JP2900571B2 - Cedar pollinosis treatment agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a peptide that activates a cedar pollen antigen-specific suppressor / inducer T cell, and a therapeutic or preventive agent for cedar pollinosis containing the peptide.

The present invention relates to a DNA probe for testing cedar pollinosis and a method for testing cedar pollinosis using the probe.

<Prior Art> Japanese cedar pollinosis, which is an allergic disease caused by cedar pollen, is known to cause rhinitis, eye and skin inflammation, asthma, or systemic allergic symptoms. In recent years, the number of patients has been rapidly increasing due to the increase in cedar pollen scattering and air pollution, and it is said that 10-20% of the population in the pollen scattering area is a potential patient.

Currently, there is no essential treatment or prevention for cedar pollinosis, and symptomatic drugs such as antiallergic drugs are prescribed, but there are also problems such as side effects, and the development of effective drugs is desired. .

Similarly, there are methods for testing hay fever, such as antibody titer testing after the onset of the disease.However, no method for prophylactically testing hay fever sensitivity has been reported. The provision of the law is also awaited.

<Problem to be solved by the present invention> Therefore, a peptide effective for the cedar pollinosis of the present invention,
It is an object of the present invention to provide a therapeutic or preventive agent for Japanese cedar pollinosis containing the peptide, and an examination and a method for Japanese cedar pollinosis.

<Means for Solving the Problems> The present inventors have conducted intensive studies in order to solve the above problems, and as a result, a peptide having a specific structure is effective for activating cedar pollen antigen-specific inhibitory T cells. Being and specific
The present inventors have found that cedar pollinosis can be effectively tested using a DNA probe, and have completed the present invention. That is,
INDUSTRIAL APPLICABILITY The present invention uses a peptide that activates a cedar pollen antigen-specific subducer / inducer T cell, a therapeutic or preventive agent for cedar pollinosis containing the peptide as an active ingredient, a DNA probe for cedar pollinosis inspection, and the probe. This is a test method for Japanese cedar pollinosis.

The major allergen causing cedar pollinosis has been identified as a protein having a molecular weight of 40,000-45,000 (Yasueda, H.et.al., J. Allergy Clin. Immunol. 71, 77-8).
6 (1983)), and the N-terminal amino acid sequence has been partially determined (Taniai, M. et al, FEBS letter 239, 329-332 (198
0)) (JP-A-1-156926).

Japanese cedar pollinosis is so-called type I allergy, and overproduction of IgE-type immunoglobulin specific to this allergen has been linked to the onset. It is also known that a genetic predisposition exists in the onset, suggesting a close correlation with the genetic polymorphism of the HLA-DQ protein (a type of human major histocompatibility antigen). That is, cedar pollinosis is a disease in which a person having a specific HLA-DQ protein on the surface layer of leukocytes such as macrophages among HLA-DQ proteins having individual differences among individuals frequently develops.

HLA protein is present on the surface of leukocytes such as macrophages and has an antigen-presenting function.HLA-DR molecules induce positive immune responses by presenting antigens to helper T cells, whereas HLA-DQ molecules Is thought to control the negative immune response by the induction of suppressor (suppressor) T cells. In individuals (humans) with low antigen presenting ability of cedar pollen allergen due to the difference in the structure of HLA-DQ molecule, suppressor T cells It is not induced sufficiently, and is said to cause a hyperimmune response (Matsushita, S. et al., J, Immunol. 138, 109 (198
7), Allergy, 38,726 (1989)).

In general, the antigen (allergen) protein is taken up by antigen-presenting cells such as macrophages, then undergoes intracellular digestion, and degraded fragments are present on the surface of macrophages and other HLA
It binds on a protein and is presented as an antigen. The fragment presented as an antigen is limited to a specific region (T cell epitope) of a part of the antigen protein due to factors such as affinity with the HLA protein. Therefore, when a partial peptide consisting of only that region is administered, the immune response to the antigen can be enhanced or suppressed without using the whole antigen, and a certain result has been achieved in animal experiments (Urban, JLet al. Cell
59,257-271 (1989)). This is thought to be linked to the creation of specific immunomodulators without side effects when using all antigens, such as binding to IgE in the case of an allergen, followed by allergic reactions such as release of chemical mediators such as histamine. I was In addition, if a helper epitope recognized by helper T cells and a suppressor epitope recognized by suppressor inducer T cells are present in different regions with one antigen, artificial immune enhancement and suppression can be effectively achieved. It is expected that it can be done.

The present inventors identified a suppressor epitope of cedar pollen allergen and administered a peptide in that region or a modified version thereof to a cedar pollinosis patient in which suppressor T cells had not been induced to efficiently induce immunosuppression. I realized that it would be linked to the essential treatment of cedar hay fever if possible, and started research.

Now, it is not always easy to identify a T cell epitope in an antigen. Therefore, as a research technique, after determining the entire amino acid sequence of the antigen, a method of preparing and searching for a large number of chemically synthesized peptides over the entire region, and constructing and using a gene that expresses a partial sequence of the antigen are performed. . However, as for the cedar pollen allergen, the amino acid sequence was not known except for a part of the N-terminal, and the gene was not isolated, so that such a method was difficult. On the other hand, attempts have been made to estimate the T cell epitope region of a specific antigen from statistical analysis of the T cell epitope structure of various known antigens. Guillet et al. Mouse IE
It has been found that there is homology with a part of the amino acid sequence of the protein, IA protein (corresponding to human HLA-DR, HLA-DQ), and proposes that this region is the site of interaction between the two. (Science, 235, 865 (1987)). The present inventors conducted a homology search with the amino acid sequence of the HLA-DQ molecule for the N-terminal amino acid sequence 22 residues of the cedar pollen allergen identified to date. Allergen N-terminal sequence is a partial sequence of HLA-DQ (182
(Residues 199 to 199) for the first time.

D is Asp, N is Asn, P is Pro, I is Ile, S is Se
r, C is Cys, W is Trp, R is Arg, G is Gly, A is Ala, Q
Is Gln, M is Met, K is Lys, L is Leu, V is Val, E is Gl
u and T each represent Thr.

9 out of 18 residues from cedar pollen antigen sequence # 2 to # 19
Considering the identity of residues and the fact that Lys corresponding to Arg # 18 belongs to the same basic amino acid, etc., the homology is very high, and Gord and Kanehisa (Nucreic Acid Research, 10,
247-263 (1982)) is calculated as -51. NBRF (National Biomedical Research Fou
HLA-DQβ was one of the most homologous among the approximately 3,000 various protein sequences registered in the databank of the ndation. Abnormally high homology between the amino acid sequence of HLA-DQ, which is one of the most important factors in the onset of cedar pollinosis, and the amino acid sequence of cedar pollen allergen itself, is onset or suppression of onset, and furthermore, cedar pollinosis This strongly suggests that this area plays a very important role in the diagnosis and testing of sclerosis. Reported HLA-D
The presence of genetic polymorphism in the sequence of this part of Qβ and the presence of at least two types of sequences suggest that individual differences in the structure of this part may be reflected in individual differences in onset. Suggests.

The present inventors have based on the above important findings (i) the same region of the cedar pollen antigen interacts with the above-mentioned region of the HLA-DQ molecule and is very closely involved in suppressor T cell induction, (Ii) HLA-DQ Genetic differences in the sequence are reflected in differences in affinity with the cedar pollen antigen sequence and have a patient-type sequence
(Iii) Suppressor T cells cannot be sufficiently induced by HLA-DQ molecules.
Either take out the epitope or modify it further to make the patient type
The intense study was conducted on the hypothesis that if the affinity with the HLA-DQ molecule is changed, suppressor T cells can be induced in a patient similarly to a healthy person and an excessive immune response can be suppressed.

To prove this hypothesis, the present inventors examined the above HLA-DQβ sequence by Southern hybridization in many cedar pollinosis patients and healthy subjects. As a result, it was found for the first time that there was a close correlation between the HLA-DQβ sequence and susceptibility to cedar pollinosis.

In short, the hypothesis was proved to be correct, and the present invention was completed.

This finding is effective in diagnosing and predicting the onset of cedar hay fever, and indicates that it provides a very important guide for the treatment and prevention of cedar hay fever.

As described repeatedly, the present invention provides a peptide that activates a cedar pollen antigen-specific suppressor inducer T cell, a therapeutic or preventive agent for cedar pollinosis containing the peptide as an active ingredient, a DNA probe for examining cedar pollinosis, and It is to provide a test method of cedar pollinosis to be used.

The structure of the peptide used in the present invention is not particularly limited as long as the peptide has the ability to activate a cedar pollen antigen-specific suppressor / inducer T cell. But,
Preferably, peptides having the following structures (1) to (7) are used. That is, (1) a peptide comprising an amino acid sequence represented by the following formula (I):

(2) A peptide comprising an amino acid sequence represented by the following formula (II).

(3) A peptide comprising an amino acid sequence represented by the following formula (III):

(4) A peptide comprising an amino acid sequence represented by the following formula (IV).

(5) A peptide having a structure in which at least one position in the amino acid sequences represented by the above formulas (I) to (IV) is substituted with one amino acid residue or peptide residue.

For example, a peptide containing a structure in which two Asn from the N-terminus are changed to Gln and the ninth to eleventh amino acid sequences from the N-terminus, ie, a structure in which Arg-Gly-Asp is changed to Lys-Ala-Ser This is the case.

(6) A peptide comprising a continuous part in the amino acid sequence represented by the above formulas (I) to (IV).

(7) N of the peptide represented by the above formulas (I) to (IV)
A peptide having one or more amino acids added to the terminal and / or C-terminal.

(8) A peptide obtained by subjecting the peptide of (1) to (7) to addition of polyethylene glycol, acetylation, and / or amidation.

The peptide sequences described in the above (1) to (8) are the N-terminal sequence of the cedar pollen antigen and the HLA-DQβ sequence as they are, or slightly modified based on them.

By the way, the peptide for activating the cedar pollen antigen-specific suppressor / inducer T cells of the present invention may be prepared by chemical synthesis such as a solid phase method, or may be prepared by a technique such as genetic engineering. good.

Now, a purified product of a peptide that activates a cedar pollen antigen-specific suppressor / inducer T cell may be administered as it is, or may contain a stabilizer such as serum albumin and an excipient such as mannitol. May be used.

Now, as an active ingredient in a therapeutic or preventive agent for cedar pollen, a peptide that activates a cedar pollen antigen-specific hand suppressor / inducer T cell is usually 0.01 to 100%, preferably 0.05 to 50%, more preferably 0.5 to 50%. What is necessary is to make it contain 5.0%.

Again, there is no problem if the therapeutic agent for cedar pollen of the present invention contains a stabilizer such as serum albumin and an excipient such as mannitol.

Thus, according to the present invention, the above-mentioned peptide found to activate cedar pollen antigen-specific suppressor inducer T cells induces specific immunosuppression in cedar pollinosis patients in which suppressor T cells are not sufficiently induced. Can be used to do.

That is, suppressor inducer T cells activate suppressor T cells. The most suitable administration method is transnasal, but transdermal, oral, ophthalmic, and injection methods can be used. When administered as a prophylactic agent before the pollen scattering time, it is effective in preventing the onset of the disease, and is also effective in improving symptoms after the onset. The dosage varies depending on the condition, but usually 0.01 mg to 1.0 g per adult per day may be administered. It should be noted that, as a reminder, the peptide of the present invention satisfies safety requirements.

Next, regarding a test DNA probe and a test method for Japanese cedar pollinosis, the present inventors have found a DNA probe for testing the gene sequence of HLA-DQβ, and have established a test method using the same. .

That is, the DNA probe of the present invention is a human histocompatibility antigen HLA
A DNA probe for examining the DQβ gene sequence, specifically the human histocompatibility antigen HLA-DQβ gene sequence, and more specifically the β2 of the human histocompatibility antigen HLA-DQβ gene sequence
180th Lev 181st Gln182th in domain domain
It is a DNA probe that hybridizes with the sequence of Ser or Asn, 183rd Pro, 184th Ile, 185th Thr or Ile 186th Val, 187th Glu.

As a specific example of a DNA probe for testing cedar pollinosis, a DNA probe having the following formulas (V) to (VIII) may be used.

Formula (V) is a DNA probe containing the Ser residue at position 182 of HLA-DQβ, formula (IV) is a DNA probe containing the Asn residue at position 182 of HLA-DQβ, and formula (VII) is 185 of HLA-DQβ. Th Thr
The DNA probe containing the residue and formula (VIII)
This is a DNA probe containing the fifth Ile residue.

Of course, the DNA probe used in the present invention has a sequence in the human histocompatibility antigen HLA-DQβ other than having the above DNA sequence, that is, Any DNA probe can be used as long as it has the property of hybridizing to DNA. For example, (V) to (VII
A DNA probe containing the DNA sequence shown in I) may be used, or a DNA probe consisting of a part of the DNA sequence may be used.

However, it is preferable to use a DNA probe having the sequences shown in the above formulas (V) to (VIII).

Usually, a combination of the probes of the formulas (V) and (VI) or the formula (VI)
Inspection is possible using any of the combinations of the probes of the formulas (I) and (VIII), but preferably the formulas (V) to (VI)
If the inspection is performed using all the probes in II), the accuracy will be higher.

Now, such a genetic test has begun to be used for testing and diagnosing a genetic disease, cancer, and the like, and can be performed in a laboratory equipped with ordinary experimental facilities such as a clinical laboratory of a medical institution.

This is a method for testing cedar pollinosis using the DNA probe of the present invention. First, as a genetic test material, DNA extracted from appropriate cells or tissues collected from a subject may be used.
It should be noted that extraction of DNA from cells and tissues may be performed by a usual method, for example, “Molecular Cloning” (J. Sambrook and TM
aniat is ed. Cold Spring Harbor Lab. USA (1979) pp28
The method described in 0) can be applied. Next, the extracted DNA is digested with an appropriate restriction enzyme, and then subjected to agarose gel electrophoresis. The resulting DNA is transferred to a filter and used as a filter for Southern hybridization. In addition, PCR (Polymerase Chain Reaction)
If the method is used, starting from a small amount of biopsy sample, for example, HLA-D
Only the target DNA sequence such as the specific region of the Qβ gene can be efficiently amplified and used conveniently. Regarding the PCR method, for example, “P
CR Technology "(HAErlich ed. Stockton Press NY
(1989)). The obtained amplified DNA fragment can be similarly used for hybridization if it is adsorbed on a filter directly or after agarose gel electrophoresis. If the above-described DNA probe labeled with an appropriate label is added to these filters and subjected to hybridization, the presence or absence and homology of the target sequence in the DNA sample can be examined. Further
A method in which a PCR-amplified DNA is directly or after cloning and then sequenced to determine the nucleotide sequence can be applied. These can be performed by the method described in the above-mentioned experimental book.

 Hereinafter, the present invention will be described based on examples.

[Example 1] T cell activation by synthetic peptide Peripheral blood was collected from a Japanese cedar pollinosis patient and a healthy sensitized person, and a T cell fraction and a macrophage fraction were separated by a conventional method. 10 μM synthetic peptide, anti-
The cells were cultured for 24 hours with the HLA-DR antibody. Peptide-induced activation of suppressor inducer T cells was measured by ³ H-thymidine uptake activity (Table 1). These activations were inhibited by anti-HLA-DQ and anti-CD4 (proteins present on the suppressor / inducer T cell surface and involved in activation) antibodies, purified cedar antigen,
Since the synthetic cedar peptide activated T cells of healthy subjects more strongly than T cells derived from patients, it can be said that the activation of suppressor / inducer T cells was detected. The results are shown in Table 1.

In addition, it is a synthetic peptide obtained by slightly modifying CP-1, CP-2, CP-3, and CP-4 based on the structure of CP-0.

[Example 2] After collecting blood from a subject, leukocytes were separated or oral mucosal cells were collected with a bamboo skewer to obtain a cell sample, and DN was obtained from these cells.
A was extracted. Two oligonucleotides, CGTGGA, chemically synthesized based on the human HLA-DQβ gene sequence
Add GACGTCTACACCTGC and GCCCAGCCCGAGGAAGATCAG
A PCR reaction was performed to amplify a DNA fragment of 660 bases between both sequences in the human HLA-DQβ gene. After agarose gel electrophoresis, the same DNA fragments were transferred to a PVDF filter, ³² P
A hybridization reaction was performed by adding the following labeled oligonucleotide probes.

Probe 1; CCTCCAGAGCCCCATC Probe 2; CCTCCAGAAC
CCCATC probe 3; CCCATCACCGTGGAGT probe 4; CCCA
TCATCGTGGAGT Probe 1 and Probe 2 are each HLA
A sequence containing a Ser residue at position 182 of DQβ (part of the sequence of (2)) and a sequence containing Asn residue (part of the sequence of (1)); probe 3 and probe 4 Are 185 each
And a sequence containing a Thr residue (part of the sequence (2))
This corresponds to the sequence containing the le residue (part of the sequence of (1)). For hybridization, the filter and the probe were reacted overnight at 48 ° C. in a reaction solution having a composition of 1M NaCl / 1% and SDS / 10% dextran sulfate. 6 × SSC after reaction (0.9MN
The filter was washed at room temperature with aCl, 0.09 M sodium citrate) and subjected to autoradiography to measure the strength of hybridization. Under these conditions, the single nucleotide difference in the DNA sequence could be distinguished. Table 2 shows the results of 53 cedar pollinosis patients and 45 healthy subjects without symptoms.

Here, for example, (1) / (1) indicates that both chromosomes have a gene encoding the sequence of (1). As shown in the results, those with the chromosome type of (2) / (2), which are extremely infrequent in healthy subjects, are significantly more frequent in patients, and conversely, are more frequently distributed in healthy subjects (1) / (1)
Patients of this type are very rare in patients. This means that the sequence of (2) is more likely to develop hay fever sensitivity than the sequence of (1), and in particular, the type (2) / (2) has a very high probability of having hay fever. It was clear that he had.

Amino acid abbreviations in the text indicate the following amino acids, respectively.

A; Ala, C; Cys, D; Asp, E; Glu, G; Gly, I; Ile, K; Lys, L; Leu, M; Met, N; Asn, P; Pro, Q; Gln, R; Arg, S; Ser, T; Thr, V; Val, W; Trp <Effect> Japanese cedar pollen antigen-specific suppressor / inducer T
It is considered that a peptide that activates cells and a drug containing the peptide as an active ingredient are effective in treating and preventing cedar pollinosis, for which radical treatment has not been possible until now. Further, the DNA probe of the present invention is effective for examination of cedar pollinosis.

Claims (5)

(57) [Claims] 1. A peptide that activates a cedar pollen antigen-specific suppressor / inducer T cell, the peptide comprising an amino acid sequence represented by any of the following formulas (I) to (IV): 2. The peptide according to claim 1, wherein at least one of the amino acid sequences represented by formulas (I) to (IV) has a structure in which one amino acid residue or peptide residue is substituted. 3. The peptide according to claim 1, which comprises a continuous part in the amino acid sequence represented by formulas (I) to (IV). 4. The peptide according to claim 1, wherein the peptide has been chemically modified by addition of polyethylene glycol, acetylation, and / or amidation. 5. An agent for treating or preventing cedar pollinosis comprising the peptide according to claim 1 as an active ingredient.