JPH0782295A - Immunodepressant containing hla-bonded oligopeptide - Google Patents

Abstract

PURPOSE:To provide a novel oligopeptide inhibiting the production of antibodies, delayed allergic reactions, etc., and useful as an immunodepressant. CONSTITUTION:An oligopeptide bonding to a human main tissue-compatible antibody: (HLA)HLA-DRB1*0405 having amino acid sequences of formula I, formula II, etc. The oligopeptide is obtained by separating lymphocytes from a patient having the DRB1*0405, establishing the lymphocytes in EB viruses, treating the obtained B cell strain with a buffer solution, fractionating the DR molecule-oligopeptide bonded product from the obtained all cell decomposition solution with an anti-DR antibody column, eluting the fractionated HLA- oligopeptide-bonded product with an acid, incubating the eluted HLA- oligopeptide-bonded product to separate the oligopeptide, and subsequently isolating and purifying the oligopeptide by ultrafiltration and column chromatography.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to HLA-binding oligopeptides, pharmaceutical compositions containing them as active ingredients, and their use in human and veterinary medicine.

[0002]

BACKGROUND OF THE INVENTION The histocompatibility antigen HLA gene region is located on the short arm of human chromosome 6, and is classified into class I (HLA-A, B, C, E, F, G) and class. It is roughly classified into II (HLA-DR, DQ, DP) genes. Class II genes consist of genes encoding the α chain or β chain of class II molecules. Class II molecule is α
It is composed of a chain and a β chain, and is expressed on the cell membrane surface of antigen-presenting cells (macrophages, dendritic cells, etc.), B cells and activated T cells. The class II molecule binds to a peptide produced by degrading the antigen taken up by the antigen-presenting cell from the extracellular fluid by the proteolytic enzyme contained in the lysosome, and is expressed on the cell surface to express CD4 ⁺ helper T cell. To present. Non-self antigen peptides and self class
Helper T cells that are activated by recognizing the II molecule together
Secretes various lymphokines to promote antibody production by B cells.

A characteristic of HLA is the presence of a high degree of individual variation (polymorphism) in the human population. Since T cells recognize individual differences in HLA molecules and show a strong immune response, HLA agreement in organ transplantation is an important factor for avoiding rejection. Furthermore, HLA polymorphisms also determine individual differences in the immune response to specific antigens by limiting the types of peptides that can bind to them and by affecting the differentiation process of T cells.

The etiology of diseases includes environmental factors and genetic factors. Diseases caused by immune disorders such as autoimmune diseases and allergies are probably multifactorial diseases that are caused by complicated intertwining of multiple environmental factors and genetic factors. As a genetic factor, the correlation between many autoimmune diseases and HLA has been clarified. That is, in rheumatoid arthritis, in white people, DR
B1 ^* 0401 and DRB1 ^* 0404 show a positive correlation with DRB1 ^* 0405 in Japanese. Insulin-dependent diabetes mellitus is DQA1 ^{* in} Caucasians, Blacks, and Japanese ^.
0301-DQB ^* 10302 and DQA1 ^* 050
It shows a positive correlation with 1-DQB1 ^* 0201. Furthermore, HLA is known to have a positive correlation with diseases such as Graves disease and Behcet's disease.

As described above, it is well known that HLA plays an important role as a genetic factor in the onset of disease, but it is known that endogenous peptides are bound to histocompatibility antigen molecules and separated. Although it is (Buus, et al, Sci
ence 242, 1045, 1988), the presence of HLA-binding oligopeptides, and the effect of the substance on the immune response have been only slightly reported (Cottens, et a.
l., WO93 / 05011, Chicz, et al., J. Exp. Me.
d., 178, 27, 1993). Therefore, the present inventors have isolated and purified HLA-oligopeptide conjugates from established human B cells, further isolated and purified oligopeptides bound to HLA, and as a result of earnestly examining the binding ability to HLA. The present inventors have surprisingly discovered that an oligopeptide having a certain amino acid sequence has an HLA-binding action, and arrived at the present invention.

[0006]

Means for Solving the Problems That is, the present invention is a human major histocompatibility complex (HLA) HLA-DRB1 ^* 0405 binding oligopeptide.

The HLA-DRB1 ^* 0405 binding oligopeptide of the present invention is specifically defined by, for example, the following formulas (I) to (VII): (V) GKPPQYIAVHVVPDQLMAFG (VI) An oligopeptide having an amino acid sequence represented by any of SDPILYRPVAVALDTKGPE (VII), and the oligopeptide of the present invention further includes 1 at the N-terminal side and / or the C-terminal side of these amino acid sequences.
Also included are oligopeptides obtained by combining 10 amino acids.

In the above formulas (I) to (VII), each alphabet represents one letter abbreviation of amino acid corresponding to L-amino acid as shown in Table 1 below.

[0009]

[Table 1]

When the oligopeptides of the present invention having the amino acid sequences represented by the above formulas (I) to (VII) are represented by using the three-letter abbreviations, SEQ ID NOs: 1 to 7 in the Sequence Listing are shown below.
Shown in.

As long as the oligopeptide of the present invention has the HLA-binding property of the present invention, one or more of the amino acids constituting the oligopeptide of the present invention may be deleted or replaced with another amino acid. Oligopeptides obtained by substitution or insertion of other amino acids are also included.

Among these, the HLA-DRB of the present invention
The 1 ^* 0405-binding oligopeptide is an oligopeptid comprising the amino acid sequence represented by the above formulas (I) to (VII), among which the above formulas (I), (II) and (III).
), (IV) and (VI), particularly oligopeptides consisting of the amino acid sequences represented by the above formulas (I) and (III) are preferred.

The oligopeptide of the present invention can be obtained as follows. As a naturally-derived substance, for example, lymphocytes are obtained from a patient having DRB1 ^* 0405, and the lymphocytes are established with EB virus to obtain a predetermined amount of B cell line. Then, the DR molecule-oligopeptide conjugate is fractionated from the whole cell lysate obtained by treating this B cell line with a buffer solution using an anti-DR antibody column. HLA collected
The oligopeptide conjugate is acid-eluted and incubated to release the target oligopeptide, and the target oligopeptide is isolated by ultrafiltration / column chromatography.
It can be purified. Alternatively, for example, the above formula (I)
Can be obtained by synthesizing oligopeptides having the amino acid sequences represented by (VII) to (VII) by a peptide synthesizer or the like by a conventionally known method. The HLA-binding oligopeptide of the present invention includes those oligopeptides thus obtained from cells and those produced by conventional publicly known peptide synthesis.

In order to maintain the maximum activity of the peptide thus obtained, the oligopeptide of the present invention should be fresh or stored at 4 ° C. within about 5 days after storage. preferable. Alternatively, the oligopeptide of the present invention can be lyophilized and stored frozen. Furthermore, the solution of the oligopeptide of the present invention may be frozen.

The present invention also relates to the human major histocompatibility complex HL
A pharmaceutical composition containing an A-DRB1 ^* 0405 binding oligopeptide as an active ingredient and an immunosuppressive agent containing a therapeutically effective amount of the oligopeptide as an active ingredient.

The pharmaceutical composition of the present invention comprises the fillers, fillers, binders, moisturizers, disintegrants, surface-active agents,
It can be prepared using a diluent such as a lubricant or an excipient, for example, a preparation for oral administration such as tablets, powders, solutions, capsules, suppositories, injections, etc., or a preparation for parenteral administration. be able to. As the excipients, diluents and the like in such preparations, conventionally known ones can be used. For example, in the case of tablets, lactose, starch, glucose,
In the case of an injection such as crystalline cellulose, water, propylene glycol, etc. may be mentioned, and if necessary, an absorption enhancer, a preservative, a coloring agent, a solubilizing agent, an interfering agent, a soothing agent may be added. it can.

In the present invention, the immunosuppressive agent of the present invention can be obtained by a known method by combining a peptide as such an active ingredient and a known appropriate excipient. Preferably, the immunosuppressive agent of the present invention is administered as a parenteral administration preparation, especially as a drip or single-dose (bolus) intravenous or local administration preparation. In the case of intravenous preparations, physiological saline, 5% dextrin-containing water, Ringer's solution, and other commercially available intravenous perfusion physiological buffer may be used depending on the patient's symptoms.

The effective amount of the HLA-binding oligopeptide of the present invention in humans or animals varies depending on, for example, the age and weight of the subject to be administered, symptoms and severity, dosage form, form and potency of the oligopeptide used, and the like. Yes, and it will eventually change depending on the intention of the doctor or veterinarian. An effective amount of the oligopeptide of the present invention is, for example, about 1 to 100 mg / kg body weight / day, preferably about 5 to 20 mg / kg body weight / day, administered by local injection such as intravenous, intramuscular, intraperitoneal or intraarticular. To be done.

Thus, the HLA-binding oligopeptide of the present invention and a pharmaceutical composition containing the HLA-binding oligopeptide as an active ingredient can be obtained. The HLA-binding oligopeptide of the present invention is treated with an antigen by an antigen-presenting cell to classify the antigen. In the process of being expressed on the cell membrane by binding to the II molecule, it substitutes for an antigen peptide, inhibits or modifies signal transduction between antigen presenting cells and helper T cells, and eventually suppresses antibody production, delayed allergic reaction, etc. It is a thing.

[0020]

The present invention will be described in detail with reference to the following examples, but these examples do not limit the scope of the present invention.

[0021]

Example 1 The method of Buus et al. (Science 242, 104
5, 1988), lymphocytes obtained from a patient having DRB1 ^* 0405 were established with the EB virus to establish a B cell line and grown until the number of cells reached about 10 ¹⁰ . Then, this established B cell line was treated with a buffer solution (1% NP40, 5,
mM Na-orthovanadate, 25 mM iodoacetamide,
The whole cell lysate was obtained by treatment with 1 mM PMSF-containing phosphate buffer (pH 7.2). This whole cell lysate was centrifuged (1
After performing 10,000 rpm for 1 hour) and passing the supernatant through a 0.45 μm membrane filter (Millipore), mouse γ-globulin-Sepharose (Pharmacia) and protein A-Sepharose column (Pierce)
Processed in. The eluate obtained by such treatment is treated with anti-DR
The DR molecule-oligopeptide conjugate was fractionated using an antibody-immobilized column (Pierce) (washing: 0.1% SD
S, 0.5% NP-40-containing phosphate buffer pH 7.2,
Phosphate buffer solution containing 1% n-octyl-glucoside, pH 7.2,
Elution: 1% n-octyl-glucoside-containing buffer solution pH 10.
5). Furthermore, the precipitate produced with 75% acetonitrile was dissolved in 2.5 M acetic acid and incubated at 37 ° C for 30 minutes to release the oligopeptide, which was subjected to ultrafiltration (molecular weight cutoff: 10 kDa), and the flow-through fraction was concentrated. Then, the desired oligopeptide was obtained. The filtration residue was saved as free DR molecule and used for the binding experiment of Example 2. The obtained oligopeptide was analyzed by HPLC (C18 column manufactured by Waters; Bondaspher
e 5 μm, 100 A, 2.1 × 150 mm) for separation (flow rate: 0.25 ml / min, elution conditions: 0.
120 min gradient elution with 06% TFA in water and 0.052% TFA in acetonitrile) (Figure 1). Based on the results obtained by subjecting each peak to a peptide sequencer using Edman degradation, a homology search was performed to determine the amino acid sequence of each peak.

Table 2 shows the relationship between the amino acid sequences corresponding to the respective HPLC peaks of the oligopeptides acid-eluted from the HLA-oligopeptide conjugate and the homology.

[0023]

[Table 2]

Based on these homology information, oligopeptides (I) having the amino acid sequences shown in Table 3
(VII) was postulated as an HLA-binding oligopeptide.

[0025]

[Table 3]

Hereinafter, in the Examples, the oligopeptides of the present invention having the peak numbers 295.17 and the like shown in Table 3 are abbreviated as "295.17 oligopeptides".

[0027]

Example 2 (1) The 295.36 oligopeptides and the like shown in Table 3 were synthesized by a peptide synthesizer. It was confirmed that each of the obtained synthetic oligopeptides had the amino acid sequence shown in Table 3, and the following examples were carried out using these synthetic oligopeptides.

(2) The solvent for the free DR molecule obtained in Example 1 was 1 mM PMSF, 0.05% NP40, 5% DM.
SO, phosphate buffer containing 0.1% NaN ₃ (pH 7.
0) and then a protease inhibitor (final concentration: 1.3
mM 1,10 phenanthroline, 73 μM pepstain A,
8 mM EDTA, 6 mM N-ethyl maleimide, 20
0 μMN, α-p-tosyl-L-lysine chloromethyl
ketone) was added. 10 nM of synthetic oligopeptides labeled with iodine 125 on 1 μM of DR molecule (29 in Table 3)
5.36, 295.24, 295.17 oligopeptide was added), and the mixture was allowed to stand at room temperature for 48 hours for reaction.

As a negative control, the irrelevant M12 peptide (YNELSGEAHKDALGKLGIDN)
Was reacted in the same manner. Then Sephadex G
-50 columns (Pharmacia, 4 × 120 mm) were used for separation and counting was performed using a gamma counter, and the (radioactivity bound to DR molecule / total radioactivity) × 100 was determined and defined as the binding rate (%).

Table 4 shows the binding rate of the labeled synthetic oligopeptide to the DR molecule.

[0031]

[Table 4]

From Table 4, the labeled forms of 295.36 oligopeptide and 295.24 oligopeptide and 295.17 oligopeptide were free DR (DRB1 ^* 040
5) Molecules with 7.5, 12.7 and 8.2% respectively
Combined In contrast, the negative control bound only 0.7%.

[0033]

Example 3 In the binding reaction of Example 2, unlabeled synthetic oligopeptide was added in a molar concentration ratio of 10 to 1,000 times to determine the binding inhibition rate (%). At this time, as a negative control, an oligopeptide that does not bind to the DR molecule (irreleva
ntM12 peptide) was added in a large excess to serve as a negative control.

FIG. 2 shows the inhibitory effect of unlabeled 295.24 oligopeptide at various molar ratios on the binding of labeled 295.24 oligopeptide and free DR (DRB1 ^* 0405) molecule.

From FIG. 2, the unlabeled 295.24 oligopeptide was labeled with the labeled 295.24 oligopeptide and the free DR (D
The binding to the RB1 ^* 0405) molecule was inhibited in a concentration-dependent manner.

Table 5 shows the inhibitory effect of various unlabeled oligopeptides listed in Table 3 on the binding of labeled 295.24 oligopeptides to free DR (DRB1 ^* 0405) molecules.

295.24, 295.36, 295.1
7, 295.29, 295.26, 295.32 and 295.21 oligopeptides were combined with labeled 295.24 oligopeptide and free DR (D
RB1 ^* 0405) molecule was inhibited. In contrast, the negative control (irrelevantM12 peptide) showed no effect.

[0038]

[Table 5]

[0039]

Example 4 Preparation of Injection A solution for injection containing 0.3 mg of 295.24 oligopeptide in 1 ml was prepared according to the following formulation.

295.24 Oligopeptide 30 mg Salt 900 mg Distilled water for injection 100 ml

[0041]

[Sequence list]

SEQ ID NO: 1 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Val Pro Ile Gln Arg Ala Val Tyr Gln Asn Val Val Val Asn Asn 1 5 10 15

SEQ ID NO: 2 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment

SEQ ID NO: 3 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Gly Ser Thr Val Phe Asp Asn Leu Pro Asn Pro Glu Ile Asp Gly Asp 1 5 10 15 Tyr Tyr Gly Trp 20

SEQ ID NO: 4 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Glu Gly Gln Leu Val Ser Ile His Ser Pro Glu Glu Gln Asp Phe Leu 1 5 10 15 Thr Lys His Ala 20

SEQ ID NO: 5 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Gly Pro Lys Pro Leu Phe Arg Arg Met Ser Ser Leu Val Gly Pro Thr 1 5 10 15 Gln Ser Phe Phe 20

SEQ ID NO: 6 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Gly Lys Pro Pro Gln Tyr Ile Ala Val His Val Val Pro Asp Gln Leu 1 5 10 15 Met Ala Phe Gly 20

SEQ ID NO: 7 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Ser Asp Pro Ile Leu Tyr Arg Pro Val Ala Val Ala Leu Asp Thr Lys 1 5 10 15 Gly Pro Glu

[0049]

[Brief description of drawings]

FIG. 1 shows the result of HPLC in Example 1.

FIG. 2 shows the inhibitory effect of the unlabeled oligopeptide on the binding between the oligopeptide (labeled) of the present invention and the free DR molecule in Example 3.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masamichi Motoki 4-3, Asahigaoka, Hino-shi, Tokyo Inside Teijin Limited, Tokyo Research Center (72) Inventor Tomohiko Higashi 4--3, Asahigaoka, Hino, Tokyo No. Teijin Limited Tokyo Research Center (72) Inventor Kenji Nagura 4-3 Asahigaoka, Hino City, Tokyo Inside Teijin Limited Tokyo Research Center (72) Inventor Keiji Komoritani 4-3 Asahigaoka, Hino City, Tokyo No. Teijin Limited Tokyo Research Center

Claims (4)

[Claims] 1. A human major histocompatibility complex (HLA) HLA.
-DRB1 ^* 0405 binding oligopeptide. Wherein the amino acid represented by any one of the following formulas (I) ~ VPIQRAVYQNVVVNN of (VII) (I) SPGTGAYYVLLN (II) GSTVFDNLPNPEIDGDYYGW (III) EGQLVSIHSPEEQDFLTKHA (IV) GPKPLFRRMSSLVGPTQSFF (V) GKPPQYIAVHVVPDQLMAFG (VI) SDPILYRPVAVALDTKGPE (VII) Claim 1 having an array
The described oligopeptide. 3. A pharmaceutical composition containing the oligopeptide according to claim 1 as an active ingredient. 4. An immunosuppressive agent containing a therapeutically effective amount of the oligopeptide according to claim 1 as an active ingredient.