JPH0912469A - Immunomodulator - Google Patents

Abstract

PURPOSE: To obtain an immunomodulator useful for treating autoimmune diseases, rejection, allergic diseases, etc., by immobilizing a peptide capable of inducing the anergy in a T cell on a carrier insoluble in a humor. CONSTITUTION: This immunomodulator is obtained by immobilizing a peptide capable of inducing an antigenic nonreactional state (anergy) in a T cell on a carrier insoluble in a humor. The synthesis of the peptide capable of inducing the energy is simple in operations and preferably synthesized according to a solid-phase synthetic method. For example, blood, a lymph or an intraarticular fluid is cited as the humor. A carrier having a hydrophilic surface and reactive groups utilizable for forming covalent bonds between the carrier and the peptide such as amino group, carboxyl group or hydroxyl group is preferred as the carrier. The shape of the carrier is preferably granular or fibrous. Furthermore, the amount of the peptide immobilized on the carrier is preferably >=3×10<-8> mol/g (carrier), more preferably 1×10<-7> to 1×10<-4> mol/g (carrier).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an immunomodulator.
INDUSTRIAL APPLICABILITY The immunomodulator of the present invention can efficiently induce anergy in T cells, so that T cells cause an immune response that causes an injury or an allergic reaction in a self-tissue or a graft. It is useful for treating allergic diseases.

[0002]

2. Description of the Related Art T cells which are in charge of immune reaction are activated only when they are presented with antigen by antigen presenting cells such as macrophages. In this case, it is necessary to form a complex consisting of three molecules of the T cell receptor on the T cell, the peptide fragment of the antigen, and the MHC (major histocompatibility complex) molecule on the antigen presenting cell. Recent studies have shown that a peptide (analog peptide) in which a part of the peptide fragment of this antigen is replaced with another amino acid forms a complex with T cell receptor and MHC molecule, but does not activate T cell, and It has been clarified that pretreatment of T cells with this analog peptide can induce an antigen non-responsive state (anaergy) to the peptide fragment of the original antigen [NATURE, 1
56, 1993].

Applying the principle of anergy, it treats diseases such as autoimmune diseases in which T cells produce an immune response causing damage to self tissues or grafts or allergic reactions, rejection at the time of organ transplantation, allergic diseases, etc. T, which produces an unfavorable immune response in the human body when
Since it is possible to selectively induce anergy only in cells, it becomes possible to effectively treat these diseases without the side effects of conventionally used immunosuppressive agents that suppress the entire immune system. From this viewpoint, attempts have been made to develop a peptide that induces anergy as a medicine (“Experimental Medicine”, Volume 12, No. 1, p. 69, 1994;
"The History of Medicine", 173, No. 7, 633, 199.
5 years).

[0004]

When an anergy-inducing peptide is directly administered to a living body, a general drawback of peptide drugs, namely low stability in the living body, causes the peptide to remain in the body fluid. There is a problem that the concentration capable of inducing anergy in T cells can be maintained only for a short time, and anergy cannot be efficiently induced in T cells.

Therefore, an object of the present invention is to provide an immunomodulator capable of significantly improving the stability of an anergy-inducing peptide and efficiently inducing anergy in T cells.

[0006]

According to the present invention, the above-mentioned object is achieved by providing an immunomodulator comprising a peptide that induces anergy in T cells immobilized on a carrier insoluble in body fluid. .

The peptide that induces anergy in T cells in the present invention (hereinafter, may be abbreviated as anergy-inducing peptide) includes a sequence capable of inducing anergy in T cells in its amino acid sequence. There is no particular limitation as long as it is one, even if there is only one sequence capable of inducing anergy contained in the amino acid sequence,
The number may be two or more. When the amino acid sequence contains two or more amino acid sequences, the sequence may be one type or two or more types.
Further, the anergy-inducing peptide immobilized on a carrier insoluble in body fluid may be only one kind or two or more kinds. The number of amino acid residues constituting the anergy-inducing peptide is not particularly limited, but it is 5 or more and 100 or less in view of low antigenicity to human body, ease of preparation, and efficiency of inducing anergy. Is preferred.

[0008] The T cells that are the subject of the present invention are preferably T cells that produce an immune response that causes an autologous tissue or graft injury or an allergic reaction. T cells that generate an immune response that causes damage to self tissues means T cells that are reactive to antigens present in self tissues, and in this case, autoimmune diseases are the main target diseases of the immunomodulator of the present invention. Become. Examples of self-organs include red blood cells, white blood cells, platelets, blood vessels, brain, heart, lungs, stomach, kidneys, liver, pancreas, spleen, small intestine, large intestine, joints, bones, muscles such as skeletal muscle and myocardium, skin, thyroid , Adrenal cortex, peripheral nerves, central nerves and the like, but are not particularly limited as long as they are self-organized. The T cell that produces an immune response that causes a disorder of the graft means a T cell that is reactive with an antigen that is present in the graft that has been transplanted into the body. In this case, the rejection reaction is the immunomodulator of the present invention. It becomes the main target disease.
Examples of the graft include organ components such as other people's heart, lungs, kidneys, liver, etc., cell components such as other people's red blood cells, platelets, lymphocytes, hematopoietic stem cells, etc. It is not particularly limited to these. The T cell that produces an immune response that causes an allergic reaction means a T cell that is reactive to an allergen, and in this case,
Allergic diseases are the main target diseases of the immunomodulator of the present invention. As allergens, various mites, fungi, those derived from microorganisms such as parasites, dogs, cats, those derived from animals such as cattle, bees, mosquitoes, those derived from insects such as cockroaches, egg white, milk, wheat, rice, Buckwheat, beans, crab, shrimp, tuna,
Examples thereof include dietary substances derived from chicken and the like, pollen of plants such as cedar, cypress, ragweed, mugwort, etc., but are not particularly limited as long as they induce allergies.

The synthesis of the anergy-inducing peptide in the present invention is carried out by a method usually used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method such as a stepwise extension method or a fragment condensation method. It is convenient to operate by a synthetic method [for example, Journal of the American Chemical Society (Journa
l of the American Chemical Society), 85, 21
49, 1963; "Biochemistry Experiment Course 1 Protein Chemistry IV Chemical Modification and Peptide Synthesis," edited by the Japanese Biochemical Society (November 15, 1972, published by Tokyo Kagaku Dojin), 2
P. 07; edited by The Biochemical Society of Japan, "Seikagaku Chemistry Laboratory Lecture 2 Protein Chemistry (2)" (May 20, 1987, Tokyo Kagaku Dojin), p. 641].

In the case of synthesizing an anergy-derived peptide by the solid phase synthesis method, for example, a polymer such as a styrene-divinylbenzene copolymer which is insoluble in the reaction solvent is directed from the C-terminal side to the N-terminal side of the desired peptide. Towards
An operation of condensing and binding a corresponding amino acid while protecting a functional group such as an α-amino group other than the α-carboxyl group of the amino acid, and forming a peptide bond such as an α-amino group in the bound amino acid. The peptide chain is extended by sequentially repeating the operation of removing the protecting group of the amino group to form a peptide chain corresponding to the desired peptide, and then the peptide chain is released from the polymer and protected. The target peptide can be obtained by removing the protecting group from the functional group present. If necessary, this peptide can be further purified by a method such as reverse phase high performance liquid chromatography to obtain a highly pure peptide.

The immunomodulator of the present invention can be produced by immobilizing the thus obtained anergy-inducing peptide on a carrier insoluble in body fluid. Here, the body fluid means a liquid component containing T cells derived from a living body, and examples thereof include blood, lymph, joint fluid, and fractionated components obtained from these. Carriers that are insoluble in body fluids include those that have a hydrophilic surface and that have reactive functional groups such as amino groups, carboxyl groups, and hydroxyl groups that can be used to form covalent bonds with peptides. preferable. Examples of the material for the carrier include synthetic polymers such as polyvinyl alcohol, polyacrylic acid and polyacrylamide, and polysaccharides such as cellulose, agarose and dextran.

Examples of the shape of the carrier which is insoluble in body fluid include particulate, fibrous, sheet, membrane and hollow fiber shapes.
It can be appropriately selected according to the usage method. When the immunomodulator of the present invention is used by filling it in a container such as a column, it is preferably in the form of particles or fibers. In this case, the carrier preferably has a large effective surface area for coming into contact with T cells in the body fluid, and is formed with an interval that allows cells contained in the body fluid to sufficiently pass therethrough. From this perspective,
The particle size of the particulate carrier is preferably in the range of 10 to 5000 μm, more preferably in the range of 50 to 2000 μm. Further, in the case of a fibrous carrier, the fiber type is preferably 1000 μm or less, more preferably 10 μm or less.

The column used as described above has an inlet portion and an outlet portion, and a body fluid passes between the inlet portion and the immunoregulatory material layer and between the outlet portion and the immunomodulatory material layer, but the immune regulator is present. It is preferable to use a filter provided with a material such as polyester that does not pass through the material. Examples of the material of the column include polyethylene, polypropylene, polycarbonate, polyester, and polymethyl methacrylate. Of these, polypropylene and polycarbonate are preferable because they can be subjected to sterilization treatment such as autoclave sterilization and γ-ray sterilization.

The immobilization of the anergy-inducing peptide on the carrier is carried out according to the method generally adopted for immobilizing the peptide or protein on the carrier. As the method, for example, the carboxyl group contained in the carrier is N-
By reacting with hydroxysuccinimide,
A method of converting to a succinimidooxycarbonyl group and reacting this with anergy-derived peptide at the amino group part (active ester method), in the presence of a condensation reagent such as dicyclohexylcarbodiimide on the amino group or carboxyl group of the carrier, anergy induction A method of condensing a carboxyl group or an amino group of a peptide (condensation method), a method of crosslinking a carrier and an anergy-derived peptide with a compound having two or more functional groups such as glutaraldehyde (carrier crosslinking method), etc. Can be mentioned. Among them, the immobilization method by the active ester method is preferable from the viewpoint of immobilizing the anergy-inducing peptide on a carrier with almost no reduction in the anergy-inducing activity of T cells.

The amount of the anergy-inducing peptide immobilized on the carrier is 3 × 10 ⁻⁸ mol / g (carrier) or more from the viewpoint of the activity of the resulting immunomodulator to induce anergy in T cells. Is preferred, and more preferably in the range of 1 × 10 ⁻⁷ to 1 × 10 ⁻⁴ mol / g (carrier).

By contacting the immunomodulator of the present invention with a body fluid containing T cells, anergy can be induced in T cells. The method of contacting the immunomodulator with the body fluid is to take the body fluid out of the body and store it in a container such as a bag and mix it with the immunomodulator, or to fill the column with the immunomodulator and let the body fluid pass therethrough. Etc., but
From the viewpoint of ease of operation, a method in which the column is filled with the immunomodulator and the body fluid is passed through the column is preferable.

In the present specification, various amino acid residues are described by the following abbreviations. Ala: L-alanine residue Arg: L-arginine residue Asp: L-aspartic acid residue Asn: L-asparagine residue Cys: L-cysteine residue Glu: L-glutamic acid residue Gln: L-glutamine residue Gly: Glycine residue His: L-histidine residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Met: L-methionine residue Phe: L-phenylalanine residue Pro: L -Proline residue Ser: L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue Further, in the present specification, a peptide is used according to a conventional method. The amino acid sequence of is described so that the N-terminal amino acid residue is located on the left side and the C-terminal amino acid residue is located on the right side.

[0018]

The present invention will be described below with reference to Examples and the like.
The present invention is not limited to these.

Reference Example 1 Formula (1): Gly Lys Lys Val Ile Thr Ser Phe Asn Glu
The peptide represented by Gly Leu Lys (SEQ ID NO: 1) was used as an automatic peptide synthesizer [Model 430A (Model Applied Applied Biosystems, USA).
430A)] by the solid phase synthesis method as described below.

A styrene-divinylbenzene copolymer having a ratio of 4-hydroxymethylphenoxymethyl group of 0.85 mmol / g (resin) [constituent ratio of styrene and divinylbenzene (molar ratio): 99: 1]. Granular resin (Applied Bios
0.28 g of HMP resin manufactured by ystems), and according to a series of operations shown in Table 1, L in the corresponding order from the C-terminal side to the N-terminal direction of the target peptide.
-Bind lysine, L-leucine, glycine, L-glutamic acid, L-asparagine, L-phenylalanine, L-serine, L-threonine, L-isoleucine, L-valine, L-lysine, L-lysine, and glycine It was In the condensation reaction, the above amino acids are N
α-9-fluorenylmethoxycarbonyl-Nε-t-
Butyloxycarbonyl-L-lysine, 9-fluorenylmethoxycarbonyl-L-leucine, 9-fluorenylmethoxycarbonylglycine, 9-fluorenylmethoxycarbonyl-L-glutamic acid γ-t-butyl ester, 9-fluor Olenylmethoxycarbonyl-L-asparagine, 9-fluorenylmethoxycarbonyl-L-
Phenylalanine, 9-fluorenylmethoxycarbonyl-Ot-butyl L-serine, 9-fluorenylmethoxycarbonyl-Ot-butyl-L-threonine, 9
-Fluorenylmethoxycarbonyl-L-isoleucine, 9-fluorenylmethoxycarbonyl-L-valine, Nα-9-fluorenylmethoxycarbonyl-Nε
-T-butyloxycarbonyl-L-lysine, Nα-9
-Fluorenylmethoxycarbonyl-N [epsilon] -t-butyloxycarbonyl-L-lysine and 9-fluorenylmethoxycarbonylglycine were used in an amount of about 2 times the molar amount of the substrate.

[0021]

[Table 1]

After the reaction procedure for all amino acids was completed, the obtained resin was washed successively with dichloromethane and methanol on a glass filter, and then vacuum dried to obtain 720 mg of a dried resin. In a vial, 720 mg dry resin and 9.5 ml trifluoroacetic acid,
0.25 ml water and 0.25 ml ethanedithiol were mixed. After standing at room temperature for 2 hours, the mixture was filtered with a glass filter, diethyl ether was added to the filtrate, and the mixture was centrifuged to obtain a white precipitate. The obtained precipitate was vacuum dried, extracted with a 2N aqueous acetic acid solution, and the extract was freeze-dried to obtain a peptide (hereinafter, this may be abbreviated as peptide 1).

The obtained peptide 1 was analyzed by high performance liquid chromatography [column: column packed with octadecylated silica gel having a particle size of 5 μm (inner diameter: 4.6 mm, length: 100 mm, TSKgel ODS-80TMCTR manufactured by Tosoh Corporation); migration Phase: A mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (the concentration of acetonitrile was gradually changed from 5% by volume to 50% by volume in 30 minutes); Flow rate: 1
ml / min; detection method: absorbance at wavelength 210 nm], a single sharp peak was shown at 11.5 min. FAB
The parent ion (M + H) ⁺ observed in the mass spectrum measurement by the (fast atom bombardment) method was 1421 (theoretical value 1420.6).
1).

Reference Example 2 In the same manner as in Example 1, the formula (2): Gly Lys Lys Val Il
e Thr Ala Phe Asn Glu Gly Leu Lys (SEQ ID NO: 2)
The peptide represented by was synthesized. However, amino acids are L-lysine, L-leucine, glycine, L-glutamic acid, L-asparagine, L-phenylalanine, L-alanine, in the corresponding order from the C-terminal side of the target peptide toward the N-terminal direction. L-threonine, L-
Isoleucine, L-valine, L-lysine, L-lysine,
And glycine was attached. In the condensation reaction, the above amino acids are Nα-9-fluorenylmethoxycarbonyl-Nε-t-butyloxycarbonyl-L-, respectively.
Lysine, 9-fluorenylmethoxycarbonyl-L-leucine, 9-fluorenylmethoxycarbonylglycine, 9-fluorenylmethoxycarbonyl-L-glutamic acid γ-t-butyl ester, 9-fluorenylmethoxycarbonyl-L -Asparagine, 9-fluorenylmethoxycarbonyl-L-phenylalanine, 9-fluorenylmethoxycarbonyl-L-alanine, 9-fluorenylmethoxycarbonyl-Ot-butyl-L-threonine, 9-fluorenyl Methoxycarbonyl-L-
Isoleucine, 9-fluorenylmethoxycarbonyl-
L-valine, Nα-9-fluorenylmethoxycarbonyl-Nε-t-butyloxycarbonyl-L-lysine,
Nα-9-fluorenylmethoxycarbonyl-Nε-t
Used as -butyloxycarbonyl-L-lysine, and 9-fluorenylmethoxycarbonylglycine.
The obtained peptide (hereinafter sometimes referred to as "peptide 2") was subjected to analytical reverse-phase high performance liquid chromatography to show a single peak at 11.0 minutes. FA
Parent ion (M +
H) ⁺ was 1405 (theoretical value 1404.61).

Example 1 Cellulose particles [Chisso Corporation] were added to 50 ml of dioxane.
Made, carboxycellulofine, particle size 125-210μm) 10
g, and to the resulting suspension, 0.5 g of N-hydroxysuccinimide and 1.0 of dicyclohexylcarbodiimide.
g was added and the mixture was shaken and stirred overnight at room temperature. The resulting mixture was washed with 0.02 mol / l phosphate buffer (pH 7.4) and suction filtered. The obtained particles were mixed with 20 ml of a 0.02 mol / l phosphate buffer solution (pH 7.4) containing 20 mg of the peptide obtained in Reference Example 1, and this mixture was stirred overnight at 4 ° C. The resulting mixture was suction-filtered, and 20 mg of the peptide obtained in Reference Example 1 was immobilized on the immunomodulating material (about 10 g).
(Hereinafter, this may be abbreviated as immunomodulator 1). When the filtrate was subjected to analytical reverse-phase high performance liquid chromatography, no unreacted peptide remained was observed (immobilization rate of peptide on the carrier: 100
%).

Test Example 1 Method of Evavold et al. [Journal of Immunology (J
ournal of Immunology) 148, 347, 199
2 years], and immunize mice with peptide 2 (corresponding to residues 64-76 of β ^d minor chain of mouse hemoglobin) to prepare various T cell clones that react with this peptide. Type T cell clone PL1
A Th1 type T cell clone having the same character as 7 was obtained. Using this T cell clone, the method of Sloan-Lancaster et al. [Nature, 363, 156, 1
[993]], and induction of anergy was investigated as follows.

Antigen-presenting cells (DCEK Hi7 fibroblasts) obtained by treating the above-mentioned T cells (5 × 10 ⁵ cells / well) with mitomycin C on a 24-well tissue culture plate.
The cells were cultured in RPMI medium at 37 ° C. for 24 hours in the presence of Immunomodulator 1 (100 mg). Further, as a control, the T cells and the antigen-presenting cells were cultured under the same conditions without adding the immunomodulator 1. After culturing, each culture was centrifuged at 3,000 rpm for 15 minutes on Ficoll Hipack manufactured by Pharmacia to separate T cells. After culturing each separated T cell in RPMI medium for 3 days, it was examined whether or not anergy was induced. That is, each T cell (2 × 10 ⁴ ) was treated with the antigen peptide Peptide 2 (10 μM) or IL-2 (20 units).
The presence of ³ H thymidine in T cells was examined by culturing for 72 hours at 37 ° C. in the presence of antigen-presenting cells (5 × 10 ⁴ cells) and ³ H thymidine (0.4 μCi). The results are shown in Table 2 below.

[0028]

[Table 2]

From Table 2, when stimulated with peptide 2 which is an antigen, the amount of ³ H thymidine uptake in T cells treated with immunomodulator 1 was drastically reduced as compared with control T cells, and thus, to peptide 2, Treated with immunomodulator 1 because it is in a tolerant state and responds similarly to control T cells when stimulated with IL-2, which is a T cell activator that is non-specific for antigen. It can be seen that the T cells subjected to the reaction have a non-responsive state specific to peptide 2, that is, anergy is induced.

[0030]

INDUSTRIAL APPLICABILITY The present invention provides an immunomodulator capable of efficiently inducing anergy in T cells.

[0031]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Gly Lys Lys Val Ile Thr Ser Phe Asn Glu Gly Leu Lys 1 5 10

SEQ ID NO: 2 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Gly Lys Lys Val Ile Thr Ala Phe Asn
Glu Gly Leu Lys 1 5
10

Claims (1)

[Claims] 1. An immunomodulator comprising a T cell anergy-inducing peptide immobilized on a carrier insoluble in body fluid.