JPH10120591A - Immunological tolerance inducing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an immunological tolerance inducing agent useful for a prevention and a treatment and safe. SOLUTION: This antigen specific immunological tolerance inducing agent is obtained by bonding a peptide consisting of 5-25 amino acids containing a b-cell epitope of B-subunit constituting A-B type toxin and/or T-cell epitope with an antigen as a subject of introducing the tolerance, though a suitable spacer. By utilizing only an essentially functional part of the immunological tolerance, an unnecessary physiological activity which a subunit of a toxin generally has, is removed to provide a safe immunological tolerance inducing agent, and useful for the prevention and treatment of an autoimmune disease, a rejection in an organ transplant and an allergy, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel agent for inducing tolerance. The immunological tolerance inducer of the present invention is useful for treating or preventing allergy, autoimmune diseases such as rheumatoid arthritis, and diseases caused by unnecessary immune reactions such as rejection at the time of transplantation.

[0002]

2. Description of the Related Art When the immune system in a living body encounters an antigen, it becomes unable to respond to the same antigen stimulus given later, which is called immune tolerance. In a normal state, immune tolerance is established for the self-constituting component, and the immune system does not cause a disorder mechanism. As described above, the term "tolerance" refers to the occurrence of a B cell response typified by antibody production and / or a T cell response typified by cytokine production or induction of cytotoxic T cells against a specific antigen. This is one of the homeostatic mechanisms of living organisms. Therefore, artificial induction of immune tolerance is considered to be effective as a treatment for autoimmune diseases caused by unnecessary immune reactions, rejection at the time of transplantation, allergies, and the like. It is already well known that oral administration of an antigen under certain conditions can induce antigen-specific immune tolerance (H.
L. Weiner et al., Annu. Rev. Immunol. 12: 809-837 (1994)). Furthermore, it is known that antigen-specific immune tolerance can be induced even by administering an antigen through the nasal mucosa (J. Mestecky
Et al., Annals of the New York Academy of Science 778:
194-201 (1996)). As described above, administration of an antigen from the intestinal tract or nasal mucosa induces immune tolerance more efficiently than when a so-called systemic antigen is administered, due to the inherent biological mechanism of the mucosal tissue. This is because the intestinal and nasal mucosal immune systems have the exclusive function of attacking and eliminating foreign substances, but also have the forgiving aspect of accepting foreign substances even if they are beneficial to them. It is thought to be derived. In other words, it is thought that there is a characteristic system that controls excessive inflammatory immune response in this dual background, and oral nasal tolerance is considered to be a phenomenon reflecting such characteristics of mucosal immunity ( AMMowat, Immuno
l. Today 8: 93-98 (1987)).

[0003] Such an immune tolerance inducing agent that actively utilizes the special properties of the mucous membrane is antigen-specific, has a wide safety margin for inducing effects, and has been unprecedented in that once-inducing induces long-term effects. It is expected to become a medicine. However, in practice, in order for oral or nasal immune tolerance to be established, the properties, concentration,
Certain conditions must be met, such as the frequency of administration. For example, it is known that, under the same conditions that oral tolerance is established when OVA (ovalbumin) is normally administered, the use of chemically modified OVA does not establish tolerance to OVA (MJM Jacobs et al., Scand. J.Im
munol. 37: 97-103 (1993); H.-J. Peng et al., Scand. J. Immu
nol. 42: 297-304 (1995)). Furthermore, it is also known that the oral immune tolerance induction efficiency changes depending on the age and administration interval during oral administration (BSLundin et al., Scand.J.Immunol. 43: 56-
63 (1996); AMCFaria et al., Immunology 78: 147-151 (199
3)). That is, a method for reliably inducing immune tolerance with any antigen by utilizing the biological mechanism inherent in the mucous membrane as described herein has not yet been established.

[0004] The antigen that has reached the mucosal surface is taken up from mucosal epithelial cells or M cells of Peyer's patches in a short time.
(P. Brandtzaeg et al., Gastroenterology 97: 1562-1584 (19
89); V. Galliaerde, Eur. J. Immunol. 25: 1385-1390 (199
Five)) . It is also known that allogeneic cells are involved in antigen uptake in the respiratory epithelium (J. Richardson
Et al., Lab. Invest. 35: 307-314 (1976)). The taken-in antigen is considered to be presented on each cell surface after being processed in a plurality of types of cells having antigen presenting ability. The T cell expressing the T cell antigen receptor having an affinity for the antigen epitope portion of the antigen thus presented.
The cells and the B cells that express the B cell surface immunoglobulin with affinity react, and a series of immune reactions occur. This series of immune responses occurs first in the mucosal region, but it has been reported that immune tolerance is established not in the local region but in the systemic immune system (G. Wildner & SRThurau, Eu
rJImmunol. 25: 1292-1297 (1995)). The immune response that leads to the establishment of immune tolerance is affected by a number of factors, such as the nature of the antigen, the type of antigen-presenting cells involved and the type of lymphocytes that react with it, and the genetic background of the individual. Therefore, in order for oral or nasal tolerance to be established, certain conditions such as the properties, concentration, and frequency of administration of the administered antigen must be satisfied, and the setting of these conditions is also determined empirically. Have been.

[0005] Holmgren & Czerkinsky discloses in WO95 / 10301 a method for increasing the efficiency of antigen absorption on the mucosal surface for the purpose of increasing the efficiency of inducing oral tolerance. Cholera toxin B subunit is a B subunit portion constituting cholera toxin which is one of AB type toxins, and is originally GM1 ganglioside (galactosyl-N-acetyl-galactosaminyl-) on mucosal surface.
(Sialyl) -galactosylglucosylceramide) and the like are known to be molecules having a function of sending the A subunit into cells by binding. Combination of genetically engineered cholera toxin B subunit (recombinant cholera toxin B subunit) efficiently induces antigen-specific oral tolerance to sheep erythrocytes or human gamma globulin or myelin basic protein (JB.Sun et al.,
Proc. Natl. Acad. Sci. USA 91: 10795-10799 (1994); JB.S
un et al., Proc. Natl. Acad. Sci. USA 93: 7196-7201 (1996);
J. Holmgren & C. Czerkinsky, WO 95/10301).

Cholera toxin B subunit (CT-B) and B subunit of heat labile enterotoxin (LT-B) are conventionally known as vaccine compositions. For example, in Anderson et al., Japanese Patent Publication No. 8-11737, the use of a non-toxic mutant LT-B as a carrier enhances the immunogenicity of a bacterial pathogen against a capsular polymer, thereby completing a bacterial pathogen vaccine composition. Is disclosed. Thus, molecules that promote binding to the cell surface, such as CT-B and LT-B,
The act of binding to the antigen as a carrier is a technique for increasing the immunogenicity of the antigen, and essentially induces an immune response opposite to the induction of immune tolerance. Holmgren & Czerkinsky mentioned above
In 95/10301, whether the effect of inducing immune tolerance by an antigen with a binding molecule to the mucosal surface such as CT-B or LT-B is really due to only an increase in the efficiency of antigen uptake on the mucosal surface It should be pointed out that it did not disclose.

[0007] The activity of CT-B and LT-B molecules themselves to cause T cell death (TONashar et al., Int. Im.
munol. 8: 731-736) and the activity of inducing helper T cells in Peyer's patches (I. Nakagawa et al., J. Infect. Dis. 173: 1428).
-1436), and even toxic (Anderson et al., Tokuhei 8-1173)
No. 7 gazette). In view of these concerns about side effects, studies have been conducted to identify T-cell epitopes and B-cell epitopes of LT-B for the purpose of vaccine development against enterotoxin.
(I. Takahashi et al., Infect.Immun. 64: 1290-1298 (199
6)). Furthermore, when the peptide itself containing both of the T-cell epitope and the B-cell epitope is orally administered, the immune response to the administered peptide is activated in the Peyer's patch, while the periphery becomes immunologically tolerant. (I. Takahashi et al., Biophy
s. Biochem. Res. Commun. 206: 414-420 (1995)). These L
Research on T-cell T-cell epitopes and B-cell epitopes is intended only for the development of vaccines using enterotoxins such as LT-B as antigens. It should be taken into account that no immune response was tested.

[0008]

The effect obtained by transmucosal administration of an antigen having a binding molecule to a mucosal surface, such as a CT-B or LT-B molecule, is due to the fact that the efficiency of antigen uptake by the mucosa is reduced. It is thought that this leads to a reduction in the number and amount of administrations, and consequently an increase in the probability of establishment of immunological tolerance, as compared to administration of the antigen alone. However, as described above, in order for immune tolerance to be established in the mucosa, two steps of the step of antigen uptake and the step of interaction with T cells and B cells are important, and the uptake efficiency is increased. In addition, it is clear that promoting interaction with T cells and B cells that work to establish immune tolerance can induce mucosal immune tolerance more essentially universally. Furthermore, when using CT-B or LT-B molecules themselves, these molecules have toxicity and non-specific physiological activity on T cells, and may cause unexpected side effects. Problems in safety have been pointed out. Thus, a more universal and safe immune tolerance inducer that is more essential and does not depend on the properties of the antigen has been desired.

That is, the problem that the present inventors are trying to solve is that an immunological tolerance inducer using CT-B molecules or LT-B molecules themselves does not retain the physiological activity inherent in the subunit of the toxin. That is what remains.
For example, 1) it forms a pentamer, 2) it has the ability to bind to the A subunit having ADP-ribosyltransferase activity and the like, 3) it has the ability to send the A subunit into cells, and 4) Furthermore, the B subunit itself has toxicity and biological activity on immunocompetent cells.

The present inventors have found that, in the amino acid sequence of these B subunits, a peptide of a specific region consisting of 5 to 25 amino acids is an essential functional part for inducing tolerance. Therefore, an object of the present invention is to utilize this peptide to not only increase the probability of establishment of immunological tolerance by promoting the efficiency of antigen uptake in the mucosa as in the prior art, but also to increase T Another object of the present invention is to provide a more essential universal and safe immune tolerance inducer by promoting interaction with cells and B cells.

[0011]

Means for Solving the Problems The inventors of the present invention not only aimed at promoting the efficiency of antigen uptake in the mucosa as in the conventional art, but also found that T cells and B cells, which act to establish tolerance in the mucosa. We worked hard on the interaction itself to establish a method for establishing antigen-specific immune tolerance in a manner that is in line with the inherent immune tolerance establishment mechanism of the living body. As a result, it has been surprisingly found that, among peptides derived from CT-B or LT-B, a peptide containing a T cell epitope or a B cell epitope can be covalently bound to an antigen to induce antigen-specific immune tolerance. The present invention has been completed. That is, A
-A peptide containing a B-cell epitope and a T-cell epitope of the B-subunit constituting the B-type toxin has a special action in mucosal immune response, and by binding the peptide having such action to an antigen, The T-cell response and B-cell response in the mucous membrane to the antigen are affected, and an antigen-specific immune tolerance is established in accordance with the inherent immune tolerance establishment mechanism of the living body.

The immunological tolerance inducer of the present invention comprises a compound represented by the following formula (1) as an active ingredient. PLA (1) In the above formula, P represents a peptide consisting of 5 to 25 amino acids, including a B cell epitope and / or a T cell epitope of the B subunit constituting AB type toxin. . L represents a spacer, and A represents an antigen of which tolerance is to be induced. That is, the compound of the active ingredient is AB
A peptide comprising 5 to 25 amino acids, including a B cell epitope and / or a T cell epitope of a B subunit constituting a type toxin, bound via a spacer to an antigen of which tolerance is to be induced, A tolerogenic drug wherein the active ingredient is present in a pharmaceutically acceptable excipient at a pharmaceutically effective dose.

[0013] When the immunological tolerance inducer of the present invention is used, for example, as shown in the Examples, the effect can be obtained with only about 1 / 1,000 of the dose required for inducing oral immunological tolerance with the antigen alone. can get. In addition, the prior art recombinant B
Unnecessary biological activity inherent in the subunit of the toxin can be removed to the utmost as compared with the one to which the subunit molecule is bound, while the effect of inducing antigen-specific immunological tolerance is at least comparable.

The term "tolerance" as used in the present invention is a phenomenon in which when the immune system in a living body encounters an antigen, it cannot respond to the same antigen stimulus given later, and is established in an antigen-specific manner. Is what you do. That is, a humoral immune response typified by antibody production, and / or a delayed type hypersensitivity reaction / antibody-dependent cytotoxicity / cytotoxicity by cytotoxic T cells against a specific antigen / natural killer Refers to a state in which a cell-mediated immune response typified by lysis of a target cell due to no cell occurs or is suppressed. It is well understood in the art that the establishment of induction of immune tolerance is not necessarily a complete immune response, but is suppressed to such an extent that it does not adversely affect the living body.

The term "AB toxin" as used in the present invention is a toxin consisting of A subunit and B subunit literally. First, the B subunit binds to a receptor of a target cell, and Assists cell membrane passage. The active A subunit enters the cytoplasm and modifies ADP-ribosylation and phosphorylation of the target functional protein,
Causes abnormal cell metabolism. For example, diphtheria toxin, Pseudomonas aeruginosa exotoxin-A, pertussis toxin, cholera toxin, toxogenic Escherichia coli heat-labile (heat-labile) enterotoxin, ciguatoxin (Shigella toxin), verotoxin and the like are "AB type toxins". include.

The term "peptide" as used in the present invention refers to AB
A series of linear amino acids comprising a B-cell epitope and / or a T-cell epitope of the B subunit constituting the type toxin, and mutually linked by a peptide bond between an α-amino group and a carboxyl group of adjacent amino acid residues Represents For example, as the B cell epitope of the heat-labile (heat-labile) enterotoxin B subunit of toxogenic Escherichia coli, the amino acid sequence sequence portion 85 to 91, the sequence portion 36 to 44, the sequence portion 64 to 73, No. 34 to 42 are known as T cell epitopes.
No. sequence sequence is known (I. Takahashi et al., Infe
ct.Immun. 64: 1290-1298 (1996)). Peptides containing such B-cell and / or T-cell epitopes can be of various lengths, whether in the neutral state (uncharged) or in salts, glucosylation, oxidation of side chains or It may or may not be modified, such as by phosphorylation. It is well understood in the art that the amino acid sequence contains acidic and basic groups, and that the particular ionization state exhibited by the peptide depends on the pH of the surrounding medium. Substituents added to amino acid side chains concomitantly, such as peptides modified by glucosyl units, lipids or inorganic ions, and modifications related to chemical conversion of the chain, such as oxidation of sulfhydryl groups, are also included in this definition. included. Thus, the term "peptide" includes the B and / or T cell epitopes of the B subunit that constitutes AB type toxin.
It is intended to include an amino acid sequence consisting of 2525 amino acids, and those having undergone the above modification that does not impair the function are also intended.

Furthermore, as long as it is useful as an immunological tolerance inducer in the present invention, the peptide of the present invention (hereinafter referred to as “the peptide”)
The present peptide) may have one or more amino acids added to its amino terminal and / or carboxyl terminal. By adding such amino acids, the antigen and the present peptide can be bound without impairing the function of the peptide. Useful amino acids for such purposes include tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof. Further, it is also possible to add a means by which the present peptide can be operably linked to another peptide immunogen or antigen by a conventional protein modification method, for example, by acetylation of the amino terminal or amidation of the carboxyl terminal.

The peptide can be prepared by a number of conventional methods. Usually, it can be prepared by a method of introducing amino acids stepwise by an organic chemical synthesis method, but can also be prepared by a genetic engineering method. A-
Although it is possible to prepare the B subunit constituting B type toxin or its content by a hydrolysis treatment using an enzyme, acid or alkali, it is possible to prepare the A subunit having an activity of causing abnormal cell metabolism. Care must be taken to avoid contamination.

Solid phase peptide synthesis or liquid phase peptide synthesis is known as an organic chemical synthesis method, and is described in detail, for example, in Nobuo Izumaya et al., "Basic and Experimental Peptide Synthesis (Maruzen)". ing. More specifically, in the liquid phase peptide synthesis method, the carboxyl group of the amino acid to be located at the C-terminus is replaced with an appropriate protecting group (for example, benzyl group (Bzl), t-butyl group (t-Bu), etc.). The amino group of the amino acid to be protected and to be located second from the C-terminus is appropriately protected (for example, t-butyloxycarbonyl group (Boc)
Protected with a benzyloxycarbonyl group (Z), etc., and dissolving these amino acids in a suitable solvent (eg, dimethylformamide (DMF), tetrahydrofuran, etc.),
(E.g., dicyclohexylcarbodiimide (DCC), etc.)
And optionally in the presence of a racemization inhibitor (eg, 1-hydroxybenzotriazole (HOBT), etc.)
About 10-24 hours.

Then, the amino protecting group of the product is
Removal using ordinary methods such as oloacetic acid gives the dipeptide.
Can be The resulting dipeptide is then replaced with a third amino acid
(This is also amino-protected) and the same as above
To give a tripeptide. In addition, similar hands
Repeat the procedure in order to bind the necessary amino acids,
To obtain the target peptide in a bound state. In addition, let it react
Peptide synthesis when the amino acid has a side chain functional group
It needs protection before the reaction. For example, for lysine
The amino group at the 6-position is protected by a tosyl group (Tos) or the like.
After completion of the final reaction, these protecting groups are catalytically reduced or fluorinated.
Removal by hydrogen (HF) etc. to obtain the desired peptide
be able to. In addition, solid-phase peptide synthesis methods use conventional techniques.
Peptide synthesizers (eg, Applied Biosys
(Thames Model 430A). This one
Method, the carboxyl terminus of the target peptide
Amino acid-bound phenylacetamidomethyl (PAM)
Resin (ie, amino acid -OCH _TwoBoc on the amino group side of -PAM)
The amino acids protected by groups are sequentially linked by automatic control,
Sample in which protecting group and PAM resin are bonded to target peptide
Can be obtained. The sample was then anisole
After any scavenger was added, HF was introduced and -2
The target peptide is released by reacting at
Can be made. The released peptide is anhydrous ether
After washing with throat, extract with water containing acetic acid and freeze-dry.
Thus, the desired peptide can be obtained. Generally, this
These synthetic peptides were purified using a synthetic peptide purification column.
Purify and recover by high performance liquid chromatography (HPLC).
And freeze-dried.

The present peptide can also be obtained by a genetic engineering method. For example, a DNA fragment encoding the amino acid sequence of interest is synthesized, this DNA fragment is inserted into an appropriate expression vector by a conventional method, an appropriate host is transformed with the expression vector, and the resulting transformant is cultured. The target peptide can be prepared by isolation and purification from the culture.

The term "epitope" as used herein refers to a site on an antigen molecule that binds to an antibody or a site that binds to a histocompatibility antigen and stimulates a T cell receptor. The former is called a B-cell epitope, and the latter is called a T-cell epitope. The smallest unit of an epitope is generally a peptide consisting of 6 to 10 amino acids, 5
It is known to be an oligosaccharide made up of ~ 8 monosaccharides. However, recent studies on the chemical structure of natural high molecular antigens and analysis of polypeptides, sugars, and lipid chains obtained by decomposing the antigens are said to have about 5 to 8 amino acids and about 4 to 6 sugars. The region containing such an epitope is preferably non-hydrophobic because it can bind to a surface immunoglobulin on B cells or a T cell receptor on T cells.

The term "spacer" as used in the present invention utilizes the above-mentioned peptide and antigen by utilizing the amino group at the N-terminal part, the carboxyl group at the C-terminal part, or the functional group of the side chain of the amino group. Are combined. More specifically, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB
S), succinimidyl 4- (N-maleimidomethyl)
Cyclohexane-1-carboxylate (SMCC), succinimidyl 4- (p-maleimidophenyl) butyrate
(SMPB) and other heterobivalent cross-linking reagents, dimethyladipimidate (DMA), dimethylpimelinimidate (DMP), dimethylsuberimidate
(DMS), disuccinimidyl suberate (DSS), dithiobis (succinimidyl propionate) (DSP), ethylene glycol bis (succinimidyl succinate) (E
GS), disuccinimidyl tartrate (DST), and the like, and can be easily carried out using a commercially available crosslinking agent, such as using a homobivalent crosslinking reagent. The term "spacer" also includes an amide bond utilizing a so-called zero-length crosslinker such as 1-ethyl-3- (3-dimethylamino) propylcarbodiimide (EDC). For details of the cross-linking agent, for example, reference can be made to "Natural Chemistry Laboratory Course 1, Protein IV, Structure-Function Relationship, Chapter 13: Cross-Linking", Hidenori Yamada et al., Tokyo Chemical Dojin, Japan, pp. 207-230. The "spacer" can also be obtained by a genetic engineering method. For example, a DNA fragment encoding the amino acid sequence of the target "peptide"
A DNA fragment encoding 1 to 4 amino acids corresponding to the "spacer" is translatably linked, and a DNA fragment encoding an amino acid sequence corresponding to the "antigen" is translatably linked. The continuous DNA fragment was inserted into an appropriate expression vector by a conventional method, an appropriate host was transformed with the expression vector, the obtained transformant was cultured, and isolated and purified from the culture, The active ingredient compound of the immune tolerance inducer of the present invention can be prepared.

The antigens used in the present invention include, for example, ovalbumin, milk, mites, mold, cedar pollen, type II collagen, myelin basic protein, insulin, erythrocytes, leukocytes, kidney cells, liver cells, skin epithelial cells or These include antigen components obtained from these, but are not particularly limited as long as they can be expected to have a therapeutic effect by inducing immune tolerance, as long as they retain the immunological activity of the antigen. It may be a peptide.

The immunological tolerance inducer of the present invention is useful for allergy of mammals including humans, autoimmune diseases such as rheumatoid arthritis, and diseases caused by unnecessary immune reactions such as rejection at the time of transplantation. It is useful for treatment and prevention of In particular, the present peptide is a peptide consisting of 5 to 25 amino acids including a B cell epitope and / or a T cell epitope of a B subunit constituting AB type toxin,
Since the subunit molecule is not used as it is, it is presumed that the safety is high.

The immunotolerant-inducing agent of the present invention may use the compound of the active ingredient alone as an immune-tolerant inducing agent.
However, they can usually be used in various dosage forms. Specific examples of such dosage forms include, for example, oral preparations, nasal preparations,
Inhalants, eye drops, vaginals, suppositories and the like can be mentioned. Formulations of these various dosage forms are in accordance with the usual method, depending on the purpose of treatment,
It can be prepared using carriers that can be usually used in the field of pharmaceutical technology, such as excipients, binders, solubilizers, solubilizers, emulsifiers, and suspending agents.

For example, when administering an oral preparation, the active ingredient is mixed with a solid or liquid non-toxic pharmaceutically acceptable carrier suitable for oral administration and administered in the form of a conventional pharmaceutical preparation. Such preparations include, for example, tablets, granules, powders, solids such as capsules, solutions, suspensions, liquids such as emulsions,
Freeze-dried preparations and the like can be mentioned, and these preparations can be prepared by conventional means on preparation. Examples of the non-toxic carrier for pharmaceuticals include, for example, pharmaceutical grade mannitol,
Contains lactose (lactose), starch (starch), magnesium stearate, sodium sucrose, cellulose, magnesium carbonate, and the like. Furthermore, white sugar, glucose,
Hydroxypropylmethylcellulose, carboxymethylcellulose, gum arabic, gelatin, magnesium metasilicate aluminate, anhydrous calcium phosphate, citric acid, trisodium citrate, hydroxypropylcellulose, sorbitol, sorbitan fatty acid ester, polyvinylpyrrolidone, vegetable oils (peanut oil, olive oil, etc.) Benzyl alcohol, propylene glycol, sucrose, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, albumin, water, physiological saline and the like. Also, if necessary, stabilizers, lubricants, wetting agents,
Conventional additives such as emulsifiers and binders can be added as appropriate. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 1.0-95%, preferably 2.5-70%, of the active compound. Also, for example, in the case of suppositories, traditional binders and carriers include, for example, polyalkalene
lycos) or triglycerides, such suppositories are 0.5
It can be formed from a mixture containing the active ingredient compound in the range of -10%, preferably 1-2%.

The tolerogenic agent of the present invention is administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and tolerogenic. The quantity to be administered depends on the subject to be treated, the capacity of the subject's immune system and the degree of protection given. The exact amount of active ingredient required to be administered depends on the judgment of the practitioner, is specific to each individual, and may vary depending on the age, weight, condition, severity of the disease, administration schedule, formulation, etc. The selection and determination are made, for example, about 0.05 to 500 mg / kg body weight per day, and the administration may be carried out several times a day.

[0029]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, in order to clarify the features of the present invention, the present invention will be described in accordance with embodiments. However, these embodiments are intended to explain various specific examples of the present invention. The invention is not limited to these examples.

[0030]

Example 1 (Synthesis) FIG. 1 shows an example of a peptide consisting of 5 to 25 amino acids, which is a B cell epitope and / or T cell epitope derived from the B subunit constituting AB type toxin.
(SEQ ID NOS: 1 to 3) were used. FIG.
Among them, CTBpeptide is a sequence of amino acid sequence 26 to 45 of cholera toxin B subunit, LTB (F)
The peptide is a sequence of amino acids 26 to 45 of the amino acid sequence of the thermolabile enterotoxin B subunit, and the LTB (I) peptide is a sequence obtained by mutating one amino acid of LTB (F) (the 17th amino acid). Phe is changed to Ile). By artificially adding cysteine to the C-terminal side of each peptide to be used, it is possible to bind to various antigens using the above-mentioned various spacers, so that cysteine is added to the C-terminal of each peptide. Have been. A peptide having the sequence shown in FIG. 1 (SEQ ID NOS: 1 to 3) was synthesized by a solid phase method using a commercially available solid phase synthesizer (manufactured by Applied Biosystems) according to a conventional method. The obtained peptide was bound to model antigen ovalbumin (OVA) and model antigen human gamma globulin (HGG) via a disulfide bond utilizing a spacer of succinimidyl 3- (2-pyridyldithio) propionate (SPDP).

Specifically, SPDP was purchased from Pharmacia (Sweden) and the binding reaction was performed according to the manual attached to the product. That is, OVA
And HGG each contain 0.1 M sodium chloride and contain
It was dissolved in 7.5 of 0.1 M sodium phosphate buffer (hereinafter referred to as sodium phosphate buffer). Sephadex G-25 to remove low molecular impurities from these protein solutions
After gel chromatography using (Pharmacia), the protein concentration was adjusted to 10 mg / ml. To this solution, SPDP, which was made into a 20 mM solution with ethanol, was added dropwise and rapidly stirring so that the molar ratio with respect to the protein was 3 to 10 times. Incubate for 30 minutes at 23 ° C with occasional stirring. Sephade with sodium phosphate buffer
Gel chromatography using xG-25 (Pharmacia) was performed again. Next, the synthesized peptide was dissolved in a sodium phosphate buffer solution, and added to the SPDP-reacted protein solution so that the molar ratio with the protein was 3 to 10 times. The mixture was reacted at 23 ° C. for 16 to 24 hours,
The protein was conjugated to the peptide. After the reaction,
The unreacted spacer and unreacted peptide were removed by gel chromatography using adex G-25 (Pharmacia), and the buffer was exchanged.
The target molecular weight fraction was recovered by gel chromatography using acryl S-300 (Pharmacia). Here, the target molecular weight is 40,000 to 60,0,0 in the case of the model antigen OVA.
00 Dalton. 150,000 for model antigen HGG
~ 300,000 Daltons.

Whether or not the collected fraction is the active ingredient compound of the immunological tolerance inducer of the present invention can be easily confirmed by a technique commonly used in the field of the present invention, for example, SDS-PAGE. When electrophoresis is performed using a commercially available gradient gel for SDS-PAGE (for example, manufactured by Daiichi Pure Chemicals Co., Ltd., Japan), the band under non-reducing conditions is compared with the band under reducing conditions. Under non-reducing conditions, bands are observed slightly larger in molecular weight than the original bands of the respective model antigens, while under reducing conditions, the original bands of the model antigen and bands derived from the peptide of 500 to 4,000 daltons are observed. I was sure that. As a result, the following six active ingredient compounds of the immune tolerance inducer of the present invention were synthesized. CTBpeptide-OVA, LTB (F) peptide-OV
A, LTB (I) peptide-OVA, CTBpeptide-H
GG, LTB (F) peptide-HGG, LTB (I) peptid
e-HGG.

[0033]

Example 2 (Evaluation in BALB / c mice) In order to evaluate the effect of orally administering the immunotolerant-inducing agent of the present invention thus prepared, antigen-specific serum and fecal extracts were used. The antibody concentration in was measured. As an example of evaluation, BALB /
A system utilizing the model antigen OVA for c mice was used. A BALB / c mouse is orally administered once with the tolerogenic agent of the present invention from the 6th week to the 7th week. A phosphate buffered saline in which the active ingredient compound of the immune tolerance inducer of the present invention is dissolved using an oral administration needle and a syringe for administration.
(PBS) 200 μl was injected into one stomach. In this example, the test was performed with eight animals per group. One week later, this time, the antigen alone is mixed with Freund's complete adjuvant to form an emulsion, and immunized by subcutaneous administration. One week later, serum and stool extracts were collected, and the concentration of antigen-specific antibodies contained therein was evaluated. The antibody concentration was measured by enzyme-linked immunosorbent assay (ELISA). The determination of antibody concentration by ELISA is well known in the art, and is described in detail, for example, in "Enzyme Immunoassay, Second Edition", Eiji Ishikawa et al., Japan Medical College, etc. That is, an antigen is adsorbed on a measurement plate, and an antigen-specific antibody in serum or stool extract that binds to the adsorbed antigen is converted to an enzyme-labeled anti-mouse IgG1 antibody, anti-mouse IgG2a antibody, or anti-mouse IgG.
The measurement was performed using an antibody or an anti-mouse IgA antibody. It is also common practice to use a biotin-labeled antibody instead of an enzyme label, and to further sensitize it by reacting with an avidin-binding enzyme. This ELISA
The antibody concentration in the original sample can be calculated back from the absorbance obtained by the above.

FIG. 2 shows an example of the result. FIG. 2 shows the percentage of antigen-specific suppression of the antibody concentration in a group of mice to which ovalbumin (OVA) was used as an antigen and a single oral administration of the inducing agent of the present invention. Here, the antigen-specific antibody concentration for each subclass in the phosphate buffered saline (PBS) administration group is represented as 100%. In addition, serum was calculated based on the measurement value at a 1,000-fold dilution, and fecal extract was calculated based on the measurement value at a 10-fold dilution. The standard error of each data is represented by an error bar. These results indicate that only 25 μg of the immunological tolerance inducer of the present invention was orally ingested only once, compared with the control group administered with phosphate buffered saline (PBS), and the antigen-specific immune response was dramatically increased. It is evident that this is suppressed.

[0035]

Comparative Example 1 In order to show that the suppression of the antibody concentration shown in Example 2 was truly antigen-specific, the total antibody concentration for each subclass was examined. The method uses the administration method and ELISA shown in Example 2 as they are, but instead of adsorbing the antigen to the measurement plate, adsorbing a commercially available anti-mouse immunoglobulin antibody. Anti-mouse immunoglobulin antibodies bind to all antibodies, regardless of antibody subclass, and are subsequently labeled with an enzyme-labeled anti-mouse IgG1, or anti-mouse IgG2a or anti-mouse IgG antibody.
By detecting with an antibody or an anti-mouse IgA antibody, the total antibody concentration for each subclass can be measured. Of course, as already described, it is common practice to use a biotin-labeled antibody instead of an enzyme label, and to further sensitize by reacting with an avidin-binding enzyme. From the absorbance obtained by this ELISA, the antibody concentration in the original sample can be calculated back.

FIG. 3 shows the results. In FIG. 3, the total antibody concentration of the group of mice to which the immune tolerance inducer of the present invention was orally administered once is shown by percentage for each subclass. The antigen-specific antibody concentration for each subclass in the phosphate buffered saline (PBS) administration group is 100%. Serum was calculated based on the value measured at 1,000-fold dilution, and fecal extract was calculated based on the value measured at 10-fold dilution. The standard error of each data is indicated by an error bar.
It can be seen that even when the immunological tolerance inducer of the present invention is administered, it has no inhibitory activity on the total antibody concentration, that is, non-specific antibody production against other than the administered antigen.

[0037]

Comparative Example 2 In the experimental system shown in Example 2, the model antigen O
It is already generally known that a single oral dose of VA, eg, 25 mg / animal, induces oral tolerance. Oral tolerance also occurs when 25 μg / animal of the model antigen ovalbumin (OVA) chemically bound to the recombinant cholera toxin B subunit is orally administered. Thus, the effects of these conventional techniques were compared with the effects of the immune tolerance inducer of the present invention. The recombinant cholera toxin B subunit binding model antigen was prepared according to the method disclosed in WO95 / 10301. The method was the administration method described in Example 2, ELISA.
Use as is. From the absorbance obtained by this ELISA, the antibody concentration in the original sample can be calculated back.

FIG. 4 shows the results. In FIG. 4, a group of mice receiving a single oral administration of 25 mg of the untreated model antigen OVA alone, 25 μm
g Antigen-specific antibody concentrations of a group of mice to which the immunological tolerance inducer of the present invention was orally administered once and a group of mice to which recombinant cholera toxin B subunit-binding antigen was once administered orally at 25 μg are shown by percentage for each subclass. . The antigen-specific antibody concentration for each subclass in the phosphate buffered saline (PBS) administration group is 100%. Serum 1,000
The measured value at the double dilution and the fecal extract were calculated based on the measured value at the 10-fold dilution. The standard error of each data is indicated by an error bar. It was revealed that a single oral ingestion of only 25 μg of the immunological tolerance inducer of the present invention caused stronger antigen-specific immunosuppression than the oral administration of 25 mg of untreated antigen alone. Furthermore, it was revealed that even when compared with the effect of the recombinant cholera toxin B subunit-binding antigen, antigen-specific immunosuppression to a similar extent or more occurred. In order to remove the undesired physiologically active sites inherent in the subunit of the toxin, which has been a problem in utilizing the CT-B molecule itself, and to maintain the target antigen-specific tolerance tolerance, It indicates that the use is effective.

[0039]

Example 3 (Evaluation in C57BL / 6 mice) To show that the effect of the tolerogenic agent of the present invention is universal, human gamma globulin (HGG) was used as an antigen for C57BL / 6 mice. In this case, the effect of inducing antigen-specific immune tolerance was examined. The evaluation method was not a measurement of the antibody concentration in the antigen-specific serum and fecal extract, but a method using the delayed-type hypersensitivity reaction (DTH). For C57BL / 6 mice, LTB was used as an example of the immunological tolerance inducing agent of the present invention from the age of 6 weeks to the age of 7 weeks.
(I) 25 μg of peptide-HGG was orally administered once. A phosphate buffered saline in which the active ingredient compound of the immune tolerance inducer of the present invention is dissolved using an oral administration needle and a syringe for administration.
(PBS) 200 μl was injected into one stomach. In this example, the test was performed with eight animals per group. One week later, this time, only the antigen was mixed with Freund's complete adjuvant to form an emulsion, and 0.5 mg of the antigen was subcutaneously administered. One week later, the antigen heat-denatured at 70 ° C for 30 minutes was dissolved in phosphate buffered saline (PBS), and the antigen 0.5m
g was injected into one hind footpad of the mouse. The opposite hind footpad was injected with PBS only. Twenty-four hours after the injection, the thickness of the paw was measured using the side injected with PBS alone as a control, and the degree of paw swelling was quantified. The measurement of an immune reaction using the DTH reaction is well known in the art, and is described in, for example, "Immunological Experimental Procedure B", edited by the Japanese Society of Immunology, Japan, The Immunology Society of Japan, pp. 813-818 and 1348-1352. page,
"Immunological Experiment Procedure X", edited by the Japan Society of Immunology, Japan, The Society of Immunology, Japan, pages 3349-3353, RGTitus & JMChiller, J.
Immunol. Meth. 45: 65-78 (1981). FIG. 5 shows the results. In FIG. 5, the vertical axis represents the paw swelling after 24 hours, minus the thickness of the control. PBS oral administration group, same 25μg compared to 25μg untreated antigen alone
g, it was shown that the use of the tolerogenic agent of the present invention resulted in antigen-specific immunosuppression in the DTH reaction.

[0040]

Example 4 (Preparation of Immune Tolerance Inducing Agent) Among the active ingredient compounds of the present invention obtained in Example 1, 10 g of lyophilized powder of LTB (I) peptide-OVA, 62 g of lactose, 20 g of corn starch, 20 g of carboxymethyl cellulose 5 g of calcium, 2 g of light anhydrous silicic acid, and 1 g of magnesium stearate were uniformly mixed and compression-molded with a tableting machine to obtain a tablet of 200 mg per tablet.
One tablet contains 20 mg of the active ingredient compound of the immune tolerance inducer of the present invention, and 3 to 6 tablets per day for an adult are divided into several doses.

[0041]

The present invention provides an immune tolerance inducing agent useful for the treatment and prevention of autoimmune diseases such as allergy and rheumatoid arthritis, and diseases caused by unnecessary immune reactions such as rejection during transplantation. In a tolerogenic agent utilizing mucosa, the purpose is not only to increase the probability of establishment of tolerability by promoting the efficiency of antigen uptake in the mucosa as in the past, but also to T cells that work to establish tolerance. By using only the essential functional part that promotes the interaction with B cells, the possibility of toxicity and the like can be reduced, and a more essential universal and safe immune tolerance inducer can be provided.

[0042]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr 1 5 10 15 Phe Lys Asn Gly Cys 20

SEQ ID NO: 2 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Tyr Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr 1 5 10 15 Phe Lys Ser Gly Cys 20

SEQ ID NO: 3 Sequence length: 21 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ser Tyr Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr 1 5 10 15 Ile Lys Ser Gly Cys 20

[Brief description of the drawings]

FIG. 1 shows an example of a peptide consisting of 5 to 25 amino acids, including a B cell epitope and / or a T cell epitope derived from the B subunit that constitutes AB type toxin.

FIG. 2 shows suppression of antigen-specific antibody production when the immune tolerance inducer of the present invention of Example 2 is orally administered.

FIG. 3 shows the total antibody concentration of each group of mice to which the immune tolerance inducer of the present invention of Comparative Example 1 was orally administered once, as a percentage.

FIG. 4: 25 mg of the untreated model antigen OVA alone of Comparative Example 2
FIG. 2 shows the inhibition of antigen-specific antibody production by single oral administration, 25 μg single oral administration of the immune tolerance inducer of the present invention, and 25 μg single oral administration of recombinant cholera toxin B subunit-binding antigen.

FIG. 5 shows the antigen-specific immunosuppressive effect of the DTH reaction of Example 3.

Claims (2)

[Claims] An immunological tolerance inducer comprising a compound represented by the general formula (1) as an active ingredient. PLA (Formula (1)) In the above formula, P is a peptide consisting of 5 to 25 amino acids, including a B cell epitope and / or a T cell epitope of the B subunit constituting AB type toxin. Represents L represents a spacer. A represents the antigen to which tolerance is to be induced. 2. The immunological tolerance inducer according to claim 1, wherein the AB toxin is cholera toxin or a heat-labile enterotoxin of Escherichia coli.