CZ122697A3 - Preparations and methods of treating disseminated sclerosis - Google Patents

Abstract

The present invention provides isolated peptides and combinations of peptides derived form myelin autoantigens such as MBP, MOG, PLP, and MAG suitable for treating multiple sclerosis, including prophylactic and therapeutic compositions and methods for preventing or treating multiple sclerosis. Preferred compositions of the invention comprise at least one isolated, purified peptide, free from all other polypeptides or contaminants, the peptide comprising an amino acid sequence, the myelin autoantigen which has T cell activity. A therapeutic composition of the invention is capable of down regulating the autoantigen specific immune response to the myelin autoantigen in a population of humans suffering from, or susceptible to multiple sclerosis, such that disease symptoms are reduced, eliminated, or reversed and/or the onset or progression of disease symptoms is prevented or slowed. Additionally, compositions and methods of the instant invention when administered in an advanced stage of disease, reverse ongoing paralysis or other signs of disease when administered during the acute phase of disease or prevents relapse when administered during remission.

Description

This application is in part a continuati on-in-part application no. USSN 08/328224, filed October 25, 1994. It is also a partial continuation application of USSN 08/300811, filed September 1, 1994, which is a partial continuation application of USSN 08/116824, filed September 3, 1993. This application is also partially a continuation application of USSN 08/241246, filed May 10, 1994. These applications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Autoimmune diseases are a major human health problem and are relatively little researched. Since there is no apparent directly responsible microbe or virus, the prevention, treatment and diagnosis of these diseases must be based on their etiology. This always involves a complex series of reactions of endogenous metabolic intermediates, structural components, cells, etc. However, with the nature, auto-immune disorders, there is always the notion that at least one autoantigen must be present when a sequence of events that produce symptoms occurs. Autoimmune demyelinating diseases such as multiple sclerosis (MS) are no exception.

MS usually occurs in the form of recurrent seizures reflecting lesions in the central nervous system (CNS). Seizures are returning, retreating and returning for many years seemingly random. The frequency of outbreaks is greatest within the first 3 to 4 years, but after the first seizure, which may be so mild that it escapes the attention of the physician and can hardly be remembered, it may not follow for 10 to 20 years. varies significantly between patients. Remi.se may be complete, especially after early seizures; often, however, remission is incomplete, and as one seizure after another follows, a gradual downward procedure follows with increasing persistent deficit. The clinical picture of MS is determined by the location of foci of demyelination in the CNS. Classical features include, impaired vision, nystagmus, poor pronunciation, decreased vibration perception and postural sense, ataxia and jitter of the limb before touch, weakness or paralysis of one or more limbs, falling out of feeling and bladder problems. Criteria for the diagnosis of clinically certain MS must include a reliable history of at least two episodes of neurological deficit and objective clinical signs of lesion on more than one CNS site. There is no known effective treatment for MS. Current therapeutic efforts are directed at improving acute episodes and preventing relapses or disease progression (Harrison's Principles of Internal Medicine 1, 12th Ed., Vol. 2, pp. 2038-2043, McGraw Hill, 1991).

The commonly used animal model for human multiple sclerosis is experimental allergic encenphalomyelitis (EAE), a demyelinating central nervous system disease that can be induced in susceptible strains of mice by immunization with myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte protein (M or synthetic peptides based on the sequences of these proteins associated with myelin. MBP is one of the presumed autoanf.i genes in multiple sclerosis (MS) and has been cpitopically mapped as in human

(1990), and in the rodent (Zamvil et al., Nature, 324: 258-260 (1986)) system. Peptides believed to contain at least one MBP (myelin basic protein) T cell epitope have been identified in WO 93/21222, EP 0 304 279, WO 91/15225, Ota et al., Letters to Natnre, 346: 183-187 (1990), Wucherpfenig et al., J. Exp. Copper.,

170: 279-290 (1994). Other peptides containing the MBP T cell epitope are identified in applications which are incorporated herein in their entirety by reference to USSN 08/328224, filed January 25, 1994, and (ISSN).

08/241246, filed May 10, 1994. MOG peptides with T cell activity are identified in USSN 08/300811, incorporated herein by reference.

Proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) are also considered to be possible autoantigens in multiple sclerosis. Studies describing the pathogenesis of autoimmune response to PLP in multiple sclerosis have been published in Trotter et al., J. Neuroimmunol. 33: 55-62 (1991); PUP T cell epitopes have been described in Pelfrey et al., -J. Nevroimmuno 1. 46: 3342 (1,993). Studies describing MAG as a potential autoantigen in multiple sclerosis are published in Johnson et al., J. Neuroimmunol. 13: 99-108 (1986).

Experimental autoimmune oncephalomyelitis (EAE) is a CD4 + T cell-mediated autoimmune disease that has some. its clinical and histological features resemble multiple sclerosis and serve as an experimental model for this and other autoimmune diseases. EAE is an inflammatory disease of the central nervous system that leads to paralysis and other neurological abnormalities. It is typically induced by purified myelin proteins and peptides. However, the EAE model is used extensively to investigate mechanisms of autoimmunity and to research potential therapeutics for autoimmune diseases.

As early as 1965, it was observed that EAE could be treated by administering MBP in a nonencephalitogenic form, presumably by inducing immunological unreactivity or tolerance (Alvord,

33, Levine, S.E. a. , 198 7 Cl .i n. Exp.

the observations were many researchers,

E.C. et al., Ann. NY Acad. Sci., 1965, 122: 3: d., Science, 1368, 161: 1155 (Bernard, C. C. Immunol., 1977, 29: 100). This earlier in the last few years added

newborns or 1 9 7 7 Cl i n. Exp. . Exp. Copper., 1989, Acad. Sci., 1991,

showing that administration of MBP and M1 peptides to adults prevents EAE (Bernard, C.C.A., 197

Immunol., 1971, 29: 100; Clayton, J.P. et al., J. Ex 169: 1681, Smekek, P.E. and d., Why. Natl: 88: 9633, Guar, A. et al., Science 1992, 258: 1491, Metzlcr, B.

d., Int. Immunol. 1993, 5: 1159, Miller, A. et al. (J.

cience,

Neuroiinnwnol. 1993, 46:73, Critchfield, J.M, et al.,

94, 263: 1139, Miller, A. et al., Proc. Naťl. Acad. Sci. USA,

1992, 89: 421). These studies suggest various routes of administration, including subcutaneous, intraperitoneal, intranasal, administration. Immunological induction: vortices by intravenous administration of peptides have been demonstrated in various antigen systems. Among the many intravenous and oral non-reactivities described herein for adults, there are limitations to the clinical utility of oral, enteral or aerosol administration of autoantigens, such as the inability to characterize the active ingredient of a therapeutic formulation once introduced into the stomach due to subsequent enzymatic degradation in the stomach. Using these methods, therefore, it is difficult to achieve predictable and reproducible therapeutic effects, not to mention the possibility of adverse side effects due to further processing of the therapeutic by an organism that may not be predictable.

The mechanism of disease prevention or induction of tolerance has been attributed in most of these cases to clonal anergy (Gaur et al., Supra), peripheral deletion (Crichfield et al., Supra), or other forms of antigen-specific tolerance. However, another mechanism by which orally administered MBP and MBP peptides can inhibit EAE appears to be TGE-β-mediated suppression (Mnllcr et al., See above, Proc. Nat'l Acad. Sci.}. MBP peptides as well as substituted MBP peptide analogs were investigated as alternative therapeutics for EAE in PLJ mice, B10.P1, and (PLJ x 3aL) Fl.Enunodom.i.nant.n.im encephalitogenic peptide for each of these strains is MBP Acl-11. which is recognized in relation to

AobAfi (Wraith, D.C. et al., Supra). MBP Acl-11 has been extensively studied by substitution analysis and its requirements for T cell recognition and binding to major histocompatibility complex (major histocompatibility and comp.lex, MHO) have been determined. The side chains of residues 3 and 6 mainly contribute to T cell recognition and side chains of residues 4 and 5 predominantly for binding to

MHC. The binding of MBP Acl-11 to Αα ^υ Λβ ^υ can be dramatically improved by various amino acid substitutions at residue 4, including alanine (Acl-1.1 [4A]) and tyrosine (Acl-11 [4Y]) (Wraith, DC et al.) supra, Fairchild, PJ et al., Int. Immunol., 1993, 5: 1151).

Substitution of residue 4, especially tyrosine, appears to improve the stability of peptide-MHC complexes formed with these peptides. Acl11 [4A]) and Acl-11 [4Y] retain the T cell receptor contact residues (TCR) required for antigenic sensation, and are more effective than MB1 Acl-11 in stimulating MBP-specific T cells in vitro . A.cl-11 [4Y] is enoepalogenic also in vivo (Acl11 [4A] is little ouvho-philtogenic for unknown reasons). It has been shown that both Acl-11 [4A] and Acl-Π [4Y] prevent EAE, and are thought to act by antigen-specific mechanisms (Smilek et al., Supra, Wraith, D.C. et al., Supra).

viz

In previous studies, MBP peptides or peptide analogs, administered in incomplete adjuvant just prior to disease onset, prevented the development of EAE (Smilek et al., Supra, Gaur et al., Supra). In a separate study using lymphocytes from MBPspecific TCR transgenic mice, adoptively transmitted EAE was prevented by early and aggressive administration of intravenous MBP prior to the onset of clinical features (Crisis et al., Supra).

These studies suggested that EAE could be prevented by injecting MBP peptides after enccphalitogenic T cells were activated, but were not concerned with whether this approach could reverse progressing paralysis (and probably active central nervous system inflammation) or prevent relapses after remission. In addition, in some other prior experiments, extremely high doses of MBP or MBP peptide were required for effective treatment. The present inventors were the first in their earlier application USSN 08/328224, filed October 25, 1994, which they continue to describe, reversing paralysis and preventing relapses after remission. Since the filing of the prior application of the present inventors in the art that MBP-derived injections is paralysis in EAE (Karin has also been discovered by many other workers by administering certain peptide analogs surprisingly, the progressing Exp. Mod., 180: 2227-2237 4).

The invention overcomes the above drawbacks and provides novel peptides, compositions and methods for treating multiple sclerosis using preparations comprising at least one peptide with an amino acid residue sequence that includes MBP T cell activity. Further, the invention addresses the unresolved problem that, in order to be generally applicable, the treatment of multiple sclerosis using peptides or peptide analogs must be effective even when applied later in the course of the disease or in its advanced stage, either during remission or during relapses following an unwanted immune response procedure.

It is therefore an object of the present invention to provide peptides and peptide combinations useful as therapeutics for multiple sclerosis including prevention of disease onset. It is a further object of the invention to identify prophylactically and therapeutically effective dose regimens and methods of administering the identified proteins, peptides, and peptide analogs for effective treatment of MS. It is further an object of the invention to identify successful treatments for late stage MS, to prevent relapses, stop disease and / or reverse the progression of MS,

SUMMARY OF THE INVENTION

The present invention provides isolated peptides and combinations of peptides derived from myeles and novel autoantigens, such as MBP, MOG, P3.P and MAG, suitable for the treatment of multiple sclerosis, including prophylactic and therapeutic agents and methods for the treatment of multiple sclerosis. Preferred formulations of the invention include at least one isolated purified peptide substantially free of all other polypeptides and impurities, comprising the myelin autoantigen amino acid sequence having T cell activity. The therapeutic composition of the invention is capable of attenuating an autoantigen-specific immune response to myelin autoantigen in a population of people suffering from or susceptible to multiple sclerosis such that the disease symptoms are suppressed, eliminated or reversed and / or the onset or progression of disease symptoms is prevented or delayed. The compositions and methods of the invention, when applied at an advanced stage of the disease, reverse the progressing paralysis or other signs of the disease when applied during the acute phase, or prevent relapse when applied during remission.

The knowledge available to those skilled in the art is the patent and scholarly literature cited herein. US patents cited herein, PCT published applications and other publications are incorporated herein by reference.

The present invention provides isolated peptides and combinations thereof derived from myelin autoantigens useful for the treatment of multiple sclerosis, as well as therapeutic compositions and methods for the treatment of multiple sclerosis. As used herein, the term "treating multiple sclerosis or" treating multiple sclerosis includes prophylactic treatment of MS-prone mammals, treatment at the onset of MS and treatment of all "advanced stages of MS" including relapsing-remitting MS, chronic progressive MS, primary progressive MS and benign MS. The therapeutic compositions of the invention include at least one purified peptide substantially free of all other proteins or impurities and comprising a defined amino acid residue sequence of myelin antigen having T cell stimulatory activity, which peptide may also be an isolated peptide. As used herein, the term "isolated" refers to a peptide which is free of other polypeptides, non-readers, starting materials or other substances and which is not conjugated to any other molecule.

The term "peptide" according to the invention refers to an undefined sequence of amino acid residues that are less than the amino acid residues of native B1. 1 Planar antigen. Preferably, the length of the peptide of the invention is at least about seven, preferably at least about 12 to 40, and in particular at least 13 to 30 amino acid residues, wherein the peptide, when derived from a protein antigen, contains less amino acids than the whole protein antigen, preferably no more more than 75% of the celeno protein antigen amino acid residues. The peptides used according to the invention have T cell activity. A peptide having "T cell activity may have one or more of the following characteristics: a) ability to induce a T cell response, e.g., stimulation (ie, proliferation or secretion of lymphokine), b) ability to induce T cell non-reactivity or decreased reactivity of respective T cell subpopulations, these cells do not participate in stimulating an immune response to an attacking autoantigen (e.g., prostreon). An anergy, tolerance, or apoptosis that modifies the profile of lymphokine secretion as compared to exposure to naturally occurring autoantigen; d) the ability to induce T suppressor cells; autoimmune disease by any mechanism or fl is derived from a non-participating antigen and has the ability to detect suppressor T cells at the site of myelin autoimmune attack, which in turn leads to suppression of immune responses at the site of myelin autoimmune attack.

Peptides comprising at least one T cell epitope have T cell activity and are capable of eliciting a T cell response, such as T cell stimulation (ie T cell proliferation or lymphokine secretion!) And / or capable of attenuating an autoantigen specific T cell response, which may lead to autoantigen-specific T cell non-reactivity or reduced levels of autoantigen-specific T cell reactivity, dpi top T cells is an essential element or smallest unit of T cell receptor recognition, where the epitope includes amino acids necessary for receptor recognition. These T cell epitopes are believed to trigger early T-cell immune response cases by binding to the respective HLA molecule on the surface of the antigen-presenting cell and stimulating the relevant T cell subpopulation. These cases lead to T cell proliferation, p lymphokine secretion, local inflammatory reactions, the arrival of additional immune cells in place, and activation of the K cell cascade leading to antibody production. In the case of an autoimmune disease, the antibodies produced are anti-autoantigen antibodies, such as MBP, resulting in clinical signs of autoimmune disease.

Peptides with a defined amino acid composition, including T cell cpitopes, can be identified for any myelin autoantigen including MBP. One method involves splitting the protein antigen into non-overlapping or overlapping peptides of the desired length and synthesizing, purifying and testing these peptides to determine whether they include at least one T cell epitope, by various assays (e.g., T cell proliferation assays, lymphokine secretion assays, and T cell non-reactivity studies). In another method, the algorithm estimates those peptides that are likely to include epitopes of T cells, and then the algorithm-estimated peptides are synthesized, purified, and tested in T cell assays or in v.ivo studies to determine whether these the estimated peptides induce T cell proliferation or lymphokine secretion, or T cell unreactivity, and are therefore likely to contain T cell epitopes. As discussed in many of the documents cited above, human T cell activity can be assayed by culturing cells derived from an individual susceptible to an autoantigen such as MBP with a peptide derived from that antigen and determining whether T cell proliferation is responding to the peptide, measured, for example, by uptake of tritiated thymidine by cells. Stimulation indices for T cell response to peptides can be calculated as the maximum number per minute (CPM) in response to the peptide divided by the control CPM. The T cell stimulation index (S.I.) equal to or greater than twice, preferably three times the background level is considered to be "positive". Positive results are used in the analysis of the potential therapeutic efficacy of the peptides discussed below and in Example 1. Preferred peptides used in the invention include at least one

PC p J., L · PJ p cells.

; ui ιλ j Λ L li PJ ci ..i Pd i i 4tT * “l 1— r—. T T -Ί 1—1 · -. V “· 1 4- Γ-. VS 1 '1 T vi in cliipujju v i Pdp .i. i. u. x

One of the algorithms for finding peptides with T cell stimulatory activity is described in Rothbard, l ^st Forum in Viroloay,

Annals of the Pasteur Institute, pp. 518-526 (December 1986),

Rothbard and Taylor, bmbo, 7: 93-100 (1988), and EP 0 304 279. These documents describe the definition of a general scheme (algorithm) for binding peptides to MHC class II, its statistical significance and the correlation of the scheme with known epitopes of I cells, as well as its successful use in finding previously unidentified T cell epitopes of various protein antigens and autoantigens. The general scheme for a peptide known to bind to MHC class II, as set forth in the above-mentioned documents, appears to contain a linear structure consisting of a charged or glycine charged amino acid followed by two hydrophobic residues. After determining whether the peptide conforms to the general scheme, it can be tested for T cell reactivity. Other algorithms that have been used to find T cell epitopes in previously undefined proteins include the algorithm described in Margalit et al., J.

Immunol. 138: 2213-2229 (1987), which amphipathic spirals.

is based on the model

Furthermore, peptides comprising "cryptic T cell epitopes that are also useful in the methods and compositions of the invention can be determined. Cryptic T cell epitopes are those determinants in the protein antigen that, due to the processing and delivery of the native protein antigen to the relevant MHC molecule, are not normally detected by the immune + + ι · ι τη <-im D nrib η rl o? -p Vn vt-. n η τ <'ί L · π im! β L-: r -r-, ^;; Γ 1λπτ ^ '\ U Ί h ttc 5 1' y υ ι,, U p / i „_1_ <A ', 1 i 1 1 x UA | J-. _>, L. J _ J .. y l_ _l_ <. · J *. It is able to induce T cell non-reactivity and, when the subject is primarily immunized with this peptide, T Punks obtained from the subject by in vitro proliferation in response to the peptide or protein antigen from which the peptide is derived. Peptides that comprise at least one cryptic epitope of T cells derived from a protein antigen are referred to herein as "cryptic peptides." To confirm the presence of cryptic T cell epitopes, a T cell proliferation assay, as known in the art, must be used in which antigen-sensitized cells are cultured in vitro in the presence of each peptide separately to identify T cell cell lines reactive with the peptide. A peptide is considered to contain at least one cryptic T cell epitope when the T cell cell line can be identified with the peptide and the T cells are capable of proliferation upon exposure to the peptide and protein antigen from which the peptide is derived.

In addition, it is not necessary that the peptide be derived from the sequence used by the method according to the amino acids of the myelin-autoantant gene, such as MBP. The method of the invention may be. any peptide comprising a defined sequence of amino acid residues that is capable of attenuating an antigen-specific immune response to the myelin autoantine gene is used. For example, peptides may be synthesized, including a defined amino acid sequence not based on the amino acid sequence of a myelin antigen that are nevertheless capable of attenuating an antigen-specific immune response, for example a peptide simulates a T cell epitope of myelin autoantigen and causes suppression of an immune response to myelin autoantigen for other reasons, for example because it is derived from a non-participating antigen. Without wishing to be bound by theory, it is believed that non-participating anti genes, which are also tissue-susceptible (but not a target of immune or autoimmune attack) may have. the ability to initiate suppressor T cells at the immune attack site, which may lead to the suppression of immune responses at the immune attack site (for example, the affected "own" i.can in the case of autoimmune disease or nasal mucosa, skin and lungs in case of allergy). Non-participating antigens include, but are not limited to, portions, antigen or autoantigen which do not themselves have an immune attack target and which have suppressor activity at the immune attack site. As used herein, the term "myelin antigen" or "myelin autoantigen" includes non-participating antigens that may have suppressor activity at the site of a myelin autoimmune attack.

Further according to the invention may be used either a compound act on that mimics a peptide capable of down regulating an antigen immune response to the myelin autoantigen specific (e.g., peptide bond _(NH-u [-CO-NCH 3] c _{T (1)} a peptidomimetic). Such compound is not may consist of other peptide bonds, but may be linked by other bonds (e.g., thioester bonds, reduced bond analogs, amide bond isosterases), provided that the non-peptide compound simulates a peptide capable of attenuating an antigen-specific immune response to the antigen in question Peptidomimetics may be based on any of the peptides of the invention, but may include, for example, an analgesic (e.g., 1 ad N -r, ethyl and radial ligaments and reduced bond analogs (NH-C _a2 [- CH.-NH-] C ₍ -n) peptide bonds.

instead of one or more normal ones

Once peptides containing a T cell epilope have been identified, it is also possible to modify the structure of these peptides for use in the invention for pressure purposes such as increasing solubility (particularly desirable if the composition is to be injected), increasing therapeutic or preventive efficacy (see further discussion) peptide analogs with increased MHC binding) or stability (e.g., ex vivo durability and resistance to proteolytic degradation in vivo) or to facilitate peptide synthesis. A modified peptide or peptide analog in which the amino acid sequence has been altered compared to the native protein sequence can be obtained, from which it is derived or compared to the unmodified peptide from which the modified peptide is derived, for example, by substitution, deletion or addition of amino acids, to modify immunogenicity, improve the solubility of the peptide, or increase the ease of peptide synthesis (e.g. matte peptide synthesis).

For example, the peptide may be modified to at least retain, if not improve, the ability to attenuate the autoimmune response in MS (e.g., by inducing T cell non-reactivity or reducing 1 cell reactivity) while retaining the ability to bind. MIIC proteins. In this case, critical binding residues for the T cell receptor can be determined by known methods (for example, by substituting each residue and determining the presence or absence of T cell reactivity). Residues that have been shown to be necessary for T cell receptor interaction may be modified by replacing the necessary amino acid with another, preferably similar, amino acid residue (conservative substitution) whose presence has been shown to increase, decrease, but not eliminate or affect T cell activity. Alternatively, those amino acid residues that are not necessary for interaction with T cell receptors may be modified by replacement with another amino acid, its incorporation may increase, decrease, but do not eliminate or affect T cell activity but do not eliminate binding to relevant MHC . Furthermore, the peptides of the invention may be modified by replacing the amino acid that has been shown to be necessary for interaction with the MHC protein complex by another, preferably similar, amino acid residue (conservative substitution) whose presence has been shown to increase, decrease, but not eliminate or affect T cell activity . Furthermore, amino acid residues that are not necessary for interaction with the MHC protein complex may be modified by replacement with another amino acid, its incorporation may increase, affect or decrease but not eliminate T cell reactivity. Non-limiting examples of preferred amino acid substitutions for non-essential amino acids include alanine, yamic acid or methyl amino acid substitutions. For example, WO 94/06628 discloses substituted peptides in which virtually any residue may be different from the substrate / substrate and the conjugates are in the amine. amino acids, ammo acids not found in nature or alanine, and yet the substituted peptide is capable of attenuating antigen

specific immune response. Another example is the work of Karin a d., J. Exp. Afed. 18C ): 2227-2237 (1,994), which discloses s t.udí e using / nnunodominant rat epitope MBP sc ickvenci amino acids 87-99, shown in FIG. 14. These 3 Π 3 1 0 Q 7 predstavuj i series 12 substituted s : Θ K V Γ i C Θ 8 7 - 9 9 e with cd The original peptides 67-99 differ j edinou substitution / daninem \ ’To each peotide position 87-99. Tvto studí ^c '

were performed to determine the predicted sites of interaction of the immunodominant peptide with MHC and TCR in a Lewis rat, as well as to find modified peptides with improved desirable characteristics for potential therapeutic use. These studies showed that replacement of lysine (K) at position 91 of peptide 87-99 with alanine (A) (see 87-99 [K> A1 in Figure 14) prevented and reversed ΕΆΕ in Lewis rats. Based on this information, one would expect that lysine (K) was replaced by aianine (At at the site of a single lysine (K) present in peptides that contain in their amino acid sequence 87-99, i.e. MBP-2, MBP- 2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5 and MBP-2.6, all shown in Figure 2, would also increase the T cell activity of the substituted peptides, for example, alanine (A) substituted MBP-2.1. for lysine (Ki at position 10 of the MBP-2.1 peptide (i.e., IENPVVHEFANIVTPRTPPPSQGKi) may have increased T cell activity resulting in improved therapeutic properties over the "parent MBP2.1 peptide".

In order to increase stability and / or reactivity, the peptides may also be modified to contain one or more of the natural butler variations in the antigen sequence.

amino acids of the protein before ymorf isms, resulting

To obtain a modified protein or peptide in the range, an oyz can be an amino acid,

NJ is also performed by substitution or ddi.ee with a non-natural amino acid or non-amino acid analogue. The peptides of the invention can be further modified using the polyethylene glycol (PEG) method of A. Sehon and co-workers (Wic et al., Supra) to produce a PEG-conjugated protein or peptide. PEG may be further added during chemical synthesis of a protein or peptide of the invention. Modifications of peptides or portions thereof may also include reduction / alkylation (Tarr in Methods of Proteinion, JE Sil et al., Humana Press, Clifton, 194 (1986), acylation (Tarr, supra), chemical notar r.

condensation with a suitable carrier (Mishell and Shiigi, ed., Sclect.

Mehod in Cellular Immunology, WHreeman, San Francisco, CA (1980), US Patent 4,939,239, or mild formalin treatment (Marsh, International Archives of Allergy and Applied Immunology, 41: 199-215 (1971)).

To facilitate purification and potentially increase the solubility of the proteins or peptides of the invention, reporter groups may be added to the peptide chain. For example, polyhistidine can be added to the peptide to purify the peptide by affinity chromatography on an ionized metal ion (Hochul, E. et al., Bi / Technol., 6: 1321-1325 (1988). If desired, specific endoprotease cleavage sites may be introduced between the reporter group and the amino acid sequence of the peptide to facilitate the isolation of peptides free of irrelevant sequences.To potentially assist, correct antigen processing of T cell epitopes in the peptide may be markedly or synthetically between regions. protease-sensitive canonical sites, containing at least one T cell epitope, can be inserted, for example, during recombinant construction of the peptide, pairs of charged amino acids, such as KK or RR, may be inserted between regions within the peptide. trypsin-type enzymes to form peptide portions containing one or more several T cell epitopes.

Another example of peptide modification is the substitution of cysteine residues preferably by serine, threonine, lucucine or glutamic acid to minimize dimerization through disulfide bonds. Further, the peptides may be modified to increase the solubility of the peptide for use in buffered aqueous solutions, such as pharmaceutically acceptable carriers or diluents, by adding functional groups to the peptide, the terminal portions of the peptide, or by not including hydrophobic regions in the peptide. For example, to increase. For example, charged amino acids or charged amino acid pairs or triplets may be added to the carboxyytic or amino acids at the end or both. Examples of charged amino acids include arginine (R), lysine (K), histidine (Η), glutamic acid (E) and aspartic acid (D). For ease of peptide synthesis, for example, for automated peptide synthesis, it may be desirable to delete or replace amino acids that make peptide synthesis more difficult or expensive. For example, if the N-terminal or C-terminal amino acid of the peptides is capable of cyclization or may be subject to degradation either during or after peptide synthesis, the amino acid may be deleted or replaced, or alternatively one or more additional amino acids may be added to block the less desirable amino acid. or carboxyterminal amino acids. The added amino acids may be derived from a native protein sequence or may be non-native amino acid residues. To enhance the above T cell activity, the peptides may be added to either the amino-terminal or carboxy-terminal end of the peptides, or both. These additional amino acids may be derived from the native protein sequence or may be non-native amino acid residues.

Preferably, the peptide formulations administered according to the invention include. a sufficient percentage. myelin autoantigen T cell epitopes (ie, at least about 20, preferably about 30, preferably about 40, and especially about 60% or more) of the total T cell reactivity to myelin autoantigen in a population of individuals who respond to an autoantigen and who have a They are included in the formulation such that the therapeutic regimen of administering the formulation to an individual with an MS of the invention results in a dampening of the autoimmune response of the MS. Several analytical schemes may be used to determine whether a peptide (a candidate therapeutic peptide of choice) or a combination of candidate peptides will contain a sufficient percentage of the total T cell reactivity of myelin autoantigen to attenuate the autoimmune response of MS in a substantial percentage of the MS population.

According to one analytical scheme (using MBP as an example), peptides containing T cell epitopes are sorted only.

the number of MBP microtiter culture lines responsive to epitope-containing peptides and the number of patients responsive thereto. Since the frequency of MBP-specific T cells in PBL of MS patients may be very low, it is often not possible to test all MBP peptides in each MS patient. Therefore, the following procedure is appropriate to determine the most appropriate therapeutic peptides as described in Example 1 below, PBLs are isolated from blood and cultures are initiated in 96 well microtiter plates. PBL is purified from fresh peripheral blood samples (approximately 7b mL) from patients with unambiguous MS using a PI cell density gradient. Microtiter cultures are initiated 2x10 ⁵ PBL per well and 10 µg / ml purified human spinal MBP in RPMI 1640 culture medium supplemented with 5% human serum AB, penicillin streptomycin and L-glutamine. From day 6-7, the cultures are supplemented with IL2. (20 U / ml) and IL4 (5 U / ml). After 11 to 13 days, the microtiter cultures are washed, resuspended in fresh medium and distributed into 12 fresh microtiter wells. Autologous frozen PBT is added as antigen-containing peanuts at ⁵ x 10 ⁴ PBL per well. Screening antigens are added in duplicate to 12 replicate wells from each microtiter culture. Medium is used as a negative control and purified human recombinant MBP at 10 pg / ml as a positive control. Each patient is also tested for reactivity with a maximum of 4 MBP peptides, each at a concentration of 1 υ μΜ. After 48 h, pulsations of 0.75 gCi of ³ H-thymine are performed and after 6 to 16 h pulsation is performed. Cultures were marked as positive for each peptide according to the following criteria: stimulation index greater than 3.0, CI'M change greater than or equal to 500, and standard deviation from mean less than CPM change. For analysis purposes, cultures are further termed &quot; peptide-positive &quot; only if they responded simultaneously to MBP and the peptide, and if they did not respond to more than one non-overlapping peptide. Groups of about 10 to 50 patients are preferably tested with each MBP peptide and each group of patients is tested with a maximum of four peptides. The peptides are then categorized according to the following criteria: 11 percent of MBP positive microtiter cultures in each patient group (total MBP reactivity) that were also positive for one of the MBP peptides, 2) percent of individuals responding to MBP in each group with at least one microtiter culture which was positive for one of the MBP peptides, wherein the individual responding to MBP is defined as a patient with at least one MBP positive microtiter culture. Individual peptides of interest are then selected if 1) they contain at least 5, preferably at least 10, and in particular at least 20% of the total MBP reactivity, and 2) the reactivity thereto is at least 20, preferably at least 30, preferably at least 40, especially at least 50 and in particular at least 60% of individuals responding to MBP. Optionally, other criteria for evaluating peptides, such as a positive index for a given peptide, may be considered. The positivity index represents both the intensity of the T cell responses to the peptide (SI1) and the frequency of the 1 'cell response to the peptide in the population of individuals responding to the myelin autoanth gene. For example, as shown in Figure 15, the positivity index 141-165 ( MBP-4) is approximately 2500, calculated based on the data presented in Example 1. The mean value of MBP-4 peptide 2.1 per MBP responder was multiplied by the percentage of MBP-4 responders in the MBP responder population.

Highly purified peptides substantially free of all other polypeptides and impurities, with defined sequence, amino acid residues comprising at least one T cell epitope used in the therapeutic compositions of the invention can be obtained synthetically by chemical synthesis using standard methods. Various methods of chemical peptide synthesis are known in the art, such as solid phase synthesis, which is fully or semi-automated in various commercially available peptide synthesizers. The synthetically produced peptides can then be purified, preferably to homogeneity, in particular to at least 90, preferably at least 95, and in particular at least 97% purity, substantially free of all other polypeptides and impurities by any of the many methods known in the art. literature for protein purification.

Especially desirable are the synthetically obtained peptides of the invention up to about 45 and preferably up to about 30 amino acid residues in length, as increasing the length may lead to difficulties in peptide synthesis. Peptides of greater length can be obtained by the recombinant DNA methods discussed below,

Peptides useful in the method of the invention may also be obtained by recombinant DNA methods in a host cell transformed with a nucleic acid sequence encoding a particular peptide. When obtained by recorabination methods, the host cells transformed with the nucleate acid encoding the desired peptide are cultured in a cell-friendly medium, and the isolated peptides may be purified from the culture medium or host cells or both by methods known in the art for peptide and protein purification, including ion exchange chromatography, ultrafiltration, electrophoresis, or immunopurification with antibodies specific for the desired peptide. Peptides obtained recombinantly may be isolated and purified, preferably to homogeneity substantially free of cellular material, other polypeptides, or culture medium, for use by the methods described above for synthetically obtained peptides.

Under certain limited conditions, the peptides may also be obtained by chemical or enzymatic cleavage of a highly purified full-length or native protein in which the chemical or enzymatic cleavage sites have been predetermined and the resulting cleavage product is reproducible. Peptides with defined amino acid sequences can be highly purified and isolated to purity substantially free of other polypeptides or impurities present in the enzymatic or chemical cleavage product by any of the procedures described above for the highly purified and isolated synthetically or recombinantly obtained peptides.

In addition, the peptides of the invention may be used in conjugates such as described in, for example, US Patent No. 130,297 (compositions with X being predetermined and d.), Wherein they are prepared therapeutically using the formula X-MHC-peptide or MHC-peptide. id-X, a functional group selected from toxins and labeling moieties, MHC is an effective portion of the MHC glycoprotein that is dissociated from the cell surface on which it normally rests, and "the peptide represents any of the peptides disclosed herein, particularly MBP or MOG, and particularly peptides 2 and 14,

Further preferred peptides of the invention comprise at least one T cell epitope of the complete protein, especially MBP or MOG. The peptides may contain tandem repeats of one epitope and / or more than one epitope.

In accordance with the methods described herein for identifying peptides containing MBP T cell activity (see discussion above and in Example 1 below), preferred peptides are MBP-derived peptides that are contemplated for therapeutic use, such as MBP-1, MBP-2, MBP -3, MBP-4 and MBP-5 shown in Fig. 2, or any portion or modification thereof. These peptides were tested for T cell activity as described in Example 1 and were shown to contain at least one epitope of T cells, as shown by the ratio of MBP positive microtiter cultures that were also positive for one of the MBP peptides (Figure 4a) and detectable. response to each of the preferred peptides in a significant percentage of MBP patients tested (Fig. 4b). The most reactive of these four peptides is MBP-4 (1.41-165), which accounts for 21% of the total MBP response and is detectable in 64% of tested MBP responders. Surprisingly, MBP-4 showed dramatically greater reactivity than the combined reactivities of MBP 141-160 and MBP 151-170, as shown in Fig. 3. The inventors of the present invention first identified this immunodominant peptide that appeared to contain multiple T epitopes cells. This novel peptide is particularly suitable for therapeutic use.

MBP-1.1, MBP-1.2, MBP-2.1, MBP-2.2, MBP2.3, MBP-2.4, MBP-2.5, MBP-2.6, and MBP-3.1 also appear to be suitable peptides for therapeutic use. These peptides are modified versions of MBP-1, MBP-2, and MBP-2, respectively. MBP-3, and are expected to have similar T cell activity to their respective "parent peptides." These peptides were

2!

modified by deletion and / or addition of amino acids in accordance with the methods of peptide modification described above, in particular for ease of synthesis.

Other peptides that have been shown to be immunodominant (i.e., have MBP T cell activity) or derived from peptides with known MBP T cell activity have been identified by the inventors or other artisans (see, e.g., USSN 08/328224, filing date) October 25, 1994, USSK

05/241246, filed May 10, 1994, WO 93/2122

EP 0 304

91 / 1522I

O:

Let tors ture

6: 1 8 2

Wuchterpfenning et al., J. Exp. Med., 170: 279-290 (1994), Martin et al., J. Immunol., (1990) 145: 540-548), Karin et al., J. Exp.

Copper. 180: 2227-2237 (1994)). Such peptides may also be suitable for therapeutic use in the compositions and methods of the invention, particularly in combination with the preferred peptides described above. Such peptides include, but are not limited to, all or part of the peptides, where the residue numbers correspond, to the amino acid residues of the human MBP protein shown in Figure 1 and having the individual amino acid sequences shown in Figure 14: 13-25, 31 -50, 61-80, 8292, 82-96, 82-97, 82-98, 82-100, 82-100, [P> Y], 83-100, 83-101,

84-97, 84-100, 85-100, 86-105, 87-99, 87-99 [91K> A], 88-100, 8899, 111-135, 1.22-140, 139-1.70, 1 41 -160, 142-1.66, 142-168, 1.461 6 0 and 15 3-17 preferably 13-25, 87-99, 87-99 [91K> A], 82-100, 82Vvh

The OOP > YJ shown in Figure 14. The peptides or preferred modifications preferably have similar or greater T cell activity in the same or greater percent of the patients tested and / or have similar or greater therapeutic efficacy in the methods of the invention than the "parent peptide from which the modified peptide was derived.

in 1 1 '--o' - j i i not parts of these

In one aspect, the invention provides therapeutic compositions comprising at least one peptide derived from myeiin antigen with T cell activity or a combination of peptides derived from myeiin antigen each having T cell activity and a pharmaceutically acceptable carrier or diluent. Preferred therapeutic compositions include a sufficient percentage of myelin autoantigen T cell activity so that when administered to an MS patient in a therapeutic regimen, preferably in a non-immunogenic form, they are capable of attenuating a myelin autoantigen-specific immune response in a population of people susceptible to such antigen-specific immune response. As used herein, the term "inhibiting" includes, but is not limited to, preventing the onset of disease symptoms, reducing the symptoms of multiple sclerosis caused by an antigen-specific immune response to MBP, or. other myelin autoantigen, in particular, reducing, turning, not progressing or relieving the symptoms. Non-progress may be characterized, but not limited to, (a) a shorter period of active disease or exacerbation, (b) less severe symptoms or disability, (c) delaying the progression of a disease in which the underlying health does not deteriorate so rapidly, (d) increasing the time between periods of active disease or exacerbation (e.g., a longer period of remission); (e) fewer relapses or exacerbations; and / or (f) slowing or stopping the progression of lesion loads detected by the MRI. Other assessment methods used by those of skill in the art include the Expanded Disability Status Scale (ED5S &gt;, Neurological Rating Scale, etc.), and the term "advanced stage" as used herein refers to any moment after clear clinical signs of an apparent disease, whether relapsing-remitting MS, chronic progressive MS, benign MS or primary progressive MS In addition to this definition, “advanced stage may include acute phases, remissions and exacerbations. As used herein, the terms generally refer to a condition in which a subject suffering from a disease (whether diagnosed or not) exhibits active symptoms or traits usually considered by those skilled in the art to be associated with a specific immune response characteristic of sclerosis. "Relapse refers to the acute phase that follows a remission. The term exacerbation, when used in the context, can also be interpreted as new and worsening symptoms or signs. “Symptoms are the indications of a disease the patient complains of. “Signs are indications observed or measured by a diagnostic. However, the terms flags and signs are used interchangeably herein unless otherwise indicated.

The therapeutic compositions of the invention preferably comprise at least one peptide or modified peptide or peptide analog comprising a T cell epitope and a pharmaceutically acceptable carrier or diluent. These preparations may preferably comprise an MBP peptide selected from the group of peptides: MBP-1, MBP-1.1, ΜΗΡΙ. 2, MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, MBP2.6, MBP-3, MBP-3.1, MBP-4, and MBP-5, preferably a peptide selected from groups comprising MBP-1.1, MBP-2.1, MBP-4 and MBP-5, and in particular MBP is an MBP-4 peptide.

The compositions of the invention may comprise at least two peptides (for example, a physical mixture of at least two peptides) each having a T cell activity and preferably comprising at least one T cell epitope of a myelin autoantigen such as MBP. These compositions may be administered in the form of a therapeutic composition with a pharmaceutically acceptable carrier or diluent.

A therapeutically effective amount of one or more of these compositions may be. to an individual suffering from MS administered simultaneously or sequentially. Preferred elements include at least one and preferably at least two peptides selected from the group consisting of MBP-1, MBP-1, MBPMB1

MBP-2.1, MBP-2.2, MBP-2.3, MRP-2.4, MBP-2.5, MBP2.6, MBP-3, MBP-3.1, MBP-4, and MBP-5, shown in Figure 2, preferably from the groups comprising MBP-1.1, MBP-1.2, MBP-2.1, MBP2.2, MBP-2.2, MBP-2.4, MBP-2.5, MBP-2.6, MBP-3.1, MBP-4 and MBP-5, and in particular from the group comprising MBP-1.1, MBP-2.1, MBP-4, and MBP5. In addition, the compositions of the invention may additionally comprise MBP-derived peptides comprising residues whose numbers correspond to the amino acid residues of the human MBP protein shown in Figure 1 and comprising the individual amino acid sequences shown in Figure 14: 13-2 5, 31-50 , 61-80, 82-92, 82-96, 82-97, 82-98,

32-100, 82-100 [100Ρ> Υ], 83-100, 83-101, 84-97, 84-100, 85-100, 86-105, 87-99, 87-99 [91Κ> Α], 88-100, 88-99, 111-135, 122-140, 139-170, 141-160, 142-166, 142-168, 146-160, and 153-170, and preferably comprise the following peptides: 13-25, 87-99, 87-99 [91K> A], 82100, 82-100 [100P> Y]. Preferably, the compositions of the invention comprise at least two peptides, wherein at least one peptide is MBP-4 and at least one peptide is selected from the group of peptides: MBP-1, MBP-1.1, MBP-1.2, MBP-2, MBP-2.1, MBP- 2.2, MBP-2.3, MBP-2.4, MBP-2.5, MBP-2.6, MBP-3, MBP-3.1, and MBP-5, as shown in Figure 2, and may further include at least one of the following peptides derived from MBP shown in FIG. 14: 13-25, 3150, 61-80, 82-92, 82-96, 82-97, 82-98, 82-100, 82-100 [100P> YJ, 83-100, 83-101, 84-97, 84-100, 85-100, 86-105, 87-99, 8799 [91K & A], 88-100, 88-99, 111-135, 122-140, 139-170 , 141-160, 142-168, 146-160 and 153-170, and in particular at least one of the following peptides: 13-25, 87-99, 87-99 [91K> AJ, 82-100, 82-100 [100P] > Yl.

Preferred compositions of the invention include the following peptides:

MBP-1, MBP-2, MBP-3, MBP-4, and MBP-5;

MBP-1.1, MBP-2.1, MBP-3, MBP-4, and MBP-5;

MBP-1.1, MBP-2, MBP-4, and MBP-5;

MBP-1, MBP-2.1, MBP-4, and MBP-5;

MBP-1, MBP-2, MBP-4, and MBP-5;

MBP-1.1, MBP-2.1, MBP-4, and MBP-5;

MBP-1.1, MBP-2.1, and MBP-4;

MBP-1, MBP-2.1, and MBp-4;

MBP-1.1, MBP-2, and MBP-4;

MBP-1.1, MBP-2.1, and MBP-5;

MBP-1.1, MBP-2.1, and MBP-3;

MBP-1 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6;

MBP-1.1 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6;

MBP-4 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6;

MBP-4 and a peptide selected from the group consisting of MBP-1 or MBP1.1;

MBP-1.1, MBP-4, and a peptide selected from the group consisting of 82-100, 82-100 [10OP> Y1, 87-99 and 87-99 [91K] AJ shown in Figure 14;

MBP-1.1, MBP-4 and a peptide selected from the group consisting of MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5 or MBP-2.6 shown in Figure 2;

MBP-1.1, MBP-5, MBP-4 and a peptide selected from the group consisting of 82-100, 82-100 [100P> Y], 87-99 and 87-99 [91K> A] shown in Figure 14;

MBP-1.1, MBP-5, MBP-4 and a peptide selected from the group consisting of MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6 shown in Figure 2;

MBP-1.1, MBP-5, MBP-4 and a peptide selected from the group consisting of 82-100, 82-100 [100P> YJ, 87-99 and 87-99 (91K> A) shown in Figure 14;

MBP-1.1, MBP-3, MBP-4 and a peptide selected from the group consisting of MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6, shown in Figure 2.

The invention also encompasses a treatment regimen comprising administering a combination of therapeutically effective peptides as a single treatment episode. These combinations of peptides may be administered simultaneously or sequentially as therapeutic compositions comprising only one peptide or several peptides. This treatment regimen does not necessarily include a physical mixture of more than one peptide, but involves a combination of peptides administered simultaneously or sequentially as a single treatment episode. Preferably, combinations of peptides also in the form of one or more preparations (each including one or more peptides) that can be administered simultaneously or sequentially as a single treatment episode include the following peptide combinations:

MBP-1, MBP-2, MBP-3, MBP-4, and MBP-5;

MBP-1.1, MBP-2.1, MBP-3, MBP-4, and MBP-5;

MBP-1.1, MBP-2, MBP-4, and MBP-5;

MBP-1, MBP-2.1, MBP-4, and MBP-5;

MBP-1, MBP-2, MBP-4, and MBP-5;

MBP-1.1, MBP-2, MBP-4 and MBP-5;

MBP-1.1, MBP-2, and MBP-4;

MBP-1

MBP-1

MBP-1

MBP-1

MBP-1

MBP-2

MBP-1

MBP-2

MBP-2.1

1, MBP-2 and MBP-2

1, MBP-2 and MBP-4;

and MBP-4;

i and MBP-5;

and MBP-3;

and a peptide selected from the group consisting of MBP-2, 2, MBP-2.3, MBP-2.4, MBP-2.5 or MBP-2.6;

and a peptide selected from the group consisting of MBP-2, 2, MBP-2.3, MBP-2.4, MBP-2.5 or MBP-2.6;

MBP-2.1

MBP-2.1

MBP-4 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6;

MBP-4 and a peptide selected from the group consisting of MBP-1 or MBP1.1;

MBP-1.1, MBP-4, and a peptide selected from the group consisting of 82-100, 82-100 (100P> ¥), 83-99 and 87-99 [91K> A] shown in Figure 14;

MBP-1.1, MBP-4 and a peptide selected from the group consisting of MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5 or MBP-2.6 shown in Figure 2;

MBP-1.1, MBP-5, MBP-4 and a peptide selected from the group consisting of 82-100, 82-100 [100P> Y], 87-99 and 87-99 [91K> A] shown in Figure 14;

MBP-1, MBP-5 MBP-4 a peptide selected from the group consisting of MBP-2.2 MBP-2.3 MBP-2. 4, MBP-2 .5 or MBP-2.6 shown in Fig. 2; MBP-1.1 MBP-5 MBP-4 a peptide selected from the group consisting of 82-100, 82-100 [100P> Y] 87-99; i 87-99 [91K> A] shown in Fig. 14; MBP-1, MBP-3 MBP-4 a peptide selected from the group consisting of MBP-2.2 MBP-2.3 MBP-2. 4, MBP-2 .5 or MBP-2.6 shown in Fig. 2.

Further, preferred formulations and preferred combinations of MBP peptides that can be administered simultaneously and / or sequentially may include any of the above formulations and additionally may further comprise a peptide comprising at least one T cell epitope derived from myelin oligodendrocyte protein (MOG), is another protein considered to be one of the autoantigens involved in multiple sclerosis

Immunol. (1976) 116: 1439-1446, Lebar and (1986) 66: 423-443, Linington and Lassman, J. Neuroiiniatinol. (1987:

17: 61-69, Lassman et al., Aot.and Neuropathol. (Berl) (1988) 75: 566576, and Sun et al., J. Immunol. (1991) 14 6:14 90-14 95). Peptides that may include MOG-derived T cell epitopes are described in LSSN 08/116824, filed September 3 (1993) and 1.JS3N (see Lebar et al., 7.

d., J. Exp. Tmmunol.

08 / 3008.1 1, ouLuiti administration of 1.9.1004 (they may be expected to be effective in the treatment of multiple sclerosis, prepared and / or administered in conjunction with the above compositions and combinations of peptides containing the T MBP cell epitope of the invention , are:

human MOG 1-13 GQFRV1GPRHPIR human MOG 103-115 HSYQEEAAMELKV human MOG 1-121 GQFRV1G PRII Pi RALGDE V

RLPCRTSPGKNATGMEVGWY R Ρ P F S RW H LY R NGK DQ D G D Q A1Έ Y GRT F, LL KDAIGE GK

VTLRIRNVRF5DEGGFTCFF

RDHSYQEEAAMELKVEDPFYW

Other epitope-containing MOG peptides were identified by the inventors using experimental analysis similar to that described above and in Example 1 (data not shown) and

include i 1] dský MOG 1-20 GQFRVTGPRHPIRALVGDEV, 1ids ký MOG í 1-30 PÍPALVGDEVELPCRISPGK, human MOG 2.1 -JO EL PG RI3 PGKNATGMEVGWY human MOG 31-50 NATGMEVGWYRPPFSRWHL, human MOG 141-160 TVGLVFLCLQYRLRGKLRAE, human MOG 151-170 YRLRGKLRAEIENLHRTFDP, human MOG 161-180 IENLHRTFDPHELRVPCWKI a human MOG 199-218 YNWLHRRLAGQFLEELRNPF.

The invention further encompasses modifications or analogs discussed above of the above-mentioned MOG peptides comprising an epitope of T cells that retain similar or greater T cell activity than the parent peptide from which the modification or analog is derived.

It is believed that formulations comprising both MOG and MBP peptides containing T cell epitopes, modifications thereof, analogs or demands thereof for oesophageal oasis and combinations of these peptides, which can be administered simultaneously or sequentially, have the advantage of maximizing buffering. effect on T cells specific for both MBP and MOG involved in autoimmune. MS response. In this way, a range of T cells that may be involved in an autoimmune response to either MBP or MOG leading to clinical manifestation of MS (demyelinating) can be targeted to attenuate and thus enhance the therapeutic effect of the compounds according to the methods of of the invention suitable peptides are to be formulated and containing an epitope of cells derived from other myelmic antigens considered to be autoantigens in MS (e.g., proteolipid protein (PLP) amyloin-glycoprotein (MAG)).

Therapeutic / prophylactic regimen of the invention (which results in reversing, preventing or delaying the onset of disease symptoms induced by the attacking auloantigen, or reducing, not progressing or ameliorating the symptoms induced by the attacking autoanligen, i.e., suppressing an autoantiger specific immune response.) comprising administering a therapeutic composition of the invention, comprising at least one isolated peptide derived from an autoantigen responsible for the condition being treated (e.g., MBP, MOG, PLP, MAG), in a non-immunogenic form. Without intending to be bound by theory, it is believed that the administration of therapeutic. of the composition according to the invention may a) induce T cell inactivity by the respective T cell subpopulations, so that they do not respond to the attacking antigen and do not participate in stimulating the immune response when exposed to the attacking protein antigen (via anergy or apoptosis), b) modify lymphokine secretion profile compared to c) cause subpopulations of 1 cells that are normally involved in the response to the attacking antigen to be withdrawn from the normal exposure sites to the sites of administration (this redistribution of T cell subpopulations may improve or decrease the ability of the immune system to stimulate an individual a conventional immune response at the site of normal exposure to the attacking antigen, resulting in a reduction or reversal of symptoms), d) induce 1 suppressor cells, or e) induce suppressor cells by non-participating antigen.

The highly purified and isolated peptides obtained as described above may be formulated into therapeutic compositions of the invention suitable for human therapy. When the therapeutic composition of the invention is to be administered by injection (e.g., ad subcutaneously, intrinsically), it is preferred that the highly purified peptide be soluble in aqueous solution at a pharmaceutically acceptable pH (i.e., a pH range of about 4 to D) so that the composition is fluid. and easy to apply with a needle. The composition also preferably comprises a pharmaceutically acceptable carrier. As used herein, the term &quot; pharmaceutically acceptable carrier &quot; refers to an environment, coatings, antibacterial and antisense agents, toxicity adjusters, buffering agents, absorption delaying or enhancing agents, surfactants and micelleformers, lipids, liposomes and liquid complexing agents, stabilizers and the like. The use of such media, and compositions for pharmaceutically active agents, is known in the art. For use in therapeutic compositions, all conventional media and compositions are contemplated as long as they are incompatible with the active compound. Other active compounds may be included in the compositions.

As mentioned above, the therapeutic compositions of the invention suitable for injectable use are preferably sterile aqueous solutions prepared by incorporating the active compound (ie, one or more of the highly purified and isolated peptides described above) in the required amount. into an appropriate vehicle with one or a combination of the above and the following ingredients as desired, followed by sterile filtration. Preferred pharmaceutically acceptable carriers include at least one vehicle such as sterile water, sodium phosphate, mannitol, sorbitol, or sodium chloride, or any combination thereof. Other suitable pharmaceutically acceptable carriers include solvents or dispersion media containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol x VA) ⁱⁿ suitable mixtures and vegetable oils. .

treatment plant

Prevention of various active substances, fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the surface treatment of microorganisms with antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid and the like. Prolonged absorption of injectable preparations may be brought about by including in the composition a delay in absorption, for example, aluminum monostearate and gelatin.

Preferred therapist compositions of the invention should be sterile, stable under the conditions of manufacture, storage, distribution and use, and protected from the contaminating action of microorganisms such as bacteria and fungi. A preferred method of preparing a therapeutic composition that preserves the integrity of the composition (i.e., prevents contamination, prolongs storage, etc.) is by preparing a formulation of the peptide and a pharmaceutically acceptable carrier or carriers so that the composition may be in the form of a lyophilized powder. an acceptable carrier, such as sterile water. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying, freeze-drying or spin drying, which provides a powder of the active ingredient plus any other desired ingredient from their previously sterile filtered solution.

therapeutic suitable for

As mentioned above, the therapeutic composition of the invention may comprise more than one isolated peptide. For administration of a plurality of active peptides, a formulation comprising a multipeptide formulation may be desirable for pharmaceutical administration to humans. The multipeptide formulation comprises at least two or more isolated peptides having a defined amino acid sequence and is capable of attenuating the antigen specific immune response. As a multipeptide formulation it may be. suitable any of the above-described compositions comprising at least two peptides. As mentioned above, highly preferred peptides include at least two of the following peptides: MBP-1, MBP-1.1, MBP-1.2

MBPMBP

1, MBP-2.2

MBP-2 .4

MBOMBP-2.6

MBP-3, MBP-3.1, MBP-4, and MBP-5. Particular considerations in preparing a multipeptide formulation include maintaining the solubility and stability of all peptides in the formulation in aqueous solution at a physiologically acceptable pH. This requires the choice of one or more pharmaceutically acceptable solvents and vehicles that are compatible with all peptides in the multipeptide formulation. Suitable vehicles include, for example, sterile water, sodium phosphate, mannitol or simultaneously sodium phosphate and mannitol. Another consideration in the case of a multipeptide formulation involves preventing peptide disruption, if necessary.

Administration of the above-described therapeutic composition to an individual in a non-immunogenic form can be accomplished using known procedures at dosages and for a time effective to inhibit the immune response of an individual treated to an MBP antigen specific MS. The inhibition of an antigen-specific immune response to a disease-related antigen in humans can be. determined clinically, if possible or subjectively (ie, the patient feels as if some or all of the symptoms associated with the disease state have been alleviated)

An effective amount of the therapeutic compositions of the invention may vary according to factors such as the degree of susceptibility of the individual to the antigen, the age, sex and weight of the individual and the ability of the peptide to induce inhibition of the antigen-specific immune response in the individual. The therapeutic composition of the invention may be administered in a non-immunogenic form by a suitable route, for example, by injection (subcutaneously, intravenously, etc.), orally, subcutaneously, inhalation, transdermally, rectally or by any combination of routes to enhance therapeutic efficacy, or by any other route. by a route known for administering therapeutic agents. It may be desirable to administer to a subject simultaneously or sequentially a therapeutically effective amount of one or more therapeutic compositions of the invention. Each of these formulations for simultaneous or sequential administration may comprise only one peptide or a multipeptide formulation as described above.

E ^J ro administering a peptide or peptide composition a non-parenteral route, the peptide may be necessary to prevent its inactivation coated with the substance or it can be administered simultaneously. For example, the peptide preparation may be administered concomitantly with enzyme inhibitors or in liposomes. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEP) and thiaaylol. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984), J. Neuroimmunol, 7:27). When the peptide is suitably protected as described above, it may be administered orally, for example with an inert diluent or an assimilable edible carrier. The peptide preparation and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets or incorporated directly into one's diet. For oral therapeutic administration, the active compound may be incorporated into vehicles and used in the form of swallowable tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. These compositions and preparations should contain at least 1% by weight of the active compound. The percentage of the composition or preparation may, of course, vary and preferably may be between about 5 and 80% by weight of the unit. The amount of active compound in these therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or compositions of the invention are prepared so that an oral dosage unit contains between about 10 µg and about 200 mg of the active compound. Tablets, lozenges, pills, capsules and the like may also contain: a binder such as gum tragacanth, acacia, corn starch or gelatin, vehicles such as dicalcium phosphate, a disintegrant such as corn starch, potato starch, alginic acid and the like, a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin, or a flavoring agent such as peppermint, methyl salrocylan, or cherry flavoring. When the dosage unit is a capsule, it may contain a liquid carrier in addition to the substances of the above type. Various other substances may be present as coatings or may otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with sugar, sugar, or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propyl paraffins as a preservative, a colorant and a flavoring such as cherry or orange flavor. Of course, any agent used in the preparation of all unit dosage forms should be pharmaceutically pure and substantially non-toxic in the amount used. The active compound may further be incorporated into sustained release formulations and formulations.

When injected (subcutaneous, iv, im, intraperitoneal) of one or more compositions of the invention, preferably about 1 gg to 3 mg, preferably about 20 gg to 1.5 mg, especially about 50 to 750 gg, and more particularly, can be administered per unit dose. about 75 to about 750 gg of each active compound (peptide). Depending on the regimen described below, doses up to 1500 gg or more may be used. It is particularly preferred to formulate parenteral formulations in dosage units for ease of administration. dosage. "Unit dose" refers to physically discrete units suitable as single doses for human subjects to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Particular requirements for the novel dosage forms of the invention are determined and directly dependent on (a) the specific characteristics of the active compound and the particular therapeutic effect achieved, and (b) the limitations of the method of formulation for such effective treatment of human subjects.

compounds for

The dosage regimen may be adjusted to achieve an optimal prophylactic or therapeutic response. For example, several divided doses may be administered over days, weeks, months, or years, or the doses may be increased or decreased proportionally with each additional injection as required by the therapeutic situation. In one preferred therapeutic regimen, subcutaneous injections of therapeutic agents are administered once daily during the acute phase and once every two days during remission for the life of the individual suffering from the disease. As alternatives, injections may be given after weeks, months or other times. The dose may remain constant with each injection or may increase or decrease with each injection. A permanent treatment program for the whole life can be optimal. As an alternative, at intervals of about three months to about one year of the initial treatment period, booster injections may be given as a single injection or may include an additional series of injections similar to the initial treatment.

Due to the highly individual immune response of individuals suffering from relapsing remitting MS, primary progressive MS, benign MS and chronic progressive MS, a thorough and variable dose regimen should be developed. The patient assessment of the intake must first be completed. A complete examination shall be performed to detect, inter alia, impaired knowledge, nyslagm, poor pronunciation, decreased perception of vibration and positional sense, limb ataxia and jitter before contact, weakness or paralysis of one or more limbs, salvation of feelings and bladder problems. The practitioner will establish, by established methods such as the EDSS, the Neurological Rating Scale or other similar known means, as well as the above discussed indications of the disease state, a baseline against which any change, including but not limited to disability control, can be measured. Although there is no typical acute or remitting phase of MS, there have been some schemes that may; to guide an experienced practitioner. As mentioned above, the frequency of relapses or acute conditions is greatest during the first 4 to 4 years of disease, but the first seizure may not be followed by another observable seizure for 10 to 20 years (although lesions detectable only by MRI may provide a different clinical picture). During typical episodes, symptoms worsen for a few days to 2 to 3 weeks and then resolve. Recovery is usually rapid within weeks, although at times it can stretch over several months.

MS is a chronic disease and presupposes continued treatment. Long-term treatment may be most effective in stopping disease transmission and disability. A dosing regimen may be appropriate every other day, at least once a week or once a month. Appropriate modifications are within the skill of the skilled artisan. Intervention at seizure onset is considered to be an important part of effective treatment.

therapies are combined with and / or with others

The severity of the seizure should be evaluated in a patient presenting with an acute stage. At. onset of the acute stage, an initial injection may be given. An obvious benefit is the initiation of c. Treatment at the earliest during the acute phase. If desired, this may be concomitant treatment with β-interferon, therapies especially designed to reduce inflammation. The patient should be observed daily after the first treatment, preferably by injection. During the acute phase, it is proposed to administer additional treatments daily or at least every third day. As with any medication, the attending physician should modify the dosage based on clinical changes that indicate a need for modification. Any such consideration is within the skill of the skilled artisan following the proposed dosage schedule and the amount administered. A dosage range of from about 75 to about 750 μα per dose provides the physician with sufficient breadth (but not inadequate amounts). Options include, but are not limited to, daily treatment during the acute phase, followed by the remission treatment described above. However, this does not preclude the possibility of more or less frequent dosing if the attending physician determines that intervention is indicated.

According to the invention it is stated that. multiple stage sclerosis can be treated with the compositions and methods of the invention. Preparations comprising, at least one peptide with T cell activity derived from myelin autoantigen are suitable for the treatment of advanced stage MS. The above-described preparations of MBP peptides, MOG peptides and combinations thereof are suitable for the treatment of advanced stage MS. As mentioned above, non-limiting examples of these peptides are: MBP-1, MBP-1.1, MBP-1.2, MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP- 2.5, MBP-2.6, MBP-3

MBP-4, MBP13-25, 31-50, 61-80, 82-92, 82-96, 82-97, 82-96, preferably. MBP-1.1; MBP-2.1; MBP-4; MBP-5; 13-25

82-100, 82-100 [100P> Y], 83-100, 83-101, 84-97, 81-100, 85-100,

86-105, 87-99, 87-99 [91K> A], 88-100, 88-99, 111-135, 122-110

142.-168, 146-160 and 153-170 a

87-99, 8739-170, 141-160, 142-166,

99 [91K> Aj, 82-100, 82-100 [100P> Y], as well as peptides derived from human MOG:

human MOG 1-13 human MOG 103-115 human MOG 1-121 human MOG 1-20 human MOG 11-30 human MOG 21-40 human MOG 31-50 human MOG 141-160. human MOG 151-170 human MOG 161- 180 human MOG 199-218

GQFRVIGPRHPIR

HSYQEEAAMELKV

GQFRVIGPRHPIRALGDEV

ELPCRTSPGKNATGMEVGWY

RPPFSRWHLYRNGKDQDGD

QAPEYRGRTE.LLKDAIGEGK

VTLRIRNVRFSDEGGFTCFF

RDH SY QE EAAME LKVE D P FYW

GQFRVIGPRHPIRALVGDEV,

P1RALVGDEVELPCRISPGK,

ELPCRISPGKNATGMEVGWY,

NATGMEVGWYRPPFSRVVHL,

TVGLVFLCLQYRLRGKLRAE,

YRLRGKLRAEIENLHRTFDP,

IENLHRTFDPHFLRVPCWKI a

YNWLHRRLAGQFLEELRNPF.

The present inventors were the first to make a surprising discovery that intervention in the midst of an oncoming seizure actually improves the condition of a subject treated by the method of the invention. At least such intervention does not worsen the subject's condition. The inventors have further shown that intervention during remission is a thrombotic immune response and does not trigger the condition as a result of such intervention. Thus, the intervention may not only cause an improvement, but it appears that when applied by the method of the invention, it is quite safe. Using the above-described EAE model, we have demonstrated that repeated intravenous administration of immunodominant MBP Acl-11 peptides successfully treats EAE induced by full MBP protein. One successful treatment (indicating T cell reactivity) has been shown to be native peptides (unsubstituted native sequence derived peptide by the procedure described herein to identify peptides containing a T cell epitope derived from protein autoantigens); suitable modified peptides can be identified by comparing the binding affinities of the modified peptide to the native peptide. A modified peptide having a binding affinity lower than the native peptide will be a suitable candidate as a peptide for the therapeutic composition of the invention. Assays for determining binding affinity can be identified from the data in USSN 03/300811 with a filing date of 1.9.1994, incorporated herein by reference. For example, Acl-11 therapy has proven successful. Ac.1-11 therapy was then compared to therapy with Acl-11 [4Y1, an analog Acl-11, which binds to AA ^{at the} AP ^with a higher affinity and Veis stability (Wraith, DC et al., Supra, and Fairchi.ld, PJ et al., supra). Interestingly, we find that Acl11 Γ4Y] is effective at a 100-fold lower dose than Acl-11 and its therapeutic effect is longer-lasting, suggesting that selected modified peptides are also suitable for the therapeutic formulation. The Applicant further demonstrates that the particular Acl-11 [4Y1] peptide tested forms in the treated mouse. an in vivo stable peptide-MHC complex. Accordingly, modified peptides that form in vivo stable peptide-MHC complexes are highly likely to be useful in the treatment of humans since their effect may greatly increase with conservative amino acid substitutions (see also Karin et al., J. Exp. Med. , 180: 2227-2237 (1994)).

We also found that the Acl-11 [4Y] MBP peptide analogue reversed onset paralysis when administered during the acute phase of EAE and prevented relapses when administered during remission. This demonstrates that selected modified peptides and selected peptide analogs can reverse active, symptom and prevent relapses in M.d.

In another model of human disease progression, we initiated intravenous demand therapy after the onset of EAE and during the acute phase. Quite surprisingly, this therapy, applied during the acute phase, reversed the emerging disease. This finding is the first of its kind. In earlier experiments by other workers, peptide therapy was initiated at a time when most mice in the treatment group had not yet exhibited signs of EAE, at least before immunization or near the onset of the disease. For example, in Gaura et al., See above, a group of 13 mice were exposed to a disease-provoking MBP regimen. Once only one of the 13 mice showed minimal signs of disease, ie one mouse had a clinical record of 1 (discussion of the clinical evaluation of the mice, see Example 2), a mixture of MBP peptides was injected intrapcritoneally into seven mice. The remaining six mice, one of which was a mouse showing signs of disease, received no C1-C18 treatment. In this case, Gaur and d. Are unable to draw meaningful conclusions from these data for mice without clinical signs of disease. The presence of a low clinical record 1 in one mouse cannot be interpreted by Lak as indicating active disease in any of the other mice. Therefore, Gaur et al. Describe treatment prior to the onset of the disease and certainly before clinical signs of the disease. In contrast, in order to be useful in clinical use in the treatment of people with MS, therapy would have to be effective even if initiated after the onset of the disease process. As a model of appropriate human therapy, we administered peptide therapy to mice, initiating 250 nmol of Acl-11 (4Y1 after the onset of the acute phase of EAE. The effect of this therapy is shown in Figure 5). Once developed, the EAE mice were assigned to the Acl-11 [4Y] peptide therapy group and to each of the three control groups (untreated, PBS or AahAfiU binding peptide OVA 323-337. Fig. 9 shows that Ac 1 therapy -11 [4 Y] at the onset of EAE leads to a dramatic improvement in disease characteristics, evident within 48 h after the initial peptide injection, in these treated mice the EAE almost disappeared by day 4 after onset and the effect of the 18-day course of peptide therapy was prolonged.

In a further surprising development, the inventors have found that intravenous peptide therapy initiated during the EAE remission phase prevents relapse. At. using EAE as a human disease model, as mentioned above, F1 mice (PLJ x SJL) again develop EAE relapsing EAE after immunization (Fritz, R.B. et al., J. Immunol., 1953, 130: 1024). EAE usually develops between 9 and 16 days after immunization and the acute phase of the disease in this model lasts approximately 3 to 20 days. During the acute phase, 20% or more of the mice may die or become moribund depending on the severity of the disease in the experiment. Most of the animals surviving the acute phase then enter a short remission phase, showing a few or no clinical signs of the disease for several days. In some experiments, mice may enter the remission phase all approximately at the same time (shown in Figure 5), but in most cases the length of the acute phase is more variable. The remission phase is followed by the first relapse phase, which generally has the same strength as the acute phase of the disease. Subsequently, surviving mice develop dew chronic chronic paralysis that persists until the end of the experiment.

In a mouse model, the Acl-11 [4Y] peptide represents remarkably effective therapy when initiated during the EAE remission phase. In order to investigate the ability of Acl-11 [4Y] to prevent EAE relapses, mice were observed during the initial episodes of paralysis that lasted 3 to 19 days. As surviving mice entered the remission period individually (defined as reducing the clinical record to i or 0 for at least two days), they were alternately assigned to two groups and intravenous therapy was initiated with 25 nmol Acl-11 [4Y] or control PBS. Mice were treated individually starting on the second day of remission. Giant. 10 shows that intravenous administration of Acl-11 [4Y], initiated during the EAE remission phase, dramatically reduced the incidence and severity of relapses, and suggests that intravenous ppt therapy may be effective, even if initiated later, during the disease.

The mechanism of intravenous peptide therapy hitherto has not been fully explained, although it has already been suggested in various other anticyclic systems that administration of the antigenic peptide intravenously prior to immunization induces anti-gene specific immune reactivity. As another proof of effectiveness

4] The therapeutics administered to MS patients were administered by i. V. Encephalitis togermic peptide or peptide analogue and found to reduce lymphoid node proliferation and IL2 production in vitro. Mice were injected intravenously with 250 nmol MBP Acl-11 ten and five days prior to immunization with MBP protein. Giant. 11a shows the ferromagnetic lymph node response of these treated mice, measured in don 10 after immunization. Pre-intravenous treatment with a non-vehicle MBP peptide Acl-11 without immunization prior to immunization reduces the subsequent proliferative T cell proliferative response to MBP Acl-11. Similar results were obtained when mice were pretreated by intravenous injections of the Acl-11 [4Y] MBP peptide analogue (data not shown). In addition, intravenous pretreatment of Acl-11 [4Y] was prevented by the production of IL2 by lymph nodes in response to MBP Acl-11 (Fig. 11b). These results confirm that intravenous pretreatment with MBP Acl-1.1 or Acl-11 [4Y1] induces T cell unreactivity on MBP Acl-11.

Thus, we investigated the efficacy of intravenous therapy with MBP peptides and peptide analogs in the treatment of reclaiming EAE and made a number of unique observations. The first and probably most important is that treatment of an autoimmune disease with one autoantigenic peptide may be effective even if it is initiated after the onset of disease traits. The specific disease characteristics used to evaluate mice are discussed in the Examples. A score of 2 or more on the Mean Clinical Score was defined as an advanced EAE stage. The results suggest that intravenous MBPs also interfere with the encephalitogenic activity of auto-aggressive immunized cells even after the cerebral blood barrier and the lymphocyte infiltration of the central nervous system have been disrupted. At no time interval, peptides were observed to at least temporarily activate, aggravate, or oxaceib the emerging disease. EAE treated after the onset of the acute phase was reversed within a few days after initiation of therapy, although the treated mice were initially heavily infected.

Histological examination confirmed that the clinical response to peptide therapy correlated with a significant decrease in lymphocyte infiltrates into the brain and spinal cord. Thus, as discussed herein, i.v. application of MBP Acl-11 [4Y] effective even at initiation up to 30 days after MBP immunization, in EAE remission phase.

Thus, the initiation of treatment during the seemingly resting phase of the disease did not again lead to exacerbations and, moreover, completely prevented all but the slightest relapses. This result is particularly interesting because there is evidence that recognition of different MBP epitopes occurs later during the immune response to MBP, and it is suggested that these epitopes may contribute to the disease (Lehmann, PV et al., Nature, 1992, 358: 155) ). The present invention suggests that administration of the peptide very early in the course of the disease would prevent the development of a later immune response to these epitopes. It is noteworthy, however, that one MBP peptide analogue effectively prevented relapses when administered after dispersal of the strong acute phase of EAE, where the immune response to MBP and possibly other ravelm antigens could advance.

In the practice of the present invention, it is preferred to select an effective therapeutic as described above. The inventors further concluded that one criterion for optimal peptide candidates would be peptides with T cell reactivity and a higher MBP binding affinity than the native peptide, which forms stable complexes with MHC class II in vivo. In particular, the inventors have found that the peptide analogue of MBP, which forms in vivo stable complexes with MHC class II, is more effective in treating EAE than Acl-11. It has previously been shown that the peptide analogue MBP Ac1.-11 [4YJ] binds to Αα "Αβ ^υ with higher affinity and that the complexes formed in vitro between Ac1-11 (4Y) and Αα" Αβ ^Μ are more stable than the complexes, formed between Acl-11 and Αα 'Αβ ^π . Acl-11 [4Y] has also been shown to prevent EAE when a large dose is inhaled prior to immunization (Mctzler, B. ad., Supra). The present invention states that a modified MBJ? the Acl-11 L4Y1 peptide analogue is at least 100-fold more potent as a therapeutic agent than the Acl-11 MBP and requires a lower dose frequency dosing schedule. This increased efficacy results in a therapeutic agent that is both more practical and more effective. We have observed that intravenous administration of Acl-11 [4YJ (but not Acl-11) results in the formation of stable peptide-MHC complexes in vivo. The improved efficacy of Acl-11 [4Y] is believed to be related to this formation of stable peptide-MHC complexes. These complexes are detectable on spleen cells by MBP-specific Acl-11-specific hybridomas for up to 10 h after injection. It is therefore believed that the formation of stable peptide-MHC complexes may be one of the hallmarks of such a therapeutically effective peptide. Surprisingly, the therapeutic effect of Acl-11 [4Y] in vivo was surprisingly present long after the pcptid-MHC complexes became undetectable on spleen cells (see Figures 6a and 9). Peptide-MHC complexes are likely to form antigen-specific functional therapeutic units that exert a long-term effect on encephalitogenic T cells, persisting even after the complexes are no longer detectable in vivo. It is not known whether these complexes exhibit. its effect peripherally or in the central nervous system. When operating in the central nervous system, the complexes may be formed or the cells carrying the complexes may migrate through the blood-brain barrier.

The invention further provides a method of treating multiple sclerosis comprising administering a therapeutically effective amount of at least one MBP peptide having T cell activity in a treatment regimen comprising a therapeutically effective amount of IFN-β.

As a result of the work described, it has been discovered that a combination of at least one peptide having T cell activity derived from myelin antigens (e.g. MBP) and IFN-β when administered. in the therapeutic regimen, it has a synergistic effect (Fig. 12c), which surprisingly reduces the clinical symptoms of EAE in mice to a far greater extent than the effect of each of the components on alleviating the symptoms of EAE, i.e.,. administered alone (Figs. 12a-b), and which is greater than would be expected for the only additive effect of the pius IFN-β peptide.

Since FAE serves as a mouse model of human MS and is induced by various myelin antigens (e.g. PLP, MBP, MOG), it is expected that a similar effect will be observed in humans. The invention therefore provides a method of treating individuals having or susceptible to multiple sclerosis, comprising administering an effective amount of a composition of the invention comprising at least one peptide with T cell activity derived from myelin antigen, preferably in a non-immunogenic formo, in a therapeutic regimen , which also includes administration of IFN-β. Preferred formulations are those of the invention described above, comprising MBP peptides as well as MBP peptides combined with MOG peptides, in conjunction with concomitant or sequential administration of IFN-β.

The administration of a composition of the invention comprising at least one peptide with myelin antigen T cell activity in a therapeutic regimen comprising IFN-β administration may be. by known procedures at dosages and for a time sufficient to effectively reduce, eliminate or prevent the symptoms associated with multiple sclerosis. The effective amount of both the peptide and IFN-β co-administered in a therapeutic regimen varies depending on the factors described above. The active compounds (e.g. MBP peptide or compositions comprising it together with IFN-β) can be administered by a suitable route as described above, for example by injection (subcutaneously, intravenously, etc.), orally, by inhalation, transdermally or rectally.

For example, about 1 µg to 3 mg, and preferably about 20 to 500 µg, of a peptide derived from myelin-antigen may be injected per dosage unit. Preferably, a dosage unit of 100 to 10,000 units of IFN-β may be injected. The dosage regimen of the two compounds may be adjusted to provide the optimal therapeutic response. For example, IFN-β may be administered concurrently with the composition of the invention, or preferably may be administered at least 5 hours apart, preferably at least 1 hour. at least 24 hours apart. The therapeutic regimen of administering antigenic peptides and IFN-β may last for several days or weeks and may be. abbreviated or extended as required by the therapeutic situation as described above.

The invention also provides a novel formulation comprising: a physical mixture of peptides with T cell activity derived from a myelin antigen, such as MBP or MOG, and 1ΈΝ-β in a pharmaceutically acceptable carrier or diluent. The composition may be used as part of a therapeutic regimen for treating or preventing multiple sclerosis in an individual.

It is generally recognized that other autoimmune diseases, such as type I diabetes and rheumatoid arthritis, also result from a specific T cell-mediated response to a particular autoantigen. Various approaches to the treatment of T cell mediated autoimmune diseases, including administering whole autoantigens or peptides derived therefrom containing T cell epitopes to a patient to attenuate the T cell mediated response, responsible for the adverse symptoms of autoimmune disease.

In addition to myelin autoantigens that play a role in MS, a number of antigens (ie autoantigens) have been found to cause disease symptoms in other autoimmune diseases such as diabetes, Graves' disease, myasthenia gravis, good pasture syndrome, psoriasis, thyroiditis and rheumatoid arthritis (ie autoantigens such as insulin, rh factor, acetylcholine receptors, thyroid cell receptors, basement membrane proteins, thyroid proteins, ICA69 (PM-1), glutamic acid decarboxylase (61K or 65K), proteolipid, collagen (type II), heat shock protein and carboxypeptidase H). He is. whereas similar compositions and methods described herein can be used in the treatment of autoimmune diseases such as rheumatoid arthritis, diabetes, myasthenia gravis, Graves' disease, good pasture syndrome, psoriasis and thyroiditis, wherein the antigen responsible for the disease is proteinaceous autoantigen.

Peptides comprising defined amino acid residue sequences having T cell activity and preferably comprising at least one T cell epitope and / or which induce T cell unreactivity or reduced T cell reactivity have been identified and isolated for many of the above autoantigens. For example, WO 94/07520 identifies peptides that are reportedly capable of attenuating; an antigen specific response to soluble type II collagen, a protein antigen believed to be an autoantigen in rheumatoid arthritis. WO 92/06704 discloses methods for identifying insulin peptides which are said to include Ί cell epitopes and which may be suitable for compositions and methods analogous to the present invention for the treatment of type I diabetes. In addition, any of the above and exemplified methods for identifying peptides may be Protein autoantigen containing epitope 1 cells, identified suitable peptides containing T cell epitopes for antigen or autoantiyen, which is believed to be responsible for any of the above diseases. Once such peptides containing the T cell epitope have been identified for the target autoanti.gen, these peptides can be used in compositions and methods analogous to those described herein for the treatment of multiple sclerosis.

Overview of pictures on the busts

Giant. 1 depicts the full-length amino acid sequence of human MBP, indicating the amino acid residue numbers referred to herein.

Giant. 2 depicts the amino acid sequences of preferred MBP-derived peptides.

Giant. 3 shows the amino acid sequences of the overlapping peptides and longer peptides used in Example 1.

Giant. 4a is a graphical representation of the percentage of total response. MBP for each peptide shown in Figure 3.

MBP reactivity was calculated based on the percentage of MBP positive m.i crotiter cultures in each individual patient group that also responded positively to one of the MBP peptides. Total MBP positive microtiter cultures were 89 to 184 in each group of 19 to 43 patients tested for each peptide.

Giant. Figure 4b is a graphical representation of the percentage of individuals responding to MBP who recognize each peptide shown in Figure 3. Patients are considered to respond to MBP if at least one of their microtiter cultures responds positively to MBP. In each group of 19 to 43 patients tested for each peptide, there were 12 to 31 patients responding to MBP. Peptide reactivity was calculated based on the percentage of responders in each group with at least one microtiter culture positive for one of the MBP peptides.

Giant. 5a is a graph of mean clinical values over 0 to 40 days after disease induction in i.v. injection of MBP Acl-11 alone as compared to control.

Giant. 5b is a graph of mean clinical values over 0 to 40 days post-disease induction in mice by iv injection of 1101 ¹ Acl-11 in combination with MBP 31-47 compared to control.

Giant. 5c is a graph of mean clinical values! for 0 to 40 days after disease induction in mice. i. injected DVA 323-339 compared to control.

Giant. 6a is a graph of mean clinical values over a period of 0 to at least 30 days after disease induction in subjects injected with either 250 nmol of Acl-11 [4Y] or Acl-11 compared to control.

Giant. 6b is a graph of mean clinical values over 0 to at least 30 days after disease induction in mice. treated with 2.5 nmol Ac.l-11 [4YJ] compared to control.

Giant. 6c is a graph of mean clinical values over 0 to at least 30 days after disease induction in mice treated

2.5 nmol Acl-11 compared to control.

Giant. 7 is a bar graph depicting mean histological values in peptide-treated mice versus control mice, where a lower value indicates a reduced number and severity of inflammatory CNS infiltrates.

Giant. 8 is a bar graph representing the relative activation of T cells at 0 to 10 h after injection of 250 nmol Acl11-4Υ] mice.

Giant. 9 is a graph showing a comparison of mouse treatment with 250 nmol Acl-11 [4Y] versus control, PBS and OVA 323-337.

Giant. 10 is a graph showing a comparison of treatment initiated during remission with 25 nmol of Acl-11 [4Y] versus control.

Giant. 11a is a graph showing. lymph node proliferation in vitro (indicated by 311 thymidine incorporation) in i. v. 250 nmol MBP Acl-11 compared to control.

Giant. 11b is a bar graph depicting the relative production of lymph node 11L2 in response to MBP Acl-11 in Aci-11 [4Y] -treated mice compared to control.

Giant. 12a is a graphical representation of an experiment showing the effect of IFN-β on EAE in two groups of 10 adult female F1 mice (SJL x TLP) in which, on day 0, EAE was induced with guinea pig MBP in complete adjuvant with addition of pertussis toxin and either only incomplete Breund's adjuvant (control) or treated on day 9, 12, 16 and 20 (indicated by the x-axis arrows) treated interperitoneally (i.e., 2000 IFN-β units, with the y-axis representing the mean clinical value (MCS) for each group (0 - no EAE clinical signs, 1 - flabby tail, 2 - partial hind limb paralysis, 3 - total hind limb paralysis, 4 partial to total hind limb paralysis, 5 moribunal condition).

Giant. 12b is a graphical representation of an experiment showing the effect of MBP peptides Ac 1-11 in two groups of 10 adult female F1 mice (SJL x TLP) in which, on day 0, EAE was induced with guinea pig MBP in complete adjuvant with addition of pertussis toxin and either administered PBS (control) or treated on day 10, 13, 17 and 21 (indicated by the x-axis arrows) treated intravenously with 250 nmol Ac 1-11, with the y-axis representing the mean clinical value for each group as shown in Fig. 12a ,

Giant. 12c is a graphical representation of an experiment showing the effect of MBP peptide Ac 1-11, administered in combination with IFN-β on EAE in two groups of 10 adult female mice (SJL x TLPiE1 that were challenged on day 0 with EAE in complete adjuvant with toxin addition) who was coughing and who was either

administered by PBS ( control) or that were on days 10, 13, 17 and 2 ') (shown open arrows on x-axis treated intravenously 250 nmol Ac 1-11 and v day 9, 12, 16 and 2 0 (shown closed arrows on x-axis treated

interperitoneally 2000 IFN-β units, with the y-axis representing the mean clinical value for each group, as shown in Figure 12a.

Giant. 13 is a graphical representation of an experiment showing the effect of different doses of IFN-β (10,000 and 2000 units, respectively) on induced EAE in two groups of 10 adult female mice (SJL x TLPγF1) on which day EAE was induced by guinea pig MBP of pertussis toxin and either treated with PBS (control) or treated on days 9, 13 and 16 (represented by closed arrows on the xi axis) treated interpretitoneally (i.p.) with 10000 and 2000 IFN-β units, respectively; the y-axis represents the mean clinical value for each group as shown in Fig. 12a.

Figure 14 shows various MBP-derived peptides that may be suitable for the compositions and methods of the invention.

Giant. 15 is a graphical representation of the ingestion index (yj axis (mean S, i / MBP responder)) multiplied by the percentage of individuals responding to each peptide (xi axis, which is derived from the same experimental data shown in FIG. 4a; and 4b and described in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

The invention is illustrated by the following non-limiting examples. examples.

Example 1

Study of Multiple Sclerosis as an Immune Response to Myelin Basic Protein in the Human Population and Peptides and Selection of MBP Peptides Suitable for Therapeutic Use

Peptide synthesis:

Peptides were synthesized using standard Fmoc / Boc chemistry and wall purification by reverse phase HPLC. Giant. 3 shows the MBP peptides used in these studies. The names of the peptides or amino acid residues are identical throughout the examples.

Protocol: Analysis of human PBL for reactivity with MBP and MBP peptides:

PBLs were purified from fresh peripheral blood samples (approximately 75 mi) from 222 patients with certain MS using Picoll density gradients. Microtiter cultures were initiated with 2 x 10 ⁵ PBL per well and 10 µg / ml purified human spinal MBP in RPMT 1640 culture medium supplemented with 5 µl human serum AB, foamyline-streptomycin and L-glutarain. From day 6-7, the cultures were supplemented with 1L2 (20 U / ml) and IL4 (5 U / ml). After 11 to 13 days, the microtiter cultures were washed, resuspended in fresh medium and divided into 12 fresh microtiter wells. Autologous frozen PBL was added as antigen-containing cells at 5 x 10 ⁴ PBL per well.

Screening antigens were added in duplicate to 12 duplicate wells from each microtiter culture. Medium as a negative control and purified human recombinant MBP at 10 ng / ml was used as a positive control. Each patient was also tested for reactivity with a maximum of 4 MBP peptides, each at a concentration of 10 μΜ. After 48 h, samples were pulsed with 0.75 nCi of ³ H-thymidine and collected after 6-16 h pulsations.

Cultures were marked as positive for each peptide according to the following criteria: stimulation index greater than 3.0, cpm change greater than or equal to 500, and standard deviation from mean less than cpm change. For analysis purposes, cultures were further termed &quot; peptide-positive &quot; only if they reacted simultaneously to MBP and the peptide and did not respond to more than one non-overlapping peptide. Reactivity with each of the peptides was tested using a minimum of 19 and a maximum of 43 patients.

Results:

On average, 6% of microtiter cultures were found to be positive for MBP reactivity for each patient (range 0 to 37 MBP positive cultures per patient). At least one microtiter culture was positive for MBP reactivity in 77% of patients and these individuals were considered to respond to MBP. The responder status did not correlate with gender, MS category (RR vs. CP), HLA-DR type, age or whether the patient was taking. or did not take Betaseron.

The MBP peptides tested included a panel. 16 ROmer (Fig. 3), overlapping by 10 amino acids and covering the entire human MBP sequence (18.5 kD isoform). Two more peptides were tested (MBP sequence (MBP 83-105 and MBP '141-165, Fig. 3). Reactivity to MBP peptides was then calculated based on the proportion of MBP positive microtiter cultures that were also positive for one of the MBP peptides Four of the peptides each accounted for at least 10% of the total reactivity to MBP (MBP 11-30, MBP 81-100, MBP 83-105 and MBP 141-165) In addition, reactivity to each of these peptides was detected in at least In addition, 77% of patients responding to MBP show reactivity to either MBP 83-10 or MBP 141-165, or both.

Of these four peptides, MBP 141-165 was the most reactive, accounting for 21% of the total reactivity to MBP, which was detectable in 64% of the responding patients tested. MBP 141165 appears to contain T cell cp-itopes from both MBP 141-1.60 and MBP 151-170 and surprisingly showed dramatically greater reactivity than the two Zmer peptides. MBP 81-100 and MBP 83-105 have a very similar sequence and probably include similar if nc identical T cell epitopes. They correspond to a region previously believed to be associated with the HLA-DR2 haplotype. Our results suggest that MS patients with both DR2 and no 13R2 respond favorably to these peptides. MBP 11-30 is an area of peptide reactivity, of which originally thought that not the MS patients often ^one recognized.

Each of the other MBP peptides accounted for less than 5% of the total MBP reactivity and was detected in 20% or less of the responding patients tested (see Figures 4a and 4b). The greatest reactivity in this group of peptides was found in MBP 111-130, which accounted for 4.5% of the MBP reactivity and was found in 10% of the responding patients tested.

Selection of preferred MBP peptides

Preferred MBP peptides were selected on the basis of two criteria:

i. The number of MS patients responding to the peptides in question (at least 75% of the test patients responding to the protein antigen recognize at least one of the peptides).

2. Intensity of T cell response to candidate peptide in MS patients (response to candidate peptide equals 40% of total antigen response).

The first criterion meets MBP 83-105 (MBP-2) and MBP 141-165 (MBP-4) (77% of responding patients recognize either or both, see Table 1 below). Together, these peptides account for 32% of the reactivity to MBP (see Table 2 below).

Table 1. Reactivity to MBP-2 and MBP-4: Individual analysis

and individuals responsive to MBP-2 MBP-4 MBP-2 and / or MBP-4 me z. i responsive to MBP 11/31 [35.5%] 20/31 [64.5%] 24/31 [77.4% J

Table 2, MBP-2 and MBP-4 reactivity: T cell line analysis

% MBP MBP positive positive MBP positive and MBP positive and MBP-2 or MBP Line i MBP-2 MBP-4 4 between. MBP positive i iniemi 20/183 [10,95%] 39/184 [21.1% 1 59/184 [32.1% _

The addition of MBP 11-30 (MBP-1) thus fulfills the second criterion since the three peptides together account for 42% of the reactivity to MBP. MBP β 1.1-130 (MBP-3) would also be a preferred peptide for therapeutic use, particularly in combination with MBP-1, MBP-2 and MBP4. MBP 81 100 appears to be equivalent to MBP 83-105 and can also be used in conjunction with other selected preferred peptides.

Example 2

Administration of peptides to mammals for treatment of EAE as a model for multiple sclerosis

Peptide synthesis:

Peptides were prepared by automated peptide synthesis (ABI 430A, Applied Biosciences, Foster City, CA) using standard 9-fluorenylmethoxycarbonyl chemistry. The peptides were purified by high pressure liquid chromatography and the amino acid composition was confirmed. The peptide sequences selected were MBP Acl-11 ASQKRPSQRHG, MBP Acl-11 [4A] ASQARPSQRHG, MBP Acl-11 [4Y] ASQYP.PSQRHG, MBI ^J 31-47 RHRDTGILDSIGRFFSG, Ova 323339 ISQAVHAAIIAEINER7G and OQAVHAAIIAEINEAG7.

MBP cleaning

MBP was prepared from guinea pigs (Keystone Biologicals, Cleveland, 0Π) using a modification of the method described in Smith, M.E., J. Neurochem., 1969, 16:83. Briefly, MBP was extracted from isolated myelin membranes using chloroform and methanol, precipitated with potassium citrate, extracted with acid, and lyophilized. SDS-PAGE analysis of this material showed a major band at an expected molecular weight of 18.5 kD.

EAE induction, evaluation and pcptid therapy

EAE was induced in mice (PJL x SJL) F1, obtained from Jackson Laboratory (Bar Harbor, ME). When mice reached 3-14 weeks of age, they were induced by EAE immunization with 50-100 µg MBP, emulsified in complete Freund's adjuvant (Gibco Laboratories, Grand Island, NY) supplemented with an additional 400 µg per M. tuberculosis H37Ra mouse (Difco Laboratories, Detroit, ME). 200 ng of cough toxin (JRH Biosciences, Lenexa, KS) was administered intravenously at the time of immunization and after 48 h. Mice were evaluated for clinical signs according to the summer scale: 1 tail paralysis, 2 - partial hind limb paralysis, total hind limb paralysis, 4 - forelimb limb paralysis, 5 moribund or dead. 5 mice were sacrificed. After death, mice were excluded from further calculations of the mid-trial. The peptides were administered intravenously as described in the individual examples below.

For lymph node proliferation assays and 1112 production, weak and para-aortic lymph node EAE lymphocyte suspensions were prepared from mice immunized as described above (pertussis toxin was not included for proliferation experiments). Lymphocytes were cultured on 5-10 ⁵ round bottom microtiter plates per well with MBP and RPMI 1640 peptides supplemented with 0.5% fresh normal mouse serum, penicillin-streptomycin, Lglutamine and 5.10 ^-5 M 2-mercaptoethanol. Cultures for proliferation assays were pulsed for 16 h with ³ H-thymidine followed by 72 h incubation. For T.L2 assays, culture supernatants were harvested for 24 h and tested for ability to promote IL2-dependent HT2 cell line growth. To differentiate IL2 production from IL4 production, HT2 biological assays were performed in the presence of a 10¾ supernatant of the monoclonal antibody IL.B.II (O'Hara, J. et al., Nature, 1985, 315: 333), which inhibits IL4-1. induced proliferation of HT2 cells.

The results are shown in Figure 9, which shows that Acl11 [4Y] reverses the course of EAE when administered after the onset of paralysis. EAE was induced in groups of 14 mice by MBP. Therapy was initiated individually in each mouse after it developed clinical signs of EAE. Data are plotted in relation to initiation of therapy for each subject (day 1 = onset of clinical signs for each individual mouse). Mice were treated intravenously on Days 1, 4, 11, and 18 of disease with PBS (open circle), 250 nmol of Acl-11 [4Y] (solid squares), or 250 nmol of Ova 323-337 (solid circles). Another control group received no treatment (open squares). Crosses indicate.

individual mice that died or were sacrificed in EAE Grade 5. The MMS value for the untreated group was 4.6 and the mortality was 64%, the MMS value for the PBS-treated group was 4.4 and the mortality was 35%. The MMS value for the Ova 323-337 treated group was 4.2 and the mortality was 21%, the MMS value for the Acl-11 [4Y] treated group was 3.2 and the mortality was 7%.

Giant. 10 shows that Acl-11f4Yj prevents EAE relapses when administered during the remission phase of the disease. EAE was induced by. MBP. Therapy was initiated on each mouse individually on the second day of remission after the initial episode of EAE. The heels are plotted in relation to initiation of therapy for each subject (day 1 = second day of remission). Groups of 6-7 surviving mice were treated intravenously on days 1, 4, 7 and 8 after commencement of remission with either PBS (open circles) or 25 nmol of Acl-11 [4Y] (solid squares). Crosses indicate individual mice that died or were sacrificed in EAE Grade 5. Initial episodes of disease prior to remission lasted 3 to 18 days (mean 9 days for both groups). The MMS value during the initial episode was similar for both groups of mice. (3.0 ρ-ro PBS treated group and 3.9 for the Acl11 [4Y1] treated group). The MMS value after treatment for the PBS group was 4.1 and the mortality was 33%. The MMS value after treatment for the peptide treated group was 0.3 and the mortality was 0%.

Giant. 11 shows that intravenous administration of Ad-11 or Acl-11 [4Y1] induces T cell unreactivity.

a) Groups of 3-4 mice were intravenously treated with either PBS (solid circles) or 250 nmol Acl-11 (solid triangles) intravenously 5 and 10 days prior to immunization with 150 µg MBP. The lymph node proliferation tests with MBP Acl-11 were performed on day 9. Controls: PBS PPD 108277 pretreatment / medium 1855 and Acl-11 PPD 88499 pretreatment / medium 1335.

(b) Groups of 4 mice were treated with either PRS or 250 nmol of Ad-11 [4Y].

Production tests of 52 lymph nodes with 150 μΜ Acl-11 or μΜ Acl-11 (4YJ) were performed on day 10.

Example 3

Advanced peptide administration

Upon immunization with MBP, mice (P JL x SJL) develop EA relapsing EAE (Fritz, RB, et al., J. Immunol., 1983, 130: 1024), making this model of EAE ideal for investigating therapeutic intervention. Although the immunodominant encephaline in the mouse: (PJL x SJL) F1 is MBP Acl-11, the subdominant epitope MBP 3147 is also encephalitogenic in the robotic strain. To determine whether one of these peptides is required for MBP-induced EAE treatment or both, the individual steps of Example 2 were repeated, except that groups of mice were treated at an advanced stage of the disease by intravenous injections of MBP Acl-11 and MBP 31-47 or Acl-11'1 alone. Groups of control mice were treated with intravenous injections of PBS or a control peptide Ova 323-339, a peptide binding to Ap ^{at L} AD, unrelated to MBP and immunogenic for mice (PLJ x SJL) Fl.

Giant. 5 shows that repeated intravenous injections of MBP Acl-11 either alone (Fig. 5a) or in combination with MBP 31-47 (Fig. 5b) reduce the incidence, intensity and mortality of EAE. Consistent with the above observations of other workers, treatment with both peptides appears to be somewhat more effective than treated with: MBP Acl-11 alone. Mice were treated with six intravenous injections of the peptide during the acute and first relapse phases. Then, mice were followed up to day 125 during the chronic remitting phase with no evidence of late disease in the MBP peptide-treated groups (data not shown). The control peptide Ova 323-339, administered intravenously, had no effect on the disease (Fig. 5c). In a separate experiment, intravenous administration of MBP 31-47 alone was not effective in treating EAE (data not shown). Giant. 5a, b and c show the efficacy of intravenous treatment of mice with EAE indicated by MBP. EAE was induced as described above by MBP in groups of 11-16 mice. Mice were treated by intravenous injection of 250 nmol of each peptide, starting on days 9, 12, 15, 21, 29 and 37. The crosses used in all figures indicate individual mice that died or were killed in EAE grade 5. The control group treated with PBS is indicated by solid circles. The results indicate that the immunodomiriant encephalogenic MBP peptide is both necessary and sufficient to treat MBE induced MBP in mice (POL x SJL) P1, as shown by the decrease in EAE intensity and mortality in treated mice.

Example 4

Multiple i.v. injection of Acl-11

Following the preparation steps of Example 2, multiple injections of Acl-11 were prepared. A single injection of peptide on day 9 delayed disease by two days and reduced mortality from 100% to 63%, but all treated mice eventually developed a strong EAE (data not shown). Three injections of peptide during the acute phase of EAE initially treated the disease effectively, but during the relapse phase the treated group of mice developed a late-onset disease (data not shown). These results, taken together, suggest that multiple intravenous injections of MBP peptides during the acute and first relapsing phase of EAE are required to treat MBP-induced disease in mice (PJh x SJL) F1. Thus, multiple injections of Acl-11 are required to reduce disease intensity in the long term. Giant. 6 shows that Acl11 [41] treats EAE at a lower dose than Ac'1-11 and has a long-lasting effect on the disease. EAE was induced by MBP in groups of 8 to 10 mice. Giant. 6a shows mice treated with i.v. on days 12 and 15 with PBS (phosphate buffered saline; open circles), 250 nmol of Acl-11 (solid squares) or 250 nmol of Acl-11f4Y] (open triangles). The MMS value for PBS-treated mice was 4.4 and the mortality was 60%. The MMS value for Acl-il-treated mice was 3.9 and the mortality was 37.5%. The MMS value for Acl-11 [4Y] treated mice was 2.1 and the mortality was 0%. Giant. 6b shows mice treated with IV. on days 12, 15, 18, 21, 24 and 2?

either PBS (open circles) or 2.5 nmol Acl-11 (solid circles). The MMS value for PBS-treated mice was 2.9 and the mortality was 20%. The MMS value for peptide-treated mice was 4.3 and the mortality was 50%.

Example 5

Treatment with an analogue of MBP peptides that forms in vivo stable complexes with MHC class II

Since peptide-MHC complexes are functional therapeutic units treating EAE, the inventors have deduced that Ac-1'1 [4Y] should be more effective in treating MBE induced by active MBP than Ac1-11. To test this hypothesis, the steps of Example 2 were performed except for the injection schedule and the dosage was changed. Fig. 6a shows that only two injections of Acl-11 [4Y] early in the course of the disease showed a longer-lasting effect than two injections of Acl-11. Giant. 6b further shows that repeated intravenous injections of Acl11L4Y] are highly effective in treating EAE at a dose of 2.5 nmol (Fig. 6c). Histological tests were then performed. Histological sections of F.AE from brain and spinal cord were prepared and stained with iiematoxylin and eosin (CVP, West Sacramento, CA). In the sections, inflammatory infiltrates were scored blindly by an observer on a scale of 1 to Efficacy. Acl-11 [4Y] was confirmed by histologist! Brain and spinal cord cross-sectional assays from peptide-treated and PBS-treated mice showing a significantly reduced number and intensity of inflammatory central nervous system infiltrates in peptide-treated mice (Fig. 7). Giant. 7 demonstrates that inflammatory infiltrates are reduced in Acl-11 [4Y] treated mice. The improved efficacy of Acl-11 [4Y] was evident.

In the above example was evident, improved efficiency Acl-11 [4Y] The inventors inferred that in vivo form stable peptide-MHC complexes between Aa ^at A3 ⁱⁿ and ACL-1114], which was injected intravenously and so causing the observed improved potency Tyr complexes formed in vivo are detectable by the 1934 hybndome, which was derived from an encephalitogenic MBP-specific T cell clone. The steps of Example 2 were followed except for the time and amount of injections and the amount of bvia

adjusted as is below. T cell hybridoma 1934 ie MBP specific Acl-11 according to Wraith, U.C., et al., Cell 1989, 59: 24 Briefly, they were hybridoma buffers

incubated at ⁵ x 10 ⁴ per well in flat-bottom microtiter plates with MBP peptides or peptide analogs. ⁵ x 10 ⁵ spleen cells (PJT x SJL) of F'1 irradiated with 3000 rads were added as antigen-containing cells. The medium was the same as in the lymph node proliferation assays, except that the serum supplement was 10% fetal calf serum instead of normal mouse serum. After 24 h, supernatants were collected and examined for their ability to promote the growth of HT2 cells. Spleens were then removed from injected mice at various times after injection of 250 nmol of Acl-11 [4Y] and splenocytes were used as peanuts containing the antigen for the Acl-11-specific hybridoma 1931. No additional peptide was added to the cultures. Giant. 8 shows that spleen cells that were pulsated in vivo. with Acl-11 [4Y1] are highly effective in activating the hybridoma of 1934. The ability to activate the hybridoma lasts for a maximum of 2 hours after injection, then begins to decline within four to six hours after injection. After 10 h after injection of Acl-11 [4Y], activation is minimal and not present after 20 h (data not shown; as shown in Fig. 8, experiments with Acl-11 hybridoma activation by complex cells show less than 1 h from injected mice. after injection (data not shown). As shown in more detail in Fig. 8, in vivo d-MHC demand complexes are detectable in Acl-1114Y-injected mice. Groups of 2 mice were intravenously at various time points prior to spleen removal (1 to 10 h). 250 nmol of Aoi-1 '[4Y] was injected. Splenocytes from injected mice were screened for the ability to activate 1934 hybridoma cells to produce TL2. As a result, the varnish of HT2 proliferation is expressed. These results support the hypothesis that the formation of stable peptide-MHC complexes in vivo contributes to the efficacy of the therapeutic analogue of MBP peptides.

Example 6

Synthesis of murine MBP peptides Acl-11

The mouse MBP peptide Acl-11 was synthesized by standard Fmoc / tBoc synthesis and purified by HPLC. The amino acid sequence of the Acl-11 peptides is as follows:

EAE induction

EAE was induced by mouse mice (SJL x PL) Fl (Jackson Labs, Bar Harbor, ME) 6-8 weeks of age by immunizing mice with 100 µg of purified guinea pig MBP in CFA (G1BCO Lab., Grand Island, NY) containing 400 pg of M. Lubercu.1 was treated with H37RA (DIFCO Lab, Detroit, MI), subcutaneously at the base of the tail. On the day of immunization and after 48 n, 200 ng of pertussis cough toxin (JIL BIOSCIENCE, Lenexa, Kansas) was administered intravenously. Mice were monitored daily for signs of disease and scored on a scale of: 0 - no clinical signs of EAE, 1 - flaccid unresponsive tail, 2 - partial hind limb paralysis, 3 total hind limb paralysis, 4 - partial to complete forelimb paralysis, 5 - moribund . Data are expressed as the average of assessments, disease intensity per day including, all animals in the group. Mice were followed for 26 days. Once the mouse died on EAE, a score was included in the calculations for all subsequent days. 5.

Effect of iFN-β on EAL

Titration was performed. experiment to determine the effects of different IFN-β doses on EAE (Fig. 13). One group of mice was on EAE induction on day 9, intraperitoneally with PBS (control), one group of mice was treated with 10,000 IFN-β (open circle) on day 9, 13 and 16 and one group of mice was on day 9, 13 and 16 2000 IFN-β (solid circle) were treated. As seen in Figure 13, EAE symptoms are similar at each time point for both doses of IFN-β, which is. suggests that for the IFN-β experiments a lower dose would be appropriate, which is preferred because the possibility of toxicity due to a higher dose of IFN-β is less likely. In the remaining experiments shown in Figures 12a to 12c, 2000 units of IFN-β were used as this dose showed an improvement in the clinical trial.

As shown in Fig. 12a, on day 9, 12, 16 and 20, a control group of PBS mice and another group of 2000 mice were treated with ΙΕΜ-β. As shown in Figure 12a, the IFN-β treated group had only slightly weaker symptoms over time than the control group.

Effect of Acd-II · murine MBB peptide on EAE

The effect of the murine MBP peptide Acl-11 was determined and the results are shown in Fig. 12b. One group of mice was treated i.p. on day 10, 13, 17 and 21 after EAE induction. PBS (control) and a second group of mice were treated i.d. with 250 nmol of Acl-11 peptide on day 10, 13, 1 'and 21. Mice. were monitored as described above. As shown in Figure 12b, mice. Ac1-1 treated treated had weaker symptoms than the control group.

Effect of treatment with a combination of Acl-11 and iFN-β on EAE

The effects of treatment with the combination of Acl-11 and IFN-β are shown in Figure 12c. One group of mice was induced. EAE was treated with PBS (control) and one group of mice was treated i.p. on days 10, 13, 17 and 21. 250 nmol of Acl-11 peptide (open arrows) and on days 9, 12, 16 and 20 i.p. 2000 unit, ΙΕΝ-β. As shown in Figure 12c, the group of mice treated with the combination of peptides and 1 ΝΝ-β showed a significant decrease in symptom strength compared to both the control group and the treatment with IFN-β alone or with the peptide shown in Figures 12a and 12b, respectively. indicates the super-effect of this combination. Thus, a treatment regimen comprising a combination of peptides and ΙΕΝ-β provides an enhanced effect leading to a reduction in the severity of EAE symptoms.

Equity rites

The skilled artisan knows, or can only, by routine experimentation, find various equivalents of the specific methods described herein. These equivalents are considered to be within the scope of the invention and are covered by the following claims.

Example 7

Administration of peptides to mammals for treatment of EAE induced by spinal cord homogenate (SCH) as a model of multiple sclerosis

Synthesis of peptides

Peptides were prepared by automated peptide synthesis (ABI 430A, Applied Biosciences, Foster City, CA) using standard 9-fluorenylmethoxycarbonyl chemistry. The peptides were purified by high pressure liquid chromatography and the amino acid composition was confirmed. The peptide sequences selected were MBP Acl-11 ASQKRPSQRHG, MBP Acl-11 [4Yj ASQARPSQRHG, MBP Acl11 [4 Y] ASQYRPSQRHG, MBP 31-47 RHRDTGILDS1GRFESG, Ova 323-339 ISQAVHAAHAE1EA33NE, and Ova 323-339 ISQAVHAAHAE1EA33EA.

MBP cleaning

SCH was prepared from guinea pigs (Keystone Biologicals, Cleveland, OH) using a modification of the method of Smith, M.E.,

J. Neurochem., 1969, 16:83. The SCH protein mixture was lyophilized and used according to dry weight.

EAE induction, evaluation and peptide therapy

EAE was induced in E1 mice (PJL x SJL) obtained from Jackson Laboratory (Bar Harbor, ME). When mice reached 8-14 weeks of age, they were induced by EAE immunization with 500-1000 µg SCK, emulsified in complete Ereund's adjuvant (Gibco Laboratories, Grand Island, NY) supplemented with an additional 400 µg per M. tubercul osis H37Ra mouse (Díco Laboratories, Detroit , MI). At the time of immunization and 48 h later, 200 ng of vomiting casia toxin (JRH Biosciences, Lenexa, KS) was administered. Mice were scored based on clinical signs according to the scale: 1. tail paralysis, 2. partial hind limb paralysis, 3. total hind limb paralysis, 4. forelimb paralysis, 5. moribund or dead. Mice rated 5 were sacrificed. After death, mice were excluded from further average clinical trial calculations. Peptides were administered intravenously as shown in the individual examples below.

The results are shown in Figure 16, which shows that Acl-11 [4Y] reverses the course of EAE when given after the onset of paralysis. EAE was induced in groups of 14 mice by SCH. Therapy was initiated individually in each mouse after it developed clinical signs of EAE. Data are plotted in relation to initiation of therapy in each subject (day 1 - onset of clinical signs for each individual mouse). Mice were treated on day 1, 3, 6 and 9 of PBS (solid squares), i.v. 250 nmol Acl11 [4Y] (open triangles) or 250 nmol AcI [4Yj (open circles). The asterisk indicates individual mice that died or were killed as a result of Grade 5 EAE.

Giant. 17 shows that Acl-11 [4Y] prevents the onset of EAE when administered before symptoms of the disease occur. EAE was induced by SCH. Therapy was initiated on each mouse individually on the eighth day after gpSCH induction of the disease. Data are plotted in relation to initiation of therapy for each subject. Groups of surviving mice were treated on days 8, 10, 13 and 17

i.v. 25 nmol Acl-11 [4Y] (open triangles) or s.c. 25 nmol Acl-11 [4Y] (solid circles). One group received no treatment (empty squares).

The results in Figures 16 and 17 show that both routes of administration are effective in preventing the onset of symptoms.

Example 8

The procedure was the same as in Example 2, but the tests for lymph node praliferation and IL2 production were not performed. EAE was induced on day 0 with gpMBP and then 3 groups of mice on day S, 10, 13 and shown graphically (open triangles) were administered Acl-11 [4Y]. The results are shown in Figure 18 as intravenous administration of subcutaneous injection (solid circles) and control (open circles). Pre-treatment (e.g., treatment before symptoms) with Acl-11 [4Y] can prevent gpMBP-induced EAE.

Example 9 cDNA encoding human MOG protein

In an initial attempt to obtain human DNA encoding a MOG protein, a human cDNA library was subjected to a polymerase reaction (PCR) using primers 3 'and 5', constructed based on the published coding sequence, of rat MOG according to Gardinier et al (see above). The human MOG sequence could not be obtained in this manner, probably due to a lack of homology at the 5 'and / or 3' end of the human and rat sequences.

Therefore, four internal rat oligonucleotides were constructed. I) and v were homologous to the upper chain.

gene (primers 94-111 and 166-183) (SEQ ID NO: 34), base 1 starting with ATG) and two were homologous to the lower chain of the gene (primers 538-555 (SEQ ID NO: 35) and 685-702). The combination of primers 166-183 (SEQ ID NO: 34) and 538-555 (SEQ ID NO: 35) was successful in performing amplification of a fragment of the expected size of approximately 400 bp from a human brain cDNA library. The sequence of these primers was:

a) 16 6-183: CAGAATCCG GGAAGAATGOCACGGGC (SEQ ID NO: 34); and

(b) 538-555: CAGC GGC C GCAC GGAG C T C T C T C T C C (SEQ ID NO: 35)

An EcoRI site is present in primers 166-383 (SEQ ID NO: 34) and a NotI site is found in primers (SEQ ID NO: 35).

The 400 bp PCR product was cloned into the expression vector pV1.1393 by digesting pVL1393 (Pharmigen CA) with EcoRI and NotI, digesting the amplified product with the same enzymes, and ligating the resulting fragments. The insert was verified by digesting several clones derived from the fused plasmids with EcoRI and NotI and sequencing the resulting 400 bp fragment of human MOG. The resulting insert is believed to lack 184 bp of sequence 5 'and 201 bp of sequence 3', based on a rat open reading frame of 738 bp in size.

Two primers were constructed from the upper and lower strands of the insert 400 bp positions 346-363:

5 '-CAGAATTCTCAGGTTCTCAGATGAAGGA-d (SEQ ID NO: 36) and

5'-AAGCGGCCGCTATCCTTCATCTGAGAACCT-3 '(SEQ ID NO: 37) wherein an EcoRI site is present in the first chain and a NotI site in the second. The underlined regions correspond to the MOG sequence.

Upper, respectively. the lower primer of human MOG 346-363 (SEQ ID NOS: 36 and 37) was used in combination with the aforementioned rat primer 5 'and 5', respectively. 3 'to amplify the missing 5' and 3 'ends of the gene from the same human brain cDNA library as used previously. A PCR product corresponding to the 3'-end of the gene was obtained, but the corresponding 5'-end was not generated.

The obtained 3'-fragment had an expected size of 400 bp and this fragment was cloned into pVL1393 and sequenced.

To obtain the 5 'portion of the gene library was a human brain heart kgtlO obtained from Cloteoh which has been previously amplified and had Liter 8.1Ο ¹⁰ pfu / ml, is subjected to the screening procedure prescribed by the manufacturer. The library was plated on 12 large plates at 30000 plaques / plate and plaques were transferred to nitrocellulose filters (2 repeating filters / plate). Twelve filters derived from 12 different plates were then hybridized with P labeled probe ^f2 corresponding! the originally cloned 400 mp internal human MOG fragment (positions 184-534). 22 strongly positive were obtained. For each positive, the stopper was removed from the original plates and incubated overnight with λ dilution buffer to wash the phage from the agar. The tube was centrifuged and the supernatant transferred.

DNA was amplified from each pool using either the direct kgLIO primer with the SstII site:

5 ^of -CTTTTGAGCáxAGTTCAGCCTGGTTAAG-3 '(SEQ ID NO: 38) and reverse primer kgtlO XhoI site:

5'-ACCTCGAGGAGGTGGCTTATGAGTATTTCTTCCAGGGTA-3 '(SEQ ID NO: 39) as well as the upper or lower strand of the internal human MOG:

5'-GT G GC A GGG AAGGTGAC1 TCAGGATC CGGAAT ¹ C-3 '(SEQ ID NO: 40) or

5EQ ID

The last two primers (SEQ ID NOs: 40 and 41 '

NO: 41) include site

BamHI (underlined in sequences) naturally present in human MOG sequences.

The primers were used in four different combinations: i) direct upper / lower internal MOG, 2) reverse lower / lower internal MOG, 3) upper internal MOG / reverse lower and 4) upper internal MOG / direct upper.

The first two combinations provided the 5'-end of the gene (up to the BamHI site) and the last two 3'-end of the gene. Both parts 5 'and 3' contain untranslated regions. Which of the two members of each combination actually results in the desired fragment depends on the orientation of the cDNA cloned into Xgt10.

The size of the obtained fragments varied in individual numbers. The five largest 5 'or 3' fragments were subcloned into the SstIII and BamHI or BamHI and XhoI sites of the SK polylinker. Three. clones from each pool were then sequenced to eliminate the presence of PCR errors. This gave the complete sequence of the gene coding region as well as the 174 bp untranslated 5 'sequence.

The complete DNA sequence obtained (SEQ ID NO: 1) and the derived amino acid sequence (SEQ ID NO: 2) are shown in Figure 1.

The human MOG gene encodes a 248 amino acid preprotein having 87% homology to 246 amino acids in the rat protein. The mature protein comprises 218 amino acids numbered in Figs. 11 to 218 (SEQ ID NO: 2). The mature protein starts with the glycine shown at position 1 and is derived from a 248 amino acid preprotein by cleavage from a presequence ranging from the MET start codon to the alanine residue immediately preceding glycine at position 1.

Example PA

Expression of Truncated Human MOG in SF-9 and E. coli Insect Cells

Expression of SF-9

The transfer vector PVL1393 containing truncated human MOG cDNA encoding amino acids 1-121 of human MOG (first 121 amino acids of SEQ ID NO: 2) was cotransfected into SF-9 cells along with the purified DNA of Baculovirus Baculogold (Pharmmgen, San Di.ego, CA). After 4 days, culture supernatant containing recombinant vortexes was collected. The recombinant virus was plaque-cleaned and subjected to three cycles of amplification to obtain a higher titer stock virus. AF-9 cells were then infected with a stock virus of MOI 2.0. Supernatant from infected cells was harvested 48 h after infection and applied to a NiNTA agarose column. Recombinant MOG protein was eluted under non-denaturing conditions using 250 mM imidazole, dialyzed against 5% propionic acid and H ₂ O and subsequently lyophilized. Protein concentration was estimated by BCA. The purified MOG protein was visualized on a Coomassie blue stained 12.5% polyacrylamide gel.

Example 10

Truncated human MOG (huMOG) was prepared according to Examples 9 and 9A. MBP (guinea pig) was prepared as described. Example 2 was followed, but EAE was induced in three separate groups of mice using 75 µg gpMBP and 100 µg huMOG (open triangles) and a combination of 75 µg gp MBP and 100 µg huMOG (solid squares). The graph in Figure 19 shows the course of the disease for each case in this example. The disease induced by MOG and MBP at the same time is more severe than for each of them alone. The results suggest that both MOG and MBP contribute to the disease.

I 'r i kl and cl i

The disease was induced as in Example 10 by huMOG + gpMBP (day 0 - disease induction). Mice were treated before symptoms of 250 nmol Acl-11 [4YJ] and control (PBS) on days 6, 8, 1, 13, 1.7, 22 and 27 (arrows indicate treatment). As shown in the graph in Figure 20, treatment with Ac1-11 [4YJ (open, squares) significantly reduced the mean clinical record compared to controls (solid squares).

Claims (41)

PATENT CLAIMS A composition for treating multiple sclerosis in a mammal, comprising at least one peptide selected from the group consisting of MBP-1, MBP-1.1, MBP-1.2, MBP-2, MBP-2.1, MBP-2.2, MBP- 2.3, MBP-2.4 MBP-2.6, MBP-3, MBP-3.1, MBP-4, and MBP-5 shown in Figure 2, or modifications or analogs or peptides thereof. The composition of claim 1, wherein said peptide is selected from the group consisting of MBP-1.1, MBP-2.1, MBP-4 and MBP-5 shown in Figure 2. The composition of claim 1, wherein said peptide is MBP-4. 4. A composition for treating multiple sclerosis in a mammal, 2.4, MBP-2.5, MBP-2.6, MBP-3, MBP-3.1, characterized in that it comprises at least one peptide derived from MBP selected from the group consisting of MBP-1, MBP-1.1, MBP-1.2, MBP-2 , MBP-2.1, MBP-2.2, MBP-2.3, MBPnm rnn MBP-4 and MB> P 'shown in Figure 2, wherein the preparation comprises at least 40% of the total T cell reactivity to MBP in a population of individuals having T cells responsive to MBP. MBP-3.1, MBP-4 and MBP-5 shown in Figure 2. A composition for treating multiple sclerosis in a mammal comprising at least two MBP peptides selected from the group consisting of MBP-1, MBP-1.1, MBP2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4 MBP-2.5 MBP-2.6 109 6. The composition of claim 5, wherein said peptides are selected from the group consisting of MBP-1.1, MBP-2.1, MBP-4 and MBP-5 shown in Figure 2. The composition of claim 5, wherein one of said peptides comprises MBP-4. The composition of claim 5, further comprising at least one peptide selected from the group consisting of amino acid sequences 13-25, 31-50, 61-80, 82-92, 82-96, 82-97, 82-98, 82-100, 82-100 [100P> Y], 83-100, 83101, 84-97, 84-100, 85-100, 86-105 , 87-99, 87-99 [91K> A], 88100, 88-99, 411-135, 122-140, 139-170, 141-160, 142-166, 142168, 146-160, and 153-170, shown in FIG. 14. The composition of claim 5, further comprising at least one peptide selected from the group consisting of amino acid sequences 13-25, 87-99, 8799 [91K> A], 82-100, 82-100 [100P> Y]. ] shown in FIG. 14. A composition for treating multiple sclerosis in a mammal, comprising at least two MBP-derived peptides, wherein the at least one peptide is MBP-4 and at least one peptide is selected from the group consisting of MBP-1, MBP-1.1, MBP- 1.2, MBP-2, MBP-2.1, MBP-2.1, MBP-2.3, MBP-2.5, MBP-2.5, MBP-2.5 6180, 82-92, 82-96, 82-97, 82-98, 82-100, 82-100 [1.00P> Y], 83 * 100 83- 101, 84-97, 84-100, 85-100, 86-105, 87-99, 87-99 [91K> A], 88100, 88-99, 11 1 -135, 122-140, 139- 1.70, 141-160, 142-166, 142168, .146-160, and 153-170, shown in Figures 2 and 14. 110 The composition of claim 10, wherein the at least one peptide is MBP-4 and the at least one peptide is selected from the group consisting of MBP-1, MBP-1.1, MBP-1.2, MBP · 2, MBP-2.1, MBP -2.2, MBP-2.3, MBP-2.4 MBP-2.6, MBP-3, MBP-3.1, MBP-5, 13-25, 87-99, 87-99 [91K > A], 82100, 82-100 [100 > Y]. 12. A composition for treating multiple sclerosis in a mammal, comprising at least two MBP peptides selected from the group consisting of MBP-1, MBP-1.1, MBP1.2, MBP-2, MBP-2.1, MBP -2.2, MBP-2.3, MBP-2.4, MBP-2.5, MBP2.6, MBP-3, MBP-3.1, MBP-4 and MBP-5, as shown in Figure 2, wherein the formulation comprises at least 40% total reactivity T cells to MBP in a population of individuals having MBP responsive T cells. 13. A composition according to claim 5 comprising a percentage. total T cell reactivity to. MBP sufficient to cause administration of the formulation to an individual with MS to attenuate the autoimmune response of MS. 14. A composition for treating multiple sclerosis in a mammal, comprising at least two MBP peptides comprising T cell epitopes, said composition being selected from the group consisting of: MBP-1, MBP-2, MBP-3, MBP-4, and MBP-5; MBP-1.1, MBP-2.1, MBP-3, MBP-4, and MBP-5; MBP-1.1, MBP-2, MBP-4, and MBP-5; MBP-1, MBP-2.1, MBP-4, and MBP-5; MBP-1, MBP-2, MBP-4, and MBP-5; 111 MBP-1.1, MBP-2.1, MBP-4, and MBP-5; MBP-1.1, MBP-2.1 and MBP-4; MBP-1, MBP-2.1, and MBP-4; MBP-1.1, MBP-2 and MBP-4; MBP-1.1, MBP-2.1 and MBP-5; MBP-1.1, MBP-2] and MBP-3; MBP-1 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MRP-2.5, or MBP-2.6; MBP-1.1 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6; MBP-4 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5 or MBP-2.6; MBP-4 and a peptide selected from the group consisting of MBP-1 or MBP1.1; MBP-1.1, MBP-4 and a peptide selected from the group consisting of 82-100, 82-100 [100P> Y], 87-99 and 87-99 [91K> A] shown in Figure 14; MBP-1.1, MBP-4 and a peptide selected from the group consisting of MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5 or MBP-2.6 shown in Figure 2; MBP-1.1, MBP-5, MBP-4 and a peptide selected from the group consisting of 82-100, 82-100 [100P> Y], 87-99 and 87-99 [91K> A] shown in FIG. 14; MBP-1.1, MBP-5, MBP-4 and a peptide selected from the group consisting of MBP-2.2, MBP-2.3, MBp-2.4, MBP-2.5 or MBP-2.6 shown in Figure 2; MBP-1.1, MBP-5, MBP-4 and a peptide selected from the group consisting of 82-100, 82-100 [100P> Y], 87-99, and 87-99 [91K> A] shown in Figure 14 ; MBP-1.1, MBP-3, MBP-4, and a peptide selected from the group consisting of MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6, as shown in Figure 2. 112 The composition of claim 5, further comprising at least one peptide with T cell activity derived from human myelin oligodendrocyte protein (MOG). 16. The composition of claim 15, wherein said MOG-derived peptide is selected from the group consisting of human MOG 1-13 GQFRVIGPRHPIR human MOG 103-135 HSYQEEAAMELKV human MOG 1-121 GQFRVIGPRHPIRALGDEV ELPCRTSPGKNATGMEVGWY RPPFSRWHLYRNGKDQDGD QAPEYRGRTELLKDAIGEGK VTLRIRNVRFSDEGGFTCFF RDHSYQEEAAMELKVEDPFYW human MOG 1-20 GQFRVIGPRHPIRALVGDEV, human MOG 11-30 PIRALVGDEVELPCRISPGK, human MOG 21-40 ELPCRISPGKNATGMEVGWY, human MOG 31-50 NATGMEVGWYRPPFSRWHL, human MOG 141-160 TVGLVFLCLQYRLRGKLRAE, 11 D MO G '151-17 0 YRDRGKLRAEIENLHRTFDP, human MOG 161-180 IENLHRTFDPHFLRVPCWKT human MOG 199-218 YNWLHRRLAGQFLEELRNPE. 17. The composition of claim 14, further comprising at least one peptide with T cell activity derived from MOG. 113 An isolated MBP-derived peptide selected from the group consisting of MBP-1.1, MBP-1.2, MBP-2.1, MBP-2.2, MBP-2.3, MBP2.4, MBP-2.5, MBP-2.6, MBP-3.1 , MBP-4 and MBP-5 shown in Figure 2, or modifications or analogs thereof. The isolated peptide of claim 18 which is MBP-4 or a modification or analogue thereof. The peptide analogue of claim 18, wherein at least one amino acid residue is replaced with alanine, glutamic acid, or methylamino acid. A peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, MBP-2.6, depicted in Figure 2, wherein the lysine (K) is replaced with alanine (AND). The peptide analogue of MBP-2.1, wherein the lysine (K) at position 10 is replaced by alanine (A), wherein the peptide analogue has the amino acid sequence DENPWIIFFANIVTPRTPPPSQGK. selected from the group of MBP-2, MBP-2.1, MBP-2.2, MBP-3, MBP-3.1, MBP-4 and the nidic bond is replaced by peptide bonds or 23. A peptide peptidomimetic comprising MBP-1, MBP-1, MBP-1.2, MBP-2.3, MBP-2.4, MBP-2.5, MBP-2.6, MBP-5, wherein at least one normal peptide is a non-peptide bond, an analog reduced by a bond analog. 24. Peptide, MBP-1.2, MBP-2, MBP-2.6, MBP-3, at least selected by addition MBP-2.1, from the group comprising MBP-1, MBP-1.1, MD n_o 11UJ. L_. ΜΒΓ 'Ί MBP-3.1, MBP-4 and MBP-5, which has been modified by one charged amino acid residue on 114 amino terminal, carboxy terminal or both ends for increased solubility of the peptides in aqueous solution. 25. A peptide selected from the group consisting of MBP-1, MBP-1.1, MBP-1.2, MBP-2, MBP-2, MBP-2 MBP-2.6, MBP-3, MBP-3.1, MBP-4, and MBP-5, which has been modified by adding at least one amino acid residue to the amino-terminal, carboxy-terminal, or both ends to increase T cell activity, said amino acid residue being derived from the native MBP protein sequence. 26. A method of treating multiple sclerosis in a mammal, comprising administering to the mammal the composition of claim 1 in an amount sufficient to inhibit an autoimmune response in multiple sclerosis. The method of claim 26, wherein the administration is selected from the group consisting of i. injection in non-immunogenic form, subcutaneous injection in non-immunogenic form, oral administration, inhalation administration, sublingual administration, transdermal administration. administration, rectal administration, or any combination thereof, 22. The method of claim 26, wherein the administration is performed subcutaneously in a non-immunogenic form. 19. A method of preventing the onset of multiple sclerosis in a mammal susceptible to multiple sclerosis, in a multiplex amount. comprising administering to the mammal a composition according to claim 1 sufficient to prevent the onset of symptoms of sclerosis 115 30. A method of treating multiple sclerosis in a mammal, comprising administering to the mammal the composition of claim 5 in an amount sufficient to control the symptoms of multiple sclerosis. 31. A method of treating multiple sclerosis in a mammal, comprising administering to the mammal the composition of claim 10 in an amount sufficient to control the symptoms of multiple sclerosis. 32. A method of treating multiple sclerosis in a mammal, comprising administering to the mammal the composition of claim 14 in an amount sufficient to control the symptoms of multiple sclerosis. multiplex u that mammals 33. A method of treating sclerosis, the composition of claim 15 in an amount sufficient to control the symptoms of multiple sclerosis. mammals, He adds J Z | '/ i η | ~ I. 1- » ·. > 1. [0009] The multiple sclerosis in a mammal is characterized in that at least two different preparations according to claim 1 are administered simultaneously or sequentially in an amount sufficient to control the symptoms of multiple sclerosis. 35. A treatment regimen for treating multiple sclerosis in a mammal, comprising administering simultaneously or postuona as a single treatment regimen of conjugated MBP-derived peptides selected from the group of peptide combinations comprising: MBP-1, MBP-2, MBP-3, MBP-4, and MBP-5; 116 MBP-1.1, MBP-2.1, MBP-3, MBP-4, and MBP-5 MBP-1.1, MBP-2, MBP-4, and MBP-5; MBP-1, MBP-2.1, MBP-4, and MBP-5; MBP-1, MBP-2, MBP-4, and MBP-5; MBP-1.1, MBP-2.1, MBP-4, and MBP-5; MBP-1.1, MBP-2.1, and MBP-4; MBP-1, MBP-2.1, and MBP-4; MBP-1.1, MBP-2, and MBP-4; MBP-1.1, MBP-2.1, and MBP-5; MBP-1.1 MBP-2.1 and MBP-3; MBP-1 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6; MBP-1.1 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6; MBP-4 and a peptide selected from the group consisting of MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5 or MBP-2.6; MBP-4 and a peptide selected from the group consisting of MBP-1 or MBP1.1; MBP-1.1, MBP-4, and a peptide selected from the group consisting of 82-100, 82-100 [100P> Y], 87-99, and 87-99 [91K> AJ] shown in Figure 14; MBP-1.1 MBP-1.1, 82-100, Fig. 14; MBP-1.1 MBP-4 and a peptide selected from the group consisting of MBP-2.2, MBP-2.4 / MBP-2.5 or MBP-6, shown in Figure 2; MBP-5, MBP-4, and a peptide selected from the group consisting of 82-100 [100P> Y], 87-99 and 87-99 [91.K> A] shown in MBP-5, MBP-4, and a peptide selected from the group consisting of MBP-2.3, MBP-2.4, MBP-2.5 or MBP-2.5, as shown in 117 MBP-1.1, MBP-5, MBP-4, and a peptide selected from the group consisting of 82-100, 82-100 [100P> Y], 87-99, and 87-99 [91K> A] shown in FIG. 14; MBP-1.1, MBP-3, MBP-4 and a peptide selected from the group consisting of MBP-2.2, MBP-2.3, MBP-2.4, MBP-2.5, or MBP-2.6, shown in Figure 2. 36. A multipeptide formulation for pharmaceutical administration to a subject with MS comprising at least two MBP peptides, each peptide being soluble and stable at a predetermined physiological acceptable pH and these the peptides are selected from groups including MBP-1, MBP-1.1 MBP-1.2, MBP-2.1 MBP-2.2 MBP-2.3 MBP-2.5 MBP-2.6, MBP-3.1 MBP-4 a MBP-5. 37. A method of treating advanced stage of multiple sclerosis in a mammal, comprising administering to the mammal a therapeutic composition comprising at least one peptide with T cell activity derived from myelin antigen, or an analogue thereof, in an amount effective to control the symptoms of multiple sclerosis. 38. The method of claim 37, wherein said myelin antigen is selected from the group consisting of myelin basic protein (MBP), myelin oligodendrocyte protein (MOG), proteolipid protein (PLP), and myelin-associated glycoprotein (MAG). ). 39. The method of claim 37, wherein said administration occurs during the acute phase of multiple sclerosis. 118 40. The method of claim 37, wherein said administration occurs during remission of multiple sclerosis symptoms, 41. The method of claim 37, wherein said peptide comprises a peptide analog having an MHC binding affinity that is greater than the MHC binding affinity of the peptide from which the analog is derived. 42. A method of treating advanced stage of multiple sclerosis in a mammal, comprising administering to the mammal a therapeutic composition comprising at least one MBP peptide selected from the group consisting of MBP-1, MBP-1.1, MBP-1.2, MBP-2, MBP- 2.1, MBP-2.2, MBP-2.3, MBP-2.5, MBP-2.5 MBP-3, MBP-3.1, MBP-4, MBP-5, 13-25, 31-50, 61-80, 82-92, 8296, 82-97, 82-98, 82-100, 82-100 [ 100P> Y] 83-100, 83-101, 8497, 84-100, 85-100, 86-105, 87-99, 87-99 [91K> A], 88-100, 88-99, 111- 135, 122-140, 139-170, 141-160, 142-166, 142-168, 146-160, and 153-170, as shown in Figures 2 and 14, or an analogue of the peptide in an amount effective to control the symptoms of multiple sclerosis . 43. The method of claim 42, wherein said peptide is selected from the group consisting of MBP-i, MBP-1.1, MBP-2, MBP-2.1, MBP-2.2, MBP-2.3, MBP-2.4, MBP2.5, MBP-2.6, MBP-3, MBP-3.1, MBP-4 5, 13-25, 87-99, 8799 [91K> A], 82-100, 82-100 [100P> Y] or analogs thereof. 44. The method of claim 37, wherein said peptide is derived from human MOG and is selected from the group consisting of: