CA2164326A1 - Cryptic peptides for use in inducing immunologic tolerance - Google Patents

Abstract

Methods of inducing immunologic tolerance in a subject, such as human, by administering a tolerizing amount of a composition comprising a cryptic peptide derived from tbe antigen and a pharmaceutically carrier are described. Compositions which include a cryptic peptide derived from a protein antigen, such as an allergen or autoantigen, can be administered to induce tolerance in a naive or pre-sensitized individual. Preferably, the composition is administered orally.

Description

-WO 94/27634 216 4 3 2 ~ PCT/AU94/00292 CRYPTIC PEPTIDES FOR USE IN INDIJCING
IMMUNOLOGIC TOLERANCE

Background of the Invention S Feeding antigens has been a classical method for inducing imml~nnlogical 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;
Mowat, A. M., et al., (1982), Immunology, 45:105; and Strobel, S., et al., (1983), Immunology, 49:451). Although regarded as being an important physiological response to dietary antigens (Mowat, A. M., (1987), Immunology Today, 8:93), it has been suggested that oral tolerance could be used to control aberrant immunological responses such as those found in autoimmune ~ e~e, Thompson, H. S. G., and Staines, A. (1990), Immunol. Today,11 :396, and allergy.
The most extensively studied model system for autoimmune disease has been that of experimental allergic encephalomyelitis (EAE). It has been shown that rats fed a tolerizing dose of myelin basic protein (MBP) prior to sen~iti7~tion can be protected from an encephalitogenic challenge with MBP (Miller, A. et al., (1991), ~ Exp. Med., 174:791;
Whitacre, C. C., et al., (1991), J. Immunol., 147:2155; and Miller, A., et al., (1992), Proc. Natl. Acad. Sci. USA, 89:421). However, there have been conflicting views as to the mech~ni.~m~ involved in inducing oral tolerance. For exarnple, Whitacre et al., (1991), J. Immunol., 147:2155, found they were unable to transfer ~u~re~ion using T cells from tolerized ~nim~l~, but showed that clonal anergy may be an important mech~ni~m for down-regulating the effector function of CD4+ MBP-reactive T cells. Alternatively, Miller, A. et al., (1991), J. Exp. Med., 174:791, have shown that suppression can be transferred to naive recipients who receive CD8+ T cells from tolerized ~nim~l~ These s~ cssel (Ts) cells through the release of a soluble cytokine were reported to be able to inhibit the in vitro response of a MBP-specific CD4+ T cell line and could also bring about a by-stander ~u~l~;ssion of unrelated T cells (Miller, A., (1991), cifed supra). The immllnoregulatory cytokine released by Ts cells was later defined as TGF-131 (Miller, A., et al. (1992) Proc. Na~l. Acad. Sci. USA 89:421).
Peptides derived from a variety of protein antigens, including bacterial and viral pathogens, ~to~ntigens~ allergens and other ~xl,e.illlental antigens such as hen egg Iysozyme (HEL), ovalburnin (OVA) and lambda lc~lessol (cl) have been e~mined for the ability to stimulate antigen-specific T cells. A wide size spectrum of peptides has been reported to serve as 1[` cell epitopes. For e~nnple, a peptide derived from Hepatitis B surface antigen (HBsAg amino acid residues 19-33) has recently been shown to stim--l~te T cell responses in a majority of human subjects who had been immlmi~rl with a recombinant hepatitis B
vaccine (Schad, V.C. et al., (1991) Seminars in Immunol., 3:217-224). A major 2~ ~32~ -2-mycobacterial antigen 65-kD protein has also been epitope-mapped (Lamb, J.R. et al., (1987) EMBO J., 6(5):1245-1249). T cell epilcl,es have been identified in the peptides comprised of amino acid residues 112-132 and 437-459 of the 65-kD protein. MBP has also been epitope-mapped in both human (Ota, K. et al., (1990) Nature, 346: 183- 187 ) and rodent (Zamvil et al., (1986) Nature, 324:258-260) systems.
T cell epitopes present in allergenic proteins have very recently been described(O'Hehir, R. et al., (1991) Ann. Rev. Immunol., 9:67-95 ). Several peptides derived from the house dust mite allergen Der p I have been shown to be T cell-reactive (Thomas, W.R., et al.
In Epitopes of Atopic Allergens Procee~1ings of Workshop from XIV Congress of the European Academy of Allergy and Clinical ~mmlm~logy, Berlin (Sept. 1989) pp. 77-82;
O'Hehir, R.E. (1991) Annual Review Immunology 9:67-95; Stewart, G.A. et al. In: Epitopes of Atopic Allergens Procee-lin~ of Workshop from XIV Congress of the European Academy of Allergy and Clinical Immunology, Berlin (Sept. 1989) pp. 41-47; and Yessel, H. et al. In:
T Cell Activation in Health and Disease: Discrimination Between Immunity and Tolerance, Conference 22-26 (Sept. 1990) Trinity College, Oxford U.K.). A T cell-stimulatory peptide derived from the short ragweed allergen ~m~ I (amino acid residues 54-65) has also been reported (Rothbard, J.B. et al., (1988) Cell, 52:515-523). Using a panel of T cell clones derived from a rye grass-allergic individual, Perez ~ ~L demons~ ed that T cell epitopes are contained within amino acid residues 191-210 of the protein allergen I~ I (Perez, M. et al., (1990) J. Biol. Chem. 265(27): 16210-16215 .

Srmm~-y of the Invention This invention pertains to methods of inducing immllnologic tolerance to a protein antigen in a subject, such as human, by ~imini~tt~ring a tolerizing amount of a composition 25 comprising at least one cryptic peptide derived from the antigen and a ph~rm~ceutically acceptable carrier. Compositions which include a cryptic peptide derived from a protein antigen, such as an allergen or ~llto~nti~en, can be ~tlmini~tered to induce tolerance in a naive or pre-sen~iti7tod individual. Preferably, the composition is ~tlminist~red orally to treat sensitivity in an individual to an allergen or allto~ntigen.
Brief D&Y~ ;ItionoftheD...~ gs Figure I is a graphic representation of the responses of T cells isolated from mice in....~ i7~d with ~ I and analyzed for response to selected peptides derived from I~er p I
by tritiated thymidine incorporation.
Figure 2a and 2b are graphic representations of the responses of T cells isolated from mice immunized with a selected peptide derived from ~ I and analyzed for response to either Der p I protein (panel a) or the a~n)p-iate peptide (panel b).

WO 94l27634 ~ 1 ~ 4 3 ~ ~ PCT/AU94/00292 Figure 3 is a schematic representation of the location of T cell epitopes recognized by mice in the 1~ I protein sequence where immlln~dominant epitopes are represented with h~tchPd squares, cryptic epitopes are represented by dotted squares and the absence of epitopes is r~lesenled by black squares.
S Figure 4 is a graphic representation of the responses of T cells isolated from mice fed with buffer (panel a), peptide GEX p57-130 (panel b), peptide GEX plO1-154 (panel c), or recombinant protein, GEX ~2 I (1-222) (panel d) followed by immunization with ~ÇLI2 I
and analyzed for response to ~ I in vitro by IL-3/GM-(: SF (panels a-d) or IL-2 production (panels e-h).
Figure S is a graphic le~lese~ ion of the responses of T cells isolated from mice fed recombinant protein GEX ~ II (1-129), peptide GEX plO1-154, or peptide GEX pl88-222 followed by immllni7~tion with Der p I and analyzed for response to 1~ I (panel a and d), peptide pl 10-131 (panel b and e), or peptide p78-100 (panel c and f) by IL-3/GM-CSF
(panels a-c) or IL-2 production (panels d-f).
Figure 6 is a graphic representation of the responses of T cells isolated from mice fed with either buffer or recombinant fusion peptide (GEX pl31-187) followed by immnni7~tion with ~2 I and analyzed for response to I~Y2 I by IL-2 production.

Detailed lDescription of the Invention This invention pertains to methods for inducing immlmologic tolerance to a protein antigen in a subject by ~-lmini~tçring at least one cryptic peptide derived from the antigen.
Protein antigens are known to contain certain ~letermin~ntx or epitopes which, upon presentation with a particular class II major histocompatibility (MHC) molecule will activate T cells of a subject upon exposure to the native protein antigen. Rather than the T cell response being limited by the presence of one or two letermin~nt~ on an antigen, it appears that the T cell response prer~ ially utilizes a selected nurnber of cletermin~ntx. Thus, a hierarchy of T cell d~ ..t usage exists for a mlllticlel~. Il~ill~ll~ protein antigen.
Accordingly, the T cell cl~l~....i ..~..lx or epitopes for a particular protein antigen can be divided into categories based on in vitro T cell proliferation assays in which protein antigen-30 primed T cells are cultured with a selected concentration of a peptide derived from the protein antigen and the amount of proliferation by the T cells in response to the peptide is determined by, for example, triti~te-l thymidine incorporation.
By this assay, a peptide is categorized as comprising an immlln~clomin~nt T cellepitope if the peptide consistently in~ es one of the highest T cell proliferative responses in 35 antigen-primed T cells in the subject tested. Relative to an immunodominant epitope, a peptide which comprises a minor T cell epitope recalls in vitro T cell proliferation to a more variable and lesser extent. Those peptides which recall T cell proliferation of less than 2 fold ~ 1~4~ 4 the background level of media alone are categorized as either not comprising a T cell epitope or comprising a cryptic T cell epitope. Cryptic epitopes are those determin~nt~ in a protein antigen which, due to processing and presentation of the native protein antigen to the a~lopliate MHC molecule, are not normally revealed to the imml-ne system. However, a 5 peptide comprising a cryptic epitope is capable oftolerizing T cells, and when a subject is primed with the peptide, T cells obtained from the subject will proliferate in vitro in response to the peptide or the protein antigen from which the peptide is derived. Peptides which comprise at least one cryptic epitope derived from a protein antigen are referred to herein as cryptic peptides. To confirm the presence of cryptic epi~ol,es in a peptide categorized by the 10 above-described assay, antigen-primed T cells are cultured in vitro in the presence of each peptide separately to establish peptide-reactive T cell lines. A peptide is considered to comprise at least one cryptic epitope if a T cell line can be established with a given peptide and T cells are capable of proliferation upon ch~llenge with the peptide and the protein antigen from which the peptide is derived.
The presence of cryptic epitopes in a protein antigen is due to a lack of exposure of certain epitopes to the immllne system which may result from normal processing of the protein antigen which fails to reveal the epitope to the a~ropliate class II MHC molecule.
~lt~rn~tively, the end product of antigen processing may be a large fragment which hides the cryptic epitope and hinders access to the MHC molecule or the T cell receptor on T cells 20 specific for the epitope. Additionally, other epitopes on the same protein antigen may compete with the cryptic epitope for binding to the same restriction element or may have a higher affinity and availability for a different restriction element, thus preventing cryptic epitope interaction with MHC.
Cryptic peptides of the invention comprise at least one cryptic epitope derived from a 25 protein antigen (i.e., the peptide comprises at least apprnximAtely 7 amino acid residues).
Such peptides can comprise as many amino acid residues as desired and preferably comprise at least about 7, more preferably at least about 15, even more preferably at least about 20 and most preferably at least about 25 amino acid residues of a protein antigen. A peptide length of about 20-40 amino acid residues is preferred as increases in length of a peptide may result 30 in difficulty in peptide synthesis as well as retention of an undesirable property (e.g., immllnoglobulin binding or enzymatic activity) due to m~int~n~nce of conformational similarity between the peptide and the protein antigen, such as an allergen from which it is derived. If desired, the amino acid sequences of one or more peptides can be produced and joined by a linker to increase sensitivity to processing by antigen-presenting cells. Such 35 linker can be any non-epitope amino acid sequence or other a~propl;ate linking or joining agent. For example, two cryptic peptides can be joined or a cryptic peptide and a peptide WO 94/27634 216 ~ 3 ~ ~ PCT/AU94/00292 compri~ an imml~nodominant or minor epitope derived from the protein antigen can be linked.
Cryptic peptides can be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid vector directing t;x~res~ion of a nucleotide sequence coding S for such peptide, or by chemical synthto~i~, or in certain limited situations by chemical cleavage of protein antigen such as an allergen. When produced by recombinant techniques, host cells transformed with nucleic acid vectors directing expression of a nucleotide sequence coding for a peptide are cultured in a medium suitable for the cells. The peptides may be secreted and harvested from a mixture of cells and cell culture medium. Alternatively, the peptide may be retained cytoplasmically and the cells harvested, Iysed and the peptide isolated a~d purified. Peptides can be isolated using techniques known in the art for purifying peptides or proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for the peptide or the protein antigen from which the peptide is derived, or a portion lhereof. The cryptic peptides described herein are isolated such that the peptide is snbst~nti~lly free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when synth~ci7.-d chemically, or obtained by chemical cleavage of a protein allergen or other protein antigen.
To obtain cryptic peptides of the invention where the T cell ~ilo~es of a protein antigen are urlknown or ill-defined, the protein structure of the antigen can be reviewed and the sequence divided into at least t~,vo peptide fragments of desired lengths. For example, the protein sequence of a protein antigen can be systematically divided into at least two non-overlapping fr~gment~ of desired length or overlapping fr~gment~ of desired length. As an illustrative example, the known amino acid sequence of Der p I, a major allergen of nermato~hz~oides ~teror~ys~inus having an amino acid sequence of 229 residues (shown in SEQ ID NO: 1), can be divided into peptide fragments of about 20-35 amino acid residues in length, with each fragment overlapping with another by about 10 amino acids. To maximize the potential of including T cell epitopes in the peptide fr~gment~, areas of overlap and length of each fragment can be designed to m~int~in the presence of T cell epitopes predicted using algo~ s (Rothbard, J. and Taylor, W.R. (1988) EMBO J. 7:93-100; and Berzofsky, J.A.
(1989) Philos. Tran* R. Soc. Lond. 323:535-544). Preferably, human T cell epitopes within a protein antigen can be predicted using known HLA class II binding specific amino acid residues. 1he res~llting peptide fragments can be produced by recombinant DNA techniques 35 or chemical synthP~
The peptide fragments derived from a protein antigen are tested to d~l~ , . li, ,e those fr~gment~ having T cell stimulating activity (i.e., proliferation, lymphokine secretion and/or W094/27634 21~432(~ PCT/AU9410~292 induction of T cell anergy/tolerization) and thus comprise at least one T cell epitope. For example, human T cell stim~ ting activity can be tested by culturing T cells obtained from a subject, such as a human, sensitive to a protein antigen (i.e., a subject which has an imml-ne response to the protein antigen) with a peptide fragment derived from the protein antigen and dett~rminin~ the presence of proliferation by T cells in response to the peptide. The presence of proliferation by T cells can be ~let~rmin~cl by, for example, uptake of tritiated thymidine.
Tmmlln~dominant T cell epitopes, minor T cell epitopes and cryptic epitopes can be identified as described in Example 3. To confirm the presence of a cryptic epitope in a selected peptide, T cells are obtained from an individual sensitive to the protein antigen and cultured with each of the cryptic peptides separately to establish peptide-reactive T cell lines.
The presence of T cell proliferation or induction of T cell tolerance in response to the peptide and the protein antigen from which the peptide is derived confirms the presence of at least one cryptic epitope in the peptide.
Cryptic peptides of the invention can be derived from a protein antigen such as an allergen or ~lto~ntigen. When derived from an allergen, the cryptic peptide can be derived from any known protein allergen, such as an allergen of the following genus: the genus Derm~topha~oides; the genus ~li~; the genus ~mbrosia; the genus T olillm; the genus Cryptomeria; the genus Alter~ria; the genus ~ ; the genus Betula; the genus Quercus: the genus ~21~; the genus ~rtemi~ia the genus Planta~o; the genus p~rietaria; the genus C~nine;
the genus Rlattella; the genus ~; the genus Peripl~neta; and the genus Sor~hl-m Cryptic peptides recognized by mice in ~ I, a major allergen of the species Derm~topha~oides ptero~ us, have been ~letermint?cl in mice and comprise amino acid residues 120-143 of I (SEQ ID NO:1), amino acid residues 144-169 of ~ I (SEQ ID NO:l) and amino acid residues 131-187 of r2çL~ I (SEQ ID NO:l).
Cryptic peptides can also be derived from protein antigens other than allergens where immunologic tolerance to an ~ to~ntigen is desired. ~l~to~ntigens from which cryptic peptides can be derived include insulin, glutamic acid decarboxylase (64K), PM- 1 and carboxypeptidase for use in treating diabetes; myelin basic protein for use in treating multiple sclerosis; rh factor for use in treating erythroblastosis fetalis; acetylcholine receptors for use in keating my~th~ni~ gravis; thyroid rece;~Lol~ for use in treating Graves Disease; basement membrane protein for use in treating Good Pasture's syndrome; and thyroid proteins for use in treating thyroiditis.
According to one aspect of this invention, cryptic peptides derived from a protein antigen are ~tlminiett?red to a subject to induce immllnnlogic tolerance in the subject to the protein antigen. ~e term subject includes living or~ni~m~ capable of mounting an immune response to a protein antigen, e.g., m~mm~ Examples of subjects include hllm~n~, rats, mice, dogs, cats, horses, cows and transgenic species thereof. Tmmlm~logic tolerance refers to a condition in a subject where a block in the development, growth or differentiation of specific Iymphocytes in the subject results upon ~lminictration of a tolerizing amount of a cryptic peptide of the invention. Tolerance results from the interaction of antigen with antigen receptors on Iymphocytes under conditions in which the Iymphocytes, instead of 5 becoming activated, are deleted or rendered unresponsive. Tolerance may also be due to the action of specific T or B Iymphocytes or other regulatory meçh~ni~m~ that prevent Iymphocyte activation. One mech~ni~m for inhibiting an immune response is the stimulation of a class of Iymphocytes, called s~p,~;sser T cells, whose principal function is to suppress the activation of specific T and B Iymphocytes. In this situation, inhibition is mediated not 10 by the antigen itself but by regulatory cells that are in~llce~l by the antigen. Another proposed meçh~ni~m for tolerance is a response by the immune system to antigen in which unique or idiotypic determin~nte of lymphocytes or antibodies specific for the antigen are targeted. This response results in a network of complementary idiotypes and antiidiotypes which block the stim~ tion of antigen-specific cells. Finally, the products of activation of B
15 and T Iyrnphocytes, namely antibodies and cytokines, respectively, are themselves capable of regulating specific immunity to result in tolerance in addition to functioning as the principle effector rnolecules of lymphocytes.
In order to induce immunologic tolerance in a subject, a tolerizing amount of a cryptic peptide derived from a protein antigen is ~timinictered to the subject. A tolerizing amount is 20 defined as a dosage of cryptic peptide necessary to induce immunologic tolerance in a subject, such as a human to the antigen from which the cryptic peptide is derived.
Tmmllnologic tolerance in a subject is indicated by non-responsiveness or ~liminution in symptoms to the protein antigen, such an an allergen or ~lto~ntigen, as determin~d by standard clinical procedures (see e.g., Varney et al,, (1990) British Medical Journal 302:265-25 269). When tolerance to an allergen is sought, such non-responsiveness includes ~liminntion in allergen in~ ecl allergic symptoms. As referred to herein, a ~liminntion in symptoms to an allergen includes any reduction in the allergic response of a subject, such as a human, to the allergen following a tre~tm~nt regimen with a cryptic peptide as described herein. This fliminntion in symptoms may be ~letermin~d subjectively in a human (e.g., the patient feels 30 more comfortable upon exposure to the allergen), or clinically, such as with a standard skin test.
Cryptic peptides derived from a protein antigen are typically ~(imini~tered to a subject in the form of a composition which includes a ph~rm~ceutically acceptable carrier or diluent.
A-lminictration of a composition of the present invention to induce immlln~logic tolerance in 35 a subject to a protein antigen can be carried out using known procedures, at dosages and for periods of time effective to tolerize the subject to the protein antigen. Effective amounts of the composition will vary according to factors such as the degree of sensiliviLy of the subject -WO 94/27634 ~ 1 ~; 4 ~ ~ ~ PCT/AU94/00292 to the antigen, the age, sex, and weight of the subject, and the ability of the cryptic peptide(s) to induce tolerance in the subject. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be ~tlmini.~tered daily or the dose may be proportionally reduced as in-lic~tçcl by the exigencies of the therapeutic 5 situation.
Cryptic peptides may be ~lmini~tered to a subject in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral ~lmini~tration, inhalation, intranasal, transdermal application, or rectal ~imini~tration. Preferred routes of ~lmini~tration to induce immlmologic tolerance in a subject are oral and intranasal ~mini~tration. See O'Hehir, R.E.
et al. (1993) Eur. J. Clin. Invest. 23(12): 763-772). Depending on the route of ~timini.~tration, the active compound (i.e., the cryptic peptide) may be coated with in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
To atlmini~t~r a cryptic peptide or peptides by enteral ~(lmini~tration, it may be 15 necessary to coat the peptide with, or co-~-lmini~ter the peptide with, a material to prevent its inactivation. For example, the cryptic peptide may be ~lmini~tered to a subject in an a~ropl;ate diluent, co-~lmini~tered with enzyme inhibitors or in an al,L,rop.iate carrier such as liposomes. Ph~rm~eutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate 20 (DEP) and trasylol. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol. 7:27). For purposes of inducing tolerance, the composition is preferably ~-lmini~tered in non-immlln-~genic form, e.g., one that does not contain adjuvant.
The active compound may also be ~rlmini~tered pale~lleldlly. Dispersions can also be 25 p~ d in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these pl~lions may contain a preservative to prevent the growth of microorg~ni~m~
Ph~rm~e~ltical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous 30 ~ ,paL~lion of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of m~nllf~ctllre and storage and must be preserved against the cont~min~ting action of microorg~ni.~m.~ such as bacteria and fungi. The carrier can be a solvent or dispersion medium co~ g, for example, water, ethanol, polyol (for example, glycerol, 35 propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be m~int~inPrl for example, by the use of a coating such as licithin, by the m~ t~ ce of the required particle size in the case of WO94/27634 ~ 64326 PCl IAU941~0292 dispersion and by the use of surfactants. Prevention of the action of microorg~ni~m~ can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, ~or example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, al.~ u.~l monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating active compound in the required amount in an a~plopliate solvent with one or a combination of ingredients enumera~ed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of slerile powders for the plc~dlion of sterile injectable solutions, the preferred methods of prcpa~dlion are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., a peptide of the invention) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
When a cryptic peptide or peptides as herein described is suitably protected, asdescribed above, the peptide may be orally ~lmini~tered, for example, with an inert diluent or an ~c~imil~hle edible carrier. The peptide and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, colllp-cs~ed into tablets, or incorporated directly into the individual's diet. For oral ~ nninictration~ the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and plc~ Lions should contain at least 1% by weight of active compound. The percentage of the compositions andp~ lions may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such compositions is such that a suitable dosage will be obtained. Preferred compositions or pl~dlions according to the present invention are prepared so that an oral dosage unit contains between from about 10 mg to about 200 mg of active compound.
As used herein "ph~rm~e~1tically acceptable carrier" includes any and all solvents, dispersion media, coatings, antih~.teri~l and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for ph~n~ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incnmp~tible with the active compound, use thereof in the composition is contemplated.
Supplementary active compounds can also be incorporated into the compositions.
It may be advantageous to form~ te compositions in dosage unit form for ease of lmini~tration and uniro,~ y of dosage. Dosage unit ffirm as used herein refers to 21 ~43~

physically discrete units suited as unitary dosages for the m~mm~ n subjects to be treated;
each unit cont~ining a predete-minP~l quantity of active compound calculated to produce the desired effect in association with the required ph~rm~ceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the 5 unique characteristics of the active compound and the particular effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the tre~tnnent of the subject.
Compositions of the invention can include one or more cryptic peptides or a cryptic peptide and a peptide comprising an immlln~dominant or minor epitope which are 10 ~(lmini~tpred to subjects, such as hllm~n~, who are naive or pre-sen~iti7çcl to the protein antigen from which the peptide is derived, at dosages and for lengths of time effective to induce tolerance in the subject to the antigen. For example, an amount of one or more of the same or of di~eLenl compositions effective to induce tolerance in a subject can be ~llmini~tered simultaneously or sequentially. A composition comprising at least two peptides 15 (e.g., a physical mixture of at least two peptides), can also be used in methods of tolerization.
For example, a cryptic peptide and a peptide comprising an immunodominant epitope can be co-~-lmini~tered.
The fact that tolerance can be incluce~l by ~tlminictering a cryptic peptide of the invention (i.e., a peptide which does not contain an epitope recognized during imml-ni7~tion 20 when the entire protein antigen is presented to a subject) is significant. Peptides effective in immlmotherapy may therefore not simply be limited to those identified by T-cell clones or polyclonal responses of sP-n~iti7Pd individuals. Adminiskation of a cryptic peptide may avoid the potential limitations inherent in ~q~lmini~tering a peptide cont~ining immlln~dominant epitopes to sçn~iti7~cl individuals. The use of cryptic peptides also offers the potential for 25 modifying immllne responses without having to redirect the development of T-cell clones which have already progressed along Thl and Th2 or equivalent ~lhw~ys.
It is also possible to modify the skucture of cryptic peptides useful in methods of the invention for such purposes as increasing solubility, enhancing therapeutic or preventive efficacy, or stability (e.g., shelf life ex vivo, and resi~t~nce to proteolytic degradation in vivo).
30 A modified peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition, to modify the ability of the peptide to induce tolerance, or to which a component has been added for the same purpose.
For example, a peptide can be modified so that it m~int~in~ the ability to induce T cell anergy or tolerance and bind MHC proteins. In this instance, critical binding residues for the 35 T cell receptor (i.e., the amino acid residues which comprise the cryptic epitope) can be let~rmined using known techniques (e.g., substitution of each residue, such as, for example, with alanine and determination of presence or absence of T cell reactivity). Those residues WO 94/27634 216 ~ ~ 2 ~ PCT/AU94/00292 shown to be essential can be modified by replacing the essçnti~l amino acid with another, preferably similar amino acid residue (a conservative substitution) whose presence is shown to çnh~nce, dimini~h, but not elimin~te or affect T cell reactivity. In addition, those amino acid residues which are not essential for T cell interaction can be modified by being replaced 5 by another amino acid whose incorporation may enhance, tlimini~h or not affect T cell reactivity, but not elimin~te binding to relevant MHC. Preferred amino acid substitutions for non-essential amino acids include, but are not limited to substitutions with ~l~nine, glutamic acid or a methyl amino acid.
~nother example of a modification of peptides is sllbstitlltion of cysteine residues 10 preferably with alanine, or glutamic acid, or alternatively with serine or threonine to minimi7P dimerization via disulfide linkages.
In order to enhance stability and/or reactivity, peptides can also be modified to incorporate one or more polymorphisms in the amino acid sequence of a protein antigen reslllting from natural allelic variation. Additionally, D-amino acids, non-natural amino acids 15 or non-arnino acid analogues can be substituted or added to produce a modified peptide within the scope of this invention. Furthermore, peptides can be modified using the polyethylene glycol (PEG) method of A. Sehon and co-workers (Wie et al. supra) to produce a peptide conjugated with PEG. Modifications of peptides can also include reduction/alkylation (Tarr in: Methods of Protein Microcharacterization, J.E. Silver ed.
20 Humana Press, Clifton, NJ, pp 155-194 (1986)); acylation (Tarr, supra); esterification (Tarr, supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in ~ellular lmmunology, WH Freeman, San Francisco, CA (1980); U.S. Patent 4,939,239); or mild formalin treatment (Marsh, (1971) International Archives of Allergy and Applied Immunology 41: 199-215).
To f~cilit~te purification and potentially increase solubility of peptides, it is possible to add reporter group(s) to the peptide backbone. For example, poly-hi~ti~line can be added to a peptide to purify the peptide on immobilized metal ion affinity chromatography (Hochuli, E. et al., (1988) Bio/Technology, 6:1321-1235). In addition, specific endoprotease cleavage sites can be introduced, if desired, between a reporter group and amino acid 30 sequences of a peptide to f~rilit~te isolation of peptides free of irrelevant sequences. In order to sllcce,s~fully tolerize a subject to a protein antigen, it may be necessary to increase the solubility of a peptide by adding functional groups to the peptide or by not including hydrophobic regions in the peptide.
To potentially aid proper antigen processing of T cell ~; ilo~es within a peptide, 35 canonical protease sensitive sites can be recombinantly or synthetically engint?ered within the peptide. For example, charged amino acid pairs, such as KK or RR, can be introduced within a peptide during recombinant construction of the peptide. The resulting peptide can be , wO 94/Z7634 '~ ~ 6 432 6 i'CT/AU94100292 rendered sensitive to cathepsin and/or other trypsin-like enzymes cleavage to generate portions of the peptide CO~ Iirlg one or more T cell epitopes. In addition, such charged amino acid residues can result in an increase in solubility of a peptide.
Site-directed mutagenesis of DNA encoding a peptide can be used to modify the structure ofthe peptide. Such methods may involve PCR (Ho et al, (1989) Gene 77:51-59) or total synthesis of mllt~te~l genes (Hostomsky, Z., et al., (1989) Biochem. Biophys. Res. Comm. 161:1056-1063). To enhance bacterial expression, the aforementioned methods can be used in conjunction with other procedures to change the eucaryotic codons in DNA constructs encoding peptides to ones preferentially used in E. ~QIi, yeast, m~mm~ n cells or other eucaryotic cells.
This invention is further illustrated by the following non-limiting examples. The co~ of all references and published patent applications cited throughout this application are hereby incorporated by reference. The following methodology described in the Materials and Methods section was used throughout the examples set forth below.
MATERIALS AND METHODS

Animals and Ant~en~
Female B10 and BALB congenic mice, and inbred C57BL/6J were purchased from the Animal Resource Centre, Murdoch, Western Australia at 6-8 weeks of age.
The house dust mite allergen Der p I was affinity purified from spent mite medium (SMM) using previously described techniques (Hoyne, G.F. et al. (1993) cited supra;
Lombardo et al. J. Immunol. 144:1353-1360 and Chapman (1989) Advances in Biosciences 74:281-295). Ovalbumin (OVA) crystalline Grade V was purchased from the Sigma Chemical ColllLpally, St. Louis, MO. Overlapping synthetic peptides derived from the published 1~ I sequence (Chua et al. (1988) J. Eicp. Med. 167:175-182) were synth~ei7ed using standard t-BOC ch~rni~try and peptides were purified by reverse phase highperformance liquid chromatography (HPLC) and the sequence of individual peptides were checked to verify identity. The peptides used in this study comprised the following dmino acid residues derived from the ~ÇLp I sequence (Chua et al. (1988) cited supra): 1-20, 13-39, 21-49, 40-60, 50-71, 61-84, 78-100, 85-109, 101-119, 110-131, 120-143, 132-157, 144-169, 158-180, 170-191, 181-204, 197-222.

Pl~alion of reconnh;n~nt protein~
Inserts encoding either the whole er p I or ~ II protein (from spent mite medium, the Commonwealth Serum Laboratories, Melbourne, Australia) or recombinant constructs (formed from the restriction endonuclease fr~gm~nt~tion of the relevant cDNA;

1-- WO 94/27634 216 4 3 ~ ~ PCTtAU94/00292 see Chua et al (1990) Int. Arch. AllergyAppl. Immunol. 91:118-123), were ligated to the p-GEX vector and transforrned into F~cherichia~Qli (Smith, D. B., and Johnson, K. S., (1988) Gene, 67:31). The procedures for the molecular cloning of these products have been described elsewhere (Chua, K. Y., et al., (1988) J. Exp. Med., 167:175 and Chua, K. Y., et al., S (1990) Int. ~rch. Allergy Appl. Immunol., 91: 124). Log phase ~ coli cells l~ rolllled with pGEX based protein or peptide constructs were in-luced to express the recombinant protein by adding 0.1 mM isopropylthiogalactosi(l~e (IPTG) (Promega) to the culture with ~h~kin~
for 60 minutes at 37C. Because large quantities of fusion peptides were required they were prepared from solubilized inclusions. Bacterial pellets were resuspended in tris buffered saline with 1 mM EDTA and transferred to a homogenizing bottle cont~ining 0.1 mm glass beads and were homogenized using a Braun MSK Homogenizer for five mimltes The lysate was removed after ultracentrifugation at 10,000 g for 10 minlltes at 4C. The pellet was washed twice with 1.75 M g~l~nitline HCL cont~ining 1 M NaCl and 1% triton-X 100 (BDH
Chemicals) by thoroughly aspirating in a pipette and then centrifugation. The pellet was then dissolved by incubating it in 8 M urea with 50 mM NaC1 and 1 mM ethylene di~minetetraacetic acid (EDTA) for 2 hours at 37C. The sarnple was dialyzed in 3-(cyclohexylamino)-propanesulfonic acid (CAPS) buffer pH 10.7 and the pH was slowly adjusted to pH 9.6. The recombin~nt material was then clarified by centrifugation at 10 000 g and the concentration of the soluble m~teri~l was estim~te-l against standard quantities of bovine serurn alburnin (BSA) using SDS-polyacrylarnide gel electrophoresis (SDS-PAGE) and staiIling with Coomassie blue. Recombinant peptides used in this study included GEX
I (amino acid residues 1-222), GEX I~ II (amino acid residues 1-129), GEX pl-14 (amino acid residues 1-14 of E2ÇL~ I), GEX p60-111 (amino acid residues 60-111 f~ I), GEX p98-140 (amino acid residues 98-140 of ~ I), GEX plO1-154 (amino acid residues 101-154 of I2~ I), GEX p57-130 (amino acid residues 57-130 of ~ I), GEX pl88-222(amino acid residues 188-222 of I2~ I)-Tntlllction of Or~l Toler~nce Mice were lightly anesthetized under ether and fed- intragastrically by a tube with 3 mg of protein or peptide on 3 consecutive days. Antigens were dissolved in CAPS buffer and arlmini.~tered in a volume of 0.2 ml. Mice were immllni7~d subcutaneously at the base of tail 7 days af~er the last feed with 100 mg of native protein em~ ified in complete Freund's Adjuvant (CFA) in a volume of 0.2 ml.

Clllhlre M~ inm Lymph node cells were cultured in Dulbecco's Modified Eagles medium (DME) supplemented with 2% fetal calf serum (FCS), 50 mM, 2-ME, 2 mM L-GI~lt~mine and 20 wO 94/27634 2 ~ ~ ~3 2 ~ PCT/AU94/00292 mg/ml ge~ lycin. FDC-P1 cells (Kelso, A., (1990), J. Immunol., 145:2167) were m~int~ined in DME + 5% FCS while CTLL-2 cells (Krillis, S. (1978) J. Immunol. 120:20) were m~int~inPd in Rosewall Park Memorial Institute (RPMI) medium + 10% FCS.

5 TCell A~,vs The periaortic and inguinal lymph nodes were collected from immlmi7Pd mice and single cell ~u~ell~ions were prepared by t;x~lessi~lg the nodes through a stainless steel wire mesh. Cells were washed and cultured at 4 x 105 cells in a volurne of 0.2 ml in DME culture medium in a 96 well flat bottom tissue culture plate. Protein or peptide antigens were added 10 at various concentrations and the cells were incubated at 37C for 24 hours. Supern~t~nt~
were collected and stored at -20C until required. The Der p I used for all in vitro assays was the allergen isolated from spent mite medium (SMM).

Lymrhokine A~ys FDC-Pl cells proliferate m~xim~lly in response to IL-3 and GM-CSF and subm~xim~lly to IFN-y or IL-4 (Kelso, A. (1990) cited supra). 2x103 cells were added in 50 ~Ll DME + 5% FCS to 50 ,ul of culture ~u~ in 96 well flat bottom tissue culture plates.
The cells were inc~lb~ted for 40 hours at 37C and then pulsed with 1 ~lCi 3H-Thymidine for another 4-6 hours at 37C. The cells were then harvested onto glass fiber filter mats and samples counted for 3H-Thymidine incull,old~ion using liquid scintillation ~e~ metry or for latter ~x~c,;lllents due to its acquisition, on a Packard matrix 9600 direct beta counter (Packard Instrllment~, Meriden, CT).
The CTLL-2 cell line will proliferate m~xim~lly with IL-2 but only poorly in thepresence of IL-4 (Kelso, A. (1990) J. Immunol. 145:2167). Supern~t~nt~ were cultured with 5000 CTLL-2 cells per well for 24 hours at 37C and pulsed with 1 ,uCi of 3H-thymidine (3H-Tdr). Cells were harvested onto glass fiber filter mats and the amount of radioactivity incorporated was determined as described above.

F.x~mrle 1 n~h,.llli~ ion of Immllnodominant, Minor ~n-l Cryptic T Cell Fcpitopes Reco~ni7~ by Mice in Der p I
It has been previously shown that H2b mice are high responders to ~ I while H2k,H2d and H2q mice are low responders (Hoyne, G. (1992) Ph.D. Thesis, T cell Recognition During Mucosal and Systemic Responses, Ulliv~ y of Western Australia). To determine the location of T-cell epitopes on I~er p I, B 10 mice were immlmi7~d subcutaneously with 100 ~g of ~ I in CFA and after 8 days the periaortic and inguinal lymph nodes were ex~mined for antigen specific lymphokine release (IL-3/GM-CSF) using a panel of overlapping peptides. In three separate ~ ;lllents the greatest response was found to ~\ WO 94/27634 216 ~ 3 2 ~ PCT/AU94/00292 peptide pl l 0-131 (amino acid residues 110-131 of ~ I) while lower responses were also seen to peptides p78-100 (amino acid residues 78-100 of ~ I) and p21 -49 (amino acid residues 21 -49 of ~ I). No other peptides could stimulate a response. An example of the results of one such experiment is shown in Figure 1 in which the following peptides were 5 used: peptide pl 10-131 ([1); and peptide p78-100 (~) and peptide p21-49 (-).
To test for cryptic epitopes, mice were immllni7~-1 with all the peptides and responses to ~çLp I and the immlmi~in~ peptide were measured in the presence of spleen adherent cells. Peptide pl20-143 (amino acid residues 120-143 of ~ÇLP I) and peptide pl44-169 (amino acid residues 144-169 of Der p I) were able to sensitize mice so they could recall 10 responses to both intact ~ I protein (Figure 2a) and the peptides (Figure 2b) respectively.
The results of Figure 2 show the mean IL-3/GM-CSF response of triplicate samples. The following peptides are shown in the Figure: peptide pl20-143 (O); peptide pl44-169 (~);
peptide pl32-157 (O); and peptide pl58-180 (X).

15 Fx~m~le 2 Tn~lnrtioll of Oral Toler~nce in ~ice by A~lmini.~tration of Fusion Peptides A number of recombinant peptides were generated by restriction enzyme digestion of I cDNA. These fragments were cloned into the pGEX t;~ression vector as describedabove and transformed into E~ ~QIi- The recombinant peptides chosen for use in this study 20 were expressed as fusions attached to the glutathione-S-transferase protein of Schi~tosom~
japol icum. The fusion proteins and peptides were solubilized from bacterial cell pellets and dialyzed into CAPS buffer pH 9.6. The recombinant peptides listed in Figure 3 were chosen on the basis of the known T-cell epitope data described above. Recombinant peptides were selected for the presence of immlln~dol,lhlani (h~trh~d squares) or cryptic epitopes (dotted 25 squares) or the absence of T cell epitopes (black squares) within the sequence. Thus, control peptides GEX pl -23 (amino acid residues 1-23 of ~ I) and GEX pl 88-222 (amino acid residues 197-222 of ~ I) did not contain any T cell epitopes. GEX p57-130 (amino acid residues 57-130 of Der p I) cont~ined two epitopes while GEX plO1-154 (amino acid residues 101-154 of ~ I) and GEX p98-140 (amino acid residues 57-130 of Der p I)30 contains the single immunodomin~nt epitope (amino acid residues 110-131), while GEX
pl 31 - 187 (amino acid residues 131 -187 of ~ I) contains the cryptic epitopes.Following a previously characterized regime for inducing oral tolerance (Hoyne, G.
F., (1993), Immunology 78:534-540), mice were fed 3 mg of fusion peptide on 3 consecutive days and after a further 7 days were immunized subcutaneously with native protein in CFA.
35 In vitro lymphokine assays were then pelrolllled 7 days later using the periaortic and inguinal lymph nodes ~timlll~t~d with either protein or synthetic peptides. Experiments were performed to show that feeding mice CAPS buffer or the recombinant GEX ~ I ( l -222) WO 94/27634 2, ~ ~ 4 3 2 ~ PCT/AU94/00292 fusion protein did not effect the IL-2 or IL-3/GM-CSF responses of mice to subcutaneous injection of OVA in CFA.
To test whether orally ~lminictered peptides could induce tolerance, control mice were fed CAPS buffer (Figure 4, panels a and e), while test ~nim~l~ received 3 mg on three consecutive days of either GEX ~ I (1-222) (Figure 4 panels d and h) or the fusion peptides GEX pS7-130 (Figure 4, panels b and f) or GEX plO1-154 (Figure 4, panels c and g). One week later the response to immuni7~tion with native Der p I in CFA was determined Mice fed CAPS buffer showed strong responses to the ~ I protein in vitro secreting both IL-3/GM-CSF (Figure 4, panel a) and IL-2 (Figure 4, panel e) in response to TCR triggering.
10 On the other hand, mice fed GEX Der p I (1-222) or either ofthe two peptides GEX pS7-130 or GEX 101-154 had depressed IL-2 responses (Figure 4, panels f-h). The more pronounced inhibition of IL-2 responses was a con~ t~nt feature of all experiment~ of this nature. Mice were also fed with GEX p98- 140 and an equal degree of tolerance induced by this peptide was found.
To ex~mine how the development of oral tolerance effected responses to T cell epitopes on the allergen mice were fed 3 mg on three consecutive days of either GEX plOl -154 or GEX p 188-222 and GEX ~ II (1 - 129) as a control. One week later mice were immuni7ecl with ~ I and the responses of draining lymph node cells were measured to the protein and peptides in vitro. The data shows the response for individual mice in each group 20 at the following antigen concentrations: ~ I, 20 ,ug/ml; and peptide pl 10- 131 and peptide p78-100, 10 ,uM. As seen in Figure 5 feeding mice either GEX pl88-222 or GEX 12~ II
(1-129) did not affect the capacity of their lymph node cells to secrete either IL-3/GM-CSF or IL-2 upon in vitro challenge with either protein or with the imm~mogenic peptides pl 10-131 or p78-100. However, in contrast, the lymphokine responses of GEX plOl-154 fed mice 25 were m~rkPrlly reduced and thus appear to have become tolerant to the whole protein (Figure 5). The tolerance induced by feeding one epitope appears to affect T cells specific for other epitopes on the allergen. Subsequent experimt?nt~ using GEX p61-100 which con~i,ls one epitope and the fusion peptide GEX p 1 -23 as a control gave the sarne result.
To determine whether a peptide co~ in~ a cryptic epitope could influence the 30 immune response, mice were fed 3mg on three consecutive days of GEX pl 31 - 187 (Figure 6 (-)) which contains the cryptic epitope found on peptide 144-169 while control mice were fed with CAPS buffer (Figure 6 (Cl)). One week later mice were immuni7~d with I2~ I in CFA. Lymph node cells were cultured in vitro with I2Ç~ I and sUpern~t~ntc assayed ~or IL-2. Each data point in Figure 6 represents the mean response of 5 ~nim~lc per group +
35 standard deviation. The responses of cryptic peptide fed mice were statistically different (p <
0.05 t-test). As shown in Figure 6 lymph node cells from control mice showed strong WO 94/27634 ~ 1 6 4 3 ~ 6 PCT/AU9~/00292 responses to Der p I in vitro by secreting IL-2, but mice fed the cryptic epitope displayed much weaker lymphokine response in vitro.
The results presented here show that feeding fusion peptides cont~ining dominant or cryptic T-cell epitopes can inhibit T cell responses to subcutaneous immllni7~tion with the S whole antigen. In the case of fusion peptides co~ g ~lomin~nt epitopes the inhibition was profound and was measured by depressed IL-2 and GM-CSF release from draining lymph node cells challenged in vitro with whole allergen or the immunodominant peptides. This included responses to peptides cont~ining residues which were not present on the fusion used for feeding. For example, feeding the fusion peptide GEX p 101 - 154 inhibited the ability of 10 Per p I immunization to induce T cells which react with the whole allergen and with synthetic peptides pl 10-131 and p78-100. This effect may therefor be mediated by a soluble factor. The inhibition was otherwise specific because it could not be induced by the Der p II
fusion protein. Similar data has been obtained by Miller, A., et al., (1991), J: E~cp. Med., 174:791; Whitacre, C. C., et al., (1991), J. Immunol., 147:2155, and Miller, A., et al., (1992), Proc. Natl. Acad. Sci. USA, 89:421, who found that oral tolerance to MBP was mediated by TGF-~31 and could be shown to suppress bystander responses in an in vitro model.Feeding two fusion proteins that did not contain T-cell epitopes did not inhibit the imm~lne responses. However, feeding the fusion peptide GEX pl 31-187 which contained the cryptic epitope found in peptide pl44-169 did significantly inhibit. The degree of inhibition was not as marked as for the fusions cont~ining dominant epitopes but presumably could be increased by extending the feeding regime or increasing the dose. Feeding the fusion peptides was also found to Sell!~iti7e T cells in the MLN so they release GM-CSF on stimlll~tion in vitro with Der p I or synthetic peptides including the cryptic peptide pl44-169 after feeding peptide GEX p 131 - 187. The presence of these sen~iti7Pd cells in oral tolerance has recently been described for OVA (Hoyne, G. F., et al., (1993), Immunology 78:534-540).

Fx~mrle 3 Determin~tion of Tmmllnodomin~nt ~rinor ~ncl Cryptic T Cell F~pitopes Reco~ni7~ by ~n Aller~ic Tn-livi~ln~l jn ner p I
To ~letermine T cell epitopes recognized by an allergic individual in the ~
I protein sequence a T cell line can be established by cnltllrin~ mite-allergic patient peripheral blood white cells in complete medium at 2 x 106/ml in the presence of 20 llg purified native Der p I/ml. After 7 days of culture at 37C in a humidified CO2 incubator the viable cells can be isolated by centrifugation with Iymphocyte separation medium (LSM, Organon Technica, Durham, NC) and cultured in complete medium cont~ining recombinant IL-2 and recombinant IL-4 for 2-3 additional weeks. When the T cells are "rested" and no longer responsive to growth factors, a secondary proliferation assay can be performed by culturing 2 432~ ~

x 104 T cells in 200 ~LI complete medium wlth 5 x 104 gamma-irradiated (3500 Rads) peripheral white blood cells as antigen presçnting cells in the presence of various concentrations of peptides derived from the intact protein. The cultures can then pulsed with tritiated thymidine (1 ~lCi/well) on day 3 and harvested onto glass fiber filters on day 4.
5 Peptides stim~ ting tritium incorporation at least 2-fold over the medium control are defined as cont~ininf~ T cell epitopes naturally exposed to the T cells when presented with the entire protein (i.e., the peptides comprise at least one minor or immunodominant epitope). Those peptides stim~ ting tritium incorporation of less than 2-fold above the medium control either do not contain a T cell epitope or contain a cryptic epitope (i.e., an epitope not normally 10 exposed to T cells when the entire protein is presented). To confirm the presence of a cryptic epitope in these peptides, T cell lines can be established by c~ rin~ peripheral blood white cells from the same individual in the presence of each peptide separately to establish peptide-reactive T cell lines. The "rested" T cells can then be challenged with each peptide and the I2~ I protein. A peptide which comprises at least one cryptic epitope is capable of 15 stim~ ting the proliferation of the T cell line in the presence of the peptide or the entire protein at a level at least 2-fold above the medium control or is capable of tolerizing T cells.

I~QUIV~T ,Fl~TS
Those skilled in the art will recognize, or be able to ascertain using no more than 20 routine experiment~tion~ many equivalents to the specific embo-liment~ of the invention described herein. Such equivalents are intended to be encomp~secl by the following claims.

94/27634 ~ ~ 6 43 2 6 PCT/AU94/00292 SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: lN~'l'l'l'U'l'~ FOR CHILD HEALTH RESEARCH
(B) STREET: P.O. BOX 885 (C) CITY: WEST PERTH
(D) STATE: W~:~l~N AUSTRALIA
(E) COUNTRY: AUSTRALIA
(F) POSTAL CODE (ZIP): 6872 (ii) TITLE OF LNv~N-llON: CRYPTIC PEPTIDES FOR USE IN IN~U~1NG
IMMUNOLOGIC TOLERANCE

(iii) NUMBER OF S~QU~N~S: 2 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy diRk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII TEXT

(V) ~UK~NL APPLICATION DATA:
(A) APPLICATION NUMBER:
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(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/072,832 (B) FILING DATE: 2-JUN-1993 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MANDRAGOURAS, AMY E.
(B) REGISTRATION NUMBER: 36,207 (C) REFERENCE/DOCKET NUMBER: IMI-037CPPC

W O 94/27634 ~ ~ ~ 4 3 2 ~ PCT/AU94/00292 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 227-7400 (B) TELEFAX: (617) 227-5941 5 ( 2) INFORMATION FOR SEQ ID NO:1:

( i ) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 834 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..738 (Xi) S~QU~N~ DESCRIPTION: SEQ ID NO:l:

Lys Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Ly& Thr Gln Phe Asp Leu Asn Ala Glu Thr Asn Ala Cy8 Ser Ile Asn Gly Asn 30 Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala W 0 94/27634 2 ~ PCT/AU94/00292 Ala Thr Glu Ser Ala Tyr Leu Ala His Arg Asn Gln Ser ~eu Asp Leu 5 GCT GA~ CAA GAA TTA GTC GAT TGT GCT TCC CAA CAC GGT TGT CAT GGT 288 Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly 0 Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro CCA AAT GCA AAC A~A ATT CGT GAA GCT TTG GCT CAA ACC CAC AGC GCT 480 Pro Asn Ala Asn Lys Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Arg His Tyr 30 Asp Gly Arg Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr 180 185 lgO

His Ala Val Asn Ile Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr _ W O 94/27634 '~ 1 ~ 4 3 ~ ~ PCT/~U94100292 TGG ATC GTA CGA AAC AGT TGG GAT ACC AAT TGG GGT GAT A~T GGT TAC 672 Trp Ile Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro 10 Tyr Val Val Ile Leu ATTTAAAATC AAAATTTTTT AGAAAATGAA TA~ATTCATT CACAAAAATT PAa~U~aA 834 (2) INFORMATION FOR SEQ ID NO:2:

(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 245 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:2:

LYB Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr 30 Gln Phe Asp Leu Asn Ala Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala WO 94/27634 216 43 PCT/AIJ94/00~9 Ala Thr Glu Ser Ala Tyr Leu Ala His Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Ala Asn Lys Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Ary His Tyr Asp Gly Arg Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro 35 Tyr Val Val Ile Leu

Claims (29)

1. A method of inducing immunologic tolerance in a subject to a protein antigen comprising administering to the subject a tolerizing amount of a composition comprising at least one cryptic peptide derived from the antigen and a pharmaceutically acceptable carrier.

2. A method of claim 1 wherein the subject is a mammal.

3. A method of claim 2 wherein the mammal is a human.

4. A method of claim 1 wherein the composition is administered orally.

5. A method of claim 1 wherein the protein antigen is an allergen.

6. A method of claim 5 wherein the allergen is of a genus selected from the group consisting of: the genus Dermatophagoides; the genus Felis; the genus Ambrosia; the genus Lolium; the genus Cryptomeria; the genus Alternaria; the genus Alder; the genus Betula; the genus Quercus; the genus Olea; the genus Artemisia; the genus Plantago; the genus Parietaria: the genus Canine; the genus Blattella; the genus Apis; the genus Periplaneta; and the genus Sorghum.

7. A method of claim 6 wherein the allergen is of the species Dermatophagoides pteronyssinus.

8. A method of claim 7 wherein the allergen is Der p. I.

9. A method of claim 1 wherein the protein antigen is an autoantigen.

10. A method of claim 9 wherein the autoantigen is selected from the group consisting of: insulin; myelin basic protein; rh factor; acetylcholine receptors; thyroid cell receptors; basement membrane proteins; thyroid proteins; PM-1; glutamic acid decarboxylase (64K); and carboxypeptidase H.

11. A method of claim 2 wherein the mammal is a mammal sensitized to the protein antigen.

12. A method of claim 1 wherein the composition further comprises a peptide comprising an immunodominant epitope derived from the protein antigen.

13. A method of inducing immunologic tolerance in a subject to an allergen comprising orally administering to the subject a tolerizing amount of a composition comprising at least one cryptic peptide derived from the allergen and a pharmaceutically acceptable carrier.

14. A method of claim 13 wherein the allergen is of a genus selected from the group consisting of: the genus Dermatophagoides; the genus Felis; the genus Ambrosia; the genus Lolium; the genus Cryptorneria; the genus Alternaria; the genus Alder; the genus Betula; the genus Quercus; the genus Olea; the genus Artemisia; the genus Plantago; the genus Parietaria; the genus Canine: the genus Blattella; the genus Apis; the genus Periplaneta; and the genus Sorghum.

15. A method of claim 14 wherein the allergen is of the species Dermatophagoides pteronyssinus.

16. A method of claim 15 wherein the allergen is Der p I.

17. A method of claim 13 wherein the subject is a human.

18. A method of claim 17 wherein the subject is a human sensitized to the allergen.

19. (Amended) A composition for inducing immunologic tolerance in a subject to a protein antigen, the composition comprising a tolerizing amount of a cryptic peptide derived from the protein antigen and a pharmaceutically acceptable carrier.

20. A composition of claim 19 in a form suitable for oral administration.

21. A composition of claim 19 wherein the protein antigen is an allergen.

22. A composition ot claim 21 wherein the allergen is of a genus selected from the group consisting of: the genus Dermatophagoides; the genus Felis; the genus Ambrosia; the genus Lolium; the genus Cryptomeria; the genus Alternaria; the genus Alder; the genus Betula; the genus Quercus; the genus Olea; the genus Artemisia; the genus Plantago; the genus Parietaria; the genus Canine; the genus Blattella; the genus Apis; the genus Periplaneta; and the genus Sorghum.

23. A composition of claim 22 wherein the allergen is of the species Dermatophagoides pteronyssinus.

24. A composition of claim 23 wherein the allergen is Der p I.

25. A composition of claim 19 wherein the protein antigen is an autoantigen.

26. A composition of claim 25 wherein the autoantigen is selected from the groupconsisting of: insulin; myelin basic protein; rh factor; acetylcholine receptors; thyroid cell receptors; basement membrane proteins; thyroid proteins; PM-1; glutamic acid decarboxylase (64K); and carboxypeptidase H.

27. A composition of claim 19 further comprising a tolerizing amount of a peptide comprising an immunodominant epitope derived from the protein antigen.

28. A composition for inducing oral tolerance in a subject to an allergen, the composition comprising a tolerizing amount of a cryptic peptide derived from the allergen and a pharmaceutically acceptable carrier, in a form suitable for oral administration.

29. A composition of claim 28 further comprising a tolerizing amount of a peptide comprising an immunodominant epitope derived from the protein antigen.