patients with spring hay fever was 83.5% for cedar, 80.0% for cypress, and 76.4% for both cedar and cypress (Okano). Mitsuhiro et al., Allergy 43, 1179-1184, 1994), 60% of Japanese cedar pollinosis patients have cypress pollen-specific IgE antibody (Yozo Saito, Treatment 78, 1571-1576, 1996). It is a general perception that cedar pollinosis patients also develop cypress pollen and vice versa.

  As for the prevention and treatment of hay fever, hay fever is an immediate type caused by an antigen-antibody reaction between pollen allergens (essentially the same as antigens that cause allergies) and specific IgE antibodies. Since it is a typical type I allergy, prevention and treatment methods theoretically corresponding to the onset mechanism of type I allergy are being carried out.

  The outline of the onset mechanism of type I allergy is as follows.

  The antigen that has entered the body is presented to helper T cells by antigen-presenting cells. As a result, B cells mature to become antibody-producing cells and produce antigen-specific IgE antibodies. This IgE antibody binds to the surface of mast cells. Next, when a new antigen enters, the antigen binds to IgE antibody on the surface of mast cells, and the stimulation releases chemical transmitters such as histamine from mast cells, causing allergic symptoms.

  There are 3 prevention and treatment methods corresponding to these processes leading to allergic symptoms: 1) avoidance of antigens causing allergy, 2) pharmacotherapy typified by antihistamine, 3) desensitization therapy with allergen There are two main prevention and treatment modalities used. However, 1) is actually difficult to implement, 2) is only symptomatic treatment, and 3) can only be expected to be the fundamental treatment of allergies, but it lacks certainty in terms of effectiveness and has serious side effects such as anaphylaxis. There is a risk associated with.

  For these reasons, peptide immunotherapy using allergen T cell epitope peptides has recently been attempted for the prevention and treatment of allergies. T cell epitopes are involved in the initiation and persistence of immune responses against protein allergens that cause clinical symptoms of allergies. These T cell epitopes are thought to trigger early events at the level of helper T cells by binding to HLA class II molecules on the surface of antigen presenting cells and stimulating related T cell subpopulations. This initial event causes T cell proliferation, lymphokine secretion, local inflammatory response, migration of proliferated immune cells to the inflammatory site and activation to the B cell cascade leading to antibody production. IgE antibodies, which are isoforms of these antibodies, are fundamentally important for the onset and persistence of allergies, and their production is affected by the properties of lymphokines secreted at the level of helper T cells in the early stage of the cascade. A T cell epitope is the basic element or the smallest unit of recognition by a T cell receptor, which contains the essential amino acids that recognize the receptor. Controlling the response of helper T cells, the key to immunosuppression, using T cell epitope peptides is thought to lead to treatment of allergic inflammation.

  For example, as a therapeutic agent for allergy using a T cell epitope peptide, a therapeutic composition containing a T cell epitope of a feline allergen (Japanese Patent Publication No. 7-505365), a T cell epitope peptide of cedar pollen Cry j 1 There are therapeutic compositions used (Japanese Patent Publication No. 8-502163), multi-epitope peptides (Japanese Patent Application No. 8-80702) in which T cell epitopes of cedar pollen allergens Cry j 1 and Cry j 2 are connected. As for cypress pollen, its main allergen Cha o 1 has a molecular weight of 45 KD and 50 KD, both isoelectric points are 6.8, and consists of a protein containing 5% sugar (Takeshi Ide et al .: Journal of Japanese Society of Pollen Science, 34, 39, 1988), but its primary structure is unknown, so the T cell epitope site on the allergen molecule has not been identified. Recently, the present inventors have succeeded in gene cloning of cypress pollen allergen, and revealed that Chao 2 exists in addition to Cha o 1 in the allergen, and each primary structure of Cha o 1 and Cha o 2 (Japanese Patent Application No. 6-335089).

  There is a coincidence period (mixing period) between the cedar and cypress pollen, and since both have common antigenicity, the symptoms caused by cedar and cypress pollen cannot be clearly distinguished. However, there are cases in which symptoms persist even after the cedar pollen season has passed, or new symptoms develop. Since most of the aerial pollen at this time is cypress pollen, these symptoms are caused by cypress pollen. It is considered a thing. Due to the current situation in which cypress trees are being planted more than cedar trees, the amount of cypress pollen scattered is increasing year by year, and it is considered that the amount of cedar pollen scattered will be higher in the future. Therefore, establishment of a comprehensive fundamental prevention / treatment method for spring tree hay fever including not only cedar pollinosis but also hinoki hay fever is desired. Immunotherapy using T-cell epitope peptides is expected to lead to the fundamental treatment of allergies, and some of the immunotherapy have already been reported for cedar pollinosis as described above. There are no reports of spring tree hay fever, including cedar and cypress.

  Under such circumstances, an object of the present invention is to provide a T cell epitope peptide useful for peptide immunotherapy for cypress pollinosis. Furthermore, an object of the present invention is to provide a T cell epitope peptide useful for peptide immunotherapy for spring tree pollinosis patients including cedar pollinosis patients having cross-reactivity with cypress pollen.

  The present inventors identified a T cell epitope site on a cypress pollen allergen molecule by stimulating a T cell line established from a cypress pollinosis patient with a synthetic overlapping peptide covering the primary structure of the cypress pollen allergen. The above problem has been solved.

  That is, the present invention specifically includes the inventions described in the claims. The present invention will be described in detail below.

  The amino acid sequence of the main allergen Cha o 1 (mature protein) of the Japanese cypress pollen allergen elucidated by the present inventors (described in Japanese Patent Application No. 6-335089) is SEQ ID NO: 1, and the amino acid sequence of Cha o 2 is It is shown in SEQ ID NO: 2, respectively. The amino acid sequence of Cha o 1 is 80% homologous to the cedar pollen allergen Cry j 1, and Cha o 2 is also 75% homologous to the cedar pollen allergen Cry j 2.

  Numerous amino acid substitutions have been found in individual allergens derived from pollen, mites and bee venoms, which are called isoallergens to each other. For example, birch tree pollen Bet v I has 11 isoallergens isolated and differing in amino acid sequence by 2-15% from each other (Swoboda, I. et al .: J. Biol. Chem. 270 : 2607 to 2613, 1995). As for Cry j 2, there are currently two isoallergens in which 6 amino acid substitutions are observed in the mature protein portion (JP-A-8-47392 and JP-A-7-70986). It is easily expected by those skilled in the art that iso allergens may exist for Cha o 1 and Cha o 2 as well, and such isoallergens are also included in Cha o 1 and Cha o 2 in the present invention. included.

  The cedar plant is classified into 9 genera, and the cypress plant is classified into 7 genera, but cedar, cequoia, metasequoia, yuyamaki (there is an independent family, cedar family, or pine family) and cypress There is a report that allergens are cross-reactive with cypress, sawara, konotegasiwa, rat eagles, and juniper in the family (Takeshi Ide et al., Allergy Clinical 11, 174-178, 1991). Allergens are widely considered to have cross-reactivity. Therefore, most of the peptides of the present invention are considered to be effective not only for cypress pollinosis but also for cedar pollinosis.

In order to obtain the T cell epitope peptide of the present invention, an overlapping peptide consisting of amino acids (12 to 20 residues) of an appropriate length covering the primary structure of Cha o 1 and Cha o 2 is synthesized. The peptide of this invention has the effect | action which stimulates and / or suppresses the activity of the T cell derived from a spring tree pollinosis patient. That is, the peptides of the present invention can induce T cell responses such as T cell proliferation or lymphokine secretion, and / or can induce T cell anergy (non-response). When identifying the T cell epitope site on the allergen molecule using T cell proliferation as an index, according to the method described in JP-A-8-47392, from the peripheral blood lymphocytes of cypress pollinosis patients, Cha o 1 and Cha o A T cell line or T cell clone that specifically reacts with 2 is established for each patient, and the T cell line or T cell clone is cultured in the presence of the individual peptides of the overlapping peptides, and T cells against the peptide are obtained. Identification is performed by measuring proliferation, eg, [ ³ H] thymidine incorporation into cells, and calculating the stimulation factor. The stimulation index (SI) of T cell response used here is calculated by dividing the cpm of [ ³ H] thymidine radioactivity incorporated into cells in the presence of peptide by cpm in the absence of peptide (control). Is calculated as This result is used to calculate the average stimulation factor for each peptide for the patient group. A peptide thus found to induce a T cell response and / or induce T cell anergy is defined as a peptide having T cell stimulating activity. Preferred T cell epitope peptides of the present invention have T cell stimulating activity and thus contain at least one T cell epitope, such as the peptide of Cha o 1 of FIG. 1 (FIGS. 2, 3, SEQ ID NO: 3). ~ 37), peptide numbers # 1-2 (SEQ ID NO: 4), # 1-4 (SEQ ID NO: 6), # 1-5 (SEQ ID NO: 7), # 1-6 (SEQ ID NO: 8), # 1-7 (SEQ ID NO: 9), # 1-8 (SEQ ID NO: 10), # 1-10 (SEQ ID NO: 12), # 1-11 (SEQ ID NO: 13) , # 1-12 (SEQ ID NO: 14), # 1-14 (SEQ ID NO: 16), # 1-15 (SEQ ID NO: 17), # 1-16 (SEQ ID NO: 18), # 1-19 ( SEQ ID NO: 21), # 1-20 (SEQ ID NO: 22), # 1-21 (SEQ ID NO: 23), # 1-22 (SEQ ID NO: 24), # 1-23 (SEQ ID NO: 25), # 1-24 (SEQ ID NO: 26), # 1-25 (SEQ ID NO: 27), # 1-26 (SEQ ID NO: 28), # 1-27 (SEQ ID NO: 29), # 1-30 (sequence) Number : 32), # 1-31 (SEQ ID NO: 33), # 1-32 (SEQ ID NO: 34), # 1-33 (SEQ ID NO: 35) and # 1-34 (SEQ ID NO: 36) (FIG. 4 ), Or peptide No. # 2-5 (SEQ ID NO: 42), # 2-7 (specifically shown in FIG. 6, FIG. 7, SEQ ID NOS: 38 to 88) of Chao 2 peptide of FIG. SEQ ID NO: 44), # 2-8 (SEQ ID NO: 45), # 2-9 (SEQ ID NO: 46), # 2-10 (SEQ ID NO: 47), # 2-11 (SEQ ID NO: 48), # 2-12 (SEQ ID NO: 49), # 2-13 (SEQ ID NO: 50), # 2-14 (SEQ ID NO: 51), # 2-15 (SEQ ID NO: 52), # 2-16 (sequence) #: 53), # 2-17 (SEQ ID NO: 54), # 2-18 (SEQ ID NO: 55), # 2-19 (SEQ ID NO: 56), # 2-20 (SEQ ID NO: 57), # 2-21 (SEQ ID NO: 58), # 2-22 (SEQ ID NO: 59), # 2-23 (SEQ ID NO: 60), # 2-24 (SEQ ID NO: 61), # 2-25 (SEQ ID NO: : 62), # 2-26 (SEQ ID NO: 63), # 2-27 (sequence) No. 64), # 2-30 (SEQ ID NO: 67), # 2-31 (SEQ ID NO: 68), # 2-32 (SEQ ID NO: 69), # 2-33 (SEQ ID NO: 70), # 2-34 (SEQ ID NO: 71), # 2-35 (SEQ ID NO: 72), # 2-36 (SEQ ID NO: 73), # 2-37 (SEQ ID NO: 74), # 2-38 (SEQ ID NO: : 75), # 2-40 (SEQ ID NO: 77), # 2-41 (SEQ ID NO: 78), # 2-42 (SEQ ID NO: 79) and # 2-43 (SEQ ID NO: 80) (FIG. 8 ), And more preferable peptides include those having an average stimulation coefficient of 2.0 or more. For example, among peptides shown in FIG. 1, peptide numbers # 1-2 (SEQ ID NO: 4), # 1-7 (sequences) Number: 9), # 1-8 (SEQ ID NO: 10), # 1-20 (SEQ ID NO: 22), # 1-22 (SEQ ID NO: 24), # 1-24 (SEQ ID NO: 26), # 1-26 (SEQ ID NO: 28), # 1-32 (SEQ ID NO: 34), # 1-33 (SEQ ID NO: 35) and peptide shown in # 1-34 (SEQ ID NO: 36), or Among the peptides of FIG. 5, peptide numbers # 2-10 (SEQ ID NO: 47), # 2-20 (SEQ ID NO: 57), # 2-21 (SEQ ID NO: 58), # 2-40 (SEQ ID NO: 77) ), # 2-41 (SEQ ID NO: 78), # 2-42 (SEQ ID NO: 79) and # 2-43 (SEQ ID NO: 80). Furthermore, suitable peptides include those having an importance index of at least 100 or more, for example, peptide numbers # 1-7 (SEQ ID NO: 9), # 1-22 (SEQ ID NO: 24) among the peptides of FIG. , # 1-32 (SEQ ID NO: 34) and # 1-33 (SEQ ID NO: 35), or peptide numbers # 2-10 (SEQ ID NO: 47) of the peptides shown in FIG. 20 (SEQ ID NO: 57), # 2-40 (SEQ ID NO: 77), # 2-41 (SEQ ID NO: 78), # 2-42 (SEQ ID NO: 79) and # 2-43 (SEQ ID NO: 80) ). Here, the importance index is obtained by multiplying the average stimulation coefficient of a certain peptide by the appearance frequency (%) of a patient showing a T cell response to the peptide.

  In order to identify an accurate epitope, a peptide having T cell stimulating activity and thus containing at least one T cell epitope is modified by deletion of an amino acid residue at either the amino terminus or carboxy terminus of the peptide. Then, the change of the T cell stimulating activity for the modified peptide is examined. In addition, when two or more peptides sharing the overlapping region exhibit T cell stimulating activity, a new T cell epitope peptide containing all or part of such peptide is prepared, and similarly T cell stimulating activity is exhibited. Can be measured.

  The T cell epitope peptides of the present invention are believed to be immunologically related by T cell cross-reactivity with Cry j 1 or Cry j 2. That is, 1) Cha o 1 and Cry j 1 are 80% homologous in amino acid sequence, and Cha o 2 and Cry j 2 are 75% homologous. 2) Cha o 1 identified in Example 5 of the present invention 1 T-cell epitope peptide # 1-2 (corresponding to amino acid sequence numbers 11 to 30 of mature Cha o 1) and Cry j 1 T-cell epitope peptide CJI-2 (amino acid number 11 of mature Cry j 1) Corresponds to ~ 30, and Japanese National Patent Publication No. 8-502163 / Figure 13), the amino acid sequence is identical except for two amino acids (Cha o 1 12th Ala is Ser in CJI-1) The 15th Asp of Cha o 1 is Ala) in CJI-1, and 3) Japanese cedar and Japanese cypress pollen share the same antigenicity. From this, the origin of the T cell epitope of the present invention is not limited to cypress, and it is considered that the T cell epitope of the present invention is effective not only for cypress pollinosis but also for cedar pollinosis.

  In addition, for the T cell epitope peptide of the present invention, amino acid residues involved in T cell receptor recognition are determined using a known technique (for example, measurement of changes in T cell stimulating activity due to substitution of the amino acid residues), Those amino acid residues that have been shown to be essential for the interaction with the T cell receptor are replaced with other amino acids, and the antigen-specific T cell stimulating activity is suppressed to allergic inflammation (T cell reactivity) Increase, lymphokine production pattern change, anergy, etc.). For example, in a human allergy model, an analog peptide in which one amino acid at the T cell recognition site on the T cell epitope peptide of cedar pollen Cry j 1 is replaced with another amino acid (the 399th Thr is Val) is T cell Proliferative response and IL-4 production were not different from wild-type peptides, but IFN-γ production, which suppresses IgE antibody production, was reported to increase (Ikagawa, S. et al .: J. Aller. Clin. Immunol. 97, 54-64, 1996). In addition, the binding motif of HLA class II molecules consists of 3-5 amino acid residues arranged in a stepping stone via 1-2 amino acids, and if these are several types of specific amino acids, Has been shown to bind to HLA class II molecules (Matsushita, S. et al .: J. Exp. Med. 180: 877-883, 1994). Amino acid residues essential for interaction with HLA class II molecules can be determined using known techniques, and the amino acid residues (HLA class II molecule binding motif) can be replaced with other amino acids to suppress allergic inflammation. . Furthermore, the peptide can be modified for the purpose of increasing the solubility of the T cell epitope peptide of the present invention and increasing the therapeutic or prophylactic effect or stability. These modifications include amino acid substitutions, deletions or additions.

  Furthermore, in the present invention, a suitable T cell epitope peptide does not bind to an IgE antibody or binds to a substantially lower extent than the natural cypress pollen allergen from which the peptide is derived binds to the IgE antibody. To do.

  The T cell epitope peptide of the present invention preferably contains at least 7 amino acid residues. In addition, these regions are linked by a linker such as Arg-Arg or Lys-Lys that is sensitive to enzymatic cleavage such as cathepsin or trypsin to increase sensitivity to processing by antigen presenting cells and contain one or more T cell epitopes. Peptide moieties can be generated. The T cell epitope of the present invention may be other Cry j 1 T cell epitope peptide (Japanese Patent Publication No. 8-502163) and / or Cry j 2 T cell epitope peptide (Japanese Patent Laid-Open No. 8-47392). It can be used in combination with a peptide.

  Peptides comprising at least one T cell epitope of the invention modulate an allergic response to an individual's allergen when administered to an individual sensitive to cypress pollen allergens and / or individuals sensitive to both cypress and cedar pollen allergens This is useful for peptide immunotherapy. In particular, the combination of the T cell epitope peptide of the present invention and the cedar pollen T cell epitope peptide is useful for peptide immunotherapy for spring tree hay fever patients represented by cedar and cypress.

The T cell epitope peptide of the present invention can also be used as a reagent for diagnosing hay fever caused by cypress pollen allergen or other tree pollen immunologically cross-reactive with the allergen. That is, about 0.1 μg / ml to 1 mg / ml, preferably about 1 to 300 μg / ml of the peptide is added to patient peripheral blood lymphocytes and cultured for 1 week, and then the amount of [ ³ H] thymidine incorporated into lymphocytes is measured. Thus, hay fever can be diagnosed. The T cell epitope peptides of the present invention can also be used to evaluate T cell function, T cell proliferation, or a combination thereof.

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

  When the T cell epitope peptide of the present invention is used for peptide immunotherapy for hay fever patients, it can be used in combination with an appropriate pharmaceutically acceptable diluent and carrier. Pollen allergy patients include cedar pollen allergen patients that are immunologically cross-reactive with cypress pollen allergens. As the administration method, a simple method such as injection (subcutaneous, intravenous injection, etc.), eye drop, nose drop, oral, inhalation, transdermal route, etc. can be used. Preferably, about 1 μg to 30 mg, more preferably about 20 μg to 10 mg is administered per dosage unit.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Overlapping peptide synthesis Based on the amino acid sequences of cypress pollen allergens Cha o 1 (SEQ ID NO: 1) and Cha o 2 (SEQ ID NO: 2), 20-residue overlaps, each containing an overlap of 10 residues Peptides (14 residues for # 1-35 (SEQ ID NO: 37) and # 2-51 (SEQ ID NO: 88)) were synthesized by Fmoc method using a peptide synthesizer PSSM-8 (Shimadzu Corporation). There were 35 overlapping peptides in Cha o 1 (FIG. 1 / SEQ ID NOs: 3-37) and 51 in Cha o 2 (FIG. 5 / SEQ ID NOs: 38-88). All the synthesized peptides were purified by high performance liquid chromatography (HPLC) using an ODS column, and the purity was 90% or more. The molecular weight of each purified product was confirmed by LASERMAT 2000 (Finnigan MAT Ltd.).

Expression of recombinant protein in Escherichia coli Amplification of cDNA by PCR from plasmid DNA (Japanese Patent Application No. 6-335089) in which Chao 1 cDNA or Chao 2 cDNA encoding cypress pollen antigen is cloned A restriction enzyme recognition site was added to the end. This DNA fragment was inserted into a histidine tag binding protein expression vector pQE9, and E. coli M15 (pREP4) was transformed. Transgene expression was confirmed by SDS-polyacrylamide gel electrophoresis for ampicillin resistant clones. The expressed protein was purified with a Ni-NTA agarose affinity column.

Establishment of T cell line
Establishment of a T cell line for Cha o 1 was performed as follows. Specifically, peripheral blood-derived lymphocytes of 19 Ala STAT (Japan DPC Corporation) or CAP-RAST (Pharmacia) cypress-positive patients were separated using a Ficoll-Paque specific gravity centrifugation method. These lymphocytes (2 × 10 ⁶ cells) were suspended in 2 ml of the same patient's plasma (10%) or human AB type serum (20%, Shoko Tsusho) supplemented RPMI1640 medium (GIBCO) and obtained in Example 2. 10 to 30 μg / ml recombinant Cha o 1 or the overlapping peptide mixture obtained in Example 1 (0.01 to 1 μM) was cultured on a 24-well plate for 3 to 10 days (37 ° C., CO ₂ incubator TABAI). When T cells activated by Cha o 1 stimulation were confirmed under a microscope, 5 U / ml IL-2 (Boehringer Mannheim) was added and cultured overnight. From the next day, after culturing for about 10 days with RPMI1640 medium supplemented with 20 U / ml IL-2, 10% or 20% human AB serum daily, and examining the specificity of the expanded T cell line, Parts were stored frozen. In addition, the establishment of a T cell line for Chao 2 was performed in the same manner for 20 cypress pollinosis patients.

Establishment of antigen-presenting cells A lymphoblastoid cell line (B cell line) in which B lymphocytes were infected with EB virus (Epstein-Barr virus, EBV) and caused transformation was established and used as antigen-presenting cells. Specifically, EBV-producing B-95-8 cells (marmoset, ATCC CRL1612) were first cultured in RPMI1640 medium supplemented with 20% inactivated fetal bovine serum (FCS, GIBCO), and the culture supernatant was filtered with a 0.22 μm sterilization filter. Filtered and stored frozen at -80 ° C. Next, 1 ml of EBV solution was added to lymphocytes (2 × 10 ⁶ ) of cypress pollinosis patients and infected at 37 ° C. for 30 minutes. EBV-infected cells were washed twice and cultured in 20% FCS-RPMI1640 medium supplemented with 200 ng / ml final concentration of cyclosporin (sand chemical) for about 20 days. After the cell mass became observable with the naked eye, the cells were further cultured in 20% FCS-RPMI 1640 medium for about 20 days, and then the cells were stored frozen until use.

Identification of T cell epitope peptide The cultured B cell line established in Example 4 was treated with 50 μg / ml mitomycin C (Sand medicine) for 30 minutes or X-ray irradiation (50 gray), followed by washing 4 times with RPMI1640 medium. did. The B cells were seeded in a 96-well plate (10,000 cells / well), and then recombinants having a final concentration of 10 μg / ml with respect to Cha o 1 and Cha o 2 were added. (Control groups include streptococcal cell wall antigen (SCW) at a final concentration of 10 μg / ml, Candida Albicans antigen (CA) at a final concentration of 10 μg / ml, tuberculin antigen (PPD) at a final concentration of 1 μg / ml. ) After that, a T cell line (20,000 cells / well) of the same patient in which a B cell line was established was seeded in each well, cultured for 48 hours, and then 0.5 μCi [ ³ H] thymidine was added to the well, and further 16 hours. Cultured. The cells were collected on a glass filter using a cell harvester (Berthold), and the amount of [ ³ H] thymidine incorporated into the cells was measured with a liquid scintillation counter to confirm the cell proliferation response.

  After confirming that the T cell line specifically proliferates to Cha o 1 or Cha o 2, each overlapping peptide (final concentration) was prepared in the same manner as described above using the T cell line established in Example 3. T cell line proliferative response to 1 μM). The average stimulation coefficient of the proliferation response of the T cell line to the overlapping peptide, the appearance frequency, and the result of the importance index calculated from them are shown in FIG. 1 and FIG.

  In addition, the proliferation response of the T cell line (N = 17) to mutant sequences (SEQ ID NOs: 89 and 90, respectively) in which one amino acid in the sequences of # 2-11 and # 2-12 was substituted was examined. The T cell stimulation activity of these two mutant sequences was 1.6 and 1.2 for the average stimulation coefficient, 16% and 11% for the appearance frequency, and 25.6 and 13.2 for the importance index, respectively. Thus, it was shown that the T cell epitope peptide of the present invention retains T cell stimulating activity even when one or more amino acids are mutated, and the activity may be further increased.

  The present invention provides a peptide comprising at least one T cell epitope of Cha o 1 which is a major allergen of cypress pollen and a peptide comprising at least one T cell epitope of Cha o 2. Furthermore, the present invention includes other tree pollen peptide fragments that immunologically show T cell cross-reactivity with the peptide. These peptides are useful for peptide immunotherapy of spring tree hay fever represented by cedar and cypress pollen.

FIG. 1 shows the T cell epitope peptide in cypress pollen allergen Cha o 1 and the importance index of the peptide. FIG. 2 shows Chao 1 overlapping peptides (# 1-1 to # 1-28). FIG. 3 shows Chao 1 overlapping peptides (# 1-29 to # 1-35). FIG. 4 shows a peptide containing the Chao 1 T cell epitope. FIG. 5 shows the T cell epitope peptide in cypress pollen allergen Cha o 2 and the importance index of the peptide. FIG. 6 shows Chao 2 overlapping peptides (# 2-1 to # 1-27). FIG. 7 shows Chao 2 overlapping peptides (# 2-28 to # 1-51). FIG. 8 shows a peptide containing the Chao 2 T cell epitope.

Claims (10)

Figure 4 peptides numbers # 1-7 (SEQ ID NO: 9) containing a peptide having the amino acid sequence as an active ingredient represented by the prophylactic or therapeutic agent for Japanese cypress pollinosis. Figure 4 peptides numbers # 1-7 (SEQ ID NO: 9) from the peptide comprising the amino acid sequence represented by, a peptide which amino acid 1 to 4 residues at only N-terminal and / or C-terminal have been deleted Te, containing Lupe peptide that have a peptide equal to or more T cell stimulating activity comprising the amino acid sequence as an active ingredient, a preventive or therapeutic agent for Japanese cypress pollinosis. Figure 4 peptides numbers # 1-7 (SEQ ID NO: 9) to the N-terminus and / or C-terminus of the peptide consisting of the amino acid sequence represented by, a peptide amino acid 10 residues are added, Peptides having the following properties:
(I) The added residue is a residue corresponding to the N-terminus and / or C-terminus of the peptide comprising the amino acid sequence in the natural Chao 1 amino acid sequence.
(Ii) T cell stimulating activity equivalent to or higher than that of the peptide comprising the amino acid sequence.
A preventive or therapeutic agent for Japanese cypress pollinosis, comprising as an active ingredient . Even without least comprising as an active ingredient the two peptides, a prophylactic or therapeutic agent for cypress pollinosis, following (a), comprise any of the peptide according to (b) or (c), and the following ( A prophylactic or therapeutic agent comprising any peptide according to d), (e) or (f).
(a) A peptide consisting of the amino acid sequence shown by peptide number # 1-7 (SEQ ID NO: 9) in FIG.
(b) a peptide having an amino acid sequence represented by the above (a) and a peptide having 1 to 4 amino acids deleted only at the N-terminus and / or C-terminus, the peptide comprising the amino acid sequence; A peptide having equivalent or higher T cell stimulating activity.
(c) A peptide having 1 to 10 amino acid residues added to the N-terminus and / or C-terminus of the peptide consisting of the amino acid sequence shown in (a) above, and having the following properties:
(I) The added residue is a residue corresponding to the N-terminus and / or C-terminus of the peptide comprising the amino acid sequence in the natural Chao 1 amino acid sequence.
(Ii) T cell stimulating activity equivalent to or higher than that of the peptide comprising the amino acid sequence.
(d) Peptide numbers # 1-2 (SEQ ID NO: 4), # 1-7 (SEQ ID NO: 9), # 1-8 (SEQ ID NO: 10), # 1-20 (SEQ ID NO: 22) in FIG. ), # 1-22 (SEQ ID NO: 24), # 1-24 (SEQ ID NO: 26), # 1-26 (SEQ ID NO: 28), # 1-32 (SEQ ID NO: 34), # 1-33 (SEQ ID NO: 35), or # 1-34 (SEQ ID NO: 36), or peptide numbers # 2-8 (SEQ ID NO: 45), # 2-9 (SEQ ID NO: 46), # 2- of FIG. 10 (SEQ ID NO: 47), # 2-11 (SEQ ID NO: 48), # 2-13 (SEQ ID NO: 50), # 2-14 (SEQ ID NO: 51), # 2-15 (SEQ ID NO: 52) ), # 2-16 (SEQ ID NO: 53), # 2-17 (SEQ ID NO: 54), # 2-18 (SEQ ID NO: 55), # 2-19 (SEQ ID NO: 56), # 2-20 (SEQ ID NO: 57), # 2-21 (SEQ ID NO: 58), # 2-22 (SEQ ID NO: 59), # 2-24 (SEQ ID NO: 61), # 2-25 (SEQ ID NO: 62) , # 2-26 (SEQ ID NO: 63), # 2-27 (SEQ ID NO: 64), # 2- 30 (SEQ ID NO: 67), # 2-31 (SEQ ID NO: 68), # 2-35 (SEQ ID NO: 72), # 2-40 (SEQ ID NO: 77), # 2-41 (SEQ ID NO: 78) ), # 2-42 (SEQ ID NO: 79) or # 2-43 (SEQ ID NO: 80).
(e) a peptide having an amino acid sequence represented by the above (d) and a peptide having 1 to 4 amino acids deleted only at the N-terminus and / or C-terminus, the peptide comprising the amino acid sequence; A peptide having equivalent or higher T cell stimulating activity.
(f) A peptide having 1 to 10 amino acid residues added to the N-terminus and / or C-terminus of the peptide consisting of the amino acid sequence shown in (d ) above, and having the following properties:
(I) The added residue is a residue corresponding to the N-terminal and / or C-terminal of a peptide comprising the amino acid sequence in the natural Cha o 1 or Cha o 2 amino acid sequence.
(Ii) T cell stimulating activity equivalent to or higher than that of the peptide comprising the amino acid sequence. The peptide numbers of (d) are # 1-2 (SEQ ID NO: 4), # 1-7 (SEQ ID NO: 9), # 1-8 (SEQ ID NO: 10), # 1-20 (SEQ ID NO: 22) , # 1-22 (SEQ ID NO: 24), # 1-24 (SEQ ID NO: 26), # 1-26 (SEQ ID NO: 28), # 1-32 (SEQ ID NO: 34), # 1-33 ( SEQ ID NO: 35), # 1-34 (SEQ ID NO: 36), # 2-10 (SEQ ID NO: 47), # 2-20 (SEQ ID NO: 57), # 2-21 (SEQ ID NO: 58), It is # 2-40 (SEQ ID NO: 77), # 2-41 (SEQ ID NO: 78), # 2-42 (SEQ ID NO: 79) or # 2-43 (SEQ ID NO: 80). The preventive or therapeutic agent for cypress pollinosis as described. The peptide numbers of (d) are # 1-7 (SEQ ID NO: 9), # 1-22 (SEQ ID NO: 24), # 1-24 (SEQ ID NO: 26), # 1-32 (SEQ ID NO: 34) , # 1-33 (SEQ ID NO: 35), # 2-10 (SEQ ID NO: 47), # 2-20 (SEQ ID NO: 57), # 2-40 (SEQ ID NO: 77), # 2-41 ( The preventive or therapeutic agent for cypress pollinosis according to claim 5, which is SEQ ID NO: 78), # 2-42 (SEQ ID NO: 79) or # 2-43 (SEQ ID NO: 80). The preventive or therapeutic agent for hinoki hay fever according to any one of claims 4 to 6 , wherein at least two peptides are bonded via a linker selected from Arg-Arg and Lys-Lys. The preventive or therapeutic agent for cypress pollinosis according to any one of claims 1 to 7 , further comprising a pharmaceutically acceptable carrier or diluent. A peptide according to any one of claims 1 to 3 , or any peptide according to (a), (b) or (c) of claim 4, and (d) of claim 4, A reagent used for diagnosis of hinoki hay fever, comprising as an active ingredient a peptide containing at least two peptides, including any peptide according to (e) or (f) . Following steps (a), (b) and (c) including, lymphocytes, cypress pollen allergens Cha o reactivity to 1 T cell epitope peptides, or Cha o 1及beauty C ha o 2 T cell epitope peptide, To measure the reactivity to.
(a) comprising the peptide according to any one of claims 1 to 3 , or the peptide according to (a), (b) or (c) of claim 4, and d) contacting the peripheral blood lymphocytes with a peptide comprising at least two peptides, including any peptide according to (e) or (f) ,
(b) culturing the mixture obtained in (a),
(c) A step of measuring the amount of [3H] thymidine incorporation into lymphocytes.

Images