SK102199A3 - Peptides comprising a t-cell epitope specific to collagen ii, their use and pharmaceutical compositions containing such peptides - Google Patents

Abstract

The invention provides an isolated peptide comprising an amino acid sequence of formula (I): A1-Xaa-Gly-A4-A5-Gly-A7-Xaa-Gly, wherein A1 represents an amino acid residue with an aromatic or aliphatic side chain, A4 represents an asparagine or arginine residue or an amino acid residue with an aromatic or aliphatic side chain, A5 represents any naturally occurring amino acid, A7 represents an amino acid with a negatively charged residue, Xaa represents any amino acid residue, and Gly represents a glycine residue; and the use of such peptides in medical therapy, particularly in the treatment of autoimmune conditions such as rheumotoid arthritis.

Description

Technical field

The present invention provides novel peptides derived from collagen CII, methods for their preparation and their use in therapy, particularly for the treatment of rheumatoid arthritis and related autoimmune diseases.

BACKGROUND OF THE INVENTION

Collagen molecules are among the major structural proteins of connective tissue. They consist of three polypeptide chains that form an extensive triple helical structure with a unique repeating amino acid sequence of X-Y-Gly. The extracellular cartilage matrix is unique in that it mainly contains type II collagen but also contains types IX and XI. CII was recently cloned from a mouse and its entire 1419 amino acid sequence was determined (Metsarantam et al., 1991, J. Biol. Chem. 266: 16862-9).

Type II collagen is believed to be hidden from cells of the immune system since it occurs only in avascular (non-bloodied) tissues such as cartilage and vitreous humor of the eye. The immune system is therefore not completely tolerant of its own type II collagen. Isolated CII protein has the ability to induce tissue-specific autoimmune disease upon injection with Freund's complete adjuvant. This induced disease is called collagen-induced arthritis (CIA). CIA is normally induced by injection of foreign CII, which induces the production of T cells and antibodies capable of recognizing the corresponding self-protein.

CIA susceptibility in mice is limited to mice with MHC IA ^q and IA ^E. The binding motifs of these molecules are similar to human motifs

DR4 and DRI. ' Therefore, CII is believed to be an autoantigen in rheumatoid arthritis (RA).

No causal therapy is currently available for the treatment of RA. Existing methods of treatment, such as e.g. The use of corticosteroids is non-specific since they generally suppress the immune response. Recent research has suggested that autoreactive T cells play an important role in the pathogenesis of RA, leading to the idea that tolerance to these pathogenic T cells by nasal / oral administration of immunogenic peptides could have therapeutic potential.

Miyahara, H. et al. (Immunology, 1995, 86: 110-115) described the use of a type II collagen fragment to suppress arthritis in mice. However, the fragment used (CII 607-621) did not contain a binding motif for any of the HLA-DR molecules related to RA in humans and cannot therefore be used as a tolerance for treating RA in humans.

WO 96/20950 discloses type II collagen peptides capable of binding to HLA DRB1 of a human MHC protein, which could be useful in the treatment of RA. However, these peptides, which are present in the stretch 273 to 404 of collagen CII protein, have only poor potency in the relevant assays for putative RA toleragens. The peptides of the present invention have significantly higher therapeutic efficacy.

SUMMARY OF THE INVENTION

The present invention provides peptides that have been found to be extremely effective in inducing immune tolerance to T cell epitopes derived from collagen CII. These peptides can be used to treat autoimmune diseases such as rheumatoid arthritis.

The present invention therefore provides an isolated peptide having or comprising the amino acid sequence of Formula (I):

A ¹ Xaa-Gly-A ⁴ -A ⁵ -Gly-A ⁷ -Xaa-Gly (I) where

A ¹ is an amino acid residue with an aromatic or aliphatic side chain

A ⁴ is an asparagine or arginine residue or an amino acid residue with an aromatic or aliphatic side chain

A ⁵ is any naturally occurring amino acid

A ⁷ is an amino acid with a negatively charged residue,

Xaa is any amino acid residue a

Gly is a glycine residue.

The peptides of the present invention are preferably peptides containing 9, 10, 11, 12, 13, 14 or 15 amino acids, more preferably 9 peptides.

The peptides of the present invention are subject to the following independent advantageous features.

A ¹ is F, I, L, A or P and most preferably F,

A ⁴ is F, I, L, A, N or P and most preferably F,

A ⁵ is K or R,

A ⁷ is E, D, Q, P or N, and most preferably Q.

Preferred peptides of the present invention include peptides either containing or having one of the following amino acid sequences:

ESG SPG ENG, PPG ADG QPG. ARG NDG QPG. QPG AKG DQG, APG AKG EAG, PTG VTG PKG, AQG SRG EPG RVG PPG ANG. PAG ASG NPG, ANG NPG PAG. TDG IPG AKG, DPG LQG PAG. SAG APG IAG, APG EKG EPG IAG APG FPG, PQG LAG QRG, FPG PRG PPG, PKG ANG DPG, APG ASG DRG, LPG ARG LTG, DAG PQG KVG. ALG APG APG, PAG ANG ECG , KQG DRG EAG, or ARG APG EPG (SEQ ID NO NO. : 1 to 25); or more preferably either RVG PPG ANG (SEQ SEQ ID NO. : 8) or ANG NPG PAG

(SEQ ID NO .: 10).

Examples of preferred peptides of the present invention also include peptides that either contain or have themselves one of the following amino acid sequences:

VCG ESG SPG ENG SPG; FAG PPG ADG QPG AKG; AAG ARG NDG QPG PAG; ADG QPG AKG DQG EAG; APG AKG EAG PTG Arg; PQG PTG VTG PKG Arg; PEG AQG SRG EPG NPG; AAG RVG PPG ANG NPG; PAG ASG NPG TDG IPG; PPG ANG NPG PAG PPG; NPG TDG IPG AKG SAG; RAG DPG LQG PAG APG; AKG SAG APG IAG APG; PAG APG ECG EPG DDG; APG IAG APG FPG PRG; PPG PQG LAG QRG IVG; APG FPG PRG PPG PQG; LAG PKG ANG DPG RPG; KQG APG ASG DRG PPG; EPG LPG ARG LTG RPG; RPG DAG PQG KVG PSG; ETG ALG APG APG PPG; PPG PAG ANG ECG EVG; PTG KQG DRG EAG AQG; or STG ARG APG EPG ETG (SEQ ID NO NO. : 26 until 52),

more preferably AAG RVG PPG ANG NPG (SEQ ID NO .: 33) and most preferably PPG ANG NPG PAG PPG (SEQ ID NO .: 35).

Preferably, the peptides of the present invention comprise a sequence of 9 to 15 amino acids present in an uninterrupted sequence in the region 701 to 721 of collagen II.

The present invention also relates to a peptide comprising a T-cell epitope specific for collagen II, which peptide comprises at least 9 amino acids in an uninterrupted contiguous sequence identical to a sequence selected from regions 110-239, 338-379, and

587 to 895 of the collagen II sequence, wherein each amino acid is optionally replaced with a functionally equivalent amino acid, wherein the peptide has formula (I) as defined above.

The invention further provides a pharmaceutical composition comprising the peptide of the present invention in association with a pharmaceutically acceptable carrier or diluent. The formulation is suitably used to provide tolerance to an autoimmune disease such as rheumatoid arthritis or relapsing polychondritis.

The invention further provides the use of a peptide of the present invention or a composition of the present invention for the manufacture of a medicament for use in the treatment of autoimmune diseases.

The invention further relates to a method of treating a human or animal suffering from an autoimmune disease, such as e.g. rheumatoid arthritis or relapsing polychondritis, the method comprising administering a therapeutically effective amount of a peptide or preparation to a human or animal of the invention.

The peptides of the present invention may be prepared by methods known to those skilled in the art. E.g. this may be by using a standard peptide bonding technique) on the Synthesis of Peptides peptide synthesizer,

University Press, 1991, (Creating Automatic The Chemical Gradual Solid Phase Condensation Using (see, e.g., Jones, J.

pp. 132-156, First edition, Oxford, and R. Epton (ed) Innovation and

Perspectives in Solid Phase Peptide Synthesis, SPCC (UK) Ltd. 1990). The preparation starts from the C-terminal amino acid, which can be obtained already cleaved to methylbenzhydrylamine, benzhydrylamine or chloromethylated resin, or to another suitable solid support. Additional amino acids are added step-by-step after their side chains have been protected. In this condensation method, alpha-amino groups of amino acids are protected by F-moc or t-Boc. Amino acid side chain protecting groups are well known in the art. The entire protected peptide is released from the chloromethylated resin by ammonolysis to give the protected amide or from methylbenzhydrylamine or benzhydrylamine resin by acidolysis.

The peptides of the present invention can also be prepared by a solution method, either by sequential condensation or by condensation of fragments (see, e.g., Jones, 1. The Chemical Synthesis of Peptides, pp. 115-131, First Edition, Oxford University Press, 1991). The amino acid with a suitably protected alpha-amino group is linked to the amino acid with a suitably protected alpha-carboxyl group (depending on the synthetic method chosen, this protection may not be necessary) using diimides, symmetrical or unsymmetrical anhydrides or other condensing agents or methods known to those skilled in the art. the methods may be either chemical or enzymatic. The alpha amino protecting group and / or the alpha-carboxyl group are removed and another suitably protected amino acid or amino acid block is attached to extend the growing peptide. A different combination of protecting groups and chemical / enzyme methods and peptide coupling / synthesis strategies may be used in each synthesis.

The peptides of the present invention are defined as peptides comprising a T-cell epitope. In other words, they are able to activate T cells. There are several known ways to determine binding strength. Preferably, the peptide of the present invention is capable of activating a T-cell with a binding force of at least 2 according to an IFN-γ release assay and / or a stimulation index value of at least 3. The IFN-γ release assay can be performed by methods known in the art or described in the examples. Similarly, the stimulation index can be determined using art-known proliferation assays, e.g. the assay described in Analysis of type II collagen reactive T cells in mice, Andersson and Holmdahl, Eur. J. Immunol. 20: 1016-1066, 1990. Preferably, the assay is performed as described in the examples of this application.

The peptides of the invention are useful in the treatment without being modified, but alternatively the peptides may also be modified, e.g. may be conjugated, i. covalently attach to a carrier system, e.g. to mucose binding structures such as e.g. cholera β-toxin, which assists absorption in the intestine.

I

The peptides of the present invention may be used for the treatment, prophylaxis or diagnosis of autoimmune diseases, and the terms therapy or treatment used herein are intended to include prophylaxis and diagnosis simultaneously.

The peptide of the present invention is an isolated peptide, that is, the peptides of the present invention do not include peptides present in the body. The peptides of the invention may be either natural or recombinantly prepared peptides or proteins, or may be synthesized chemically as described in this application.

The compounds of the invention may be administered in doses ranging from 10 µg to 10 mg per day, either as a single dose or divided into 2-4 doses per day. The unit dosage form contains a dose of 2.5 µg to 10 mg of the compound of the present invention. The compound of the invention may be administered by the intranasal route in the form of a solution, suspension, HFA aerosol or in the form of a dry powder formulation, e.g. using a Turbuhaler® applicator, or in a systemic manner, e.g. orally in the form of tablets, pills, capsules, syrups, powders, granules or also parenterally in the form of sterile parenteral solutions or suspensions, or also rectally in the form of suppositories.

The compounds of the present invention may be administered alone or in the form of a pharmaceutical composition comprising a compound of the invention in combination with a pharmaceutically acceptable diluent, adjuvant or carrier. Particularly preferred are those compounds which do not contain substances capable of eliciting adverse effects, e.g. allergic reaction.

Powder formulations and pressurized packaged HFA aerosols of the compounds of the invention may be administered by nasal inhalation. For inhalation, the compound of the invention is finely divided. The comminuted compound preferably has particles with a statistical mean diameter (by weight) of 10 µm and is suspended in a mixture of propellants with the aid of a dispersant such as e.g. a C8-C20 fatty acid (e.g., oleic acid) or a salt thereof, a bile acid, a phospholipid, an alkyl saccharide, a perfluorinated or polyethoxylated surfactant, or another pharmaceutically acceptable dispersing agent.

The compounds of the present invention may also be administered via a dry powder inhaler. Such an inhaler may be either a single dose or multi dose inhaler and may be an breath actuated dry powder inhaler.

One method of preparation is to admix the finely divided compound of the invention with a carrier, e.g. mono-, dialbo, or polysaccharide, sugar alcohol or other polyols. Suitable carriers are sugars, e.g. lactose, glucose, raffinose, malesitose, lactitol, maltitol, trehalose, sucrose, mannitol and starch. Alternatively, the finely divided compound may be coated (coated) with another substance. The powder mixture can also be filled into hard gelatin capsules, each containing the desired dose of the active ingredient.

The pharmaceutical composition comprising the compound of the present invention is preferably in the form of tablets, pills, capsules, syrups, powder or granules for oral administration or in the form of sterile solutions or suspensions for parenteral administration or in the form of suppositories for rectal administration.

For oral administration, the active ingredient may be admixed with an adjuvant or carrier, e.g. lactose, sucrose, sorbitol, mannitol, or starches such as potato, corn starch or amylopectin, cellulose derivatives, a binder such as e.g. gelatin or polyvinylpyrrolidone and a lubricant such as e.g.

magnesium stearate, calcium stearate, polyethylene glycol, waxes, paraffin, and the like, and then compressed into tablets.

If coated tablets are desired, the core prepared as described above may be coated with a concentrated sugar solution which may contain gum arabic, gelatin, talc, titanium dioxide, and the like. Alternatively, the tablet may be coated with a suitable polymer dissolved in a volatile organic solvent.

For the preparation of soft gelatin capsules, the compound of the present invention may be admixed e.g. with vegetable oil or polyethylene glycol. Hard gelatine capsules may contain a granulate of the compound with either the above-described tablet excipients, e.g. lactose, sucrose, sorbitol, mannitol, starches, cellulose derivatives or gelatin. Liquid or semi-solid drug forms can also be filled into hard gelatine capsules.

Liquid preparations for oral administration may be in the form of syrups or suspensions, e.g. in the form of solutions containing a compound of the invention and a sugar, and a mixture of ethanol, water, glycerol and propylene glycol. Such liquid preparations may optionally contain coloring agents, flavoring and perfuming agents, saccharin and carboxymethylcellulose as a thickening agent, or other excipients known to those skilled in the art.

The present invention is further illustrated by the following non-limiting examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

The peptide CII sequences shown in Table 1, each consisting of 15 amino acids, were synthesized using an SMPS 350 automatic synthesizer (Zinsser, Frankfurt / Main,

Germany) chemically with Fmoc (Atherton, Sheppard, Solid Phase Peptide Synthesis - A Practical Approach, IRL Press, Oxford). Each peptide was acetylated at the N-terminus and amidated at the C-terminus. Peptide quality was assessed by HPLC and the expected molecular weight was confirmed by mass spectroscopy of the peptide sample.

The stretches of amino acids that are shown in bold are so-called. The core sequence, i. stretches of amino acids that bind most strongly compared to the binding force structurally close to related peptides.

Table 1

peptide amino acid remains peptide amino acid residues 110-124 SPG ESG SPG ENG SPG 635-649 FAG PPG ADG QPG AKG 140-154 AAG ARG NDG QPG PAG 641-655 ADG QPG AKG DQG EAG 161-175 GPG FPG APG AKG EAG 665-679 PSG APG PQG PTG VTG 170-184 APG AKG EAG PTG ARG 677-691 PQG PTG VTG PKG ARG 185-199 PEG AQG SRG 701-715 AAG RGG PPG ANG NPG 203-217 PAG ASG NPG TDG IPG 707-721 PPG ANG NPG PAG PPG 209-223 NPG TDG IPG AKG SAG 740-754 RAG DPG LQG PAG APG 218-232 AKG SAG APG 749-763 PAG APG ECG EPG DDG 224-238 APG IAG APG FPG PRG 770-784 PPG PQG LAG QRC IVG 230-244 APG FPG PRG PPG PQC 779-793 QPC IVG LPG 338-352 LAG PKG ANG DPG RPG 806-820 KGG APG ASG DRG PPG 353-367 EPG LPG ARG LTG RPG 821-835 PVG PPG LTG PAG EPG 365-379 RPG DAG PQG KVG PSG 857-871 ETG ALG APG 593-607 PPG PAG ANG ECG EVG 881-895 PTG KQG DRG EAG AQG 614-628 STG ARG APG EPG ETG

Example 2 ’

Mouse CII was extracted from the sword lobe of the sternum in a known manner by pepsin digestion and then further purified by salting. Rat CII was again purified from Swarm's chondrosarcoma in a known manner. The collagens were dissolved in 0.1 M acetic acid. The collagen that was used to restimulate the primary lymph node cells was denatured for 30 minutes by incubation at 56 ° C.

Example 3

To test for proliferative response and cytokine release by lymphoid node cells, male mice (B10.QX DBA / 1) F1 were immunized in the hindlimb foot with 50 µg of mouse CII or a synthetic peptide emulsified in CFA (containing H37Ra) at 7-10 weeks of age. , Difco, Detroit, MI). Arthritis was induced in the same 7 to 10-week-old mice by immunizing the mice at the root of the tail with a dose of 100 µl of rat CII emulsified CFA prepared according to Example 2.

After 5 weeks, the immune response of the mice was boosted with a dose of 50 µg of CII emulsified in a 1: 1 weight ratio of IFA (Difco, Detroit, MI). Lymph node cells were aspirated from strongly arthritic joints, pooled, and tested in a proliferation assay for reactivity with a panel of mouse CII peptides prepared according to Example 1 as described below.

Cytokine release was tested in 50 μΐ of supernatant from induced primary lymph node cell cultures that were restimulated in vitro with a panel of CII peptides prepared according to Example 1 using IFN-γ and IL-4 mini kits (Endogen, Cambridge, MA). The results are shown in Table 2.

Table 2

peptide ARE YOU IFN-γ release (ng / ml) peptide ARE YOU IFN-γ release (ng / ml) 110-124 2.2 2.2 635-649 2.7 0.4 140-154 2.1 1.5 641-655 2.0 0.9 161-175 3.1 4.2 665-679 3.5 1.5 170-184 1.5 677-691 2.2 2.9 185-199 4.1 1.9 701-715 18.0 2.5 203-217 3.6 6.5 707-721 19.4 5.8 209-223 5.2 740-754 2.8 2.4 218-232 3.9 2.3 749-763 4.8 1.0 224-238 5.0 4.4 770-784 2.7 1.4 230-244 3.9 5.8 779-793 3.9 338-352 4.4 2.0 806-820 2.3 353-367 3.1 1.5 821-835 1.8 365-379 2.8 857-871 2.4 0.3 593-607 2.9 0.7 881-895 2.6 0, 6 614-628 2.7 2.3

Example 4

Either T or B cells were harvested from the lymph node cells induced by mouse CII using a magnetically activated cell sorter (MACS) with superparamagnetic microspheres conjugated to rat monoclonal antibody L3T4 (CD4) or rat anti-mouse antibody B220 CD45R) (both antibodies from Milteny) GmbH, Bergish Gladbach, Germany) essentially as recommended by the manufacturer, except that PBS / BSA buffer was replaced with PBS containing 2% FCS, 5 mM EDTA, 50 μΜ 2-ME and mM HEPES. Fractions were analyzed with a FACScan flow cytometer (Becton Dickinson) using a conjugate (FITC-anti-mouse CD3-8) to label (stain) T cells and a conjugate (FITC-anti-mouse light chain k) to label B cells (Pharmigen, San Diego). , CA). The cells were washed three times with serum-free DMEM after separation and then used in the proliferation assay of Example 3. Enriched CD4 + T cells as antigen-presenting cells were mixed with spleen cells from syngeneic mice at a 1: 2 weight ratio. AP cells were used as a suspension of single spleen cells treated with 0.84% NH ₄ Cl at pH 7.4 to lyse the red blood cells.

Table 3

Cell type CPM x 10 ' ³ unfractionated 13.6 CD4 + enriched 21.4 CD4 + impoverished 0.3 CD45R enriched 12.5 CD45R impoverished 0.4

J

LIST OF SEQUENCES SEQ ID NO NO. 1: ESG SPG ENG sequence SEQ ID NO. 2: PPG ADG QPG sequence SEQ ID NO. 3: ARG NDG QPG sequence SEQ ID NO. 4: QPG AKG DQG sequence SEQ ID NO. 5: APG AKG EAG sequence SEQ ID NO. 6: PTG VTG PKG sequence SEQ ID NO. 7: AQG SRG EPG sequence SEQ ID NO. 8: RVG PPG ANG sequence SEQ ID NO. 9: PAG ASG NPG sequence SEQ ID NO. 10 : ANG NPG PAG sequence SEQ ID NO. 11 : TDG IPG AKG sequence SEQ ID NO. 12 : DPG LQG PAG sequence SEQ ID NO. 13 : SAG APG IAG sequence SEQ ID NO. 14 : APG ECG EPG sequence SEQ ID NO. 15 : IAG APG FPG sequence SEQ ID NO. 16 : PQG LAG QRG sequence SEQ ID NO. 17 : FPG PRG PPG sequence SEQ ID NO. 18 : PKG ANG DPG sequence SEQ ID NO. 19 APG ASG DRG sequence SEQ ID NO. 20 : LPG ARG LTG sequence SEQ ID NO. 21 : DAG PQG KVG sequence SEQ ID NO. 22 : APG APG sequence SEQ ID NO. 23 : PAG ANG ECG sequence SEQ ID NO. 24 : KQG DRG EAG sequence SEQ ID NO. 25 : ARG APG EPG

sequence SEQ ID NO. : 26: VCG ESG SPG ENG SPG; sequence SEQ ID NO. : 27: FAG PPG ADG QPG AKG; sequence SEQ ID NO. : 28: AAG ARG NDG QPG PAG; sequence SEQ ID NO. : 29: ADG QPG AKG DQG EAG; sequence SEQ ID NO. : 30: APG AKG EAG PTG Arg; sequence SEQ ID NO. : 31: PQG PTG VTG PKG Arg; sequence SEQ ID NO. : 32: PEG AQG SRG EPG NPG; sequence SEQ ID NO. : 33: AAG RVG PPG ANG NPG; sequence SEQ ID NO .: 34: PAG ASG NPG TDG IPG; sequence SEQ ID NO. : 35: PPG ANG NPG PAG PPG; sequence SEQ ID NO .: 36: NPG TDG IPG AKG SAG; sequence SEQ ID NO .: 37: RAG DPG LQG PAG APG; sequence SEQ ID NO. : 38: AKG SAG APG IAG APG; sequence SEQ ID NO. : 39: PAG APG ECG EPG DDG; sequence SEQ ID NO. : 40: APG IAG APG FPG PRG; sequence SEQ ID NO. : 41: PPG PQG LAG QRG IVG; sequence SEQ ID NO. : 42: APG FPG PRG PPG PQG; sequence SEQ ID NO .: 43: LAG PKG ANG DPG RPG; sequence SEQ ID NO. : 44: KQG APG ASG DRG PPG; sequence SEQ ID NO. : 45: EPG LPG ARG LTG RPG; sequence SEQ ID NO. : 46: RPG DAG PQG KVG PSG; sequence SEQ ID NO .: 47: EXG ALG APG APG PPG; sequence SEQ ID NO. : 48: PPG PAG ANG ECG EVG; sequence SEQ ID NO .: 49: PTG KQG DRG EAG AQG; sequence SEQ ID NO .: 50: STG ARG APG EPG ETG sequence SEQ ID NO .: 51: AAG RVG PPG ANG NPG; sequence SEQ ID NO. : 52: PPG ANG NPG PAG PPG

Claims (18)

An isolated peptide having the amino acid sequence of formula (I): A ¹ -Xaa-Gly-A ⁴ -A ^{5 *} -Gly-A ⁷ -Xaa-Gly (I) where A ¹ is an amino acid residue with an aromatic or aliphatic side chain A ⁴ is an asparagine or arginine residue or an amino acid residue with an aromatic or aliphatic side chain A ⁵ is any naturally occurring amino acid A ⁷ is an amino acid with a negatively charged residue, Xaa is any amino acid residue a Gly is a glycine residue. An isolated peptide comprising the amino acid sequence of any of the peptides of claim 1. A peptide according to claim 2 comprising 10 to 15 amino acids. The peptide of claim 1, wherein A ⁵ is a lysine or arginine residue and A ⁷ is a glutamic acid residue. 5. An isolated peptide having any of the following sequences: ESG SPG ENG PPG ADG QPG ARG NDG QPG. QPG AKG DQG, APG AKG EAG, PTG VTG PKG, AQG SRG EPG RVG PPG ANG. PAG ASG NPG, ANG NPG PAG. TDG IPH AKG, DPG LQG PAG. SAG APG IAG, APG ECG EPG IAG APG FPG, PQG LAG QRG, FPG PRG PPG, PKG ANG DPG, APG ASG DRG, LPG ARG LTG, DAG PQG KVG. ALG APG APG, PAG ANG ECG KQG DRG EAG, or ARG APG EPG (Sequence Sequence) SEQ ID NO .: 1 to 25). An isolated peptide comprising Any peptide according to claim 3 amino acid sequence An isolated peptide having any sequence: from the following VCG ESG SPG ENG SPG; FAG PPG ADG QPG AKG; AAG ARG NDG QPG PAG; ADG QPG AKG DQG EAG; APG AKG EAG PTG Arg; PQG PTG VTG PKG Arg; PEG AQG SRG EPG NPG; AAG RVG PPG ANG NPG; PAG ASG NPG TDG IPG; PPG ANG NPG PAG PPG; NPG TDG IPG AKG SAG; RAG DPG LQG PAG APG; AKG SAG APG IAG APG; PAG APG ECG EPG DDG; APG FPG PRG PPG PQG; PPG PQG LAG QRG IVC; APG FPG PRG PPG PQG; LAG PKG ANG DPG RPG; KQG APG ASG DRG PPG; EPG LPG ARG LTG RPG; RPG DAG PQG KVG PSG; ETG ALG APG APG PPG; PPG PAG ANG ECG EVG; PTG KQG DRG EAG AQG; or STG ARG APG EPG ETG (SEQ ID NO .: 26 An isolated peptide that is a fragment of 10 to 14 amino acids from any of the peptides of claim 7. A peptide according to any one of the preceding claims, which binds to T cells with a binding force of at least 2 in an IFN-γ release assay. A peptide according to any one of the preceding claims, which binds to T cells with a stimulation index of at least 3. The peptide of any one of the preceding claims, wherein the peptide sequence is found in the collagen II sequence at regions 110 to 239, 338 to 379 or 587 to 895. The peptide of claim 11, wherein the peptide sequence is found at region 701 to 721 of the collagen II sequence. The isolated peptide of any preceding claim for use in therapy. The peptide of claim 13, wherein the treatment comprises induction of tolerance. Use of an isolated peptide according to any one of the preceding claims for the manufacture of a medicament for use in the treatment of autoimmune diseases. The use of claim 10, wherein the autoimmune disease is rheumatoid arthritis. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 12 in association with a pharmaceutically acceptable carrier or diluent. A method of treating a human or animal suffering from or susceptible to an autoimmune disease, comprising administering to a human or animal a therapeutically effective amount of a peptide of any one of claims 1 to 12 or a composition of claim 17. 19. A peptide comprising a T-cell epitope specific for collagen II and comprising at least nine amino acids of the same sequence and in a continuous sequence of the sequences 110-239, 338-379 and 587-895 of collagen II, each amino acid optionally being replaced a functionally equivalent amino acid and wherein the peptide has the formula A ¹ -Xaa-Gly-A ⁴ -A ⁵ -Gly-A ⁷ -Xaa-Gly (I) where A ¹ is an amino acid residue with an aromatic or aliphatic side chain A ⁴ is an asparagine or arginine residue or an amino acid residue with an aromatic or aliphatic side chain A ⁵ is any naturally occurring amino acid A ⁷ is an amino acid with a negatively charged residue, Xaa is any amino acid residue a Gly is a glycine residue.