JPH09509135A - Cryptic peptides for use in inducing immune tolerance - Google Patents

Abstract

  (57) [Summary] Described is a method of inducing immunological tolerance in a patient such as a human by administering a tolerizing amount of a composition comprising a cryptotic peptide derived from an antigen and a pharmaceutically acceptable carrier. Compositions containing cryptic peptides derived from protein antigens such as allergens or self-antigens can be administered to induce tolerance in naive or presensitized individuals. Preferably, this composition is administered orally.

Description

Detailed Description of the Invention                 For use in inducing immune tolerance                       Cryptic peptideBackground of the Invention   Providing antigens is a classical way to induce immune unresponsiveness or oral tolerance (Asherson, G.L. et al. (1977), Cell Immunol., 33: 145; Asherson, G.L. et al. 1979), Immunology, 36: 449; Challacombe, S.J. and Tomasi, T.J. (1980), J. Exp. .Med., 152: 1459; Bruce, M.G. and Ferguson, A. (1986), Immunology, 57: 627; Mow. at, A. (1982), Immunology, 45: 105; and Strobel, S. et al. (1983), Immunology, 49:45. 1). An important physiological response to dietary antigens (Mowat, A.M. (1987), Immu nology Today, 8:93), taking oral tolerance into account in autoimmune diseases. Ectopic immune response as found (Thompson, H.S.G. and Staines, A. (1990), Imm unol.Today, 11: 396) and allergies have been suggested. .   The most widely studied model system for autoimmune disease is experimental allergic encephalomyelitis. (EAE) model system. Tolerizing amount of myelin basic protein (MB P) fed rats can be protected from encephalomyelitis challenge with MBP (Miller, A. et al. (1991), J. Immunol., 147: 2155 and Miller, A. et al. (1992), Proc. Natl. Acad. Sci. USA, 89: 421. ). However, conflicting views have emerged regarding the mechanisms involved in the induction of oral tolerance. Have been. For example, Whitacre et al. (1991), J. Immunol., 147: 2155 Inability to transfer suppression using T cells from domesticated animals , But cloned anergy CD4⁺MBP-reactive T cell effect It has been shown that it may be an important mechanism for down-regulating tractor function. Or Mi ller, A. et al. (1991) J. Exp. Med., 174: 791, CD8 from suppression-tolerized animals.⁺T It has been shown that the cells can be transferred to a natural recipient. these Suppressor (T) cells of MBP are specific for MBP due to the release of soluble cytokines. CD⁺Reportedly able to block the in vitro response of T cell lines, irrelevant Can also cause by-stander suppression of T cells in Deaf (Miller, A. (1991) supra). Immunomodulatory cytokines released by T cells Was later defined as TGF-β1 (Miller, A. et al. (1992) Proc. Natl. Acad. Sc. i.USA 89: 421).   Bacterial and viral pathogens, autoantibodies, allergens and other experimental antigens such as Chicken egg lysozyme (HEL), ovalbumin (OVA) and lambda repress Peptides derived from various protein antigens including sir (c1) Tested for their ability to stimulate vesicles Was. It has been reported that proteins with a wide size spectrum act as T cell epitopes. It has been tell. For example, peptides derived from hepatitis B surface antigen (HBsAg Minoic acid residues 19-33) were recently identified in human patients immunized with recombinant hepatitis B vaccine. Have been shown to stimulate T cell responses in the majority of individuals (Schad, V.C. et al. (1991)  Seminars in Immunol., 3: 217-224). 65kD protein of major mycobacterial antigen The quality was also epitope mapped (Lamb, J.R. et al. (1987) EMBO J., 6 (5): 1245-1249. ). From the 112-132 and 437-459 amino acid residues of this 65 kD protein T-cell epitopes have been identified in the following peptides. MBP is also human ( Ota, K. et al. (1990) Nature, 346: 183-187) and rodents (Zamvil et al. (1986) Nature, 324). : 258-260).   The T cell epitopes present in allergenic proteins have been most recently described (O'He hir, R. et al. (1991) Ann. Rev. Immunol., 9: 67-95). Indoor dust mite allergenDerpFrom I Several derived peptides were shown to be T cell reactive (Thomas, W. R. et al., European Conference on Allergy and Clinical Immunology X IV (September 1989, Berlin) From Epitopes of Atopic Allergens Proceedings of Workshop, pages 77-82; O'Hehir, R.E. (1991) Annual Review Immunology 9: 67-95; Stewart, G.A. Etc. Epi from European Academic Conference XIV on Allergy and Clinical Immunology (September 1989, Berlin) topes of Atopic Allergens 41-47 in Proceedings of Workshop; and Yessel, H. et al. Conference 22-26 (September 1990, Trinity College, Oxford, UK) T Cell Activat ion in Health and Disease). T derived from the short ragweed allergen AmbaI A cell stimulating peptide (amino acid residues 54-65) was also reported (Rothbard, J.B. Et al. (1988) Cell, 52: 515-523). T cells derived from individuals with ryegrass allergy Using a panel of clones, Perez et al. Show that the T cell epitope is the protein allergen L It was shown to be contained within amino acid residues 191-210 of olpI (Perez, M. et al. (1 990) J. Biol. Chem. 265 (27): 16210-16215).Summary of the Invention   This invention improves the tolerance of a protein antigen in a patient, for example, a human patient, to its immunological tolerance. At least one cryptic peptide derived from a source and a pharmaceutically acceptable key Related to a method of inducing by administering a tolerizing amount of a composition containing a carrier You. Cryptic peptides derived from protein antigens such as allergens or self-antigens Administration of a composition containing tid induces tolerance in naive or presensitized individuals. You can guide. Preferably, this composition is administered orally to administer in an individual. Treat sensitivity to allergens or self-antigens.Brief description of the drawings   Figure 1DerpIsolated from mice immunized with I,DerpSelect from I For the response to specific peptides by tritiated thymidine incorporation. 3 is a graphical representation of T cell response.   2a and 2b showDerpMice immunized with selected peptides derived from I Isolated,DerpTo I protein (panel a) or the appropriate peptide (panel b) 3 is a graphical representation of T cell responses analyzed for response.   FIG. 3 shows T cell epitopes recognized by mice.DerpPosition in the I protein sequence A schematic representation of the location where immunodominant epitopes are represented by parallel-lined squares. , Cryptic epitopes are represented by dotted squares, indicating that there is no epitope. It is represented by a black square.   FIG. 4 shows buffer (panel a), peptide GEXp57-130 (panel b), Peptide GEXp101-154 (panel c), or recombinant protein GEXDerpI (1-222) (panel d) was fed and thenDerpFrom mice immunized with I Isolated and in vitroDerpRegarding the response to I, IL-3 / GM-CSF ( Response of T cells analyzed by panels a-d) or IL-2 production (panels e-h) Is a graph display.   FIG. 5 shows the recombinant protein GEXDerpII (1-129), peptide GEXp101 -154, or the peptide GEXp188-222, followed byDerpFree with I Quarantine Isolated from theDerpI (panels a and d), peptide p110-131 ( Panel b and e) or the response to peptide p78-100 (panels C and f). Regarding the answer, IL-3 / CSF (panels a to c) or IL-2 production (panels d to c) FIG. 6 is a graphical representation of T cell responses analyzed according to f).   Figure 6 shows either buffer or recombinant fusion peptide (GEXp131-187) Feed and thenDerpIsolated from mice immunized with I,DerpResponse to II Is a graphical representation of the response of T cells analyzed by IL-2 production.Detailed description of the invention   This invention induced immunological tolerance to a protein antigen in a patient from that antigen To induce by administering at least one cryptographic peptide Method. Protein antigens are specific class II major histocompatibility complex Upon exposure to its native protein antigen when presented with the (MHC) molecule Known to contain certain determinants or epitopes that activate T cells in patients ing. The T cell response may be limited by the presence of one or two determinants on the antigen Nonetheless, the T cell response appears to preferentially utilize some of the selected determinants. Thus, a hierarchy of T cell determinant utilization exists for multideterminant protein antigens. Obedience A specific protein antigen Based on an in vitro T cell proliferation assay Can be divided into categories, and in the proliferation assay, protein antigen Lyme T cells with selected concentrations of peptides derived from their protein antigens In culture, the amount of proliferation by T cells in response to the peptide can be determined, eg It is measured by the incorporation of midine.   This assay showed that if the peptide was antigen primed in the patients tested, If one consistently induces one of the best T cell proliferative responses in The peptides are categorized into categories containing immunodominant T cell epitopes. Immunodominance d Compared to pitopes, peptides containing minor T cell epitopes are B) regenerates more variable and low levels of T cell proliferation. Medium only Peptides that regenerate T cell proliferation less than twice background levels are T cells Categories without epitopes or with Cryptic T cell epitopes Classified into. Cryptic epitopes process natural protein antigens And in protein antigens not normally presented to the immune system due to presentation to the appropriate MHC molecule Is the determinant of. However, peptides containing cryptic epitopes Patients that can tolerize T cells and prime the patient with the peptide T cells obtained from E. coli in response to the peptide or protein antigen from which the peptide was derived. Proliferate in vitro. From protein antigen A peptide containing at least one cryptic epitope derived is Is called a cryptic peptide. In peptides classified by the above assay In order to confirm the presence of a cryptic epitope of Cells were cultured in vitro in the presence of each peptide separately and the peptide-reactive T cells were incubated. Establish a cell line. If you can establish a T cell line using a given peptide, And T cells are charen with the peptide or the protein antigen from which the peptide is derived If the peptide is capable of growing in It is considered to contain an epitope.   The presence of cryptic epitopes in protein antigens is exempt from certain epitopes. Due to lack of exposure to the epidemiological system, epidemics against suitable class II MHC molecules It may result from the normal processing of protein antigens that may not exhibit a taupe. Or anti The final product of the original processing hides the cryptic epitopes, Access to T cell receptors on T cells specific for offspring or epitopes thereof It can be a large piece that interferes with scanning. In addition, other epitopes on the same protein antigen are: Can compete for binding to the same restriction element as the cryptic epitope, or Can have a higher affinity and availability for different restriction elements, Therefore, it blocks the interaction of cryptic epitopes with MHC.   Is the cryptic peptide of this invention a protein antigen? From at least one cryptic epitope (ie, this peptic Contains at least about 7 amino acid residues). Such peptides are as desired It may contain amino acid residues of many protein antigens, preferably at least about 7 , More preferably at least about 15, even more preferably at least about 20 and Most preferably it can comprise at least about 25 amino acid residues. 20-40 A peptide length of about amino acid residues is preferable. Because the increase in peptide length , Peptide synthesis is difficult, and peptide and protein antigens (such as Undesired features due to the retention of conformational similarities between the incoming allergens) Because it can result in retention of sex (eg, immunoglobulin binding activity or enzymatic activity). You. If desired, the amino acid sequence of one or more peptides can be generated and Binding to increase susceptibility to processing by antigen-presenting cells Can be done. Such linker may be any non-epitope amino acid sequence or other suitable It may be a binder or binder. For example, linking two types of cryptic peptides Can or can be derived from protein allergens with cryptic peptides It can be linked to peptides containing immunodominant or minor epitopes.   A cryptic peptide is a nucleotide sequence encoding such a peptide. Recombinant D in host cells transformed with a nucleic acid vector directing expression NA technique Allergens, etc. by surgery, by chemical synthesis, or in some limited circumstances Can be produced by chemical cleavage of the protein antigen of Raw by recombinant technology Nucleic acid which, if made, directs the expression of the nucleotide sequence encoding the peptide. Culturing a host cell transformed with a vector in a medium suitable for that cell I do. These peptides are secreted and taken from the mixture of cells and cell culture medium. Rukoto can. Alternatively, the peptide is retained in the cytoplasm and the cells are harvested Then, the peptide can be isolated and purified by dissolving. Peptide, peptide Or can be isolated using techniques known in the art for purifying proteins, The techniques include ion exchange chromatography, gel filtration chromatography, and Ultrafiltration, electrophoresis and peptide or protein antigens from which this peptide was derived or Includes immunoaffinity purification using antibodies specific for that portion . The cryptic peptides described herein are produced by recombinant DNA technology In some cases, it does not contain cellular substances or culture medium, Chemical allergen or other protein antigen, if obtained by chemical cleavage Isolate to be substantially free of precursors and other chemicals.   When the T cell epitope of the protein antigen is unknown or unclear, the crypte of the present invention is used. The protein structure of the antigen and obtain the desired sequence in order to obtain the desired peptide. Length of Can be divided into at least two peptide fragments. For example, protein antigen Protein sequences of at least two non-overlapping fragments of desired length or desired Can be divided into overlapping pieces of length. As an example for explanation, 229 Has the amino acid sequence of the residue (shown in SEQ ID NO: 1)Dermatophagoides pterony ssinus ofDerpThe known amino acid sequence of the I major allergen is approximately 22-35 amino acids. Peptide fragments that have a residue length and each fragment overlaps with other fragments by about 10 amino acids. Can be divided into pieces. Include T cell epitopes in these peptide fragments To maximize the likelihood of the T cell epitopes predicted using the algorithm Overlapping regions and the length of each fragment can be designed to maintain their presence ( Rothbard, J. and Taylor, W.R. (1988) EMBO J. 7: 93-100; and Berzofsky, J.A. (198 9) Philos.Trans.R.Soc.Lond.323: 535-544). Preferably human in the protein antigen The T cell epitopes are pre-defined using known HLA class II binding specific amino acid residues. I can think. Recombinant DNA technology or chemical synthesis of the resulting peptide fragments Can be generated by   Peptide fragments derived from protein antigens were tested for T cell stimulatory activity (ie, proliferation). , Lymphokine secretion and / or induction of T cell anergy / tolerance) Decided. For example, patients such as humans who have a human T cell stimulating activity and are sensitive to protein antigens. (Ie immunity to the protein antigen T cells obtained from patients with a response) were treated with peptide fragments derived from this protein antigen. Measuring the presence of proliferation by T cells in response to the protein in culture with strips Can be tested by The presence of proliferation by T cells can be determined, for example, by tritium. It can be measured by the incorporation of thymidine chloride.   Immunodominant T cell epitope, minor T cell epitope and cryptic enzyme The pitope can be identified as described in Example 3. In selected peptides Sensitive to its protein antigen to confirm the presence of a cryptic epitope in Cells from each individual and cultured separately with each cryptogenic peptide Establish a Tide reactive T cell line. Peptides and proteins derived from the peptides The presence of an induction of T cell proliferation or T cell tolerance in response to a primordial phenotype is a minor Ensure the presence of at least one cryptic epitope.   The cryptic peptide of this invention is a protein anti-protein such as an allergen or an autoantigen. It can be derived from Hara. If you derive from an allergen, Cryptic Pep Tide from any known protein allergen, such as allergens of the following genera You can induce:DermatophagoidesGenus,Felis Genus,AmbrosiaGenus,LoliumGenus ,Cryptomeria Genus,AlternariaGenus,Alder Genus,BetulaGenus,Quercus Genus,OleaGenus,Artemisia  Genus,PlantagoGenus,ParietariaGenus,CanineGenus,Blattella Genus,ApisGenus,Periplaneta  Genus, andSorghum Genus.Dermatophagoidespteronyssinus Major species Allergen,DerpThe cryptic peptide recognized by the mouse in I Measured atDerp120-143 amino acid residues of I (SEq ID NO: 1),Derp 144-169 amino acid residues of I (SEQ ID NO: 1) andDerpI (SEQ ID NO: 1) Amino acid residues 131-187 of   Cryptic peptides can also be used when tolerance to self-antigens is desired. It can also be derived from protein antigens other than allergens. Cryptic Peptic The autoantigens from which the liver can be derived include insulin for the treatment of diabetes and deca-glutamate. Ruboxylase (64K), PM-1 and carboxypeptidase, multiple sclerosis Basic protein for the treatment of dementia, rh factor for the treatment of fetal erythroblastosis, myasthenia gravis Acetylcholine Receptor for the treatment of Graves 'disease, Thyroid Receptor for the treatment of Graves' disease , Basement membrane protein for the treatment of Goodpasture's syndrome, and thyroid for the treatment of thyroiditis Contains proteins.   According to one aspect of the present invention, a cryptic peptide derived from a protein antigen is To induce immunological tolerance to the protein antigen in that patient. The term patient refers to a patient who is able to mount an immune response to a protein antigen. An object such as a mammal is included. Examples of patients include humans, rats, mice, dogs, This includes cats, horses, cows and transgenic species thereof. Exemption Epidemiological tolerance refers to the block of development, growth or differentiation of specific lymphocytes within a patient. Refers to the condition of a patient caused by the administration of a tolerated dose of a clear cryptic peptide. Generous Is the antigen under conditions in which lymphocytes are deleted or rendered unresponsive without being activated. Resulting from the interaction with the antigen receptors on lymphocytes. Tolerance is also a specific T Or by other regulatory mechanisms that block the action of B lymphocytes or lymphocyte activation There is also a possibility. One mechanism for blocking the immune response is called suppressor T cells. One class of lymphocytes that are exposed (the main function of which is the specific T and B lymphocytes Suppressing activation) is a stimulus. In this situation, blocking is the antigen It is mediated by regulatory cells that are induced by the antigen but not by themselves. other The proposed mechanism of tolerance is the unique or isotypic determinants of lymphocytes or The original specific antibody is the response by the immune system to the targeted antigen. This response The answer is that this response consists of a network of complementary idiotypes and antigen-specific cells. It produces an anti-idiotype that blocks irritation. Finally, the activity of B and T lymphocytes The products of sexualization, the antibody and the cytokine itself, are the principle of lymphocytes, respectively. In addition to functioning as an effector molecule, it regulates specific immunity and causes tolerance Rukoto can.   Crypti derived from protein antigens to induce immune tolerance in patients A tolerizing amount of luc peptide is administered to the patient. Tolerizing dose in patients such as humans Krip Clips required to induce tolerance to the antigen from which the tick peptide was derived Defined as the dose of tick peptide. Immune tolerance in patients is standard Unresponsiveness to protein antigens or reduced symptom as measured by bed procedure (See, for example, Varney et al. (1990) British Medical Journal 302: 265-269. No). If tolerance to an allergen is sought, such non-responsiveness may include alleles. Includes a reduction in gen-induced allergic symptoms. In this case, allergen The symptom reduction for Any reduction in allergic response to allergens after treatment regimen with tide included. This reduction in symptoms can be subjectively measured in humans (eg. If, for example, the patient feels more comfortable when exposed to the allergen), or It can be measured clinically such as by using a standard skin test.   Cryptic peptides derived from protein antigens are typically produced in patients. It is administered in the form of a composition containing a pharmaceutically acceptable carrier or diluent. To the patient Administration of the compositions of the invention to induce immune tolerance to protein antigens Effective dose to tolerate a patient to its protein antigens using known procedures And can be done during the period. An effective amount of this composition is for the patient's antigen. Factors such as degree of susceptibility, age, sex, and weight, and cryptic peptides Tolerance in patients with It depends on the ability to induce. Dosage regimen to give optimal therapeutic response Can be adjusted to For example, daily or divided doses It can be reduced depending on the urgency of the medical treatment situation.   Cryptic peptides can be prepared by conventional methods, such as injection (subcutaneous, intravenous, etc.), oral Administration to patients by administration, inhalation, intranasal administration, transdermal application, or rectal administration Can be done. The preferred route of administration for inducing immune tolerance in a patient is oral administration. And intranasal administration. O'Hehir, R.E. et al. (1993) Eur.J.Clin.Invest.23 (12): 763-7 See 72. Depending on the route of administration, the active compound (ie, the cryptic peptide) Coated with an enzyme that can inactivate this compound, a substance that protects it from production and other natural conditions You can do it.   In order to administer a cryptic peptide by enteral administration, Must be coated or co-administered with a substance that blocks its inactivation. Can be For example, the cryptic peptide may be administered to a patient in a suitable diluent. , Co-administered with enzyme inhibitors, or in a suitable carrier such as liposomes You can Pharmaceutically acceptable diluents include salt solutions and aqueous buffer solutions. You. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophore. Includes sulfate (DEP) and trasilol. Liposomes in oil-in-water Water CGF emulsion and conventional liposome Included (Strejan et al. (1984) J. Neuroimmunol. 7:27). For the purpose of inducing tolerance For this purpose, the composition preferably comprises a non-immunogenic form, for example an adjuvant. Do not administer   The active compound may also be administered parenterally. Glycerol, liquid polyethylene Dispersions can also be prepared with glycols, and mixtures thereof, and oils. Under ordinary conditions of storage and use, these preparations inhibit the growth of microorganisms. Preservatives for can be included.   Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and Included are sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Everything In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. Must. It must be stable under the conditions of manufacture and storage. And must be protected against contamination by microorganisms such as bacteria and mold. Cat The rear may be a solvent or dispersion medium, for example water, ethanol, polyol. (Eg glycerol, propylene glycol and liquid polyethylene glycol Etc.), suitable mixtures thereof and vegetable oils. Suitable fluidity, for example The particle size required for dispersion is maintained by using a coating such as lecithin And by the use of surfactants. Microorganism The inhibition of the action of And antifungal agents such as paraben, chlorobutanol, phenol, ascorbine It can be achieved by acid, thimerosal and the like. Often in the composition, Isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, chloride It is preferable to include sodium. Agents that delay absorption in the composition, such as aluminum Injectable composition containing aluminum monostearate and gelatin Prolonged absorption of can be achieved.   Sterile injectable solutions of the active compound in the required amount in the appropriate solvent in one of the ingredients Alternatively, it can be prepared by mixing (if necessary) with the combination and then sterilizing filtration. Rukoto can. In general, the dispersion comprises the active compound, the basic dispersion medium and any of the above. Prepared by mixing in a sterile vehicle containing the other required ingredients. Aseptic injection In the case of sterile powders for the preparation of sprayable solutions, the preferred method of preparation is the active ingredient (ie Peptide of the invention) to which any desired ingredient from the above sterile filtered solution was added. Vacuum drying and freeze drying to produce a powder.   The cryptic peptides described herein, if properly protected as described above, Peptides, eg with an inert diluent or an assimilable edible carrier It can be administered orally. This peptide and other ingredients may be hard or soft Encapsulate shell gelatin capsules, compress into tablets, or mix directly into your personal diet. You can do it. For oral administration, the active compound is mixed with excipients and ingestible tablets, buccal tablets, troches Used in the form of chi, capsules, elixirs, suspensions, syrups, wafers, etc. You can Such compositions and preparations should contain at least 1% by weight of active compound. Should be included. The percentages of this composition and preparation may, of course, vary. The unit of about 5 to 80% by weight is convenient. Of the active compound in such compositions The amount should be such that an optimum dosage will be obtained. Preferred compositions or preparations according to the invention Are prepared so that an oral dosage unit contains about 10-200 mg of active compound.   As used herein, "pharmaceutically acceptable carrier" includes any and all soluble ingredients. Media, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc. Is included. Media and agents for such pharmaceutically active substances are well known in the art. Is knowledge. Except where the conventional medium or drug is incompatible with the active compound. Is contemplated for use in the composition. Mixing supplementary active compounds with these compositions You can also.   It is advantageous to formulate the composition in dosage unit form for ease of administration and uniform dosage. Can be. Dosage unit, as used herein, refers to the mammalian patient being treated. A physically separate unit suitable as a unit dosage to achieve the desired effect. A predetermined amount of the active compound calculated to produce Including with Ah No. The specification of the dosage unit form of this invention is defined by (a) the unique properties and reach of the active compound. The effects to be achieved and (b) the essential technique of synthesizing such active compounds for the treatment of patients. Dictated by the limits and directly dependent on them.   The composition of this invention comprises one or more cryptographic peptides to be administered to a patient such as a human. Can include a peptide, or a cryptic peptide and an immunodominant or minor -A peptide containing an epitope can be included (the patient is The patient has been unsensitized or presensitized to the protein antigen that has arrived, and the dosage and duration are And sufficient to induce tolerance to this antigen). For example, a kind Simultaneously administer an effective amount of the same or different compositions to induce tolerance in a patient. Or sequentially. Composition comprising at least two peptides (Eg, a physical mixture of at least two peptides) is also a method of tolerization. Can be used. For example, cryptic peptides and immunodominant epitopes Can be co-administered.   The cryptic peptide of this invention (ie, the complete protein antigen was given to the patient Peptide containing no epitope recognized in immunization). The fact that tolerance can be induced by and is important. For immunotherapy Peptides that are effective in, therefore, are simply T-cell clones or the po- sition of sensitized individuals. It is not limited to the one recognized by the reclonal response. Cryptic Peptide administration to individuals sensitized with peptides containing immunodominant epitopes It avoids potential inherent limitations in doing things. Cryptic The use of peptides is already_h1 and T_hT going along 2 or an equivalent route The possibility of modifying the immune response without the need to redirect the development of cell clones Also provide.   Increased solubility, therapeutic or protective effect or stability (eg in vitro shelf life) And to increase the resistance to proteolysis in vivo). It is also possible to modify the structure of cryptic peptides useful in the method of the invention It is. Amino acid substitutions, deletions or deletions to modify the ability of the peptide to induce tolerance. Is a modification in which the amino acid sequence has been changed by addition, or a component has been added for the same purpose. A modified peptide can be produced.   For example, a peptide that induces T cell anergy or tolerance induces MHC protein Can be modified to maintain the ability to bind to. In this example, Binding residues important for vesicle receptors (ie Amino acid residues) by known techniques (eg, substitution of each residue with, for example, alanine, and It can be determined by measuring the presence or absence of T cell reactivity). To be mandatory The indicated residues are replaced by their essential amino acids and other, preferably similar amino acid residues (where Amino acids whose presence increases or decreases T cell reactivity but is not eliminated or affects It can be modified by substitution with (residue) (conservative substitution). Further, T Amino acid residues that are not essential for cell interaction are replaced by other amino acids Increase, decrease or do not affect the reactivity, but increase binding to relevant MHC It can be modified by substituting non-excluded amino acids). Nonessential net Suitable amino acid substitutions for non-acids include, but are not limited to, alanine, glutamic acid. Or includes substitution with a methyl amino acid.   Another example of peptide modification minimizes dimer formation by disulfide bonds A cysteine residue for preferably alanine or glutamic acid, or serine or Substitution with threonine.   To increase stability and / or reactivity, the peptides can be modified to Taking one or more polymorphisms in the amino acid sequence of a protein antigen resulting from a genetic mutation It can be crowded. Furthermore, D-amino acids, unnatural amino acids or non-amino acid analogs Can be substituted or added to produce modified peptides within the scope of this invention. come. In addition, the peptide was synthesized by A. Sehon and a collaborator (Wie et al. Modified to produce PEG-conjugated peptides using the glycol glycol (PEG) method Rukoto can. Peptide modification is performed by reduction / alkylation (Tarr: Methods of Prot ein Microcharacterization, edited by J.E.Silver, New Jersey, Clifton, Humana Pre ss, pages 155-194 (1986)), acylation (Tarr, supra), esterification (Tarr, supra). ), Chemical coupling to a suitable carrier (edited by Mishell and Shiigi, Sel ected Methods in Cellular Immunology, San Francisco, CA, WH Free man (1980); U.S. Pat. No. 4,939,239) or mild formalin treatment (M arsh, (1971) International Archives of Allergy and Applied Immunology 41: 199-215) can also be included.   To facilitate purification of peptides and potentially increase their solubility, the Porter groups can be added to the peptide backbone. For example, polyhistidine Metal ion affinity chromatography with immobilized peptide in addition to peptide (Hochuli, E. et al. (1988) Bio / Technology, 6: 1321-1235) I can do it. In addition, if desired, a specific endoprotease cleavage site may be reported. Of peptides containing no irrelevant sequence by introducing between peptide group and peptide sequence Can be facilitated. To successfully tolerize patients to protein antigens In addition, do not add a functional group to the peptide or introduce a hydrophobic region into the peptide. It may be necessary to increase the solubility of the peptide.   Potentially aids in proper antigen processing of T cell epitopes within peptides Standard protease sensitive sites in order to recombinantly or synthetically Can be crafted by For example, a pair of charged amino acids such as KK or RR During the recombinant construction of the peptide Can be introduced in Chid. The resulting peptide is cathepsin and / Or other sensitizing trypsin-like enzyme cleavage to one or more T cell epitopes A portion of the containing peptide can be generated. Furthermore, such charged amino acid residues are , Can result in increased solubility of the peptide.   Using the site-directed mutagenesis method of the DNA encoding the peptide, The structure can be modified. Such a method is described in PCR (Ho et al. (1989) Gene 77:51. -59) or total synthesis of mutant genes (Hostomsky, Z. et al. (1989) Biochem. Biophys. Res. . Comm. 161: 1056-1063). In order to increase bacterial expression, Method in combination with other procedures to eukaryotes in DNA constructs encoding peptides. Preferential codons in E. coli, yeast, mammalian or other eukaryotic cells It can be changed to the one used.   The invention is further illustrated by the following non-limiting examples. Cited in this application All cited references and published patent applications are incorporated herein by reference. Incorporate. The methods described in the Materials and Methods section below are used in the examples below. Was.                             Materials and methodsAnimals and antigens   Female B10 and BALB congenic mice and the same Inbred C57BL / 6J (6-8 weeks old) at Murdo, Western Australia I bought it from Animal Resource Center, ch.   Indoor dust mite allergenDerpFrom the tick-depleted mite medium (SMM), previously described Technology (Hoyne, G.F. et al. (1993) supra; Lombardo et al. J. Immunol. 144: 1353-1360 and Chapman (1989) Advances in Biosciences 74: 281-295). E. Crystalline ovalbumin (OVA) Grade V, St. Louis, Missouri Purchased from Sigma Chemical Company. PublishedDerpI sequence (Chua et al. (1988) J.Ex. p.Med. 167: 175-182), using overlapping synthetic peptides as standard t-BOC chemistry. The peptide was synthesized by reverse phase high performance liquid chromatography (HPLC). After purification, the sequence of each peptide was checked to confirm its identity. In this study The peptide used isDerpI sequence (Chua et al. (1988) supra) Contains mino acid residues: 1-20, 13-39, 21-49, 40-60, 50- 71, 61-84, 78-100, 85-109, 101-119, 110-1 31, 120-143, 132-157, 144-169, 158-180, 1 70-191, 181-204, 197-222.Preparation of recombinant protein   allDerpI orDerpII protein-encoding insert (Melb, Australia) ourne, Commonwealth Serum Laboratories, from exhausted mite media) or recombinant constructs (related cDNA) Formed from restriction endonuclease fragments of A; Chua et al. (1990) Int. Arch. Al lergy Appl.Immunol. 91: 118-123) was ligated to the pGEX vector. Transformed into enterobacteria (Smith, D.B. and Johnson, K.S. (1988) Gene, 67:31. ). Procedures for molecular cloning of these products are described elsewhere (Chua. , K.Y. (1988) J. Exp. Med., 167: 175 and Chua, K.Y. Et al. (1990) Int. Arch. Allergy Appl.Immunol., 91: 124). pGEX-based protein or peptide constructs The transformed logarithmic phase E. coli was treated with 0.1 mM isopropyl thiogalactosider. By adding IPTG (Promega) to the culture and shaking at 37 ° C for 60 minutes. Induced expression of recombinant protein. Since a large amount of fusion peptide was needed, They were prepared from solubilized contents. Bacterial pellet containing 1 mM EDTA Resuspend in Tris-buffered saline and homogenize with 0.1 mm glass beads Transfer to an Iz bottle and homogenize for 5 minutes using a Braun MSK homogenizer. I did it. After ultracentrifugation at 10,000g for 10 minutes at 4 ° C, collect the lysate. Issued. The pellet was washed with 1 M NaCl and 1% Triton-X100 (BDH Chemical s) containing 1.75M guanidine HCL by careful pipetting Washed twice, then centrifuged. The pellet was then washed with 50 mM NaCl and And 1 mM 2 hours at 37 ° C in 8M urea containing ethylenediaminetetraacetic acid (EDTA) It was dissolved by incubating. This sample was treated with 3- (cyclohexylamido). No) -Propane sulfonic acid (CAPS) buffer solution (pH 10.7) is dialyzed to adjust the pH. Adjusted slowly to 9.6. The recombinant material is then centrifuged at 10,000 g Clarified by the standard method, and the concentration of the soluble substance was changed to the standard amount of bovine serum albumin (BSA). For SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Evaluation was performed using staining with Macy Blue. The recombinant peptides used in this study were: GEXDerpI (amino acid residues 1-222), GEXDerpII (amino acid residues 1 to 1 29), GEXp1-14 (DerpI amino acid residues 1-14), GEXp60- 111 (DerpI amino acid residues 60-111), GEXp98-140 (DerpI Amino acid residues 98-140), GEXp101-154 (DerpI amino acid residue Groups 101-154), GEXp57-130 (DerpAmino acid residues 57 to 13 of I 0), GEXp188-222 (DerpI amino acid residues 188-222).Induction of oral tolerance   The mice were lightly anesthetized under ether and the tube was intragastrically administered with 3 mg of protein or peptide. Cide was fed for 3 consecutive days. Dissolve antigen in CAPS buffer and add 0.2 ml It was administered as a product. Mice were placed at the base of the tail 7 days after the last feeding, with a volume of 0.2 m. Complete Freund's adjuvant at l Immunized subcutaneously with 100 mg of native protein emulsified in (CFA).Culture medium   Lymph node cells were supplemented with 2% fetal calf serum (FCS), 50 mM 2-ME, 2 mM   Dulbecco's modified ai supplemented with L-glutamine and 20 mg / ml gentamicin The cells were cultured in a medium (DME). FDC-P1 cells (Kelso, A. (1990), J. Imm unol., 145: 2167) in DME + 5% FCS, while CTLL-2 cells (Kr illis, S. (1978) J. Immunol. 120: 20) Rosewall Park Memorial Institute ( RPMI) medium + 10% FCS.T cell assay   Peraortic and inguinal lymph nodes were taken from immunized mice and these nodes were scanned. An isolated cell suspension was prepared by passing through a stainless steel wire mesh. cell Wash the cells in a 96-well flat bottom tissue culture plate, 4 × 10 in nutrient medium^FiveCultured in cells. Protein or peptide antigen at various concentrations And the cells were incubated at 37 ° C. for 24 hours. Need to collect supernatant Stored at -20 ° C. Used for all in vitro assaysDerpI is , An allergen isolated from exhausted mite medium (SMM).Lymphokine assay   FDC-P1 cells respond to IL-3 and GM-CSF. In response, it proliferated most, followed by IFN-γ or IL-4 (Ke lso, A. (1990) supra). 2 × 10^ThreeCells were incubated with 50 μl DME + 5% FCS for 9 Added to 50 μl culture supernatant in a 6-well flat bottom tissue culture plate. These cells Were incubated for 40 hours at 37 ° C., then 1 μCi^ThreeWith H-thymidine Pulse labeling was performed at 37 ° C. for another 4 to 6 hours. The cells are then filtered through a glass fiber filter Collect on a mat and use a sample using liquid scintillation spectrometry. Or Packard Matrix 9600 Direct Base to acquire it for later experiments. At Counter (Connecticut, Meriden, Packard Instruments),^ThreeH- The uptake of thymidine was counted.   The CTLL-2 cell line grows maximally on IL-2, but in the presence of IL-4. It grows only slightly (Kelso, A. (1990) J. Immunol. 145: 2167). Transfer the supernatant to a well Cultivated with 5,000 CTLL-2 cells for 24 hours at 37 ° C.^ThreeH -Thymidine (^ThreeH-Tdr) was pulse-labeled. Cells, glass fiber filter The amount of radioactivity incorporated was measured on a mat and measured as above.Example 1   In Der p I recognized by the mouse           Immunodominance, minor and cryptic           Measurement of T cell epitope   H2^bMouseDerpH2 is a high response animal to I^k, H2^dAnd H2^qMa Usu was previously shown to be a poor responder (Hoyne, G. (1992) Ph.D. Thesis, T.  cell Recognition During Mucosal and Systemic Responses, Western Austral ia university).DerpTo determine the location of T cell epitopes on I, B10 mice In 100 μg of CFADerpImmunized subcutaneously with I, and 8 days later, around the aorta and rat Radial lymph nodes, using a panel of overlapping peptides to release antigen-specific lymphokine ( IL-3 / GM-CSF). Maximum response in three separate experiments The answer is peptide p110-131 (DerpFor amino acid residues 110 to 131) of I On the other hand, the lowest response is also the peptide p78-100 (DerpAmino acid residue of I 78-100) and p21-49 (DerpFor amino acid residues 21 to 49) of I I was seen. Other peptides were unable to stimulate the response. Of such an experiment An example of one result is shown in Figure 1, where the following peptide was used: peptide p1 10-131 (□), peptide p78-100 (Δ) and peptide p21-49 (●).   Mice were tested for all peptides to test for cryptotic epitopes. Immunize withDerpI and immunity Responses to activated peptides were measured in the presence of splenic adherent cells. Peptide p12 0-143 (DerpI amino acid residues 120-143) and peptide p144-1 69 (DerpAmino acid residues 144 to 169) of I sensitize mice and CompleteDerpFor I protein (Fig. 2a) and peptide (Fig. 2b) respectively The response can be played back. The results in Figure 2 show the average IL of triplicate samples. -3 / GM-CSF response is shown. The following peptides are shown in this figure: : Peptide p120-143 (□), peptide p144-169 (Δ), pepti Do p132-157 (o) and peptide p158-180 (x).Example 2   In mice by administration of fusion protein           Induction of oral tolerance   Some recombinant peptides,DerpIt was generated by restriction enzyme digestion of IcDNA. These fragments were cloned into pGEX expression vector as described above and transformed into E. coli. Was transformed into. The recombinant peptides selected for this study wereSchistosom a  japonicum Fused to the glutathione-S-transferase protein of Escherichia coli Was expressed as. Soluble these fusion proteins and peptides from bacterial cell pellets And was dialyzed into CAPS buffer (pH 9.6). These sets listed in FIG. A replacement peptide was selected based on the known T cell epitope data above. Was. Recombinant peptide is immunodominant within its sequence (paralleled squares), clip Presence of tick epitope (dotted square) or absence of T cell epitope (black) Selected square). Therefore, the control peptide GEXp1-23 (DerpOf I Amino acid residues 1 to 23) and GEXp188-222 (DerpAmino acid residue 1 of I 97-222) did not contain any T cell epitopes. GEXp 57-130 (DerpAmino acid residues 57-130 of I) contain two epitopes. However, on the other hand, GEXp101-154 (DerpI amino acid residues 101-154 ) And GEXp98-140 (DerpAmino acid residues 57-130 of I are a single immunity Containing a dominant epitope (amino acid residues 110 to 131), GEXp131-18 7 (DerpAmino acid residues 131-187 of I contain a cryptic epitope I'm out.   A previously characterized regimen for induction of oral tolerance (Hoyne, G.F. (1993), Imm. unology 78: 534-540), mice received 3 mg of fusion peptide for 3 consecutive days. After 7 days of challenge, the mice were immunized subcutaneously with native protein in CFA. Then 7 days Later, periaortic and inguinal lymph nodes stimulated with either protein or synthetic peptides Was used to perform the in vitro lhokine assay. CAPS buffer for mice Liquid or recombinant GEXDerpProviding an I (1-222) fusion protein in CFA IL-2 or IL-3 / G in mice for subcutaneous injection of OVA Experiments were performed to show that it had no effect on the M-CSF response.   To test whether orally administered peptides can induce tolerance, control mice were tested. Mice were given CAPS buffer (FIG. 4, panels a and e), while test animals were given 3 mg of GEXDerpI (1-222) (Fig. 4 panels d and h) or fusion pepti GEXp57-130 (FIG. 4, panels b and f) or GEXp101-154 (FIG. 4, panels c and g) were given for 3 consecutive days. One week later, natural in CFADerp The response to immunization with I was measured. Mice given CAPS buffer IsDerpA strong response to I protein, IL-3 in response to TCR triggering / GM-CSF (FIG. 4, panel a) and IL-2 (FIG. 4, panel e) In vitro secretion was demonstrated. On the other hand, GEXDerpI (1-222) or two GEXp57-130 or GEXp101-154 The resulting mice had a decreased IL-2 response (Figure 4, panels fh). IL- The more pronounced inhibition of the 2 response was a consistent feature of all experiments of this nature. When mice were given GEXp98-140, they were induced by this peptide. A similar degree of tolerance was found.   How Oral Tolerance Development Affects Responses to T Cell Epitopes on Allergens 3 mg of GEXp101-154 or GEXp188 to see if it resonated − 222 and GEXDerpII (1-129) (for control) was given to the mice for 3 consecutive days. Was. One week later,DerpImmunize with I to produce a depleting lymph node cell response Measured in vitro for proteins and peptides. Data are for the next antigen concentration Shown are responses for individual mice in each group at:DerpI, 20 μg / ml Peptide p110-131 and peptide p78-100, 10 μM. Shown in Figure 5 As described above, the mouse was treated with GEXp188-222 or GEX.DerpII (1-129) Giving either a protein or immunogenic peptide p110-131 or p In vitro challenge with 78-100 those lymph node cells Does not affect its ability to secrete IL-3 / GM-CSF or IL-2. But However, in contrast, lymphokine responses of mice fed GEXp101-154. The answers were significantly reduced and therefore appeared to be tolerized to all proteins (Fig. 5). . Tolerance induced by feeding one epitope is associated with other epitopes on the allergen. Seems to affect T cells specific to the loop. One subsequent epitome Group containing GEXp61-100 and fusion peptide GEXp1-23 (for control) Experiments with gave the same results.   Determine whether peptides containing cryptic epitopes can influence the immune response In order to determine the amount of crypts found in 3 mg of peptide 144-169, Tea GEXp131-187 (Fig. 6 (●)) containing the epitope for 3 consecutive days On the other hand, the control mouse was given the CAPS buffer (FIG. 6 (□)). One week later , Mice in CFADerpImmunized with I. Lymph node cells in vitro soDerpCultured with I and the supernatant was assayed for IL-2. Each data in Figure 6 Points represent mean response ± standard deviation of 5 animals per group. Cryptic peptide The response of the mice given a difference was statistically different (p <0.05 t-test). Figure As shown in Fig. 6, lymph node cells from control mice were stimulated by IL-2 secretion. In vitroDerpI showed a strong response to A given mouse displayed a much weaker lymphokine response in vitro.   These results provided here indicate that either dominant or cryptotic T cell epitopes have been identified. Feeding a fusion peptide containing a T cell response to subcutaneous immunization with whole antigen It can be prevented. In the case of a fusion peptide containing a dominant epitope , This inhibition is significant, with allergens or immunodominant peptides in vitro Reduced IL-2 and GM-CSF release from challenged lymph node cell depletion Measured by expulsion. This is a pattern containing residues that are not present on the fusion used for feeding. Included a response to Petit. For example, feeding the fusion peptide GEXp101-154 What to do isDerpI immunization of allergens and total Ability to induce T cells that react with adult peptides p110-131 and p78-100 Stopped the power. Therefore, this effect may be mediated by soluble factors. Or, This prevention isDerpIt is specific because it cannot be induced by the II fusion protein. Similar Miller, A. et al. (1991) J. Exp. Med., 174: 791; Whitacre, C.C. et al. (1990). J. Immunol. 147: 2155; and Miller, A. et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 421. And oral tolerance to MBP is mediated by TGF-β1 Found that it could be shown to suppress bystander responses in an in vitro model. Was.   Feeding two fusion proteins that do not contain T-cell epitopes enhances the immune response. Didn't stop. However, the clip found in peptide p144-169 Feeding a fusion peptide GEXp131-187 containing a tick epitope Blocked significantly. The degree of inhibition is significant for fusions containing dominant epitopes. Not bad, probably increased by extending feeding regimen or increasing dose Could be done. Feeding these fusion peptides also resulted in MLN After sensitizing T cells, they fed the peptide GEXp131-187Derp I or a synthetic peptide containing the cryptic peptide p144-169 -It was also found to release GM-CSF when stimulated in vitro. . Presence of these sensitized cells in oral tolerance Have recently been described for OVA (Hoyne, G.F. et al. (1993), Immunology 78: 534. -540).Example 3   Recognized by allergic individuals           Immunodominance in Derpl, minor and           Determination of cryptotic T cell epitopes   Recognized by allergic individualsDerpT cell epitope in the I protein sequence In order to determine the × 10⁶/ Ml, 20 μg / ml purified naturalDerpCulture in the presence of I. The T cell line can be established by Moistening CO₂In the incubator After culturing at 37 ° C for 7 days, the viable cells were treated with lymphocyte separation medium (LSM). , Organon Technica, Durham, North Carolina) Isolated and further for 2-3 weeks in complete medium containing recombinant IL-2 and recombinant IL-4 Can be cultured. T cells are "resting" and no longer respond to growth factors 2 × 10 if not sex^Four5 × 10 5 T cells in 200 μl complete medium.^Four With gamma-irradiated (3500 rad) peripheral leukocytes (antigen presenting cells) of By culturing in the presence of peptides derived from different concentrations of the complete protein. Two proliferation assays can be performed. These cultures were then cultured on day three. Pulse labeled with lithiated thymidine (1 μCi / well) and on day 4, glass fiber Wei Can be collected on the filter. Less tritium uptake than medium controls A peptide that stimulates more than twice as much as a complete protein (ie, less peptide With one minor or immunodominant epitope). And includes T cell epitopes that are exposed to T cells. Twice as much as the medium control Peptides that stimulate less tritium uptake do not contain T cell epitopes Or a cryptic peptide (ie, usually given the complete protein, , Epitopes that are not exposed to T cells). Crypti in these peptides Peripheral leukocytes from the same individual to confirm the presence of Separate culturing to establish peptide-reactive T cell lines in the presence of tide Can establish a T cell line. The "resting" T cells are then added to each pellet. Petit andDerpYou can challenge with I protein. At least one chestnut Peptide containing a petit epitope is the existence of the peptide or complete protein. To stimulate the proliferation of T cell lines at levels at least two times higher than the medium control under Or T cells can be tolerized.Equivalent   One of ordinary skill in the art will recognize many equivalents of the specific embodiments of the invention described herein, or This can be confirmed using routine experiments. Such equivalents are defined in the claims below. Included in It is what is done.

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Claims (1)

[Claims] 1. A method of inducing immunological tolerance to a protein antigen in a patient, the method comprising administering to the patient a tolerizing amount of a composition comprising at least one cryptogenic peptide derived from the antigen and a pharmaceutically acceptable carrier. The above method, including. 2. The method of claim 1, wherein the patient is a mammal. 3. The method of claim 2, wherein the mammal is a human. 4. The method of claim 1, wherein the composition is administered orally. 5. The method according to claim 1, wherein the protein antigen is an allergen. 6. The method of claim 5, wherein the allergen is of a genus selected from the group consisting of: Dermatophagoides , Felis , Ambrosia , Lolium , Cryptomeria , Alte rnaria , Alder , Betula , Quercus. Genus, Olea , Artemisia , Plantago , Parietaria , Canine , Blattella , Apis , Periplaneta , and So rghum . 7. The method according to claim 7, wherein the allergen is of the species Dermatophagoides pteronyssinus . 8. The method according to claim 7, wherein the allergen is Der p I. 9. The method according to claim 1, wherein the protein antigen is an autoantigen. 10. The self-antigen is selected from the group consisting of insulin, myelin basic protein, rh factor, acetylcholine receptor, thyroid cell receptor, basement membrane protein, thyroid protein, PM-1, glutamate decarboxylase (64K) and carboxypeptidase H. Item 9. The method according to Item 9. 11. The method according to claim 2, wherein the mammal is a mammal susceptible to a protein antigen. 12. The method of claim 1, wherein the composition further comprises a peptide comprising an immunodominant epitope derived from a protein antigen. 13. A method of inducing immune tolerance to an allergen in a patient, comprising orally administering to the patient a tolerizing amount of a composition comprising at least one cryptogenic peptide derived from the allergen and a pharmaceutically acceptable carrier. The above method. 14． 14. The method of claim 13, wherein the allergen is of a genus selected from the group consisting of: Dermatophagoides , Felis , Ambrosia , Lolium , Cryptomeria , Alte rnaria , Alder , Betula , Quercus. Genus, Olea , Artemisia , Plantago , Parietaria , Canine , Blattella , Apis , Periplaneta , and So rghum . 15. 15. The method of claim 14, wherein the allergen is of the species Dermatophagoides pteronyssinus . 16. 16. The method of claim 15, wherein the allergen is Der p I. 17． 14. The method of claim 13, wherein the patient is a human. 18. 18. The method of claim 17, wherein the patient is a human susceptible to allergen. 19. A composition for inducing immunological tolerance to a protein antigen in a patient, which comprises a tolerizing amount of a cryptic peptide derived from the protein antigen and a pharmaceutically acceptable carrier. 20. 20. The composition of claim 19, in a form suitable for oral administration. 21. 20. The composition of claim 19, wherein the protein antigen is an allergen. 22. 22. The composition of claim 21, wherein the allergen is of the genus selected from the group consisting of: Dermatophagoides , Felis , Ambrosia , Lolium , Cryptomeria , Alte rnaria , Alder , Betula , Quercus. Genus, Olea , Artemisia , Plantago , Parietaria , Canine , Blattella , Apis , Periplaneta , and So rghum . 23. The composition of claim 22, wherein the allergen is of the species Dermatophagoides pteronyssinus . 24. 24. The composition of claim 23, wherein the allergen is Der p I. 25. 20. The composition of claim 19, wherein the protein antigen is a self antigen. 26. The self-antigen is selected from the group consisting of insulin, myelin basic protein, rh factor, acetylcholine receptor, thyroid cell receptor, basement membrane protein, thyroid protein, PM-1, glutamate decarboxylase (64K) and carboxypeptidase H. Item 26. The composition according to Item 25. 27. 20. The composition of claim 19, further comprising a tolerizing amount of a peptide comprising an immunodominant epitope derived from a protein antigen. 28. A composition for inducing oral tolerance to an allergen in a patient, said composition comprising a tolerizing amount of a cryptotic peptide derived from the allergen and a pharmaceutically acceptable carrier, said composition being suitable for oral administration. Stuff. 29. 29. The composition of claim 28, further comprising a tolerizing amount of a peptide comprising an immunodominant epitope derived from a protein antigen.