WO2003000718A2

WO2003000718A2 - Polypeptides comprising t cell epitopes from alpha-fodrin and the uses thereof in treatment of sjögren's syndrome

Info

Publication number: WO2003000718A2
Application number: PCT/JP2001/008221
Authority: WO
Inventors: Yoshio Hayashi
Original assignee: Fujisawa Pharmaceutical Co., Ltd.
Priority date: 2001-06-22
Filing date: 2001-09-21
Publication date: 2003-01-03
Also published as: WO2003000718A3; AU2001288087A1; AUPR589701A0

Abstract

The present invention provides a polypeptide consisting of a partial amino acid sequence of α-fodrin containing an amino acid sequence depicted in SEQ ID No:1, SEQ ID No:2 or SEQ ID No:4, its mutant, particularly, an epitope analogue containing a sequence depicted in SEQ ID No:3, and use thereof.

Description

DESCRIPTION

NOVEL PEPTIDE AND USE THEREOF TECHNICAL FIELD

The present invention relates to a novel peptide and use thereof. More particularly, the present invention relates to a pathogenic T cell epitope in Sjδgren's syndrome and an analogue thereof.

BACKGROUND ART Sjδgren's syndrome (SS) is a mysterious autoimmune disorder characterized by lymphocytic infiltrates and destruction of the salivary and lacrimal glands, and systemic production of autoantibodies to the ribonucleoprotein (RNP) particles SS-A/Ro and SS-B/La (Chan et al., J. Clin. Invest . 87, 68-76 (1991); Kruize et al., Immunol . Today 16, 557-559 (1995)). Moreover, in many cases SS-A/Ro- or SS-B/La-positive patients have complications such as systemic lupus erythematosus and rheumatism (Chan et al., (1991), supra} Chan et al., Nucleic Acids Research, 17, 2233-2244, (1989)). The role of different genetic or environmental factors in initiating the autoimmune reactivity is still unclear. Although the specificity of cytotoxic T lymphocyte (CTL) function has been an important issue of organ-specific autoimmune response, the mechanisms responsible for tissue destruction in SS remain to be elucidated. Recent study has suggested that a cleavage product of 120 KDa α-fodrin may be an important autoantigen in the pathogenesis of SS (Haneji et al., Science 276, 604-607 (1997); Yanagi et al., Eur. J. Immunol . 28, 3336-3345 (1998) ) . Sera from patients with SS reacted positively with purified 120 KDa antigen, and proliferative response of peripheral blood lymphocytes (PBMC) from SS patients to the purified autoantigen was detected, but not from systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) patients, and healthy controls. These results indicate that the anti-120 KDa α-fodrin immune responses play an essential role on the development of primary SS but the mechanisms underlying the immunodominant epitope recognition remain unclear.

DISCLOSURE OF THE INVENTION It is therefore an object of the present invention to identify the autoreactive T cell epitope in Sjδgren's syndrome and to provide this epitope peptide. Another object of the present invention is to provide an analogue of this peptide and a pharmaceutical composition containing the analogue peptide, particularly a pharmaceutical composition for the prophylaxis and treatment of Sjδgren's syndrome.

The present inventors have identified the pathogenic T cell epitope involved in antigen-specific immune response in urine model of human SS and established autoreactive T cell lines that recognize synthetic N-terminal portion of α-fodrin (AFN) , which produce Thl cytokines and show significant cytotoxic activities. That is, the present inventors have determined the pathogenic T cell epitope which allows for the regulation of autoimmune response to α-fodrin in SS, and found that an analogue peptide obtained by introducing a mutation into a specific part of the epitope peptide is useful for the peptide therapy of autoimmune diseases such as Sjδgren's syndrome. Accordingly, the present invention provides the following.

(1) A polypeptide consisting of a partial amino acid sequence of α-fodrin containing an amino acid sequence depicted in SEQ ID No : 1.

(2) A polypeptide consisting of a partial amino acid sequence of α-fodrin containing an amino acid sequence depicted in SEQ ID No: 2.

(3) A polypeptide consisting of a partial amino acid sequence of α-fodrin containing an amino acid sequence depicted in SEQ ID

No: 4.

(4) A polypeptide comprising the same amino acid sequence depicted in SEQ ID No:l, SEQ ID No: 2 or SEQ ID No: 4 except that one to several amino acids is (are) substituted, deleted, added or inserted, which can function as a T cell epitope.

(5) A reagent for diagnosis of Sjδgren's syndrome, which comprises the polypeptide of any of (1) to (4) above. (6) A pharmaceutical composition comprising the polypeptide of any of (1) to (4) above.

(7) A gene encoding a polypeptide of the following (a) or (b) :

(a) a polypeptide consisting of a partial amino acid sequence of α-fodrin containing an amino acid sequence depicted in SEQ ID No:l, SEQ ID No: 2 or SEQ ID No: 4,

(b) a polypeptide comprising the same amino acid sequence depicted in SEQ ID No:l, SEQ ID No: 2 or SEQ ID No: 4 except that one to several amino acids is (are) substituted, deleted, added or inserted, which can function as a T cell epitope. (8) A polypeptide comprising the same amino acid sequence depicted in SEQ ID No:l, SEQ ID No:2 or SEQ ID No: 4 except that one to several amino acids is (are) substituted, deleted, added or inserted, which polypeptide binds with an MHC class-II molecule and suppresses a proliferative responses of autoreactive T cell. (9) The polypeptide of (8) above, wherein the amino acid sequence is obtained by substituting one amino acid of the amino acid sequence depicted in SEQ ID No:l, SEQ ID No: 2 or SEQ ID No: 4. (10) The polypeptide of (8) above, wherein the amino acid sequence is that depicted in SEQ ID No:3. (11) A gene encoding a polypeptide of any of (8) to (10) above.

(12) A pharmaceutical composition comprising a polypeptide of any of (8) to (10) above as an active ingredient.

(13) The pharmaceutical composition of (12) above, which is for the prophylaxis and treatment of Sjδgren's syndrome. (14) A method of the prophylaxis and treatment of Sjδgren's syndrome, which comprises administering an effective amount of a polypeptide of any of (8) to (10) above to a patient. (15) Use of a polypetide of any of (8) to (10) above for the manufacture of an agent for the prophylaxis and treatment of Sjδgren' s syndrome.

(16) A commercial package comprising a pharmaceutical composition of (13) above, and a written matter associated therewith, the written matter stating that the pharmaceutical composition can or should be used for the prophylaxis and treatment of Sjδgren's syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows results of analogue peptide-based therapy. Fig. 2 shows results of analogue peptide-based therapy.

Fig. 3 shows results of induction of oral tolerance in mice. Fig. 4 shows results of induction of oral tolerance in mice.

DETAILED DESCRIPTION OF THE INVENTION The polypeptide consisting of a partial amino acid sequence of α-fodrin used in the present invention is not subject to any particular limitation as regards biological species, molecular weight (number of amino acids) and the like, as long as it is an autoreactive T cell epitope or a polypeptide containing the epitope, or a substance having an equivalent action (hereinafter these are also generally referred to simply as an epitope peptide) . In the present invention, an epitope has a structure recognized by a T cell receptor. It is considered that an attack on the epitope present in the α-fodrin protein of an individual himself by a pathogenic T cell is a factor to the onset of an autoimmune disease such as Sjδgren's syndrome etc. This epitope contains an amino acid sequence specifically depicted in SEQ ID No:l, preferably SEQ ID No: 2. The epitope peptide can be prepared in the present invention by chemical synthesis, purification from various organs, or by genetic engineering. As long as the function of a T cell epitope is maintained, this amino acid sequence may have one to several amino acids added to the N- terminal and/or C-terminal of the sequence. When it is particularly produced by genetic engineering, the epitope peptide may be mutated (substitution, insertion, addition or deletion) as long as the function of an epitope can be maintained. It is also possible to provide the epitope peptide as a protein by adding or inserting a longer polypeptide chai (s) to or in an epitope. Moreover, side chains of the constituent amino acids of the above-mentioned epitope peptides may be protected by suitable protective groups [e.g. Cι_₆ acyl groups such as formyl, acetyl, benzoyl, etc. (preferably Cχ-₆ alkanoyl) ] unless the function of a T cell epitope is lost.

The epitope peptide of the present invention, can be also obtained by decomposing a human α-fodrin protein by protease etc. The protease that can be used advantageously includes trypsin, chymotrypsin, and calpain (preferably calpain) . Besides these, an α-fodrin protein derived from any tissue (e.g., brain) or cells from warm-blooded animals (e.g. guinea pig, rat, mouse, rabbit, swine, sheep, cattle, monkey, etc.) can be used as a starting material. The cleavage of α-fodrin protein with a protease can be achieved by allowing them to react in a buffer solution of pH from about 6 to about 8 at from about 10 to about 50 °C

(preferably from about 30 to about 45°C) for from about 30 minutes to about 5 hours.

Conveniently, it can be produced according to the peptide synthesis method to be mentioned later. Of the epitope peptides of the present invention, examples of the mutated amino acid by substitution, insertion, addition or deletion include those that underwent addition of amino acids, deletion of constituent amino acids, or substitution of different amino acids for constituent amino acids, to the extent that the epitope peptide can maintain the function of a T cell epitope. Examples of the mutated epitope peptide obtained by addition of amino acid(s) include that obtained by adding at least one amino acid added to the amino acid sequence depicted in SEQ ID No:l, SEQ ID No:2 or SEQ ID No: 4. Examples of the mutated epitope obtained by deletion of constituent amino acid(s) include that obtained by deleting at least one α-fodrin constituent amino acid from the amino acid sequence depicted in SEQ ID No:l, SEQ ID No: 2 or SEQ ID No: 4. Examples of the mutated epitope obtained by substitution of amino acid(s) include that obtained by substituting at least one α-fodrin constituent amino acid by a different amino acid. The number of amino acid to be added is at least one, but it could be any number as long as the amino acid sequence does not lose the function of a T cell epitope. The number of amino acid to be deleted is at least one, but it could be any number as long as the amino acid sequence does not lose the function as a T cell epitope. When the constituent amino acid is substituted by a different amino acid, the number of the substituted amino acid is at least one, but it could be any number as long as the amino acid sequence does not lose the function of a T cell epitope. When an epitope peptide is mutated, a method generally known in this field is used or two or more such methods are used in an appropriate combination. The epitope peptide of the present invention can be easily produced by the peptide synthesis method.

The amino acid sequence of human α-fodrin protein and its full length sequence, the amino acid sequence of its protein fragment and the DNA sequence encoding the protein fragment are disclosed in Journal of Biological Chemistry, 265, 4427-4433, (1990) .

The method for chemical synthesis of the epitope peptide of the present invention may be whichever of solid-phase synthesis and liquid-phase synthesis. That is, a partial peptide or amino acid capable of constituting the epitope peptide of the present invention is condensed with the residual part, and, when the resulting product has a protecting group, the protecting group is removed to produce the objective peptide. In addition, when the number of amino acids is small, the epitope peptide can be easily prepared by one cycle of peptide synthesis. Alternatively, a polypeptide having a relatively long amino acid sequence is synthesized and cleaved at the N-terminal and/or C-terminal to give a desired peptide. The base polypeptide having a long amino acid sequence can be chemically synthesized by peptide synthesis, or by genetic engineering based on a DNA sequence encoding the base polypeptide, according to a known method or a combination of known methods (USP No. 6,121,057). The condensation and deprotection of amino acid or peptide can be carried out by, for example, the methods described in the following literatures (1)- (5) .

(1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966) . (2) Schroeder and Luebke, The Peptide, Academic Press. New York (1965) .

(3) Izumiya, N. et al., Peptide Gosei no Kiso to Jikken (Fundamentals and Experiments in Peptide Synthesis) , Maruzen, Ltd. (1975) . (4) Yatori, H. and Sakakibara, S., Seikagaku Jikken Koza 1 (Biochemical Experiment Series 1), Tanpakushitu no Kagaku (Protein Chemistry) , p.205 (1977).

(5) Yatori, H. (ed.), Zoku Iyakuhin no Kaihatsu (Drug Development, Continued), vol. 14, Peptide Synthesis, Hirokawa Shoten. After completion of the reaction, the epitope peptide of the present invention can be purified and isolated by using such techniques as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. in a suitable combination. Introduction of a mutation into an epitope peptide may result in the loss of the function of an epitope, depending on the site and the kind of mutation, and may sometimes suppress the reactivity of T cell to the epitope peptide. For example, the present invention provides a polypeptide having the same amino acid sequence depicted in SEQ ID No:l except that one to several amino acids underwent substitution, deletion, addition or insertion, and have a low autoreactive T cell growth stimulant action and an MHC class-II binding capability (hereinafter this polypeptide is to be also referred to as an epitope analogue) .

The epitope analogue of the present invention can be chemically synthesized in the same manner as in the above- mentioned epitope peptide. An autoreactive T cell reacts with self-components, which are specifically autoreactive CTL (cytotoxic T lymphocyte) and the like. An autoreactive T cell can be generally induced and prepared by a method known in this field. An autoimmune disease is caused by an attack on the self-components by the T cell. If the growth of the autoreactive T cell can be suppressed, it leads to the prophylaxis and treatment of autoimmune diseases.

When compared to an epitope peptide, the epitope analogue of the present invention maintains an MHC class-II binding capability but suppresses proliferative responses of the autoreactive T cell. By the "suppresses proliferative responses of the autoreactive T cell" is meant that it shows a significantly lower proliferation of autoreactive T cell as compared to that by the use of an epitope peptide, and is not subject to any particular limitation. Preferably, the proliferation stimulating action is completely absent or very little if there is found any. The level of the proliferation of autoreactive T cell can be measured by a method known in this field. For example, the CTL-induced T lymphocyte growth stimulant action can be determined by ³H thymidine uptake test etc. in the presence of a given amount of an epitope analogue. The MHC class- II binding assay was modified from the protocol previously described (Wauben et al., J. Immunol . 152, 4211-4220 (1994)). The binding with an MHC class-II molecule can be determined by assessing the relative affinity of peptide using class II (I-A^q) spleen cells from NFS/sld mice. The preferable relative affinity is more than 30%. More preferably, the relative affinity is more than 55%. And the most relative affinity is more than 80%. The epitope peptide and epitope analogue of the present invention can be used as a pharmaceutical composition containing either of them as an agent for the prophylaxis and treatment of autoimmune disease, such as systemic lupus erythematosus, rheumatoid arthritis, Sjδgren's syndrome and the like, and particularly as an agent for the prophylaxis and treatment of

Sjδgren's syndrome. More specifically, by the administration of the epitope peptide or epitope analogue of the present invention, inflammation observed in the salivary and lacrimal glands in autoimmune diseases, particularly Sjδgren's syndrome, can be treated or prevented. In particular, the epitope peptide of the present invention can establish the tolerance to autoantigen before and after the onset of the disease. When an epitope analogue is administered, the growth of an autoreactive T cell observed with an autoimmune disease can be suppressed. Moreover, the administration of the epitope analogue results in the suppression of autoantibody. The epitope peptide and epitope analogue of the present invention can be administered as a pharmaceutical composition containing either of them along with a known immunosuppressant or an anti-inflammatory agent, for the prophylaxis and treatment of autoimmune diseases, particularly as an agent for the prophylaxis and treatment of Sjδgren's syndrome.

For application as a medicine, the epitope peptide or epitope analogue of the present invention can be administered either as it is in a bulk form or in a dosage form containing it in combination with a pharmaceutically acceptable carrier, excipient or diluent (e.g. as an injection, tablets, capsules, a liquid, an ointment, or a suppository etc.) to warm-blooded animals (e.g. human etc.) safely by the oral or other route. Injections can be prepared by using physiological saline or an aqueous solution of glucose or the like in the routine manner. Tablets, capsules, and other dosage forms can also be manufactured by the established corresponding pharmaceutical procedures. As a medicine, an epitope peptide or epitope analogue of the present invention is administered to warm-blooded animals in the doses selected from the daily dose range of from about 0.01 ng to about 1000 mg/kg, preferably from about 0.1 ng to about 10 mg/kg, more preferable from about 1 ng to about 100 μg/kg, more preferably from about 10 ng to about 10 μg/kg, taking the route of administration, clinical symptoms, and other factors into consideration.

The epitope peptide of the present invention can be used for the diagnosis of autoimmune diseases inclusive of Sjδgren's syndrome. Specifically, by detecting and assaying autoantibodies to the α-fodrin or α-fodrin fragment protein in the blood of a warm-blooded animal, the diagnosis, disease staging, or prediction of onset of autoimmune disease, particularly Sjδgren's syndrome can be successfully accomplished. The detection and assay of autoantibodies to α-fodrin and α-fodrin fragment protein can be made typically by the following method. The epitope peptide of the present invention (hereinafter referred to collectively as antigen peptide) is coupled to a matrix such as cellulose beads in the routine manner. Then, the sample to be assayed is added and allowed to react at a given temperature

(from about 4°C to about 40°C) for a given time. After this reaction mixture is washed thoroughly, an antibody specifically binding to antibodies of the same species as that of the sample and labeled with a fluorescent substance, a chromogenic substance, an enzyme, or a radioisotope is added and allowed to react at a given temperature (from about 4°C to about 40°C) for a given time. The reaction mixture is then washed thoroughly and, where necessary, a substrate for the enzyme is added and allowed to react at a given temperature (from about 4°C to about 40°C) for a given time. Then, the optical density, intensity of fluorescence, or scintillation count of the reaction product is determined. The carrier to retain the epitope peptide and the method for retaining the peptide on the carrier are known.

The assay method is, for example, as follows.

(1) A sample to be assayed is added to an antigen peptide immobilized on a matrix to conduct an antigen-antibody reaction. Then, a conjugate of a label enzyme such as peroxidase with an antibody binding to the antibodies of the same species as that of the sample (enzyme-labeled antibody) is added and reacted.

(2) To the reaction product obtained in (1), a substrate for label enzyme is added and the optical density or intensity of the color development of the reaction product is measured to calculate the enzymatic activity of the reaction product.

(3) The above steps (1) and (2) are followed using standard solutions of an anti-α-fodrin or α-fodrin fragment protein antibody occurring in the serum taken beforehand from a patient with Sjδgren's syndrome, to construct a standard curve correlating the amount of the anti-α-fodrin or α-fodrin fragment protein antibody with the optical density or intensity of the color development.

(4) Using the serum from a different patient with Sjδgren's syndrome or the risk of developing the syndrome, the above steps (1) and (2) are repeated and the measured optical density or intensity of color development are compared with the above standard curve to find the amount of the patient' s anti-α-fodrin or α-fodrin fragment antibody.

Where, in this specification, amino acids are designated with abbreviations, the abbreviations recommended by IUPAC-IUB Commission on Biochemical Nomenclature or those used commonly in the art are employed. Some examples are listed below. Any amino acid that may exist in an optically active form means an L- compound, unless otherwise indicated. alanine arginine aspartic acid cysteine glutamine glutamic acid glycine isoleucine : leucine lysine methionine proline serine : threonine

valine

The SEQ ID No. mentioned under the title of Sequence Listing in this specification represents the following sequence. [SEQ ID No:l] Amino acid sequence of human α-fodrin comprising amino acids

303 - 318. [SEQ ID No: 2]

Amino acid sequence of human α-fodrin comprising amino acids

304 - 317. [SEQ ID No: 3]

Amino acid sequence of human α-fodrin comprising amino acids 303 - 318, wherein the 308th gultamic acid is substituted by alanine . [SEQ ID No: 4] Amino acid sequence of human α-fodrin comprising amino acids 301 - 320. [SEQ ID No: 5]

Amino acid sequence of human α-fodrin comprising amino acids 306 - 315. [SEQ ID No : 6]

Amino acid sequence of human α-fodrin comprising amino acids 1 - 20. [SEQ ID No: 7]

Amino acid sequence of human α-fodrin comprising amino acids 303 - 318, wherein the 303rd aspartic acid is substituted by alanine. [SEQ ID No: 8] Amino acid sequence of human α-fodrin comprising amino acids

303 - 318, wherein the 304th leucine is substituted by alanine. [SEQ ID No: 9] Amino acid sequence of human α-fodrin comprising amino acids 303 - 318, wherein the 307th leucine is substituted by alanine. [SEQ ID No: 10]

Amino acid sequence of human α-fodrin comprising amino acids 303 - 318, wherein the 309th aspartic acid is substituted by alanine .

[SEQ ID No: 11] Amino acid sequence of human α-fodrin comprising amino acids

303 - 318, wherein the 310th lysine is substituted by alanine.

[SEQ ID No: 12] Amino acid sequence of human α-fodrin comprising amino acids 303 - 318, wherein the 311th valine is substituted by alanine. [SEQ ID No: 13]

Amino acid sequence of human α-fodrin comprising amino acids 303 - 318, wherein the 312th lysine is substituted by alanine.

[SEQ ID No: 14]

Amino acid sequence of human α-fodrin comprising amino acids 303 - 318, wherein the 314th leucine is substituted by alanine.

[SEQ ID No: 15] Amino acid sequence of human α-fodrin comprising amino acids 303 - 318, wherein the 315th cysteine is substituted by alanine. [SEQ ID No: 16] Amino acid sequence of human α-fodrin comprising amino acids 303 - 318, wherein the 317th glutamic acid is substituted by alanine. The present invention is explained in more detail in the following by way of Examples, to which the present invention is not limited. Examples

Example 1 : Identification of the pathogenic T cell epitope (1) To determine the autoreactive T cell peptide epitope which allows for the regulation of autoimmune response to α-fodrin in SS, a fused protein containing a partial fragment of human α- fodrin was prepared and examined for a proliferative responses of T-cell. Materials and Methods (1) Mice

It is known that when thymectomized on day 3 after birth, NFS/ si d mice develop Sjδgren's syndrome in 4-20 weeks after operation. Moreover, the symptoms observed then are known to well reflect the manifestations of primary Sjόgren's syndrome.

NFS/N strains carrying the mutant gene sld (Hayashi et al., Am. J. Pathol . , 132, 187-191 (1988)) were reared in specific pathogen-free mouse colony. Thy ectomy was performed on day 3 after birth (3d-Tx) in NFS/ sld mice. (2) Recombinant protein of human α-fodrin fragment

The DNAs coding for human α-fodrin protein and fragment thereof are known [Journal of Biological Chemistry, 265, 4427- 4433 (1990)). A human α-fodrin fragment was produced using JS-1 cDNA (1-1784 bp) , 2.7A cDNA (2258-4884 bp) and 3'DA cDNA (3963- 7083 bp) . The JS-1 cDNA, the 2.7A cDNA and the 3'DA cDNA were completely digested with the restriction enzyme EcoRI and each ligated to the EcoRI site of E. coli pGEX-4Ts vector (Pharmacia) . According to the manufacturer' s instructions for this vector, the vector was introduced into E. coli and the fusion protein of glutathione S-transferase (GST) and α-fodrin fragment protein was harvested. According to the instructions, this fusion protein was digested with thrombin and the α-fodrin fragment protein was isolated and purified independently of glutathione S-transferase. (3) PCR and single-strand conformation polymorphisms (SSCP)

LNCs (5xl0⁵/ml) were cultured with JS-1, 2.7A, 3'DA, or GST recombinant proteins at a concentration of 5 μg/ml for 4 days in RPMI 1640 with 1 U/ml of recombinant IL-2 and 10% FCS. Total RNA was prepared with Isogen (Nippon Gene, Co., Tokyo) . cDNA synthesis and PCR were carried out using method as described (Furukawa et al., Br. J. Rheumatol . , 35, 1223-1230 (1996)). Amplification was performed with Taq polymerase with 5' primer specific for TCRVβ6 and Vβ8 genes and 3' primer specific for TCR Cβ gene (Furukawa et al., (1996), supra) . Amplified DNA was diluted at 1:20 in a denaturing solution and held at 90 °C for 2 min. The diluted sample (2 μl) was electrophoresed in non- denaturing 5% polyacrylamide gels containing 10% glycerol. The gel was run at 35W constant power for 2 hrs. After electrophoresis, the DNA was transferred to immobilon-S (Millipore Intertech, Bedford, MA) and hybridized with biotinylated Cβ probe (Furukawa et al., (1996), supra) , streptavidine, biotinylated alkaline phosphatase, and a chemiluminescent substrate system (Millipore Intertech) . (4) Steps and Results

Spleen cells and regional lymph node cells (LNCs) from 3d- Tx NFS/ sld mice (8 week-old) were cultured with recombinant α- fodrin fusion protein (JS-1, 2.7A, and 3'DA) . A significantly increased proliferation was detected in LNCs with JS-1 recombinant protein, and spleen cells with 2.7A protein, but not in non-Tx NFS/sld mice. To investigate the comparison in clonotypes of infiltrating T cells in vivo and autoantigen- stimulated LNCs in vitro, the combination of RT-PCR and subsequent SSCP analysis was used to discriminate the diversities including in the D, J, and N regions (Matsuoka et al . , J. Immunol . , 151, 1691-1701 (1993); Yamamoto et al., Int . Immunol, 4, 1219-1225 (1992); Hayashi et al., Arthritis Rheum. , 38, 1077-1084 (1995) ) . The dominant and common bands in both infiltrating T cell in the salivary glands at 8 weeks and JS-1-stimulated LNCs were detected in the products of Vβ6 genes, but not of Vβ8 genes. In contrast, the amplified DNA from the TCR Vβ chain of 2.7A- stimulated cells developed as a smear following PCR and SSCP analysis. These results indicate that JS-1-reactive T cells were present in the autoimmune lesions of murine SS model. Thus, JS-1 protein was determined as an appropriate α-fodrin autoantigen. Example 2:Identi ication of the pathogenic T cell epitope (2)

(1) Synthetic peptides Peptides were synthesized using TBOC chemistry on a model

430A peptide synthesizer (Applied Biosystems, Foster City, CA) . The peptides were purified by reverse-phase HPLC, and all peptides were analyzed for purity by mass spectrometry.

(2) Steps and Results Epitope scanning was determined by using variant peptides

(Brocke et al., Nature, 379, 343-346 (1996); Quaratino et al., J. Exp. Med. , 183, 349-358 (1996); Wahren-Herienius et al., (1999), supra) that were truncated gradually from the N and C termini of the JS-1 epitope. Synthetic peptides which were designed to be 20 amino acid residues in length, overlapping by 5 amino acid residues were generated (pl-p45 from N terminal side) . First, mixtures of 5 peptides in each pool were tested in a proliferation assay using LNCs at 5 weeks. Significant proliferative responses were seen with JS-1 peptide mixture (p21- p25, and p31-p35) , but not with others. We next examined proliferative response to individual peptide. The epitope specificity was demonstrated to response to JS-1 peptide p21 (N- terminal portion of α-fodrin (from the 301st to 320th inclusive of the amino acid of AFN:AFN₃₀ι-32o) / p31 (AFN451-470) and p32 ( FN4₆₆_ ₄₈₅) . The ability of LNCs to respond peptide was assessed by interleukin-2 (IL-2), interferon-γ (IFN-γ) , and IL-4 production, as detected by sandwich ELISA (Haneji et al., (1997), supra; Yanagi et al., (1998), supra) . We found that culture supernatants from p21 (AFN₃₀₁-₃₂₀) "Stimulated LNCs contained high levels of IL-2 and IFN-γ, but negligible levels of IL-4. Culture supernatants from p31- and p32-stimulated LNCs contained lower amounts of IL-2 and IFN-γ. Thus, N-terminal portion of α-fodrin p21 (AFN₃0₁-32₀) is an important T cell epitope, accounting for the increased concentration of Thl cytokines in these cultures. Peptides were further tested for their ability to initiate a proliferative response and cytokine production, indicating that an appropriate T cell epitope peptide capable of inducing SS lesions is a peptide p21-16 [AFN₃₀₃-₃₁₈, SEQ ID No:l] or p21-14 [AFN₃₀₄-₃₁₇, SEQ ID No: 2] (see Table 1) .

Table 1

Peptide _, . . , Proliferation __τ _ __ .

Ammo acid sequence IL-2 IFN-γ IL-4 No.

ERDLAALEDKVKALCAEADR

No.21-20 9228 . 7 104 .5 78 . 9 ud*

[SEQ ID No: 4]

DLAALEDKVKALCAEA

No.21-16 20010. 1 95. 9 104 . 1 ud*

[SEQ ID No:l]

LAALEDKVKALCAE

No.21-14 19844 . 4 86. 6 94 . 8 ud*

[SEQ ID No: 2]

ALEDKVKALC

No.21-10 3852 . 9 6. 6 ud* ud*

[SEQ ID No: 5]

MDPSGVKVLETAEDIQERRQ

No.l 2658 . 9 ud* ud* ud*

[SEQ ID No : 6]

*ud, undetectable

As used herein, by the ^Λp21-16" is meant a modified p21 containing 16 amino acids, which is prepared by truncating 2 amino acids each from the N terminal and C terminal of p21 peptide, and by the "p21-14" is meant a modified p21 containing 14 amino acids, which is prepared by truncating 3 amino acids each from the N terminal and C terminal of p21 peptide. Example 3:Establishemnt of autoreactive T cell lines

(1) Autoreactive T cell line

LNCs from 3d-Tx NFS/sld mice (5-week-old) were cultured with α-fodrin peptides (10 μg/ml) in RPMI 1640 supplemented with 10% fetal calf serum (FCS) , 10 mM HEPES and lOOU/100 μg per ml penicillin/streptomycin in 96-well plates at lxlO⁶ cells/well. On day 3, IL-2 (Gemzyme Diagnostics, Cambridge, MA) was added and cells were fed with media containing 0.5 ng/ml of IL-2 every 3 day. On day 14, an aliquot was analyzed for reactivity to α- fodrin peptides. lxlO⁴ cells from each cell lines were cocultured with 1x10⁴ irradiated autologous splenocytes in duplicate for 72 hrs. α-fodrin specific T cell lines (stimulation index>3) were retested using synthetic peptides. (2) Cytotoxicity assay

Cytotoxic assays were done as described (Saito et al., J. Immunol . , 162, 2488-2492 (1999)), using peptide-pulsed (10 μg/ml^"1) MSG cells (Saito et al., (1999), supra) labeled with [⁵¹Cr] sodium chromate as target at a 1:50 target: effector ratio. (3) Procedures and Results

To establish α-fodrin peptide-specific T cell line, limiting dilution analysis (LDA) (Bieganowska' et al., J. Exp. Med. , 185, 1585-1594 (1997); Selin et al., Immunity, 11, 733-742 (1999) ) was performed on LNCs from 3d-Tx NFS/sld mice at 5 weeks in the presence of p21 (AFN301-320) t p31 (AFN451-470) and p32 (AFN₆6-485) and irradiated syngeneic spleen cells. We succeed in isolating three strongly proliferative T cell lines from p21-stimulated LNCs, but not from p31- and p32-stimulated cells. The majority of these autoreactive T cells were CD4+ T cells bearing Vβ6 containing Thl cytokines such as IL-2 and IFN-γ, but not IL-4. The TCRVβ usage and third complementary determining regions (CDR3) sequence of three T cell lines were determined by RT-PCR amplification and sequencing of the PCR products (Sanger et al., Proc. Natl . Acad. Sci . , 74, 5463-5467 (1977)). Interestingly, these sequences are homologous to VDJβ sequences of the T cells from affected glands of SS model mice at 8 weeks. It should be noted that a perfect match in Vβ6-CDR3 gene was observed between the T cells infiltrating into salivary glands and the expression of three AFN₃₀₁--₃₂₀-specific T cell lines. We further confirmed that the T cell lines have significant cytotoxicity when tested in ⁵¹Cr-release assay against mouse salivary gland (MSG) cells (Wahren-Herienius et al., Immunol . Today, 20, 234-240 (1999)). To analyze whether these autoreactive T cells cause autoimmune lesions, each line was transferred intraperitoneally (i.p.) into irradiated (20 Gy) non-Tx NFS/sld mice at 4 weeks. Organ-specific autoimmune lesions in the salivary and lacrimal glands developed at 8 and 12 weeks after the i.p. injection in all mice, and tissue-infiltrating lymphocytes were positive for CD4 and Vβ6. Moreover, the inflammatory lesions in the salivary and lacrimal glands were associated with "sicca syndrome" showing decreased secretion of saliva and tear (Saito et al., (1999), supra) . Example 4 :Alanine scanning mutagenesis and Class II binding studies

(1) Class II binding

The peptide-MHC-binding assay was modified from the protocol previously described (Wauben et al., J. Immunol . , 152, 4211-4220 (1994)). The relative affinity of peptide was determined using class II (I-A^q)⁺ spleen cells from NFS/sld mouse. I-A^q+ cells (lxloVwell) were incubated at 37°C for 24 hrs in 96- well microtiter plates in a total volume of 100 μl culture medium containing 700 pg/ml of IFN-γ. 20 μg/ml of FITC-labeled AFN₃₀₃-3₁₈ and 5 μg/ml of unlabelled AF ₃₀₃-₃₁₈ or alanine-substituted analogues were added for 75 min. Cells were fixed in 1% paraformaldehyde, and were incubated with biotynilated anti-I-A^q+ antibody (PharMingen, San Diego, CA) on ice. lxlO⁴ cells from each sample were analyzed on EPICS (Coulter, Miami, FL) . (2) Procedures and Results

To assess the individual contribution to binding of residue in the epitope motif and its proximity, we performed alanine scanning mutagenesis (Manning et al., Immunity, 8, 413-425 (1998); Wither et al., J. Immunol . , 162, 2113-2122 (1999)) of the p21-16 (AFN₃₀₃-3₁₈) peptide, which is capable of inducing autoimmune lesions. The results of proliferation assays and class II binding on the AFN30₃-₃₁8 peptide are shown in Table 2.

Table 2

* The relative affinity of peptides was determined using class II (I-A^q)⁺ spleen cells from NFS/sld mice. ** nd, not done.

Class II binding studies indicate that the two amino acid substitutions of MHC contact points (V₃₁₁ & E₃₁₇) within epitope are sufficient to alter peptide binding and MHC restriction. Moreover, it is striking that only two TCR contact points (D₃₀₃ & E308) are essential for initiation of antigen-specific T cell response .

Example 5:Analogue peptide-based therapy (1) Analogue-peptide treatment Four-week-old female 3d-Tx NFS/sld mice were injected subcutaneously on the neck with 5 μg/head of the analogue peptide (E308^→A) -incorporated liposome (n=12) once a week until week 7. Mice were analyzed at 8 weeks and compared with 3d-Tx NFS/sld mice treated with control peptide (E317→A) -incorporated liposome (n=7) . (2) Confocal immunofluorescence analysis

IFN-γ-stimulated MSG cells (Wahren-Herienius et al., (1999), supra) were fixed with 1% paraformaldehyde, and were incubated with mAb to I-A^q molecule (PharMingen) , and FITC-labeled AF30₃-₃₁8 peptide. The labeled second antibody was Texas Red-conjugated goat anti-mouse IgG (molecular probes, Eugene OR) . For microscopy, a LEICA TCSNT laser scanning microscope (Nussloch, Germany) was used. (3) Procedures and Results

The effects of an analogue peptide (E₃₀₈ ^→A) -based subcutaneous vaccinations through incorporation with liposomes into murine SS model were examined (Haneji, N. et al., Journal of Immunology 153, p.2769 (1994) or Haneji, N. et al., Science vol.275, p.604 (25 April 1997). Inhibitory effects were clearly observed by the treatment with an analogue peptide (E₃₀₈ ^→Α) - incorporated liposome (Fig. 1) . Protein immunoblot analysis demonstrated that autoantibody production to the 120 kDa α-fodrin was inhibited in sera from mice treated with analogue peptide. The average saliva and tear volume of the SS model mice treated with analogue peptide were significantly higher than those of untreated mice (Fig. 2) . Significant inhibitory effects on proliferative response of LNCs were observed in analogue-peptide- treated mice. The activation markers (CD44^high, CD45RB^lo, Mel- 14^low) were down-regulated in LNCs gated on CD4 from treated mice with analogue peptide. In in vitro studies, colocalization of FITC-labeled AFN₃₀₃-3₁₈ peptide and I-A^q molecule was observed in the IFN-γ stimulated MSG cells. Example 6:Induction of oral tolerance in Tx-NFS/sld mice

(1) Fodrin-peptide fragment treatment

The fodrin peptide (JS-1 p21-20/5 μg/head/week) was orally administrated to 16-month-old female Tx NFS/sld mice (n=6) with a margen tube once a week and 4 times in total. At one month from the start of the administration, the mice were evaluated for oral tolerance, and the obtained test data was compared with that of female Tx NFS/sld mice treated with PBS buffer.

(2) Results Induction of oral tolerance was clearly observed by the treatment with the fodrin peptide JS-1 p21-20 (Fig. 3) . The average saliva and tear volume of the mice treated with the fodrin peptide were significantly higher than those of control mice (Fig. 4) .

This application is based on Application No. PR5897/01 filed in Australia, the content of which is incorporated hereinto by reference.

Claims

(1) A polypeptide consisting of a partial amino acid sequence of α-fodrin containing an amino acid sequence depicted in SEQ ID No:l.

(3) A polypeptide consisting of a partial amino acid sequence of α-fodrin containing an amino acid sequence depicted in SEQ ID No: 4.

(5) A reagent for diagnosis of Sjδgren's syndrome, which comprises the polypeptide of any of claim 1 to claim 4.

(6) A pharmaceutical composition comprising the polypeptide of any of claim 1 to claim 4.

(7) A gene encoding a polypeptide of the following (a) or (b) :

(a) a polypeptide consisting of a partial amino acid sequence of α-fodrin containing an amino acid sequence depicted in SEQ ID No:l, SEQ ID No:2 or SEQ ID No:4,

(b) a polypeptide comprising the same amino acid sequence depicted in SEQ ID No:l, SEQ ID No: 2 or SEQ ID No: except that one to several amino acids is (are) substituted, deleted, added or inserted, which can function as a T cell epitope.

(8) A polypeptide comprising the same amino acid sequence depicted in SEQ ID No:l, SEQ ID No: 2 or SEQ ID No: 4 except that one to several amino acids is (are) substituted, deleted, added or inserted, which polypeptide binds with an MHC class-II molecule and suppresses a proliferative responses of autoreactive T cell.

(9) The polypeptide of claim 8, wherein the amino acid sequence is obtained by substituting one amino acid of the amino acid sequence depicted in SEQ ID No:l, SEQ ID No: 2 or SEQ ID No: 4.

(10) The polypeptide of claim 8, wherein the amino acid sequence is that depicted in SEQ ID No : 3.

(11) A gene encoding a polypeptide of any of claim 8 to claim 10.

(12) A pharmaceutical composition comprising a polypeptide of any of claim 8 to claim 10 as an active ingredient.

(13) The pharmaceutical composition of claim 12, which is for the prophylaxis and treatment of Sjδgren's syndrome.

(14) A method of the prophylaxis and treatment of Sjδgren's syndrome, which comprises administering an effective amount of a polypeptide of any of claim 8 to claim 10 to a patient.

(15) Use of a polypeptide of any of claim 8 to claim 10 for the manufacture of an agent for the prophylaxis and treatment of

Sjδgren' s syndrome .

(16) A commercial package comprising a pharmaceutical composition of claim 13, and a written matter associated therewith, the written matter stating that the pharmaceutical composition can or should be used for the prophylaxis and treatment of Sjδgren's syndrome .