CA2251584A1 - Autoantigen and proteins structurally related thereto for use in immunotherapy of autoimmune diseases - Google Patents

Abstract

The present invention relates to the use of autoantigen HC gp-39, and proteins c omprising an amino acid sequence which exhibits at least 50 % homology with the amino acid sequence of HC gp-39, more p articular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDR-FLCTHIIYSFANISND (SEQ ID NO:1) in antigen-specific t reatment of articular cartilage destruction in autoimmune diseases in mammals to induce systemic tolerance of th e immune system. The autoantigen HC gp-39, and the arthritogenic proteins comprising an amino acid sequence which exhibits at l east 50 % homology with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDR-FLCTHIIYSFANISND (SEQ ID NO:1) are also suitable to induce arthritis in animals, preferably mice. The invention furthermore relates to pharmaceutical composition s comprising said autoantigen and/or said arthritogenic proteins, a diagnostic method for the detection of autoreactive T cells in a tes t sample and test kits to be used in said method.

Description

CA 022~1~84 1998-12-13 WO 97/40149 PCT/Er97101903 -I -AUTOANTIGEN AND PROTEINS STRUCTUR~LLY RELATED THERETO FOR USE IN
IMMUNOTHERAPY OF AUTOIMMUNE DISEASES

The invention relates to a novel autoantigen and proteins related thereto, their use in treatment of chronic destruction of articular cartilage in autoimmune ~iice~ses~ ph~rm~l~.e~ltical compositions comprising said ~--to~ntigen and/or proteins, a diagnostic method for the detection of aulo,cactive T cells in a test sample and test kits to be used in said method.
The immlm~ system is established on a principle of discrimination between foreign antigens (non-self antigens) and ~lto~nti~enS (self antigens, derived from the individuals own body) achieved by a build in tolerance against the autoantigens.
The immllne system protects individuals against foreign antigens and responds toexposure to a foreign antigen by activating specific cells such as T- and B lymphocytes and producing soluble factors like interleukins, antibodies and complement factors. The antigen to which the immune system responds is degraded by the antigen ,~lese~ g cells (APCs) and a fragment of the antigen is ~ . ssed on the cell surface associated with a major histocompatibility complex (MHC) class II glycoprotein. The MHC-glycoprotein-antigen-fragment complex is presented to a T cell which by virtue of its T cell receptor recognizes the antigen fragment conjointly with the MHC class II protein to which it is bound. The T cell becomes activated, i.e. proliferates and/or produces interleukins, resulting in the expansion of the activated lymphocytes directed to the antigen under attack (Grey et al., Sci. Am., ~1:38-46, 1989).
Self antigens are also continuously processed and presented as antigen fragments by the MHC glycoproteins to T cells (Jardetsky et al., Nature ~:326-329, 1991). Self recognition thus is intrinsic to the immune system. Under normal circ~-m~t~rlces the immune system is tolerant to self antigens and activation of the immune response by these self antigens is avoided.
When tolerance to self antigens is lost, the immune system becomes activated against one or more self antigens, resulting in the activation of autoreactive T cells and the production of autoantibodies. This phenomenon is referred to as ~uloi~ nity. As the immune response in general is destructive, i.e. meant to destroy the invasive foreign antigen, autoimmune responses can cause destruction of the body's own tissue.

The contribution of T cells to autoimrnune diseases has been established by several S studies. In mice, experimental autoirnmune encephalomyelitis (EAE) is mediated by a highly restricted group of T cells, linked by their specificity for a single epitope of myelin basic protein (MBP) complexed to an MHC class II molecule. In the Lewis rat, a species with high susceptibility to various autoirnmune fli~e~ces, disease has been shown to be mediated by T
cells. In h~lm~nc autoimmune diceaces are also thought to be associated with the development of 10 auto-aggressive T cells.

A destructive autoimmlme response has been implicated in various ~lice~ce~ such as rhellm~toid arthritis (R~), in which the integrity of articular cartilage is destroyed by a chronic infl~mm~tory process resulting from the presence of large numbers of aclivated lymphocytes 15 and MHC class II ~x~les~ g cells. The mere presence of cartilage appears neces~.y for s~ in~ the local infl~mm~tory response: it has been shown that cartilage degradation is associated with the activity of cartilage-responsive autoreactive T cells in RA (Sigall et al., Clin. Exp. Rheumat. 6:59, 1988; Glant et al., Biochem. Soc. Trans. 18:796, 1990; Burmester et al., Rheumatoid arthritis Smolen, Kalden, Maini (Eds) Springer-Verlag Berlin Heidelberg, 20 1992). Furthermore, removal of cartilage from RA patients by surgery was shown to reduce the infl~mm~tory process (G.S. Panayi et al, Clin. Exp. Rheumatol. 11 (suppl.8): S1-S8, 1993). The cartilage proteins are therefore considered to be target ~llto~ntigens which are competent of stimul~ting T cells. Activation of these autoreactive T cells leads to development of autoimmune ~li.ce~ce. However, the identification of the autoantigenic components that play a 25 role in the onset of rheumatoid arthritis has so far remained elusory.

The infl~mm~tory response resulting in the destruction of the cartilage can be treated by several drugs, such as for example steroid drugs. However, these drugs are oftenimmunosuppressive drugs that are nonspecific and have toxic side effects. The disadvantages of 30 nonspecific irnmuno~ plcssion makes this a highly unfavourable therapy.

CA 022~1~84 1998-12-13 The antigen-specific, nontoxic immuno~u~les~ion therapy provides a very attractive altemative for the nonspecific immunosuppression. This antigen-specific therapy involves the tre~tment of patients with the target autoantigen or antigens having an amino acid sequence which exhibits sequence homology with the amino acid sequence of the target ~ to~ntigen, or 5 synthetic T cell-reactive peptides derived from said autoantigen or said antigen. These synthetic peptides correspond to T cell epitopes of the a~lto~ntigen and can be used to induce specific T
cell tolerance both to themselves and to the a~lto~ntigen. Although it seems paradoxical to desensitize the immune system with the very same antigen responsible for acliv~illg the immune system, the controlled aclmin.ctration of the target (auto)antigen can be very effective in 10 desensitization of the immune system. Desensitization or immlmt)logical tolerance of the ilnl~luile system is based on the long-observed phenomenon that ~nim~l~ which have been fed or have inhaled an antigen or epitope are less capable of developing a systemic immlme response towards said antigen or epitope when said antigen or epitope is introduced via a systemic route.
To effectively use the tolerance therapy to treat the T cell mediated cartilage destruction, 15 there is a great need to identify the responsible ~llto~ntigen or antigens having an amino acid sequence which exhibits sequence homology with the amino acid sequence of the target ~ut-~ntigen~ to desensitize patients against the ~lto~ntigen that is activating the T cells responsible for the infl~mm~tQry process.

It is an object of the invention to provide the ~llto~ntigen~ and proteins having an amino acid sequence which exhibit sufficient homology with the amino acid sequence of the ~l~to~n1igen, said ~lto~ntigen and/or protein being suitable to induce specific T cell tolerance to the responsible cartilage antigen in patients ~urre,ing from T cell-mediated cartilage destruction.
It is a further object of the invention to provide arthritic animal testmodels which are suitable for use in screening for novel drugs to :iU~plCSS arthritic symptoms. It is another object of the invention to provide a method for detecting autoreactive T cells involved in the destruction of articular cartilage and test l~its to be used in said method.

It was surprisingly found that Human Cartilage glycoprotein 39 (herein after referred to as HC gp-39) is a target ~lto~ntigen in RA patients which activates specific T cells, thus c~ ing or me~ ting the infl~mm~tory process. The arthritogenic nature of HC gp-39 was subst~nti~te~l in the Balb/c mouse. A single, subcutaneous injection of said protein in Balb/c mice was able to initiate arthritic signs in the ~nim~lc. The course of the HC gp-39- induced disease was characterized by relapses occuring periodically in fore paws and/or hind paws and gradually developed from a mild arthritis into a more severe form. Also, a symmetrical 5 distribution of afflicted joints was observed which is, together with the observation of recurrent relapses and nodule formation, reminiscent of disease progression in arthritis, especially RA.
Even more surpri~ingly it was found that a~lmini~tration of HC gp-39 resulted inimmunological tolerance and, more importantly, in delayed and/or ~u~.cssed arthritic development.
It was furthermore surprisingly found that proteins comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39 are able to induce arthritis when injected to ~nim~l~ In particular proteins comrri~ing an amino acid sequence which exhibits at least 50% homology with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND (SEQ ID NO: 1 ) were able to induce arthritis in ~nim~l~, in the same way as described for HC gp-39.
Preferably, these arthritogenic proteins comprise an amino acid sequence which exhibits at least 70%, more preferably 80%, most ~rere~ably 90% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND (SEQ ID NO: 1).
The percentage of sequence homology of the arthritogenic ploleills accoldillg to the invention with the arnino acid sequnece of SEQ ID NO: 1 is understood to be measuered via generally used sequence comparison programmes such as FASTA (W.R. Pearson and D.J.
T.ipm~n, Proc. Natl. Acad. Sci. USA, 85, 2444-2448, 1988).
Suitable arthritogenic proteins according to the invention are for example pig heparine-binding 38kDa protein, bovine 39 kDa whey protein, human YKL-39 protein, murine breast regressing 39kDa protein (brp39), human oviduct-specific glycoprotein, murine oviduct-specific glycoprotein, hamster oviduct-specific glycoprotein, bovine oviduct-specific glycoprotein, human chitotriosidase precursor protein and murine secretory protein YM-l precursor. The arthritogenic proteins according to the invention are very suitable for inducing systemic tolerance of the immune system to homologous a~lto~ntigens and can be used to delay and/or ~UlJplGss arthritic development in m~mm~l~

~ . .

CA 022~1~84 1998-12-13 HC gp-39 is present in serum of both patients and healthy adults, although the serum concentration of the protein is about twice as much in patients as compared to healthy adults.
Furthermore, mRNA coding for HC gp-39 can be found in synovial specimens or cartilage 5 obtained from RA patients, whereas cartilage of healthy adults, obtained at surgery, does not contain a significant amount of said mRNA. When articular chondrocytes and synovial cells are cult~red, their major secretory product becomes HC gp-39 (Hakala et al., J. Biol. Chem., Vol.
268, 34:25803, 1993). The arthritogenic nature of HC gp-39 was neither described nor suggested in the Hakala et al publication, nor in any other publication.
Proteins of which the amino acid sequence exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particul;ar with the arnino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYS~ANISND (SEQ ID NO: 1) have been described. The identification of pig heparine-binding 38kDa protein is described in Shackelton et al. (1995), J.Biol.Chem. Vol. 270, No. 22, 13076-13083, however no function of the protein was identified. The isolation and characterization of bovine 39 kDa whey protein is described in J.J. Rejman et al. (1988), Biochem.Biophys.Res.Comm. Vol. 150, No. 1, 329-334.
Murine breast regressing 39kDa protein (brp39) is described in Morrison and Leder, 1994, Oncogene 9, 3417. Cloning of the cDNA encoding human oviduct-specific glycoprotein and the coll~,sponding amino acid sequence is described in Arias et al. (1994), Biology of Reproduction 51, 6~5-694. Other m~mm~ n oviduct-specific glycoproteins such as murine- and h~m~tP.r oviduct-specific glycoprotein are disclosed in JP-A-07107979, Kinosei Peptide Kenkyusho KK.
The purification and molecular cloning of the bovine oviduct-specific glycoprotein is described in Y. Sendai et al, 1994, Biol. of Reprod. 50, 927-934. Human chitotriosidase prec~ or protein is secreted by activated human marcophages and the cloning of the corresponding cDNA and amino acid sequence is described in Boot et al. (1995), J.Biol.Chem. Vol. 270, No. 44, 26252-~ 26256. The amino acid sequence of murine secretory protein YM-l precursor was submitted to EMBL Data Library, June 1992, Accession No. M94584 by Chang et al., unpublished. The amino acid sequence of human chondrocyte protein YKL-39 is described in Hu et al. (1996), J.Biol.Chem. Vol 271, No. 32, 19415-19420. None of the afore-mentioned publications however hint or suggest towards the arthritogenic nature of the proteins according to the invention nor to the fact that these proteins can be used as a medical substance in a therapy to -. . . , ~ . .

CA 022~1~84 1998-12-13 induce specific T-cell tolerance to HC gp-39 in m~mm~l~, more specifically man, suffering from T-cell mediated cartilage destruction, such as for example arthritis, more specifically rheumatoid arthritis.

Thus, according to the invention HC gp-39, and proteins comprising an amino acidsequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQY~EGDGSCFPDAL-DRFLCTHIIYSFANISND (SEQ ID NO: 1) are very suitable for use in a therapy to induce specific T-cell tolerance to HC gp-39 in patients suffering from T-cell mediated cartilage destruction, such as for example arthritis, more specifically rhellm~toid arthritis. Also within the scope of the invention are fragments of HC gp-39 and the arthritogenic proteins according to the invention, which are able to induce T-cell specific tolerance to the HC gp-39 anto~ntigen in the cartilage under attack.
WO 95/01995 and WO 95/02188 describe the diagnostic use of HC gp-39 as a marker for RA, however the arthritogenic nature of HC gp-39 is neither disclosed nor suggested.
Nowhere do they hint or suggest towards the use of HC gp-39, or fr~rnPnt~ thereof or T-cell reactive peptides according to the present invention in the antigen specific therapy to induce T-cell specific tolerance to the HC gp-39 in the cartilage under attack.

HC gp-39, arthritogenic proteins comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSC-FPDALDRFLCTHIIYSFANISND
(SEQ ID NO: 1), and fragments derived from said arthritogenic proteins according to the invention can be prepared with the aid of recombinant DNA techniques. For this purpose, a nucleic acid sequence which codes for HC gp-39 or a protein comprising an arnino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDR-FLCTHIIYSFANISND (SEQ ID NO: 1) or a fragment derived from the proteins according to the invention or a multimer of said fragment is inserted into an e~p~ession vector. Suitable expression vectors are, amongst others, plasmids, cosmids, viruses and YAC's (Yeast Artificial CA 022~1~84 1998-12-13 Chromosomes) which comprise the necessary control regions for replication and expression.
The expression vector can be brought to ~ ssion in a host cell. Suitable host cells are, for instance, bacteria, yeast cells and m~mm~ n cells. Such techniques are well kno~vn in the art (Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory S Press, Cold Spring Harbor, 1989).
Using these recombinant DNA techniques bovine whey protein, the sequence of which has been disclosed herein, can be pl~a.ed in a host cell.
Thus, the present invention also provides for isolated cDNA encoding the bovine whey protein as indicated by SEQ ID NO: 3 as well as the bovine whey protein according to SEQ ID
10 NO: 2. It will be clear that also fragments can be prepared once given the teaç~ing~ of the present invention. The term "fragment" refers to any sequence of amino acids that is part of the protein, having common elements of origin, structure and mech~ni~m of action that are within the scope of the present invention.

Suitable fragments derived from the arthritogenic proteins acco~ g to the invention can also be ~r~ed by means of one of the lcnown organic chemical methods for peptide synthe~i~ The organic chemical methods for peptide synthesis are considered to include the coupling of the required amino acids by means of a con-len~tion reaction, either in homogeneous phase or with the aid of a so-called solid phase. The most common methods for 20 the above cond~n~tion reactions are: the carbodiimide method, the azide method, the mixed anhydride method and the method using activated esters, such as described in The Peptides, Analysis, Synthçsj~, Biology Vol. 1-3 (Ed. Gross, E. and Meienhofer, J.) 1979, 1980, 1981 (Ac~ernic Press, Inc.).
Preparation of suitable fragments of above-mentioned HC gp-39 and arthritogenic 25 proteins according to the invention using the "solid phase" is for instance described in J. Amer.
Chem. Soc. 85:2149 (1963) and Int. J. Peptide Protein Res. 35:161-214 (1990).
A particulary suitable solid phase is, for example, the p-alkoxybenzyl alcohol resin (4-hydroxy-methyl-phenoxy-methyl-copolystrene-1% divinylbenzene resin), described by Wang (1974) J. Arn. Chem. Soc. 2~:1328. After synthesis the peptides can be split from this solid 30 phase under mild conditions.

CA 022~1~84 1998-12-13 After synthesis of the desired amino acid sequence, detaching of the peptide from the resin follows, for example, with trifluoroacetic acid, co.~ g scavengers, for example triisopropyl silane, anisole or ethanedithiol,thioanisol.
The reactive groups which may not participate in the con~1~n~tion reaction are, as stated 5 effectively protected by groups which can be removed agains very easily by hydrolisis with the aid of acid, base or reduction. A more extensive account of possible protecting groups can be found in The Peptides, Analysis, Synthesis, Biology, Vol. I - 9 (Eds. Gross, Udenfriend and Meienhofer) 1979 - 1987 (~c~lemic Press, Inc.). The protective groups can be split off by various conventional methods, depending on the nature of the particular group, for example 10 with the aid of trifluoroacetic acid or by mild reduction, for example with hydrogen and a catalyst, such as palladium, or with HBr in glacial acetic acid.

Although it seems paradoxical to desensitize the immune system with the very same 15 antigen les~ollsible for activating the immlme system, this des~x;L;7~l;0n is based on the long-observed phenomenon that ~nim~l~ which have been fed or have inhaled an antigen or epitope are less capable of developping a systemic immune ~I,onse towards said antigen or epitope when said antigen or epitope is introduced via a systemic route. Hence, controlled a~mini~tration of HC gp-39, and/or proteins comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND
(SEQ ID NO: 1) can be effective in desen~iti7~tion of the immlme system. Fragm~nt~ of the proteins according to the invention that are capable to desensitize patients against HC gp-39 are also within the scope of the invention.
According to the invention, patients in which the cartilage is under attack of autoresponsive T cells can be treated with a pharmaceutical composition comprising HC gp-39, one or more proteins comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRF~CTHIIYSFANISND (SEQ ID NO: 1) or one or more fragrnents derived from a protein according to the invention and a ph~ celltical . . .

CA 022~1~84 1998-12-13 _g acceptable carrier in order to make the specific autoreactive T cells of these patients tolerant to the HC gp-39 in the cartilage under attack and to (liminish the infl~mm~tory response.
Suitable proteins to be used in a pharmaceutical composition according to the invention are proteins comprising an amino acid sequence which exhibits at least 70%, preferably 80%, 5 more preferably 90% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCT-HIIYSFANISND (SEQ ID NO: 1).
Very suitable proteins to be used in a ph~rrn~ceutical composition according to the invention are for example pig heparine-binding 38kDa protein, bovine 39 kDa whey protein, 10 murine breast regressing 39 kDa protein (brp39), murine oviduct-specific glycoprotein, h~rnster oviduct-specific glycoprotein, bovine oviduct-specific glycoprotein, human YKL-39, human oviduct-specific glycoprotein, human chitotriosidase precursos protein and murine secretory proteinYM-l precursor.
Also very suitable to be used in a ph~rrn~r.elltical composition according to the 15 invention are DNA (~ ression)vectors comprising DNA which ~ncotles for HC gp-39 or a protein comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequenceYKLVCYYTSWS-QYREGDGSCFPDALDRFLCTHIIYSFANISND (SEQ ID NO: 1) or one or more fragrnents derived from the proteins according to the invention. Upon delivery the 20 DNA (e~les~ion)vector can provide by ~ es~ion a level of the l~cumbinant HC gp-39 or protein comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the arnino acid sequence YKLVCYYTSWSQYREGDGSCFP-DALDRFLCTHIIYSFANISND (SEQ ID NO: I ) or fragments according to the invention which is similar to the level which would be achieved by 25 direct ~rlministration of a ph~ ceutical composition comprising the HC gp-39 protein or peptides.
The autoantigen HC gp-39, arthritogenic proteins comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the arnino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDR-30 FLCTHIIYSFANISND (SEQ ID NO: 1) and fragments derived from said arthritogenic proteinsaccording to the invention have the advantage that they have a specific tolerizing effect on the . .

CA 022~1~84 1998-12-13 WO 97/40149 PCT/EP97tO1903 autoreactive T cells thus leaving the other components of the immune system intact as compared to the nonspecific ~u~ cs~ive effect of the immunosuppressive steroid drugs.
Treatment with the autoantigen or the proteins comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular 5 with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND (SEQ ID NO: 1) or fragments according to the invention will be safe and no toxic side effects will occur.
Tolerance can be attained by atlmini~tP.ring high or low doses of the ~utoAntigen or proteins according to the invention. The amount of ~llto~ntigen or protein will depend on the 10 route of A~lmini~tration, the time of ~-lministration~ the age of the patient as well as general health conditions and diet.
In general, a dosage of 0.01 to 1000 ',lg of protein per kg body weight, preferably 0.5 to 500 ',lg, more preferably 0.1 to 100 ~lg of protein can be used.
Ph~rm~e~ltical acceptable carriers are well known to those skilled in the art and include, 15 for example, sterile salin, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil and water. Other carriers may be, for example MHC class ~I
molecules, if desired embedded in liposomes.
In addition the pharmaceutical composition according to the invention may comprise one or more adjuvants. Suitable adjuvants include, amongst others, ~luminium hydroxide, 20 aluminium phosphate, amphigen, tocophenols, monophosphenyl lipid A, ~ lyl dipeptide and saponins such as Quill A. The amount of adjuvant depends on the nature of the adjuvant itself.
Furthermore the pharmaceutical composition according to the invention may comprise one or more stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, 25 sucrosedextrin and glucose, proteins such as albumin or casein, and buffers like aL~aline phosphates.
Suitable ~lmini.~tration routes are intrarnuscular injections, subcutaneous injections, intravenous injections or intraperitoneal injections, oral and intranasal A~mini~tration~ Oral and intranasal A-lrnini.ctration are preferred ~imini.~tration routes.

CA 022~1~84 1998-12-13 Due to its arthritogenic nature, HC gp-39 as well as the proteins comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDR-FLCTHIIYSFANISND (SEQ ID NO: l) can be 5 used to induce clinical arthritis in ~nim~l~ Upon ~lmini~tration of small amounts of HC gp-39 or one or more proteins comprising an amino acid sequence which exhibits at least 50%
homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGD-GSCFPDALDRFLCTHIIYSFANISND (SEQ ID NO:
l) or one or more frgaments of the proteins according to the invention, arthritic signs will lO develop in said ~nim~1~ res1~1ting in a disease pattern rçmini~cent of disease progression in arthritis, especially rhs11m~toid arthritis. When Balb/c mice were injected subcutaneously with HC gp-39 protein or a protein comprising an amino acid sequence which exhibits at least 50%
homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND (SEQ ID NO:
l), the ~nim~1.c developed arthritic signs. The course of the HC gp-39-inrl11( ed disease as well as the disease in-ln~ed by the protein comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND (SEQ ID
NO: l) was characterized by relapses occuring periodically in fore paws and/or hind paws and the gradually development from a mild arthritis into a more severe form. Also, a symmetrical distribution of afflicted joints was observed which is, together with the observation of ~e~,ullcllt relapses and nodule formation, reminiscent of disease progression in arthritis, especially RA.
Thus, these afflicted 2nim~1~ provide an adequate animal model to study the mec~ni~m underlying the initiation and progression of arthritic development. Additionally, said afflicted ~nim~1~ can be used to search for new drugs to treat arthritis and to study the effect of these drugs upon the arthritic development. Preferably mice are used as animal model for arthritis, especially rheumatoid athritis.
To induce arthritis in said ~nim~1~, suitable amounts of HC gp-39 or one or more of the proteins according to the invention have to be ~tlmini~tered Suitable amounts are O.l-lO00 ~,lg, preferably l - l 00 ~lg, more preferably l 0-50 llg per kg body weight. The amount of HC gp-39 or proteins comprising an amino acid sequence which exhibits at least 50% homology with the CA 022~l~84 l998- l2- l3 WO g7/40149 PCT/EP97/01903 amino acid sequence of HC gp-39, more in particular with the amino acid sequenceYKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND (SEQ ID NO: 1) or fragments thereof will depend on the route of adminstration, time of a~rninistration and the type of animal used. Suitable ~ nin~tration routes are the same as described before. To induce the S effect of arthritis inducton, the HC gp-39 protein, or proteins comprising an arnino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLC-THIIYSFANISND (SEQ ID NO: 1) or peptides according to the invention may comprise one or more stabilizers or adjuvants as 10 described before.

HC gp-39, the proteins coln~lisil~g an atnino acid sequence which exhibits at least 50%
homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND (SEQ ID NO: I) 15 or fragments thereof according to the invention are also very suitable for use in a diagnostic method to detect the presence of activated autoreactive T cells involved in the chronic infl~mm~tion of the articular cartilage.
The diagnostic method accordillg to the invention comprises the following steps:a) isolation of the peripheral blood mononuclear cells (PBMC) from a blood sample of 20 an individual, b) culture said PBMC under suitable conditions, c) incubation of said PBMC culture in the presence of the ~uto~ti~en or proteinsaccording to the invention and fragments thereof, and d) detection of a response of T cells, for exarnple a proliferative response, indicating the 25 presence of activated autoreactive T cells in the individual.
In case of detection of a response by measuring the proliferative response of the autoreactive T cells, the incorporation of a radioisotope such as for example 3H-thymidine is a measure for the proliferation. A response of the autoreactive T cells present in the PBMC can also be detected by measuring the cytokine release with cytokine-specific ELISA, or the~0 cytotoxicity with 5~Chromium release. Another detection method is the measulelllent of ession of activation markers by FACS analysis, for example of Il-2R. A diagnostic CA 022~1~84 1998-12-13 composition comprising one or more of the peptides according to the invention and a suitable detecting agent thus forrns part of the invention. Depending on the type of dection, the detection agent can be a radioisotope, an enzyme, or antibodies specific for cell surface or activation markers.
S Also within the scope of the invention are test kits which comprise one or more peptides according to the invention. These test kits are suitable for use in a diagnostic method according to the invention.
Thus, the present invention provides for a method to detect whether ~ltoag~,~ssi~e T
cells reactive towards HC gp-39 are present in patients suffering from T-cell mediated cartilage destruction such as for example arthritis, in particular rh~llm~toid arthritis. If HC gp-39-specific T cells are present, tolerization of these T cells with a ph~rm~ceutical compostion comprising HC gp-39 or one or more arthritogenic proteins comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence of HC gp-39, more in particular with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYS-FANISND (SEQ ID NO: 1) or peptides according to the present invention or combinations thereof can delay or su~ ess arthritis development.

The following examples are illustrative for the invention and should in no way be interpreted as limiting the scope of the invention.
LEGENDS TO THE FIGI~RES

Figure 1: Initiation and progression of arthritis in HC gp-39 tolerized and non-tolerized Balb/c mice. AS = total arthritic score of the afflicted ~tlim~l~ per day following sensitization. N
= number of afflicted ~nim~l~ per day following sensitization.

Figure 2: Initiation and progression of arthritis in Balb/c mice upon a~lmini~tration of 0.2 llg, 1 ~,lg, 5 ,ug and 25 ~lg bovine 39 kDa whey protein respectively. Initiation/progression is presented as accumulative score (total arthritic score of afflicted ~nim~l~) per day following sensitization.

~ . . ~

F.THODS
Purification of EIC gp-39 from the MG63 osteos~rcoma cell line S MG63 cells (human osteosarcoma ATCC CRL 1427) were cultured in cell factories in DMEM/HAM's F12 serum free medium. HC gp-39 was purified from the culture supernatant by heparin affinty chromatography folllowed by super dex 75 chromatography. Purity was checked by SDS-PAGE. In addition, N-t~nnin~l amino acid sequencing confirmed that the purified protein was identical to the protein described by Hakala et al.
Arthritogenicity of HC gp-39 in Balb/c mice 10 or 50 ~lg of purified HC gp-39 in a 100 Ill volume PBS (0.5 M NaCI, 0.01 M sodium phosphate buffer, pH 7.5) mixed 1:1 in incomplete Freunds adjuvant (IFA) was injected subcutaneously in the chest region in 2 x 4 female Balb/c mice (Harlan CPB, Zeist, The Netherlands) whereas 4 controls were injected with PBS (1:1 in IFA). Mice were eY~mined daily for clinical signs of arthritis. Severity of arthritis was ~sessed by scoring each paw from 0-3 (according to the article by Glant et al). In short, score 0 = no changes, score 1 = erythema and swelling, score 2 = swelling and a~pe~al~ce of defor nities, score 3 = immobility due to loss of flexion and extension.
Tolerance induction by intranasal administration of HC g~39 Twenty eight llg of protein was ~-lministered intranasally (2 x 10 ~11) to 10 female Balb/c mice (anesthetized lightly with Enflurance) using a PT45 micro conduit and a Hamilton syringe. Antigen was a-lminiitered on day -15, -10 and -5 prior to arthritis induction. Controls (n = 10) were submitted to the same procedure but recieved the vehicle (PBS) only (Table 1).
Imrnunological tolerance was evaluated by measuring delayed type hypersensitivity (DTH) responses following sensitization as described above on day 0, using 10 llg of protein.
Sensitization on day 0 was followed by an injection of 10 ~lg HC gp-39 in 50 ~,lg volume in the left hind footpad on day 8 (challenge). DTH reactions were measured as the increase in footpad ([swelling left (mrn x 10-3) -swelling right (mm x 10~3)]/swelling right (rnm x 10-3)) x 100%.

WO 97t40149 PCTIEP97101903 The footpad swelling was measured using an inhouse designed !umeter at 0, 24 and 48 hr after challenge.
Tolerance to arthritis induction in these same mice was then further monitored until day 31 following sensitization and mice were examined daily for clinical signs. Severity of arthritis 5 was assessed as mentioned above.

Table 1. Tolerization scheme.
DAY HC gp-39 tolerized non-tolerized -15 28 ~,lg HC gp-39 intra nasal PBS
-10 28 llg HC gp-39 intra nasal PBS
-5 28 ~lg HC gp-39 intra nasal PBS

O 10 llg HC gp-39 subcutaneous 10 ~Ig HC gp-39 sub~ neQus 8 ro ~lg HC gp-39 footpad 10 ',lg HC gp-39 footpad 9 24 hour DTH 24 hour DTH
48 hour DTH 48 hour DTH

0-31 score arthritic signs score arthritic signs DTH = delayed type h~,c,~t,.~ilivity. PBS = O.5 MNaCI, O.01 M.sodium phospha~e buffer, pH 7.5 E~F.~U~ TS
~thritogenicity of HC gp-39 Following one injection of 50 ~ug HC gp-39 mixed with IFA, all mice gradually developed a severe arthritis (Table 2). Signs of arthritis were observed first at day 15-20 after sen.~iti7.~tion, in the fore paws of 3 out of 4 ~nim~l~. The mouse that did not show any signs in 15 the fore paws developed arthritis in the hind paws by day 34 upon sensitization. The course of HC gp-39-induced disease was characterized by relapses occuring periodically in fore paws or hind paws and gradually develloped from mild arthritis into a more severe arthritis (disease progression was followed for 62 days). Very often (>50%) a symmetrical distribution of afflicted joints was observed, me~ning that both fore paws or both hind paws showed arthritic 20 signs at the same time.

, . " .. . ... ... .... . . .

Arthritis induction was similarly achieved using 10 ~lg instead of 50 ~g HC gp-39 mixed with IFA. Arthritic scores, however, were somewhat lower (data not shown). Three out of 4 mice developed a severe arthritis. One mouse showed only mild signs during the duration of the experiment. All through the length of the experiment control mice showed no signs of 5 arthritis.
In aggregate, both 10 and 50 lug of protein were sufficient to induce a progressive arthritis in Balb/c mice. The chronic nature of arthritis induction by HC gp-39, characterized by reculTent relapses in addition to symmetrical affliction of joints is remini.cce~t of disease progression in rhellm~toid arthritis (RA).
Table 2. Initiation and progression of arthritis in HC ~p-39 sensitized Balb/c mice.
sensitization (n = 4)arthritis onset (day) arthritis signs FP HP none mild severe PBS contr. - - 4 0 0 10 ~g HC gp-39 -, 13, 15, 57 32, 34, 36, 43 0 1 3 50~gHC gp-39 13, 15,15, 48 29, 34, 34, 53 0 0 4 FP = fore paw. HP = hind paw. contr. = controls. Arthritis signs: none: score = O, mild: score per animal does not exceed 2, severe: score per animal is > 3.

Immunological tolerance measured in the DTH assay Control mice injected with HC gp-39 at day 0 showed a strong, antigen-specific DTH
response, which suggests that a cellular immune response to HC gp-39 was elicited upon sensitization (Table 3). Intranasal ~t1mini~tration of HC gp-39, however, completely abrogated DTH responses upon challenge with the ~-to~ntigen, thereby showing that the HC gp-39-20 specific T-cells were indeed tolerized.
Notably, 4 out of 10 ~nim~l~ of the non-tolerized group developed ar~ritis in the ankle adjacent to the site of challenge. In contrast, the tolerized group did not develop an arthritis in the joints neighbouring the challenged site, thereby suggesting that immlmological tolerance to HC gp-39 results in protection against arthritis development.

. .

Table 3. DTH responses to HC gp-39 following tolerization by nasal administration.
teatment mean % swelling arthritis ankle O hr 24 hr 48 hr controls (n = 10) -1.3 31.4 38.6 4/10 tolc ~e.l(n=9) -0.05 3.7 1.5 0/9 Tolerance to arthritis induction or pro6. e~ on S The HC gp-39 tolerized and the non-tolerized Balb/c mice were then filrther monitored for initiation and progression of arthritis.
In all mice of the control (non-tolerized) group, disease was initi~tecl upon sensitization with HC gp-39 (Table 4). Seven mice gradually developed a severe arthrtis whereas three mice showed only mild signs (highest score 2). In contrast, five mice of the HC gp-39-tolerized group were protected against disease development during the course of the experiment.
Furthermore, two ~nim~l~ of the tolerized group showed only mild signs for brief periods of time. Three ~nim~l~ developed a more severe arthritis with scores of 4.
Int~ Lingly, in tolerized ~nim~l~, arthritis onset was delayed in both hind and fore paws by a minimllm of 7-9 days respectively (Figure 1). Although fewer ~nim~l~ were affected in the tolerized group, the arthritic score per animal of the fore paws was comparabe to the arthritic score in the non-tolerized ~nim~l~. The arthritic score per animal of the hind paws however, was somewhat lower in the tolerized ~nim~l~ (Figure 1).

Table 4. Initiation and progression of arthritis in HC gp-39-tolerized and non-tolerized Balb/c mice.
animals arthritis onset (day) arthritis signs none mild severe controls (n = 10) 13-1~ 0 3 7 tolerized (n = 10) 23-24 5 2 3 Onset arrhritis: first signs appear - hlghest number of animals affected. Arthritis signs: no signs. score = 0, mild.
score per animal does not exceed 2, severe. score per animal is > 4.

WO 97/40149 PCT~EP97/01903 -18-The experiments above demonstrate the arthritogenic nature of HC gp-39 in Balb/cmice. The course of HC gp-39-in(l11ce(1 disease was characterized by relapses occuring periodically in fore paws and/or hind paws and gradually developed from a mild arthritis into a 5 more severe form. Also, a symmetrical distribution of afflicted joints was observed which is together with the observation of le~;ulr~lll relapses, r~mini~cent of disease progression in rh~ m~t(~id arthritis. The fierce arthritogenic nature of HC gp-39 was illustrated by a single, subcutaneous injection of 10 or 50 llg of protein which initiated arthritic signs in all ~nim~1~
That HC gp-39 specific T cells are indeed elicited in response to HC gp-39 sensitization was lO shown by induction of HC gp-39-specific DTH responses. These data were further confirmed by the demonstration of HC gp-39-specific jn vitro proliferative responses in ~n;m~l~
immunized in the footpad with HC gp-39 (data not shown). Importantly, non-tolerized ~nim~lc developed arthritis in the ankle neighbouring the injection site, thereby indeed suggesting an involvement of HC gp-39-specific T cells in arthritis induction.
Intranasal a-lmini~tration of peptide antigen has been used to induce antigen-specific lI.C tolerance. The experiments showed that h~ asal a~lmini~tration of HC gp-39 leads to immunological non-responsiveness. DTH responses following s~n~iti7~tion were completely abrogated in HC gp-39-tolerized mice whereas control mice showed an antigen-specific swelling. These observations indicate that a~lmin~tration of HC gp-39 leads to peripheral~0 immune tolerance.
In non-tolerized ~nim~1~ DTH responses were accompanied by arthritis in the ankle (adjacent to the challenged site) in four out of ten mice. In contrast, the ankles of HC gp-39-tolerized ~nim~1~ were indeed fully protected, thereby suggesting that autoreactive T-cells have been effectively silenced. The notion that tolerization with HC gp-39 protects against disease 25 developement was taken further by the observation that 5 out of lO ~nim~1~ in the tolerized group were entirely protected throughout the length of the experiment. Although the other five anim~1~ in the group did eventually develop clinical signs, the onset of arthritis was considerably delayed. Hence it can be concluded that HC gp-39-specific T cells are involved in the arthritogenic process and more importantly, that by tolerization of these T cells with a 30 pharmaceutical composition according to the present invention arthritis development can be delayed or ~upplessed.

. .

FXAMPl,F 2 M~THQDS

Arthritogenicity of Bovine 39 kDa whey protein in Balb/c mice 0.2, 1, 5 or 25 ~g of purified Bovine 39 kDa whey protein in a 100 ~11 volume (0.5 M
NaCl, O.OlM sodium phosphate buffer, pH 7.5) mixed 1:1 in incomplete Freunds adjuvant (IFA) was injected subcutaneously in the chest region in 4 x 10 female Balb/c mice (Charles River, Sulzfeld, Germany) whereas 10 controls were injected with PBS (1:1 in IFA). Mice were eY~mined every other day for clinical sigrls of arthritis. Severity of arthritis was ~sçssed by 10 scoring each paw from 0-3 (according to the article by Glant et al). In short, score 0 = no changes, score 1 = erythema and/or mild swelling, score 2 = severe swelling and/or appearance of deformities, score 3 = immobility due to loss of flexion and extension.

Cloning of the bovine whey gp39 cDNA
15 A Cow m~mm~ry gland Lambda Bluemid(-) cDNA library (oligo dT and random-primed, custom made by Clontech Laboratories Inc.) was amplified using the XLl Blue MRF strain as a host and library plating and preparation of filter replicas were performed acco.dil,g to the m~nllf~ctmers instructions. Filters were hybridised with a PvuII fragment from HC gp-39 cDNA after 32p labelling using an oligolabelling kit (Pharmacia). Positive plaques were 20 amplified, rescreened and insert DNA of the co~re~ollding phages were sequenced using a Ther nosequenase kit (Amersham). The sequence is indicated in SEQ ID NO: 3. At nucleotide position 954 one clone contained a C whereas another contained a T.

25 ~F.~U~ TS

Arthritogenicity of Bovine 39 kI)a whey protein Following one injection of 25 llg Bovine 39 kDa whey protein mixed with IFA, all mice gradually developed arthritis (Table 5). Signs of arthritis were observed first at 8-20 days after sensitization in fore- or hind paws. The course of Bovine 39 kDa whey protein-in~ ced arthritis 30 was characterized by relapses occurring periodically in hind paws and gradually developed from ... ... , .. ..... . ~

CA 022~l~84 l998- l2- l3 mild arthritis into a more severe arthritis (disease progression was followed for 70 days). Very often a symmetrical distribution of afflicted joints was observed, meaning that both fore paws or both hind paws showed arthritic signs at the same time.
Arthritis induction was similarly achieved using 5, 1 or 0.2 instead of 25 llg Bovine 39 5 kDa whey protein mixed with IFA.
Mice induced with either I or 0.2 llg Bovine 39 kDa whey protein developed a more mild form of arthritis: 7 and 6 mice respectively developed a mild arthritis, whereas only 3 and 4 mice developed severe arthritis.
At the end of the experiment, cumulative scores per scoring day for each group of mice 10were calculated (Figure 2). Following immuni7~tion with 25~1g Bovine 39 kDa whey protein, a maximum score of 27 (25 ~lg) was reached after 30 days, followed by distinct relapses. When lower dosages of Bovine 39 kDa whey protein were used for immunization, ~nim~l~ showed a somewhat lower arthritis score compared with mice induced with the 2511g dose. Although lower scores were achieved, a relapsing pattern of arthritis was found in all groups.
15In control, PBS treated mice sometimes a slight and transient swelling of hind paws was observed. This phenomenon was also observed in naive, non-injected ~nim~l~ This mean cumulative swelling per scoring day for these groups of mice had a mean value of 3.4 and was considered as biological background variation.
In conclusion, Bovine 39 kDa whey protein (both 25, 5, 1 and 0.2 llg) is capable to 20 induce a progressive arthritis in female Balb/c mice. The chronic nature of arthritis induction by Bovine 39 kDa whey protein, characterized by recurrent relapses in addition to syrnmetrical affliction of joints, is reminiscent of disease progression in rheumatoid arthritis (RA).

WO 97/40149 PCT~EP97/01903 Table 5. Initiation and progression of arthritis in Bovine 39 kDa whey protein sensitized Balb/c mice.
sensitization (n=10) arthritis onset (day) arthritis signs FP HP none mild severe 0.2 ~g B 39 kl~a whey -,-,-,-,-, 8,14,14,14,18, 0 6 4 protein ,,,14,18 18,18,18,30,54 1 ~gB 39 kl~awheyprotein -,-.-,-,-, 8,8,8,8,8, 0 7 3 8,12,12,16,18 8,14,16,18,18 5 ~g B 39 kl)a whey protein -,-,-,-,-, 6,6,8,10,10 0 0 10 8,10,10,12,14 16,16,18,20,30 25 ~g B 39 kDa whey -,-,-,-,-, 8,8,10,12,12 0 1 9 protein ,,,-,12 14,14,18,18,20 FP = fore paw, HP = hind paw. Arthritis signs: none. score = O, mild: score per ammal does not exceed 2, severe:
score per animal > 3.
s ..... . .. . ..

CA 0225l584 l998-l2-l3 WO97/40149 PCT~P97/01903 SEQUENCE LISTING

(l) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: AKZO NOBEL N.V.
(B) STREET: Velperweg 76 (C) CITY: Arnhem (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP): 6824 BM
(G) TELEPHONE: 0412 666376 (H) TELEFAX: 0412 650592 (I) TELEX: 37503 akpha nl (ii) TITLE OF INVENTION: Novel peptides derived from autoantigen for use in immunotherapy of autoimmune diseases (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM:
tA) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.0, Version #l.30 (EPO) (2) INFORMATION FOR SEQ ID NO: l:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:

Tyr Lys Leu Val Cys Tyr Tyr Thr Ser Trp Ser Gln Tyr Arg Glu Gly l 5 l0 15 .

CA 022~1~84 1998-12-13 WO97/40149 ~CT~P97/01903 Asp Gly Ser Cys Phe Pro Asp Ala Leu A5p Arg Phe Leu Cys Thr His Ile Ile Tyr Ser Phe Ala Asn Ile Ser Asn Asp (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 383 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Gly Leu Arg Ala Ala Gln Thr Gly Phe Val Val Leu Val Leu Leu l 5 l0 15 Gln Ser Cys Ala Ala Tyr Lys Leu Ile Cys Tyr Tyr Thr Ser Trp Ser Gln Tyr Arg Glu Gly Asp Gly Ser Cys Phe Pro Asp Ala Ile Asp Pro Phe Leu Cys Thr His Val Ile Tyr Ser Phe Ala Asn Ile Ser Asn Asn Glu Ile Asp Thr Trp Glu Trp Asn Asp Val Thr Leu Tyr Asp Thr Leu Asn Thr Leu Lys Asn Arg Asn Pro Asn heu Lys Thr Leu Leu Ser Val Gly Gly Trp Asn Phe Gly Ser Gln Arg Phe Ser Lys Ile Ala Ser Lys l00 105 ll0 CA 022~1~84 1998-12-13 WO97/40149 PCT~P97/01903 Thr Arg Ser Arg Arg Thr Phe Ile Lys Ser Val Pro Pro Phe Leu Arg Thr His Gly Phe Asp Gly Leu Asp Leu Ala Trp Leu Tyr Pro Gly Trp Arg Asp Lys Arg His Leu Thr Thr Leu Val Lys Glu Met Lys Ala Glu Phe Val Arg Glu Ala Gln Ala Gly Thr Glu Gln Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Lys Ile Ala Ile Asp Arg Gly Tyr Asp Ile Ala Gln Ile Ser Arg His Leu Asp Phe Ile Ser Leu Leu Thr Tyr Asp Phe His Gly Ala Trp Arg Gln Thr Val Gly His His Ser Pro Leu Phe Arg Gly Gln Glu Asp Ala Ser Ser Asp Arg Phe Ser Asn Ala Asp Tyr Ala Val Ser Tyr Met Leu Arg Leu Gly Ala Pro Ala Asn Lys Leu Val Met Gly Ile Pro Thr Phe Gly Arg Ser Tyr Thr Leu Ala Ser Ser Lys Thr Asp Val Gly Ala Pro Ile Ser Gly Pro Gly Ile Pro Gly Gln Phe Thr Lys Glu Lys Gly Ile Leu Ala Tyr Tyr Glu Ile Cys Asp Phe Leu His Gly Ala Thr Thr His Arg Phe Arg Asp Gln Gln Val Pro Tyr Ala Thr Lys Gly Asn Gln Trp Val Ala Tyr Asp Asp Gln Glu Ser Val Lys Asn CA 022~1~84 1998-12-13 WO97/40149 PCT~P97/01903 Lys Ala Arg Tyr Leu Lys Asn Arg Gln Leu Ala Gly Ala Met Val Trp Ala Leu Asp Leu Asp Asp Phe Arg Gly Thr Phe Cys Gly Gln Asn Leu Ala Phe Pro Leu Thr Ser Ala Ile Lys Asp Val Leu Ala Glu Val (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 1152 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GCATACAAGC TGATCTGCTA CTACACCAGC TGGTCCCAGT ACCGGGAGGG TGATGGGAGC l20 TGCTTCCCAG ACGCCATCGA CCCCTTCCTG TGCACCCATG TCATCTACAG CTTTGCCAAC l80 ~ l'~'l'AAGGG AAGCCCAAGC AGGCACAGAG CAGCTTCTGC TCAGTGCAGC AGTAccTGcA 540 ., . . . ~ . .....

CA 022~l~84 l998-l2-l3 WO97/40149 PCT~P97/01903 CCC~l~lllC GAGGCCAGGA AGATGCAAGT TCTGACAGAT TCAGTAACGC TGACTACGCT 720 GTGAGCTACA TGCTGAGGCT GGGG&CTCCA GCCAATAAGC TG&TGATGGG TATCCCCACT 780 TTTG&&AGGA &CTACACTCT GGCCTCTTCC AAGACAGATG TG&GAGCCCC CATCTCAGG& 840 CCAGGAATTC CAGGCCAGTT CACCAAG&AG AAAG&GATCC TTGCCTATTA TGAGATCTGT 900 GACTTCCTCC AC&&AGCCAC CACCCACAGA TTCCGTGACC AGCAGGTCCC CTATGCCACC 960 AAGGGCAACC AGTG&GTGGC GTATGACGAC CAGGAGAGTG TCAAAAACAA GGCACGGTAC 1020 CTGAAGAACA G& QGCTGGC TGGCGCCATG GTGTGGGCCC TGGACTTG&A TGACTTCCGG 1080

Claims (13)

Claims

1. Protein comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND
(SEQ ID NO: 1) for use as a therapeutical substance.

2. Use according to claim 1, characterized in that said protein is bovine 39 kDa whey protein or human YKL-39 protein.

3. Pharaceutical composition comprising one or more proteins comprising an amino acid sequence which exhibits at least 50% homology with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND (SEQ ID NO: 1), and a pharmaceutical acceptable carrier.

4. Parmaceutical composition according to claim 3, characterized in that said protein is bovine 39 kDa whey protein or human YKL-39 protein.

5. Use of the protein comprising an amino acid sequence which exhibits at least 50%
homology with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND
(SEQ ID NO: 1) for the manufacture of a pharmaceutical preparation for the induction of specific T-cell tolerance to the homologous autoantigen in mammals suffering from T-cell mediated cartilage destruction.

6. Use according to claim 5, characterized in that said protein is bovine 39 kDa whey protein or human YKL-39 protein.

7. One or more proteins comprising an amino acid sequence which exhibits at least 50%
homology with the amino acid sequence YKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISND
(SEQ ID NO: 1) for use in a method to induce arthritis in animals, preferably mice.

8. A method according to claim 7, characterized in that said protein is bovine 39 kDa whey protein or human YKL-39 protein.

9. Animals which suffer from arthritis, whereby said arthritis was induced by the method according to claim 7 or 8.

10. Use of the animals according to claim 9 for use in the screening of novel drugs for the treatment of arthritis, especially rheumatoid arthritis.

11. Protein having the amino acid sequence of SEQ ID NO: 2.

12. Isolated DNA encoding the amino acid sequence according to claim 11.

13. Isolated DNA according to claim 12 having the DNA sequence of SEQ ID NO: 3.