JPH10509136A - Polypeptides and their use in the treatment and prevention of auto-immune diseases - Google Patents

Abstract

  (57) [Summary] The present invention relates to a polypeptide fragment comprising the sequence VVNKIRG (SEQ ID 107) and comprising 9 or more amino acid residues, its use in the prevention, diagnosis and treatment of autoimmune diseases such as rheumatoid arthritis and the production of said fragments. Provide a way.

Description

DETAILED DESCRIPTION OF THE INVENTION       Polypeptides and their use in the treatment and prevention of auto-immune diseases   The present invention relates to polypeptide fragments, auto-immune diseases such as rheumatoid Its use in the prevention, diagnosis and treatment of arthritis and the production of these fragments It is about the law.   An autoimmune disease is a similarity between a foreign molecule or antigen and the organism's own molecular structure. It is thought to happen. The chronic condition of arthritis is the joint, especially the connection of the body It is believed that autoimmunity against tissue components is involved.   Rheumatoid arthritis (RA) is the most common form of arthritis that indicates the development of autoimmunity. (Elson et al., Autoimmunity, 1992,13: Outlined in 327 ). The disease is the third most common illness in the elderly and has pain and distress It brings great suffering. Often there is an association between HLA-DR4 and RA And this is implicated in the involvement of T-cells in the disease (Stasn ey), New Eng. J. Med., 1978,298: 869 and Watanabe et al., J. Ex. Me d., 1989, 169:2263] and the existence of a genetic contribution to the disease. However, recent twin studies [Silman et al., Brit. J. Rheumatol. , 1993,32: 903] suggests that the upper limit of the genetic contribution is only 15% did. Thus, by definition, the main factor contributing to the induction of RA is environmental There will be. This claim suggests that the incidence of RA among South Africans has shifted from villages to towns. Higher as you move [Solomon et al., Ann. rheum. Dis., 1975,Three Four : 128] and abnormal immune response to microorganisms in patients with this disease The fact that the answer increases [Deighton et al., Brit. J. Rheumatol., 1992,31: 241]. Such considerations suggest that RA is Caused by potentially homologous bacterial or viral antigens [McCulloch et al .: Clin. Ex p. Immunol., 1993,92: 1 outlined).   It has been shown to be useful in examining the environmental factors that contribute to this disease. One model is pristane-induced arthritis (PIA). This model is Intraperitoneal injection of finoil pristane (2,6,10,14-tetramethylpentadecane) Some of the mice that have become chronic T-cells after about 60-200 days, depending on the strain of the mouse Dependent on the discovery that they develop dependent inflammatory arthritis (Potter r) M., J. Immunol., 1981, 127: 1591; Bedwell et al. Immunol. 1987, 25: 393; Wooley et al., Arthritis. Rheum., 1987, 32: 1022; Wooley et al., Arthritis. Rheum., 1989, 32: 1022; and Levit t) et al. Rheumatol., 1992, 19: 1342]. The course of the PIA is thus similar to RA Other established animal models such as adjuvant arthritis, strep This arthritis is distinguished from Tococcus cell wall arthritis and collagen-induced arthritis. I'm different. Histopathological arthritis is synovial cell hyperplasia with cellular infiltration and cartilage erosion. Formation and formation of pannus (Bedwell et al.) , J. et al. Immunol., 1987,twenty five:393; Hopkins et al., Rheumatol. Int., 1984,Five : 21; and Thompson et al., Imm. Let., 1993,36:227].   Recent studies have established that the microbial environment affects the development of the PIA. A Specific pathogen-free (SPF) mice raised under aseptic conditions in isolators Be resistant to the onset of IA while returning such animals to their previous environment. Then restores the susceptibility of the disease [Thompson et al., Imm. Let., 1 993, 36: 227]. Endogenous gut flora among susceptible and refractory mice Are different [Thompson et al., Imm. Let., 1993,36:227] but this change Whether it affects susceptibility to the disease, or how it actually It is unknown whether mice are predisposed to PIA when exposed to PIA. I However, sera from PIA-affected mice develop the same age of the disease. Immune-dominant mice were compared to pristane-injected mice or High concentration of antibodies against the C. bacterium 65kD heat shock protein (hsp65) It is known that   In many infectious diseases, such as tuberculosis and leishmaniasis, heat shock It has long been recognized that protein (hsp) is an immunodominant antigen. I was These infectious diseases have abnormalities similar to those observed in RA. Properties, such as high agalactosyl-IgG concentrations, organs involved and It may indicate the range of existing autoantibodies. Environmental factors are not clear in RA. Of paramount importance, microbial drugs and consequently hsps are relevant. Was.   hsps are categorized into genes according to their molecular weight and sequence homology within each group. It is. For example, the hsp60 (60 kD) gene cluster includes hsp65 (mycobacteria) and hsp58 ( Mammals).   Spleen T-cells from mice suffering from arthritis have normal or related age-matched In vitro in response to hsp65 than T-cells from mice without adenitis It was found to grow more actively. In addition, mouse for Pristine Challenge Previously immunized with hsp65 in incomplete Freund's adjuvant (IFA) In some cases, no onset of the disease will be seen [Thompson et al. I mmunol., 1990, 20: 2479 and Thompson et al., Autoimmunity, 1991,11 : 89]. This protective effect is specific to hsp65,E. StiffGroE1 equivalent Or not elicited by other unrelated antigens. Flaws [Thompson et al., Eur. J. Immunol., 1990,20: 2479] and also the antigen It cannot be attributed to competition [Baker et al., Autoimmunity, 199 2,14: 73]. These discoveries are based on exposure to microbiota in the environment, Can be sensitized to hsp, and in the process, Increase the likelihood that induction of arthritis is necessary. If so, by hsp65 It is anticipated that there will be an association between sensitization and susceptibility to PIA. There will be. Experiments by the applicant suggest that this hypothesis is correct.   One possibility to explain how PIA can arise from such sensitization Sex is determined by an epitope on hsp65 of a microorganism that cross-reacts with self (mammalian) hsp58. Is to promote immune responsiveness to pristane (Thompson Et al., Imm. Let., 1993,36:227; and Thompson et al., Eur. J. Immunol. , 1990,20: 2479]. This suggests that hsps are unusually high throughout the prokaryotic and eukaryotic worlds. Despite sequence conservation, it is the predominant antigen in immune responsiveness to microorganisms The fact that it is reliable [Cohen et al., Immunol. Today , 1991,12, P. 105]. Thus, each microbial hsp is capable of responding to any animal with an immune system. And scattered self epitopes. Furthermore, these are the normal components of all cells A component, but its synthesis is increased by many different forms of cellular stress . hsp 58 was detected in joints of patients with RA [Karlsso n-Parra) et al., Scand. J. Immunol., 1990,31: 283], and Mau suffering from PIA -Derived T-cells react with joint extracts [Thompson et al., Eur. J. Im munol., 1990,20: 2479] indicates that hsp58 is a target in the joints of mice with PIA. It is reasonable to assume that the antigen is This hypothesis tells the PIA Motion protected from the development of arthritis in affected mice and pre-immunization with hsp 65 High immune response to the 65 kD mycobacterial heat shock protein. Enables explanation of the paradox of showing responsiveness. Of mice with PIA It is anticipated that only one should develop an autoimmune response against the 65 kD group of hsps. On the other hand, the response of mice pre-immunized with hsp65 is limited to microbial-specific determinants Should be. In other words, induced by immunization with hps65 in IFA Response differs from that elicited by sensitization by environmental / intestinal microbes. You.   T-cell mediated responses to mycobacterial antigens have been demonstrated in experimental animal models and in humans. It has been implicated in the etiology of inflammatory arthritis in both. . Rat Adjuvant Seki In arthritis, the disease affects the 65 kD mycobacterial heat shock protein. It was established that the disease could be triggered by a T-cell clone specific for this. Also, 65 Pre-immunization with a kD-specific T cell line or the hsp itself allows for subsequent adjuvants. Protect rats from inducing arthritis (Van Eden Nature, 1988, 331: 171 and Holoshitz et al., Science, 1983, 219: 56).   The epitope recognized by the arthritogenic T cell clone is amino acids 180-18 8 is localized. EP-A-322990 has the amino acid sequence 172-19 of bacterial hsp 65 And polypeptides for inducing resistance to autoimmune diseases The use of the polypeptide as an immunogen is described. WO 92/04049 isMaiko Bacterium tuberculosis Protein A peptide containing the amino acid sequence corresponding to positions 180-186 of hsp 65 may be For example, it is effective for prevention and treatment of autoimmune arthritis.   Using the PIA model, autoimmune responses to antigens that cross-react with hsp 65 Pristane-induced arthritis (Thompson (Th. ompson) et al., Eur. J. Immunology, 1990, 20: 2479-2484). In addition, by hsp 65 Pre-immunization alters the specificity for this antigen or the immune responsiveness to the antigen Protecting mice from the development of pristane-induced arthritis through altered capacity It is shown.   After further study using this PIA model, the applicant surprisingly found that (A region completely different from and far away from the region described in WO 92/04049). In addition, certain regions of the microbial protein hsp 65 may provide a protective response against arthritis. Found to be effective.   The present invention also provides polypeptides having nine or more amino acid residues. The polypeptide has the following sequence:                     VVNKIRG (SEQ ID 107) Or a homologue thereof or a functional equivalent or analog thereof.   The polypeptide preferably has up to a total of 21 amino acid residues, and more Preferably, it may contain 9 to 11 amino acid residues.   A 7-mer sequence as defined above (or a homologue thereof or a functional equivalent thereof or Analogs) are hereinafter also referred to as "motifs" of the present invention. This motif is a microorganism (Mycobacteria) Corresponds to hsp 65 sequences 261-267.   The invention also has up to 21 amino acid residues comprising the sequence: Provide a polypeptide consisting of the sequence of:                   VVNKIRGTFKS (SEQ ID 108) Here, the sequences include their homologs or their functional equivalents or analogs. Up The polypeptide sequence described corresponds to the sequence 261-271 of the microorganism hsp 65. Microbial hsp 65 Is described, for example, in EP-A-262 710. This reference isMaiko Mycobacterium bovis ) Derived microorganism hsp 65 However, there is substantial sequence homology between the microbial hsp 65s (generally greater than 60%, Up to 98%), so that the corresponding region of any microorganism hsp 65 can be identified, And it falls within the scope of the present invention.   Examples of polypeptides of the present invention also include those of the microbial hsp65 protein containing the motif. Fragments, such as the amino acid sequences 251-267 (SEQ ID 51).   The polypeptides of the present invention, when used in a pre-immunization treatment, may cause autoimmune diseases and disorders. It has been shown to have a preventive effect, especially on the development of RA. Like this These presented polypeptides have T_HIs expected to induce two cells These polypeptides are also useful in treating RA in light of the fact that Would. The present invention further relates to the prevention or treatment of patients against autoimmune diseases such as RA. A vaccine is also provided for immunization for the purpose, said vaccine comprising a vaccine as described above. Contains repeptide.   The polypeptide may be useful parenterally, for example, subcutaneously, intramuscularly, intravenously or intraperitoneally. Is administered.   The polypeptides of the present invention may also be administered transmucosally, for example, orally or nasally. Alternatively, it can be administered as a suppository.   The polypeptide of the present invention may be used in combination with a pharmaceutically acceptable carrier or excipient. Advantageously, it is administered as a pharmaceutical composition. Such compositions are described in the present invention. Constitute a further aspect of.   Suitable carriers include liquid carriers such as oils or water.   Suitable carriers also include solid carriers, such as tablets for oral administration. Or a formulation including suspension or suppository.   Compositions or formulations containing a polypeptide of the present invention as an active ingredient are readily available Suitable for nasal administration by field inhaler, atomizer or nebulizer It can be.   The polypeptide of the present invention can be produced using various methods apparent to those skilled in the art. You. For example, using a known technique, the microorganism hsp 65 is fragmented and then similarly fragmented. Obtaining a predetermined fragment by purification using a method known in the field Can be manufactured. However, the peptides obtained in this way are not very accurate It does not have any predetermined length.   The polypeptides of the present invention can also be obtained using recombinant DNA technology. You. A nucleotide sequence encoding the predetermined polypeptide Utilizing a vector system that produces expression, it can be incorporated into a suitable host.   However, polypeptide sequences can be obtained by standard chemical methods or obtained in the art. It can also be produced by a complete synthetic method using a possible peptide synthesizer. You.   As used herein, the expression "homologue" refers to the described polypeptide. At least 60%, preferably at least 70% and most preferably A peptide having an amino acid sequence that is at least 80% homologous is meant. Also, the table The current "functional equivalent" or "mimetic" Peptides or other organic chemicals that produce similar effects as the compounds of the present invention in vivo Means any chemical that can be In particular, such compounds are Produces a prophylactic immunogen response, which can be tested by testing as described in the Examples below. Used in a pristane-induced arthritis model, using all hsp 65 Better than the response achieved.   Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. Will be done.   FIG. 1 shows the oversequence corresponding to the complete sequence of the microorganism hsp 65 used in the examples. 1 is a peptide library containing a pool of 11 groups of wrapping peptides.   FIG. 2 shows a pool of 11 groups of overlapping peptides as defined in FIG. 6 arthritic mice (top panel), 6 protected mice (middle panel) Figure) Comparison of the proliferative responses of T cells from each of the six normal mice (n = 6) FIG.   FIG. 3 shows in vitro hsp65 using peptides from the microorganism hsp65. 2 shows the results of experiments to examine the stimulation of T cells from pre-immunized mice FIG.   FIG. 4 shows the protection of mice pre-immunized with several polypeptides against PIA. It is a figure showing the result of the research for measuring control.   FIG. 5 shows that 60 days after injection of pristane (D = 60), It is a figure which shows the therapeutic effect by immunizing with a polypeptide.   FIG. 6 contains the motif of the present invention 10 days before pristane injection (D = -10). It is a figure which shows the preventive effect by immunizing with a polypeptide.   FIG. 7 shows that 20 μg of biotin-treated polypeptide (hsp 261-271: SEQ ID 108 ), An increasing amount of “cold” virus hemagglutinin is converted to human EBV-form FIG. 3 shows the effect of adding to class II receptors on a transgenic B-cell line. You.   FIG. 8 shows biomarkers for class II receptors on human EBV-transformed B-cell lines. Dose-dependent binding of the tin-treated polypeptide (hsp 261-271: SEQ ID 108). FIG.   FIG. 9 shows stimulation with the peptide fragment hsp 261-271 or all hsp 65 of the present invention. FIG. 2 is a diagram showing cytokine profiles of T cells obtained.   The following examples illustrate the invention.                                 Example 1   PIA mice, mice protected by hsp 65 and normal age matched mice   Of T cells derived fromanimal   Unless otherwise stated, 4-8 week old male CBA / Igb mice were used. CBA / Igb Mau Used the (101 species XCBA) F1 hybrid backcrossed to CBA mice, In Qing, it was obtained by selecting mice with the Igb allotype.Induction of arthritis by pristane   One group of 6 mice was treated with Emma in incomplete Freund's adjuvant (IFA). Immunization with 50 μg of Mycobacterium hsp 65 by intraperitoneal route did. This group constituted the protected group of mice. 10 days later, this group and 6 more Group of mice received 0.5 ml of pristane twice intraperitoneally at 50 day intervals (WI., Aldrich Chemical Co., Milwaukee) Co.)], and induced arthritis. The last group of "normal" mice served as controls Bred.Synthetic peptides used as antigens in immunization studies   10 representing the complete sequence of the microorganism hsp 65, corresponding in length to 15 to 19 amino acids. A library consisting of six overlapping peptides was simultaneously multiplexed-peptidic. Solid phase synthesis [Houghton R.A., Proc. Natl. Acad. Sci. USA, 1985 82: 5131] and polyamide resin [Arshady et al., J. Am. Chem. Soc. Perkin Trans., 1981, I.529] and FMOC. Complete la The library is shown in FIG. Complete the peptide with trifluoroacetic acid and Extracted from the resin using a simple scavenger, and solvent evaporation and methanol Isolated by precipitation with toluene and diethyl ether. Purity, amino acid analysis And checked by HPLC. Irrelevant control antigen BSA and human Ig G was also used with the mitogen ConA.   Eleven antigens were prepared, each of which was identified by the polypeptides shown as groups 1-11 in FIG. Contains one pool of the tide group.Preparation of T-cells and APC for culture   After 200 days, the spleen of each mouse was removed under aseptic conditions, and 20 mM HEPES (pH 7.2; In a Petri dish containing RPMI-1640 supplemented with Flow Labs) A cell suspension was prepared. The splenocytes were buffered with Tris (Tris: pH 7.2) at 0.83% (w / v) NH_FourErythrocytes were removed by treatment with Cl 2 solution. After washing, remove cells by 1.25X1 0⁷The cells were suspended in RPMI-1640 HEPES at a concentration of cells / ml. T cells of immune response animals Panning method such as Engleman (Engleman) [Engleman (E ngleman) et al. Immunol., 1981, 127: 2124]. Briefly explain A 10cm Petri dish (GB, Sterilin Ltd., Hanslow) Coated with 5 ml of a 0.5 mg / ml mouse γ-globulin solution in PBS for 2 hours at room temperature. After washing once with PBS, Petri dishes were washed with a 5/100 dilution of rabbit anti-mouse Ig serum. Incubate overnight at 4 ° C with ml. After washing, 8 ml of the spleen cell suspension (1 X10⁸Cells) are poured into the mouse Ig-rabbit anti-mouse Ig coated Petri dishes and incubated at room temperature. Incubated for 40 minutes. The non-adherent cells are then gently aspirated and subsequently cultured. Washed in the ground. These cells were then used as a T cell-rich fraction. 85% purity Was achieved by flow cytometry (FACScan, GB, Oxford) Using Becton Dickinson Ltd.), anti-Thy 1.2 staining Evaluated by color. Normal mouse splenocytes were used as antigen presenting cells. This In these embodiments, the APC was converted from a cesium source (GB, Gravatomui, Gosport). Industries (Gravatom Industries) irradiated 1000 rad.Culture and proliferation assays   This was performed as described in Thompson et al., Supra. The medium used was 4 mM L-glutamine (Flow), 100 U / ml benzylpenicillin (GB, Leanford Glaxo Ltd.), 100 μg / ml streptomycin Sulfate (GB, Evans Medical Ltd. of Greenford), 5 × 10^-FiveM 2-mercaptoethanol (Sigma), 20 mM HEPES and Improvement of Eagle's medium supplemented with 0.5% fresh normal mouse serum (αMEM ) (Flow). These cultures provide various antigens at 92.5-10 μg / ml. 2 ml volume in a 24-well plate (Flow), with or without, 1.25X10 / ml⁶Individual purified spleen T-cells + 1.25 x 10⁶Including APC Was. Some cultures are also stored in round-bottom 96-well plates (Flow). The volume was 200 μl. All cultures are 5% CO_TwoAnd 95% air in a humid atmosphere3 Incubated at 7 ° C.   After the indicated incubation time, 100 μl of each of the 2 ml cultures Transfer the three pulls to a 96-well round bottom culture plate (Flow) 2mCi^ThreeH-thymidine (specific activity 70-85 Ci / mM; GB, Amersham Amersham Exposure for 6 hours by International Corporation (Amersham International Ltd.). Next The cells were then transferred to a multiple sample harvester (Norway, Lier) (Skatron AS) using glass fiber filter mat (GB, Medstone Fatman (Whatman Ltd.) and assembled in newly synthesized DNA. Crowded^ThreeH-thymidine is converted to LKB lane using known liquid scintillation procedures. Rackbeta counter (LKB-Walak, Uppsala, Sweden) lac) Ltd. Pharmacia). Stimulate these results Index (S.I. = test value cp divided by cpm for control without antigen) m) (FIG. 2). The positive stimulus is at most^ThreeH-thymidine Uptake was 30,000 counts.   From these results and specifically from those obtained from the hsp 65 protected group, group 6 It is evident that the polypeptide of the above results in the most significant stimulation of T cells.   This conclusion is based on the results obtained from the hsp 65 protected group in FIG. Gained by showing the most consistent increase in S.I. Was done. For example, assuming that the baseline is approximately S.I. = 3, Group 6 has six results. Five of them match, that is, exceed the value. The pools of the other groups Did not give such repeatable results.                                 Example 2   Inhibition of T cells from hsp65 protected mice in the presence of polypeptide antigen   Proliferation in b   According to the results of Example 1 above, preferred peptides in group 6 (hsp 261-271) The motifs (hsp 11-26 and hs Peptides without p 251-266) were also included in this test.   Groups of hsp 65 pre-immunized or PIA-protected animals were prepared according to the procedure described in Example 1. Prepared. Following essentially the same procedure, spleen cells from the mouse are cultured and then And the stimulation index was measured as described in Example 1. Conclusion The results are shown as a graph in FIG.   FIG. 3 shows three sets of results, each of which is (1) normal (2) protected animals (imm65) previously immunized with hsp65 and (3) pristane Presents data on the proliferation of T cells in cells from animals with arthritis (PIA) ing. For each set, the columns are as follows: (1) All hsp65 (2) Fragment hsp 251-266 (3) fragment hsp 261-271 according to the invention (4) Fragment hsp 11-16   From this experiment, data sets 2 and 3 (imm65 and PIA) were generated by all hsp65 Of a polypeptide of the invention essentially equivalent to that produced by the stimulation, It is clear that it has demonstrated its ability to induce T-cell proliferation. This used 2 shows that the peptide contains an immunodominant T-cell epitope. Such an immunodominance Epitopes are important for immunological purposes.                                 Example 3   Immunization with the microbial hsp65 fragment protects mice against PIAanimal   Unless otherwise stated, 4-8 week old male CBA / Igb mice as described in Example 1 Used.Immunization of animals   Groups of mice were immunized intraperitoneally 10 days prior to the Pristane challenge as follows: It has become epidemic.   50 μg of each peptide was administered as an emulsion in IFA. Pre-immunization of group 3 The polypeptide fragments used in the above were from UK, Chesir and Northwich. By Cambridge Research Biochemicals Created.Induction of arthritis by pristane   Next, 0.5 ml of pristane was intraperitoneally injected twice at 50-day intervals as described in Example 1. Arthritis was induced by intravenous injection. The animal is administered at various time points. The ankle joints were examined for the incidence of arthritis. The final incidence rate is Evaluation was made 200 days after injection of the stun. This arthritis causes the ankle joint to It was evaluated by measuring with a rotometer. In CBA / Igb mice, Joint size is 3.0-4.0 mm compared to a normal joint that is in the range of 2.5-2.8 mm Was in the range. However, this difference is easily distinguishable, and thus in most experiments Assessed the joints visually and by whether they suffered from arthritis (Tonp Thompson et al., Eur. J. Immunol., 1990, 20: 2479 and Barker Et al., Autoimmunity, 1992, 14:73).   The percentage of animals in each group affected by arthritis after a period of 200 days is shown in FIG. You. The peptide region corresponding to regions 261-271 was compared to total hsp65 in mice. To achieve improved protection against RA in the Columns:                     VVNKIRGTFKS (SEQ ID 108) Has proven to be effective in achieving advantageous effects.   The effect of hsp peptide fragment 261-271 on pristane-induced arthritis (of the present invention) Further evidence of the effect (including the motif) is shown in FIGS.   FIG. 5 evaluates the therapeutic efficacy of the peptides of the invention in treating PIA. The result of the experiment performed for this is shown. 50 μg of pepti as emulsion in IFA Was administered by intraperitoneal route (ip) to each mouse at 60 days of age. 50 days after this treatment Two pristane doses were given at intervals. Once on day 0 and once on day 50 Injected. Consider 60 days as the stage immediately before the onset / onset of PIA. Surprised In addition, the polypeptide 261-271 completely prevents the occurrence of PIA symptoms in all mice. It was found to prevent. Arthritis incidence (%) was assessed visually and histologically. Evaluation Both were evaluated.   FIG. 6 evaluates the preventive effect of the peptide according to the present invention in the prevention of PIA. The experimental results for the following are shown. In this case, the peptide will be available at 10 The drug was administered the day before (-10 days). Subsequently, two pristane injections are given at 50 day intervals, ie One injection on day 0 and one injection on day 50. These results indicate that the peptide of the present invention Has shown that pre-immunization with Cd significantly reduces the rate of arthritis.   Evidence for the ability of the polypeptides of the present invention to bind to MHC group II receptors Is shown in FIGS. 7 and 8.   In FIG. 7, 20 μg of Group II on a human EBV-transformed B-cell line Increased amount of binding to the binding of the biotin-treated peptide fragment hsp 261-271 9 shows the effect of adding “cold” viral hemagglutinin. FIG. 7 shows that biotin at a certain concentration Biotin-untreated ("cold") effects on the rate of binding by treated hsp 261-271 ) Viral hemagglutinin (which binds to group II receptors, but its identification is (Impossible) dose escalation effects. These results indicate that group II receptors Showing good binding capacity / affinity for the "cold" viral blood Agglutinin binds (%) by biotin-treated hsp 261-271 in a dose-dependent manner. Supported by data showing no inhibition.   FIG. 8 shows the dose of biotin-treated hsp 261-271 on SW or SF cell lines. Shows dependent binding. The SWB-cell line has a higher affinity for the peptide And the binding peak (about 60%) is seen at 50 μg peptide. Of the peptide At higher doses, binding rates decrease. SFB-cell lines are rarely used up to 50 μg. Does not bind to the peptide at all, after which the binding rate increases with the concentration of peptide used Increase. Interestingly, the data in FIGS. 7 and 8 show that at low concentrations, Viral hemagglutinin is actually the biotin-treated peptide against both cell lines. Enhance binding (eg, at 20 μg peptide concentration for the SW cell line Looking at the increase shown in FIG. 8, the binding is increased by 20 μg viral hemagglutinin, Looking at the SF cell line at a peptide concentration of 20 μg, the binding was 0 to 30 Increase by 5050 μg viral hemagglutinin.   7 and 8, the binding rate to the B-cell population was determined by extravidin (e Measured using xtrAvidin-FITC (Sigma) and FACS analysis.   FIG. 9 shows the site of mouse-derived T-cells in response to various stimulation protocols. 4 shows the results of an experiment for measuring the Cain profile.   The first two sets of data in FIG. 3 show pre-immunization with native hsp65 prior to pristane injection. And either the peptide hsp 261-271 (hsp (PEP)) or the natural hsp (hsp (hsp)) Cytokine profile of T-cells re-stimulated with APS while co-cultured with APS Represents a rule. The data presented in Sets 3 and 4 show that pristane was injected and then Stimulate with peptide 261-267 (IPP (pep)) or inject pristane and Stimulated by natural hsp (IPP (hsp)), while co-cultivating with APS Represents the protocol.   In each case, particularly interesting and significant data is represented by a second bar, Shows production of the Th2 anti-inflammatory cytokine IL-4. hs (hsp) p (pep) resulted in an almost 3-fold increase in IL-4, and IPP (pep) compared to IPP (hsp). It is important to note that this results in an approximately 2-fold increase in IL-4. each In some cases, measurement of the amount of cytokine released was performed by ELISA.   The role of microbial hsps in both inducing arthritis and defending against the disease is Applicants believe that it may be in the form of antigen presentation. Depends on this And T_HTwo cells are induced, spreading determinants lead to pristane-induced arthritis Or T_HTwo cells are triggered and lead to defense due to repertoire restriction It is. This will devise ways to prevent or treat arthritis and other autoimmune diseases. The possibility arises.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), OA (BF, BJ, CF, CG , CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AM, AT, AU, BB, BG, BR, BY, CA, C H, CN, CZ, DE, DK, EE, ES, FI, GB , GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG, M K, MN, MW, MX, NO, NZ, PL, PT, RO , RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, US, UZ, VN

Claims (1)

[Claims] 1. Sequence: VVNKIRG (SEQ ID 107) or its homologues or functional equivalents Or a polypeptide consisting of 9 to 21 amino acid residues, including polypeptides or analogs. 2. Polypeptide according to claim 1, consisting of 2.9 or 10 amino acid residues. Chid. 3. The polypeptide according to claim 1, consisting of 11 amino acid residues. 4. Sequence: VVNKIRGTFKS (SEQ ID 108) or its homolog or its functional equivalent 2. The polypeptide according to claim 1, comprising a product or analog. 5. Sequence: VVNKIRGTFKS (SEQ ID 108) or its homolog or its functional equivalent 4. The polypeptide according to claim 3, comprising a product or analog. 6. Claims 1 to 5 in combination with a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition comprising the polypeptide according to any one of the preceding items. 7. Use of the pharmaceutical composition according to claim 6 for preventing an autoimmune disease. for. 8. Use of the pharmaceutical composition according to claim 6 for treating an autoimmune disease. for. 9. A method for producing a polypeptide according to any one of claims 1 to 5. To fragment the microbial heat shock protein hsp65 and obtain the predetermined And isolating and / or purifying the polypeptide fragment. The above method. Ten. The method according to any one of claims 1 to 5, which uses a recombinant DNA technology. A method for producing a polypeptide, comprising a nucleotide encoding a predetermined polypeptide. Incorporating the nucleotide sequence into a suitable host using a vector system, the polypeptide And the step of isolating and / or purifying the polypeptide The above method, comprising: 11． A method for producing a polypeptide according to any one of claims 1 to 5. The amino acid or amino acid sequence is linked by chemical synthesis to The above method, comprising the step of producing a repeptide. 12． Claims 1 to 5 for the manufacture of a medicament for the prevention of auto-immune diseases. Use of the polypeptide according to any one of the claims. 13. The method according to claims 1 to 5, for producing a medicament for treating an auto-immune disease. Use of the polypeptide according to any one of the claims. 14. A patient suffering from an auto-immune disease is described in any one of claims 1 to 5. Or a pharmacological composition according to claim 6 in an effective amount thereof. And a method for preventing the disease. 15． A patient suffering from an auto-immune disease is described in any one of claims 1 to 5. Or a pharmacological composition according to claim 6 in an effective amount thereof. A method for treating the disease, comprising the step of administering the disease.