WO2008131599A1

WO2008131599A1 - T-cell receptor vaccines materials and methods

Info

Publication number: WO2008131599A1
Application number: PCT/CN2007/001490
Authority: WO
Inventors: Jingwu Z. Zhang; Kowkkeung Walter Ho; Dongqing Zhang; Wei Sun
Original assignee: Maxx Genetech Co., Ltd.
Priority date: 2007-04-30
Filing date: 2007-04-30
Publication date: 2008-11-06

Abstract

Provided are improved T cell receptor vaccines and methods for their production. Methods for treating T cell associated rheumatoid arthritis using T cell receptor vaccines are also provided.

Description

T-CELL RECEPTOR VACCINES MATERIALS AND METHOT)S

Technical Field

[0001] The present invention relates to the field of molecular biology and particularly relates to T-cell receptor specific sequences and methods for diagnosing and treating rheumatoid arthritis.

Background of the Invention

[0002] T cell receptors are found on T cells. T cell receptors comprise α and β chains, with β chains comprising the following regions from N-terminus to C-terminus: Vβ-Dβ-Jβ- Cβ. T cell receptors naturally vary in the Vβ-Dβ-Jβ regions. When an antigen is presented to the T cells by an antigen-presenting cell (APC), a T cell receptor with variable regions (including Vβ-Dβ-Jβ) that so happen to recognize the antigen binds to the antigen on the APC. The T cell bearing the T cell receptor then undergoes activation (clonal expansion). [0003] Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovium of the peripheral joints, in which T cells are thought to play an important role in the pathogenesis (Lee et al., 2001 and Lipsky et al., 1998). This is supported by marked infiltration and accumulation of ThI pro-inflammatory cells in the synovial membrane of RA in close association with MHC Class II genes, including DR4 (genotypes B 1*0404 and DRBl*0401) and DQ (DQBl*0302 and DQBl*0301), in Caucasian RA patients (Kerlan- Candon et al., 2001; MacGregor et al., 1995; and Fries et al., 2002). Further supporting evidence includes skewing of cytokine environment in favor of T cell-mediated inflammation and clonal expansion of infiltrating T cells in the affected joints (Dolhain et al., 1996; Berner et al., 2000; Davis et al., 2001; Goronzy et al., 1994; Struyk et al., 1993; Gonzalez-Quintial et al., 1996; and Alam et al., 1996). However, the antigen specificity of the infiltrating T cells in rheumatoid synovium is unknown. Several self antigens, including collagen type II, heat shock proteins and others, are implicated in RA based on T cell reactivity to these antigens in patients with RA (Londei et al., 1989; Pope et al., 1989; Devereux et al., 1991; and Res et al., 1994). Microbial antigens, such as mycobacterial antigens and staphylococcal superantigens, may also contribute to T cell activation in RA (Paliard et al., 1991 and Holoshitz et al., 1986). However, it is unclear whether T cell responses to any of these antigens are clinically relevant to RA.

[0004] In the absence of an eliciting antigen(s) associated with RA, attempts have been made to identify T cell receptor (TCR) structural features characteristic of infiltrating T cells derived from synovial fluid or synovial membrane of RA patients. It was hoped that a common TCR structural associated with rheumatoid synovium may provide better understanding of the mechanism whereby these infiltrating T cells are activated and perpetuated in the synovium and may potentially lead to novel therapeutic strategies. As the T cell receptor repertoire is shaped by the genetic background of the individual and the response to self or environmental antigens, antigen-driven stimulation in the context of similar MHC Class II molecules leads to oligoclonal expansion of T cells utilizing common V-D-J segments. On the other hand, T cell activation induced by superantigen stimulation is characterized by polyclonal expansion of a particular TCR Vβ gene family with different D-J segments.

Summary of the Invention

[0005] The present invention relates to a substantially pure and isolated peptide of about

10 to about 20 amino acids that comprises an amino acid sequence selected from the group consisting of SEQ JJD NO: 1, SEQ JD NO: 2, SEQ ID NO: 3 and an immunogenic variant of

SEQ JX) NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0006] In an aspect of the invention the peptide can have an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ JX) NO: 2, SEQ ID NO: 3 and an immunogenic variant of SEQ ID NO: 1, SEQ JX) NO: 2, or SEQ JX) NO: 3. The immunogenic variant can comprise a peptide that includes an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ JX) NO: 6.

[0007] The present invention also relates to a vaccine for treating rheumatoid arthritis.

The vaccine comprises at least one isolated immunogenic peptide having an amino acid sequence corresponding to about 10 to about 20 contiguous amino acids of at least one of T cell receptor variable beta 14 (Vβl4) or T cell receptor variable beta 16 (Vβl6) of an individual suffering from rheumatoid arthritis.

[0008] In an aspect of the invention, the vaccine can comprise a peptide or combination of peptides that include at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ JX) NO: 2, SEQ ID NO: 3 and an immunogenic variant of SEQ ID

NO: 1, SEQ JX⁾ NO: 2, or SEQ TX⁾ NO: 3.

[0009] In another aspect of the invention, the vaccine can comprise a first peptide that includes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and a second peptide with a different amino acid sequence as the first peptide and selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [0010] In another aspect of the invention, the vaccine can include a peptide that has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ED NO: 2, SEQ ID NO: 3, and an immunogenic variant of SEQ ED NO: 1, SEQ ED NO: 2, or SEQ ED NO: 3. The immunogenic variant can comprise a peptide that includes an amino acid sequence selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 5, and SEQ ED NO: 6.

[0011] In yet another aspect, the vaccine can include an adjuvant that upon administration to a subject stimulates an immune response. The adjuvant can comprise at least of one of alum or Incomplete Freund's adjuvant.

[0012] The present invention further relates to a method of treating rheumatoid arthritis in subject suffering from rheumatoid arthritis. The method comprises administering to the subject an effective amount of an immunogenic T cell receptor peptide or combination of immunogenic T cell receptor peptides to elicit an immune response. The peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and immunogenic variants thereof.

[0013] The present invention also relates to a pharmaceutical composition for suppressing pathogenic T cell response in an individual suffering from rheumatoid arthritis. The pharmaceutical composition includes an immunologically effective amount of the immunogenic peptide or combination of immunogenic peptides having an amino acid sequence corresponding to about 10 to about 20 contiguous amino acids of at least one of T cell receptor variable beta 14 (Vβl4) or T cell receptor variable beta 16 (Vβl6) of an individual suffering from rheumatoid arthritis.

[0014] In an aspect of the invention, the pharmaceutical composition can comprise a peptide or combination of peptides that include at least one amino acid sequence selected from the group consisting of SEQ ED NO: 1, SEQ ED NO: 2, SEQ ID NO: 3 and an immunogenic variant of SEQ ED NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The immunogenic variant can comprise a peptide that includes an amino acid sequence selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 5, and SEQ ED NO: 6. -A-

[0015] In another aspect of the invention, the pharmaceutical composition can comprise a first peptide that includes an amino acid sequence selected from the group consisting of SEQ ED NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and a second peptide with a different amino acid sequence as the first peptide and selected from the group consisting of SEQ ID NO: 1, SEQ ED NO: 2, SEQ ID NO: 3, and an immunogenic variant of SEQ ED NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0016] In yet another aspect, the pharmaceutical composition can include an adjuvant that upon administration to a subject stimulates an immune response. The adjuvant can comprise at least of one of alum or Incomplete Freund's adjuvant. The pharmaceutical composition can also include a pharmaceutically acceptable carrier.

[0017] The present invention further relates to a vaccine for treating rheumatoid arthritis. The vaccine includes a nucleic acid having a nucleotide sequence that encodes a peptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1, SEQ ED NO: 2, SEQ ID NO: 3, and an immunogenic variant of SEQ ED NO: 1, SEQ ED NO: 2, or SEQ ED NO: 3. The nucleic acid can be provided in an expression vector. [0018] The present invention further relates to a method of preventing or treating rheumatoid arthritis in an individual. The method comprises administering to the individual an expression vector comprising a nucleic acid having a nucleotide sequence encoding a peptide having an amino acid sequence corresponding to about 10 to about 20 contiguous amino acids of at least one T cell receptor variable beta 14 (Vβl4) or T cell receptor variable beta 16 (Vβl6). The nucleotide is expressed in the individual at a level sufficient to elicit an immune response against the encoded peptide thereby preventing onset of rheumatoid arthritis or treating rheumatoid arthritis in the individual.

[0019] In an aspect of the invention, the nucleic acid can have a nucleotide sequence that encodes a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ LD NO: 2, SEQ ED NO: 3, and an immunogenic variant of SEQ ED NO: 1, SEQ ED NO: 2, or SEQ ED NO: 3.

[0020] In another aspect of the invention, the nucleic acid can encode a peptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1, SEQ ED NO: 2, SEQ ED NO: 3 and an immunogenic variant of SEQ ED NO: 1, SEQ ED NO: 2, or SEQ ED NO: 3. The immunogenic variant can comprise at least one peptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6,

[0021] The present invention also relates to a method of detecting rheumatoid arthritis in an individual suspected of having rheumatoid arthritis. The method comprises obtaining a tissue sample from the suspected individual and measuring the level of at least one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ DD NO: 3 or an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 in the sample. The presence of at least one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or an immunogenic variant of SEQ ID NO: I₃ SEQ ID NO: 2, or SEQ ID NO: 3 in the sample indicates a possible detection of rheumatoid arthritis in the suspected individual.

[0022] Another aspect of the invention relates to a method of detecting rheumatoid arthritis in an individual suspected of having rheumatoid arthritis. The method includes the step of generating a probe complementary to at least one nucleic acid having a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. A tissue sample is obtained from the suspected individual. The probe is then mixed with the tissue sample. A positive hybridization signal indicates a possible detection of rheumatoid arthritis in the suspected individual.

[0023] Another aspect of the invention relates to a method of detecting rheumatoid arthritis in an individual suspected of having rheumatoid arthritis. The method includes the step of obtaining a tissue sample from the subject and measuring in the sample the expression of at least one nucleic acid having a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. The level of expression of the at least one nucleic acid having a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicating a possible detection of rheumatoid arthritis in the suspected individual.

Brief Description of the Drawings

[0024] The foregoing and other features of the present invention will become apparent to those skilled in the art to which the present invention relates upon reading the following description with reference to the accompanying drawings, in which: [0025] Fig. 1 is a plot illustrating that DBA/1 mice were protected from collagen induced arthritis through vaccination of Vβl4 and Vβl6 peptides before immunization of bovine type II collagen. Group of DBA/1 mice (Group 4 and 5, eight for each group) were challenged with 20 nmol of peptide intradermal at -8, 0 and +8 days with regard to primary bovine type II collagen injection.

[0026] Fig. 2 is a plot illustrating that immunization of Vβ 14 and Vβ 16 peptides reduced the severity of incidence of CIA in DBA/1 mice (Group 6 and 7). Vβl4 and Vβl6 peptides were injected at 10, 20 and 30 days after immunization of bovine type II collagen.

Detailed Description

[0027] To aid in the understanding of the invention, the following terms are defined below.

[0028] The terms "polypeptide" and "peptide" are used interchangeably throughout the specification and designate a linear series of amino acids connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids. Polypeptides can be a variety of lengths, either in their natural (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications. It is well understod in the art that amino acid sequences contain acidic and basic groups, and that the particular ionization state exhibited by the peptide is dependent on the pH of the surrounding medium when the peptide is in solution, or that of the medium from which it was obtained if the peptide is in solid form. Also included in the definition are peptides modified by additional substituents attached to the amino acids side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversions of the chains, such as oxidation of sulfahydryl groups. Thus, "polypeptide" or its equivalent terms is intended to include the appropriate amino acid sequence referenced, subject to those of the foregoing modifications which do not destroy its functionality.

[0029] As used herein, the term "peptide fragment" or "fragment of peptide" means an immunogenically effective subset of the amino acid sequence that comprises a T cell receptor (TCR). The term is intended to include such fragments in conjunction with or combined with additional sequences or moieties, as for example where the peptide is coupled to other amino acid sequences or to a carrier. The terms "fragment" and "peptide" can, therefore, be used interchangeably since a peptide will be the most common fragment of the TCR.

[0030] Reference herein to a "fragment," "portion" or "segment" of a TCR does not mean that the composition must be derived from intact TCRs. Such "fragments," "portions" or

"segments" can be produced by various means well-known to those skilled in the art, such as, for example, manual or automatic peptide synthesis, various methods of cloning or enzymatic treatment of a whole TCR.

[0031] As used herein when referring to the relationship between peptide fragments of the invention and sequences of TCRs, "corresponding to" means that the peptide fragment has an amino acid sequence, which is sufficiently homologous to a TCR sequence or fragment thereof to stimulate an effective regulatory response in the individual. The sequence, however, need not be identical to the TCR sequence as shown, for instance, in the

Example.

[0032] By "immunogenically effective", it is meant an amount of the T cell receptor or fragment thereof which effectively elicits an immune response to prevent or treat a T cell mediated pathology or an unregulated T cell clonal replication in an individual. Such amounts will vary between species and individuals depending on many factors for which one skilled in the art can determine.

[0033] By "substantially pure," it is meant that the TCR peptide is substantially free of other biochemical moieties with which it is normally associated in nature. Such substantially pure TCRs or fragments thereof, for instance, can be synthesized, produced recombinantly by means known to those skilled in the art. In addition, whole TCRs can be enzymatically treated to produce such fragments.

[0034] As used herein, "individual" or "subject" means any vertebrate, including humans, capable of having a T cell mediated pathology or unregulated clonal T cell replication and is used interchangeably with "vertebrate."

[0035] The present invention generally relates to isolated and substantially pure TCR peptides that can be provided in a vaccine or pharmaceutical composition and used in an immunotherapy method for preventing, ameliorating or treating T cell-mediated rheumatoid arthritis. The immunotherapy method of the present invention avoids many of the problems associated with previously suggested methods of treatment. By vaccinating, rather than passively administering heterologous antibodies, the host's own immune system is mobilized to suppress the autoaggressive T cells. Thus, the suppression is persistent and may involve any or all immunological mechanisms in effecting that suppression. This multi-faceted response is more effective than the uni-dimensional suppression achieved by passive administration of monoclonal antibodies or ex vivo-derived regulatory T cell clones, which requires a highly individualized therapeutic approach because of MHC non-identity among humans in order to avoid graft versus host reactions. The methods of the present invention are also more effective than vaccination with attenuated disease-inducing T cells that lack specificity for the protective antigen on the surface of a particular T cell as well as the variable induction of immunity to that antigen. In addition, vaccination with attenuated T cells is plagued by the same labor intensiveness and need for individualized therapies as noted above for ex vivo derived regulatory T cell clones.

[0036] The isolated and substantially pure peptides of the present invention comprise TCRs or immunogenic fragments thereof from specific T cells that mediate rheumatoid arthritis. The peptides can have an amino sequence that corresponds to a portion or fragment of the amino acid sequence of at least one of T cell receptor variable beta (Vβl4) or T cell receptor variable beta 16 (Vβl6) of an individual suffering from rheumatoid arthritis. [0037] "Variable beta 14 (Vβl4)" refers to a specific human β chain variable region of T cell receptors. Vβl4 has the following amino acid sequence:

MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYM SWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRK EKRNFPLILESPSPNQTSLYFCASS. (SEQ ID NO: 13)

[0038] "Variable beta 16 (Vβl6)" refers to a specific human β chain variable region of T cell receptors. Vβl6 chain has the following amino acid sequence:

EAGVTQFPSHSVTEKGQTVTLRCDPISGHDNLYWYRRVMGKEIKFLLHFVKE SKQDESGMPNNRFLAERTGGTYSTLKVQPAELED SGVYFCASS. (SEQ ID NO: 14). [0039] The portion of the amino acid sequence of the T cell receptor variable beta 14 (Vβl4) or T cell receptor variable beta 16 (Vβl6) to which the vaccine peptide corresponds to can be about 10 to about 20 amino acids in length. The peptides of the invention can therefore have lengths, for example, between about 10 to about 20 amino acids. [0040] The peptide can be homogenous, for example, a single peptide, or can be composed of more than one type of peptide, each of which corresponds to a different portion of the T cell receptor variable beta 14 (Vβl4) or T cell receptor variable beta 16 (Vβl6). The vaccine peptides can be of variable length so long as they can elicit an immune response. [0041] It will be appreciated that the isolated peptides need not have exactly the same amino acid sequence as the corresponding portion of the Vβl4 or Vβl6 peptides and that immungenic improvements or variations to the amino acid sequence can be made, generally using the Vβl4 or Vβl6 amino acid sequence as a starting point. Modifications and changes may be made in the structure of such a peptide and still obtain a molecule having like or otherwise desirable immunogenic characteristics. For example, certain amino acids may be substituted for other amino acids in the peptide without appreciable loss of interactive binding capacity.

[0042] Since it is the immunogenic capacity and nature of the peptide that defines that peptide's immunogenic activity, certain amino acid sequence substitutions can be made in the amino acid sequence (or of course, the underlying DNA sequence) and nevertheless obtain a peptide with like immunogenic properties. It is thus contemplated that slight changes may be made in the sequence of the peptides without appreciable loss of their biological utility or activity.

[0043] It is also well understood by the skilled artisan that there is a limit to the number of changes that may be made within a defined portion of the peptide, which results in a molecule with an acceptable level of immunogenic activity. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.

[0044] In an aspect of the present invention, the peptide or combination peptides can comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and an immunogenic variant of SEQ ED NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. SEQ ID NO: 1 corresponds to 12 contiguous amino acids of T cell receptor variable beta 14 (Vβl4). SEQ ID NO: 2 corresponds to 12 contiguous amino acids of T cell receptor variable beta 16 (Vβl6). SEQ ID NO: 3 corresponds to 12 contiguous amino acids of T cell receptor variable beta 16 (Vβl6). [0045] Immunogenic variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 mean amino acid sequences comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 having additions, deletions or substitutions that do not substantially effect the ability of the sequence to elicit an immune response against the desired T cell receptor sequence. Such sequences commonly have at least one, two, three, four or more amino acid additions or deletions adjacent to the N or C ends or within the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. A portion or segment of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 can be used so long as it is sufficiently characteristic of the desired T cell receptor or fragment thereof to cause an effective immune response against desired T cell receptors, but not against undesired T cell receptors. Such variations in the sequence can easily be made, for example by synthesizing an alternative sequence having at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The alternate sequence can then be tested, for example by immunizing a vertebrate, to determine its effectiveness.

[0046] By way of example, an immunogenic variant of SEQ ID NO: 1 is a peptide having an amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 4 comprises the amino acid sequence of SEQ ID NO: 1 as well as two additional amino acids at the N and C ends of the sequence and an additional amino acid within the sequence. An example of an immunogenic variant of SEQ ID NO: 2 is a peptide having an amino acid sequence of SEQ ID NO: 5. SEQ ID NO: 5 comprises the amino acid sequence of SEQ ID NO: 2 as well as two additional amino acids at the N and C ends of the sequence and an additional amino acid within the sequence. An example of an immunogenic variant of SEQ ID NO: 3 is a peptide having an amino acid sequence of SEQ ID NO: 6. SEQ ID NO: 6 comprises the amino acid sequence of SEQ ID NO: 3 as well as an additional amino acid within the sequence. [0047] The peptides in accordance with the invention can be used to immunize an individual having rheumatoid arthritis. The immune response generated in the individual can neutralize or kill T cells expressing Vβl4 or Vβl6 and, thus, prevent or treat the deleterious effects of such Vβl4 or Vβl6 bearing T cells. Moreover, to the extent that Vβl4 or Vβl6 is common to T cell receptors on pathogenic T cells mediating other autoimmune diseases or autoimmune diseases in general, such vaccines can also be effective in ameliorating such other autoimmune diseases.

[0048] In rheumatoid arthritis studies described in the Example, the results indicate that immunization of mice with vaccines comprising at least one of SEQ ID NO: 1 or SEQ ID NO: 2 substantially reduced the severity of collagen-induced arthritis in the mice. Thus, peptides comprising about 10 to about 20 amino acids and corresponding to the Vβl4 or Vβlό region can be used as vaccines of the present invention.

[0049] The peptides in accordance with the present invention can be produced, either by recombinant techniques or by synthetic methods. The peptides in accordance with the present invention may be synthesized using conventional peptide synthesis or any of a number of other protocols well known in the art. For example, the peptides may be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifϊeld, 1963, J. Am. Chem. Soc. 85:2149. During synthesis, N-α-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal to an insoluble polymeric support, e.g., polystyrene beads. The peptides are synthesized by linking an amino group of an N-α-deprotected amino acid to an α-carboxyl group of an N-α-protected amino acid that has been activated by reacting it with a reagent, such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N-α-protecting groups include Boc which is acid labile and Fmoc which is base labile. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art (see Atherton et al., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer- Verlag).

[0050] If desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the sequence of the peptides. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, α- amino isobutyric acid, Aib, i.e. 2-amino isobutync acid, 4-aminobutyric acid, γ- Abu, 2- amino butyric acid, Abu, 6-amino hexanoic acid ε-Ahx, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β -methyl amino acids, C α -methyl amino acids, N α -methyl amino acids, and amino acid analogs in general.

[0051] Purification of the resulting peptides is accomplished using conventional procedures, such as preparative "PLC" using gel permeation, partition and/or ion exchange chromatography. The techniques, choice of appropriate matrices and buffers are well known in the art (Atherton et al, 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press). In an aspect of the invention, the synthetic peptide prepared is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% pure. [0052] As an alternative to synthetic production, the immunogenic peptides of the present invention can produced by recombinant means. Once the nucleotide sequence encoding an immunogenic peptide has been identified, the nucleotide sequence, or a fragment thereof can be obtained and cloned into an expression vector for recombinant expression. The expression vector can then be introduced into a host cell for propagation of the peptide. [0053] Any recombinant immunogenic peptide useful in the complexes of the invention is preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% pure. The DNA may be obtained by DNA amplification or molecular cloning directly from a tissue, cell culture, or cloned DNA (e.g., a DNA "library") using standard molecular biology techniques (see, e.g., Methods in Enzymology, 1987, volume 154, Academic Press; Sambrook et al. 1989, Molecular Cloning—A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York; and Current Protocols in Molecular Biology, Ausubel et al. (eds.), Greene Publishing Associates and Wiley lnterscience, New York, each of which is incorporated herein by reference in its entirety). Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, the immunogenic gene can be cloned into a suitable vector for propagation of the gene. [0054] The DNA can be amplified from genomic or cDNA by polymerase chain reaction (PCR) amplification using primers designed from the known nucleotide sequence of a related or homologous immunogenic gene. PCR is used to amplify the desired nucleotide sequence in a DNA clone or a genomic or cDNA library, prior to selection. PCR can be carried out, e.g., by use of a thermal cycler and Taq polymerase (Gene Amp). The polymerase chain reaction (PCR) is commonly used for obtaining genes or gene fragments of interest. For example, a nucleotide sequence encoding an immunogenic peptide of any desired length can be generated using PCR primers that flank the nucleotide sequence encoding the immunogenic peptide. Alternatively, an immunogenic peptide gene sequence can be cleaved at appropriate sites with restriction endonuclease(s) if such sites are available, releasing a fragment of DNA encoding the immunogenic peptide or a derivative thereof. If convenient restriction sites are not available, they may be created in the appropriate positions by site- directed mutagenesis and/or DNA amplification methods known in the art (see, for example, Shankrppa et al, 1992, PCR Method Appl. 1:277-278). The DNA fragment that encodes the immunogenic peptide is then isolated, and ligated into an appropriate expression vector, care being taken to ensure that the proper translation reading frame is maintained. [0055] In an alternative embodiment, for the molecular cloning of an immunogenic peptide gene from genomic DNA, DNA fragments are generated to form a genomic library. Since some of the sequences encoding related immunogenic peptides are available and can be purified and labeled, the cloned DNA fragments in the genomic DNA library may be screened by nucleic acid hybridization to a labeled probe (fenton and Davis, 1977, Science 196: 180; Grunstein and Hogness, 1975, Proc. Natl Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify an appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map. [0056] Alternatives to isolating the immunogenic peptide genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence or synthesizing a cDNA to the mRNA which encodes the immunogenic peptide. For example, RNA for cDNA cloning of the immunogenic peptide gene can be isolated from cells which express the immunogenic peptide. A cDNA library may be generated by methods known in the art and screened by methods, such as those disclosed for screening a genomic DNA library. If an antibody to the immunogenic peptide is available, the immunogenic peptide may be identified by binding of a labeled antibody to the immunogenic peptide synthesizing clones.

[0057] In an aspect of the invention, the at least one peptide or combination of peptides can be formulated into pharmaceutical compositions or vaccines and be administered to a host exhibiting or at risk of exhibiting a rheumatoid arthritis autoimmune response. The pharmaceutical composition or vaccine will generally comprise an effective amount of the at least one peptide dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The effective amount of the peptide can be an amount or unit dosage, which when administered to the subject, is effective to elicit an immune response in the subject to the T- cells having Vβl4 or Vβl6 and, thus, prevent or treat the deleterious effects of such Vβl4 or Vβl6 bearing T cells. This amount can vary depending on the specific pharmaceutical composition or vaccine formulation.

[0058] The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. Veterinary uses are equally included within the invention and "pharmaceutically acceptable" formulations include formulations for both clinical and/or veterinary use. [0059] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. For human administration, preparations can meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards. Supplementary active ingredients can also be incorporated into the compositions.

[0060] "Unit dosage" formulations are those containing a dose or sub-dose of the administered ingredient adapted for a particular timed delivery. Exemplary "unit dosage"formulations are those containing a daily dose or unit or daily sub-dose or a weekly dose or unit or weekly sub-dose and the like. For example, the peptide can be provided in the vaccine or pharmaceutical composition at a dosage of about 10 μg/ml to about 10 mg/ml. [0061] The peptides of the present invention will most often be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, transdermal, or other such routes, including direct injection into the site (e.g., synovial fluid administration, bolus injection or continuous infusion). The preparation of an aqueous composition that contains such a peptide will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

[0062] The pharmaceutical composition forms for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form should be sterile and fluid to the extent that syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. [0063] The pharmaceutical or vaccine can also include or be administered in conjunction with an adjuvant to increase the immunogenicity of the vaccine. The adjuvant used can be any adjuvant known for peptide- or protein-based vaccines. For example, the adjuvant can include inorganic adjuvants in gel form, such as aluminium hydroxide/aluminium phosphate, ALUM, and calcium phosphate; bacterial adjuvants, such as monophosphoryl lipid A and muramyl peptide; particulate adjuvants, such as the so-called ISCOMS ("immunostimulatory complexes", liposomes and biodegradable microspheres; adjuvants based on oil emulsions and emulsifiers such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), SAF, saponines (such as QS-21;), squalene/squalane; synthetic adjuvants, such as non- ionic block copolymers, muramyl peptide analogues, synthetic lipid A, synthetic polynucleotides, and polycationic adjuvants.

[0064] Other adjuvants that can be used in the invention can be found in A Compendium of Vaccine Adjuvants and Excipients (2nd Edition), Vogel, F., Powell, M., and Alving, C, in Vaccine Design— The Subunit and Adjuvant Approach, Powell, M., Newman, M., Burdman, J., Editors, Plenum Press, New Yorlς 1995, pp. 141-227, and 2nd Meeting on Novel Adjuvants Currently In/Close to Human Clinical Testing, World Health Organization- Organization Mondiale de Ia Sante Foundation Merieux, Annecy, France, 5-7 Jun. 2000, Kenney, 111., Rabinovich, N. R., Pichyangkul, S., Price, V., and Engers, H., Vaccine, 20 (2002) 2155-63. All of which are incorporated herein by reference. [0065] Under ordinary conditions of storage and use, such preparations can contain a preservative to prevent the growth of microorganisms. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum mono stear ate and gelatin. [0066] Prior to or upon formulation, the peptides of the present invention can be dialyzed to remove undesired small molecular weight molecules, and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. Sterile injectable solutions are prepared by incorporating the active agents in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0067] It will be appreciated that the immunogenic peptide of the present invention can be formulated in other pharmaceutically acceptable forms, such as tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, topical formulations, liposomal forms and the like. Thus, for example, the peptides may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs. [0068] Pharmaceutical "slow release" capsules or "sustained release" compositions or preparations may also be used and are generally applicable. Slow release formulations are generally designed to give a constant drug level over an extended period and may be used to peptides in accordance with the present invention. The slow release formulations are typically implanted.

[0069] The present invention also relates to a method of treating T-cell mediated rheumatoid arthritis in a subject using the immunogenic peptides. In the method, vaccines or pharmaceutical compositions comprising the peptides of the present invention are administered to the subject by conventional methods, in dosages which are sufficient to elicit an immunological response in the individual to the peptides. Such dosages can be easily determined by those skilled in the art. By way of example, the peptide can be provided in the vaccine or pharmaceutical composition at a dosage of about 10 μg/ml to about 10 mg/ml. [0070] The subject can comprise individuals that include T-cells expressing Vβl4 or Vβl6 and, thus, prevent or treat the deleterious effects of such Vβl4 or Vβl6 bearing T cells. These subjects can be individuals displaying symptoms of rheumatoid arthritis. [0071] The vaccine or pharmaceutical composition can be administered to the subject prior to, during, or after the onset of rheumatoid arthritis. The frequency of administration will depend on the formulation and dosage of the vaccine as well as the subject's immune response to the vaccine.

[0072] The present invention further relates an isolated and substantially pure DNA or RNA nucleic acid that can be provided in a vaccine or pharmaceutical composition and used in an immunotherapy method for preventing, ameliorating, or treating T cell-mediated rheumatoid arthritis. The isolated and substantially pure DNA or RNA nucleic acid can comprise an isolated nucleic acid molecule that is capable of encoding an immunogenic peptide that corresponds to a portion or fragment of the amino acid sequence of at least one ofVβH or Vβlό.

[0073] In an aspect of the invention, the isolated and substantially pure nucleic acid can have a nucleotide sequence that corresponds to a portion or fragment of the Vβl4 or Vβlό gene that encodes an immunogenic peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and immunogenic variants of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. For example, the DNA fragment can have a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9. [0074] SEQ ID NO: 7 comprises a 36 nucleotide sequence that encodes SEQ ID NO: 1. SEQ ID NO: 8 comprises a 36 nucleotide sequence that encodes SEQ ID NO: 2. SEQ ID NO: 9 comprises a 36 nucleotide sequence that encodes SEQ ID NO: 3. [0075] Other nucleic acid molecules within the invention are nucleic acid variants that encode the about 10 to about 20 amino acid immunogenic variant peptides of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. Such nucleic acid variants may be, e.g., a naturally occurring allelic variant of a fragment of a native Vβ 14 or Vβlό gene, a homolog of a fragment of a native Vβ 14 or Vβlό gene, or a non-naturally occurring variant of a fragment of a native Vβl4 or Vβl6 gene. These nucleic acid variants have a nucleotide sequence that differs from a native Vβl4 or Vβl6 gene in one or more bases. For example, the nucleotide sequence of such nucleic acid variants can feature a deletion, addition, or substitution of one or more nucleotides of a native Vβl4 or Vβlό gene. Nucleic acid insertions are preferably of about 1 to 10 contiguous nucleotides, and deletions are preferably of about 1 to 30 contiguous nucleotides.

[0076] Examples of nucleic acid variants encoding such immunogenic variants of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are ones having a nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. SEQ ID NO: 10 comprises a 51 nucleotide sequence that encodes a peptide having an amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 11 comprises a 51 nucleotide sequence that encodes a peptide having an amino acid sequence of SEQ ID NO: 5. SEQ ID NO: 12 comprises a 36 nucleotide sequence that encodes a peptide having an amino acid sequence of SEQ ID NO: 6. [0077] Nucleic acids that hybridize under stringent conditions to one of the foregoing nucleic acids can also be used in the invention. For example, such nucleic acids can be those that hybridize to one of the foregoing nucleic acids under low stringency conditions, moderate stringency conditions, or high stringency conditions.

[0078] The nucleic acids of the invention can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The nucleic acids within the invention may additionally include other appended groups, such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. U.S.A.

86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO 88/09810, published Dec. 15, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al. (1988) BioTechniques 6:958- 976) or intercalating agents. (See, e.g, Zon (1988) Pharm. Res. 5:539-549). To this end, the nucleic acids may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc. [0079] The DNA or RNA nucleic acids can be used in a gene therapy method of treating or preventing a T cell mediated rheumatoid arthritis. In this method, the DNA or RNA nucleic acid encoding the immunogenic fragment is first inserted into an appropriate delivery system and then administered to the subject in a vaccine or pharmaceutical composition. The delivery system can include a vector, such as a plasmid or viral vector (e.g., retroviral or adenoviral vectors) modified for gene therapy purposes and of purity and pharmaceutical acceptability. Such vectors may incorporate nucleic acid encoding the present immunogenic peptide for expression in the target cells, such as endothelial cells, bone marrow cells, blood cells, liver (or other organ) cells, muscle cells, fibroblasts, or other cells. Additionally, one may use a mixture of such vectors, such as those containing genes for one or more Vβl4 or

Vβl6 fragments. Gene therapy may also involve a vector containing more than one nucleic acid for the desired immunogenic peptide.

[0080] The expression vectors can include one or more promoters upstream of the inserted nucleic acid that act to promote transcription of the nucleic acid and to thus promote expression of the encoded peptide. In an aspect of the invention, the promoter can include, for example, β-actin promoter, S V40 early and late promoter, immunoglobulin promoter, human cytomegalovirus promoter, and retroviral LTRs.

[0081] Alternatively, the nucleic acid may be placed within a pharmaceutically acceptable carrier to facilitate cellular uptake, such as a lipid solution carrier

(e.g., a charged lipid), a liposome, or polypeptide carrier (e.g., polylysine. A review article on gene therapy is Verma, Scientific American, November 1990, pages 68-84 which is herein incorporated by reference.)

[0082] The desired nucleic acid may also be first placed within a cell, and the cell may be administered to a patient (such as a transplanted tissue) or the desired nucleic acid may be administered directly to the patient for uptake in vivo. The cells to be transferred to the recipient may be cultured using one or more factors affecting the growth or proliferation of such cells, such as SCF.

[0083] Administration of DNA or RNA nucleic acid of the present invention to the target cell may be accomplished by formation of a dispersion of particles, or an aerosol. Typically some type of bulking agent can be involved, and a carrier, such as a lipid or polypeptide.

These materials can be pharmaceutically acceptable. One may use a nebulizer for such delivery, such an ultrasonic or dry powder nebulizer. Alternatively, one may use a propellant based system, such as a metered dose inhaler, which can deliver liquid or a suspension of particles.

[0084] For gene therapy dosages, one can generally use between one copy and several thousand copies of the present nucleic acid per cell, depending on the vector, the expression system, the age, weight and condition of the recipient and other factors, which will be apparent to those skilled in the art. [0085] The expression vector can be provided in a vaccine or pharmaceutical composition and administered to the individual subcutaneously, intradermally, intravenously, or orally, for example, to the muscle tissue or spinal fluid of the individual. The nucleic acid is then expressed at a level sufficient to elicit an immune response against the encoded peptide thereby preventing onset of rheumatoid arthritis or treating rheumatoid arthritis in the individual.

[0086] In one aspect of the invention, a solution comprising about 10 to about 100 μg of a DNA or RNA plasmid including at least one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 is injected into the muscle tissue of an individual. The in vivo expression of the desired gene can be tested by determining an increased production of the encoded polypeptide by the subject according to methods known in the art or as described, for example, in Wolff et al., Science 247: 1465-1468 (1990). [0087] It is believed that the treated cells will respond to the direct injection of DNA or RNA by expressing the encoded immunogenic peptide for at least about 60 days. Thus, the desired immunogenic peptide can be effectively expressed by the cells of the individual as an alternative to vaccinating with such immunogenic peptides.

[0088] The present invention is further directed to a method of detecting rheumatoid arthritis in an individual. The method includes obtaining a tissue sample from a suspected individual. The expression level of at least one peptide having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 can be measured in the sample. The presence of the at least one peptide having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ED NO: 3, or an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sample can indicate a possible detection of rheumatoid arthritis in the suspected individual. [0089] In aspect of the method, the expression level of at least one peptide having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 can be measured by generating an antibody to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The antibody can be tagged with a detectable moiety and mixed with the tissue sample. The tissue sample can be obtained from synovial fluid, synovial lesion tissue, or peripheral blood of the suspected individual. A positive signal indicates possible detection of rheumatoid arthritis in the subject.

[0090] Another method of detecting rheumatoid arthritis in an individual suspected of having rheumatoid arthritis includes the step of obtaining a tissue sample from the subject and measuring in the sample the expression level of at least one nucleic acid having a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. The level of expression of the at least one nucleotide having a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicates a possible detection of rheumatoid arthritis in the suspected individual. [0091] Another method of detecting rheumatoid arthritis includes generating a probe complementary to a DNA fragment having a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. A tissue sample is then obtained from the suspected individual. The probe is then mixed with the tissue sample. In this method a positive hybridization signal indicates a possible detection of rheumatoid arthritis in the suspected individual. In an aspect of the invention, the tissue sample can be obtained from synovial fluid, synovial lesion tissue, or peripheral blood.

[0092] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE

Study of preventive and therapeutic effects of immunization with T cell receptor Vβ chain peptides on collagen-induced arthritis in DBA/1 mice

[0093] Rheumatoid arthritis (RA) is a T cell mediated chronic autoimmune disease characterized by inflammation of the synovium of the peripheral joints. This is supported by marked infiltration and accumulation of ThI pro-inflammatory cells in the synovial membrane of RA in close association with MHC Class II genes. Previous analysis of TCR V gene repertoire from the synovial lesion samples from RA patients and controls showed that there was highly skewed Vβl4 and Vβl6 usage in synovial T cells of RA (Sun et al., 2005 & US Patent Application 10/612,468, both herein incorporated by reference in their entirety). [0094] Two specific peptide sequences, each being 12 amino acids long, were selected from human Vβl4 andVβlό regions to study the preventive and therapeutic effects of collagen induced arthritis animal model.

Material & Methods Mice

[0095] 6-8 weeks old, male, DBA/1 mice were purchased from Shanghai Institute of Material Medical, Chinese Academy of Sciences. Mice were maintained at the SPF graded animal facility.

Induction of CIA in DBA/1 mice

[0096] Bovine type-II collagen: The CII used in the study was purchased from the Chondrex. CII was stored lyophilized in a freezer before use. CII was dissolved in 0.05mol/L acetate acid to reach concentration of 2mg/ml by general rotation and then emulsified in complete Freund's adjuvant (CFA, Sigma) with one to one portion for immunization. [0097] Arthritis was induced in DBA/1 male mice after injection of 200 μg of heterologous bovine type II collagen subcutaneously in the tail. 21 days after primary immunization, mice were then again immunized with 200 μg of bovine CII emulsified in incomplete Freund's adjuvant (IFA, Sigma).

Peptide Synthesis

[0098] Peptides were syntheses by solid phase synthesis. Amino acid sequences of peptides are listed followings:

1) human TCR Vβl4: IYYSMNVEVTDK (12aa, MW=1461) (SEQ ID NO: 1)

2) human TCR Vβl6: LLHFVKESKQDE (12aa, MW=1472) (SEQ ID NO: 2)

Immunizations against CIA with human TCR Vβ 14 and Vβ 16 peptides

[0099] Mice were divided into groups 1-8 (Table 1). Group 4 and 5 mice were immunized with 20 nmol of TCR Vβl4 and Vβl6 peptides emulsified with CFA (Sigma) intradermal 8 days before the first immunization of bovine CII, the same day (0 day) and 8 day after first immunization of bovine type II collagen.

[00100] Group 6 and 7 mice were immunized with 20 nmol of TCR Vβl4 and Vβl6 peptide emulsified with CFA (Sigma) through intradermal 10 days, 20 days and 30 days after first immunization of bovine type II collagen. [00101] The mice closely monitored for the development of arthritis symptoms for 9 weeks (64 days) after first immunization of bovine type II collagen.

TABLE l

Arthritis score analysis

[00102] Disease was assessed by measuring of paw thickness. Score 0 represents normal; score 1 represents mild redness and swelling of the ankle or wrist; score 2 represents moderate redness and swelling of ankle and wrist; score 3 represents severe redness and swelling of the entire paw including digits. Mean arthritis index was based on sum up of all four paws with a maximum score of 12 for each animal.

Results

[00103] Fig. 1 shows that the DBA/1 mice were protected and from CIA through vaccination of Vβl4 and Vβlό peptides before immunization of CII. Group of DBA/1 mice

(Group 4 and 5, eight for each group) were challenged with 20 nmol of peptide intradermal at

-8, 0 and +8 days with regard to primary CII injection.

[00104] Fig. 2 shows that Immunization of Vβl4 and Vβl6 peptides reduced the severity of incidence of CIA in DBA/1 mice (Group 6 and 7). Vβl4 and Vβl6 peptides were injected at 10, 20 and 30 days after immunization of bovine type II collagen.

[00105] Table 2 shows the vaccine with Vβl4 and Vβlό peptides prevents CIA in

DBA/1 mice. Incidence and arthritis severity was measured at 37 days after primary immunization of CII. DBA/1 mice were challenged with 20nmol of Vβl4 or Vβlό peptides at -8, 0 and 8 days with respect of CII immunization. TABLE 2

CIA

Treatment Incidence Arthritis severity

Control (PBS/IF A) 4/5 (80%) 5.0

TCR Vβl4-p 2/8 (20%) 2.3

TCRVβl6-p 3/8 (30%) 3.0

[00106] The studies showed that the immunization of mice with human TCR Vβl4 and Vβl6 peptides reduced the severity and incidence of the arthritis in DBA/1 mice. The results indicate that human Vβl4 or Vβl6 peptides are candidates for clinical application in treating RA in human by suppressing pathogenic T cells.

[00107] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims.

Claims

Having described the invention, the following is claimed:

1. A substantially pure and isolated peptide of about 10 to about 20 amnio acids comprising: an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

2. The peptide of claim 1, having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3,

3. The peptide of claim 1, the immunogenic variant comprising at least one peptide including an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

4. A vaccine for treating rheumatoid arthritis comprising: at least one isolated immunogenic peptide or combination of peptides, the peptide having an amino acid sequence corresponding to about 10 to about 20 contiguous amino acids of at least one of T cell receptor variable beta 14 (Vβl4) or T cell receptor variable beta 16 (Vβlό) of an individual suffering from rheumatoid arthritis.

5. The vaccine of claim 4, comprising at least one peptide or combination of peptides including an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ED NO: 2, SEQ ID NO: 3 and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

6. The vaccine of claim 4, the amino acid sequence of the peptide corresponding to about 10 to about 20 contiguous amino acids of a T cell receptor variable beta 14 (Vβl4).

7. The vaccine of claim 4, the amino acid sequence of the peptide corresponding to about 10 to about 20 contiguous amino acids of a T cell receptor variable beta 16 (Vβl6).

8. The vaccine of claim 4, including a first peptide having an amino acid sequence corresponding to about 10 to about 20 contiguous amino acids of the T cell receptor variable beta 16 (Vβl6) and a second peptide having an amino acid sequence corresponding to about 10 to about 20 contiguous amino acids of the T cell receptor variable beta 14 (Vβl4).

9. The vaccine of claim 4, the peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

10. The vaccine of claim 4, the immunogenic variant comprising at least one peptide having an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

11. The vaccine of claim 4, further comprising an adjuvant that upon administration to a subject stimulates an immune response.

12. The vaccine of claim 11, the adjuvant comprising at least one of alum or Incomplete Freund's adjuvant.

13. A method of treating or preventing rheumatoid arthritis in a subject suffering from rheumatoid arthritis, comprising the step of: administering to the subject an effective amount of at least one immunogenic T cell receptor peptide or combination of immunogenic T cell receptor peptides to elicit an immune response, the peptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and immunogenic variants thereof.

14. A pharmaceutical composition for suppressing pathogenic T cell response in an individual suffering from rheumatoid arthritis, comprising: an immunologically effective amount of an immunogenic peptide or combination of immunogenic peptides having an amino acid sequence corresponding to about 10 to about 20 contiguous amino acids of at least one of a T cell receptor variable beta 14 (Vβl4) or T cell receptor variable beta 16 (Vβl6) of an individual suffering from rheumatoid arthritis.

15. The pharmaceutical composition of claim 14, comprising at least one peptide or combination of peptides having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

16. The pharmaceutical composition of claim 14, the amino acid sequence of the peptide corresponding to about 10 to about 20 contiguous amino acids of a T cell receptor variable beta 14 (Vβl4).

17. The pharmaceutical composition of claim 14, the amino acid sequence of the peptide corresponding to about 10 to about 20 contiguous amino acids of a T cell receptor variable beta 16 (Vβl6).

18. The pharmaceutical composition of claim 14, the immunogenic variant comprising at least one peptide having an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

19. The pharmaceutical composition of claim 14, further comprising an adjuvant that upon administration to a subject stimulates an immune response.

20. The pharmaceutical composition of claim 19, further comprising a pharmaceutically acceptable carrier.

21. A vaccine for treating rheumatoid arthritis comprising a nucleic acid having a nucleotide sequence encoding a peptide having an amino acid sequence selected from the group consisting of SEQ DD NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

22. The vaccine of claim 21, the nucleic acid being provided in an expression vector.

23. A method of preventing or treating rheumatoid arthritis in an individual, comprising the steps of: (a) administering to the individual an expression vector comprising a nucleic acid having a nucleotide sequence encoding a peptide having an amino acid sequence corresponding to about 10 to about 20 contiguous amino acids of at least one T cell receptor variable beta 14 (Vβl4) or T cell receptor variable beta 16 (Vβl6); and (b) expressing the nucleic acid in the individual, the nucleic acid being expressed at a level sufficient to elicit an immune response against the encoded peptide thereby preventing onset of rheumatoid arthritis or treating rheumatoid arthritis in the individual.

24. The method of claim 23, the nucleic acid having a nucleotide sequence encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

25. A method of detecting rheumatoid arthritis in an individual suspected of having rheumatoid arthritis, comprising the steps of: obtaining a tissue sample from the suspected individual; and measuring the level of at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or an immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 in the sample, wherein the presence of at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or immunogenic variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 in the sample indicating a possible detection of rheumatoid arthritis in the suspected individual.

26. The method of claim 25, wherein the tissue sample is obtained from synovial fluid, synovial lesion tissue, or peripheral blood of the suspected and normal individuals.

27. A method of detecting rheumatoid arthritis in an individual suspected of having rheumatoid arthritis, comprising the steps of: obtaining a tissue sample from the suspected individual; measuring the expression level of at least one nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, wherein the level expression of at least one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicates a possible detection of rheumatoid arthritis in the suspected individual.

28. The method of claim 27, wherein the tissue sample is obtained from synovial fluid, synovial lesion tissue, or peripheral blood of the suspected and normal individuals.

29. The method of claim 27, the expression level be measured by generating a probe complementary to a nucleic acid having a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; mixing the probe with the tissue sample; and detecting the hybridization of the probe.