JP2002513558A - Myelin basic protein peptide and use thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates generally to the treatment of autoimmune diseases of the central nervous system characterized by demyelination. In particular, the present invention relates to novel peptides derived from human myelin basic protein (MBP). When complexed with the appropriate major histocompatibility complex (MHC) molecule, these peptides can be used to treat multiple sclerosis and other demyelinating autoimmune diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates generally to the treatment of central nervous system autoimmune diseases characterized by demyelination. In particular, the present invention relates to novel peptides derived from human myelin basic protein. These peptides, alone or complexed with appropriate major histocompatibility complex (MHC) molecules, can be used as agents for the treatment of multiple sclerosis and other demyelinating autoimmune diseases.

BACKGROUND OF THE INVENTION Multiple sclerosis (MS) is a chronic, inflammatory autoimmune disease of the human central nervous system (CNS), demyelination, and macrophages, plasma cells and T cells in the CNS (Allen (1991) Path)
theory of Multiple Sclerosis, p.341, McA
lpine's Multiple Scrolls; Matthewse
Et al., Churchill Livingstone, Edinburgh).
MS is an autoimmune disease directed at myelin and myelin-producing oligodendrocytes. In MS and other demyelinating diseases, T cell clones specific for myelin, a component of myelin basic protein (MBP), result in demyelination of the neural sheath in the CNS. In addition to MBP-restricted T-lymphocyte clones, demyelinating inflammatory foci can contain a large number of unrestricted immune cells that can mediate tissue damage (Zamvil (199)
0) Ann. Rev .. Immunol. 8: 579-621; Wucherpf
enning (1991) Immunol. Today 12: 277-282
Martin (1992) J .; Immunol. 148: 1359-1366
Martin (1992) Ann. Rev .. Immunol. 10: 153-
187).

[0003] Major histocompatibility complex (MHC) class II (DR) molecules are antigen-treated /
It is a heterodimer displayed on the cell surface of a presenting cell (APC). In demyelinating diseases, APC class II molecules are derived from myelin and M-derived oligodendrocytes.
BP self-peptide "presents" to helper (CD4 +) T lymphocytes.
This Class II molecule just "presents" the peptide to T cells by binding the peptide in a "peptide binding site" or groove located structurally at the end of the molecule furthest from the cell membrane. The peptide (or antigen) binding site also
Termed "antigen binding pocket" or "MHC groove" (Brown (1993) Na
true 364: 33-39; Stern (1994) Structure
2: 245-251).

[0004] The binding of a peptide to the peptide binding site depends on the peptide and class II
It depends on both primary amino acid sequences of the molecule. Only high affinity binding between the peptide and the class II molecule polypeptide forms a complex that is displayed extracellularly on T cells. The affinity, or strength, of this intermolecular interaction is determined by the same factors present for all peptide: polypeptide binding reactions (eg, conformation, hydrophobic binding, charge, ionic interactions). The restrictions imposed by the primary sequence are further limited by size restrictions; the peptide binding site
It is believed that it can accommodate peptides ranging in length from about 8 to about 12 amino acids ("Agretope" is the portion of the peptide that is recognized by MHC molecules). As a result, different portions of the antigenic polypeptide are typically presented by different Class II molecules. Thus, when a peptide is internalized by APC and proteolytically processed into fragments, only a single peptide or a small number of peptides will bind with high affinity to a particular Class II molecule (ie, , "Specifically binds"). Only a limited number of Class II molecules are expressed in an individual, and only a limited repertoire of peptides derived from a polypeptide antigen is presented.

[0005] For example, in the case of MS, a single region of the MBP is involved in binding a limited set of class II molecules and assists in generating anti-myelin autoreactive T cell clones. In some MS patients, the putative pathogenic "immune-dominant" MBP peptide and the class II molecules to which it binds are DRB1 ^* 1501 and / or DRB5 ^* 0.
Residues 83-, which binds to any of the class II DR alleles designated as 101
It is suggested that there are 102 MBP peptides (MBP 83-102) (other class II alleles and MBP peptides can also mediate MS or other demyelinating diseases in different individuals).

[0006] Recognition of the peptide: class II molecular complex by helper CD4 + T lymphocytes is mediated by the binding of the T cell clone-specific receptor (TCR) to this complex. The affinity of the TCR for this complex is determined by its attraction to both class II molecules and peptides occupying the antigen binding pocket (the portion of the peptide recognized by the TCR is referred to as an "epitope").
High affinity TCR binding to the APC complex activates T cells and their clonal expansion and immunoregulatory cytokines (which can be stimulatory or immunosuppressive)
Induces the secretion of Thus, there is amplification of the immune response specific for the antigenic peptide (epitope) presented by the class II polypeptide. If the peptide is a self peptide, the response can be an autoimmune response.

[0007] Interfering with the ability of the peptide: class II complex to bind to the TCR may inhibit or suppress the development of an autoimmune response. For example, administration of antibodies to MHC class II polypeptides can interfere with complex: TCR binding and the resulting pathogenic immune response. Antibodies to autoreactive TCRs or T cell clones can have the same effect.

[0008] Alternative immunosuppression strategies may be used to take advantage of the need for costimulatory signals for T cells by APCs. To activate CD4 + T cells,
Class II: TCR binding by peptides is not sufficient. An additional "costimulatory" signal is required (not an MHC molecule). Typically, the required costimulatory signal is provided by APC cell surface proteins. Importantly, the interaction of Class II: peptides with TCR without APC costimulation not only does not induce T cell activation, but also re-challenge, known as anergy in vitro and tolerance in vivo. An antigen-specific non-responsive state also occurs (Boussiotis (1994) Curr. Opin.
Immunol. 6: 797-807; Park (1997) Eur. J. Im
munol. 27: 1082-1090). This suppression and re-challenge non-responsiveness has been postulated to be due to T cell clonal anergy or to non-responsiveness induced by immunosuppressive cytokines (Schwartz (198).
9) Cell 57: 1073-1081; Quill (1987) J. Am. Imm
unol. 138: 3704-3712).

A 20 amino acid long MBP 83-102 may be immunodominant, but binds class II molecules and / or T cell receptors with higher affinity Naturally processed MBP peptides in some individuals , Myelin or peptide MBP
83-102 synthetic or recombinant variants may be therapeutically effective. Such peptides are better reagents for inhibiting pathogenic autoimmune responses (eg, via their administration as a tolerogenic inducible soluble class II: peptide conjugate). The present invention meets these and other needs by providing novel MBP peptides that can bind to Class II molecules and TCRs.

[0010] Current treatment of autoimmune diseases mainly consists of treatment of symptoms, but does not mediate the etiology of the disease. A broad spectrum of immunosuppressive agents with a number of undesirable side effects are typically used. Improper treatments currently available prevent or suppress MHC-restricted immune responses, but identify new agents that avoid unwanted side effects (eg, non-specific suppression of the individual's overall immune response) Identify the urgent need for Compounds that can selectively suppress autoimmune responses at the level of helper CD4 + T cells provide a safer and more effective treatment. The present invention meets these and other needs by providing novel MBP peptides and peptide: class II molecular complexes.

SUMMARY OF THE INVENTION The present invention provides an isolated myelin basic protein (MBP) peptide, which has an amino acid sequence

[0012]

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[0013] wherein X is any amino acid. 1
In one embodiment, the isolated myelin basic protein peptide has an amino acid sequence

[0014]

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Or by having conservative substitutions thereof. The isolated myelin basic protein peptide can be linked to a heterologous sequence. In one embodiment, the linkage of the myelin basic protein peptide to the heterologous sequence results in a fusion protein.

The present invention also provides an amino acid sequence

[0017]

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There is provided an isolated myelin basic protein peptide that specifically binds to an antibody directed against the peptide characterized by having

[0019] In another embodiment, the present invention relates to an isolated nucleic acid encoding a myelin basic protein peptide, wherein the peptide has the amino acid sequence

[0020]

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Provides an isolated nucleic acid wherein X is any amino acid. This encoded myelin basic protein peptide has the amino acid sequence

[0022]

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Alternatively, it can be characterized by having conservative substitutions thereof. In one embodiment, the isolated nucleic acid contains SEQ ID NO: 1.

The present invention also provides a composition comprising an MHC class II complex capable of binding to a T cell receptor, wherein the complex comprises: substantially enough MHC to form an antigen binding pocket An MHC class II polypeptide containing the extracellular domain of a class II molecule, wherein this component is encoded by an allele associated with an autoimmune disease directed against myelin basic protein, wherein the class II An MHC class II polypeptide, wherein the components are soluble under physiological conditions in the absence of detergents or lipids; and amino acid sequences

[0025]

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Wherein X is any amino acid and R ₁ is Asn or Gln; wherein the myelin basic protein peptide comprises But MHC class I
Binds to the component I antigen binding pocket. This myelin basic protein peptide has an amino acid sequence

[0027]

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Or have conservative substitutions thereof. The composition may contain a fusion protein and / or effector composition. In alternative embodiments, the autoimmune disease is multiple sclerosis, or the class II molecule polypeptide is HLA D
Contains the antigen binding pocket of R2.

The present invention relates to a pharmaceutically acceptable carrier and an amino acid sequence wherein X is any amino acid.

[0030]

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There is provided a pharmaceutical composition comprising a pharmacologically effective amount of a peptide characterized by having

The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmacologically effective amount of a composition comprising an MHC class II complex capable of binding to a T cell receptor. The complex is substantially an MHC class II polypeptide containing the extracellular domain of an MHC class II molecule sufficient to form an antigen binding pocket, wherein the component comprises An MHC class II polypeptide encoded by an allele associated with an autoimmune disease directed at the subject, wherein the class II component is soluble under physiological conditions in the absence of detergents or lipids; and Amino acid sequence

[0033]

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Wherein X is any amino acid and R ₁ is Asn or Gln; wherein the myelin basic protein peptide is But MHC class I
Binds to the component I antigen binding pocket.

The present invention is directed to an antibody that is specifically immunoreactive with a myelin basic protein peptide under immunologically reactive conditions, wherein the peptide has the amino acid sequence

[0036]

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An antibody is provided, characterized by having the following: In another embodiment, the invention relates to a myelin basic protein peptide comprising a peptide encoded by a nucleic acid encoding a myelin basic protein peptide, wherein the peptide has an amino acid sequence

[0038]

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Wherein the antibody is specifically immunoreactive under immunologically reactive conditions against a peptide wherein X is any amino acid.

The present invention is a method of inhibiting a T cell-mediated immune response to a myelin basic protein in a subject, comprising providing the subject with an isolated myelin basic protein peptide (MBP). Administering the T cell-mediated immune response in an effective amount to treat the T cell mediated immune response, wherein the peptide has the amino acid sequence

[0041]

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Wherein X is a peptide wherein any amino acid is provided.

The present invention is a method of inhibiting a T cell mediated immune response to a myelin basic protein in a subject, comprising providing the subject with an MHC class II complex capable of binding to a T cell receptor. Administering a composition comprising the MHC class II polypeptide, comprising an extracellular domain of an MHC class II molecule sufficient to form an antigen binding pocket, comprising: , Wherein this component is encoded by an allele associated with an autoimmune disease directed against myelin basic protein, wherein the class II component is expressed under physiological conditions in the absence of detergents or lipids. A soluble, MHC class II polypeptide; and an amino acid sequence

[0044]

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Wherein X is any amino acid and R ₁ is Asn or Gln; wherein the myelin basic protein peptide comprises But MHC class I
A method is provided that binds to the antigen binding pocket of the I component.

The present invention provides a method of inhibiting a T cell mediated immune response to a myelin basic protein in a subject, wherein the T cell mediated immune response results in a pathology to the nervous system. provide. In one embodiment, the pathology of the nervous system is
Characterized as multiple sclerosis.

The present invention is a method for identifying a T cell epitope on an antigen, wherein the antigen comprises M
When bound to the antigen binding pocket of an HC class II molecule, the antigen can bind to a T cell receptor, and such binding triggers an extracellular acidification reaction by T cells that express the T cell receptor. The method comprises the steps of: a) providing a composition containing the T cell epitope bound to the antigen binding pocket of an MHC class II molecule; b) providing a T cell expressing the T cell receptor Contacting with an epitope; and c) measuring the extracellular acidification, wherein a change in the extracellular acidification indicates binding of the T cell epitope to the T cell receptor. , Provide a way. In one embodiment, the change in extracellular acidification is measured using a microphysiometer.

A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification, drawings and claims.

All documents, patents and patent applications cited herein are expressly incorporated herein by reference for all purposes.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods and compositions that can be used to inhibit aspects of the immune system that are responsible for unwanted autoimmunity. The present invention relates to autoimmune-mediated demyelinating diseases (
This is the first description of a novel set of peptides derived from human myelin basic protein (MBP) that can be used in the treatment of multiple sclerosis (MS) in particular. The present invention provides, for example, therapeutic compositions containing novel MBP peptides for inducing oral or systemic tolerance. Also provided is a soluble MBP peptide: Class II molecule conjugate for targeting T cells that mediate an autoimmune response. This complex is a self-reactive T cell receptor that can bind to the MBP: class II complex (
TCR) by targeting T cell clones reactive with MBP. Binding of the MBP: Class II complex to T cells is therapeutically effective for inhibiting or treating the development of an autoimmune demyelinating disease. Although the present invention is not limited by any particular mechanism of action, the compositions of the present invention can induce a T-cell clonal anergy and tolerance, or induce a cytokine-mediated immunosuppressive immune response, for example. By doing so, it is pharmaceutically active.

The present invention relates to a pharmaceutically acceptable carrier and an amino acid sequence wherein X is any amino acid.

[0052]

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There is provided a pharmaceutical composition comprising a pharmacologically effective amount of a peptide characterized by having Administration of the peptide alone can be used to induce oral tolerance (a systemic, antigen-specific, immunological overresponse event resulting from oral administration of the protein). The mechanism by which oral tolerance occurs depends on the amount of antigen (peptide) administered. Low doses are effective in inducing regulatory immunosuppressive helper CD4 + T cells.
Higher doses favor clonal deficits and anergy (tolerance). Regulatory T cells induced by low doses of oral antigen, when restimulated, secrete immunosuppressive cytokines that suppress inflammatory responses in an antigen-nonspecific manner. Orally administered autoantigens are widely used in a wide variety of experimental autoimmune diseases (the most widely studied animal models for MS, mouse demyelinating disease experimental autoimmune encephalomyelitis (E
AE)). Mouse MBP peptides have been used to generate antigen-specific oral tolerance and to prevent the development of acute EAE (Bitar (1988) Cell. Immun. 112: 364-3).
70; Higgins (1988) J. Am. of Immun. 140: 440-4
45; Koury (1992) J. M .; Exp. Med. 176: 1355-13
64; Whitcre (1996) Clin. Immunol. Immuno
pathol. 80: S31 to S39). In one study, oral administration of bovine myelin to MS patients induced T cells that secreted the immunosuppressive cytokine TGF-β-1 (Fukaura (1996) J. Clin. Invest.
. 98: 70-77). Thus, the novel human MBP peptides of the present invention may be effective in generating an antigen-specific immunosuppressive response, such as in inducing oral tolerance.

The present invention also provides a purified complex comprising at least an effective antigen binding portion of a dimeric class II molecule or a single chain (monomeric) subunit of class II and an MBP peptide of the present invention. About the body. An "effective antigen-binding moiety" is a dimer necessary to bind the MBP peptide with sufficient affinity to form a complex that is recognized by the autoreactive TCR and can specifically bind to it. Or monomer class I
Minimum amount of I molecule. The conjugates of the invention containing the MBP peptide and the appropriate Class II molecule can both be covalently or non-covalently bound or otherwise associated. A third component, such as an "effector" component, can also be included in a conjugate of the invention. The effector component is a cytotoxic agent (eg,
Toxins, radioisotopes, apoptosis inducers, etc.) and can selectively eliminate or neutralize targeted autoimmune T cell populations.

In another aspect, the invention relates to a pharmaceutical composition, wherein the peptides and conjugates of the invention are active ingredients. These compositions are used to downregulate or eliminate autoreactive components of the immune system and treat autoimmune (demyelinating) T cell-mediated immune responses. The present invention provides a pharmaceutically acceptable carrier,
And a pharmaceutical composition comprising a pharmacologically effective amount of a MBP: Class II molecular conjugate. This complex selectively binds to MBP autoreactive T cells. The present invention is not limited to any means by which the composition is pharmaceutically active, but the peptides and conjugates can induce clonal anergy / tolerance or induce a cytokine-mediated immunosuppressive immune response I can do it. This complex can be further conjugated to a cytotoxic agent to specifically remove targeted T cells.

I. Nucleic Acids Encoding MBP Peptides and MHC Class II DR Alleles and Recombinant Expression of These Nucleic Acids The present invention provides novel myelin basic protein (MBP) peptides, and at least Provided are complexes with MHC class II polypeptides containing sufficient extracellular domain of class II molecules to form an antigen binding pocket, as well as nucleic acids encoding these MBP peptides and class II molecule polypeptides. In addition to providing synthetic forms of MBP and Class II nucleic acids, the invention provides recombinantly produced nucleic acids, MBP peptides, and Class II polypeptides. Since the nucleic acids encoding these peptides and proteins can be expressed in vivo or in vitro, the present invention provides various means for expressing these sequences, including expression cassettes, vectors, cell lines, transgenic plants and animals, and the like. I will provide a.

The present invention can be practiced in combination with any of the methods or protocols known in the art that are well described in the scientific and patent literature. Accordingly, only some general techniques are described before discussing exemplary methodologies and examples for the novel reagents and methods of the present invention. General Techniques Genes and nucleic acids encoding the MBP peptides and MBP: Class II complexes of the present invention, which can be RNA, cDNA, genomic DNA, or hybrids thereof, can be obtained from a variety of sources. Detached; genetically engineered;
It can be expressed recombinantly; or it can be synthesized in vitro. MBP and M of the present invention
The nucleic acid encoding the BP: Class II complex may be expressed in transgenic plants and animals, transformed cells and transformed cell lines, transformed cell lysates, or may be partially purified. It can be in pure or substantially pure form. Techniques for nucleic acid manipulation of genes encoding MBP and MBP: Class II complexes of the invention (eg, site-directed mutagenesis,
Preparation of library, subcloning into expression vector, labeling of probe,
DNA sequencing, DNA hybridization) are described in the scientific and patent literature. For example, Sambrook, Ed., MOLECULAR C
LONGING: A LABORATORY MANUAL (2nd edition), 1st to 3rd
Vol., Cold Spring Harbor Laboratory, (198
9) ("Sambrook"); CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, Ausubel, Ed. John Wil
eye & Sons, Inc. , New York (1997) (“Ausu
bel "); and LABORATORY TECHNIQUES IN B
IOCHEMISTRY AND MOLECULAR BIOLOGY: HY
BRIDZATION WITH NUCLEIC ACID PROBES
, Part 1, Theory AND Nucleic Acid Prepara
ed., edited by Tijssen, Elsevier, N .; Y. (1993) ("T
ijssen "). Sequencing methods typically use dideoxy sequencing (Sequenase, US Biochemical), but other kits and methods are available and are well known to those skilled in the art.

[0058] Nucleic acids and proteins are detected, isolated, and quantified by any of a number of common means well known to those skilled in the art, according to the teachings and methods of the invention described herein. . These include, for example, analytical biochemical methods (eg, NMR, spectrophotometry, radiometry, electrophoresis, capillary-electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and high speed Diffusion chromatography), various immunological methods (eg, liquid or gel precipitation, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Immunofluorescence assay,
Southern analysis, Northern analysis, dot blot analysis), gel electrophoresis, RT-PC
R, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.

[0059] Mutations can be introduced into nucleic acids by various conventional techniques well described in the scientific and patent literature. For example, one rapid method for efficiently performing site-directed mutagenesis is the overlapping extension polymerase chain reaction (OE-PCR) (Urban (1997) Nucleic Acids Res. 25: 2).
227-2228).

MHC Class II DR Allele The present invention provides a nucleic acid encoding a novel myelin basic protein (MBP) peptide and at least the extracellular domain of a MHC class II molecule sufficient to form an antigen binding pocket. And nucleic acids encoding MHC class II polypeptides. The present invention relates to human DR class II alleles (particularly DRB1 ^* 1501 and DRB1 ⁾ encoding polypeptides capable of binding to the MPB peptides of the invention.
5 ^* 0101 (eg, Weber (1993) Proc. Natl. Acad)
. Sci. 90: 11049-11053). However, the complexes of the present invention are not limited to these DR alleles or their corresponding Class II polypeptides. MBP of the present invention:
A class II complex includes any polypeptide comprising an MHC class II antigen binding pocket, or a functional equivalent thereof. These polypeptides are the M
It can bind to the BP peptide with sufficient affinity to be used in the method of the present invention. Means for identifying such DR alleles and class II polypeptides known are well known in the scientific and patent literature, for example, through electronic data banks (Medline, GenBank, etc.). MHC encoding a polypeptide having an antigen binding pocket capable of binding a specific peptide
Means for identifying Class II DR alleles (eg, the MBP peptides of the invention) are also well known in the scientific and patent literature (eg, Rammen).
see (1995) Immunogenetics 41: 178-228; S
inigaglia (1994) Curr. Opin. Immunol. 6: 5
See 2-56).

[0061] In one embodiment of the invention, the class II: peptide conjugate is water-soluble. Water solubility can be manipulated in Class II polypeptides by deleting transmembrane domain (typically hydrophobic) amino acid residues. This is D
It is most effectively achieved by recombinantly redesigning the R allele and expressing truncated class II molecules. Deletion of the transmembrane domain may be due to the complete deletion of residues or by recombinantly redesigning the class II coding sequence to replace hydrophobic residues with hydrophilic residues. This also facilitates recovery of the water-soluble class II polypeptide after recombinant expression. For example, transmembrane inactivated Class II can be secreted directly into the recombinant culture medium of a recombinant expression host.
Additional deletions of cytoplasmic or extracellular residues may be effective in removing potential immunogenic epitopes. In one embodiment, the nucleic acid binds to the minimal peptide binding site / TCR binding polypeptide (ie, binds with sufficient affinity to the MBP peptide of the invention, is recognized by a suitable self-reactive TCR, and (Only the amount of polypeptide necessary to be used in the method of the invention). Peptide binding polypeptides may be in the form of a minimal peptide binding site molecule, isolated or recombinant full length, modified, or truncated Class II
In any form, such as a molecule, it can be in dimeric or monomeric form.

[0062] Additional class II polymorphic DR alleles can be identified and characterized using a variety of methods. This method includes: i) computer search of DNA databases for DNA containing sequences conserved for the class II DR molecules described above, ii) from known DR gene sequences for mRNA, cDNA or DNA sequences or libraries Hybridization with the probe of
ii) PCR or other signal or target amplification techniques, using primers complementary to highly conserved regions in different DR genes.

[0063] Nucleic acid amplification methods can also be used to identify, isolate and generate class II polymorphic alleles. Suitable amplification methods include, but are not limited to: polymerase chain reaction (PCR) (PCR PROTOCOLS,
A GUIDE TO METHODS AND APPLICATIONS,
Innis, Ed., Academic Press, N.M. Y. (1990), and PCR STRATEGIES (1995), edited by Innis, Academic.
Press Inc. , N .; Y. (Innis)); Ligase chain reaction (LC
R) (Wu (1989) Genomics 4: 560; Landegren (
1988) Science 241: 1077; Barringer (1990).
) Gene 89: 117); Transcription amplification (Kwoh (1989) Proc. Na).
tl. Acad. Sci. USA 86: 1173); and self-sustaining sequence replication (Guatelli (1990) Proc. Natl. Acad. Sci. US).
A, 87: 1874); Qβ replicase amplification (Smith (1997) JC).
lin. Microbiol. 35: 1477-1149, automated Qβ replicase amplification assay; Burg (1996) Mol. Cell. Probes 1
0: 257-271) and other RNA polymerase-mediated techniques (eg, NA
SBA, Cangene, Mississauga, Ontario); Ber
ger (1987) Methods Enzymol. 152: 307-316
, Sambrook, Tijssen, and Ausubel, and Mul
lis (1987), U.S. Patent Nos. 4,683,195 and 4,683,
See also No. 202).

Sequencing of Nucleic Acids Sequencing of isolated MBP-encoding nucleic acids and Class II-encoding nucleic acids is used, for example, to identify and characterize allelic DR species encoding polypeptides capable of binding to MBP peptides. Confirming the sequence of a nucleic acid that has been synthetically produced or cloned; confirming a mutation, and the like. MBP coding sequence and class I
The I coding sequence may be inserted as an insert in the vector, as an insert released and isolated from the vector, or in any of various other forms (ie, amplification products).
Can be sequenced. MBP coding inserts and class 11 coding inserts can be released from the vector by restriction enzymes, amplified by PCR, or transcribed by a polymerase. Based on the N-terminus or C-terminus to sequence the insert to identify the full length coding sequence;
Alternatively, primers based on insertion points in the original phage or other vector can be used. Various nucleic acid sequencing techniques are well known and have been described in the scientific and patent literature. See, for example, Rosenthal (1987) supra; Alinghaus (1997) Anal. Chem. 69: 3747-3
753; Dubiley (1997) Nucleic Acids Res. 2
5: 2259-2265.

Expression of Recombinant MBP Peptides and Class II Polypeptides The present invention provides methods and reagents for the recombinant expression of novel MBP peptides and class II molecules used in the conjugates of the present invention. . The nucleic acids of the invention can be introduced into the genome or into the cytoplasm or nucleus of a cell and expressed using a variety of conventional techniques. These techniques are fully described in the scientific and patent literature. For example, Roberts (1987) Nature 328:
731; Berger (1987) supra; Schneider (1995) Pr.
otein Expr. Purif. 6435: 10; Sambrook, Ti
See jssen or Ausubel. Product information from manufacturers of biological reagents and laboratory equipment also provides information on known biological methods. The promoters and vectors used in the present invention can be isolated from natural sources, obtained from sources such as ATCC or GenBank libraries, or synthesized according to the synthetic methods described herein or It can be prepared by recombinant methods. Some selected exemplary general and specific teaching examples of such techniques are set forth below.

Vectors and Transcription Control Elements The present invention provides methods and reagents for making the novel nucleic acids described herein, but uses novel expression cassettes, vectors, transgenic plants and animals. Further provided are methods and reagents for expressing these nucleic acids using constitutive and inducible transcriptional and translational cis-acting (eg, promoters and enhancers) and trans-acting control elements. Expression of a nucleic acid encoding a native, recombinant or synthetic MBP peptide or polypeptide is operably linked to a promoter (which may be tissue-specific, constitutive or inducible) in its coding region. Alternatively, it can be accomplished by incorporating the construct into an expression cassette (eg, an expression vector) and introducing the resulting construct into an in vitro reaction system or appropriate host cell or organism. Synthetic procedures can also be used to produce any of the nucleic acids of the present invention. Transcriptional and translational control elements include transcriptional and translational initiation sequences, promoters and enhancers, transcriptional and translational terminators, polyadenylation sequences, and other sequences useful for transcribing DNA into RNA. In constructing the recombinant expression cassettes, vectors and transgenics of the present invention, a promoter fragment may be used to direct the expression of a desired nucleic acid in all tissues of a plant or animal. Promoters that drive expression continuously under physiological conditions are termed "constitutive" promoters and are active under most environmental conditions and conditions of development or cell differentiation. The expression system may include at least one independent terminator sequence, a sequence that enables replication of the cassette in vivo (eg, a plant, eukaryote, or prokaryote or a combination thereof) (eg, a shuttle vector) and The expression system of choice (eg, plant, eukaryotic, or prokaryotic)
Contains a selectable marker for To ensure proper polypeptide expression under various conditions, a polyadenylation region at the 3 'end of the coding region may be included.

The nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses, for example, expressed transiently in cells using an episomal expression system. Expression vectors capable of expressing proteins are well known in the art. The selectable marker can be incorporated into expression cassettes and vectors to provide a selectable phenotype in transformed cells and sequences encoding episomal maintenance and replication, such that integration into the host genome is not required. For example, the marker encodes antibiotic resistance (especially resistance to chloramphenicol, kanamycin, G418, bleomycin, hygromycin) or herbicide resistance (eg, resistance to chlorosulfuron or Basta). Can enable selection of those cells to be transformed with the desired DNA sequence. For example, Blondel
t-Rouault (1997) Gene 190: 315-317; Aubr
echt (1997) J. Am. Pharmacol. Exp. Ther. 281: 9
See 92-997. Since a selectable marker gene that confers resistance to a substrate such as neomycin or hygromycin can be utilized only in tissue culture, a chemoresistance gene can also be used as a selectable marker in vitro and in vivo.

Production of Transformants and Transgenic Plants and Animals The present invention provides various in vivo systems that express the MBP peptides, class II polypeptides and peptide: class II complexes of the present invention. As this system,
Transformed cells and transgenic plants and animals are included.
In vivo expression systems that can be used include bacteria, yeast, insects (baculovirus)
, Plant and mammalian cell lines. The system used depends on various factors, including the activity and amount desired.

There are several well-known methods for introducing nucleic acids into animals, plants, bacteria and other cells. These are processes often referred to as "transformations," any of which can be used in the methods of the present invention (eg, Sambrook, Ausu)
bel or Tijssen). In vivo expression systems and techniques for transforming a wide variety of animal and plant cells are well known and described in the technical and scientific literature. For example, for plant cells, Weising,
Ann. Rev .. Genet. 22: 421-478 (1988), and for animal and bacterial cells, see Sambrook or Tijssen.

II. MBP Peptides and MHC Class II DR Polypeptides The present invention relates to novel myelin basic protein (MBP) peptides, which have sufficient affinity to be recognized by suitable self-reactive TCRs. Provided are Class II polypeptides comprising at least the amount of polypeptide required to bind MBP, and Class II: MBP peptide complexes. The present invention provides isolated (from natural sources), synthetic and recombinantly produced forms of MBP peptides and Class II polypeptides. These peptides and proteins can be expressed recombinantly in vitro or in vivo. The peptides, polypeptides and conjugates of the invention can be made and isolated using any method known in the art. And the present invention provides some exemplary means for producing such proteins. Furthermore, means for making Class II: peptide conjugates include:
For example, U.S. Patent Nos. (USPN) 5,194,425, issued March 16, 1993; 5,130,297, issued July 14, 1992; 5,284.
No. 5,935, issued on Feb. 8, 1994; 5,260,422, issued on Nov. 9, 1993; and 5,468,481 issued on Nov. 21, 1995. .

The MBP peptides and class II: peptide conjugates of the invention can be synthesized in whole or in part using chemical methods well known in the art (eg, Carut).
hers (1980) Nucleic Acids Res. Symp. Ser
. 215-223; Horn (1980) Nucleic Acids Res
. Symp. Ser. 225-232; Banga, A .; K. , Therape
Utic Peptides and Proteins, Formulati
on, Processing and Delivery Systems (1
995) Technology Publishing Co. , Lancast
er, PA (see "Banga")). For example, peptide synthesis can be performed using various solid-phase techniques (eg, Robert (1995) Science).
ce 269: 202; Merrifield (1997) Methods E
nzymol. 289: 3-13), and automatic synthesis, for example,
ABI 431A Peptide Synthesizer (Perkin
Elmer) can be achieved according to the manufacturer's instructions.

The newly synthesized peptide was prepared using high performance liquid chromatography (HPLC
) And can be substantially purified (eg, Creighto
n, Proteins, Structures and Molecular
Principles, WH Freeman and Co, New York
k NY, 1983). The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing (eg, Edman degradation procedure; Creight)
on, supra). Laser absorption mass spectrometry (MALDI-MS) also assesses the progress of peptide synthesis at all necessary levels (automated assembly, cleavage and deprotection chemistry, RP-HPLC analysis and purification, and confirmation of the structure of the final product) (Moore (1997) Methods Enzymol.
289: 520-542). Electrospray ionization mass spectrometry is a simple and rapid method for confirming proper peptide synthesis and identifying most synthesis by-products (Burdick (1997) Methods Enzymol. 289).
: 499-519).

In addition, the amino acid sequences of the peptides and polypeptides of the present invention or any portion thereof may be changed during direct synthesis and / or may be incorporated into other proteins or any portion thereof using chemical methods. Combined with the sequence from which it is derived, it can produce a mutant polypeptide. The modified peptides and proteins of the present invention
In addition to manipulation of the nucleic acid encoding the sequence (eg, site-directed mutagenesis), it can be generated by introducing unnatural amino acid side chains by chemical modification of the polypeptide (eg, for general methodology, see Paetzel (1997) J.B.
iol. Chem. 272: 9994-10003). As another example, the partial specific uptake of the biotin-containing amino acid biocytin is described in Gallivan (1997) Chem. Biol. 4:
739-749; for site-specific incorporation of unnatural amino acids into proteins in vivo, see, eg, Liu (1997) Proc. Nat
l. Acad. Sci. USA 94: 10092-10097;
Also, Koh (1997) Biochemistry 36: 11314-113.
See also 22.

Class II polypeptides suitable for use in the present invention also include suitable D
It can be isolated from natural sources, such as cell lines expressing the R allele or patients of the appropriate genotype, using various techniques well known in the art. For example, these cells can be solubilized by treatment with papain, by treatment with 3M KCl, or by treatment with a detergent. Class II from lymphocytes
Detergent extraction of proteins followed by affinity purification can also be used.
The detergent may then be dialyzed or selectively bound beads (eg, Bio Bead).
s). The molecule may be from any cell that expresses the class II molecule of interest, such as B or T lymphocytes from an individual with the appropriate genotype (eg, an individual with a demyelinating autoimmune disease). It can be obtained by isolation.
Suitable MHC molecules can be isolated from B cells or T cells that have been immortalized by transformation (eg, transformation of B cells with a replication-defective Epstein-Barr virus using techniques known in the art). .

[0075] Isolation of individual subunits from an isolated Class II molecule is readily accomplished using standard techniques known to those skilled in the art. For example, α and β subunits from class II molecules can be separated using SDS / PAGE and electroelution (eg, Rothenhausler (1990) Proc. N
atl. Acad. Sci. USA 87: 352-354; Gorga (19
87) J. Biol. Chem. 262: 16087-16094; Dornm.
air (1989) Cold Spring Harbor Symp. Qua
nt. Biol. 54: 409-416). One skilled in the art will recognize many other standard methods for separating molecules (eg, ion exchange chromatography, size exclusion chromatography, gel permeation chromatography, HPLC, RP-HP
(LC or affinity chromatography) can be used. See, for example, Banga.

In one embodiment, an additional “effector” composition can be linked to a complex of the invention to inhibit or eliminate an autoimmune response. This molecule
Effector "moieties include, for example, toxins, chemotherapeutics, cytotoxic T lymphocytes (C
TL) Emission of antibodies, lipases, or toxic radioisotopes against surface molecules (eg, radioisotopes (eg, yttrium-90, phosphorus-32, lead-212,
Iodine-131, or gamma irradiation from palladium-109). Numerous protein toxins are well known in the art and include, for example, ricin, diphtheria, gelonin, pseudomonas toxin and abrin. Chemotherapeutic agents include, for example, doxorubicin, daunorubicin, methotrexate,
Cytotoxins, and antisense RNA. Antibiotics can also be used. In some cases, the toxin or other effector component is entrapped in a delivery system such as a liposome or dextran carrier; in these cases, either the active ingredient or the carrier is a class II: peptide complex Can be combined.

In preparing pharmaceutical compositions of the invention, it may often be desirable to modify these peptides or conjugates to alter their pharmacokinetics and biodistribution (discussed further below). For example, a suitable method for increasing the serum half-life of the complex includes a treatment to remove carbohydrates involved in removing the complex from the bloodstream. Preferably, substantially all of the carbohydrate portion is removed by the treatment. When at least about 75%, preferably about 90%, and most preferably about 99% of the carbohydrate moieties are removed, substantially all of the carbohydrate moieties are removed. Conjugation with soluble macromolecules (eg, proteins, polysaccharides or synthetic polymers (eg, polyethylene glycol)) is also effective. Other methods include protection of the complex in vesicles composed of substances such as proteins, lipids (eg, liposomes, discussed below), carbohydrates or synthetic polymers.

Complex Formation Class II polypeptide: MBP peptide complexes can be formed by any standard means known in the art. For example, a peptide of the invention can be non-covalently associated with an antigen binding site, for example, simply by mixing the two components. They can also be covalently attached to the antigen binding pocket using standard procedures, for example, with photoaffinity labels (eg, Hall (1985) Bi).
chemistry 24: 5702-5711; Leuscher (199
0) J. Biol. Chem. 265: 11177-11184; Wraith
(1989) Cell 59: 247-255). Other modes of linkage include, for example, via a carbohydrate / lecithin group to a glycoprotein, a class II
Of the α- and / or β-chains as carbohydrate moieties (Husa
in (1995) Biochem. Mol. Biol. Int. 36: 669-
677). A dehydration reaction with carbodiimide can also be used. Heterobifunctional linkers (eg, N-hydroxysuccinimide ester (NHS), N-
Succinimidyl 3- (2-pyridyldithio) propionate (SPDP), substituted 2-iminothiolanes, glutaraldehyde, and the like can also be used (eg, Traut (1995) Biochem. Cell Biol. 73).
: 949-958; Haselgrubler (1995) Bioconjug.
Chem. 6: 242-248; Carroll (1994) Bioconj.
ug. Chem. 5: 248-256). Alternatively, Class II:
Peptide complexes can be designed as one continuous recombinant polypeptide. P
CT Publication No. WO 96/40944, December 19, 1996; WO 96/40
194, December 19, 1996; and WO 97/04360, January 1, 1997.
See January 6.

[0079] Effector or other fusion protein components (as discussed above, such as for purification) may also be attached to the complex using these methods. The sequence from which the complex is prepared depends on the components in each situation. For example, in an exemplary protocol, a peptide moiety and an MHC subunit component are non-covalently associated by contacting (eg, by mixing) the peptide with the MHC subunit component. The effector is then covalently linked. Alternatively, the effector and MHC subunit can be first conjugated, and the conjugate can be complexed with the MBP peptide component. If the effector is itself a protein, the entire complex can be made directly from appropriately encoding DNA using recombinant methods.

Evaluation of the Complex The peptides of the invention and the MBP: Class II complex can be assayed using various in vitro models well known in the art. As noted above, TCR binding by Class II: peptides is not sufficient to activate CD4 + T cells. Additional "co-stimulation" signals are needed. The interaction of the class II: peptide complexes of the present invention with the TCR lacks a costimulatory signal. Therefore,
An unresponsive state of antigen-specific T cells is induced (Boussiotis (1
994) Curr. Opin. Immunol. 6: 797-807; Park
(1997) Eur. J. Immunol. 27: 1082-1090). This "tolerance" or "anergy" immunosuppression and re-challenge non-responsiveness is due to T
It can be caused by cellular clonal anergy, by non-responsiveness induced by immunosuppressive cytokines, or both (Schwar
tz (1989) Cell 57: 1073-11081; Quill (1987).
J.). Immunol. 138: 3704-3712).

[0081] In one exemplary in vitro system, the complex is incubated with autoreactive T cells, and the cells are re-challenged with MBP. Peripheral blood T cells or myelin (M) from patients with demyelinating diseases (eg, MS)
BP) reactive T cell clones can be used. T cells can be isolated prior to analysis. Autoreactive T cells are treated with antigen (MB) in vitro before administering putative tolerogens.
P or myelin) (using syngeneic APC). “Resting” T cells (cells that have not been stimulated in vitro) can be used. T cells can be treated with various amounts of the conjugates of the invention over various lengths of time. Binding of this complex to MBP-reactive T cells results in inhibition or elimination of the in vitro autoimmune response.

The degree of immunosuppression or non-rechallenge responsiveness (ie, tolerance, anergy) is measured by monitoring cell proliferation, cell metabolism, secretion of cytokines or lymphokines or any form of cell activation. obtain. T cell activation can be measured by various means well known in the art. For example, T cell proliferation is assessed, for example, as measured by ³ H thymidine incorporation or 3- (4,5-dimethyl-thiazole-2-7 ′)-2,5-diphenyltetrazolium bromide (MTT) incorporation. Can be done. (See, for example, Liu (1997) J. Am.
Neurochem. 69: 581-593). Alternatively, the immunosuppressive efficacy of class II: peptide complexes can be assessed by measuring cytokine transcription, translation or secretion, as T cells synthesize and secrete cytokines upon activation. Accordingly, various cytokines and lymphokines, such as interleukins, interferons (IFNs) (eg, γINF),
Tumor necrosis factor (TNF) (eg, TNFβ) and the like can be quantified. Cell death can also be monitored. This is because prolonged incubation of quiescent T cells with soluble MHC-class II peptide complexes has been observed to result in T cell apoptosis (Arimilli (1996) Im).
munol. and Cell Biol. 74: 96-104). Cell death
It can be measured by various known procedures, such as dye exclusion transmission. Apoptosis can be assessed using, for example, cellular DNA fragmentation, observation (using transmission electron microscopy), detection and quantification of apoptosis-related proteins (eg, bcl-2, etc.) (eg, Arimilli (1996) supra). checking).

A preferred method of assessing the immunosuppressive efficacy of a class II: peptide complex of the present invention is to use a microphysiometer to measure the rate of production of acidic metabolites in T cells. Through the use of. Using this device, the effect of the interaction of this complex with the TCR can be quickly and easily detected.
The earliest measurable event in T cell activation following stimulation by a TCR: Class II: peptide complex binding reaction is an increase in lymphocyte metabolism. This is reflected in its acidic by-products. Microscopic physical instruments measure the acidity of the major catabolic products, lactic acid and carbon dioxide, in mammalian cells. Very slight changes in the acidity of the culture medium bathing small amounts of cells can be easily determined using a photo-addressable potentiometric sensor. The rate of acidification is used as a measure of the catabolic rate of the cell being assayed. For example, Parce (1989) Science
ce 246: 243-247; Owicki (1990) Proc. Natl
. Acad. Sci. USA 87: 4007-4011; Renschler.
(1995) Cancer Res. 55: 5642-5647; Beeson
(1996) J. Amer. Exp. Med. 184: 777-782.

The addition of the MBP peptide or myelin polypeptide to the mixture of autoreactive T cells and syngeneic APCs is achieved by adding class I bound to autoreactive TCRs and APCs.
I: Increased acid release due to antigen-specific binding to the MBP peptide complex. Thus, the immunosuppressive ability of the MBP peptide or class II: MBP peptide complex of the present invention is determined by first adding the complex to autoreactive T cell / APC cultures, followed by antigen (MBP or myelin). ) Can be assessed by challenge. The lack or relative decrease in cell activation, which is indicative of immunosuppression, can be measured by the decrease or lack of extracellular acidification.

In one embodiment, the conjugate of the invention with an effector component is used. This treatment can be two-fold: the individual is first treated with a class II: MBP peptide complex to down regulate the immune system, and further down regulation is achieved by treatment with the complex and the effector component.

(III. Detection and Purification of Polypeptide) The present invention also provides methods and reagents for isolating, detecting or quantifying the MBP peptide and the class II polypeptide complex of the present invention by various methods. I do.

Antibodies In one embodiment, the invention provides an antibody that is specifically immunoreactive with an MBP peptide of the invention under immunological reaction conditions. These Abs are:
It can be used in the isolation, detection or quantification of an MBP peptide or complex of the invention. Methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art and have been described in the scientific and patent literature. For example, Co
Ligan, CURRENT PROTOCOLS IN IMMUNOLOG
Y, Wiley / Greene, NY (1991); States (ed.) BAS
IC AND CLINICAL IMMUNOLOGY (7th edition) Language
Medical Publications, Los Altos, CA ("
States ”); Goding, MONOCLONAL ANTIBODIE
S: PRINCIPLES AND PRACTICE (2nd edition) Academ
ic Press, New York, NY (1986); Kohler (19)
75) Nature 256: 495; Harlow (1988) ANTIBO.
DIES, A LABORATORY MANUAL, Cold Spring
See Harbor Publications, New York.
Such techniques include selection of antibodies from a library of recombinant antibodies displayed in phage ("phage display libraries") or the like in cells. Huse (1989) Science 246: 12
75; Ward (1989) Nature 341: 544; Hoogenbo.
om (1997) Trends Biotechnol. 15: 62-70; K
atz (1997) Annu. Rev .. Biophys. Biomol. Str
oct. 26: 27-45. Recombinant antibodies are available from Nordhaug
(1997) J. Am. Immunol. Methods 204: 77-87; Bo
der (1997) "Yeast surface display for
screening combinatorial polypeptide
libraries, "Nat. Biotechnol. 15: 553-557
Can be expressed in mammalian cells by means of a transient expression vector or a permanent expression vector.

(Purification of MBP Peptide and Complex) The MBP peptide and the complex of the present invention can be purified by the method and reagent of the present invention.
From a variety of sources, natural sources, selected synthetic or recombinant expression systems (eg, plant cells, larval homogenates, bacterial cells, yeast, mammalian cells, human cells,
Depending on the tissue culture medium, transgenic plants and animals), it is possible to purify to substantially pure. General information on standard purification procedures is well known in the patent and scientific literature, as described above;
Scopes, R.A. K. , Protein Purification: Pri
nchiples and Practice, 2nd edition, Springer Ve
rlag, (1987), Banga, Ausubel, and Sambrook.
See k.

Fusion Proteins MBP peptides and conjugated polypeptides can also be used to facilitate cell killing (eg, using effector factors as described above), protein detection, purification, or other applications. Can be expressed as a protein having one or more additional polypeptide domains linked to it. Domains that facilitate detection and purification include, for example, metal-chelating peptides (eg, polyhistidine regions (tract) and histidine-tryptophan modules) that allow for purification on immobilized metals, and purification on immobilized immunoglobulins. Protein A domain, as well as a FLAGS extension / affinity purification system (Im
munex Corp. (Seattle, WA). A cleavable linker sequence between the purification domain and the plant disease resistant polypeptide (eg, Factor Xa or enterokinase (Invitrogen, Sa
n Diego, CA)) may be useful to facilitate purification. For example, one expression vector contains a polypeptide-encoding nucleic acid sequence linked to six histidine residues, followed by a thioredoxin and an enterokinase cleavage site (eg, Williams (1995) Biochemist).
ry 34: 1787-1797). Histidine residues facilitate detection and purification, while enterokinase cleavage sites provide a means for purifying the desired protein from the rest of the fusion protein. Vectors encoding the fusion proteins and techniques relating to the application of the fusion proteins are well described in the scientific and patent literature. For example, Kroll (1993), DNA Ce
ll. Biol. , 12: 441-53.

IV. MBP Peptides and Conjugates: Formulation and Administration of Pharmaceutical Compositions The MBP peptides and class II: peptide conjugates of the present invention are typically used to form pharmaceutical compositions. It is combined with a pharmaceutically acceptable carrier (excipient). Pharmaceutically acceptable carriers include, for example, physiologically acceptable compounds that act to stabilize a pharmaceutical composition of the invention or to increase or decrease its rate of absorption or clearance. May be included. Physiologically acceptable compounds include, for example, sugars (eg, glucose, sucrose, or dextran), antioxidants (eg, ascorbic acid or glutathione)
Chelating agents, compositions that reduce the clearance or hydrolysis of low molecular weight proteins, peptide or polypeptide complexes, or excipients or other stabilizers, and / or buffers. Detergents can also be used to stabilize the pharmaceutical composition or to increase or decrease the absorption of the pharmaceutical composition. See below. Exemplary detergents include liposomal carriers. Pharmaceutically acceptable carriers and formulations for peptides and polypeptides are known to those of skill in the art and have been described in detail in the scientific and patent literature. For example, Remington's Pharmaceutical Sc
update, Mack Publishing Company, Ea
stone, Pennsylvania ("Remington's") and B
See ana.

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art will appreciate that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the protein or polypeptide of the present invention, and on its particular physicochemical characteristics. to understand.

(Aqueous Solutions for Enteral, Parenteral, or Transmucosal Administration) Compositions for administration generally include a pharmaceutically acceptable carrier, preferably when the composition is water-soluble. , An aqueous carrier) dissolved in a solution of the peptide or polypeptide of the present invention. Examples of aqueous solutions that can be used in formulations for enteral, parenteral, or transmucosal drug delivery include, for example, water, saline, phosphate buffered saline, Hanks's solution, Ringer's solution, dextrose / Saline, glucose solution and the like. The formulation may include pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions. Such auxiliary substances include, for example, buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.
Additives may also include additional active ingredients such as bactericides or stabilizers. For example, the solution may include sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, or triethanolamine oleate. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. MBP in these formulations
The concentration of the peptide and / or class II: peptide conjugate can vary widely and is selected based on the particular mode of administration selected and the needs of the patient, primarily based on fluid volume, viscosity, weight, etc. Is done.

Solid Formulations for Enteral Delivery Solid formulations can be used for enteral (oral) administration. They are, for example,
It may be formulated as a pill, tablet, powder, or capsule. For solid compositions, conventional non-toxic solid carriers can be used. These carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, sodium carbonate, and the like. For oral administration, the pharmaceutically acceptable non-toxic compositions contain any of the commonly used excipients (e.g., the carriers listed previously) and generally 10% to 95% of the active ingredient (MBP peptide Or a complex). Non-solid formulations may also be used for enteral administration. The carrier can be selected from various oils. Such oils include oils of petroleum, animal, plant or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil, etc.). Suitable pharmaceutical excipients include, for example, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, barley, rice, wheat, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, chloride Sodium, dried skim milk powder, glycerol, propylene glycol, water, ethanol and the like.

The MBP peptide and polypeptide conjugates of the present invention, when administered orally,
It is recognized that digestion must be protected. This is typically by complexing the peptide or polypeptide complex with a composition that renders it resistant to acid and enzymatic hydrolysis, or Or by packaging in a carrier that is resistant to liposomes (eg, liposomes). Means of protecting the compound from digestion are well-known in the art. For example, Fix (1996) Pharm Res.
13: 1760-1764; Samanen (1996) J. Am. Pharm. Ph
armacol. 48: 119-135; U.S. Patent No. 5,391,377, which describes a lipid composition for oral delivery of a therapeutic agent (liposome delivery is described in further detail below). Is done).

Topical Formulations for Transdermal / Transmucosal Delivery Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salt derivatives and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration may be by nasal spray or with suppositories. For example, Ban
ga, Chapter 10; Sayani (1996) "Systemic deliver.
ry of peptide and proteins cross ad
sortive mucosae "Crit. Rev .. Ther. Drug.
Carrier Syst. 13: 85-184. For topical transdermal administration, the drug is formulated in ointments, creams, salves, powders, and gels. Transdermal delivery systems may also include, for example, patches. See, for example, Banga, Chapter 9.

[0096] Peptide and polypeptide conjugates can also be administered by sustained delivery or release mechanisms. This can deliver the formulation inside. For example, biodegradable microspheres or capsules, or compositions (eg, MBP peptides: Class I
Other biodegradable polymer forms capable of sustained delivery of the I polypeptide conjugate) can be included in the formulations of the invention (eg, Putney (1998) Nat. Bio.
technology. 16: 153-157).

Formulations for Inhaled Delivery For inhalation, peptides or polypeptides can be delivered using any system known in the art. Such systems include, for example, dry powder aerosols, liquid delivery systems, air jet nebulizers, propellant systems, and the like. For example, Patton (1998) Biotechniques 1
6: 141-143; products and inhalation delivery systems for polypeptide macromolecules are described below (eg, Dura Pharmaceuticals (
(San Diego, CA), Aradigm (Hayward, CA), Ae
rogen (Santa Clara, CA), Inhale Therape
utic Systems (San Carlos, CA), etc.).

For example, a pharmaceutical formulation can be administered in the form of an aerosol or mist. For aerosol administration, the formulation may be supplied in finely divided form along with a surfactant and propellant. The surfactant is preferably soluble in the propellant.
Representative of such agents are fatty acids containing 6-22 carbon atoms (e.g., caproic, octanoic, lauric, palmitic, stearic, linoleic,
Aliphatic polyhydric alcohols or cyclic anhydrides thereof (eg, ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, hexitol anhydride (derived from sorbitol) of linolenic acid, olesteric acid, and oleic acid) )), And polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycerides, can be used. The surfactant may comprise from 0.1% to 20%, preferably from 0.25% to 5% by weight of the composition. The balance of the formulation is usually propellant. Liquefied propellants are typically gaseous at room temperature conditions and condensed under pressure. Among the suitable liquefied propellants are lower alkanes containing up to 5 carbon atoms (
For example, butane and propane), and are preferably fluorinated alkanes or fluorochlorinated alkanes. The mixtures described above can also be used. In making an aerosol, a container equipped with a suitable valve is charged with a suitable propellant, including the finely divided compound and a surfactant. Thus, until the components are released by the action of the valve,
Maintained at elevated pressure. For example, Edwards (1997) "Large
poor particles for pulmonary drug
delivery "Science 276: 1868-1871.

In another embodiment, the device for delivering the formulation to respiratory tissue is an inhaler, where the formulation volatilizes. Other liquid delivery systems include, for example, an air jet nebulizer.

Other Formulations In preparing the medicaments of the present invention, various formulation modifications can be used and manipulated to alter pharmacokinetics and biodistribution. Numerous methods for altering pharmacokinetics and biodistribution are known to those skilled in the art. Examples of such methods include the protection of complexes in vesicles composed of substances (eg, proteins, lipids (eg, liposomes, see below), carbohydrates, or synthetic polymers (discussed above)). Is mentioned. For a general discussion of pharmacokinetics, see, for example, Re
See Mington's, Chapters 37-39, or Banga, Chapter 6.

Delivery Routes The peptide and polypeptide conjugates used in the methods of the invention can be delivered alone or as a pharmaceutical composition by any means known in the art. Such means include, for example, systemic, regional, or local; intra-arterial, intrathecal (IT), intravenous (IV), parenteral, intrapleural, topical (topica).
l), subcutaneously, intratracheally (eg, by oral or local administration)
Transmucosal (eg, oral mucosa, bladder mucosa, vaginal mucosa, uterine mucosa, rectal mucosa, nasal mucosa). Actual methods for preparing administrable compositions are known, or will be apparent, to those skilled in this art and have been described in detail in the scientific and patent literature. For example, Remington's
s and Banga. Particularly preferred modes of administration include intra-arterial or intrathecal (IT) injections, particularly those that have a "regional effect" such as to focus on specific organs, such as the brain and CNS. (Eg, Gurun (1997) Anesth An).
alg. 85: 317-323). For example, if it is desirable to deliver the peptide or polypeptide conjugate of the invention directly to the brain, intra-carotid artery injection. Parenteral administration is useful when high systemic dosages are required.
The preferred route of delivery. Enteral administration is a preferred method when administration of the peptide to induce oral tolerance is for therapeutic purposes (eg, Kennedy (
1997) J. Amer. Immunol. 159: 1036-1044; Kent (19
97) Ann. NY Acad. Sci. 815: 412-422). Actual methods for preparing parenterally administrable compositions are known, or will be apparent, to those skilled in this art and are described in detail below (e.g.,
Remington's, Banga Chapter 7). Also, Bai (1997) J
. Neuroimmunol. 80: 65-75; Warren (1997) J.
. Neurol. Sci. 152: 31-38; Tonegawa (1997).
J. Exp. Med. 186: 507-515.

Treatment Regimens; Pharmacokinetics Pharmaceutical compositions may be administered in various unit dosage forms, depending on the method of administration. Dosages for representative peptide and polypeptide pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisory in nature and will be adjusted depending on the particular therapeutic situation, patient tolerance, and the like. The amount of MBP peptide or class II: peptide conjugate sufficient to accomplish this is defined as "therapeutically effective dose." The effective dosing schedule and amount for this use (ie, the “dosing regimen”) depends on various factors. Such factors include the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health,
The patient's physical condition, age, pharmaceutical formulation and concentration of the active agent and the like are included. In calculating the dosage regimen for a patient, the mode of administration also is taken into account. The dosing regimen must also take into account pharmacokinetics (ie, the rate of absorption of the pharmaceutical composition), bioavailability, metabolism, clearance, and the like. For example,
The latest version of Remington's; Egleton (1997) "Bioava
irability and transport of peptide a
nd peptide drugs into the brain "Pept
ids 18: 1431-1439; Langer (1990) Science
e 249: 1527-1533.

In therapeutic applications, the compositions are administered to a patient suffering from a demyelinating disease in an amount sufficient to cure or at least partially arrest the disease and / or its complications. Is done. An amount sufficient to accomplish this is defined as "therapeutically effective dose." Effective amounts for this use will depend on the severity of the disease, the general state of the patient's health, the frequency and route of administration, the judgment of the clinician, and the like. For example, in one embodiment, the dosage for intravenous (IV) administration of a soluble class II: peptide conjugate pharmaceutical composition is over several hours (typically 1, 3, or 6 hours). Administered from about 0.01 mg / hr to about 1.0 mg / hr, which can be repeated for several weeks in intermittent cycles. Significantly higher dosages (eg, about 10 m
g / ml) can be used, especially if the drug is to be administered to an isolated site and not into the bloodstream. For example, it may be administered to a body cavity or a lumen of an organ (eg, cerebrospinal fluid (CSF)).

Dosage may be adjusted by assessing the reduction or amelioration of symptoms or by subject criteria (
For example, analysis of blood or histopathological specimens) can be determined empirically. For example, in MS, the disease is characterized by various complaints and findings of CNS dysfunction. This is accompanied by remissions and persistent relapses. Onset is usually insidious. The most frequently presented symptoms are one or more of the ends, trunk,
Or paresthesia on one side of the face; weakness or awkwardness of the legs or hands (clu
or visual impairment (eg, partial blindness and pain in one eye, diplegia, blurred vision, scotoma). Other common early symptoms are instantaneous eye palsy, transient weakness at one or more ends, slight stiffness or unusual fatigue in the limbs, slight gait disturbance, difficulty adjusting the bladder, dizziness, Or mild emotional disorders; all are evidence of diffuse CNS involvement, and often occur months or years before the disease is recognized. Successful treatment can also be monitored by histopathology. In MS, plaques or islets of demyelination with oligodendrocyte destruction and perivascular inflammation predominate throughout the CNS, mainly in white matter, and in the lateral and dorsal columns, especially in the cervical and dorsal regions. It deflects into the optic nerve and the perivascular area. Areas in the midbrain, pons, and cerebellum are also affected, and gray matter in both the cerebellum and cords can be affected. Cell bodies and axons are usually protected, especially in early lesions. Later, axons can be destroyed, especially in long areas, and fibrotic gliosis gives this area a "sclerotic" appearance. Both early and late lesions can be found simultaneously. Accordingly, the compositions of the invention are administered to prevent the progression of the disease and to reduce the onset, frequency, or severity of these or other symptoms.

[0105] In one exemplary embodiment, the dosage is from about 0.5 mg / kg to about 25 mg / kg.
kg, and in a preferred embodiment from about 3 mg / kg to about 15 mg / kg. In another exemplary embodiment, the unit dosage is between about 0.01 to 1000 mg / dose, and in a preferred embodiment, about 10 to about 100 mg / dose. See also U.S. Patent No. 5,468,481, issued November 21, 1995. In another embodiment, the peptide is administered enterally to induce oral tolerance at a dosage ranging from 10 to 2500 μg / day, and in a preferred embodiment at about 20 to 50 μg / day. For example, Barnett (1998) Arthritis
Rheum. 41: 290-297. Here, 20, 100,
Oral administration of cartilage-derived type II collagen (to ameliorate arthritis) at a dosage range of 500 or 2,500 μg / day showed the best results with 20 μg / day.

Pharmaceutical compositions comprising the peptides and conjugates of the invention can be administered alone or in combination with other therapeutic treatments. Single or multiple doses of the composition can be administered, depending on the dosage and frequency, as required and tolerated by the patient.

Liposomal Formulations The present invention provides for drugs that include the transmembrane domain of the class II subunit of the complex. In one embodiment, the pharmaceutical formulation comprising the Class II: MBP peptide conjugate is incorporated into a lipid monolayer or bilayer. The invention also provides formulations wherein the water-soluble peptide or conjugate is attached to a monolayer or bilayer surface. For example, the peptides can be attached to hydrazide-PEG- (distearoylphosphatidyl) ethanolamine-containing liposomes (eg, Zalipsk)
y (1995) Bioconjug. Chem. 6: 705-708). Any form of liposome or lipid membrane can be used, such as a flat lipid membrane or a cell membrane of intact cells (eg, red blood cells). Liposomal formulations are obtained by any means. These means include intravenous administration, transdermal administration (
For example, Vutla (1996) J. Mol. Pharm. Sci. 85: 5-8), transmucosal administration, or oral administration. The present invention also provides pharmaceutical preparations in which the peptides and / or conjugates of the invention are incorporated into micelles and / or liposomes (eg, Suntres (1994) J. Ph.
arm. Pharmacol. 46: 23-28; Woodle (1992) P
harm. Res. 9: 260-265).

[0108] Liposomes and liposome formulations can be prepared according to standard methods, and are also well known in the art. For example, Remington's; A
kimaru (1995) Cytokines Mol. Ther. 1: 197
-210; Alving (1995) Immunol. Rev .. 145: 5-3
1; Szoka (1980) Ann. Rev .. Biophys. Bioeng.
9: 467; U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. In one embodiment, the liposomes of the invention typically comprise a class II: peptide complex located on the surface of the liposome, in a manner such that the complex is available for interaction with the TCR. Usually, the transmembrane region is first taken into the film at the time of forming the film. Liposomes can also be used to target desired drugs (eg, toxins or chemotherapeutic agents) to specific self-reactive T cells.

[0109] Liposomal charge is an important determinant in liposome clearance from the blood. Here, negatively charged liposomes are more rapidly taken up by the reticuloendothelial cell line (Juliano (1975) Biochem. Biophy.
s. Res. Commun. 63: 651) and thus have a shorter half-life in the bloodstream. Incorporation of the phosphatidylethanolamine derivative increases circulation time by preventing liposome aggregation. For example, incorporation of N- (ω-carboxy) acylamide-phosphatidylethanolamine into large unilamellar vesicles of L-α-distearoylphosphatidylcholine dramatically increases liposome circulatory life in vivo (eg, Ahl (1997) Biochim. Bi.
ophys. Acta 1329: 370-382). Liposomes with long circulating half-lives may typically be desired for therapeutic and diagnostic applications. For example, liposomes that can be maintained in the bloodstream for up to 8, 12, or 24 hours are particularly preferred embodiments of the present invention.

Typically, liposomes are prepared with about 5 to 15 mol% of a negatively charged phospholipid (eg, phosphatidylglycerol, phosphatidylserine, or phosphatidyl-inositol). Negatively charged phospholipids (eg,
Phosphatidylglycerol) also prevents spontaneous liposome aggregation,
Thus, it serves to minimize the risk of forming liposome aggregates that are smaller than the size. Membrane-rigidi at a concentration of at least about 50 mol%
fying agent (e.g., sphingomyelin or saturated neutral phospholipids), and 5-15 mol% of monosialyl gangliosides are generally present in the bloodstream, as described in U.S. Patent No. 4,837,028. It may provide for increased circulation of the liposome preparation.

In addition, liposome suspensions can contain lipid defense factors that protect lipids against free radical damage and lipid peroxide damage on storage. Lipid-soluble free radical quencher (eg, α-tocophenol and water-soluble iron-specific chelating agent (eg, ferrioxyanine (ferrioxi))
anine)) is preferred.

The formulations of the present invention may comprise multi-layered vesicles of different sizes. In this method,
The vesicle-forming lipid is dissolved in a suitable organic solvent or solvent system and dried under vacuum or with an inert gas to form a thin lipid film. If desired, the film can be redissolved in a suitable solvent (eg, tertiary butanol) and then lyophilized to a more homogeneous lipid mixture, which is a powder-like form that is more easily hydrated. ). The film is covered with an aqueous solution of the peptide or polypeptide complex and can be hydrated, typically with stirring for 15-60 minutes. The size distribution of the resulting multilamellar vesicles can be changed to smaller sizes by hydration of the lipid under more vigorous stirring conditions or by the addition of a solubilizing detergent (eg, deoxycholate). The hydration medium contains the peptide or complex at the desired concentration in the internal volume of the liposome in the final liposome suspension. Typically, the drug solution contains 10 to 100 mg / ml of a peptide or conjugate of the invention in a buffered saline solution.

After preparation of the liposomes, the liposomes can be sized to achieve the desired size range and relatively narrow size distribution of the liposomes. One preferred size range is about 0.2-0.4 micron, which allows the liposome suspension to be sterilized by filtration through a conventional filter (typically a 0.22 micron filter). enable. The filter sterilization method uses liposomes of about 0.2
When scaled down to ~ 0.4 microns, it can be implemented on a high throughput basis. Several techniques are available for sizing liposomes to a desired size (see, for example, US Pat. No. 4,737,323). By sonication of the liposome suspension either by tank or probe sonication,
Causes a progressive size reduction into smaller unilamellar vesicles of less than 0.05 microns in size. Homogenization is another method that relies on the sharing of shear energy from large liposome fragments to smaller liposome fragments. In a typical homogenization procedure, the multilamellar vesicles are re-passed through a standard emulsion homogenizer until a selected liposome size (typically between about 0.1-0.5 microns) is observed. Circulated. In both methods, the particle size distribution can be monitored by conventional laser beam particle size discrimination. Extrusion of liposomes through small pore polycarbonate or asymmetric ceramic membranes is also an effective method for reducing liposome size to a relatively well-defined size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired liposome size distribution is achieved. Liposomes can be successfully extruded through a smaller pore membrane to achieve a progressive reduction in liposome size.

Even under the most efficient methods of encapsulation, liposome suspensions sorted in initial size can contain up to 50% or more complex in free (unencapsulated) form. If a particular formulation is desired, several methods are available for removing unentrapped compounds from the liposome suspension. In one method, the liposomes in this suspension are pelleted by high-speed centrifugation, leaving free compounds and very small liposomes in the supernatant. Another method involves concentrating the suspension by ultrafiltration, and then resuspending the concentrated liposomes in an exchange medium. Alternatively, large liposome particles can be separated from solute molecules using gel filtration. Following this treatment, the liposome suspension can be administered, for example, intravenously, intraperitoneally (IP), transdermally, or transmucosally.
l) The concentration can be as desired for use in administration. This involves resuspending the liposomes in a suitable volume of a suitable medium (where the liposomes have been concentrated, for example, by centrifugation or ultrafiltration), or concentrating this suspension ( Here, the total suspension volume was increased by the step of drug removal). This suspension is then sterilized by filtration, as described above. These liposomes, including peptides or class II: peptide complexes, can be administered parenterally or topically at dosages that vary according to, for example, the mode of administration, the drug being delivered, the particular disease being treated, and the like. Can be administered.

Micelles are commonly used in the art to increase the solubility of molecules having non-polar regions. Thus, those skilled in the art will recognize that micelles are useful in the compositions of the present invention. Micelles containing the conjugates of the invention are prepared according to methods well known in the art (see, eg, Remington's, Chapter 20). Micelles containing the peptides and / or conjugates of the present invention typically comprise a standard surfactant or detergent.
ent). Micelles are formed in aqueous solution by surfactants (molecules containing a hydrophobic moiety and one or more ionic or otherwise strong hydrophilic groups). As the concentration of solid surfactant increases, the monolayer adsorbed at the air / water or glass / water interface packs tightly, so that further occupancy is already present in the two monolayers at the interface. Requires excessive compression of active molecules. An increase in the amount of surfactant dissolved above that concentration causes an equal amount of new molecules to aggregate into micelles. Suitable surfactants include sodium laurate, sodium oleate, sodium lauryl sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and PL
URONIC F-127 (registered trademark) (Wyandotte Chemical)
ls Corp. ). Preferred surfactants are nonionic polyoxyethylene and polyoxypropylene detergents suitable for IV injection (eg, PL
URONIC F-127 (registered trademark), n-octyl-α-D-glucopyranoside, etc.). Phospholipids, such as those described for use in generating liposomes, can also be used for micelle formation. In some embodiments of the invention, the class II subunits of the complex include a lipophilic transmembrane region and a relatively hydrophilic extracellular domain, such that the mixed micelles share a common surfactant or phosphorous. It can be formed in the presence of lipids and subunits. The mixed micelles of the present invention may comprise any combination of subunits, phospholipids and / or surfactants. Thus, micelles may comprise subunits and detergents, subunits in combination with both phospholipids and detergents, or subunits and phospholipids.

[0116] Kits may also be provided for therapeutic or diagnostic use. In one embodiment, the pharmaceutical formulation of the invention is in lyophilized form, which can be placed in a container. Complexes that may or may not also bind to the label or toxin include buffers such as Tris, phosphate, carbonate, etc., stabilizers, antibiotics, inactive proteins such as serum albumin, and the like. Included in the kit with a set of instructions for use. Generally, these materials will be present in less than about 5% by weight, based on the amount of conjugate, and will usually again be present in a total amount of at least about 0.001% by weight, based on protein concentration. It is often desirable to include an inert bulking agent or excipient to dilute the active ingredient, where the excipient can be present from about 1% to 99% by weight of the total composition. If the assay employs an antibody that can bind to the complex, it will usually be in a separate vial. Typically, the antibodies are conjugated to a label and formulated by techniques well known in the art.

Definitions To facilitate understanding of the invention, a number of terms are defined below.

The term “antibody” refers to a polypeptide substantially encoded by one or more immunoglobulin genes or a genus or subgenus of a polypeptide of the invention as described above. A fragment or synthetic or recombinant analog thereof that specifically binds and recognizes substances and antigens.

The term “conservative substitution” refers to a change in the amino acid composition of a peptide or protein, such as a polypeptide comprising an MBP peptide sequence of the invention, that does not substantially alter the activity of the peptide or protein. This may be a conservatively modified alteration of a particular amino acid sequence, ie, an amino acid substitution of an amino acid that is not important for protein activity, or has similar properties of an amino acid (eg,
Including substitutions with other amino acids (acidic, basic, positively or negatively charged, polar or non-polar, etc.), such that substitutions of important amino acids do not substantially alter activity. Polypeptide sequences of the invention implicitly include conservatively substituted variants thereof. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for each other: 1)
Alanine (a), serine (S), threonine (T); 2) aspartic acid (D)
Glutamic acid (E); 3) asparagine (N); glutamine (Q); 4) arginine (R); lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V And 6) phenylalanine (F), tyrosine (
Y), tryptophan (W) (Creightton (1984) Protein)
s, W.S. H. See also Freeman and Company). One skilled in the art will recognize that the substitutions identified above are not the only possible conservative substitutions. For example, for some purposes, all charged amino acids, even if they are positive or negative, may be considered to be conservative substitutions for one another. In addition, individual substitutions, deletions or additions that alter, add or delete one amino acid or a small percentage of amino acids in the encoded sequence are also referred to as "
Conservatively modified modifications "may be considered. The term "conservative substitution" also refers to changes in the nucleic acid sequence such that the substitution does not substantially alter the expected activity of the nucleic acid, e.g., the peptide or protein encoded by the nucleic acid. Does not change the activity of Nucleic acid sequences of the invention implicitly include conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences. In particular, degenerate codon substitution is achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer (1991)
) Nucleic Acid Res. 19: 5081; Ohtsuka (19
85) J. Amer. Biol. Chem. 260: 2605-2608; Rossoli
ni (1994) Mol. Cell. Probes 8: 91-98).

“Fusion protein” refers to a composition comprising at least one polypeptide or peptide domain that binds a second domain. The second domain can be a polypeptide, peptide, polysaccharide, and the like. A “fusion” can be a bond created by a chemical bond or by charge (electrostatic attraction, ie, salt bridges, H bonds, etc.) interactions. If the polypeptide is recombinant, a "fusion protein" can be translated from the common message. Alternatively, the composition of the domain can be linked by any chemical or electrostatic means.

“Heterologous sequence” refers to any amino acid or nucleic acid sequence that is not a myelin basic protein sequence.

As used herein, “isolated” refers to, for example, MBP peptide, class II:
When referring to a molecule or composition, such as a peptide complex, or nucleic acid, the molecule or composition is associated with a protein, other nucleic acid (eg, RNA), in vivo or in its naturally occurring state, or other Separated from at least one other compound, such as a contaminant. Thus, a polypeptide or nucleic acid is considered isolated if it is isolated from any other component with which it naturally associates, such as in a cell extract (eg, a cell membrane). However, an isolated composition can also be substantially pure. An isolated composition can be in a homogeneous state and can be in a dry or aqueous solution. Purity and homogeneity can be determined by, for example, polyacrylamide gel electrophoresis (SDS-PAGE).
Or it can be determined using analytical chemistry techniques such as high performance liquid chromatography (HPLC).

The term “nucleic acid” or “nucleic acid sequence” refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single-stranded or double-stranded form. The term encompasses nucleic acids, ie, oligonucleotides, that include a known analog of a natural nucleotide having similar or improved binding properties for a desired purpose, such as a reference nucleic acid. The term also includes nucleic acids that are metabolized in a manner similar to the naturally occurring nucleotides, or that are metabolized at an improved rate over the desired purpose. The term also includes nucleic acid-like structures with a synthetic backbone. The DNA backbone analogs provided by the present invention include phosphodiesters, phosphorothioates, phosphorodithioates, methylphosphonates, phosphoramidates, alkyl phosphotriesters, sulfamates, 3'-thioacetals, methylene (methylimino), 3'- Including N-carbamates, morpholino carbamates, and peptide nucleic acids (PNA); Oligonucleoti
des and Analogues, a Practical Approa
ch. Edited by Eckstein, IRL Press, Oxford Univ
severity Press (1991); Antisense Strateg
ies, Annals of the New York Academy
f Sciences, volume 600, edited by Basega and Denhardt (
NYAS 1992); Milligan (1993) J. Mol. Med. Chem.
36: 1923-1937; Antisense Research and
See Applications (1993, CRC Press). P
NA comprises a non-ionic backbone such as N- (2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/3915.
4; Meta (1997) Toxicol Appl Pharmacol 1
44: 189-197. Other synthetic scaffolds encompassed by this term include methylphosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukuup (1997) Bioc).
hemistry 36: 8692-8698) and benzylphosphonate linkage (Samstag (1996) Antisense Nucleic Ac).
id Drug Dev 6: 153-156). The term nucleic acid refers to a gene,
Used interchangeably with cDNA, mRNA, oligonucleotide primers, probes and amplification products.

The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a subject.
A pharmaceutical composition of the invention is a formulation comprising a pharmaceutically effective amount of a MBP peptide and / or a class II: peptide conjugate, and a pharmaceutically acceptable carrier.

The terms “treat” and “treatment” refer to prophylactic and therapeutic treatments that are administered to a subject. Prophylactic treatment can be performed on individuals who do not have the disease or condition, who do not show signs of the disease or condition, or who show only early signs of the disease or condition.
Including administering a treatment for the purpose of reducing or eliminating the risk of acquiring the disease or condition, or reducing or eliminating a symptom or pathological condition resulting from or associated with the disease or condition. . The present invention provides a method for treating a T cell-mediated immune response to myelin basic protein in a subject with an MBP peptide and / or a class II: peptide conjugate, for an individual considered at high risk for developing a demyelinating disease or condition. Methods for treating by administration are provided. Therapeutic treatment also prevents, eliminates, reduces or eliminates any condition or condition in an individual who exhibits signs of the condition or disease, or is at risk of developing or causing such a condition or disease. Administering the treatment for the purpose. Thus, the methods of the present invention provide a means of treating a demyelinating disease and preventing, eliminating, eliminating or otherwise reducing the clinical symptoms and tissue pathology of the disease, as discussed above. . A “pharmacologically effective amount” is an amount of a compound that is administered to an individual to effectively treat the individual prophylactically or therapeutically against the disease or condition.

The examples and embodiments described herein are for illustrative purposes only, and in view of that, various modifications or alterations will be suggested to those skilled in the art and It is to be understood that they should be included within the text as well as the appended claims.

EXAMPLES The following examples are provided by way of illustration and do not limit the invention described in the claims.

Example 1 Identification of Core Structure and Critical TCR Contact Residues in Antigenic MBP Peptide by Measuring T Cell Activation Signals Pharmaceutical formulations containing these compositions and methods of treating a T cell mediated immune response to MPB in a subject by administering these novel compositions are provided. The following example demonstrates the novel MBP peptide comprising a core TCR recognition sequence amino acid residue involved in the binding of an autoreactive TCR associated with a demyelinating disease, such as MS, by the MBP peptide and appropriate class II molecules. The identification will be described in detail.

A new, minimal structure and key residues for its interaction with the self-reactive TCR have been identified for the human myelin basic protein (MBP) peptide.
Synthetic peptide analogs were identified as DRB5 expressing MHC class II (DR2) molecules.
* Incubated with TCR on 0101 restriction transformed human T cell clone (SS8T). Microphysiological meter (m
A real-time cellular response to a T cell activation event stimulated by binding of a class II: peptide to the TCR was measured using a microphysiometer. This microphysiometer monitored changes in extracellular acidification rates in response to MBP peptide: Class II polypeptide binding to self-reactive TCRs. The increase in extracellular acidification rate is a direct consequence of early T cell signaling events, such as high affinity TCR binding to class II: peptide complexes. Culture SS
8T cells were treated with N- (amino) -terminal and C- (carboxy) -terminal truncated MB
The P-peptide was separately exposed in a microphysiometer chamber to determine the minimum amino acid residues required for a T cell response. MBP with one alanine substitution in parallel
Peptide analogs were tested in this assay to identify key amino acid residues involved in TCR interactions. As described below, a minimum core length of 10 amino acid residues was determined to correspond to peptide MBP (91-100) (SEQ ID NO: 38), and residues F-91, K-93, N- 94, I-95 and V-96 are T
It was determined to be essential for CR interaction. Acidification rate measurements correlated well with increased levels of gamma interferon (IFN) and tumor necrosis factor (TNF) alpha cytokine production.

[0130] Herpesvirus simyli (HVS) immortalized human T cells were used in an in vitro assay to identify novel MBP peptides of the invention. HVS T cells are
Expresses the same level of TCR as normal human T cells without any loss of antigen recognition (
Weber (1993) Proc. Natl. Acad. Sci. USA 90
: 11049). Specifically, the H, used in these assays, referred to as SS8T,
VS transformed human T cell clones were generated from MS patients. SS8T has been previously described by HLA class II DR2 (DRB5 * 0101) polypeptide and MB.
It was characterized as being restricted to the P (84-102) peptide (Webe (1
993) described above. MBP having an N-acetylated tyrosine residue at the N-terminus (84-
102) (Ac-MBP (83-102) Y83)
When complexed with HLA DR2 (DRB5 * 0101), it was characterized as being recognized by SS8T transformed T cells (Mukku (1995) Mol. I.
mmunol. 32: 555; Arimilli (1995) J. Mol. Biol. C
hem. 270: 971). To identify the minimum length and key TCR contact residues of this MBP (83-102) Y83 peptide, different truncated peptides and alanine analog peptides were synthesized. The various peptides used in this study were
This is shown systematically in FIGS. 1A and 1B. As an alanine analog peptide, one amino acid was replaced with alanine for each residue in the MBP (90-102) Y83 sequence.

The key MBP peptide amino acid residues required for TCR engagement in this model system are two factors: peptide residues responsive to binding to amino acid residues of the MHC class II binding groove (peptide agretope) And the interaction of some of the amino acid residues of this peptide with the TCR (epitope). To determine the minimum length of the immunodominant epitope, various truncated peptides were incubated with SS8 T cells at different concentrations for 48 hours at 37 ° C. in microtiter plates. After incubating the T cells with the peptide, the culture was incubated with monoclonal antibodies (mAbs) (anti-human γIFN mAb and rabbit anti-human γIFN polyclonal Ab, Endogen Inc., Woburn, respectively) for the presence of secreted γIFN and TNFβ. MA; peroxidase-conjugated goat IgG and rabbit IgG, Jackson Immunoresearch
ch Laboratories, West Grove, PA; human γIFN
, Boehringer Mannheim, Indianapolis, IN
Anti-human TNF-β mAb, goat anti-human TNF-β polyclonal and recombinant human TNF-β, R & D Systems Inc, Minneapolis
, MN). In parallel analysis, SS8T
Cells were immobilized in agarose and exposed to various truncated peptides in a microphysiometer chamber to monitor their rate of extracellular acidification.

Peptides prepared for these studies were acetylated at the N (amino) -terminus and amidated at the C (carboxy) -terminus. Sequence Ac-YDEN
MBP with PVVHFFKNIVTPRTPP (SEQ ID NO: 1) (83-10
2) a peptide; and the sequence Ac-GFGYGGRASDYKSAHKGFKG (
The MBP (124-143) peptide having SEQ ID NO: 37) was applied to
Sidechain protected Fmo on Biosystems 431A automated peptide synthesizer
Synthesized by standard solid-phase method using c-amino acid. The deprotected crude peptide was purified by reverse phase HPLC, and the homogeneity and identity of the purified peptide was confirmed by mass spectroscopy. All truncated and alanine analog MBP peptides (FIG. 1) were synthesized by solid phase peptide synthesis using Fmoc chemistry. All chemicals, including Rink amide MBRA resin and side chain protected Fmoc amino acids, were obtained from Nova Biochem, San Diego, CA. HBTU / HOBt or PyBOP activation chemistry was described by Luu (1996).
) Int. J. Peptide Protein Res. 47:91, ABI431A automated peptide synthesizer or ABIMED / GILSO
Employed for protected amino acid coupling on a NAMS 422 multiple peptide synthesizer. After synthesizing the peptides on the solid phase, they were converted to 5% of 4% as a scavenger.
Cleavage with TFA containing -methoxybenzenethiol and 5% 4-methylmercaptophenol. The crude peptide was added to a pentane: acetone mixture (4: 1
, V: v) and isolated by centrifugation. The peptide was washed three times with a pentane: acetone mixture, then with pentane and dried in vacuo. Peptides were purified by reverse phase HPLC using a C18 column; pure fractions were pooled and lyophilized. Peptides were characterized by electrospray mass spectroscopy.

The T cells were treated with 2 mM L-glutamine, 100 units / ml penicillin, 100 u
The cells were cultured at 37 ° C. in RPMI 1640 medium supplemented with g / ml streptomycin, 10% fetal bovine serum and 50 units / ml human recombinant IL-2 (rIL-2). Every other day, cells were transferred to fresh culture medium. Different concentrations of various peptides were added at a density of 20,000 cells / 200 μl / well in the absence of rIL-2.
Incubated with cells in microtiter tissue culture plates. 4 at 37 ° C
After an 8-hour incubation, cultures were collected from each well and tested for γIFN and TNFβ cytokines. The detection of γIFN is performed by Arimilli
(1995) Ab ELISA as described above. For detection of TNFβ, Nunc Maxisorb 96-well plates were coated with a concentration of 0.5 ug / well of anti-human TNFβ mAb and incubated at 4 ° C. overnight. Wells were blocked with 0.1% bovine serum albumin and samples were incubated for 2 hours at room temperature. A standard curve was prepared using recombinant human TNFβ
Produced by using a dilution range of 0.01 ng / ml. Goat anti-human TNFβ was then added at a concentration of 1 ug / ml and the plates were incubated at 25 ° C. for a further 2 hours. The wells are washed three times and 3,3 ',
5,5'-tetramethylbenzidine (TMB, Moss, Inc, Pasade)
na, MD) prior to color development with HRP-conjugated mouse anti-goat Ab and 1 ug / ml
For 1 hour at 21 ° C. The reaction was stopped with 2N sulfuric acid in 5 minutes and the absorbance was measured at 450 nm.

The newly cultured SS8T cells were transformed with low melting point agarose (Molecullar D
devices Corp. Nag et al., Sunnyvale, CA).
Immobilized in microphysiological meter cell capsules as described in 1992. Briefly, T cells were quiescent for 2 days after IL-2 pulse application. Cells were counted and suspended in serum-free loading medium (low buffered PRM1 1640 with 10% fatty acid free, endotoxin free BSA). Cells were collected by centrifugation and resuspended at a concentration of 3 × 10 5 T cells per 7.5 μl loading medium. Low melting agarose, thawed and stored at 37 ° C, was added at 2 / 7.5 μl.
. A 5 μl concentration was added to the suspended cells. 10 μl of agarose / cell mixture
Immediately, the cell capsule cap (Mo) held in a 12-well culture plate
Lecμlar Devices Corp. A) spotted in the center. After 5 minutes, 2 ml of loading medium was placed in a capsule cap on solidified agarose and the membrane insert was placed on the cells. The assembled cell capsules are placed in a Cytosensor chamber at 37 ° C. and 10% BS / ml
A, but without added HEPES or bicarbonate, low buffered RPMI 1
Perfused with 640 medium at 50 μl per minute. Extracellular acidification measurement was performed using Cyt
McConnell (1992) Sci in an oscillator microphysiology meter.
ence 257: 1906 by collecting 45 second voltage titration measurements every 2 minutes. The acidification rate data (uV / sec) was calibrated to 100% prior to cell stimulation, which allowed comparison of data from cells in another chamber.

FIGS. 2A, 2B and 2C show the results of these analyses, with the first six N-terminal truncated peptides: MBP (84-102) (SEQ ID NO: 2), MBP (85-1).
02) (SEQ ID NO: 3), MBP (86-102) (SEQ ID NO: 4), MBP (87
-102) (SEQ ID NO: 5), MBP (88-102) (SEQ ID NO: 6), and
MBP (89-102) (SEQ ID NO: 7) shows increased levels of γIFN, TNFβ and extracellular acidification rates. SS8 T cells exposed to these peptides show a 2- to 4-fold increase in γIFN and a 3- to 4-fold increase in TNFβ production in a dose-dependent manner (FIGS. 2A, 2B). Similarly, an increase in the acidification rate at 118% of the basal rate was observed within 10 minutes (FIG. 2C), with amino acid residues 84, 85, 86, 87, 88, and 88 89 is not important for TCR recognition in SS8 T cells. ΓIFN and T measured after 48 hours (h)
NFβ cytokine concentration correlated well with the rate of acidification.

FIGS. 2D, 2E and 2F show the following five N-terminal truncated MPBs (90-102
) (SEQ ID NO: 8), MBP (91-102) (SEQ ID NO: 9), MBP (92-1)
02) (SEQ ID NO: 10), MBP (93-102) (SEQ ID NO: 11), and M
FIG. 9 shows the results of comparing γ (IFN, TNFβ and acidification rate of BP (94-102) (SEQ ID NO: 12) peptide. Of these, amino acid residue 91, residue 92
, And truncation of residue 93 did not produce γIFN, TNFβ cytokines, and acidification rates. This indicates that these residues are important in T cell stimulation. Since then, one study has suggested that residue 92 (F-92) is important for binding to class II (Wucherpfennig (1994).
J.). Exp. Med. 179: 279). Class II: MBP (93-102
) (SEQ ID NO: 11) The lack of a T cell response by the complex is due to a lack of peptide binding to class II polypeptides or to important TCR contact residues (epitope residues).
Due to the lack of

In another assay, SS8 T cells were transformed with the six C-terminal truncated peptide MBP (8
3-101) (SEQ ID NO: 13), MBP (83-100) (SEQ ID NO: 14), M
BP (83-99) (SEQ ID NO: 15), MBP (83-98) (SEQ ID NO: 16)
, MBP (83-97) (SEQ ID NO: 17), and MBP (83-96) (SEQ ID NO: 18). As shown in FIGS. 3A, 3B, and 3C, only the parent sequence (SEQ ID NO: 1) and MBP (83-101) (SEQ ID NO: 13) showed an increase in γIFN, TNFβ, or acidification rate. This indicates that the C-terminal amino acid residue 100, residue 99, residue 98, residue 97 and residue 96 are important in MBP TCR stimulation. MBP (83-100
) (SEQ ID NO: 14) and MBP (83-99) (SEQ ID NO: 15) peptides produced small amounts of γIFN and TNFβ. 5 additional C-terminal truncated peptides MBP (83-95) (SEQ ID NO: 19), MBP (83-94) (SEQ ID NO: 20), MBP (83-93) (SEQ ID NO: 21), MBP (83-92) ) (SEQ ID NO: 22), and a similar analysis of MBP (83-91) (SEQ ID NO: 23)
did not show any stimulation of γIFN, TNFβ, or an increase in the rate of acidification (
(FIGS. 3D, 3E and 3F). These data include amino acid residue 92, residue 93,
Residues 94 and 95 are shown to be important in peptide T cell recognition. The result of both N-terminal and C-terminal truncation is FFKNIVTPRT (MB
P (91-100) (SEQ ID NO: 38)), when presented by Class II DRB5 * 0101, indicates that it is the minimum core sequence for TCR stimulation by MBP.

The contribution of individual amino acids in peptides towards TCR engagement can be studied by various methods, including D amino acid analogs, substitution of conserved amino acid residues, exchange of charge on amino acids, and alanine substitution. Alanine substitution is more common.
Because it is the simplest side-chain amino acid with a chiral center, and it has only a small interference in the secondary structure of polypeptides and proteins. It is based on a comparison of evolutionarily related proteins,
It is a general substitution for all but aromatic amino acids. Therefore, the important TCR contact residues were defined using an alanine substitution strategy. Various analog peptides from MBP (90-102) (SEQ ID NO: 8) with a single amino acid substitution using alanine were incubated with SS8 T cells. Cultures were tested for γIFN, TNFβ and compared for acidification rates. 4A, 4B and 4C show two peptides MBP (83-102) A90 (SEQ ID NO: 24) and MBP (83
-102) When A92 (SEQ ID NO: 26) was presented to SS8 T cells by class II DRB5 * 0101, γIFN, TNF with increased acidification rate
It shows that β could be induced. MBP (83-102) A91 (SEQ ID NO: 25),
MDP (83-102) A93 (SEQ ID NO: 27), MDP (83-102) A9
4 (SEQ ID NO: 28), and the MDP (83-102) A95 (SEQ ID NO: 29) peptide did not induce cytokines and showed no increase in acidification rate. This indicates that amino acid residues F-91, K-93, N-94, and I-95 are important in T cell stimulation. In one study, MBP (84-1)
02) In the peptide, residues F-91 and K-93 are secondary and N-9
4 was suggested to be a major TCR contact residue (Vergelli (199).
7) J.I. Immunol. 158: 3746). The current data shows that residue F-91
, K-93, N-94, and I-95 are identified in this system as MBP (84-102).
It indicates that this is an important TCR contact residue in the peptide.

Alanine analog MBP (83-102) A97 (SEQ ID NO: 31), MBP (
83-102) A98 (SEQ ID NO: 32), MBP (83-102) A99 (SEQ ID NO: 33), MBP (83-102) A100 (SEQ ID NO: 34), MBP (83
-102) A101 (SEQ ID NO: 35) and MBP (83-102) A102
(SEQ ID NO: 36) shows increased levels of γIFN with increased acidification rates,
2 shows TNFβ cytokines. MBP (83-102) A96 (SEQ ID NO: 30)
Only did not induce cytokines and showed no increase in acidification rate (FIGS. 4D, 4E and 4F). This experiment indicates that V-96 is also a key residue for T cell recognition. As shown in FIG. 4F, peptide MDP (8
3-102) Except for A96 (SEQ ID NO: 30), all peptides show an intermediate increase in acidification rate within 10 minutes. Therefore, the acidification rate associated with the alanine analog peptide correlated with γIFN and TNFβ production by SS8 T cells. In another study, N-94 and V96 showed that DRB5 * 0101 and MB
It was suggested to be a TCR contact residue for the P (84-102) peptide restricted T cell clone (Wucherpfennig (1994) supra).

In all experiments, MBP (124-143) (SEQ ID NO: 37) was used as a negative control. Although the MBP (124-143) peptide has high affinity for binding to DR2 (Mukku (1995) supra), DRB
Does not stimulate 5 * 0101 restricted T cell clones (Arimilli (1995)
Described above).

FIG. 5A shows the core TCR recognition sequence as MBP (91-100) (SEQ ID NO: 38).
) (Area surrounded by a box). As described above, this peptide was identified by measuring the rate of extracellular acidification in SS8T cells using truncated peptides and by the γIFN, TNFβ cytokine response. FIG. 5B shows that the key amino acid residues involved in TCR contacts are F-91, K-93, N-
Shown (by arrows) as 94, 1-95, and V-96. As described above, these amino acids were identified by the extracellular acidification rate in SS8T cells and the γIFN, TNFβ cytokine response by using an alanine analog peptide.

Prior to T cell stimulation, to indicate a requirement for a peptide that binds to a class II polypeptide, ie, T cell stimulation is a direct result of TCR engagement by MHC peptide complexes formed on the cell surface. A study was performed to determine whether or not due to direct binding of the peptide to the TCR. Ab blocking experiments were performed using anti-TCR mAb and anti-class II mAb (monomorphic human HLA DR).
Hybridoma cell line L243, which produces a mAb to the molecule, is available from American
an Type Cμture Collection, Bethesda,
(Obtained from MD). Transformed SS8T human T cells carrying class II on their surface were confirmed by flow cytometry. SS8 T cells were incubated with either anti-TCR or anti-DR Ab to block TCR and class II sites. The cells were then washed and placed in a microphysiology chamber and exposed to 10 ug / ml of the MBP (83-102) (SEQ ID NO: 1) peptide. Blocking the TCR or class II with specific mAbs resulted in complete inhibition in the rate of acidification. These results also indicate that peptide binding to class II molecules on the cell surface is a rapid cell activation event (McConnell (1995) Proc. Natl. Aca).
d. Sci. ScL USA 92: 2750). After high affinity binding of the TCR to the peptide: class II polypeptide complex, an initial cell activation event (signal) occurs within 10 minutes as measured by extracellular acidification on a microphysiometer.
Late cell activation events (signals) are measured within 48 hours; typical late activation events measured in these studies were γIFN and TNFβ cytokine responses. Measurement of early and late activation events is equivalent in identifying peptides that are important for TCR recognition, as was done to identify novel MBP peptides of the invention.

In summary, these studies were based on the amino acid sequence Phe-X-Lys-Asn-Il.
A new family of MBP peptides has been identified, characterized by having e-Val-XX-X-Thr-XX (where X is any amino acid). These peptides are also included in the novel Class II: MBP complexes of the present invention. These complexes comprise an MHC class II complex capable of binding a T cell receptor, comprising: an MHC class comprising the extracellular domain of an MHC class II molecule sufficient to form an antigen binding pocket II polypeptide, and amino acid sequence Phe-X-Lys-R1-Ile-Val-XX-X-Thr-
XX (where X is any amino acid and R1 is Asn or Gln) consisting essentially of a myelin basic protein;
The peptide is bound to the MHC class II component antigen binding pocket.

[Brief description of the drawings]

FIG. 1A shows the MBP peptides used in the study described in Example 1. FIG.

FIG. 1B shows MBP peptides with various alanine-substituted residues used in the study described in Example 1.

2A, 2B and 2C show the results of the analysis described in Example 1. FIG. This result indicates that γ-IFN and TNF were obtained using six N-terminal-deleted MBP peptides, respectively.
Demonstrates increased levels of β and extracellular acidification rates. 2D, 2E and 2
F shows the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates using five N-terminal truncated MBP peptides, respectively.

2A, 2B and 2C show the results of the analysis described in Example 1. This result indicates that γ-IFN and TNF were obtained using six N-terminal-deleted MBP peptides, respectively.
Demonstrates increased levels of β and extracellular acidification rates. 2D, 2E and 2
F shows the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates using five N-terminal truncated MBP peptides, respectively.

2A, 2B and 2C show the results of the analysis described in Example 1. FIG. This result indicates that γ-IFN and TNF were obtained using six N-terminal-deleted MBP peptides, respectively.
Demonstrates increased levels of β and extracellular acidification rates. 2D, 2E and 2
F shows the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates using five N-terminal truncated MBP peptides, respectively.

2A, 2B and 2C show the results of the analysis described in Example 1. This result indicates that γ-IFN and TNF were obtained using six N-terminal-deleted MBP peptides, respectively.
Demonstrates increased levels of β and extracellular acidification rates. 2D, 2E and 2
F shows the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates using five N-terminal truncated MBP peptides, respectively.

2A, 2B and 2C show the results of the analysis described in Example 1. This result indicates that γ-IFN and TNF were obtained using six N-terminal-deleted MBP peptides, respectively.
Demonstrates increased levels of β and extracellular acidification rates. 2D, 2E and 2
F shows the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates using five N-terminal truncated MBP peptides, respectively.

2A, 2B and 2C show the results of the analysis described in Example 1. FIG. This result indicates that γ-IFN and TNF were obtained using six N-terminal-deleted MBP peptides, respectively.
Demonstrates increased levels of β and extracellular acidification rates. 2D, 2E and 2
F shows the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates using five N-terminal truncated MBP peptides, respectively.

FIGS. 3A, 3B and 3C show the results of the analysis described in Example 1. FIGS. This analysis shows that γ-IFN, T, respectively, induced by the C-terminally deleted MBP peptide.
Compare NF-β and acidification rate. 3D, 3E and 3F show the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates, respectively, induced by the C-terminally deleted MBP peptide.

3A, 3B and 3C show the results of the analysis described in Example 1. This analysis shows that γ-IFN, T, respectively, induced by the C-terminally deleted MBP peptide.
Compare NF-β and acidification rate. 3D, 3E and 3F show the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates, respectively, induced by the C-terminally deleted MBP peptide.

3A, 3B and 3C show the results of the analysis described in Example 1. This analysis shows that γ-IFN, T, respectively, induced by the C-terminally deleted MBP peptide.
Compare NF-β and acidification rate. 3D, 3E and 3F show the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates, respectively, induced by the C-terminally deleted MBP peptide.

3A, 3B and 3C show the results of the analysis described in Example 1. This analysis shows that γ-IFN, T, respectively, induced by the C-terminally deleted MBP peptide.
Compare NF-β and acidification rate. 3D, 3E and 3F show the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates, respectively, induced by the C-terminally deleted MBP peptide.

3A, 3B and 3C show the results of the analysis described in Example 1. This analysis shows that γ-IFN, T, respectively, induced by the C-terminally deleted MBP peptide.
Compare NF-β and acidification rate. 3D, 3E and 3F show the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates, respectively, induced by the C-terminally deleted MBP peptide.

3A, 3B and 3C show the results of the analysis described in Example 1. This analysis shows that γ-IFN, T, respectively, induced by the C-terminally deleted MBP peptide.
Compare NF-β and acidification rate. 3D, 3E and 3F show the results of the analysis described in Example 1. This analysis compares the γ-IFN, TNF-β and acidification rates, respectively, induced by the C-terminally deleted MBP peptide.

4A, 4B and 4C show the results of the analysis described in Example 1. FIG. This result demonstrates the MBP peptide residues required for induction of γ-IFN and TNF-β, together with the acidification rate, respectively, when presented to SS8T cells by Class II DRB5 ^* 0101. FIGS. 4D, 4E and 4F show class II DRB5 ^* 0.
When presented to SS8 T cells by G.101, γ, along with the acidification rate, respectively.
-Demonstrates MBP peptide residues required for induction of IFN, TNF-β.

4A, 4B and 4C show the results of the analysis described in Example 1. This result demonstrates the MBP peptide residues required for induction of γ-IFN and TNF-β, together with the acidification rate, respectively, when presented to SS8T cells by Class II DRB5 ^* 0101. FIGS. 4D, 4E and 4F show class II DRB5 ^* 0.
When presented to SS8 T cells by G.101, γ, along with the acidification rate, respectively.
-Demonstrates MBP peptide residues required for induction of IFN, TNF-β.

4A, 4B and 4C show the results of the analysis described in Example 1. This result demonstrates the MBP peptide residues required for induction of γ-IFN and TNF-β, together with the acidification rate, respectively, when presented to SS8T cells by Class II DRB5 ^* 0101. FIGS. 4D, 4E and 4F show class II DRB5 ^* 0.
When presented to SS8 T cells by G.101, γ, along with the acidification rate, respectively.
-Demonstrates MBP peptide residues required for induction of IFN, TNF-β.

4A, 4B and 4C show the results of the analysis described in Example 1. This result demonstrates the MBP peptide residues required for induction of γ-IFN and TNF-β, together with the acidification rate, respectively, when presented to SS8T cells by Class II DRB5 ^* 0101. FIGS. 4D, 4E and 4F show class II DRB5 ^* 0.
When presented to SS8 T cells by G.101, γ, along with the acidification rate, respectively.
-Demonstrates MBP peptide residues required for induction of IFN, TNF-β.

FIGS. 4A, 4B and 4C show the results of the analysis described in Example 1. FIGS. This result demonstrates the MBP peptide residues required for induction of γ-IFN and TNF-β, together with the acidification rate, respectively, when presented to SS8T cells by Class II DRB5 ^* 0101. FIGS. 4D, 4E and 4F show class II DRB5 ^* 0.
When presented to SS8 T cells by G.101, γ, along with the acidification rate, respectively.
-Demonstrates MBP peptide residues required for induction of IFN, TNF-β.

4A, 4B and 4C show the results of the analysis described in Example 1. FIG. This result demonstrates the MBP peptide residues required for induction of γ-IFN and TNF-β, together with the acidification rate, respectively, when presented to SS8T cells by Class II DRB5 ^* 0101. FIGS. 4D, 4E and 4F show class II DRB5 ^* 0.
When presented to SS8 T cells by G.101, γ with acidification rate respectively
-Demonstrates MBP peptide residues required for induction of IFN, TNF-β.

FIG. 5A shows MBPs (91 to 100) (in a highlighted area)
SEQ ID NO: 38) shows the core TCR recognition sequence. FIG. 5B shows F-91, K-
Key MBP amino acid residues involved in TCR contacts are indicated (by arrows) as 93, N-94, 1-95, and V-96.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 43/00 C07K 16/18 4H045 C07K 14/47 16/28 16/18 16/40 16/28 19 / 00 16/40 C12Q 1/02 19/00 C12N 5/00 B C12N 15/09 ZNA A61K 37/02 C12Q 1/02 C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE , DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML) , MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB , GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ , VN, YU, ZA, ZWF terms (reference) 4B024 AA01 BA21 BA46 CA07 CA09 GA18 4B063 QA05 QQ79 QR90 QS36 QX05 QX10 4B065 AA93X AA93Y CA24 CA25 CA44 4C084 AA02 AA07 BA01 BA02 CA18 ACA23 CA01 CA02 ACA EE01 4H045 AA10 AA11 AA20 AA30 BA16 CA40 DA01 DA21 EA21 FA72

Claims (23)

[Claims] Claims 1. An isolated myelin basic protein (MBP) peptide, wherein the peptide has the amino acid sequence Wherein X is any amino acid. 2. The isolated myelin basic protein peptide of claim 1, wherein said peptide has the amino acid sequence Or a peptide characterized by having conservative substitutions thereof. 3. The isolated myelin basic protein peptide of claim 1, which is linked to a heterologous sequence. 4. The isolated myelin basic protein peptide of claim 3, which is a fusion protein. 5. An amino acid sequence An isolated myelin basic protein peptide that specifically binds to an antibody directed against the peptide characterized by having 6. An isolated nucleic acid encoding a myelin basic protein peptide, wherein the peptide has the amino acid sequence Wherein X is any amino acid. 7. The isolated nucleic acid of claim 6, wherein the encoded myelin basic protein peptide has the amino acid sequence Or an isolated nucleic acid characterized by having conservative substitutions thereof. 8. The isolated nucleic acid of claim 6, wherein said nucleic acid comprises SEQ ID NO: 1. 9. A composition comprising an MHC class II complex capable of binding to a T cell receptor, wherein the complex comprises substantially any of the following: a sufficient number of MHC class II molecules to form an antigen binding pocket. An MHC class II polypeptide containing an extracellular domain, wherein the component is encoded by an allele associated with an autoimmune disease directed against myelin basic protein, wherein the class II component comprises: An MHC class II polypeptide that is soluble under physiological conditions in the absence of detergents or lipids; and an amino acid sequence Wherein X is any amino acid and R ₁ is Asn or Gln; wherein the myelin basic protein peptide is MHC class I
A composition bound to the antigen binding pocket of the I component. 10. The composition according to claim 9, wherein the myelin basic protein peptide has an amino acid sequence Or having a conservative substitution thereof. 11. The composition according to claim 9, which is a fusion protein. 12. The composition according to claim 9, further comprising an effector component. 13. The composition of claim 9, wherein the autoimmune disease directed to myelin basic protein is multiple sclerosis. 14. The composition of claim 9, wherein said Class II
A composition wherein the polypeptide contains the antigen binding pocket of HLA DR2. 15. A pharmaceutically acceptable carrier and an amino acid sequence wherein X is any amino acid. A pharmaceutical composition comprising a pharmacologically effective amount of a peptide characterized by having a. 16. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmacologically effective amount of the composition of claim 9. 17. An antibody that is specifically immunoreactive under conditions immunologically reactive to a myelin basic protein peptide, wherein said peptide has the amino acid sequence An antibody, characterized by having 18. An antibody that is specifically immunoreactive with a myelin basic protein peptide containing the peptide encoded by the nucleic acid of claim 6 under immunologically reactive conditions. 19. A method of inhibiting a T cell-mediated immune response to a myelin basic protein in a subject, wherein the method comprises administering to the subject the peptide of claim 1 or the peptide of claim 9. Administering the described composition in an effective amount to treat the T cell mediated immune response. 20. The method of claim 19, wherein said T cell mediated immune response results in a lesion to the nervous system. 21. The method of claim 19, wherein the lesion on the nervous system is characterized as multiple sclerosis. 22. A method of identifying a T cell epitope on an antigen, wherein the antigen binds to a THC antigen binding pocket of an MHC class II molecule.
A T cell that expresses the T cell receptor.
Inducing an extracellular acidification reaction by the cells, the method comprising the steps of: a) providing a composition containing the T cell epitope bound to an antigen binding pocket of an MHC class II molecule; b) Contacting a T cell expressing the T cell receptor with the epitope; and c) measuring the extracellular acidification, wherein the alteration of the extracellular acidification is a T cell to the T cell receptor. Showing the binding of a cellular epitope. 23. The method of claim 22, wherein the change in extracellular acidification is measured using a microphysiometer.