JPH07118295A - T-cell epitope peptide in cedar pollen allergen and analog peptide thereof - Google Patents

Abstract

PURPOSE:To obtain an analog peptide effective for treating/preventing pollinosis, by substituting a part of the amino acid sequence of a T-cell epitope peptide in cedar pollen antigen for other amino acids. CONSTITUTION:Using ceder pollen antigen-specific T-cell clone and T-cell line or the peripheral blood T-lymphocytes of patients with pollinosis, the T-cell epitope in cedar pollen allergen is identified, and the analog peptide is synthesized by substituting a part of the amino acid sequence of a peptide containing the epitope for other amino acids. T-cell response is tested using this analog peptide.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide useful for the treatment and prevention of allergic conditions caused by cedar pollen (hereinafter referred to as cedar pollinosis).

[0002]

2. Description of the Related Art The number of cedar pollinosis patients has been rapidly increasing since 1970, and it is said that the number is about 10 million, which is a serious social problem. Cedar pollinosis presents with allergic conjunctivitis such as eye pain and hyperemia, sneezing, nasal allergy such as stuffy nose, and nasal allergy is more severe than those derived from other allergens. As a treatment method for Japanese cedar pollinosis, administration of antiallergic agents such as antihistamines and steroid hormones has been conventionally performed. In addition, attempts have been made to cure cedar pollinosis by desensitizing the cedar pollen allergen.

In order to develop a therapeutic drug using a T cell epitope or a B cell epitope of cedar pollen, it is necessary to analyze the structure of the cedar pollen allergen. The major allergen of cedar pollen was isolated and purified by Yasuda et al.
Was named (SBP) (Yasueda, H., et al., J. Aller
gy Clin. Immunol. 71, 77-86, 1983). This SBP has a molecular weight of 45 to 50 kDa and is currently called CryjI according to the WHO nomenclature. After that, in the process of separation and purification of CryjI, Cr
CryjII having a molecular weight of 37 kDa, which is different in antigenicity from yjI, was isolated (Taniai, M. et al. FEBS Letters 239, 329-33.
2, 1988, Sakaguchi, M. et al. Allergy 45, 309-312,
1990).

For Cryj I, cDNA has been cloned and its deduced entire amino acid sequence has been elucidated.
I and its fragments have been reported to be useful in therapy and diagnosis (WO93 / 01213, "ALLERGENIC PROTEINS
AND PEPTIDES JAPANESE CEDAR POLLEN).

[0005] As an example in which T cell activation could be inhibited by using an analog peptide of T cell epitope peptide, an autoimmune encephalomyelitis model mouse was provided with an epitope part of myelin basic protein, which is an autoantigen. As a result of administration of the modified analog peptide, it was reported that this analog peptide inhibited the activation of T cells that recognize myelin basic protein and prevented its development (Urbain, JL, Horvath, SJ, and Hood). , L .: Aut
oimmune T cells: immune recognition of normal and
variant peptide epitopes and petide-based therapy.
Cell 59, 257-270, 1989).

[0006]

The drugs currently used for cedar pollinosis are mainly antihistamines that are non-specific for antigens, and they are symptomatic drugs, not radical therapeutic drugs. If a therapeutic drug targeting a T cell epitope is developed, an antigen-specific therapeutic drug will be obtained. It is an object of the present invention to provide a peptide targeting a T cell response to a cedar pollen allergen, which is useful for treating and preventing cedar pollinosis.

[0007]

The present invention provides a T cell epitope peptide that is common to cedar pollinosis and is involved in its development, an HLA class II restricted molecule involved in antigen recognition of cedar pollen allergen-specific T cell clones. , And analog peptides that modify T cell response function are disclosed.

To develop a therapeutic drug targeting a T cell epitope involved in cedar pollinosis, synthesis of overlapping peptides covering the entire region of pollen allergen, cedar pollen allergen-specific T cell clone and T cell line , Establishment of B cell line transformed with Epstein-Barr virus (EBV) (antigen-presenting cells), measurement of antigen-presenting ability of cedar pollen allergen overlap peptide or its analog peptide (lymphocyte proliferation reaction) and various It is necessary to carry out each step of measuring the productivity of cytokines, especially IFN-γ, IL-2, IL-4, IL-10, and identifying HLA class II restricted molecules.

<Synthesis of Overlapping Peptides> Overlapping peptides covering the entire region of cedar pollen allergen are synthesized. In the case of using CryjI as a cedar pollen allergen, based on the entire amino acid sequence of CryjI elucidated by the present inventors (Japanese Patent Application No. 5-170451, see FIG. 1), from N-terminal Asp to C-terminal Cys. Overlapping peptides covering 353 amino acid residues are synthesized. The amino acid residues are 9 to 21 residues, and the overlapping portion is 8 to 15 residues.

<Establishment of T cell clones and T cell lines> To isolate lymphocytes from the peripheral blood of cedar pollen patients,
Usually, a method is used in which the above blood is layered on a Ficoll-Paque solution (Pharmacia), centrifuged at 400 × g for 30 minutes at room temperature, and an intermediate layer suspended in a white band is collected with a capillary pipette. The lymphocytes obtained were placed in a 24-well culture plate and
X 10 ⁶ cells / well are seeded and cultured with 50 μg / ml of purified pollen allergen in a 5% CO ₂ incubator at 37 ° C. for 7 to 10 days to activate T cells. As the culture medium, RPMI1640 liquid medium containing 10 to 15% human AB serum is used (hereinafter, the culture medium has the same composition). IL-2 is not added when the initial culture and the antigen presenting ability of T cells are measured. The limiting dilution method may be used to clone the target T cells from the culture solution. However, the present inventors spread the lymphocytes stimulated for 7 days on a culture dish, and then used a micropipette under a microscope. A method (selection method) of picking up activated T cells one by one is adopted. Activated T cells are small and large, and can be sorted.

T cells screened in this way
The clone was previously mitomycin C (50-100 μg / ml)
EBV-transformed B cell line or peripheral cells inactivated by
5 × 10 blood lymphocytes (from the same patient)^Five/ Well seeded
Transferred to a 96-well round-bottomed culture plate and purified with 25 μg / ml
Powder allergen and 20 U / ml IL-2 (or 0.2 μg / ml PHA)
Culture for 12 to 14 days in the presence of. One of the proliferating T cells
Subsequent screening of T cell clones retaining antigen specificity
Select a loan. This T cell clone (2 x 10 ^Four/ U
And mitomycin C-treated EBV transform
B cell line or peripheral blood lymphocytes (1 x 10^Five/ We
2) and 2) were seeded on two 96-well round bottom plates, and 25 μg /
7 days in the presence of 20 ml / ml of purified pollen allergen and 20 U / ml of IL-2
Incubate. Use the first plate for the assay,
The second plate was used for harvesting T cell clones
It The first plate was 0.
5 μCi [³Add [H] thymidine with a microsyringe and
After culturing for ~ 24 hours, harvest the cells by harvester treatment.
Liquid scintillation counter with adsorption to the filter
With [³Intracellular uptake of [H] thymidine was measured to determine antigen specificity.
Mark wells containing T cell clones carrying
It Assay from the second plate after 7 days of culture
The wells corresponding to the marked wells of the plate
T cell clones that retain their original specificity are transformed into secondary screenin
To go.

The T cell clone obtained was the same as the previous one.
Culture in 24-well culture plates for 7 days in the presence of purified pollen allergen and IL-2 with antigen presenting cells. By repeating the culture cycle of antigen addition at 7-day intervals, pollen allergen-specific T cell clones can be obtained.

It is generally considered that T cell clones that react with an antigen having a high T cell activating ability are easily obtained, but the reverse is difficult to obtain. The cedar pollen allergen has a lower T cell activating ability than the allergen generally used as an experimental model, and thus it is difficult to establish a T cell clone. For the first time, there was a report on the preparation of a cedar pollen allergen-specific T cell clone at an allergic society conference held in May 1993, but after screening about 100 T cells, only one clone was finally obtained. Absent.

<B cell line (antigen presenting cell) by EBV
Established> Ficoll-Paque specific gravity from peripheral blood of Japanese cedar pollinator
Lymphocytes obtained by cardiac method (1 x 10 ⁶) Is an EBV-producing cell
B95-8 culture supernatant (1 x 10⁶pfu) with 100 μg / ml
Contains 10% fetal calf serum in the presence of cyclosporin A
Cultured in RPMI1640 medium and transformed by EBV
Established B cell line.

<Measurement of antigen-presenting ability> Search for T cell epitopes was carried out in the presence of overlapping peptides covering the entire region of cedar pollen allergen, in the presence of peripheral blood T lymphocytes of cedar pollinosis patients or T cell clones. Alternatively, a T cell line (reactive cell) and an EBV transform B cell line inactivated by mitomycin C treatment or a peripheral blood T lymphocyte (antigen-presenting cell) were mixed and cultured, and incorporated into the reactive cell [ It is performed by measuring the amount of ³ H] thymidine with a liquid scintillation counter. As the culture medium, RPMI1640 culture medium containing 10 to 15% human AB serum is used.

<Identification of HLA class II restricted molecules> Involved in antigen recognition of cedar pollen allergen-specific T cell clones
In order to identify HLA class II-restricted molecules, a monoclonal antibody with 10-fold serial dilution was prepared in a mixed culture system of T cell clones and antigen-presenting cells in the presence of T cell epitope peptides.
HLA-DR, -DQ and -DP antibodies are added respectively, and growth inhibition of T cell clone in each peptide is examined. Furthermore, for L cells
It is possible to introduce various combinations of HLA class II restricted molecule genes and use them as antigen-presenting cells. Using this method, more detailed analysis of HLA class II restricted molecules becomes possible.

<Synthesis of Analog Peptides> A large number of analog peptides in which a part of the amino acid sequence of a peptide containing a T cell epitope that specifically recognizes cedar pollen allergen is substituted with other amino acids were synthesized, and T cells against these peptides were synthesized. The proliferative response of the An analog peptide that does not react with T cells is HLA class I in the body due to competitive inhibition.
Together with the I-restricted molecule, it suppresses the activation of T cells by the actual antigen-presented T cell epitope. Alternatively, by acting on T cells, the state of non-responsiveness to T cells (T-cell anerg
y). Peptides that react with T cells induce the production of IFN-γ. These analog peptides can be a therapeutic agent for cedar pollinosis that is specific to cedar pollen allergens.

[0018]

EXAMPLES The present invention will now be described in detail based on examples, but the present invention is not limited thereto.

<Purification of CryjI> CryjI is an ion exchange column using DE52 and CM52 (manufactured by Whatman) gels from cedar pollen by the method of Yasueda et al. (J. Allergy Clin. Immunol., 71: 77-86, 1983). Purified by chromatography.

<Synthesis of Overlap Peptides> Peptide Synthesizer PSSM-8 (manufactured by Shimadzu Corporation) was used for peptide synthesis. Based on the primary structure of CryjI shown in FIGS. 1 and 2, it is shifted by 5 residues from the N-terminal side.
Up to 21 mer was synthesized based on the mer. Also, for peptides 335 to 346 residues, an analog peptide was synthesized by substituting the amino acid residues in each configuration with different amino acids.

<Establishment of B Cell Line> Peripheral blood lymphocytes (1 × 10 ⁶ ) obtained by the Ficll-paque gravity centrifuge method were treated with E of about 1 × 10 ⁶ pfu.
Cells were infected with virus by incubation with BV for 1 hour at 37 ° C. When the virus-infected cells are transferred to a 24-well culture plate and cultured for about 2 weeks in the presence of 100 ng / ml of cyclosporin A, B cell colonies will appear. At this point it was split in half and transferred to new wells. When this operation is sequentially repeated for subculture, B cells capable of self-proliferation may appear. After expanding the cells in the well containing the self-proliferating B cells and confirming the proliferation, the cells were transferred to a 25 cm ² culture flask and further cultured for 30 to 50 days to obtain an EBV transform B cell line. B
Some of the cell lines were cryopreserved.

<Establishment of CryjI-specific T cell clones and lines> 30 types of T cell clones from cedar pollinosis patients
Ten types of T cell lines were established. Each of these T cells showed a proliferative response to CryjI, but did not proliferate against other streptococcal cell wall antigens (SCW), Candida Albicans antigen (CA), and purified tuberculin antigen (PPD). From this, it is considered that the prepared T cell clones and T cell lines are T cells that recognize CryjI and can react specifically with allergen. Less than,
The details will be described.

From heparinized peripheral blood of a Japanese cedar pollinosis patient
Ficoll-paque (Pharmacia-LKB) by gravity centrifuge
Peripheral blood lymphocytes were obtained. 2 × 10 this lymphocyte⁶/ Ue
In a 24-well culture plate for 20 to 5
The cells were cultured in the presence of 0 μg / ml of purified CryjI for 7 days. That
Tissues activated by rugen stimulation
And use a micropipette under the microscope to separate them one by one.
I picked up one. This cell was previously prepared with mitomycin C (Kyowa
Fermented) treated autologous peripheral blood lymphocytes (5 x 10^Five/ U
L) seeded in a 96-well round bottom plate, 25 μg
CryjI (20 ml / ml) and IL-2 (20 U / ml) for another 12 days
It was Proliferating T cell clone (2 x 10 ^Four/ Well)
And mitomycin C-treated autologous peripheral blood lymphocytes (1 x 10^Five
/ Well) in 96-well flat bottom culture plates
And 25 μg / ml CryjI was added. At this time, exactly the same
Two types of culture plates were prepared. The first plate is cultivated
On the second day of feeding, 0.5 μCi [³[H] Thymidine is pulsed for another 18
After culturing for a long time, cells are harvested and liquid scintillation is performed.
At the counter³Measures intracellular uptake of [H] thymidine
To possess CryjI specificity by
Elected. The second plate was cultured for 7 days, then Cryj
Select only T cells that have I-specificity and set the cell number to 4
× 10^Five/ Auto-treated with mitomycin C according to well
My peripheral blood lymphocytes (2 x 10⁶/ Well) or stock in EBV
B cell line (5 x 10^Five/ Well) with 25 μg / ml
Cryj I and 20 U / ml IL-2 were added and 24-well culture was performed.
Incubated on plate. Repeat this culture every 7 days
This maintained CryjI-specific T cell clones. T
Cell lines from peripheral blood lymphocytes stimulated by CryjI for 7 days
Viable T cells were separated by Ficoll-paque gravity centrifuge
Stimulates once every 7 days under the same conditions as clones
Maintained by.

<Measurement of Overlapping Peptide Antigen Presenting Capability> Ten kinds of established T cell clones and one kind of T cell line were cultured with cedar pollen allergen overlapping peptide to determine the epitope. . The results are shown in Fig. 1. There are 7 identified T cell epitopes in CryjI, and the amino acid sequence is 61-7.
5, 91-105, 106-120, 146-160, 211-225, 326-340, 335
It was located at -346. The details of the method are as follows.
T cell clones or T cell lines cultured for 7 to 10 days after antigen stimulation with CryjI were adjusted to 2 × 10 ⁴ / well, and autologous peripheral blood lymphocytes or EBV treated with mitomycin C as antigen-presenting cells. Transform B cell lines were seeded in 96-well flat bottom culture plates at 2 × 10 ⁵ / well or 5 × 10 ⁴ / well and 25 μg
After adding CryjI / ml or overlapping peptide of 1 μM, the cells were cultured for 2 days. Then add 0.5 μCi to each well.
[ ³ H] thymidine was pulsed, and the cells were further cultured for 18 hours. Cells were captured on a glass filter and taken up inside the cells
The amount of [ ³ H] thymidine was measured with a liquid scintillation counter.

<Identification of HLA class II restricted molecule> HLA class II restricted by using T cell clone recognizing Cryj I amino acid residue 327-346 epitope peptide and self antigen presenting cell or L cell incorporating HLA class II gene The molecule was identified. HLA class II typing is Histocompatibil
It was performed according to the standard protocol described in City Testing 1991. Proliferation response system using T cell clone
Anti-HLA-DR, -DQ, and -DP monoclonal antibodies diluted in 10-fold series were added, and the proliferative response of T cell clones was investigated. Furthermore, L cells incorporating various HLA class II type genes were used as antigen-presenting cells to investigate the proliferative response of T cell clones. Anti-HLA-DR, -DQ, -D
The restraint molecule was determined to be the HLA-DR molecule by suppressing the proliferative response using the P monoclonal antibody. Furthermore, by using L cells as the antigen presenting cells, it was revealed that the antigen presentation of the 327-346 epitope peptide was carried out by the molecule (DR52) derived from the DRB3 ^* 0301 gene.

<Measurement of antigen-presenting ability of analog peptide> 32
A peptide was synthesized in which the N- and carboxyl-terminal amino acids of the 7-346 epitope peptide were sequentially reduced by one residue. T specifically recognizes an epitope in this peptide
The peptide was added to the culture system of cell clones and antigen-presenting cells. T cell clone consists of peptide 335-346 1
It reacted with a 2mer peptide, but did not react with shorter peptides. In other words, the T cell clone is
It means that the peptide consisting of er is identified. Then, an analog peptide in which amino acid substitution was introduced into the 335-346 epitope peptide was synthesized, and the proliferative response of T cell clone was investigated (FIG. 3). When amino acid substitutions were introduced at the amino acid sequences 340 and 341, the T cell clone could not recognize these peptides at all. The amino acid sequence 33
When amino acid substitutions were introduced at positions 7, 338, 339, 342, 344, and 345, there were peptides that the T clone could not recognize.

These analog peptides may competitively inhibit the binding of the true epitope peptide to the restricted molecule during antigen presentation. Further, it is possible that a certain analog peptide binds to a restricted molecule and causes T cells that react in an epitope-specific manner to fall into an immunologically unresponsive (T-cell Anergy) state. Actually, using T cell clone and 335-346 epitope peptide as a model, an amino acid substitution-introduced analog peptide was synthesized and the reactivity of T cell clone was investigated. Some peptides cannot be recognized by T cell clones at all, and some have only a partial proliferation response. That is, in the 335-346 epitope peptide, the amino acids at positions 337-345 are HLA class II.
It is predicted to be an important amino acid that binds to a restricted molecule or provides antigen information to the T cell receptor.
Therefore, it is considered that these analog peptides have an action of impairing the original function of T cells that react in an epitope-specific manner. This result means that activation of T cells that recognize the epitope can be suppressed by introducing an amino acid substitution into the epitope peptide. 335-346 Epitope peptides 337, 338, 3
T cells proliferate against an analog peptide in which the amino acids at positions 39, 342, 344, and 345 are replaced with other amino acids. Of these peptides, for example, Thr at the amino acid at position 339 is Va
The analog peptide substituted with l induces IFN-γ production. These analog peptides improve the balance of cytokines involved in cedar pollen allergy, and are useful as therapeutic / preventive agents for cedar pollinosis having a new mechanism of action.

[0028]

EFFECTS OF THE INVENTION Peptides that react with cedar pollen allergen-specific T cells make it possible to analyze specific HLA class II-restricted T cell responses and the cytokine-producing ability involved in them, and to identify the mechanism of action of cedar pollinosis. It is useful for elucidation. Furthermore,
Synthesized analog peptides in which a part of the amino acid sequence of the peptide is substituted, cloned into T cells against these peptides, T cell line or cedar pollinosis patient peripheral blood T
Measure reactivity with lymphocytes. Analog peptides that do not react with T cells competitively inhibit the binding of T cell epitope peptides to HLA class II antigens in the human body. Alternatively, by binding to HLA class II antigen, HLA
T cells that respond to class II restriction are unresponsive (T cell a
energy). The analog peptides that react with T cells are
It modifies T cell function and induces the production of IFN-γ. IFN
-Γ acts suppressively on Th2 cells. Such analog peptides are specific to cedar pollen allergens and do not disturb the normal immune response to other foreign antigens,
It is expected as a therapeutic / preventive drug for cedar pollinosis with a new mechanism of action.

[Brief description of drawings]

FIG. 1 is a diagram showing the entire amino acid sequence of CryjI (first half).

FIG. 2 is a diagram showing the entire amino acid sequence of CryjI (second half).

FIG. 3 shows the proliferative response of T cell clones to the 335-346 epitope peptide and amino acid substitution pseudoepitope. The letter after the peptide number indicates the substituted amino acid (single letter notation).

Front page continuation (72) Inventor Naoki Komiyama 540 Narita, Odawara, Kanagawa Prefecture, Meiji Dairy Co., Ltd.Health Science Research Institute (72) Inventor Kikousuke, 540, Narita, Odawara, Kanagawa, Meiji Dairy Co., Ltd., Health Science Research Institute

Claims (7)

[Claims] 1. A peptide reactive with cedar pollen allergen-specific T cell clones and / or T cell lines and / or peripheral blood T lymphocytes of cedar pollinosis patients. 2. The peptide according to claim 1, wherein the cedar pollen allergen is CryjI. 3. An analog peptide obtained by substituting a part of the amino acid sequence of the peptide according to claim 1, which is a CryjI-specific T cell clone and / or T cell line and / or cedar pollinosis patient peripheral blood T lymphocytes. An analog peptide that does not react with. 4. An analog peptide obtained by substituting a part of the amino acid sequence of the peptide according to claim 1, which is a CryjI-specific T cell clone and / or T cell line and / or cedar pollinosis peripheral blood T lymphocytes. An analog peptide that reacts and leads to increased interferon γ production. 5. The peptide according to claim 1, which has at least one of the following amino acid sequences. Gly Ala Thr Arg Asp Arg Pro Leu Trp Ile Ile Phe Ser Gly Asn Thr Phe Asp Gly Arg Gly Ala Gln Val Tyr Ile Gly Asn Gly Gly Pro Cys Val Phe Ile Lys Arg Val Ser Asn Val Ile Ile His Gly His Pro Gln Asp Gly Asp Ala Leu Thr Leu Arg Thr Ala Thr Asn Lys Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asn Ala Phe Asn Val Glu Asn Gly Asn Ala Thr Pro Gln Leu Thr Lys Thr Pro Gln Leu Thr Lys Asn Ala Gly Val Leu Thr 6. The analog peptide according to claim 3, which has at least one of the following amino acid sequences. Thr Pro Glu Leu Thr Lys Asn Ala Gly Val Leu Thr Thr Pro Gln Ser Thr Lys Asn Ala Gly Val Leu Thr Thr Pro Gln Leu Ser Lys Asn Ala Gly Val Leu Thr Thr Pro Gln Leu Thr Ala Asn Ala Gly Val Leu Thr Thr Pro Gln Leu Thr Arg Asn Ala Gly Val Leu Thr Thr Pro Gln Leu Thr His Asn Ala Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Ala Ala Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Gln Ala Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Asp Ala Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Ser Ala Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Thr Ala Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Asn Thr Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Asn Val Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Asn Ala Gly Ser Leu Thr Thr Pro Gln Leu Thr Lys Asn Ala Gly Ala Leu Thr Thr Pro Gln Leu Thr Lys Asn Ala Gly Val Ser Thr Thr Pro Gln Leu Thr Lys Asn Ala Gly Val Val Thr 7. The analog peptide according to claim 4, which has at least one of the following amino acid sequences. Thr Pro Asn Leu Thr Lys Asn Ala Gly Val Leu Thr Thr Pro Gln Ile Thr Lys Asn Ala Gly Val Leu Thr Thr Pro Gln Val Thr Lys Asn Ala Gly Val Leu Thr Thr Pro Gln Leu Val Lys Asn Ala Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Asn Gly Gly Val Leu Thr Thr Pro Gln Leu Thr Lys Asn Ala Gly Ile Leu Thr Thr Pro Gln Leu Thr Lys Asn Ala Gly Leu Leu Thr Thr Pro Gln Leu Thr Lys Asn Ala Gly Thr Leu Thr Thr Pro Gln Leu Thr Lys Asn Ala Gly Val Ile Thr