WO2004098489A2

WO2004098489A2 - Compositions and methods for modulation of specific epitopes of hsp60

Info

Publication number: WO2004098489A2
Application number: PCT/IL2004/000404
Authority: WO
Inventors: Hubert Kolb
Original assignee: Peptor Ltd.
Priority date: 2003-05-12
Filing date: 2004-05-12
Publication date: 2004-11-18
Also published as: WO2004098489A3

Abstract

Distinct epitopes of HSP60 are identified to modulate binding and activation of macrophages. One epitope, localized to residues 335-366, and more specifically to residues 254-265, of human HSP60 binds LPS and by presenting it to toll receptor-4 complex immunostimulates the cells, and a second epitope, localized to residues 481-500 of HSP60, is responsible for binding to the macrophage cell surface. The present invention relates to novel compounds, capable of inhibiting the binding of those peptidic regions to LPS or to macrophages. The present invention further relates to pharmaceutical compositions comprising these novel compounds, useful for prevention or treatment of inflammatory and autoimmune diseases and disorders.

Description

COMPOSITIONS AND METHODS FOR MODULATION OF SPECIFIC

EPITOPES OF HSP60

FIELD OF THE INVENTION The present invention relates to agents capable of inhibiting the binding of a first specific epitope of heat shock protein 60 (HSP60) to lipopolysaccharide (LPS) and to agents capable of inhibiting the binding of a second specific epitope of HSP60 to macrophages. The present invention further relates to pharmaceutical compositions comprising such agents, useful for prevention or treatment of inflammatory disorders and autoimmune diseases.

BACKGROUND OF THE INVENTION

Heat Shock Proteins

Heat shock proteins (HSPs) are highly conserved proteins expressed in all pro- and eukaryotic cells. They are involved in many important cellular processes such as correct folding of newly synthesized proteins and subunit assembly and therefore are termed molecular chaperones (Hard, F. U., 1996, Nature 381, 571-579; Bukau, et al., 2000, Cell

101, 119-122). Under stressful or non-physiological conditions like high temperature, ultraviolet radiation, and viral or bacterial infection, cellular HSP synthesis is up-regulated. HSPs exert cytoprotective functions such as preventing the aggregation of denatured proteins, initiating their refolding or proteolytic degradation (Singh-Jasuja, et al., 2001, Biol.

Chem. 382, 629-636). According to their molecular weight, HSPs are divided into six subfamilies: small HSPs, HSP40, HSP60, HSP70, HSP90 and HSP100. They are located in the cytosol (HSP70, HSP90, HSP 100), in the endoplasmic reticulum (HSP70, HSP90) or in mitochondria (HSP60) (Fink, A., 1999, Physiol. Rev. 79, 425).

Recently, the HSP60, HSP70, and HSP90 subfamilies have attracted increasing attention because of their potential roles in immunologically relevant processes. Several studies have identified HSPs as targets of immune responses during microbial infections (Kiessling, et al. 1991, Immunol. Rev. 121, 91-111; Zugel, U., and Kaufinann, S. H., 1999, Immunobiology 201, 22-35). Because of the high sequence homology between microbial HSPs and endogenous HSPs derived from damaged or stressed tissue, immunological cross- reactivity was suggested to contribute to the development of autoimmune disorders including rheumatoid arthritis and diabetes ( Holoshitz et al., 1986, Lancet 2, 305-309; Elias et al, 1991, Proc. Natl. Acad. Sci. U.S.A 88, 3088-3091; Abulafia-Lapid et al, 1999, J. Autoimmun. 12, 121-129). Hsp60 is a mitochondrial chaperone with a major role in protein folding and unfolding as well as translocation of proteins into mitochondria. Hsp60 is found in the cell cytosol under stressful and inflammatory conditions; infection or elevated cytokine levels will induce the cellular stress response. Therefore, it is not surprising that HSP60 is a highly immunogenic protein: it is the "common antigen" of gram-negative bacteria. Immunological reactivity to both bacterial and autologous-HSP60 is highly prevalent in the general population, since the pathogen-directed immune response can easily convert into an autoimmune response due to the high homology.

T-cell responses to multiple HSP60 epitopes are present in various autoimmune and inflammatory diseases including type 1 diabetes, rheumatoid and juvenile arthritis, multiple sclerosis, ankylosing spondylitis, pelvic inflammation-associated infertility, inflammatory bowel disease, atherosclerosis, graft rejection and more. The immune system reacts to HSP60 epitopes that are either cross-reactive between the human and bacterial analogues, or idiosyncratic.

Human HSP60 has been reported to induce pro-inflammatory reactivity in human and murine innate immune cells, such as macrophages and dendritic cells (DC) (Chen et al., 1999, J. Immunol. 162, 3212-3219; Kol et al, 1999, J. Clin. Invest. 103, 571-577; Vabulas et al., 2001, J. Biol. Chem. 276, 31332-31339). This response includes the release of the inflammatory mediators tumor necrosis factor α (TNFα), nitric oxide (NO), and interleukin 6 (IL-6). In addition, HSP60 was found to induce gene expression of the cytokines IL- 12(p70) and IL-15, promoting a T helper 1 (Thl) phenotype. These findings suggest a role of HSP60 as danger signal for the innate immune system. Recently, the binding receptor for human HSP60 on macrophages has been characterized (Habich et al., 2002, J. Immunol. 168, 569-576).

It was recently discovered that HSP60 is a putative endogenous activator of Toll-like receptors in mammals (Ohashi et al., 2000, J. Immunol. 164, 558-61), while the previously described ligands for Toll-like receptors in mammalian cells are of microbial origin, which is in line with a function of these receptors in innate immune responses. This finding suggests that Toll-like receptors may not only have a function in innate immune defense against microbial pathogens but also serve physiological functions by interacting with endogenous ligands.

Further approaches to identify the potential HSP60 binding receptor have shown that HSP60 binding to macrophages is independent of Toll-like receptor (TLR) 4, which was found to be involved in LPS signaling (Poltorak et al, 1998, Science 282, 2085-2088; Chow et al., 1999, J Biol. Chem. 274, 10689-10692). In contrast, it was found that the presence of a functionally active TLR4 protein is essential for HSP60 signaling (Ohashi et al., 2000, J. Immunol. 164, 558-561). These observations suggest that the process of HSP60 binding and signaling involves different ligand-receptor interactions.

Studies have indicated that TLR4 and CD 14 are involved in the inflammatory signaling of HSP60 (Kol et al, 2000, J. Immunol. 164, 13-17), but that TLR4 is not involved in HSP60 binding (Habich et al., 2002, J. Immunol. 168, 569-576). These observations suggest that the interaction of HSP60 with macrophages is a highly complex process. Similar observations were made for other HSPs. Recently, studies of the LPS binding factor C in the LAL assay and of mutated variants have shown that potent LPS binding is mediated by the tetrapeptide LKGK (Tan et al. FASEB. J. 14: 1801-1813, 2000).

So far, the potential epitopes of HSP60 required for or involved in receptor binding and immunostimulation are unknown. Nowhere in the background art are epitopes of HSP60, identified as responsible for binding to the receptor on macrophages or for the induction of inflammatory mediators in these cells. Nowhere in the background art it is determined whether HSP60 epitopes involved in receptor binding and in eliciting a pro- inflammatory response are the same or different.

Use of Heat shock proteins in therapy Many disclosures claim uses of heat shock proteins or fragments thereof as immune modulators in diagnosis, treatment or prevention of autoimmune diseases. Most of these disclosures relate to heat shock protein 60 also known previously as HSP65, or fragments of this protein.

For example, the particular protein produced by the human body during development of IDDM, which serves as a diagnostic marker for the incipient outbreak of

IDDM, is the human heat shock protein having a size of about 65 KD (human HSP65) or an antigen cross-reactive therewith as disclosed in EP 0417271, and in US patents 5,114,844; 5,671,848; 5,578,303 and 5,780,034. It has been disclosed that fragments of this HSP60 protein may serve as therapeutically useful entities in preventing or alleviating IDDM and host vs. graft disease (US patents 6,180,103 and 5,993,803 and WO 96/19236, WO 97/01959 and WO 98/08536). In addition, fragments of HSP60 may be used as carriers for development of synthetic vaccines by increasing the immunogenicity of poorly immunogenic antigens as disclosed in US patents 5,736,146 and 5,869,058.

European Patent No. 0262710 discloses polypeptides useful for alleviation, treatment, and diagnosis of autoimmune arthritis and similar autoimmune diseases. The claimed polypeptides are derived from bacterial protein named "Antigen A" which was identified later as mycobacterial HSP60.

WO 92/04049 discloses peptides of at least seven amino acids homologous to a fragment of Mycobacterium tuberculosis HSP60, which inhibit T-lymphocytes activation and proliferation and can protect from immune reactions and immune-related disease. WO 89/12455 and WO 94/29459, disclose the use of stress proteins and analogs for producing or enhancing an immune response or for inducing immune tolerance, for prophylaxis or therapy of autoimmune diseases and for treating or preventing infectious or cancers. A fusion protein is claimed comprising a stress protein fused to a protein against which an immune response is desired. WO 95/25744 discloses microbial stress protein fragments containing epitopes homologous to related mammalian epitopes - used to treat and prevent inflammatory autoimmune diseases and to prevent transplant rejection. The protective epitopes are located in short peptides comprising 5-15 amino acid sequences regions of stress proteins, that are highly conserved between microorganisms and animals. WO 97/11966 and WO 96/10039 disclose polypeptides of up to 21 amino acids, derived from microbial heat shock protein which are useful for prophylaxis or treatment of autoimmune diseases especially arthritis.

WO 96/16083 discloses a peptide 25 amino acids long, derived from the 10 kD heat shock protein (HSP 10) of Mycobacterium tuberculosis which is useful in pharmaceutical products for the treatment of inflammatory pathologies, especially rheumatoid arthritis.

WO 91/02542 discloses the use of antigenic and/or immuno-regulatory material derived from mycobacterium vaccae and specifically HSP60, for treating chronic inflammatory disorders caused or accompanied by an abnormally high release of IL-6 and/or TNFα.

WO 96/18646 discloses peptides of 9-20 amino acids derived from Mycobacterial HSP60 used for treatment or prevention of autoimmune CNS diseases, e.g. multiple sclerosis, chronic inflammatory CNS disease and primary brain tumors.

WO 94/02509 discloses peptides of 7-30 amino acids derived from DR3 -restricted epitope of Mycobacterial HSP60 used for treatment of HLA-DR3 related autoimmune diseases. WO 00/27870 discloses peptides derived from Mycobacterial and rat HSP60 and vaccines comprising such peptides for immunization against autoimmune and inflammatory diseases.

US 5,958,416 describes heats shock protein peptides and methods for modulating autoimmune central nervous system diseases. WO 01/43691 discloses fragments and antagonists of Hsp60, capable of reducing or prevention the induction of a pro-inflammatory immune response of cells of the innate immune system by HSP60, for treatment of inflammatory and autoimmune diseases. The compounds disclosed inhibit the binding of HSP60 to the toll-like-receptor, and therefore reduce or prevent the induction of a consequent pro-inflammatory response. WO 03/063759 discloses peptides and peptide analogs of heat shock proteins capable of interacting directly with dendritic cells which are useful for prevention or treatment of either inflammatory disorders and autoimmune diseases or malignancies, viral infections and allergy.

Magainins Magainins were previously disclosed as antimicrobial peptides, that can inhibit, prevent or destroy the growth or proliferation of microbes, such as bacteria, fungi, viruses or the like (U.S. 4,962,277, U.S. 5,221,664).

The magainins (U.S. 5,856,127), from the skin of the African clawed frog (Xenopus laevus), are some of the smallest natural antimicrobial peptides yet discovered, ranging from 21-27 amino acids in length (Zasloff 1987, Proc. Natl. Acad. Sci. USA, 84 5449-53). These cationic peptides form an amphipathic, single α-helix which can span a cell membrane. It. is hypothesized that these molecules form ion channels in the microbial cell's membrane which the cell cannot control, eventually leading to lysis of the cell. The .α-helix is essential for activity and changes in the amino acid sequence which stabilize this helical structure enhance the molecule's lytic activity against selected bacteria (Chen et al. 1988, FEBS Letters 236, 462-466). The magainins are of interest because of their ability to lyse bacterial and yeast cells but not animal cells (Soravia et al. 1988, FEBS Letters 228, 337-340), suggesting good potential for use in agricultural and forest plant species.

Magainin II amide binds to bacterial cell wall constituents, mostly to negatively charged phospholipids, including LPS (Reed et al, 2003, J. Biol. Chem. 278, 31853-31860; Bandholtz et al., 2003, Cell. Moll. Life Sci., 60, 422-429). Nowhere in the background art is it taught or suggested that such or similar agents may be used for treatment of inflammatory non-infectious diseases or autoimmune diseases.

The involvement of the heat shock proteins in modulation of immune responses is now well established, however, nowhere in the background art is it taught or suggested which particular region within the HSP60 sequence is responsible for its immunostimulatory activity via binding to LPS and presenting to the TLR4 complex, or whether its specific inhibition may lead to suppression of HSP60 proinflammatory activity, nor which specific region in HSP60 is responsible for its binding to macrophages.

SUMMARY OF THE INVENTION

The present invention discloses certain novel agents and compositions capable of modulating specific epitopes of the human heat shock protein 60 (HSP60) molecule having the sequence set forth in SEQ ID NO:l. One aspect of the present invention relates to amino acids 481-500 of human HSP60 comprising the sequence KNAGVEGSLIVEKIMQSSSE having the sequence set forth in SEQ ID NO:2, which is now identified as a mediator region responsible for binding of human HSP60 to macrophages. The invention further discloses fragments, analogs and functional derivatives of said region and other novel agents capable of mimicking this region thereby inhibiting said binding of HSP60 to macrophages.

According to specific embodiments, the present invention provides immunomodulatory agents capable of inhibiting the binding of HSP60 to macrophages, the binding mediated by at least part of amino acids 481-500 of HSP60 having the sequence set forth in SEQ ID NO:2.

According to one embodiment the immunomodulatory agent is a peptide fragment, analog, mimetic or functional derivative of at least part of amino acids 481-500 of HSP60 having the sequence set forth in SEQ ID NO:2, that is capable of binding to macrophages and inhibiting their binding to human HSP60.

According to another embodiment the immunomodulatory agent is an antibody or antibody fragment or a peptide derived from the binding site of said antibody, capable of binding to at least part of amino acids 481-500 of HSP60 having the sequence set forth in SEQ ID NO:2, and inhibiting its binding to macrophages.

According to yet another embodiment the immunomodulatory agent is a small organic molecule, capable of binding to at least part of amino acids 481-500 of HSP60 having the sequence set forth in SEQ ID NO:2, and inhibiting its binding to macrophages. Another aspect of the present invention relates to the region of HSP60 comprising amino acids 335-366 of human HSP60 having the sequence EDVQPHDLGKVGEVIVTKDDAMLLKGKGDKAQ set forth in SEQ ID NO:3. This sequence was unexpectedly identified herein as comprising an epitope involved in LPS- binding and responsible for the LPS, the binding mediated proinflammatory activity of HSP60. The present invention provides novel anti-inflammatory agents capable of blocking binding of HSP60 to LPS, the binding mediated by at least part of amino acids 335-366 of HSP60.

According to one embodiment the anti-inflammatory agent is a peptide fragment, analog, mimetic or functional derivative of at least part of amino acid sequence 335-366 of human HSP60 having the sequence set forth in SEQ ID NO:3, and is capable of binding to LPS and inhibiting its binding to human HSP60.

According to another embodiment the anti-inflammatory agent is an antibody or antibody fragment or a peptide derived from the binding site of said antibody, capable of binding to at least part of amino acid sequence 335-366 of human HSP60 having the sequence set forth in SEQ ID NO: 3, and inhibiting its binding to LPS.

According to yet another embodiment the anti-inflammatory agent is a small organic molecule capable of binding to LPS and inhibiting its binding to human HSP60.

According to a specific embodiment amino acid residues 354-365 of human HSP60 having the sequence DAMLLKGKGDKA having the sequence set forth in SEQ ID NO:4, are involved in LPS-binding. The invention provides anti-inflammatory agents such as fragments, mimetics, analogs, salts, and functional derivatives of said peptide which interfere with the binding of LPS to HSP60.

According to one embodiment the anti-inflammatory agent is a peptide fragment, analog, mimetic or functional derivative of at least part of amino acids 354-365 of human HSP60 having the sequence set forth in SEQ ID NO:4, and is capable of binding to LPS and inhibiting its binding to human HSP60.

According to another embodiment the anti-inflammatory agent is an antibody or antibody fragment or a peptide derived from the binding site of said antibody, capable of binding to at least part of amino acids 354-365 of human HSP60 having the sequence set forth in SEQ ID NO:4, and inhibiting its binding to LPS. According to yet another embodiment the anti-inflammatory agent is a small organic molecule capable of binding to LPS and inhibiting its binding to human HSP60.

According to a specific embodiment the anti-inflammatory agent is capable of attenuating the immunostimulatory action of HSP60 by inhibiting its ability to present bound LPS to the Toll receptor 4 (TLR4) complex. According to further embodiments the invention provides pharmaceutical compositions comprising at least one immunomodulatory or anti-inflammatory agent according to the invention.

According to one aspect the pharmaceutical compositions comprise at least one immunomodulatory agent capable of inhibiting the binding of HSP60 to macrophages, the binding mediated by at least part of amino acids 481-500 of HSP60 having the sequence set forth in SEQ ID NO:2. The formulation of said agent into a pharmaceutical composition further comprises the addition of a pharmaceutically acceptable carrier, excipient and/or diluent.

According to another aspect the pharmaceutical compositions comprise at least one anti-inflammatory agent capable of inhibiting the binding of HSP60 to LPS, the binding mediated by at least part of amino acids 335-366 of human HSP60 having the sequence set forth in SEQ ID: NO:3. The formulation of said agent into a pharmaceutical composition further comprises the addition of a pharmaceutically acceptable carrier, excipient and/or diluent.

According to yet another aspect the pharmaceutical compositions comprise at least one anti-inflammatory agent capable of inliibiting the binding of human HSP60 to LPS, the binding mediated by at least part of amino acids 354-365 of human HSP60 having the sequence set forth in SEQ ID NO:4. The formulation of said agent into a pharmaceutical composition further comprises the addition of a pharmaceutically acceptable carrier, excipient and/or diluent. Another aspect of the present application relates to the discovery that magainin II amide having the sequence GIGKFLHSAKKFGKAFVGEIMNS-NH₂ set forth in SEQ ID NO:5, interferes with the immunostimulatory property of HSP60. According to one embodiment the present invention discloses the use of magainin II amide, and analogs, fragments, salts and functional derivatives thereof for inhibition of the pro-inflammatory reactivity of HSP60. According to yet another embodiment the present invention provides pharmaceutical compositions comprising magainin II amide and analogs, fractions, salts, and functional derivatives thereof as an active ingredient for inhibiting the pro-inflammatory activity of HSP60 in the innate immune system and for treatment of diseases and disorders associated with abnormal levels of HSP60, particularly inflammatory and autoimmune diseases.

The agents of the present invention are useful as active ingredients in pharmaceutical compositions for the prevention or treatment of diseases and disorders involving abnormal

HSP60 levels their etiology or pathology. These diseases and disorders comprise autoimmune diseases and inflammatory reactions of the joints, the skin, the mucous membranes and inner organs.

Inflammatory diseases that can be treated by the agents and methods of the present invention include, but are not limited to: rheumatic diseases including rheumatoid arthritis, osteoarthritis, psoriatic arthritis, juvenile arthritis, Reiter's syndrome, infectious arthritis, ankylosing spondylitis, systemic lupus erythematosus, and gout; inflammatory diseases of the gastrointestinal tract including the oral cavity, inflammation associated with gingivitis or periodontal disease, inflammation associated with inflammatory bowel disease, Crohn's disease; inflammatory diseases of the respiratory tract including obstructive bronchitis, bronchial asthma, inflammatory pulmonary disease; inflammatory vascular and cardiac diseases; inflammatory allergic diseases; inflammatory dermatological diseases including psoriasis and inflammatory skin diseases.

Autoimmune diseases that can be treated by the agents and methods of the present invention include, but are not limited to, insulin dependent diabetes mellitus (i.e., IDDM, or autoimmune diabetes), multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, psoriasis, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia purpura, rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bulbous pemphigoid, discoid lupus, ulcerative colitis, Crohn's disease and dense deposit disease. The efficacy of the compositions of the present invention to treating diseases may be established in animals that develop spontaneous or induced models for such diseases, such as, for example non-obese diabetic (NOD) mice for IDDM and experimental autoimmune encephalomyelitis (EAE) for multiple sclerosis.

Another aspect of the present invention is to provide methods of treatment of an individual in need thereof by administering pharmaceutical compositions comprising an agent according to the present invention. These pharmaceutical compositions may be administered by any suitable route of administration, including orally, topically, transdermally or systemically as well as via nasal or oral ingestion. Preferred modes of administration include but are not limited to parenteral routes such as intravenous (i.v.) intramuscular (i.m.), and intraperitoneally (i.p.) injections, as well as nasal or oral ingestion. As it is known to those skilled in the art the pharmaceutical compositions may be administered alone or in conjunction with additional treatments for the conditions to be treated.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will better be understood in relation to the drawings and detailed description of the preferred embodiments which follow:

FIGURE 1: Demonstrates the effect of 20-mer Peptides on HSP60 Binding to J774A.1 cells.

FIGURE 2: Depicts the effect of Different 20-mer Peptides on HSP60 Binding (A) and TNFα Production (B) on J774A.1 cells.

FIGURE 3: displays nitrite formation (A) and HSP60 binding (B) in cultures of BMM from C57BL/10ScSn and from TLR4-deficient C57BL/10ScCr mice.

FIGURE 4: Shows the effect of treatment with proteinase K (A), heat (B), and PmB (C) on HSP60-stimulated TNFα production in J774A.1 macrophages.

FIGURE 5: Presents the effect of magainin I, magainin II and magainin II Amide on HSP60-stimulated TNFα production. FIGURE 6: Dot blot experiments analyzing the effect of mixtures (A) and of individual (B) 20-mer peptides on the binding of antibody clone 4B9/89 to HSP60.

FIGURE 7: Binding of [³H]LPS to different proteins.

FIGURE 8: Dose dependence and inhibition of [³H]LPS-binding to HSP60.

FIGURE 9: Effect of anti-HSP60 antibodies on [3H]LPS-binding to HSP60. FIGURE 10: Effect of pep354-365 on [³H]LPS-binding to HSP60.

DETAILED DESCRIPTION OF THE INVENTION

Human heat shock protein 60 has been shown to bind to the surface of innate immune cells and to elicit pro-inflammatory responses. The present invention discloses the identification of epitopes of HSP60 responsible for evoking these reactions.

It is disclosed that binding of HSP60 to its receptor is not sufficient to mediate the macrophage stimulatory effect and that macrophage activation, but not binding of HSP60 is TLR4- and CD14-dependent. Rigid controls demonstrated that the only macrophage-activity compound in the recombinant HSP60 preparation is tightly linked to the HSP60 molecule. Inhibition studies with a cationic peptide suggested that the immunostimulatory epitope resembles or mimics LPS. The disclosure of the present invention that different regions of the HSP60 molecule are involved in binding and activation of macrophages allow the definition, identification and characterization of inhibitors of the immunoregulatory functions of HSP60. When using a peptide library to characterize epitope(s) of HSP60 involved in binding to macrophages, a peptide corresponding to aa residues 481-500 of the human HSP60 sequence was identified. This peptide mediated strong dose-dependent inhibition of HSP60 binding to macrophages. Significant inhibition of binding was already reached at low micromolar concentrations of the peptide pep481-500. No inhibitory effect, even at 125 μM, was observed for the overlapping peptides pep471-490 or pep491-510.

Analysis of several monoclonal antibodies against HSP60 recognizing different regions of the native HSP60 molecule (amino acid residues 1-200, 335-366 / 484-547, and 383-447) revealed that none blocked HSP60 binding, whereas the pro-inflammatory activity of HSP60 could be inhibited by only one antibody, clone 4B9/89 that has been described to either bind to region of aa residues 335-366 or to aa residues 484-547 of the human HSP60 sequence (Sharif et al., 1992, Arthritis Rheum., 35, 1427-1433). This suggests that region 335-366 of the HSP60, having the sequence

EDVQPHDLGKVGEVIVTKDDAMLLKGKGDKAQ, harbors the immunostimulatory epitope while region 481-500, having the sequence KNAGVEGSLIVEKIMQSSSE, the binding epitope.

It is now disclosed that binding of HSP60 to its receptor is not sufficient to mediate the macrophage stimulatory effect.

In the present invention it is demonstrated for the first time that the macrophage- stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and that it can be neutralized by the lipopolysaccharide (LPS)-binding peptide magainin II amide. Moreover, it was demonstrated that HSP60 specifically binds ³H-labeled LPS. [³H]LPS-binding to HSP60 was saturable and displaced by the unlabeled ligand. It is also found that the potent LPS binding mediated by the tetrapeptide sequence LKGK is also present in human HSP60 at positions 358-361.

According to one approach to characterize epitopes of HSP60 involved in receptor binding and activation of macrophages, peptides corresponding to overlapping 20-mer fragments of human HSP60 were screened for their ability to block binding to macrophages. A peptide corresponding to the amino acids 481 to 500 of the human HSP60 having the sequence KNAGVEGSLIVEKIMQSSSE was identified. This peptide mediated a strong dose dependent inhibition of HSP60 binding to macrophages. Significant inhibition of the binding was already reached at micromolar concentrations of the peptide pep481-500. No inhibitory effect, even at 125 μM, was observed for the overlapping peptides pep471-490 or pep491-510. This indicates a major contribution of the region around amino acid 490 to cell surface binding of HSP60. Interestingly, an excess of the inhibitory peptide pep481-500 failed to affect HSP60-stimulated induction of inflammatory mediators, as shown by TNFα and NO production. Also, none of the other peptides of the HSP60 sequence was found to block macrophage activation by HSP60. This suggested that multiple epitopes or that conformational epitopes are involved.

Further insight was gained from inhibition studies with mAbs binding to different regions of the HSP60 molecule. One mAb, clone 4B9/89, which caused a dose dependent inhibition of the HSP60-stimulated TNFα production, was identified. Clone 4B9/89 recognizes the amino acids 335-366 or 484-547 of the human HSP60 sequence (Sharif et al, ibid). Since the antibody did not block pep481-500-dependent cell surface binding of HSP60, it seems probable that the region 335-366 (comprising the sequence EDVQPHDLGKVGEVIVTKDDAMLLKGKGDKAQ), accounts for the macrophage- stimulatory property of HSP60. The present invention discloses that macrophage stimulation is mediated by a defined region of the HSP60 molecule, and this region does not contain the epitope involved in cell surface binding of HSP60.

Previous studies had indicated that TLR4 and CD 14 are involved in the inflammatory signaling of HSP60 and that TLR4 is not involved in HSP60 binding. This issue was reassessed by studying in parallel HSP60 binding and macrophage stimulation.

HSP60 failed to evoke an inflammatory response in BMM from TLR4-defective mice, although normal binding of HSP60 to the cell surface was observed.

The present invention is based on part on the unexpected discovery that an LPS binding region is located within the HSP60 sequence. According to one aspect of the invention it is now disclosed that amino acids 335-366 of human HSP60 comprises an LPS binding sequence that exerts its immunostimulatory action by presenting bound LPS to the

TLR4 complex.

The findings made in the present invention implicate a physiological role of human

HSP60 as LPS-binding protein and identified a defined region of HSP60, aa residues 354- 365, involved in LPS-binding. The results demonstrate that the capacity of human HSP60 to activate innate immune cells depends on LPS, specifically bound to the chaperone. Previous investigations have shown that the induction of a pro-inflammatory response by HSP60 is mediated via the TLR4/CD14 receptor complex in macrophages and DC, which is also involved in LPS-signaling. Because of the similarity of the immunostimulatory properties of HSP60 and LPS, it had been suggested that endotoxin contaminations might be responsible for the observed inflammatory effects of the stress protein preparations. The results of the present invention revealed that the immunostimulatory property of human HSP60 is sensitive to protease treatment or heat denaturation, but cannot be inhibited by the potent LPS-binding peptide PmB. More importantly, when small amounts of LPS were added to HSP60 as defined contamination, the immunostimulatory properties became protease resistant, heat resistant and PmB sensitive reflecting the amount of LPS admixed. These experiments excluded a role of free contaminating LPS, but indicated that the immunostimulatory principle is tightly linked to the HSP60 molecule.

Further insight was gained from studies with LPS-binding peptides other than PmB. In the present invention magainin II amide is identified as a peptide that interferes with the immunostimulatory property of human HSP60. The specificity was verified in that two closely related defensins, magainin I and II did not block macrophage activation by HSP60. Conversely, magainin II amide did not inhibit macrophage activation via an HSP60/LPS-independent pathway, as shown by control experiments with MALP-2, which is known to act via TLR2 (Kraus, E., and Femfert, U., 1976, Hoppe Seylers Z. Physiol. Chem. 357, 937-947). Taken together, the findings indicate that macrophage stimulation by human

HSP60 is not due to free contaminating LPS, but to LPS or structurally related molecules binding tightly to the surface of the chaperone.

The findings were confirmed by the analysis of the binding of radioactive-labeled LPS to low endotoxin human HSP60 in a binding assay. For the first time a direct binding of LPS to HSP60, which was specific, saturable and competable by unlabeled LPS was demonstrated. Saturable binding of LPS to HSP60 was reached at 1 μM LPS, with a KD of ~ 300 nM. At concentrations higher than 5 μM of LPS, a further significant increase in LPS- binding values was observed. This observation could be explained by increased aggregation of LPS molecules at higher concentrations. Amphipathic molecules like LPS usually occurs as monomers in solution. However, at higher concentrations of LPS changes in physical properties take place, resulting in the formation of organized aggregates, i.e. micelles or lamellar structures. Another explanation for the finding could be the existence of additional low-affinity binding sites on HSP60 for LPS. By the analysis of the results we determined an occupancy ratio of approximately 2 mol LPS : 1 mol HSP60.

Interestingly, two other chaperones, mammalian HSP70 and HSP90, have also been reported to function as receptors for LPS (Kol et al, 2000, ibid, Wang et al., 1998, J. Biol. Chem. 273, 24309-24313). Moreover, it has been shown that HSP70 binds to other bioactive lipids and that the recognition of HSP70 by a CD14-containing receptor cluster is dependent on HSP70-lipid association (Akashi et al, 2000, Biochem. Biophys. Res. Commun. 268, 172-177). Although HSPs are typically regarded as being intracellular, they can be expressed on the surface of mononuclear cells and endothelial cells (Vabulas et al., 2002, ibid; Becker et al, 2002, J. Cell. Biol. 158, 1277-1285). Moreover, soluble autologous HSP60 and HSP70 have been identified in the circulation of human individuals where serum concentrations reached the microgram level (Asea et al. ibid; Wallin et al., 2002, Trends. Immunol. 23, 130-135). Furthermore, LPS concentrations in blood range from 20 - 100 pg in the periphery to 1 ng close to the gut (Bausinger et al, 2002, Eur. J. Immunol. 32, 3708-3713; Gao, B., and Tsan, M. F., 2003, J. Biol. Chem. 278, 174-179), and therefore are sufficiently high to allow loading of extracellular HSP60 with LPS, even in the absence of bacterial infections, under physiological conditions.

The ability of HSP60 to bind LPS and present it to the TLR4/CD14 receptor complex represents a new meaningful biological property that my be used in designing agents capable of fighting inflammation and other conditions which involves harmful LPS activity.

The property of mammalian HSP60 to act as a danger antigen (Byrd et al, 1999, Proc. Natl. Acad. Sci. U.S.A 96, 5645-5650, Triantafilou et al., 2001, Nat. Immunol. 2, 338-345; Pfeiffer et al., 2001, Eur. J. Immunol. 31, 3153-3164), includes the element to serve as a sensor for danger signals and to present such structures to the innate immune system. The concept of HSP60 acting as a sensor for microbial structures may also be extend to other mammalian chaperones. HSP70 also has been observed to bind LPS in a tight and PmB- resistant manner (Knolle et al., 1999, J. Immunol. 162, 1401-1407). High-affinity and specific binding of LPS to glycoprotein 96 (gp96) has been shown by a recent study (Matsuzaki et al., 1997) Biochim. Biophys. Acta 1327, 119-130). Furthermore, HSP90 has been suggested to act as primary receptor for immunostimulatory bacterial CpG DNA and to deliver these ligands to the TLR9 complex (Vorland et al., 1999, Scand. J. Infect. Dis. 31, 467-473). An important biological function of mammalian chaperones seems possible, i.e. the improvement of the recognition of microbial structures by innate immune cells.

Furthermore, the present invention provides identification of the epitope region of the human HSP60 molecule responsible for LPS-binding. Inhibition studies with commercially available monoclonal antibodies directed against different epitopes of the native human HSP60 molecule were performed. Only one mAb, clone 4B9/89, which caused a dose-dependent inhibition of HSP60-stimulated TNFα production from J774A.1 macrophages, was identified. This antibody was reported to bind to region aa335-366 of the human HSP60 sequence (Matsuzaki et al., 1999, FEBS Lett . 449, 221-4). Subsequently, the potential inhibitory effect of this antibody on LPS-binding to human HSP60 in the radioactive [³H]LPS-binding assay was analyzed. The results of these experiments revealed that mAb clone 4B9/89 strongly inhibited [³H]LPS-binding to HSP60 by more than 90%, thereby indicating that the region aa335-366 of the HSP60 molecule is involved in LPS- binding. By screening selected 20-mer peptides, covering the region aa331-380 of human HSP60, in the [³H]LPS-binding assay, the LPS-binding epitope region could be further restricted to the region aa351-370 of the HSP60 molecule. Only peptide pep351-370 strongly inhibited LPS-binding to HSP60, whereas none of the adjacent 20-mer peptides did. Finally, the inhibition of [ H]LPS-binding to HSP60 by a 13-mer peptide, covering the region aa354-365 of the human HSP60 sequence was tested. It was found that this peptide dose dependently decreased [³H]LPS-binding to HSP60 to about 30% when tested in a range of 0.5 μM to 15 μM.

Taken together, these findings indicate that the region aa354-365 of the HSP60 molecule is involved in specific binding of LPS.

By searching for sequence homologies between the human HSP60 region aa354- 365 and other LPS-binding proteins, Factor C, present in the lysate of amebocytes of the horseshoe crab Limulus polyphemus (Tan et al., 2000, FASEB J. 14, 1801-1813) was identified. Factor C is a serine protease that initiates a coagulation cascade in the hemo lymph of Limulus after contact to LPS. Recognition of LPS by Factor C obviously depends on the presence of several high-affinity LPS-binding regions on the protease, so called sushi domains (Ho, B. 1983, Microbio. Lett. 24, 81-84; Muta, T. and Iwanaga, S., 1996, Prog. Mol. Subcell. Biol. 15, 154-189). By the analysis of peptides derived from the sequence of sushi domain 1 and 3, they found that a modification of sushi domain 1 -derived peptide SI (replacement of two amino acid with lysine residues, termed SΔl) resulted in an improvement of its LPS neutralization potential and LPS-binding affinity compared to the wildtype SI peptide (Tan et al. ibid). By sequence alignments of the LPS-binding region aa353-365 of HSP60 with peptide SI and SΔl the amino acid motifs LKG (SEQ ID NO:6) and LKGK (SEQ ID NO:7), respectively, displaying 100% identity was identified. The sequence motifs LKG and LKGK have also been found in other mammalian HSPs, but not in microbial HSP60 as depicted in Table I.

Table I. Sequence alignments of Factor C-derived sushi peptides (SI, SΔl) versus HSPs

Additional search for sequence homologies in human LPS-binding proteins, like

LPS-binding protein (LBP) or bactericidal/permeability-increasing protein (BPI) did not result in the identification of sequence identities. However, BPI and LBP, which are both members of the BPI protein family (Elsbach, P. and Weiss, J., 1998, Curr. Opin. Immunol. 10, 45-49; Weiss, J. 2003, Biochem. Soc. Transactions 31, 785-790), are known to share only 45% primary structural identity, but are closely similar in their tertiary structure (Beamer et al., 1997, Science 276, 1861-1864; Iovine et al., 2002, J. Biol. Chem. 277, 7970- 7978). Taken together, the present invention demonstrated for the first time a specific binding site for LPS on the human HSP60 molecule, namely region aa354-365, which is suggested to be involved in macrophage stimulation. Furthermore, this new data demonstrate that this region of HSP60 is different from the epitope responsible for receptor binding on macrophages, previously identified herein as region aa481-550 on the HSP60 molecule. In mammalians the existence of proteins like HSP60, specialized for the recognition and binding of microbial structures may serve important biological functions, i.e. the improvement of the efficiency of LPS recognition by innate immune cells.

The possible contribution of LPS to immunostimulatory properties of HSPs (Wallin et al. ibid) deserves special comments. Two recent studies demonstrated that human HSP70 might be heavily contaminated with LPS, accounting for its immunostimulatory activity. This activity could be blocked by PmB (Bausinger et al. ibid, Gao ibid). Unfortunately, these works did not study the nature of PmB resistant stimulatory activities of HSP70, which is seen at higher HSP70 concentrations that applied by these groups. The issue is not easily resolved, since not all LPS species are neutralized by PmB. Furthermore, HSP90 and HSP70 were found to be able to bind to LPS (Byrd et al. ibid, Triantafilou et al., ibid). Moreover, it has been shown that HSP70 binds to other bioactive lipids and that the recognition of HSP70 by a CD14-containing receptor cluster is dependent on the HSP70- lipid association (Pfeiffer et al, ibid). Based on these findings, it has been suggested that HSPs function as LPS binding and recognition enhancing proteins, delivering LPS to its receptor complex including TLR4 and CD 14 (Wallin et al. ibid). It is now disclosed that stress induced endogenous HSPs interact with microbial products or immunostimulatory endogenous lipids to activate macrophages. LPS concentrations in blood range from 20 - 100 pg in the periphery to 1 ng close to the gut (Knolle et al., ibid), and therefore are sufficiently high to allow loading of extracellular HSP with LPS, in the absence of bacterial infections. It was surprisingly found that of several HSP60 antibodies tested, only one interfered with the macrophage-stimulatory activity of HSP60. If indeed LPS or other microbial compounds represent the immunostimulatory epitope of HSP60, this is restricted to a single region of the HSP60 molecule. This strongly argues against non specific adsorption of LPS onto the HSP60 surface and "presentation" to CD14/TLR4, but suggests a specific site responsible for the pro-inflammatory property of HSP60. This site either directly interacts with CD14/TLR4 or presents immunostimulatory compounds to the LPS receptor complex on macrophages. Experiments with different concentrations of PmB and artificial mixtures of E. coli LPS and HSP60 excluded even a partial inhibition of HSP60- mediated immunostimulation by the LPS neutralizing agent. Heat treatment of HSP60 was also done in the presence of low concentrations of LPS to exclude the possibility that LPS might be trapped by the insoluble denatured protein colloid. Magainin II amide was identified in the present invention as a peptide that interferes with the immunostimulatory property of HSP60. The specificity was verified in that two closely related defensins, magainin I and II did not block macrophage activation by HSP60. Conversely, magainin II amide did not inhibit macrophage activation via an HSP60/LPS- independent pathway. Magainin II amide is a cationic peptide and binds to bacterial cell wall constituents, mostly to negatively charged phospholipids, including LPS (Matsuzaki et al, 1997 ibid; Vorland et al, ibid). Most interestingly, according to the finding of the present invention, magainin II amide inhibits the immunostimulatory activity of HSP60 and also of LPS. It is disclosed that the macrophage activating epitope of HSP60 either is a LPS- like molecule binding to a specific site on the HSP60 surface and not recognized by PmB, or the epitope on HSP60 consists of negatively charged side chains of amino acids of the HSP60 sequence with a conformation mimicking the charge pattern of LPS. In parallel studies, magainin II amide failed to interfere with the binding of HSP60 to the macrophage surface. This lends further support to the concept that two different epitopes of HSP60 are involved in surface binding and delivering an activation signal to the CD14/TLR4 receptor complex.

Terminology and definitions:

The term "heat shock protein " relates to any member of heat shock proteins family also known as chaperones. The term "heat shock protein" also referred to "stress protein" a term that was used in the past to such molecules.

"Functional derivatives" of the peptides of the invention as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide, do not confer toxic properties on compositions containing it and do not adversely affect the antigenic properties thereof.

These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed by reaction with acyl moieties.

As used herein, "functional" includes reference to an activity sufficient to produce a desired effect. A "functional peptide" will have the activity to achieve a desired result, such as cytokine inhibition or induction. Alternatively, a functional peptide will provide the cell with a beneficial or therapeutic effect, such as induction of release of a specific mediator. Thus reference to a particular peptide or "functional peptide" includes the naturally occurring peptide sequence or a peptide that has the substantially the same activity as the naturally occurring sequence. "Functional peptides" of the invention also include modified peptides (with amino acid substitutions, both conservative and non-conservative) that have the same activity as a wild-type or unmodified peptide. "Salts" of the peptides of the invention contemplated by the invention are physiologically acceptable organic and inorganic salts.

The term "analog" further indicates a molecule which has the amino acid sequence according to the invention except for one or more amino acid changes. Analogs according to the present invention may comprise also peptide mimetics. "Peptide mimetic" means that a peptide according to the invention is modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with other covalent bond. A peptidomimetic according to the present invention may optionally comprises at least one bond which is an amide-replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The design of appropriate "analogs" may be computer assisted.

The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies) and antibody fragments so long as they exhibit the desired biological activity. "Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to antibodies that are highly specific, being directed against a single antigenic site. The monoclonal antibodies to be used in accordance with the present invention may be made by recombinant DNA methods (see, e.g., U.S. Patent 4,816,567 of Cabilly et al.).

The term "framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined. The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR". The CDRs are primarily responsible for binding to an epitope of an antigen. The extent of FRs and CDRs has been precisely defined (see, Kabat et al, ibid).

As used herein, the term "humanized antibody" refers to an antibody comprising a framework region from a human antibody and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin. Parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. Importantly, the humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDRs. For further details, see e.g. U.S. Pat. No. 5,225,539 assigned to Medical Research Council, UK.

The expression "human antibody" is intended to mean an antibody encoded by a gene actually occurring in a human, or an allele, variant or mutant thereof.

As used herein and in the claims, the phrase "therapeutically effective amount" means that amount of peptide or peptide analog or composition comprising same to administer to a host to achieve the desired results for the indications disclosed herein.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Pharmaceutical compositions may also include one or more additional active ingredients.

Certain abbreviations are used herein to describe this invention and the manner of making and using it. For instance, aa/s refers to amino acid/s, BSA refers to bovine serum albumin, DC refers to dendritic cells, ELISA refers to enzyme-linked immunosorbent assay, FCS refers to fetal calf serum, HSP refers to heat shock protein, IDDM refers to Insulin- dependent Diabetes Mellitus, IFN refers to interferon-gamma, IL- refers to interleukin, IP- 10 refers to interferon-inducible protein 10, kD refers to Kilo Dalton, LAL refers to Limulus amebocyte lysate, LPS refers to lipopolysaccharide, mAbs refers to monoclonal antibodies, MALP-2 refers to macrophage-activating lipopeptide-2, NO refers to nitric oxide, PI refers to L-A-phosphatidylinositol, PMA refers to phorbol myristyl acetate, PmB refers to polymyxin B, SD refers to standard deviation, Tlr refers to toll-like-receptor, Tlr2 refers to Toll-like receptor 2, Tlr4 refers to Toll-like receptor 4, TNF refers to tumor necrosis factor alpha.

The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent and convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. The D isomers are indicated by "D" before the residue abbreviation.

Conservative substitution of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions includes replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration into the central nervous system, targeting to specific cell populations and the like. One of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Target Inflammatory Disorders and Diseases

Inflammatory diseases and disorders that can be treated by the methods of the present invention include, but are not limited to, inflammation of the joints, the skin, the mucous membranes and inner organs; Rheumatic diseases including rheumatoid arthritis, osteoarthritis, psoriatic arthritis, juvenile arthritis, Reiter's syndrome, infectious arthritis, ankylosing spondylitis, systemic lupus erythematosus, and gout; Inflammatory diseases of the gastrointestinal tract including the oral cavity, inflammation associated with gingivitis or periodontal disease, inflammation associated with Inflammatory Bowel Disease, Crohn's disease; Inflammatory diseases of the respiratory tract including obstructive bronchitis, bronchial asthma, inflammatory pulmonary disease; Inflammatory vascular and cardiac diseases; Inflammatory allergic diseases; Inflammatory dermatological diseases including psoriasis and inflammatory skin diseases.

The methods of the present invention can be used to treat such inflammatory diseases and disorders by inhibiting the proinflammatory and immunostimulatory activity of HSP60, through inhibiting its binding to LPS, or by means of inliibiting its binding to macrophages.

Target Autoimmune Diseases

Autoimmune diseases that can be treated by the methods of the present invention include, but are not limited to, insulin dependent diabetes mellitus (i.e., IDDM, or autoimmune diabetes), multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, psoriasis, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia purpura, rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bulbous pemphigoid, discoid lupus, ulcerative colitis, Crohn's disease and dense deposit disease. The diseases set forth above, as referred to herein, include those exhibited by animal models for such diseases, such as, for example non-obese diabetic (NOD) mice for IDDM and experimental autoimmune encephalomyelitis (EAE) mice for multiple sclerosis.

The methods of the present invention can be used to treat such autoimmune diseases by reducing or eliminating the immune response to the patient's own (self) tissue, or, alternatively, by reducing or eliminating a pre-existing autoimmune response directed at tissues or organs transplanted to replace self tissues or organs damaged by the autoimmune response.

Pharmacology

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants for example polyethylene glycol are generally known in the art.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the variants for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the peptide and a suitable powder base such as lactose or starch.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable natural or synthetic carriers are well known in the art (Pillai et al, Curr. Opin. Chem. Biol. 5, 447, 2001). Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Apart from other considerations, wherein the novel active ingredients are peptides, peptide analogs or cells, dictates that the formulation be suitable for delivery of these type of compounds. Clearly, peptides are less suitable for oral administration due to susceptibility to digestion by gastric acids or intestinal enzymes. The preferred routes of administration of peptides are intra-articular, intravenous, intramuscular, subcutaneous, intradermal, or intrathecal. A more preferred route is by direct injection at or near the site of disorder or disease.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC^_Q (the concentration which provides 50% inhibition) and the LD50

(lethal dose causing death in 50 % of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (e.g. Fingl, et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.

The following examples are intended to illustrate how to make and use the compounds and methods of this invention and are in no way to be construed as a limitation. Although the invention will now be described in conjunction with specific embodiments thereof, it is evident that many modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such modifications and variations that fall within the spirit and broad scope of the amended claims. EXAMPLES General methods:

Mice and Cell Lines:

C57BL/10ScCr mice, lacking the complete TLR4 protein and C57BL/10ScSn mice with normal TLR4 protein expression (Poltorak et al., 1998, Science 282, 2085-2088), were kindly provided by Dr. M. Freudenberg (Max Planck Institute for Immunology, Freiburg, Germany).

Mouse bone marrow cells were obtained by flushing femurs and tibias with ice-cold PBS and washed by centrifugation (500 x g, 5 min). A total of 3.5 x 106 bone marrow cells were incubated in tissue culture dishes in 10 ml Pluznik medium containing 5% horse serum, 15% fetal calf serum (FCS, Gibco-BRL, Life Technologies, Rockville, CA), 15% L929 cell-conditioned medium and 65% RPMI 1640 (PAA Laboratories GmbH, Linz, Austria) supplemented with 10% FCS, ampicillin (25 mg/1), penicillin (120 mg/1), streptomycin (270 mg/1), 1 mM sodium pyruvate, 2 mM glutamine, non essential amino acids (10 ml/1, 100 x), 24 mM NaHCO3 and 10 mM HEPES (Burgess). After 6 to 7 days, adherent bone marrow-derived macrophages (BMM) were detached by accutase (PAA Laboratories GmbH), followed by washing with RPMI 1640 (500 x g, 5 min), and were used for further studies.

The mouse macrophage cell line J774A.1 was purchased from the German Collection of Microorganisms and Cell Culture (Braunschweig, Germany). J774A.1 cells were cultured in RPMI 1640 medium (PAA Laboratories GmbH, Linz, Austria) supplemented with 10% fetal calf serum (FCS, Gibco-BRL, Life Technologies, Rockville, CA), ampicillin (25 mg/1), penicillin (120 mg/1), streptomycin (270 mg/1), 1 mM sodium pyruvate, 2 mM glutamine, non essential amino acids (10 ml/1, 100 x), 24 mM NaHCO₃ and 10 mM HEPES.

Reagents:

Recombinant human HSP60 was obtained from Peptor Ltd. (Rehovot, Israel). Low endotoxin recombinant human HSP60 (<0.05 EU/μg protein) was from StressGen Biotechnologies (Victoria, BC, Canada). A set of overlapping peptides (pep) of 20 amino acids, spanning the human HSP60 sequence from the amino acids 1 to 560, were from the Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.

Synthetic peptides of 13 amino acids, pep354-365 (corresponding to aa residues 354-365 of the human HSP60 sequence, N-terminally one amino acid was added for technical reasons) and an rmrelated control peptide, were from Eurogentec Deutschland GmbH (Cologne, Germany).

Escherichia coli O26:B6 LPS, Tritirachium album proteinase K, polymyxin B

(PmB), magainin I, magainin II, magainin II amide and ovalbumin (OVA) were from Sigma

(Taufkirchen, Germany). Transferrin was from Molecular Probes (Leiden, The

Netherlands). E. coli K12 LCD25 [³H]LPS and unlabeled LPS were from List Biological Laboratories Inc. (Campbell, CA).

The immunostimulatory oligonucleotide ODN1668 5'-TCCATGA CGTTCCTGATGCT-3' containing a CpG motif (21) was from Life Technologies (Karlsruhe, Germany). Murine anti-human HSP60 monoclonal antibodies (mAbs) were from StressGen Biotechnologies (Victoria, BC, Canada; clone LK1), Dianova (Hamburg, Germany; clone 4B9/89) and BD Transduction Laboratories (San Diego, CA; clone 24). Goat anti-mouse IgG antibody was used as isotype control for all murine monoclonal antibodies (Sigma).

The macrophage-activating lipopeptide (MALP)-2 was kindly provided by Dr. P. Mϋhlradt (Immunobiology and Structure Research Groups, Gesellschaft fur Biotechnologische Forschung, Braunschweig, Germany) (22).

Murine anti-human HSP60 monoclonal antibodies (mAbs) were from StressGen Biotechnologies (clone LK1), Dianova (Hamburg, Germany; clone 4B9/89) and BD Transduction Laboratories (San Diego, CA; clone 24). Goat anti-mouse IgG antibody was used as isotype control for all murine monoclonal antibodies (Sigma). Recombinant human HSP60 from Peptor Inc. used in the macrophage-stimulating assays was tested for its endotoxin content by quantitative Limulus amebocyte lysate (LAL) assay (BioWhittaker, Verviers, Belgium) and was been found to contain <1 EU/μg protein. The LPS batch used in these assays gave a reactivity of 0.01 EU per pg. All other substances used in macrophage-stimulating assays showed no reactivity in the LAL assay. Protein Labeling:

Labeling of human HSP60 with fluorescence dye was performed as described previously (14) using the Alexa Fluor488 Protein Labeling kit (Molecular Probes, Leiden, The Netherlands). Briefly, 1 mg of HSP60 was incubated with Alexa Fluor488 in 0.1 M sodium bicarbonate for 1 h at room temperature, followed by incubation for 3 h at 4 °C. Unconjugated dye was removed by extensive dialysis in PBS. By this method one protein molecule was found to bind 6-9 Alexa488 molecules as calculated from OD measurements at 280 nm and 494 nm.

HSP60 Binding and Inhibition Studies:

After two days of continuous culture, J774A.1 cells were gently detached with accutase. The cells were centrifuged at 500 x g for 5 min (4 °C) and resuspended in PBS with 1% BSA for the binding assay (4 °C). Macrophages (1 x 106 cells/ml) were incubated in a total volume of 100 μl in the presence of 350 nM HSP60-Alexa488 for 45 min on ice for the binding studies. Subsequently, cells were washed with PBS/1% BSA and resuspended in PBS containing 1% paraformaldehyde.

For the inhibition studies the macrophages were preincubated with unlabeled HSP60, the different peptides, magainins or PI in the indicated concentrations for 15 min at room temperature. Then Alexa488-labeled HSP60 was added and the incubation was continued for another 45 min on ice.

In the experiments using different anti-HSP60 antibodies, HSP60-Alexa488 was preincubated together with the mAbs for 30 min at room temperature, the mixture was added to the cells and the incubation was continued for another 45 min on ice. After washing and fixation steps, the samples were evaluated using a FACSCalibur flow cytometer (BD Bioscience, Rockville, CA).

Cell surface binding of Alexa488-labeled HSP60 was calculated using the geometric mean fluorescence value after subtracting the autofluorescence of the cells. Stimulation of Macrophages for TNFα and NO Production:

For the stimulation of TNFα production, J774A.1 cells were adjusted to a density of

1 x 10⁶ cells/ml and seeded in the wells of flat-bottom 96-well plates (200 μl/well). After incubation for 18 h (37 °C, 5% CO₂), LPS, recombinant human HSP60 (Peptor Inc.) or

MALP-2 was added to the macrophage cultures. After another 6 h of incubation, supernatants were collected and stored at -20 °C until analysis.

To test their effects on the macrophage-stimulatory properties of LPS, HSP60 or MALP-2, the different magainins were preincubated with the cells (30 min, 37 °C).

When analyzing the effect of anti-HSP60 antibodies, HSP60 was preincubated with the antibodies for 30 min at room temperature and then added to the cells. Heat treatment of LPS or HSP60 was performed by boiling the substances for 10 min before addition to the cells.

For protease or PmB treatment, LPS or HSP60 were incubated with proteinase K (1 h, 37 °C) or PmB (1 h, 4 °C) prior to addition to the cells. The amounts of TNFα in culture supernatants were quantified by sandwich ELISA using an OptEIA mouse TNFα Set (BD PharMingen, San Diego, CA) as described previously (10, 14). The TNFα content was calculated by using a standard curve obtained with recombinant mouse TNFα with substrate solution as a blank.

Radioactive [³H]LPS-Binding Assay:

[³H]LPS (specific activity 0.695 μCi per μg LPS) binding was determined by incubating low endotoxin recombinant human HSP60 (StressGen Biotechnologies, 1 or 5 μg/ml corresponding to 17 and 83 nM, respectively) with 500 nM [³H]LPS for 30 min at 4 °C in a final volume of50 μl of 50 mM Tris (pH 7).

When analyzing the effects of unlabeled LPS or antibodies on [³H]LPS-binding to HSP60, these substances were preincubated (30 min, 4 °C and room temperature, respectively) with HSP60 before [³H]LPS was added. When analyzing the effect of different peptides on [³H]LPS-binding to HSP60, peptides were preincubated (30 min, 4 °C) with [³H]LPS before HSP60 was added. Bound versus free [ H]LPS was assayed by vacuum filtration of the binding reactions on Protran nitrocellulose membranes (Schleicher and Schuell Bioscience Inc., Dassel, Germany). Vacuum filtration was performed with a Bio-Dot microfiltration apparatus (Bio-Rad Laboratories GmbH, Munich, Germany). Filters were rapidly washed with a total volume of 4.5 ml ice-cold 50 mM Tris (pH 7), placed in 1 ml of scintillation fluid (Ultima Gold, PerkinElmer Life Sciences GmbH, Rodgau-Jϋgesheim, Germany), mixed and the retained radioactivity was quantified by liquid scintillation counting.

All binding reactions were corrected by subtraction of background values (membrane-bound [ H]LPS in the absence of protein).

TNFα Measurements: The amounts of TNFα in culture supernatants were quantified by sandwich ELISA using an OptEIA mouse TNFα Set (BD PharMingen, San Diego, CA) as previously described (11, 14). TNFα content was calculated by using a standard curve obtained with recombinant mouse TNFα with substrate solution as a blank. Dot blot analysis:

For dot blot analysis 140 ng human HSP60 or 7.5 μg of single 20-mer peptides with

10 amino acids overlap corresponding to the regions aa321-380 or aa471-560 of human

HSP60 were spotted onto Hybond ECL membranes (Roche Diagnostics, Mannheim,

Germany). After blocking in 5% skim milk solution, membranes were incubated with murine anti-human HSP60 antibody clone 4B9/89 (0.2 μg/ml, overnight).

In another approach antibody clone 4B9/89 (0.5 μg/ml) was preincubated with a mixture of 20-mer peptides (1 μM) covering the regions aal41-230, aa321-380 or aa471- 560, with the individual 20-mer peptides (10 μM) or with PBS (control) for 1 h. Subsequently, these mixtures were incubated with human HSP60 dotted onto membranes for 30 min. The detection was performed with rabbit peroxidase-labeled anti-mouse IgG antibody (1 μg/ml, 45 min, DakoCytomation, Hamburg, Germany) using the ECL detection system (Amersham Pharmacia Biotech, Freiburg, Germany). Quantitative analysis was performed by Lumi-Imager (Boehringer, Mannheim) and shown as BLU.

Measurement of NO Production: The amount of NO released by macrophages was assessed by the determination of accumulated nitrite (NO2-) in cell-free supernatants using the colorimetric Griess reaction, as described previously (Chen et al, 1999, J. Immunol. 162, 3212-3219; Wood et al.,1990, Biochem. Biophys. Res. Commun. 170, 80-88). The amount of accumulated nitrite in the samples was quantified by a standard curve obtained with NaNO₂. Statistical Analysis:

Data were expressed as mean values ± SD. Statistical analysis was performed using the Student t test, two-tailed. Differences were considered statistically significant with p < 0.05. Example 1: Identification of HSP60 Epitopes Involved in Receptor Binding and Induction of Inflammatory Mediators

Competition experiments with a set of overlapping 20-mer peptides spanning the amino acid 1 to 560 of human HSP60, were performed to identify regions of the chaperone involved in surface binding or the induction of a pro-inflammatory cytokine response from macrophages. J774A.1 cells were preincubated with different 20-mer peptides (125 μM, 15 min, room temperature), spanning the human HSP60 sequence from amino acid 1 to 560. Subsequently, 350 nM Alexa488-labeled HSP60 was added and the incubation was continued for another 45 min on ice. The analysis was performed by flow cytometry. Inhibition of HSP60-Alexa488 binding is indicated as %. At the high concentration of 125 μM, only one peptide was found to induce relevant inhibition of HSP60-Alexa488 binding, i.e. more than 50%, pep481-500 (Figure 1). As shown in Figure 2 A, preincubation of J774A.1 cells with increasing concentrations of pep481-500 (1.75 - 125μM) resulted in significant (p < 0.05) inhibition already at 1.75 μM followed by a dose dependent decrease of HSP60-Alexa488 binding from 61% to 27%. The adjacent peptides, peρ471-490 and ρep491-510, did not compete with HSP60-Alexa488 binding.

In parallel, peptides were analyzed for their capacity to interfere with the immunostimulatory effects of HSP60. Peptides were added to cultures of macrophages, which were subsequently stimulated with HSP60 for the release of TNFα and NO. In (A) J774A.1 cells were incubated in the absence (autofluorescence) or presence of 350 nM Alexa488-labeled HSP60 (HSP60*) for 45 min on ice. For competition studies J774A.1 cells were preincubated with unlabeled HSP60, peρ481-500, peρ471-490 or pep491-510 at the indicated concentrations (15 min, room temperature) followed by the incubation with HSP60*. The analysis was performed by flow cytometry and the binding of HSP60* was set to 100%. Error bars represent mean + S.D. of tliree independent experiments. Significant differences to binding of HSP60* alone are indicated as *, p < 0.05. In (B) J774A.1 cells were preincubated with pep481-500, pep471-490 or pep491-510 at the indicated concentrations (15 min). Subsequently medium or HSP60 (3 μg/ml corresponding to 0.05 μM) were added. After 6 h of incubation TNFα concentrations in the cell culture supernatants were measured by sandwich ELISA. Data represent the mean values of TNFα (ng/ml) + S.D. of three independent experiments, each performed in triplicate. The effect of 20-mer Peptides on HSP60 Binding (A) and TNFα Production (B) on J774A.1 cells is described in figure 2. None of the peptides induced significant inhibition of the production of either compound. A more detailed analysis was performed with peptides pep471-490, pep481-500 and pep491-510. Pep481-500 did not inhibit HSP60-induced TNFα production when tested in the concentration range of 3.5 - 50 μM, although there was a dose dependent trend. The adjacent peptides, pep471-490 and pep491-510 also showed no inhibitory effect on HSP60-induced TNFα production (Figure 2B).

In order to achieve stronger steric hindrance of HSP60 regions and to affect also conformational epitopes, the effect of commercially available mAbs, directed against different epitopes of the native human HSP60 protein, was investigated on HSP60-induced TNFα production in J774A.1 cells, and in parallel on the binding of HSP60 to these cells. Clone 24 (aa residues 1-200), clone 4B9/89 (aa residues 335-366 or aa residues 484-547) and clone LK1 (aa residues 383-447), were tested as shown in Table II. When analyzing these antibodies at concentrations of 8 and 20 μg/ml in the binding assay, none of them competed with the binding of HSP60-Alexa488 to the cells.

TABLE II. Effect of anti-HSP60 antibodies on HSP60-induced TNFa production and HSP60 binding to macrophages

HSP60 (3 μg/ml) or Alexa488-labeled HSP60 (350 nM) were preincubated with the different mAbs to HSP60 (30 min) and then added to J774A.1 cells. The inhibitory effects of the antibodies on HSP60-induced TNFa production and on binding of HSP60-Alexa488 were analyzed by sandwich ELISA and by FACS analysis, respectively. TNFa production induced by HSP60 alone and binding of HSP60-Alexa488 alone was set 100%. The data represent mean values ± S.D. of three independent experiments (in the case of TNFa production performed in triplicate). Significant differences to HSP60-induced TNFa production and to binding of HSP60-Alexa488 are indicated as *, p < 0.05.

As shown, preincubation of HSP60 with mAb clone 4B9/89 (suggested to recognize either the region aa335-366 or aa484-547 of human HSP60) resulted in a significant (p < 0.05) dose dependent decrease of TNFα production to 42 ± 7% of the TNFα production induced by HSP60 alone (set as 100%) in J774A.1 macrophages. None of the other tested mAbs to HSP60 or the IgG isotype control could inhibit the HSP60-induced TNFα release in J774A.1 cells. When testing these antibodies for their effect on HSP60-Alexa488 binding to J774A.1 macrophages, none of the mAbs could compete with the binding of HSP60- Alexa488. Taken together, these results indicate that the regions of HSP60 involved in eliciting a pro-inflammatory signal is not responsible for binding to the macrophage surface.

Example 2: Binding of HSP60 is not Sufficient for Immunostimulatory Effect Role of TLR4 and CD14

The role of TLR4 was reassessed in parallel binding and NO secretion studies using BMM of C57BL/10ScCr mice, which do not express TLR4. Nitrite formation (A) and HSP60 binding (B) in cultures of BMM from C57BL/10ScSn and from TLR4- deficient C57BL/10ScCr mice is shown in figure 3. (A) BMM were incubated with medium, LPS (10 ng/ml), HSP60 (10 mg/ml) or ODN1668 (30 μg/ml) as indicated. After 24 h of cultivation, the nitrite concentrations in the cell supernatants were determined by the Griess reaction. The data represent the mean concentrations of nitrite + SD of three independent experiments, each performed in triplicate. (B) BMM were incubated with Alexa488-labeled HSP60 (HSP60*) at the indicated concentrations for 45 min on ice. For competition studies the cells were preincubated with unlabeled 5.1 μM HSP60 (15 min, room temperature) followed by the incubation with HSP60*. The analysis was performed by flow cytometry and binding of HSP60* was calculated using the geometric mean of fluorescence value after subtracting the autofluorescence of the cells.

As shown in Figure 3 A, HSP60 elicited no secretion of NO in these macrophages, while cells of the control strain C57BL/10ScSn did respond. In these experiments LPS and the oligonucleotide ODN1668, containing an immunostimulatory CpG motif, served as controls, since LPS signaling is TLR4-dependent (18, 19), whereas ODN1668 signaling is TLR9-dependent (24). As expected, 10 ng/ml LPS induced a strong formation of nitrite in supernatants of C57BL/10ScSn-derived BMM (35.49 ± 1.02 μM), while there was nearly no nitrite formation in C57BL/10ScCr-derived BMM after LPS stimulation (0.90 ± 0.33 μM). In contrast, ODN1668 induced high amounts of nitrite in the BMM cultures of both mouse strains. In parallel, the binding of HSP60-Alexa488 to macrophages of both mouse strains was analyzed (Figure 3 B). Binding of HSP60 to BMM of TLR4-deficient C57BL/10ScCr mice occurred with comparable intensity and similar dose dependency as with BMM of the control strain. The role of CD 14 in HSP60 binding and signaling was analyzed by using PI, an inhibitor of CD 14. Stimulation of J774A.1 cells with HSP60 in the presence of increasing doses of PI (0 - 300 μg/ml) resulted in a dose dependent decrease of TNFα production from 26.79 ± 2.31 ng/ml to less than 4 ng/ml as shown in Table III. As a control, CD14- independent stimulation of macrophages by the mycoplasma lipopeptide MALP-2 was analyzed. Even at the higher concentrations of PI (30 and 300 μg/ml) no inhibition of MALP-2-induced TNFα production was observed. By contrast, binding of HSP60- Alexa488 to J774A.1 cells was not inhibited by PI. Interestingly, at higher concentrations of PI an increase of HSP60-Alexa488 binding was observed. These results indicate that CD 14 is involved in HSP60 signaling, whereas it does not contribute to HSP60 binding.

TABLE III. Effect of phospathidylinositol on HSP60-Induced TNFα production and HSP60 binding to macrophages.

n.t. not tested

J774A.1 cells were preincubated with phospathidylinositol at the indicated concentrations (15 min), followed by the addition of 3 μg/ml HSP60 or 1 ng/ml MALP-2 ("Production of TNFa") or 350 nM HSP60-Alexa488 ("Maximal binding ofHSP60-Alexa488"). HSP60- or MALP-2-stimulated TNFα production and binding of HSP60-Alexa488 were analyzed by sandwich ELISA and by FACS analysis, respectively. The data represent mean values ± S.D. of three independent experiments (in the case of TNFα production performed in triplicate). Significant differences to HSP60- and MALP-2-induced TNFα production and to binding of HSP60-Alexa488 are indicated as *, p < 0.05.

Example 3: Characterization of the Macrophage- Stimulatory Epitope of HSP60

In a first series of experiments it has been verified that the immunostimulatory principle of HSP60 was tightly associated with the chaperone molecule. The effect of treatment with proteinase K (A), heat (B), and PmB (C) on HSP60-stimulated TNFα production in J774A.1 macrophages is depicted in figure 4. (A) Cells were incubated with medium, 10 μg/ml untreated or proteinase K-treated (10 μg/ml, 1 h, 37 °C) HSP60, 1 ng/ml untreated or proteinase K-treated LPS or 10 μg/ml HSP60 and 1 ng/ml LPS (untreated or proteinase K-treated). (B) Cells were incubated with medium, 3 μg/ml untreated or heat- treated (boiling, 10 min) HSP60, 1 ng/ml untreated or heat-treated LPS or 3 μg/ml HSP60 and 1 ng/ml LPS (untreated or heat-treated). (C) Cells were incubated with medium or LPS (10 ng/ml) and HSP60 (10 μg/ml) pretreated with different concentrations of PmB (1 h, 4°C). (D) Cells were incubated with medium, 10 μg/ml untreated or PmB-treated (10 μg/ml) HSP60, 10 ng/ml untreated or PmB-treated LPS or 10 μg/ml HSP60 and 10 ng/ml LPS (untreated or PmB-treated). After 6 h of incubation the TNFα concentrations in the cell culture supernatants were measured by sandwich ELISA. Data represent the mean values of TNFα (ng/ml) + S.D. of three independent experiments, each performed in triplicate. Significant differences to the corresponding untreated control group are indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001. As shown in Figure 4 A the HSP60 preparation lost most of its macrophage- stimulatory activity after exposure to the protease. Protease treatment did not alter the capacity of 1 ng/ml LPS to stimulate TNFα production. As a further control, synthetic mixtures of HSP60 and LPS were prepared and treated with proteinase K. Cells incubated with an untreated combination of HSP60 and LPS secreted significantly higher amounts of TNFα (22.11 ± 0.39 ng/ml) compared to macrophages incubated with untreated LPS alone (18.59 ± 0.95 ng/ml, p = 0.004). After proteinase K treatment of the mixture the TNFα production dropped to a similar level as induced with proteinase K-treated LPS alone. Next, the chaperone molecule was denatured by boiling for 10 min. This led to complete abolishment of the macrophage-stimulatory activity of the HSP60 preparation (Figure 4 B). By contrast, J774A.1 cells incubated with untreated or heat-treated LPS (1 ng/ml) produced comparable high levels of TNFα (14.17 ± 0.46 ng/ml and 11.99 ± 0.69 ng/ml, respectively). Since denatured protein might bind or trap LPS, the experiment was repeated with a mixture of HSP60 and LPS. Cells exposed to the untreated mixture of HSP60 and LPS released significantly higher amounts of TNFα (17.90 ± 0.58 ng/ml) compared to those from macrophages exposed to untreated LPS alone (14.17 ± 0.46 ng/ml, p = 0.001). After heat treatment of the mixture the TNFα production dropped to the same level (12.20 ± 1.07 ng/ml) as induced with heat-treated LPS alone (11.99 ± 0.69 ng/ml). Taken together, these experiments indicate that a surface structure of HSP60 accounts for the macrophage-stimulatory activity. This structure might be tightly bound LPS or an endogenous peptide structure interacting with CD 14 and TLR4. The inhibitory capacity of several compounds known to bind LPS or LPS-like structures was therefore analyzed. First, the effects of the LPS inhibitor PmB was tested by studying dose dependency and using artificial mixtures of HSP60 and LPS. As shown in Figure 4 C, preincubation of LPS with PmB dose dependently decreased TNFα release, almost reaching background levels at 10 μg/ml PmB. In contrast, preincubation with 0.1 - 10 μg/ml PmB did not suppress the capacity of HSP60 to stimulate TNFα production. As a further control, macrophages were stimulated with a mixture of HSP60 and LPS (Figure 4 D). Cells exposed to the untreated mixture of HSP60 and LPS released significantly higher amounts of TNFα (6.93 ± 0.42 ng/ml) compared to those from macrophages exposed to untreated HSP60 alone (3.51 ± 0.09 ng/ml, p = 0.0002). After preincubation of the mixture with PmB the TNFα production dropped to a similar level (4.28 ± 0.42 ng/ml) as induced with PmB treated HSP60 alone (2.89 ± 0.25 ng/ml).

Example 4: LPS binding agents

Additional analysis were performed with three closely related LPS binding defensins of the magainin family, magainin I, magainin II and magainin II amide. The effect of magainin I, magainin II and magainin II Amide on HSP60-stimulated TNFα production is described in figure 5. J774A.1 cells were preincubated with increasing concentrations of magainin I (A, D), magainin II (B, D) or magainin II amide (C, D) for 15 min. Subsequently medium, 1 ng/ml LPS (A, B, C), 6 μg/ml HSP60 (A, B, C) or 1 ng/ml MALP-2 (D) were added. After 6 h of incubation the TNFα concentrations in the culture supernatants were measured by sandwich ELISA. Data represent the mean values of TNFα (ng/ml) + S.D. of three independent experiments, each performed in triplicate. Significant differences to TNFα production induced by LPS, HSP60 or MALP-2 alone are indicated as *, p < 0.05; **, p< 0.01; ***, p < 0.001.

As shown in Figure 5 (A, B), increasing concentrations of magainin I or magainin II (0, 1, 3, and 10 μg/ml) failed to suppress HSP60- or LPS-induced TNFα production from

J774A.1 cells. The addition of magainin II amide resulted in a dose dependent decrease of the TNFα production (Figure 5 C). A significant (p < 0.01) decrease of the HSP60- stimulated TNFα production was already observed at a concentration of 1 μg/ml magainin II amide (23.26 ± 1.46 ng/ml). At a concentration of 3 and 10 μg/ml magainin II amide, the TNFα response to HSP60 was reduced to 21.83 ± 0.54 ng/ml and 13.87 ± 2.23 ng/ml (p < 0.001) corresponding to an inhibitory effect of 26% and 53%. The stimulatory effect of LPS could also be inhibited dose dependently by magainin II amide. Preincubation of J774A.1 cells with increasing doses of magainin II amide resulted in a reduction of TNFα production to 2.00 ± 0.11 ng/ml at a concentration of 10 μg/ml magainin II amide. Essentially the same findings were observed with NO as parameter of macrophage activation (data not shown). To exclude a non specific inhibitory activity of magainins, MALP-2 was used as a TLR4- independent stimulus of TNFα secretion. None of the different magainins was found to inhibit MALP-2-induced TNFα production in J774A.1 cells (Figure 5 D). Interestingly, preincubation of the cells with 10 μg/ml magainin II resulted in an enhanced MALP -2- induced TNFα production. The potential inhibitory effect of the different magainins on the HSP60-Alexa488 binding to J774A.1 cells was further analyzed by FACS (Table IV). The results of these experiments showed that none of the tested magainins was able to compete with HSP60-Alexa488 for binding, supporting our assumption of the existence of different epitopes in the HSP60 protein responsible for receptor binding and macrophage activation.

TABLE IV. Effect of magainin I, magainin II and magainin II amide on HSP60 binding

J774A.1 cells were preincubated with the different magainins at the indicated concentrations (15min), followed by the addition of 350 nM HSP60-Alexa488. Binding of HSP60- Alexa488 were analyzed by FACS and binding of HSP60-Alexa488 alone was set 100%. The data represent mean values ± S.D. of three independent experiments.

Example 5: Analyzing the epitope specificity of HSP60 antibody clone 4B9/89

Since the HSP60 antibody clone 4B9/89 is suggested to recognize either the HSP60 region aa335-366 or the region aa484-547 (Sharif et al., ibid), including the putative binding region, the epitope specificity of this antibody was further analyzed. Dot blot experiments analyzing the effect of mixtures of distinct 20-mer peptides on the binding of antibody clone 4B9/89 to HSP60 (figure 6A) revealed that preincubation of the peptide mixture covering the region aa321-380 of HSP60 with antibody clone 4B9/89 resulted in the inhibition of antibody binding to HSP60. Neither the mixture of peptides from a region not recognized by the antibody (aa residues 141-230), nor the peptide mixture covering the region aa471- 560 inhibited antibody binding to HSP60. Next, we investigated the effect of the individual 20-mer peptides on the binding of antibody clone 4B9/89 to HSP60 by dot blot analysis (figure 6B). Preincubation of pep361-380 with antibody clone 4B9/89 inhibited binding of the antibody to HSP60, whereas none of the other peptides interfered with antibody binding to HSP60. These results were confirmed by additional dot blot analysis with single 20-mer peptides, which showed that only the peptide corresponding to region aa361-380 was recognized by antibody clone 4B9/89 (2.06 x 106 BLU) in a similar intensity as human HSP60 (2.65 x 106 BLU). None of the other tested 20-mer peptides was detected by antibody clone 4B9/89 (< 0.1 x 106 BLU). By assigning the epitope specificity of clone 4B9/89 to region aa335-366, the location of the binding epitope of HSP60 to the C-terminal region of the molecule was confirmed.

Example 6: HSP60 specifically Binds LPS

Based on the above findings, we analyzed the capacity of HSP60 to bind LPS by a radioactive binding assay. In this assay, [ H]LPS and low endotoxin recombinant human HSP60 were mixed in solution and free versus HSP60-bound [³H]LPS was separated by vacuum filtration on a nitrocellulose membrane. [ H]LPS (500 nM) was incubated in the absence (background control) or presence of 83 nM (corresponding to 5 μg/ml) of HSP60, OVA or transferrin for 30 min at 4 °C. Bound [³H]LPS was separated from free [³H]LPS by vacuum filtration. Data represent the mean values of bound [³H]LPS shown as counts per minute + S.D. of two independent experiments. Significant differences to bound [³H]LPS in the absence of protein (background control) are indicated as ***, p < 0.001. As shown in Figure 7, binding of [³H]LPS to HSP60 could be demonstrated. Incubation of 83 nM HSP60 with 500 nM [³H]LPS resulted in a significant (p < 0.001) increase of bound [³H]LPS from 547 ± 24 cpm (background control, membrane-bound [³H]LPS in the absence of protein) to 5683 ± 270 cpm in the presence of HSP60. By contrast, binding of [³H]LPS to the same amounts of OVA or transferrin was in the range of the background control. Furthermore, dose dependence and inhibition studies of [³H]LPS-binding to HSP60 were performed as shown in figure 8. A, Low endotoxin HSP60 (17 nM) was incubated with increasing concentrations of [³H]LPS for 30 min at 4 °C as indicated. Bound [³H]LPS was separated from free ligand by vacuum filtration. B, Low endotoxin HSP60 (83 nM) was preincubated with increasing concentrations of unlabeled LPS for 30 min at 4 °C as indicated. Subsequently 500 nM [³H]LPS was added and the incubation was continued for another 30 min at 4 °C. Bound [ H]LPS was separated from free ligand by vacuum filtration. HSP60- bound [³H]LPS in the absence of unlabeled LPS was set 100%. Data represent the mean values of bound [³H]LPS minus background (bound [³H]LPS alone) shown as (A) counts per minute ± S.D. or as (B) % ± S.D. of two to three independent experiments. Significant differences to HSP60 in the absence of [³H]LPS (A) or to HSP60-bound [³H]LPS in the absence of unlabeled LPS (B) are indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001. As shown binding of LPS to HSP60 (17 nM) dose dependently increased up to 1 μM [³H]LPS (2874 ± 1494 cpm), where saturation of LPS-binding was reached (Figure 8 A). At concentrations higher than 5 μM [³H]LPS, a further considerable increase of LPS-binding values was observed, which was most likely caused by self-aggregation of LPS molecules, independent of direct HSP60/LPS interactions. Next, inhibition of [³H]LPS-binding to HSP60 was investigated by unlabeled LPS as an additional proof for the specificity of this interaction (Figure 8B). Binding of [³H]LPS (500 nM) to HSP60 (83 nM) in the absence of unlabeled LPS was set as 100%. Preincubation of HSP60 with increasing doses of unlabeled LPS in the range of 1 μM up to 18 μM (2-fold - 36-fold molar excess) resulted in an increased inhibition of [³H]LPS-binding to HSP60 up to 80 %. At concentrations higher than 18 μM of unlabeled LPS the inhibitory effect remained around 92%. Taken together, the results of these experiments demonstrate a direct and specific binding of LPS to the HSP60 molecule.

Example 7: Identification of the LPS-Binding Site on the HSP60 Molecule

Attempted to identify the potential binding region of LPS on the HSP60 molecule were performed. In a first approach the effect of several commercially available mAbs, directed against different epitopes of the native human HSP60 protein, i.e. clone 24 (aa residues 1-200), clone 4B9/89 (aa residues 335-366) or clone LK1 (aa residues 383-447), on HSP60-induced TNFα production from J774A.1 cells, was investigated (Table V). HSP60 (3 μg/ml) was preincubated with different mAbs to HSP60 (30 min, room temperature) and then added to J774A.1 cells. TNFα production was analyzed by sandwich ELISA and TNFα production induced by HSP60 alone was set 100%. The data represent mean values ± S.D. of three independent experiments each performed in triplicate. Significant differences to HSP60-induced TNFα production are indicated as *, p < 0.05; **, p < 0.01. When analyzing these antibodies in three different concentrations (1, 8 and 20 μg/ml), preincubation of HSP60 with mAb clone 4B9/89 resulted in a significant (p < 0.01) and dose-dependent decrease of TNFα production to 42 ± 7% of the TNFα production induced by HSP60 alone (set as 100%). None of the other tested mAbs to HSP60 or the IgG isotype control inhibited the HSP60-induced TNFα release from J774A.1 cells. These findings indicated that the region aa335-366 of the HSP60 molecule is involved in interaction with LPS.

Table V. Effect of anti-HSP60 antibodies on HSP60-induced TNFα production from macrophages

Anti-human-HSP60 C oncentration Production of TNFα antibody (μg/ml) (%)

C lone Recognized Epitope

— — 100

4B9/89 aa335-366 1 89 ± 10

8 59 + 6** 20 42 ± η * *

Clone 24 aal-200 1 113 + 20

8 129 + 10* 20 122 ± 9*

LK1 aa383-447 1 98 ± 20

8 91 ± 10

20 102 ± 15

IgG isotype 1 114 ± 8 contro 1 8 101 + 13

20 111 ± 13

The effect of mAb clone 4B9/89 on the binding of [³H]LPS to HSP60 in the radioactive [³H]LPS-binding assay was tested (Figure 9). Low endotoxin HSP60 (83 nM) was preincubated with different concentrations of mAbs clone 4B9/89 or clone LK1 for 30 min at room temperature as indicated. Subsequently 500 nM [³H]LPS was added and the incubation was continued for another 30 min at 4 °C. Bound [ H]LPS was separated from free ligand by vacuum filtration. HSP60-bound [ H]LPS was set 100%. Data represent the mean values of bound [³H]LPS minus antibody-bound [³H]LPS in the absence of HSP60 shown as % + S.D. of four independent experiments. Significant differences to HSP60- bound [³H]LPS in the absence of antibodies are indicated as ***, p < 0.001. Preincubation of low endotoxin human HSP60 (83 nM) with different concentrations of the antibody (1, 10 and 25 μg/ml) resulted in a significant (p < 0.001) decrease of LPS-binding to HSP60 to 9 ± 5%. By contrast, mAb clone LK1 used as control did not inhibit LPS-binding to HSP60.

In a more detailed analysis, screening of individual 20-mer peptides with an overlap of 10 amino acids, covering the human HSP60 region aa331-380, which includes the region recognized by the mAb clone 4B9/89, for inhibiting [³H]LPS-binding to HSP60 was performed (Table VI). Preincubation of 75 μM pep351-370 with 500 nM [³H]LPS resulted in a significant (p < 0.001) decrease of LPS-binding to low endotoxin HSP60, i.e. to 54 ± 17%. By contrast, none of the other peptides interfered with LPS-binding to HSP60. This result restricted the region of interest to amino acid residues 351-370 of the HSP60 molecule involved in LPS-binding.

Table VI. Effect of selected 20-mer peptides on [³H]LPS binding to HSP60

^aHSP60 epitope recognized by mAb clone 4B9/89

500 nM [3H]LPS was incubated with 75 μM of the different 20-mer peptides for 30 min at 4 °C. Subsequently 83 nM HSP60 was added and the incubation was continued for 30 min at 4 °C. HSP60-bound [3H]LPS in the absence of peptides was set 100%. Data represent the mean values of bound [3H]LPS minus background (bound [3H]LPS alone) shown as % ± S.D. of two to six independent experiments. Significant differences to HSP60-bound [3H]LPS in the absence of peptides are indicated as ***, p < 0.001. Mean values of peptide- bound [3H]LPS were in the range of 23 ± 17% (data not shown).

Finally, the effect of a 13-mer peptide covering the region aa354-365 of the human HSP60 molecule was analyzed. 500nM of [ H]LPS were incubated with different concentrations of pep354-365 or an unrelated 13-mer control peptide for 30 min at 4 °C as indicated. Subsequently 83 nM HSP60 was added and the incubation was continued for 30 min at 4 °C. HSP60-bound [³H]LPS was set 100%. Data represent the mean values of bound [³H]LPS minus peptide-bound [³H]LPS in the absence of HSP60 shown as % + S.D. of two independent experiments. Significant differences to HSP60-bound [³H]LPS are indicated as *, p < 0.05; **, p < 0.01. As shown in Figure 10 preincubation of 500 nM [³H]LPS with increasing concentrations of pep354-365 in the range of 0.5 μM up to 15 μM resulted in a significant (p < 0.05) and dose-dependent decrease of LPS-binding to low endotoxin HSP60 from 100% (HSP60 alone) to 32% + 5%. At higher concentrations (35 - 100 μM) the inhibitory effect remained stable around 65%. By contrast, an unrelated control protein did not interfere with [³H]LPS-binding to HSP60. Taken together, these findings indicate that the region aa354-365 of the HSP60 molecule is involved in specific binding of LPS.

While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, rather the scope, spirit and concept of the invention will be more readily understood by reference to the claims which follow.

Claims

THE CLAIMS

1. An immunomodulatory agent capable of inhibiting binding of human heat shock protein 60 (HSP60) to macrophages, comprising a composition selected from: a. a peptide fragment, analog, mimetic or functional derivative of amino acids 481-500 of human HSP60 having the sequence set forth in SEQ ID NO:2, that is capable of binding to macrophages and inhibiting their binding to human HSP60; b. an antibody or antibody fragment or a peptide derived from the binding site of said antibody, capable of binding to at least part of amino acids

481-500 of human HSP60 having the sequence set forth in SEQ ID NO:2, and inhibiting its binding to macrophages; and c. a small organic molecule, capable of binding to at least part of amino acids 481-500 of human HSP60 having the sequence set forth in SEQ ID NO:2, and inhibiting its binding to macrophages.

2. An anti-inflammatory agent capable of inhibiting binding of HSP60 to lipopolysaccharide (LPS), comprising a composition selected from: a. a peptide fragment, analog, mimetic or functional derivative of at least part of amino acids 335-366 of HSP60 having the sequence set forth in SEQ ID NO:3, and is capable of binding to LPS and inhibit its binding to human HSP60; b. an antibody or antibody fragment or a peptide derived from the binding site of said antibody, and is capable of binding to at least part of amino acids 335-366 of HSP60 having the sequence set forth in SEQ ID NO:3, and inhibiting its binding to LPS; and c. a small organic molecule capable of binding to LPS and inhibit its binding to human HSP60.

3. The anti-inflammatory agent of claim 2 wherein the binding of HSP60 to LPS is mediated by amino acids 354-365 of HSP60 having the sequence set forth in SEQ ID NO:4.

4. A pharmaceutical composition comprising as an active ingredient at least one immunomodulatory agent capable of inhibiting the binding of HSP60 to macrophages, comprising a composition selected from: a. a peptide fragment, analog, mimetic or functional derivative of amino acids 481-500 of human HSP60 having the sequence set forth in SEQ ID

NO:2, that is capable of binding to macrophages and inhibiting their binding to human HSP60; b. an antibody or antibody fragment or a peptide derived from the binding site of said antibody, capable of binding to at least part of amino acids 481-500 of human HSP60 having the sequence set forth in SEQ ID NO:2, and inhibiting its binding to macrophages; and c. a small organic molecule, capable of binding to at least part of amino acids 481-500 of human HSP60 having the sequence set forth in SEQ ID NO:2, and inhibiting its binding to macrophages; and an acceptable carrier, excipient and/or diluent.

5. A pharmaceutical composition comprising as an active ingredient at least one anti- inflammatory agent capable of inhibiting the binding of HSP60 to LPS, comprising a compound selected from: a. a peptide fragment, analog, mimetic or functional derivative of at least part of amino acids 335-366 of HSP60 having the sequence set forth in

SEQ ID NO:3, and is capable of binding to LPS and inhibit its binding to human HSP60; b. an antibody or antibody fragment or a peptide derived from the binding site of said antibody, and is capable of binding to at least part of amino acids 335-366 of HSP60 having the sequence set forth in SEQ ID NO:3, and inhibiting its binding to LPS; and c. a small organic molecule capable of binding to LPS and inhibit its binding to human HSP60; and an acceptable carrier, excipient and/or diluent.

6. A pharmaceutical composition comprising as an active ingredient at least one anti- inflammatory agent capable of inhibiting the binding of HSP60 to LPS, comprising a compound selected from: a. a peptide fragment, analog, mimetic or functional derivative of at least part of amino acids 335-366 of HSP60 having the sequence set forth in SEQ ID NO:3, and is capable of binding to LPS and inhibit its binding to human HSP60; b. an antibody or antibody fragment or a peptide derived from the binding site of said antibody, and is capable of binding to at least part of amino acids 335-366 of HSP60 having the sequence set forth in SEQ ID NO:3, and inhibiting its binding to LPS; and c. a small organic molecule capable of binding to LPS and inhibit its binding to human HSP60; and an acceptable carrier, excipient and/or diluent.

7. A method of treating an individual in need thereof, suffering from a disorder or disease involving abnormal levels of HSP60, comprising administering a pharmaceutical composition comprising any of the agents of claims 1-3, and an acceptable carrier, excipient and/or diluent.

8. The method of claim 7 wherein the disease is selected from inflammatory disease and autoimmune disease.

9. The method of any one of claims 7 and 8 wherein the agent is magainin II amide having the sequence set forth in SEQ ID NO:5, or an analog, fragment, salt or functional derivative thereof.

10. The method of any one of claims 8 and 9 wherein the inflammatory disease is selected from the group consisting of: rheumatic disease including rheumatoid arthritis, osteoarthritis, psoriatic arthritis, juvenile arthritis, Reiter's syndrome, infectious arthritis, ankylosing spondylitis, systemic lupus erythematosus, and gout; inflammatory disease of the gastrointestinal tract including the oral cavity, inflammation associated with gingivitis or periodontal disease, inflammation associated with inflammatory bowel disease, Crohn's disease; inflammatory disease of the respiratory tract including obstructive bronchitis, bronchial asthma, inflammatory pulmonary disease; inflammatory vascular and cardiac disease; inflammatory allergic disease; inflammatory dermatological disease including psoriasis and inflammatory skin disease.

11. The method of any one of claims 8 and 9 wherein the autoimmune disease is selected from the group consisting of: insulin dependent diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, psoriasis, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia purpura, rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bulbous pemphigoid, discoid lupus, ulcerative colitis, Crohn's disease and dense deposit disease.

12. Use of peptide, or analogs, salts and functional derivatives thereof comprising at least part of amino acids 481-500 of human HSP60 having the sequence set forth in SEQ ID NO:2, for inhibiting the binding of HSP60 to macrophages.

13. Use of a peptide, or an analog, salt or functional derivative thereof corresponding to at least part of amino acids 335-366 of human HSP60 having the sequence set forth in SEQ ID NO:3, for inhibiting the binding of LPS to HSP60..

14. Use of a peptide, or an analog, salt or functional derivative thereof corresponding to at least part of amino acids 354-365 of human HSP60 having the sequence set forth in SEQ ID NO:4, for inhibiting the binding of LPS to HSP60.

15. Use of magainin II amide having the sequence set forth in SEQ ID NO:2, SEQ ID NO: 5, or an analog, fragment, salt or functional derivative thereof for inhibition of the pro-inflammatory or immunostimulatory reactivity of HSP60.

16. Use of magainin II amide, or an analog, fragment, salt or functional derivative thereof as an active ingredient for the preparation of a pharmaceutical composition for treatment of a disorder or disease involving abnormal levels of HSP60.

17. The use according to claim 16 wherein the disease is selected from inflammatory disease and autoimmune disease.

18. The use according to claim 17 wherein the inflammatory disease is selected from the group consisting of: rheumatic disease including rheumatoid arthritis, osteoarthritis, psoriatic arthritis, juvenile arthritis, Reiter's syndrome, infectious arthritis, ankylosing spondylitis, systemic lupus erythematosus, and gout; inflammatory disease of the gastrointestinal tract including the oral cavity, inflammation associated with gingivitis or periodontal disease, inflammation associated with inflammatory bowel disease, Crohn's disease; inflammatory disease of the respiratory tract including obstructive bronchitis, bronchial asthma, inflammatory pulmonary disease; inflammatory vascular and cardiac disease; inflammatory allergic disease; inflammatory dermatological disease including psoriasis and inflammatory skin disease.

19. The use according to claim 17 wherein the autoimmune disease is selected from the group consisting of: insulin dependent diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, psoriasis, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia purpura, rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bulbous pemphigoid, discoid lupus, ulcerative colitis, Crohn's disease and dense deposit disease.