WO2016074079A1 - Novel polypeptide for the prevention of chronic inflammation - Google Patents

Abstract

The present invention provides a polypeptide comprising a functional osteoprotegerin (OPG) amino acid sequence linked to a functional Receptor Activator of Nuclear Factor κΒ (RANK) amino acid sequence, and method of use for the treatment and/or prevention of tissue destruction by RANKL-expressing neutrophils, macrophages and T lymphocytes.

Description

NOVEL POLYPEPTIDE FOR THE PREVENTION OF CHRONIC INFLAMMATION

FIELD OF THE INVENTION

[0001] The present invention provides a novel compound and method of use for the treatment and/or prevention of pathological conditions provoked by inflammatory cells that express RANKL.

BACKGROUND OF THE INVENTION

[0002] The association of inflammation and bone loss is present in several clinical conditions like septic arthritis, osteomyelitis, chronic inflammatory arthropathies (i.e. rheumatoid and psoriatic arthritis), as well as periodontitis. In most of these conditions, inflammatory bone loss starts early in the disease process and is a cause of considerable patient morbidity. These destructive bony lesions result from excessive activity of the monocyte-derived bone resorbing cells, osteoclasts. Osteoclast differentiation and function are both regulated by the molecular couple RANK (Receptor Activator of NF-Kappa B) and its ligand, RANKL. RANK is a membrane protein expressed by osteoclasts and dendritic cells. RANKL, belonging to the TNF family, was discovered as a stromal cell-associated protein that required direct cell-to-cell interactions with osteoclasts, and as a soluble protein. Immune cells, such as activated T lymphocytes, also synthesize and release functionally active RANKL. Excessive RANKL concentrations are present in local and systemic inflammatory conditions associated with excessive bone resorption. The decoy receptor of RANKL, osteoprotegerin (OPG), negatively regulates osteoclasts. Indeed, the RANKL RANK/OPG system is the final common pathway through which most osteotropic factors modulate their effects on bone.

[0003] Neutrophils are the most abundant circulating leukocytes and are the first cells to migrate to inflammatory sites. They have a wide range of effector functions owing to preformed cytoplasmic and membrane proteins, enzymes and newly synthesized lipid and protein mediators that enable them to respond to diverse environmental triggers. Neutrophils are, thus, an essential arm of the immune system, mounting the initial inflammatory response and participating in host defense. Peripheral blood neutrophils are certainly the most studied type of neutrophils. However, during inflammation, these leukocytes leave the circulation and enter new habitats, the tissues where they are exposed for extended periods to multiple factors like cytokines, endogenous growth factors, bacterial products as well as multiple other local products. Indeed, the activity of infiltrating neutrophils has been intimately linked to disease evolution in a variety of clinical conditions. Thus, these cells contribute not only to acute inflammatory reactions but also to the evolution of a variety of chronic inflammatory diseases.

[0004] From this point of view, the inventor recently isolated and characterized a subset of neutrophils with a functional profile that is markedly different from that of freshly isolated neutrophils (Chakravarti A et a/., Lab Invest (2009) 89:1084-1099). Constituting 8-17% of the global neutrophil populations exposed to the cytokines, these cells have substantially increased life span persisting beyond 72 hours. They have a unique inflammatory profile seen in the pattern of surface marker expression, production of superoxide anions (0₂-), phagocytosis, leukotriene biosynthesis, chemotactic responses and degranulation. In addition, this subset produces substantial amounts of cytokines like IL-1 , IL-1 receptor antagonist (IL-1 Ra), and IL-8 and interacts strongly with resident stromal cells. Thus, certain inflammatory conditions can induce a phenotypic 'switch' in the circulating neutrophil towards a resident neutrophil with different and new functions.

[0005] Note that certain of these factors, like the powerful chemoattractants leukotriene B₄ and IL-8 produced by inflammatory neutrophils, lead to amplify the local recruitment of neutrophils and the inflammatory process. Moreover, the production of other major phlogogenic factors, like IL-1 β for instance, by these inflammatory neutrophils greatly enhance the local inflammatory process with deleterious consequences like destruction of joint and bone tissues.

[0006] However, neutrophils possess the necessary pathway(s) to control, at least in part, their destructive capacity through certain phosphatases like the protein-tyrosine phosphatase SHP-1. The biological significance of SHP-1 as a negative regulator in immunity is seen in the severe autoimmunity developed by motheaten mice. These SHP-1 deficient mice also manifested abnormal effector functions of neutrophils. As a consequence, it could be very useful during inflammation to naturally amplify this negative regulatory pathway. Interestingly, if SHP-1 inhibitors presently exist, no pharmacological activators of the protein-tyrosine phosphatase SHP-1 are available.

[0007] Normal neutrophils express the mRNA for RANKL, but only very low RANKL protein as surface membrane and intra-cellular RANK-L, with no soluble extracellular RANKL (Poubelle PE, et a/., Arthritis Res Ther (2007) 9:R25). Inflammatory neutrophils can express greatly RANKL at their surface, and inflammatory milieu (synovial fluids from chronic inflammatory arthropathies, TLR [toll-like receptors] ligands, cytokines and growth factors...) can induce RANKL expression at the membrane by normal neutrophils in vitro (Chakravarti A, et a/., Blood (2009) 114:1633-1644). The fact that inflammatory and blood neutrophils express RANKL as cell-associated and membrane materials, and that neutrophils incubated in vitro for up to 4 days generated no soluble RANKL has allowed to consider neutrophils as a new cell type which generate RANKL without any release in the extra-cellular milieu. From that point of view, neutrophils are different from other cell types such as osteoblasts, fibroblasts or T lymphocytes which produce RANKL and release soluble RANKL after stimulation (Kanamaru F, et at., Immunol Lett (2004) 94:239-246 and Wong BR, et at., J Exp Med (1997) 186:2075-2080. Membrane-bound RANKL was also found to signal within the neutrophil itself, activating SHP-1 through tyrosine phosphorylation (Chakravarti A, et at., Blood (2009) 114:1633-1644). The functional association of a membrane-bound ligand to membrane-bound receptors, termed "reverse signaling", is seen in many members of the TNF superfamily (Bazzoni F, Beutler B., N Engl J Med (1996) 334:1717-1725). As a type II integral membrane protein, RANKL would require the association with molecular adaptors to link it to intracellular signaling cascades. For instance, increased production of interferon-γ by activated T cells, mediated by RANKL signaling, demonstrates the biological significance of this system (Chen NJ, et al., J Immunol (2001) 166:270-276).

[0008] Accelerated bone resorption is presently considered an early event in the pathogenesis of several septic and inflammatory diseases, where RANKL mediates increased osteoclastogenesis and, consequently, abnormal bone erosions. Neutrophils are the predominant infiltrating cells in this stage and are present in large numbers in areas of osteolysis and adjacent inflammatory sites. Human, as well as murine, neutrophils strongly upregulate their expression of membrane RANKL after TLR stimulation, and have thus the capacity to activate osteoclastic bone resorption through neutrophil-osteoclast interactions (Chakravarti A, et a/., Blood (2009) 114:1633-1644). As a corollary, RANKL increased at the surface of activated neutrophils links this cell type to inflammatory bone destruction through the cell population's capacity to directly activate osteoclastogenesis. Given the predominance and number of neutrophils at sites of rapid bone loss as well as in flare-ups of chronic diseases, these cells might constitute a non-negligible source of RANKL in pathological bone lesions, as well as in the various conditions of neutrophil-to-cell interactions (i.e. neutrophil interactions with dendritic cells (Bennouna S, Denkers EY., J Immunol (2005) 174:4845-4851 and van Gisbergen KP, et al., J Exp Med (2005) 201 :1281-1292)). [0009] Other immune cells like T lymphocytes and macrophages can also regulate SHP-1 expression through RANKL activation, thereby downregulating the abnormal chronic inflammatory process related to the disordered target.

[0010] For example, such activated T lymphocytes have been found in mammary cancer and prostate cancer to stimulate metastasis through RANKL-RANK signaling (Tan W, Zhang W, Strasner A, et al., Nature (2011) 470:548-553).

[0011] Negative regulation of the neutrophil / T lymphocytes / macrophages production of cytokines by RANKL-activated SHP-1 could be of particular importance during inflammation and cancers where great numbers of activated immune cells strongly affect adjacent tissues.

SUMMARY OF THE INVENTION

[0012] In a first aspect, the present invention therefore provides a novel polypeptide comprising a functional OPG amino acid sequence linked to a functional RANK amino acid sequence.

[0013] In a second aspect, the present invention provides a polypeptide selected from the group consisting of: X-OPG_{(22-62) to (154-194)}- Y-RANK_{(30 -70) to (157-187)} and X'-

RANK_{(30 -70) to (157-187)}-Y'-OPG _{(22-62) to (154-194)}-

[0014] In a further aspect, the present invention provides use and method for the treatment of inflammation comprising the administration of a novel polypeptide as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1 is a schematic representation of the RANK-RANKL-OPG system.

[0016] Fig. 2 is an illustration of the signaling pathways controlled by Src homology 2 (SH2) domain containing protein tyrosine phosphatase 1 (SHP-1).

[0017] Fig. 3 illustrates the competing positive (a) and negative (b) feedback pathways during T cell activation, where TCR is effectively engaged by high-affinity agonistic pMHC complexes, and where TCR is inefficiently triggered by an equal number of low-affinity antagonistic pMHC complexes promoting only SHP-1 phosphorylation without effectively stimulating ERK activation.

[0018] Fig. 4 illustrates the effects of KPP-1 on RANKL-expressing inflammatory cells.

[0019] Figs. 5A and 5B illustrates the effect of novel polypeptides A and B on IL-8 production by RANKL-expressing neutrophils. [0020] Figs. 6A and 6B illustrate the expression of RANKL in activated Jurkat T cells by immunofluorescence (6A), and analysis of protein tyrosine phosphorylation through RANKL by thin layer chromatography (6B) when activated Jurkat T cells were further incubated in the presence of vehicle, RANK+OPG, RANK or OPG.

[0021] Figs. 7A and 7B illustrate the expression of RANKL in human macrophages activated by a rheumatoid arthritis (RA) synovial fluid by immunofluorescence (7A), and analysis of protein tyrosine phosphorylation through RANKL by thin layer chromatography (7B) when activated macrophages were further incubated in the presence of vehicle, RANK+OPG, RANK or OPG.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations:

[0022] COPD: chronic obstructive pulmonary disease; I L- 1 β : lnterleukin-1 beta; IL-8: lnterleukin-8; IFN-γ: Interferon-gamma; OC: osteoclast; OPG: osteoprotegerin; RANK: Receptor Activator of NF-Kappa B; RANKL: receptor activator of NF-Kappa B-ligand; SF: synovial fluid; SHP-1 : Src homology domain-containing cytosolic phosphatase 1 ; TNF: tumor necrosis factor; TLR: Toll-like receptor; TRAP: tartrate- resistant acid phosphatase.

[0023] The present invention is based on a new therapeutic concept that uses the natural capacity of inflammatory cells directly implicated in the tissue lesions to downregulate the abnormal chronic inflammatory process related to the disorders targeted. This new concept has emerged from recently discovered in-depth mechanisms that explain the pathologic role of certain leukocytes from chronic inflammatory disorders. The main leukocytes implicated are neutrophils, macrophages and T lymphocytes which can, when activated, express a functional receptor RANKL with intra-cellular signaling.

The RANK-RANKL-OPG system

[0024] In the 90s it was discovered that bone remodeling was governed by a triad ultimately named RANK-RANKL-OPG that consisted of a ligand, receptor activator of NF-KB ligand (RANKL), a receptor, RANK, and a soluble decoy receptor, osteoprotegerin (OPG) (see Fig. 1). OPG belongs to the TNF receptor family and is a physiologically inhibitor of osteoclastic bone resorption. Very importantly, these regulatory factors were also shown to be implicated in interactions and functions of immune cells. RANK belongs to the TNF receptor family and is a transmembrane protein expressed by osteoclasts that mediates osteoclast survival, differentiation and activity when activated by RANKL. RANK is also expressed in cells of the immune system including dendritic cells and monocytes.

[0025] RANKL belongs to the TNF ligand superfamily and is the specific and unique ligand for RANK. RANKL is a type II transmembrane protein with a short cytoplasmic tail and transmembrane region that is linked to the extracellular domain by a stalk region. Three RANKL isoforms have been identified: RANKL1 that represents the complete protein RANKL, RANKL2 that has a shorter intracellular domain, and RANKL3 that does not have the intracellular or transmembrane domains and that acts as a soluble form protein. Surface RANKL (RANKL1) and its soluble form (RANKL3) are tightly regulated and increased expression of RANKL is associated with different pathologies.

[0026] To date, increased RANKL expression at the surface of cells as well as soluble RANKL is associated with abnormal inflammatory bone resorption of rheumatoid and psoriatic arthritis, periodontitis, osteolysis of multiple myeloma, expansion of cancer cells, or metastasis of breast cancer.

Neutrophils

[0027] Neutrophils are the most abundant circulating leukocytes and are the first cells to migrate to inflammatory sites. They have a wide range of effector functions owing to preformed cytoplasmic and membrane proteins (i.e. adhesion molecules), enzymes (i.e. NADPH oxidase, myeloperoxydase, metalloproteinases, hydrolases) and newly synthesized lipid and protein mediators (i.e. leukotriene 134/platelet- activating factor, and cytokines, respectively) that enable them to respond to diverse environmental triggers. During inflammation, these leukocytes leave the circulation and enter new habitats, the tissues where they are exposed for extended periods to multiple factors like cytokines, endogenous growth factors, bacterial products as well as multiple other local products. Indeed, the activity of infiltrating neutrophils has been intimately linked to disease evolution in a variety of clinical conditions. Thus, these cells contribute not only to acute inflammatory reactions but also to the evolution of a variety of chronic inflammatory diseases.

[0028] Normal neutrophils express the mRNA for RANKL, but only very low RANKL protein as surface membrane and intra-cellular RANK-L, with no soluble extracellular RANKL. Inflammatory neutrophils can express greatly RANKL at their surface, and inflammatory milieu (synovial fluids from chronic inflammatory arthropathies, TLR [toll-like receptors] ligands, cytokines and growth factors...) can induce RANKL expression at the membrane by normal neutrophils in vitro. [0029] Negative regulation of the neutrophil production of cytokines by RANKL- activated SHP-1 could be of particular importance during inflammation where great numbers of activated neutrophils strongly affect adjacent tissues. Since the sequential addition of OPG and RANK fragments to RANKL-expressing neutrophils drives these neutrophils to increase their phosphatase SHP-1 phosphorylation with a downregulatory impact on IL-1 β and IL-8 (Chakravarti A, et a/., Blood (2009) 114:1633-1644), the inventor hypothesized that the addition of combined sequences of OPG and RANK fragments could have in vitro and in vivo effects appropriate in the treatment of chronic inflammatory pathologies where neutrophils are implicated and express RANKL.

Tyrosine phosphatase SHP-1

[0030] Protein tyrosine phosphorylation plays a variety of significant roles in cell signaling transduction, physiological functions, and pathological processes. Phosphorylation and dephosphorylation are important post-translational protein modifications which lead to changes in protein functions. Protein kinases and phosphatases play counterbalancing roles that regulate these protein functions. Among phosphatases, two cytoplasmic protein tyrosine phosphatases (PTPs), composed of two Src homology 2 (SH2) NH2-terminal domains and a C-terminal protein-tyrosine phosphatase domain and referred to as SHP-1 and SHP-2, govern multiple cellular functions. SHP-1 (SH2 domain-containing phosphatase 1 , a 64 kDa protein) is an intracytoplasmic PTP expressed almost exclusively in hematopoietic cells, with limited expression occurring in some epithelial lineages. SHP-1 plays an important role as a major negative regulator of activation delivered through a variety of surface receptors, such as B- and T-antigen receptors, cytokine/growth factor receptors, and NK inhibitory receptors (Fig. 2). SHP-1 acts by dephosphorylating the receptors and receptor-associated tyrosine kinases.

[0031] The importance of SHP-1 is highlighted by the phenotype of the SHP-1- deficient moth-eaten mice which die at several weeks of age from inflammation of the lung, resulting from uncontrolled granulocyte proliferation and infiltration. They have a profound immunological dysfunction, also manifested in T cells. In T lymphocytes, SHP-1 is essential for attenuation of T cell receptor (TCR) signaling after initiation of the response (Fig. 3).

[0032] Indeed, SHP-1 plays a role in the differential response to agonist/antagonist peptides. Many signaling molecules downstream of TCR are targets of SHP-1 - mediated dephosphorylation. T lymphocytes

[0033] The human immune system is a complex versatile system that is aimed at protecting the host against invaders through the innate and adaptive immunity. Inappropriate activation of either the innate (granulocytes, NK cells) or the adaptive (T and B cells) arm of the immune system may lead to diseases. Lymphocytes play a key role in the initiation and maintenance of inflammation. Several chronic inflammatory diseases resulting from T cell-mediated autoimmunity, such as rheumatoid arthritis, psoriasis, Crohn's disease, show persistent inflammation with deleterious effects on various types of tissues. Similarly, there is an intricate relationship between chronic inflammatory reactions, stimulation of proliferation, and cancer development (Balkwill F, and Coussens LM, Nature (2004) 431 :405-406). Tumor antigen-specific T lymphocytes can infiltrate cancer tissues and can have a role in immune surveillance. However, it has been shown in animal models that inflammatory T cells can promote cancer and its spread.

[0034] T lymphocytes that do not express RANKL spontaneously, have the capacity to express RANKL at their surface when activated only. Interestingly, such RANKL- expressing activated T lymphocytes have been shown to be responsible for abnormal bone resorption in rheumatoid arthritis by stimulating directly RANK-expressing osteoclasts (Kong YY, et a/., Nature (1999) 402:304-309), and for promoting breast cancer metastasis (Tan W, et a/., Nature (2011) 470:548-553).

Anti-RANKL blocking antibodies or RANKL-activating KPP-1

[0035] A corollary of the great involvement of RANKL in different pathologies is the therapeutic importance of controling abnormal RANKL expression. Presently, the sole compound available to control such an abnormal RANKL expression is a biologic that targets soluble RANKL through its inhibition by an anti-human RANKL antibody named Denosumab.

[0036] Another way to control the functions of activated leukocytes which have an abnormal expression of RANKL can be, not to inhibit directly RANKL, but to activate the inhibitory phosphatase SHP-1 present in RANKL-expressing activated leukocytes like neutrophils and T lymphocytes involved in the chronic inflammatory process of various pathologies (see above). KPP-1 has this capacity to activate SHP-1 linked to surface RANKL greatly expressed by inflammatory leukocytes (Fig. 4). Moreover, it is important to stress that, in parallel to the simultaneous activation of the inhibitory phosphatase SHP-1 expressed by myeloid cells and the reduction of the inflammatory response of the cells targeted by KPP-1 , the occupancy of RANKL receptor by KPP-1 renders surface RANKL inefficient for activating cells that express surface RANK. Finally, the production of KPP-1 that is genetically synthesized costs much less in comparison to the production of humanized antibodies like Denosumab. It is also possible that KPP-1 and Denosumab will be given together to increase the efficacy of controling RANKL-associated deleterious effects.

In summary

[0037] Myeloid cells, like neutrophils and T lymphocytes, implicated in deleterious lesions of chronic inflammatory diseases, breast and lung cancers, can overexpress RANKL and can be used directly to reduce their pro-inflammatory potential through an increase of the inhibitory phosphatase SHP-1 linked to surface RANKL. This inhibitory pathway specifically activated through RANKL by KPP-1 is present only in activated leukocytes of the pathologic process of the diseases targeted, that avoids activation of RANKL expressed by other types of cells.

[0038] Monocytes and macrophages, which are also myeloid cells greatly implicated in the chronic inflammatory process, can abnormally express surface RANKL in chronic inflammatory arthritis (Crotti TN, et a/., Ann Rheum Dis (2002) 61 :1047- 1054). Normal monocyte-macrophages cultured in vitro in the presence of rheumatoid synovial fluids or of breast cancer cells (MDA-231) strongly increased their surface expression of RANKL. As a corollary, inflammatory monocyte- macrophages could be another potential target for KPP-1.

[0039] Since the sequential addition of OPG and RANK fragments to RANKL- expressing neutrophils drives these neutrophils to increase their phosphatase SHP-1 with a downregulatory impact on IL-1 β and IL-8, it was hypothesized that the addition of a chimeric polypeptide containing both active sequences could have an effect more efficient than the peptides added separately to amplify this neutrophil SHP-1 response. To test this hypothesis, 2 novel polypeptides were genetically synthesized and were named KPP-1 A (SEQ ID NO:6) and KPP-1 B (SEQ ID NO:7) corresponding respectively to the following peptidic sequences: Gly-Ser-OPG_22-194-Pro-Glu-Phe-Pro- RANK_30-187 and Gly-Ser-RANK_30-187-Pro-Glu-Phe-Pro-OPG_22-194.

Chimeric novel polypeptide

[0040] Based on the SHP-1 stimulatory effect of the combination OPG peptide + RANK peptide in RANKL-expressing neutrophils that lead to a significant decrease of pro-inflammatory cytokines like IL-1 β and IL-8 (Chakravarti A, et a/., Blood (2009) 114:1633-1644), the inventor designed and synthesized genetically peptidic chimeras that comprise the two peptides associated together by the addition of a few amino- acids. It is possible to obtain other chimeric polypeptides with peptides smaller than those actually present in the examples.

[0041] In a first aspect, the present invention therefore provides a chimeric novel polypeptide comprising a functional OPG amino acid sequence linked to a functional RANK amino acid sequence.

[0042] Particularly, a functional RANK amino acid sequence is a sequence having a N-terminal from amino acid 30 to amino acid 187 when taken from the amino acid sequence of RANK (defined by protein accession number: Q9Y6Q6) and defined herein as SEQ ID NO:2. More particularly, a functional RANK amino acid sequence is a sequence having a N-terminal from amino acid 30 to amino acid 187 from SEQ ID NO:2.

[0043] Particularly, with respect to the method of the invention as defined herein, the chimeric novel polypeptide is a molecule activating endogenous expression of membrane-bound RANKL in neutrophils. More particularly, the molecule activating endogenous expression of membrane-bound RANKL in neutrophils, macrophages or T-lymphocytes comprises a RANK peptide or a peptidic derivative thereof. Still more particularly, the RANK peptide is defined as amino acids 30 to 187 of the RANK protein (SEQ ID NO:2). Most particularly, the RANK peptidic derivative may consist of amino acids 30 to 187 optionally truncated by one up to 40 amino acids at either end of the peptide such that it is defined as any length from amino acid 30 to 70 at the N-terminal and any length from amino acid 157 to 187 at the C-terminal.

[0044] Particularly, a functional OPG amino acid sequence is a sequence having a N-terminal from amino acid 22 to amino acid 194 when taken from the amino acid sequence of OPG (defined by protein accession number: 000300) and defined herein as SEQ ID NO:4. More particularly, a functional OPG amino acid sequence is a sequence having a N-terminal from amino acid 22 to amino acid 194 from SEQ ID NO:4.

[0045] Particularly, with respect to the method of the invention as defined herein, the ligand is a molecule increasing phosphorylation of SHP-1. More particularly, the ligand comprises an OPG peptide or a peptidic derivative thereof. Still more particularly, the OPG peptide is defined as amino acids 22 to 194 of the OPG protein. Most particularly, the OPG peptidic derivatives may consist of: amino acids 22 to 187 optionally truncated by one up to 40 amino acids at either end of the peptide such that it is defined as any length from amino acid 23 to 62 at the N-terminal and any length from amino acid 154 to 193 at the C-terminal. [0046] In a second aspect, the present invention provides a peptide selected from the group consisting of: X-OPG_22-194-Y-RANK_30-187 and X-RANK_30-187-YOPG_22-194.

[0047] Particularly, X is an amino acid linker sequence that comprises 1 , 2 or 3 amino acids, particularly amino acids that constitute a restriction site for ease of cleavage. More particularly, X is made out of 1 , 2 or 3 amino acids selected from: Gly, Ala, Ser or conservative substitutions thereof. Most particularly, X is made out of 1 or 2 amino acids selected from: Gly, and Ser or conservative substitutions thereof.

[0048] Particularly, Y is an amino acid linker sequence that comprises 3 to 10 amino acids. More particularly, Y is made out of 3 to 10 amino acids selected from: Pro, Phe, Glu, or conservative substitutions thereof. Most particularly, Y is made out of 3, 4, 5 or 6 amino acids selected from: Pro, Phe, Glu, or conservative substitutions thereof.

[0049] As used herein, the term "conservative substitution" denotes the replacement of an amino acid residue by another, biologically similar residue. It is well known in the art that the amino acids within the same conservative group can typically substitute for one another without substantially affecting the function of a protein. For the purpose of the present invention, such conservative groups are set forth in Table 1 based on shared properties:

[0050] Particularly, X is Gly-Ser or conservative substitutions thereof. More particularly, Y is Pro-Glu-Phe-Pro or conservative substitutions thereof.

[0051] Particularly, the present invention provides a peptide selected from the group consisting of: Gly-Ser-OPG_22-194-Pro-Glu-Phe-Pro-RANK_30-187 (SEQ ID NO:6) and Gly-Ser-RANK_30-187-Pro-Glu-Phe-Pro-OPG_22-194(SEQ ID NO:8).

[0052] More particularly, the present invention provides a peptide such as : Gly-Ser- OPG_22-194-Pro-Glu-Phe-Pro-RANK_30-187 (SEQ ID NO:6).

Therapeutic Targets

[0053] From the preliminary results performed with products derived from the present invention, the inventor identified several potential therapeutic targets: Rheumatoid arthritis and psoriatic arthritis (Neutrophils (Poubelle PE, et a/., Arthritis Res Ther (2007) 9:R25; and Pillinger MH, and Abramson SB, Rheum Dis Clin North Am (1995) 21 :691-714), Lymphocytes (Miranda-Carus ME, et at., Arthritis Rheum (2006) 54:1151-1164), Monocytes-macrophages (Crotti TN, et at., Ann Rheum Dis (2002) 61 :1047-1054)); Breast Cancer (Neutrophils (Houghton AM., Cell Cycle (2010) 9:1732-1737; and Houghton AM, et at., Nat Med (2010) 16:219-223), Lymphocytes (Tan W, et a/., Nature (2011) 470:548-553), Monocytes-macrophages (Blot E, et a/., Br J Cancer (2003) 88:1207-1212)); Lung Cancer (Neutrophils (De Larco JE, et a/., Clin Cancer Res (2004) 10:4895-4900; and Wislez M, et a/., Clin Cancer Res (2007) 13:3518-3527), Lymphocytes (Woo EY, et al., J Immunol (2002) 168:4272-4276), Monocytes-macrophages (White ES, et a/., J Immunol (2001) 166:7549-7555)); Chronic Obstructive Pulmonary Disease (COPD) (Neutrophils (Stockley RA. Chest (2002) 121 :151S-155S; and Quint JK, and Wedzicha JA, J Allergy Clin Immunol (2007) 119:1065-1071), Lymphocytes (Gadgil A, and Duncan SR, Int J Chron Obstruct Pulmon Dis (2008) 3:531-541), Monocytes-macrophages (Murugan V, and Peck MJ, Exp Lung Res (2009) 35:439-485)); Crohn's Disease (Neutrophils (Maor I, et al., Dig Dis Sci (2008) 53:2208-2214), Lymphocytes (Fuss IJ, et al., J Immunol (1996) 157:1261-1270), Monocytes-macrophages (Zhou L, et al., Cell Mol Life Sci (2009) 66:192-202)).

[0054] With respect to the method and use of the invention as defined herein, the use and method for the treatment or prevention may be applicable to any condition involving neutrophil- and/or T Lymphocyte- and/or macrophage-dependent inflammation, particularly chronic inflammation. More particularly, the method of the invention may be applied to treat or prevent conditions such as, for example: rheumatoid arthritis (RA), psoriatic arthritis, and bone lesions induced by immune cell activation (such as, for example: osteomyelitis, periodontitis, periprosthetic osteolysis), psoriasis, breast cancer, lung cancer, chronic obstructive pulmonary disease (COPD), Inflammatory Bowel Disease (IBD) like Crohn's disease.

[0055] In a further aspect, the present invention provides the use a novel polypeptide as defined herein for the treatment of inflammation.

[0056] More particularly, there is provided the use of the novel polypeptide as defined herein for the treatment of rheumatoid arthritis (RA), psoriatic arthritis, and bone lesions induced by immune cell activation (such as, for example: osteomyelitis, periodontitis, periprosthetic osteolysis), psoriasis, breast cancer, lung cancer, chronic obstructive pulmonary disease (COPD), Inflammatory Bowel Disease (IBD) like Crohn's disease.

[0057] In a further aspect, the present invention provides a method for the treatment of inflammation comprising the administration of a novel polypeptide as defined herein.

[0058] More particularly, there is provided a method for the treatment of at least one of: rheumatoid arthritis (RA), psoriatic arthritis, and bone lesions induced by immune cell activation (such as, for example: osteomyelitis, periodontitis, periprosthetic osteolysis), psoriasis, breast cancer, lung cancer, chronic obstructive pulmonary disease (COPD), and Inflammatory Bowel Disease (IBD) like Crohn's disease, comprising the administration of a novel polypeptide as defined herein.

Mode of administration

[0059] The present invention also contemplates medicaments, and methods of making same, many of which methods are well known in pharmaceutical practice. For example, the novel polypeptides of the invention can be formulated into various forms for administration to mucous membranes, into intra-articular areas, intraperitoneally, intravascularly, topically, subcutaneously, and via suppository. Such medicaments may be formulated together with suitable carriers, excipients, binders, fillers, and the like into dosage forms, with each form comprising a fraction or a multiple of the daily dose required in order to achieve the desired treatment result. Particularly, the clinical treatment may use sequential parenteral injections (subcutaneous route) to avoid peptidic digestion in the Gl tract.

[0060] Alternatively, the compounds of the invention may be formulated for buccal administration such as, for example, sublingual administration. Such formulations may take the form of chewing delivery systems such as chewing gum or chew tablets.

[0061] It will also be appreciated that various combinations of the preceding elements may be made to provide other efficacious peptides, compositions, and methods according to the present invention.

[0062] The present disclosure will be more readily understood by referring to the following examples which are given to illustrate embodiments rather than to limit its scope.

EXAMPLES

Example 1- EFFECT OF KPP-1A AND KPP-1B ON SHP-1

[0063] To test the possible functional effect of KPP-1A and KPP-1 B on the amplification of the activity of phosphatase SHP-1 , 2 types of experiments were conducted by using ex vivo RANKL-expressing neutrophils of synovial fluid from active chronic inflammatory arthropathies and by inducing in vitro RANKL expression by normal neutrophils incubated in the presence of RANKL expression activators, as described (Chakravarti A, et a/., Blood (2009) 114:1633-1644). The intensity of RANKL expression by these neutrophils was routinely evaluated by cytofluorometry. The results are summarized in Figs. 5A and 5B. In Fig. 5A: Normal human blood neutrophils (5x10⁶/ml) were incubated with LPS for 2 days. They were then incubated in the presence of vehicle (Ctl), RANK+OPG 1 μg/ml each (R+O), KPP-1A or KPP- 1 B 1 μg/ml each for 24 hours. After centrifugation, acellular supernatants were collected and frozen at - 80°C until assayed for IL-8 measurement by ELISA. Results are expressed as means±SEM (n=3). Neutrophils after 2 days with LPS expressed 12% RANKL as analyzed by FACScan. In Fig. 5B: Synovial fluid neutrophils from chronic inflammatory arthritis were purified by Percoll gradient centrifugation and incubated in the presence of vehicle (Ctl), RANK+OPG 1 μg/ml each (R+O), KPP-1A or KPP-1 B 1 μg/ml each for 24 hours. After centrifugation, acellular supernatants were collected and frozen at - 80°C until assayed for IL-8 measurement by ELISA. Results are expressed as means. Ex vivo synovial fluid neutrophils expressed 43% RANKL as analyzed by FACScan.

[0064] KPP-1A showed a negative regulatory effect on IL-8 production by RANKL- expressing neutrophils in both experimental conditions. In contrast, no inhibitory effect was observed for KPP-1 B; to date, no explanation is available for this discrepancy.

Example 2- METHOD OF PRODUCTION OF THE NOVEL POLYPEPTIDES

[0065] a) Peptidic sequences to be synthesized and coupled:

- human OPG sequence: from aa 22 to aa 194 (SEQ ID NO:4)

- human RANK sequence: from aa 30 to aa 187 (SEQ ID NO:2)

[0066] To perform the adequate production of both novel polypeptides (KPP-1A, KPP-1 B: see details below), 2 (two) aa (G = glycine; S = serine) were added at the beginning and 4 (four) aa (E = glutamate, F = phenylalanine, and P = proline) were added between the first and the second subpeptides human 0PG_22-194 and human RANK_30-187

[0067] A) Sequence of the novel polypeptide OPG-RANK (named KPP-1A):

Nucleotidic sequence (OPG Accession number: AB002146 / RANK Gene ID: 8792):

5' GGA TCC GAA ACG TTT CCT CCA AAG TAC CTT CAT TAT GAC GAA GAA ACC TCT CAT CAG CTG TTG TGT GAC AAA TGT CCT CCT GGT ACC TAC CTA AAA CAA CAC TGT ACA GCA AAG TGG AAG ACC GTG TGC GCC CCT TGC CCT GAC CAC TAC TAC ACA GAC AGC TGG CAC ACC AGT GAC GAG TGT CTA TAC TGC AGC CCC GTG TGC AAG GAG CTG CAG TAC GTC AAG CAG GAG TGC AAT CGC ACC CAC AAC CGC GTG TGC GAA TGC AAG GAA GGG CGC TAC CTT GAG ATA GAG TTC TGC TTG AAA CAT AGG AGC TGC CCT CCT GGA TTT GGA GTG GTG CAA GCT GGA ACC CCA GAG CGA AAT ACA GTT TGC AAA AGA TGT CCA GAT GGG TTC TTC TCA AAT GAG ACG TCA TCT AAA GCA CCC TGT AGA AAA CAC ACA AAT TGC AGT GTC TTT GGT CTC CTG CTA ACT CAG AAA GGA AAT GCA ACA CAC GAC AAC ATA TGT TCC GGA AAC AGT GAA TCA ACT CAA AAA CCG GAA TTC CCG ATC GCT CCT CCA TGT ACC AGT GAG AAG CAT TAT GAG CAT CTG GGA CGG TGC TGT AAC AAA TGT GAA CCA GGA AAG TAC ATG TCT TCT AAA TGC ACT ACT ACC TCT GAC AGT GTA TGT CTG CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG AAT GAA GAA GAT AAA TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG GCC CTG GTG GCC GTG GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC GCG TGC ACG GCT GGG TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC CGC AAC ACC GAG TGC GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG CTC AAC AAG GAC ACA GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT GAT GCC TTT TCC TCC ACG GAC AAA TGC AGA CCC TGG ACC AAC TGT ACC TTC CTT GGA AAG AGA GTA GAA CAT CAT GGG ACA 3' (SEQ ID N0:5)

[0068] Protidic sequence (OPG protein accession number: 000300 / RANK protein accession number: Q9Y6Q6):

GSETFPPKYLHYDEETSHQLLCDKCPPGTYLKQHCTAKW KTVCAPCPDHYYTDSWHTSDECLYCSPVCKELQYVKQEC NRTHNRVCECKEGRYLEIEFCLKHRSCPPGFGVVQAGTP ERNTVCKRCPDGFFSNETSSKAPCRKHTNCSVFGLLLTQ KGNATHDNICSGNSESTQKEEFEIAPPCTSEKHYEHLGRC CNKCEPGKY Met SSKCTTTSDSVCLPCGPDEYLDSWNEED KCLLHKVCDTGKALVAVVAGNSTTPRRCACTAGYHWSQD CECCRRNTECAPGLGAQHPLQLNKDTVCKPCLAGYFSDA FSSTDKCRPWTNCTFLGKRVEHHGT (SEQ ID NO:6)

Sites of restriction in italics

Start of the sequence OPG/RANK

End of the sequence OPG/RANK

AA added to the sequences (P = proline^

[0069] B) Sequence of the novel polypeptide RANK-OPG (named KPP-1B):

Nucleotidic sequence (RANK Gene ID: 8792 / OPG Accession number: AB002146):

5'-GGA TCC ATC GCT CCT CCA TGT ACC AGT GAG AAG CAT TAT GAG CAT CTG GGA CGG TGC TGT AAC AAA TGT GAA CCA GGA AAG TAC ATG TCT TCT AAA TGC ACT ACT ACC TCT GAC AGT GTA TGT CTG CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG AAT GAA GAA GAT AAA TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG GCC CTG GTG GCC GTG GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC GCG TGC ACG GCT GGG TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC CGC AAC ACC GAG TGC GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG CTC AAC AAG GAC ACA GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT GAT GCC TTT TCC TCC ACG GAC AAA TGC AGA CCC TGG ACC AAC TGT ACC TTC CTT GGA AAG AGA GTA GAA CAT CAT GGG ACA CCG GAA TTC CCG GAA ACG TTT CCT CCA AAG TAC CTT CAT TAT GAC GAA GAA ACC TCT CAT CAG CTG TTG TGT GAC AAA TGT CCT CCT GGT ACC TAC CTA AAA CAA CAC TGT ACA GCA AAG TGG AAG ACC GTG TGC GCC CCT TGC CCT GAC CAC TAC TAC ACA GAC AGC TGG CAC ACC AGT GAC GAG TGT CTA TAC TGC AGC CCC GTG TGC AAG GAG CTG CAG TAC GTC AAG CAG GAG TGC AAT CGC ACC CAC AAC CGC GTG TGC GAA TGC AAG GAA GGG CGC TAC CTT GAG ATA GAG TTC TGC TTG AAA CAT AGG AGC TGC CCT CCT GGA TTT GGA GTG GTG CAA GCT GGA ACC CCA GAG CGA AAT ACA GTT TGC AAA AGA TGT CCA GAT GGG TTC TTC TCA AAT GAG ACG TCA TCT AAA GCA CCC TGT AGA AAA CAC ACA AAT TGC AGT GTC TTT GGT CTC CTG CTA ACT CAG AAA GGA AAT GCA ACA CAC GAC AAC ATA TGT TCC GGA AAC AGT GAA TCA ACT CAA AAA-3' (SEQ ID N0:7)

[0070] Protidic sequence (RANK protein accession number : Q9Y6Q6 / OPG protein accession number : 000300):

GSIAPPCTSEKHYEHLGRCCNKCEPGKY Met SSKCTTTSDS VCLPCGPDEYLDSWNEEDKCLLHKVCDTGKALVAVVAGN STTPRRCACTAGYHWSQDCECCRRNTECAPGLGAQHPL QLNKDTVCKPCLAGYFSDAFSSTDKCRPWTNCTFLGKRV EHHGTBEFBETFPPKYLHYDEETSHQLLCDKCPPGTYLKQ HCTAKWKTVCAPCPDHYYTDSWHTSDECLYCSPVCKELQ YVKQECNRTHNRVCECKEGRYLEIEFCLKHRSCPPGFGV VQAGTPERNTVCKRCPDGFFSNETSSKAPCRKHTNCSVF GLLLTQKGNATHDNICSGNSESTQK (SEQ ID NO:8)

Sites of restriction in italics

Start of the sequence RANK/OPG

End of the sequence RANK/OPG

AA added to the sequences (P = proline)

[0071] b) RT-PCR amplification of OPG (active site) and RANK (extracellular portion) fragments: KPP-1A: KPP-1B:

BamH1-OPG-P-EcoR1 BamH1-RANK-P-EcoR1

EcoR1-P-RANK-Xho1 EcoR1-P-OPG-Xho1

[0072] c) Fragment digestion by EcoR1 and ligation of the 2 fragments to obtain:

KPP-1A: KPP-1B:

BamH 1 -OPG-P-EcoR1 -P-RANK-Xho1 BamH 1 -RANK-P-EcoR1 -P-OPG-Xho1

[0073] d) Digestion of the novel polypeptides (A and B) by BamH1 and Xho1 , and then insertion in Bamhl and Xho1 sites of the pGEX4T-1 plasmid. Plasmid amplification in DH5a bacteria, and sequencing of nucleotidic sequences of the respective plasmids.

[0074] Primers used for PCR amplification:

KPP-1A:

BamHI- opg (Forward) 5' GGA TCC GAA ACG TTT CCT CCA AAG TAC CTT C 3' (SEQ ID NO:9)

OPG-p-EcoRI (Reverse) 5' GAA TTC CGG TTT TTG AGT TGA TTC ACT GTT TC 3' (SEQ ID NO: 10)

EcoRI-p- RANK (Forward) 5' GAA TTC CCG ATC GCT CCT CCA TGT ACC AGT GAG 3' (SEQ ID NO:11)

RANK-STOP-XHOI (Reverse) 5' CTC GAG TCA TGT CCC ATG ATG TTC TAC TCT ctt tc 3' (SEQ ID NO: 12)

KPP-1 B:

. BamHI- RANK (Forward) 5' GGA TCC ATC GCT CCT CCA TGT ACC AGT GAG 3' (SEQ ID NO: 13)

. RANK-p-EcoRI (Reverse) 5' GAA TTC CGG TGT CCC ATG ATG TTC TAC TCT ctt tc 3' (SEQ ID NO: 14)

• EcoRI-p- OPG (Forward) 5' GAA TTC CCG GAA ACG TTT CCT CCA AAG TAC CTT C 3' (SEQ ID NO: 15)

• OPG-stop-XHOI (Reverse) 5' GAG CTC TCA TTT TTG AGT TGA TTC ACT GTT TC 3' (SEQ ID NO: 16) [0075] Properties of the novel polypeptides

Number of amino acids: 339 Molecular weight: 37927.7

Theoretical pi: 6.96

[0076] Amino acid composition:

Ala (A) 15 4.4%

Arg(R) 14 4.1%

Asn(N) 14 4.1%

Asp(D) 16 4.7%

Cys(C) 37 10.9%

Gin (Q) 10 2.9%

Glu (E) 24 7.1%

Gly(G) 21 6.2%

His (H) 16 4.7%

lie (I) 3 0.9%

Leu (L) 22 6.5%

Lys(K) 25 7.4%

Met(M) 1 0.3%

Phe(F) 10 2.9%

Pro(P) 24 7.1%

Ser(S) 24 7.1%

Thr(T) 30 8.8%

Trp(W) 5 1.5%

Tyr(Y) 13 3.8%

Val(V) 15 4.4%

Pyl(O) 0 0.0%

Sec(U) 0 0.0%

(B) 0 0.0%

(Z) 0 0.0%

(X) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 40 Total number of positively charged residues (Arg + Lys): 39 [0077] Atomic composition: Carbon C 1622

Hydrogen H 2491

Nitrogen N 467

Oxygen O 511

Sulfur S 38

Formula: C-₁₆₂₂H₂₄₉₁ N₄₆₇O₅₁₁S₃₈

Total number of atoms: 5129

[0078] Extinction coefficients:

Extinction coefficients are in units of M ¹ cm ¹, at 280 nm measured in water.

Ext. coefficient 49120

Abs 0.1% (=1 g/l) 1.295, assuming ALL Cys residues appear as half cystines

Ext. coefficient 46870

Abs 0.1% (=1 g/l) 1.236, assuming NO Cys residues appear as half cystines [0079] Estimated half-life:

The N-terminal of the sequence considered is G (Gly).

The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).

>20 hours (yeast, in vivo).

>10 hours (Escherichia coli, in vivo).

[0080] Instability index:

The instability index (II) is computed to be 62.42

This classifies the protein as unstable.

[0081] Aliphatic index: 46.02

Grand average of hydropathicity (GRAVY): -0.692

[0082] e) Production of novel polypeptides in the bacteria BL21 star™ (Invitrogen)

• Transformation of BL21 star™ with pGEX4T-1-Polyp-A or pGEX4T-1-Polyp-B and selection of positive clone(s) with ampicillin resistance.

• 3 transformants were selected for each novel polypeptide and incubated O/N at 37°C in 5 ml LB medium supplemented with 100 μg/ml of ampicillin and grew until the OD₆₀₀ reached 0.6 to 1.0.

• Those cultures were used to inoculate fresh LB medium containing 100 μg/ml ampicilin to an OD₆₀₀ of 0.1.

• Cultures were induced with 0.5 mM IPTG when they reached OD₆₀₀ of 0.4 for 3 hours. • Cultures were centrifuged and pellets were sonicated 30 sec in PBS 0.6% Triton X-100^® and then centrifuged.

• Clone supernatants were then analyzed by western blot with a mouse anti- GST antibody.

• The best clone was chosen for each novel polypeptide.

[0083] f) Batch purification

• For each ml of bacteria supernatant sonicated, 250 μΙ of glutathione sepharose 4B beads (50% slurry = 50% PBS-50% beads) were added and incubated overnight at 4°C.

• Beads were washed: one time with 10 bead-volume of PBS 0.6% Triton X- 100; 4 times with 10 bead-volume of PBS 0.1% Triton X-114 and 3 times with 10 bead-volume of PBS.

• Beads were then incubated with 250 μΙ of elution buffer (10 mM reduced glutathione in 50 mM Tris, pH 8) for 15 min at RT. Three different elutions were done and analyzed by western blot.

• Eluates containing Chimera were pooled and measure of LPS was assessed (Limulus assay).

Example 3- REGULATION OF SHP-1 EXPRESSION THROUGH RANKL ACTIVATION IN

HUMAN T LYMPHOCYTES AND MACROPHAGES IN VITRO.

[0084] To evaluate in human T lymphocytes and macrophages the possible activation of the phosphatase SHP-1 , as previously demonstrated in activated human neutrophils (Chakravarti A, et al., Blood (2009) 114:1633-1644), activated Jurkat cells (immortalized T cells from an acute T cell leukemia, ATCC^® Number: CRL-2063™) and activated macrophages were studied for their protein tyrosine phosphorylation after stimulation of RANKL expressed at their surface.

[0085] To increase RANKL expression, Jurkat cells were activated by phorbol myristate acetate (PMA) and ionophore A23187 for 24 hrs before addition of vehicle (Cont), RANK+OPG peptides, RANK or OPG peptides alone for 60 min. Their RANKL expression was evaluated by immunofluorescence (Figs. 6A). Cells were recovered at various times to assess their protein tyrosine phosphorylation (Methods are detailed in Chakravarti A, et a/., Blood (2009) 114:1633-1644). A 72 kDa band that corresponded to SHP-1 was, at best, highlighted when T cells were stimulated by RANK+OPG (Fig. 6B). This was the only condition where SHP-1 tyrosine phosphorylation was observed during 60 min. These data indicate that RANKL activation by RANK+OPG peptides in T lymphocytes was associated with a sustained activation of the phosphatase SHP-1.

[0086] To increase RANKL expression, human macrophages were activated by RA (rheumatoid arthritis) synovial fluid for 24 hrs before addition of vehicle (Cont), RANK+OPG peptides, RANK or OPG peptides alone for 60 min. As above, RANKL expression was verified (Fig. 7A) and cells were recovered to evaluate their protein tyrosine phosphorylation. Similarly, a 72 kDa band that corresponded to SHP-1 was highlighted after 20 min (Fig. 7B). RANK+OPG and RANK conditions seemed equivalent, OPG alone had no effect. These data indicate that RANKL activation by RANK+OPG peptides and RANK peptide in macrophages was associated with activation of the phosphatase SHP-1.

[0087] In conclusion, leukocytes such as T lymphocytes and macrophages express a functional RANKL receptor at their surface when they are activated since its specific stimulation is associated with SHP-1 tyrosine phosphorylation. As a corollary, their production of pro-inflammatory factors like cytokines could be downregulated by active SHP-1 as demonstrated in activated neutrophils (Chakravarti A, et a/., Blood (2009) 114:1633-1644).

[0088] The demonstration of an agonistic effect of the combination of OPG + RANK peptides on RANKL-expressing neutrophils is original and novel, mainly because it activates a protein-tyrosine phosphatase SHP-1 which has negative regulatory functions in immunity, inflammation and neutrophils, macrophages and T lymphocytes. This interesting effect is more prominent when RANKL-expressing cells were incubated in the presence of the novel polypeptide KPP-1A.

[0089] To optimize this specific therapeutic effect (natural negative regulatory pathway of inflammation through the protein-tyrosine phosphatase SHP-1), a selective amplification, i.e. treatment with KPP-1A, is required by activating RANKL- expressing cells present in active chronic inflammatory diseases and other inflammatory conditions provoked by RANKL-expressing immune cells.

[0090] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

1. A polypeptide comprising a functional OPG amino acid sequence linked to a functional RANK amino acid sequence, wherein said polypeptide activates RANKL.

2. A peptide selected from the group consisting of: X-OPG_(22-62) to _(154.194)- Y- RANIK _(30-70) to _(157-i87), wherein each of X and Y is an amino acid linker sequence that comprises 1 to 10 amino acids, selected from: Gly, Ala, Pro, Phe, Glu, or Ser, or a conservative substitution thereof.

3. The peptide of claim 1 or 2, wherein the RANK peptide portion comprises a sequence having from amino acid 30 to amino acid 187 as defined according to amino acid sequence of RANK (defined by protein accession number : Q9Y6Q6)

4. The peptide of claim 1 , 2 or 3, wherein the RANK amino acid sequence is a sequence having amino acid 30 to amino acid 187 as defined in SEQ ID NO:2.

5. The peptide of claim 1 to 4, wherein RANK consists of: amino acids 30 to 187 optionally truncated by one up to 40 amino acids at either end of the peptide such that it is defined as any length from amino acid 30 to 70 at the N-terminal and any length from amino acid 157 to 187 at the C-terminal.

6. The peptide of claim 1 to 5, wherein the OPG amino acid sequence is as set forth in SEQ ID NO:4.

7. The peptide of claim 1 to 6, wherein the OPG amino acid sequence is a sequence having amino acid 22 to amino acid 194 as defined in SEQ ID NO:4.

8. The peptide of claim 1 to 7, wherein the OPG peptidic derivative may consist of: amino acids 22 to 187 optionally truncated by one up to 40 amino acids at either end of the peptide such that it is defined as any length from amino acid 23 to 62 at the N-terminal and any length from amino acid 154 to 193 at the C-terminal.

9. A peptide consisting of X-OPG_22-194-Y-RANK_30-187, wherein Xand Y are as defined in claim 2.

10. The peptide of claim 9, wherein X is Gly-Ser or conservative substitutions thereof.

11. The peptide of claim 9 or 10, wherein Y is Pro-Glu-Phe-Pro or conservative substitutions thereof.

12. Use of a peptide as defined in any one of claims 1 to 11 , for the treatment or prevention of a condition involving neutrophil- and/or T Lymphocyte- and/or macrophage-dependent inflammation, particularly chronic inflammation in which activated immune cells express RANKL.

13. The use according to claim 12, wherein said condition is selected from the group consisting of: rheumatoid arthritis (RA), psoriatic arthritis, and bone lesions induced by immune cell activation (such as, for example: osteomyelitis, periodontitis, periprosthetic osteolysis), psoriasis, breast cancer, lung cancer, chronic obstructive pulmonary disease (COPD), Inflammatory Bowel Disease (IBD) like Crohn's disease.

14. A method for the treatment or prevention of inflammatory condition comprising the administration of a polypeptide as defined in any one of claims 1 to 11.

15. The method of treatment according to claim 14, wherein the inflammatory condition is selected from the group consisting of: rheumatoid arthritis (RA), psoriatic arthritis, and bone lesions induced by immune cell activation (such as, for example: osteomyelitis, periodontitis, periprosthetic osteolysis), psoriasis, breast cancer, lung cancer, chronic obstructive pulmonary disease (COPD), Inflammatory Bowel Disease (IBD) like Crohn's disease.

16. A formulation for buccal delivery of a peptide as defined in any one of claims 1 to 11 , comprising said peptide in admixture with an excipient suitable for a chewing gum or a chew tablet.

17. The method of treatment according to claim 14, wherein said peptide is formulated in a chewing gum or chew tablet for buccal delivery.

18. A peptide as defined by SEQ ID NO:6.