Eotaxin-2 Inhibitors In The Treatment Of Fibromyalgia

TECHNOLOGICAL FIELD

The present invention concerns the use of inhibitors of eotaxin-2

(CCL24), in particular anti eotaxin-2 antibodies, in the treatment of

fibromyalgia.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

1. Clauw DJ. Jama 2014; 311(15): 1547-55.

2. Wolfe F, Clauw DJ, Fitzcharles MA, Goldenberg DL, Katz RS, Mease P, et al. Arthritis care & research 2010; 62(5):600-10.

3. Hauser W, Thieme K, Turk DC. European Journal of Pain 2010;

14(1):5-10.

4. Ablin J, Amital H, Ehrenfeld M, Aloush V, Elkayam O, Langevitz

P, et al. Harefuah. 2013; 152(12):742-7, 51, 50.

5. Ablin J, Fitzcharles M-A, Buskila D, Shir Y, Sommer C, Hauser W.

Evidence-Based Complementary and Alternative Medicine 2013; 2013.

6. Garcia JJ, Cidoncha A, Bote ME, Hinchado MD, Ortega E. Annals of Clinical Biochemistry: An international journal of biochemistry and laboratory medicine. 2014; 51(5):576-81.

7. Zhang Z, Cherryholmes G, Mao A, Marek C, Longmate J, Kalos M,

et al. Experimental biology and medicine. 2008;233(9): 1171-80.

8. Wang H, Buchner M, Moser MT, Daniel V, Schiltenwolf M. The Clinical journal of pain. 2009;25(l): l-4.

9. Wang H, Moser M, Schiltenwolf M, Buchner M. The Journal of rheumatology. 2008;35(7): 1366-70.

10. Wallace D, Linker- Israeli M, Hallegua D, Silverman S, Silver D, Weisman M. Rheumatology. 2001 ;40(7):743-9.

11. Ortega E, Bote M, Giraldo E, Garcia J. Scandinavian journal of medicine & science in sports. 2012;22(1): 104-12.

12. WO 2010/086854. 13. Mor A et al 2013 WJCD. Vol. 3 No. 4: 339-346.

14. Ablin M. Entin-Meer V. Aloush S. et al. 2010; V161(2):276-83.

15. Mausner-Fainberg K, Kami A, George J, et al. World J. Immunol.

2013 Mar; 3(1):7-14.

16. WO 2015/132790.

17. Wolfe F, Clauw DJ, Fitzcharles M-A, Goldenberg DL, Hauser W, Katz RS, et al. Fibromyalgia criteria and severity scales for clinical and epidemiological studies: a modification of the ACR Preliminary Diagnostic Criteria for Fibromyalgia. The Journal of rheumatology. 2011 ;38(6): 1113- 22.

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

Fibromyalgia syndrome (FMS) is a chronic disorder defined by widespread pain, often accompanied by fatigue, cognitive dysfunction, and depression. The disease affects up to one in 20 patients in primary care (1). Establishing diagnosis and evaluating effects of therapy in patients with FMS may be difficult because of the multifaceted nature of the syndrome and overlap with other chronically painful conditions (2) and to date there are no validated biochemical markers for either the diagnosis or the severity assessment of FMS patients.

Current treatment is aimed at suppressing and controlling both pain and somatic symptoms (3, 4). Pharmacological treatment of FMS includes pain relievers, antidepressants and anti-seizure drugs. Patients response is extremely heterogeneous and most patients require a combination of several medications as well as non- pharmacological treatments, in order to achieve improvement (5) . Currently, no clear marker is correlated with the disease progression therefore there is no key indicator to guide a specific mode of treatment.

Although the etiology remains unclear, characteristic alterations in the pattern of sleep and changes in neuroendocrine transmitters such as serotonin, substance P, growth hormone and Cortisol suggest that regulation of the autonomic and neuro-endocrine system appears to be the basis of the syndrome. Garcia et al (6) demonstrate that FMS patients have higher serum concentrations of certain cytokines, in particular the chemokines (chemotactic cytokines) thymus and activation-regulated chemokine (TARC/CCL17), monokine induced by gamma- interferon (MIG/CXCL9), macrophage-derived chemokine (MDC/CCL22), interferon- inducible T-cell alpha chemoattractant (I- TAC/CXCL11), and eotaxin (CCL11), as compared to healthy subjects. Garcia et al further suggest that these chemokines can serve as biomarkers in the diagnosis of FMS.

Zhang et al. reported high levels of MCP-1 and eotaxin in FMS patients (7).

In addition, most FMS patients were shown to have elevated concentrations of serum IL-8 (8, 9), of IL-6 in the supernatants of peripheral blood mononuclear cells (PBMC) (10) and of IL-Ιβ, TNFa, IL-6 and IL-10 in the supernatants of isolated monocytes (11).

None of the above publications showed increased levels of eotaxin-2 in FMS patients or suggested that inhibition of eotaxin-2 may have positive implications on the treatment of FMS.

Eotaxin-2 is a chemokine that promotes cell trafficking and regulates inflammatory activities through the CCR3 complex, especially by inducing chemotaxis of eosinophils, fibroblasts and T-cells. Eotaxin-2 is produced by several inflammatory cells and its receptor CCR3 is present on eosinophils, T-cells, monocytes and fibroblasts.

Eotaxin-2 is known to be involved in allergy conditions as there is a significant increase in the levels of Eotaxin-2 during the allergic response.

WO 2010/086854 (12) discloses antibodies directed against eotaxin-2 that were effective in animal models of cardiovascular, autoimmune and inflammatory diseases.

Mor et al (13) show that a monoclonal antibody prepared against eotaxin-2 attenuated adhesion of lymphocytes to fibronectin and potently inhibited their migration towards VEGF. The antibodies also significantly reduced atherosclerotic plaques in ApoE knock-out mice. As described in Ablin et al. (14) and Mausner et al. (15) inhibition of Eotaxin-2 demonstrated a protective effect in a rat model of Rheumatoid arthritis and in a mouse model of experimental autoimmune encephalomyelitis.

WO 2015/132790 (16) discloses isolated poiyspecific antibodies directed to a unique epitope in the chemokine eotaxin 2, whereby the antibodies bind additional CCR3-binding chemokines. These antibodies were shown to be effective in inhibiting fibrotic and inflammatory features in murine models of systemic sclerosis and idiopathic pulmonary fibrosis (IPF).

GENERAL DESCRIPTION

The present invention describes methods and compositions for treating, or ameliorating the symptoms of FMS by inhibiting the high serum levels of eotaxin- 2 associated with the disease. In particular embodiments, the present invention provides antibodies for use in the treatment of FMS.

Accordingly, in a first of its aspects, the present invention provides an isolated antibody that binds to eotaxin-2, or any antigen-binding fragment thereof that retains the activity of the antibody, for use in the treatment of Fibromyalgia syndrome (FMS).

In one embodiment, said antibody is a fully humanized anti eotaxin-2 antibody comprising a heavy chain variable region comprising:

a) the complementary determining region VH CDR1 comprising the

amino acid sequence denoted by SEQ ID NO. 1 or a variant thereof; b) the complementary determining region VH CDR2 comprising the

amino acid sequence denoted by SEQ ID NO. 2 or a variant thereof; and

c) the complementary determining region VH CDR3 comprising the

amino acid sequence denoted by SEQ ID NO. 3 or a variant thereof; and

a light chain variable region comprising

d) the complementary determining region VK CDR1 comprising the

amino acid sequence denoted by SEQ ID NO. 4 or a variant thereof; e) the complementary determining region VK CDR2 comprising the

amino acid sequence denoted by SEQ ID NO. 5 or a variant thereof; and f) the complementary determining region VK CDR3 comprising the amino acid sequence denoted by SEQ ID NO. 6 or a variant thereof.

In one embodiment, said anti eotaxin-2 antibody is a fully humanized antibody comprising the heavy chain variable region denoted by SEQ ID NO:7 or a variant thereof and the light chain variable region denoted by SEQ ID NO: 8 or a variant thereof.

In one embodiment, said antigen-binding fragment thereof is selected from the group consisting of Fv, single chain Fv (scFv), heavy chain variable region capable of binding the antigen, light chain variable region capable of binding the antigen, Fab, F(ab)2' and any combination thereof.

In another aspect, the present invention provides a pharmaceutical composition comprising a fully humanized antibody, or any antigen-binding fragment thereof that retains the activity of the antibody, wherein said antibody comprises a heavy chain variable region comprising:

a) the complementary determining region VH CDR1 comprising the amino acid sequence denoted by SEQ ID NO. 1 or a variant thereof; b) the complementary determining region VH CDR2 comprising the amino acid sequence denoted by SEQ ID NO. 2 or a variant thereof; and

c) the complementary determining region VH CDR3 comprising the amino acid sequence denoted by SEQ ID NO. 3 or a variant thereof; and

a light chain variable region comprising

d) the complementary determining region VK CDR1 comprising the amino acid sequence denoted by SEQ ID NO. 4 or a variant thereof; e) the complementary determining region VK CDR2 comprising the amino acid sequence denoted by SEQ ID NO. 5 or a variant thereof; and

f) the complementary determining region VK CDR3 comprising the amino acid sequence denoted by SEQ ID NO. 6 or a variant thereof; and a pharmaceutically acceptable carrier; wherein said pharmaceutical composition is for use in the treatment of Fibromyalgia syndrome (FMS).

In another aspect, the present invention provides a method of treating Fibromyalgia syndrome (FMS) comprising administering to a patient in need thereof a therapeutically acceptable amount of an antibody that binds to eotaxin-2, or any antigen-binding fragment thereof that retains the activity of the antibody.

In one embodiment, said antibody that binds to eotaxin-2 is a fully humanized antibody, or any antigen-binding fragment thereof that retains the activity of the antibody, wherein said antibody comprises a heavy chain variable region comprising:

a) the complementary determining region VH CDR1 comprising the amino acid sequence denoted by SEQ ID NO. 1 or a variant thereof; b) the complementary determining region VH CDR2 comprising the amino acid sequence denoted by SEQ ID NO. 2 or a variant thereof; and

c) the complementary determining region VH CDR3 comprising the amino acid sequence denoted by SEQ ID NO. 3 or a variant thereof; and

a light chain variable region comprising

d) the complementary determining region VK CDR1 comprising the amino acid sequence denoted by SEQ ID NO. 4 or a variant thereof; e) the complementary determining region VK CDR2 comprising the amino acid sequence denoted by SEQ ID NO. 5 or a variant thereof; and

f) the complementary determining region VK CDR3 comprising the amino acid sequence denoted by SEQ ID NO. 6 or a variant thereof, or the pharmaceutical composition of the invention.

In one embodiment, said antibody that binds to eotaxin-2, is an isolated polyspecific antibody, or any antigen-binding fragment thereof, that binds to at least two CCR3 -binding chemokines.

In one embodiment, said antibody is a monoclonal antibody.

In one embodiment, said monoclonal antibody is a chimeric antibody, a human antibody, a humanized antibody or a fully humanized antibody. In one embodiment, said antigen-binding fragment thereof is selected from the group consisting of Fv, single chain Fv (scFv), heavy chain variable region capable of binding the antigen, light chain variable region capable of binding the antigen, Fab, F(ab)2' and any combination thereof.

In one embodiment, said at least two CCR3-binding chemokines are selected from the group consisting of Eotaxin 1, Eotaxin-2, Rantes and MCP-3.

In one embodiment, said antibody binds Eotaxin 1 , Eotaxin-2, Rantes and

MCP-3.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 is a graph showing the circulating systemic levels of Eotaxin 2 (CCL24) in 52 FMS patients, compared with control (20 healthy subjects), as measured by Elisa. The results are presented in picograms per milliliter (pv-P value).

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention is based on the surprising finding that FMS is associated with high levels of eotaxin 2 (CCL24) in the sera of patients. It is therefore postulated by the inventors that reducing the levels of eotaxin-2 in the patient's sera using an eotaxin-2 antagonist, such as an anti eotaxin 2 antibody, can treat or ameliorate the symptoms of Fibromyalgia syndrome (FMS).

Therefore, in one embodiment the present invention provides an antibody that binds eotaxin-2 (also referred to as an anti eotaxin-2 antibody), for use in the treatment or amelioration of symptoms of FMS.

In one embodiment, the anti eotaxin2 antibody is a monoclonal antibody that is directed to a conformational epitope in the eotaxin 2 (CCL24) polypeptide and which binds and inhibits the activity of eotaxin-2 (as well as additional chemotactic agents, the proinflammatory CCR3 binding chemokines: Eotaxin 1, Rantes and MCP-3/1). In certain embodiments said anti eotaxin-2 antibody is a murine monoclonal antibody (termed herein CM101, US 9,067,989) and its humanized version HCM101 (WO 2015/132790). These antibodies were implicated for use in the treatment of fibrotic diseases, autoimmune inflammatory disorders, monocyte related disorders or allergic atopic disorders. US9,067,989 and WO 2015/132790 are both incorporated herein by reference.

The present invention provides for the first time a novel use for anti eotaxin- 2 antibodies in the treatment of FMS.

Therefore, in a first of its aspects the present invention provides an isolated anti eotaxin-2 antibody, or any antigen-binding fragment thereof that retains the binding activity of the antibody, for use in the treatment of FMS.

The terms "eotaxin 2 " (eosinophil chemotactic protein 2), "CCL24" (Chemokine (C-C motif) ligand 24) or "MPIF-2 " (myeloid progenitor inhibitory factor 2) are used interchangeably and refer to a cytokine belonging to the CC chemokine family which is encoded by the human CCL24 gene, located on human chromosome 7. CCL24 interacts with chemokine receptor CCR3. CCL24 activity includes induction of chemotaxis in eosinophils, basophils, T lymphocytes and neutrophils, as well as induction of angiogenic and migratory responses in endothelial and smooth muscle cells.

In certain embodiment, the anti eotaxin-2 antibody of the invention is a poly-specific antibody. As used herein the term "poly-specific (or polyspecific) antibody" refers to poly reactive antibodies which are able to recognize multiple antigens, specifically the invention encompasses poly-specific antibodies which are able to recognize several, different, proinflammatory CCR3-binding chemokines. Specifically the invention relates to an antibody that was generated against eotaxin 2 and was found subsequently to bind and effectively attenuate the activity of additional chemokines (for example, eotaxin 1 , Rantes and MCP- 3). In certain embodiments, the poly-specific antibody of the invention binds to the various chemokines with similar affinities. In other embodiments, the antibody has differential binding affinity to the various chemokines. In one specific embodiment the antibody binds with a higher affinity to eotaxin 2 than to the other tested chemokines. Apparently, the poly-specific antibodies recognize a cross- reactive epitope in these proinflammatory CCR3-binding chemokines. The term "CCR3-binding chemokines" as herein defined refers to any chemokine that binds to the protein CCR3 and encompasses for example but not limited to Eotaxin 1, Eotaxin-2, Eotaxin-3, Rantes, MCP-3 and MCP-4. It should be emphasized that some of the CCR3 -binding chemokines also bind additional chemokine receptors, e.g. CCR1, CCR2 or CCR5.

CCR3 (C-C chemokine receptor type 3) is a protein that in humans is encoded by the CCR3 gene. CCR3 has also recently been designated CD193 (cluster of differentiation 193). The protein encoded by this gene is a receptor for C-C type chemokines. It is a 7-transmembrane G protein-coupled receptor which is expressed by eosinophils as well as by a wide array of cell types including macrophages and endothelial cells. This receptor binds and responds to a variety of chemokines, including eotaxin (also termed eotaxin 1 or CCL11), eotaxin-2 (CCL24), eotaxin-3 (CCL26), MCP-3 (CCL7), MCP-4 (CCL13), and RANTES (CCL5).

Thus in some embodiments, the isolated polyspecific antibody for use of the invention is wherein the at least two CCR3-binding chemokines are selected from the group consisting of Eotaxin 1, Eotaxin-2, Rantes and MCP-3.

The term "Eotaxin 1" (also known as C-C motif chemokine 11 and eosinophil chemotactic protein) as herein defined refers to a protein that in humans is encoded by the CCL11 gene. Eotaxin 1 selectively recruits eosinophils by inducing their chemotaxis, and therefore, is implicated in allergic responses. The effects of Eotaxin 1 are mediated by its binding to a G-protein-linked receptor known as a chemokine receptor, including CCR2, CCR3 and CCR5.

The term "Rantes" (regulated on activation, normal T cell expressed and secreted), also termed chemokine (C-C motif) ligand 5 (CCL5) as herein defined refers to a protein which in humans is encoded by the CCL5 gene. Rantes is an 8 kDa protein classified as a chemotactic cytokine or chemokine for T cells, eosinophils, and basophils, and plays an active role in recruiting leukocytes into inflammatory sites. With the help of particular cytokines (i.e., IL-2 and IFN- gamma) that are released by T cells, Rantes also induces the proliferation and activation of certain natural-killer (NK) cells to form CHAK (CC-Chemokine- activated killer) cells. Rantes binds, inter alia, the CCR3 receptor.

The term "MCP-3" (monocyte-specific chemokine 3) also termed chemokine (C-C motif) ligand 7 (CCL7) as herein defined is classified among the subfamily of chemokines known as CC chemokines. MCP-3 specifically attracts monocytes, and regulates macrophages function. It is produced by certain tumor cell lines and by macrophages. This chemokine is located on chromosome 17 in humans, in a large cluster containing many other CC chemokines.

The present invention thus provides an isolated polyspecific antibody, or any antigen-binding fragment thereof, that binds to at least two CCR3 -binding chemokines for use in the treatment of Fibromyalgia syndrome (FMS).

The terms "Fibromyalgia" and "Fibromyalgia syndrome (FMS)" are used interchangeably herein to denote a medical condition characterized by chronic widespread pain and a heightened pain response to pressure. Other symptoms include feeling tired to a degree that normal activities are affected, sleep problems, and troubles with memory. Some people also report restless leg syndrome, bowel or bladder problems, numbness and tingling, and sensitivity to noise, lights or temperature. Fibromyalgia is frequently associated with depression, anxiety, and posttraumatic stress disorder. Other types of chronic pain are also frequently present.

The cause of fibromyalgia is unknown but believed to involve a combination of genetic and environmental factors with half the risk attributed to each. The condition runs in families and many genes are believed to be involved. Environmental factors may include psychological stress, trauma, and certain infections. The pain appears to result

from processes in the central nervous system and the condition is referred to as a "central sensitization syndrome". Fibromyalgia is recognized as a disorder by the US National Institutes of Health and the American College of Rheumatology. There is no specific diagnostic test. Diagnosis involves first ruling out other potential causes and verifying that a set number of symptoms are present.

Fibromyalgia is estimated to affect 2-8% of the population. Females are affected about two times more often than males. Rates appear similar in different areas of the world and among different cultures.

As indicated above, the present invention provides isolated antibodies that bind to eotaxin-2 (and optionally to at least one additional proinflammatory CCR3- binding chemokine) for use in the treatment of Fibromyalgia syndrome (FMS). The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or functional fragments thereof that specifically bind and recognize an antigen, namely eotaxin-2.

In a preferred embodiment the antibody of the invention is a monoclonal antibody. The term "monoclonal antibody", "monoclonal antibodies" or "mAb" as herein defined refers to a population of substantially homogenous antibodies, i.e., the individual antibodies comprising the population are identical except for possibly naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are directed against a single antigenic site.

Monoclonal antibodies may be prepared and purified by any method known in the art. For example, monoclonal antibodies may be prepared from B cells taken from the spleen or lymph nodes of immunized animals (e.g. rats or mice), by fusion with immortalized B cells under conditions which favor the growth of hybrid cells.

Immunization of mice may be carried out for example as described in WO 2010/086854. Briefly, immunization of mice may be carried out for example by primary sub-cutaneous (s.c.) immunization with the desired antigen, namely with a CCR3- binding chemokine, e.g. eotaxin 2, or with a fragment of a CCR3-binding chemokine comprising a conformational epitope in the N-loop (e.g. 50μg) emulsified with complete Freund's adjuvant. Two subcutaneous booster injections with the antigen (e.g. 50μg) emulsified with incomplete Freund's adjuvant are then administered every 2 weeks. The mice with the highest neutralizing antibody titer receive an additional intravenous (i.v.) boost of the antigen (e.g. 5μg) in PBS four days prior to spleen removal.

After the final boost (e.g. four days), the spleen of the mouse with the highest neutralizing antibody titer is removed and splenocytes are fused to mouse myeloma cells (e.g. NS0 cells) using polyethylene glycol, as previously described (Kohler, G. and Milstein, C. (1975) Nature 256: 495-497). After fusion, the hybridoma cells are selected by growing the cells in HAT (hypoxantine- aminopterin-thymidine) medium. Cell clones are then screened for specific antibody production, for example using the ELISA assays described below.

Purification of monoclonal antibodies may be based for example on affinity chromatography, namely, using an affinity column to which the specific epitope is conjugated. An exemplary antibody structural unit comprises a tetramer, as known in the art. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" and one "heavy chain". The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen (or epitope) recognition.

Thus, the terms "heavy chain variable region" (VH) and "light chain variable region" (VL) refer to these heavy and light chains, respectively. More specifically, the variable region is subdivided into hypervariable and framework (FR) regions. Hypervariable regions have a high ratio of different amino acids in a given position, relative to the most common amino acid in that position. Four FR regions which have more stable amino acids sequences separate the hypervariable regions. The hypervariable regions directly contact a portion of the antigen's surface. For this reason, hypervariable regions are herein referred to as "complementarity determining regions" , or "CDRs".

From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDRl, FR2, CDR2, FR3, CDR3 and FR4. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDRl, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.

Thus, the complementarity determining regions CDRH1, CDRH2 and CDRH3 refer to the three complementarity determining regions starting from the N-terminus of the antibody's heavy chain and the complementarity determining regions CDRLl, CDRL2 and CDRL3 refer to the three complementarity determining regions starting from the N-terminus of the antibody's light chain.

In some embodiments the isolated anti eotaxin-2 monoclonal antibody is a chimeric antibody, a human antibody, a humanized antibody or a fully humanized antibody.

The term "chimeric" antibodies as herein defined refers to antibodies in which a portion of the heavy and/or light chain is derived from a particular species, while the remainder of the chain(s) is derived from another species, as well as fragments of such antibodies, which exhibit the same biological activity. Chimeric antibodies may be prepared by any method known in the art, for example as described below.

A murine -human chimeric antibody may be prepared by the amplification and cloning of murine VH and VL genes, encoding the antibody variable regions, followed by murine -human chimeric antibody expression. To this end, total RNA is isolated from murine anti-eotaxin 2 hybridoma cells that are shown to secrete antibodies with the desired characteristics and cDNA is synthesized using oligo (dT)i5 primer, M-MLV and AMV reverse transcriptases. Amplification of the heavy and the light variable genes (VH and VL) may be carried out using a panel of primers directed at the 5' terminus of framework 1 of each gene, essentially as described in Benhar and Reiter (Benhar, I. and Reiter, Y. (2002) Curr. Protoc. Immunol. Chapter 10: Unit 10 19B), and to the constant region (CHI or Ck, respectively) at the 3' end.

The variable genes are then re-amplified using non-degenerate primers introducing restriction sites at both ends for cloning, for example, into a pCMV- based antibody expression vector.

The amplified heavy and light variable genes are separately purified, digested and cloned into appropriate mammalian full-length Ig expression vectors, providing each chain with a corresponding signal-peptide and constant gene, resulting in IgGl/k murine human chimeric antibody expression.

The term "humanized" antibodies traditionally refers to forms of non- human (for example, murine) antibodies that contain a human-derived immunoglobulin framework with minimal sequences derived from non-human immunoglobulin at the CDRs and optionally at additional relevant positions. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and activity.

As used herein the term "fully humanized" antibodies relates to antibodies designed to have only human sequences. The fully humanized antibodies of the present invention were prepared using the Composite Human Antibodies™ technology that minimizes immunogenicity of the antibodies in patients. In this humanization technology multiple sequence segments derived from variable (V) regions of unrelated human antibodies are used as acceptors for the complementarity determining regions (CDRs) of the starting antibodies. Through careful selection of human sequence segments and the application of in silico tools, CD4+ T cell epitopes are avoided so the risk of immunogenicity is reduced compared to standard humanized antibodies whilst antibody affinity and specificity is maintained. Such antibodies contain only human sequences and are thus defined as "fully humanized".

The term "human antibody" as used herein refers to an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies known in the art. This definition specifically excludes a humanized antibody that comprises non-human antigen-binding residues.

Preparation of humanized and human antibodies is well known in the art. Antibodies may also be prepared using phage display. As known in the art, antibody phage display (APD) is based on genetic engineering of bacteriophages and repeated rounds of antigen-guided selection and phage propagation.

The APD process begins with antibody-library preparation, by preparation of quality RNA from the cell source chosen (e.g., peripheral blood mononuclear cells). This RNA is reverse-transcribed into cDNA, which is used for PCR of the VH and VL chains of the encoded antibodies. This step is followed by ligation of the variable heavy (VH) and variable light (VL) PCR products into a phage display vector, culminating in analysis of clones of mAbs.

For preparing large quantities of the antibody (either chimeric, humanized, fully humanized or human), a stable cell line expressing the antibody can be prepared, by transfecting cells (e.g. CHO cells) with the Ig expression vector containing both heavy and light chains of the antibody. The antibodies may then be manufactured in a state of the art single -use bioreactor system. The antibodies may be purified to clinical grade using well established monoclonal antibody purification methods. Highly anti eotaxin-2 antibody producing clones may be then selected and expanded based on antibody levels in the supernatant, as tested by any method known in the art, for example, an eotaxin-2 specific ELISA assay, as detailed herein below. A master cell bank, developed for the specific clone, may serve as the starting growing material for all clinical grade batches. In some embodiments the present invention provides an isolated anti eotaxin-2 murine antibody (termed herein CM101), or any antigen-binding fragment thereof, for use in the treatment of FMS, wherein said antibody is selected from the group consisting of:

a. a monoclonal antibody comprising a heavy chain encoded by a nucleic acid having at least 90% homology with SEQ ID NO: 11 and a light chain encoded by a nucleic acid having at least 90% homology with SEQ ID NO: 12, or a fragment thereof which retains the binding activity of the antibody; and b. a monoclonal antibody secreted by hybridoma D8 (ECACC Accession No. D 809081702), or a fragment thereof which retains the binding activity of the antibody.

The nucleic acid sequence encoding for the murine antibody CM101 heavy chain is denoted as SEQ ID NO: 11 :

GGGCAGCAGANCCGGGGCNGNGGATAGACAGANGGGGGNNGNCGTTT

TGGCTGAGGAGACGGTGACTGAGGTTCCTTGACCCCAGTTGTCCATAGC

GTAGCTACTACCGTAGGAATGACTTGCACAGAAATATGTAGCCGTGTC

CTCATTTCTGAGGTTGTTGATCTGCAAATAGGCAGTGCTGGCAGAGGTT

TCCAAAGAGAGGGCAAACCGTCCCTTGAAGTCATCAGTATATGTTGGC

TCTCCATTGTAGGTGTTGATCCAGCCCATCCACTTTAAACCCTTTCCTGG

AGCCTGCTTTACCCAGTTCATTCCAGAGTTTGTGAAGGGATACCCAGAA

GCCCTGCAGGAGATCTTGACTGTGTCTCCAGGCTTCTTCAGCTCANGTC

CAGACTGCACCAACTGGATCTGGGCCATGGCCNGCTA

The nucleic acid sequence encoding for the murine antibody CM 101 light chain (kappa) is denoted as SEQ ID NO: 12:

GGGCCAATGGNNGAGGACGCGGATGGGGGTGTCGNNGTGCCTTNGTCG

NNNNCTNNTTGNNCANCNTCNACNNCNNNNANNNNANNGNNNNNTGN

AANANNGATGGNNNTNNNCNACANNNTGGNNTCCTNNNNNNNTNNNN

TGNNNNNGACNNCANANACANNNNCNACNNNATGANCNNCNNNCNNN

NNTTGANNNNNGNCNANTATGAACNANNNAANNNNNNTACCTGNNAN

GCCACTCACAAGACATCA The invention also encompasses humanized and fully humanized versions of the antibody CM101.

In some embodiments the present invention provides a fully humanized isolated anti eotaxin2 antibody (termed herein HCMlOl), or any antigen-binding fragment thereof, for use in the treatment of FMS, wherein said antibody comprises a heavy chain variable region comprising:

a) the complementary determining region VH CDRl comprising the amino acid sequence denoted by SEQ ID NO. 1 or a variant thereof;

b) the complementary determining region VH CDR2 comprising the amino acid sequence denoted by SEQ ID NO. 2 or a variant thereof; and

c) the complementary determining region VH CDR3 comprising the amino acid sequence denoted by SEQ ID NO. 3 or a variant thereof; and

a light chain variable region comprising

d) the complementary determining region VK CDRl comprising the amino acid sequence denoted by SEQ ID NO. 4 or a variant thereof;

e) the complementary determining region VK CDR2 comprising the amino acid sequence denoted by SEQ ID NO. 5 or a variant thereof; and

f) the complementary determining region VK CDR3 comprising the amino acid sequence denoted by SEQ ID NO. 6 or a variant thereof.

In accordance with the invention VH CDRl comprises the amino acid sequence NSGMN denoted by SEQ ID NO. 1, or a variant thereof; VH CDR2 comprises the amino acid sequence WINTYNGEPTYTDDFKG denoted by SEQ ID NO. 2, or a variant thereof, VH CDR3 comprises the amino acid sequence HSYGSSYAMDN denoted by SEQ ID NO. 3 or a variant thereof; VK CDRl comprises the amino acid sequence KASQSVDYDGDSYMN denoted by SEQ ID NO. 4 or a variant thereof; VK CDR2 comprises the amino acid sequence VASNLKS denoted by SEQ ID NO. 5 or a variant thereof; and VK CDR3 comprises the amino acid sequence QQSNEEPWT denoted by SEQ ID NO. 6 or a variant thereof.

The above CDR sequences CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 denoted by SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively are also presented in the context of their respective heavy and light chains sequences: The amino acid sequence of the heavy chain of the isolated fully humanized anti eotaxin 2 monoclonal antibody exemplified herein is denoted by SEQ ID NO: 7 and is of the amino acid sequence:

QIQLVQSGPELKKPGASVKVSCRASGYPFTNSGMNWVKQAPGKGLKWM

GWINTYNGEPTYTDDFKGRFAFSLETSASTAYLQINNLRNEDTATYFCASH

SYGSSYAMDNWGQGTSVTVSS

The amino acid sequence of the light chain of the isolated humanized anti eotaxin 2 monoclonal antibody exemplified herein is denoted herein by SEQ ID NO: 8 and is of the amino acid sequence:

DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKL LIYVASNLKSGIPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEEPWTFG GGTKVEIK

Therefore in further embodiemtns the isolated antibody for use according to the invention is wherein said antibody is a fully humanized antibody comprising the heavy chain variable region denoted by SEQ ID NO:7 or a variant thereof and the light chain variable region denoted by SEQ ID NO: 8 or a variant thereof.

In another embodiment, the isolated antibody for use according to the invention is wherein said antibody is a fully humanized antibody comprising a heavy chain variable region encoded by a nucleic acid sequence denoted by SEQ ID NO: 9 or a variant thereof and a light chain variable region encoded by a nucleic acid sequence denoted by SEQ ID NO: 10 or a variant thereof.

SEQ ID NO: 9:

CAGATCCAATTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGCC

TCAGTCAAGGTCTCCTGCAGGGCTTCTGGGTATCCCTTCACAAACTCTG

GAATGAACTGGGTAAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATG

GGCTGGATCAACACCTACAATGGAGAGCCAACATATACTGATGACT

TCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTA

TTTGCAGATCAACAACCTCAGAAATGAGGACACGGCTACATATTTCTGT

GCAAGTCATTCCTACGGTAGTAGCTACGCTATGGACAACTGGGGTC

AAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO: 10:

GACATTGTGCTGACCCAATCTCCAGACTCTTTGGCTGTGTCTCTAGGGG AGAGGGCCACCATCAACTGCAAGGCCAGCCAAAGTGTTGATTATGAT

GGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACC

CAAACTCCTCATCTATGTTGCATCCAATCTAAAATCTGGCATCCCAGC

CAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGC

AGCCTGCAGCCTGAGGATTTTGCAACCTATTACTGTCAGCAAAGTAAT

GAGGAACCGTGGACGTTCGGTGGAGGCACCAAGGTGGAAATCAAA

*CDR nucleotide sequences are bolded and underlined.

The present invention also encompasses variants of the heavy and light chain variable regions. The variants may include mutations in the complementarity determining regions of the heavy and light chains which do not alter the activity of the antibodies herein described, or in the framework region.

By the term "variant" it is meant sequences of amino acids or nucleotides different from the sequences specifically identified herein, in which one or more amino acid residues or nucleotides are deleted, substituted or added.

It should be appreciated that by the term "added ", as used herein it is meant any addition of amino acid residues to the sequences described herein.

Variants encompass various amino acid substitutions. An amino acid "substitution" is the result of replacing one amino acid with another amino acid which has similar or different structural and/or chemical properties. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

Typically, variants encompass conservative amino acid substitutions. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

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);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M).

Conservative nucleic acid substitutions are nucleic acid substitutions resulting in conservative amino acid substitutions as defined above.

Variants in accordance with the invention also encompass non-polar to polar amino acid substitutions and vice-versa.

As used herein, the term "amino acid" or "amino acid residue" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

Variant sequences refer to amino acid or nucleic acids sequences that may be characterized by the percentage of the identity of their amino acid or nucleotide sequences with the amino acid or nucleotide sequences described herein (for example, the amino acid or nucleotide sequences of the heavy and light chains of the antibodies herein described).

In some embodiments, variant sequences as herein defined refer to nucleic acid sequences that encode the heavy and light chain variable regions, each having a sequence of nucleotides with at least 70% or 75% of sequence identity, around 80% or

85% of sequence identity, around 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the sequences of the heavy and light chain variable regions described herein.

By the term "activity of the antibodies" it is meant the ability of the antibodies to bind eotaxin-2, and preferably to inhibit a biological function mediated by eotaxin-2, for example inhibition of cell recruitment or chemotaxis (for example recruitment or chemotaxis of eosinophils or monocytes or fibroblasts). The biological functions can be measured in vivo or in vitro using methods well known in the art.

The binding of the antibody of the invention to eotain-2 may be measured for example using ELISA assays.

The present invention further encompasses any antigen-binding fragments of the isolated monoclonal antibody of the invention. Such antigen-binding fragments may be for example Fab and F (ab')2, which are capable of binding antigen. Such fragments may be produced by any method known in the art, for example by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

Thus in some embodiments the isolated anti eotaxin-2 monoclonal antibody according to the invention is wherein said antibody is an antibody fragment selected from the group consisting of Fv, single chain Fv (scFv), heavy chain variable region capable of binding the antigen, light chain variable region capable of binding the antigen, Fab, F(ab)2' and any combination thereof.

The antigen-binding fragment of the antibody exhibits the same biological activity of the whole antibody, namely it retains the binding activity of the whole antibody, and is also termed herein a "functional fragment".

In another one of its aspects the present invention provides an isolated nucleic

acid molecule comprising a nucleotide sequence encoding an antibody or any antigen- binding fragment thereof according to the invention, wherein said antibody is for use in the treatment of FMS.

The term "nucleic acid" or "nucleic acid molecule" as herein defined refers to a polymer of nucleotides, which may be either single- or double-stranded, which is a polynucleotide such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double- stranded polynucleotides. The term DNA used herein also encompasses cDNA, i.e. complementary or copy DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase).

The invention further provides an expression vector comprising the isolated nucleic acid molecule as herein defined.

"Expression vector" sometimes referred to as "expression vehicle" or "expression construct", as used herein, encompasses vectors such as plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles, which enable the integration of DNA fragments into the genome of the host. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. The expression vector in accordance with the invention may be competent with expression in bacterial, yeast, or mammalian host cells, to name but few.

In yet another one of its aspects the present invention provides a host cell transfected with the isolated nucleic acid molecule according to the invention or with the expression vector according to the invention.

The term "host cells" as used herein refers to cells which are susceptible to the introduction of the isolated nucleic acid molecule according to the invention or with the expression vector according to the invention. Preferably, said cells are mammalian cells, for example CHO cells or NSO cells. Transfection of the isolated nucleic acid molecule or the expression vector according to the invention to the host cell may be performed by any method known in the art.

In yet another one of its aspects the present invention provides an immunoconjugate comprising the antibody or any antigen-binding fragment thereof according to the invention and an additional therapeutic agent, as defined herein below.

The term "immunoconjugate" as herein defined refers to an antibody or any antigen-binding fragment thereof according to the invention that is conjugated (linked or joined) to an additional agent. Immunoconjugates may be prepared by any method known to a person skilled in the art, for example, by cross-linking the additional agent to the antibody according to the invention or by recombinant DNA methods.

The anti eotaxin-2 antibody of the invention may be administered in combination with at least one additional therapeutic agent.

The term "additional therapeutic agent" used herein refers to any agent that may be used for treating FMS. In accordance with certain embodiments said at least one additional therapeutic agent is selected from a group consisting of chemotherapeutics, cytokines, peptides, antibodies and antibiotics.

In certain embodiments, said additional therapeutic agent includes, but is not limited to pain relievers (e.g. Acetamirophen, Ibuprofen, Naproxen sodium, Tramadol), Antidepressants (e.g. Duloxetine, Venlafaxine, Milnacipran, Amitriptyline, Fluoxetine, Paroxetine), Anti-seizure drugs (e.g. Gabapentin and Pregabalin) and muscle relaxants (e.g. Diazepam).

In certain embodiments the additional therapeutic agent is an additional antibody. The term "additional antibody" as herein defined refers to an antibody, which is not the antibody according to the invention, which may be used in combination with the antibody of the invention.

The present invention further provides a pharmaceutical composition comprising as an active ingredient the isolated anti eotaxin-2 antibody of the invention, or any antigen-binding fragment thereof or the immunoconjugate as herein defined and a pharmaceutically acceptable carrier, excipient or diluents, wherein said pharmaceutical composition is for use in the treatment of Fibromyalgia syndrome (FMS).

The "pharmaceutical composition''' of the invention generally comprises the antibody or any antigen-binding fragment thereof as herein defined and a buffering agent, an agent which adjusts the osmolarity of the composition and optionally, one or more pharmaceutically acceptable carriers, excipients and/or diluents as known in the art.

As used herein the term "pharmaceutically acceptable carrier, excipient or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

In other embodiments the pharmaceutical composition according to the invention further comprises an additional therapeutic agent. Non-limiting example of additional therapeutic agents include pain relievers (e.g. Acetamirophen, Ibuprofen, Naproxen sodium, Tramadol), Antidepressants (e.g. Duloxetine, Venlafaxine, Milnacipran, Amitriptyline, Fluoxetine, Paroxetine), Anti-seizure drugs (e.g. Gabapentin and Pregabalin) and muscle relaxants (e.g. Diazepam).

In specific embodiments the present invention relates to a pharmaceutical composition comprising an isolated anti eotaxin-2 fully humanized antibody, or any antigen- binding fragment thereof, wherein the antibody comprises a heavy chain variable region of the amino acid sequence denoted by SEQ ID NO. 7 or a variant thereof and a light chain variable region of the amino acid sequence denoted by SEQ ID NO. 8, or a variant thereof, wherein said pharmaceutical composition is for use in the treatment of Fibromyalgia syndrome (FMS).

Further provided is a method of prophylaxis, treatment or amelioration of FMS, comprising administering to a subject in need thereof a therapeutically effective amount of the isolated poly-specific antibody or any antigen-binding fragment thereof or the pharmaceutical composition according to the invention.

In some embodiments the invention provides a method of prophylaxis, treatment or amelioration of FMS, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated anti eotaxin-2 antibody, or any antigen-binding fragment thereof, or the pharmaceutical composition according to the invention.

The terms "subject" or "patient" are used interchangeably and refer to a subject that may benefit from the present invention such as a mammal (e.g. canine, feline, ovine, porcine, equine, bovine, or human). In one specific embodiment the patient is human. By the term "prophylaxis" as herein defined it is meant to provide a "preventive treatment" or "prophylactic treatment", namely acting in a protective manner, to defend against or prevent the appearance of a symptom of FMS, or FMS disease onset.

It is to be understood that the terms "treat", "treating", "treatment or forms thereof, as used herein, mean reducing, preventing, curing, reversing, ameliorating, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease or a condition or delaying the onset of one or more clinical indications of FMS.

Administration according to the present invention may be performed by any of the following routes: oral administration, intravenous, intramuscular, intraperitoneal, intratechal or subcutaneous injection; intrarectal administration; intranasal administration, ocular administration or topical administration.

In specific embodiments administration according to the present invention may be performed intravenously. In other specific embodiments administration may be performed intraperitoneally. In other specific embodiments administration may be performed by inhalation.

The antibodies or antibody fragments as herein defined, any pharmaceutical compositions comprising the same or any conjugates comprising them may be administered to a subject prior to or post disease onset.

Thus in some embodiments the method of prophylaxis, treatment or amelioration of FMS according to the invention is where said isolated anti eotaxin- 2 antibody or any antigen-binding fragment thereof according to the invention, or pharmaceutical composition according to the invention is administered to said subject prior to or after disease onset.

A "therapeutically effective amount" of the isolated monoclonal antibody or any antigen-binding fragment thereof according to the invention, or the pharmaceutical composition according to the invention for purposes herein defined is determined by such considerations as are known in the art in order to cure, arrest or at least alleviate or ameliorate the medical condition. For any preparation used in the methods of the invention, the dosage or the therapeutically effective amount can be estimated initially from in vitro cell culture assays or based on animal models such as the animal models detailed herein. In some embodiments the therapeutically effective amount in accordance with the invention is in the range of 0.01 to 100 mg/kg.

In other embodiments the therapeutically effective amount in accordance with the invention is in the range of 0.01 to 40 mg/kg, 0.1 to 40 mg/kg, 1 to 10 mg/kg, or 5 to

10 mg/kg.

In other embodiments the isolated anti eotaxin-2 antibody or any antigen- binding fragment thereof according to the invention or pharmaceutical composition according to the invention is administered to the subject as a single dose or multiple doses.

The term "subject in need thereof' in the context of the present invention refers to mammals and in particular to human subjects suffering from FMS.

For example, FMS patients are recruited as described in the Example below and are treated with a single dose of HCM101. The patients are then folio wed-up for 2 months. Patients are treated with the antibody at a dose range of 5-10mg/kg.

The present invention further provides the isolated humanized poly-specific antibody or any antigen-binding fragment thereof according to the invention or the pharmaceutical composition according to the invention for use in a method of prophylaxis, treatment or amelioration of FMS.

In specific embodiments the invention provides an isolated anti eotaxin-2 antibody, or any antigen-binding fragment thereof according to the invention or the pharmaceutical composition according to the invention for use in a method of prophylaxis, treatment or amelioration of FMS.

In specific embodiments the invention provides an isolated fully humanized anti eotaxin2 antibody, or any antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region of the amino acid sequence denoted by SEQ ID NO. 7 or a variant thereof and a light chain variable region of the amino acid sequence denoted by SEQ ID NO. 8, or a variant thereof for use in a method of prophylaxis, treatment or amelioration of FMS.

It is appreciated that the term "purified" or "isolated" refers to molecules, such as amino acid or nucleic acid sequences, peptides, polypeptides or antibodies that are removed from their natural environment, isolated or separated. An "isolated antibody" is therefore a purified antibody. As used herein, the term "purified" or "to purify" also refers to the removal of contaminants from a sample. EXAMPLES

Elevated levels of eotaxin 2 were found in the serum of patients with FMS

In the following experiment, the levels of eotaxin 2 were determined in sera of patients with primary FMS.

Patients:

Patients were examined by a physician, in order to verify the diagnosis of FMS according to the American College of Rheumatology (ACR) updated diagnostic criteria (17) and were screened for alternative diagnoses such as inflammatory joint disease. Additional information was obtained from the patients, including for example, Widespread pain index (WPI) documenting extent of widespread pain, and Symptoms severity score (SSS) documenting severity of associated symptoms e.g. fatigue.

Preparation of samples: whole blood samples were obtained and kept at room temperature for 30 min. The whole blood samples were then centrifuged at 3000rpm for 15 minutes. The serum was deposited into a new tube and could be kept frozen at -80°C or stored at 4°C for subsequent analysis.

Eotaxin 2 analysis:

Levels of Eotaxin 2 were determined in the sera of the FMS patients and in healthy donors using the following Elisa kit: Quantikine human CCL24/Eotaxin-2, (R&D solutions #DCC240B), according to the manufacturer's protocol.

Briefly, ΙΟΟμΙ of each tested sample and a standard (present in the kit) were placed in appropriate wells. The samples were incubated for 2.5 hours at room temperature or overnight at 4°C with gentle shaking. The solution was discarded and the well was washed 4 times with 300μ1 IX Wash Solution. After the last wash, any remaining IX Wash Buffer was removed by aspirating or decanting. Next, ΙΟΟμΙ of the detection antibody (for example, IX Biotinylated Eotaxin-2 Detection Antibody) was added to each well and incubated for 1 hour at room temperature with gentle shaking. The solution was discarded and the wells were washed 4 times with the Wash solution as described above. ΙΟΟμΙ of IX HRP- Streptavidin solution was added to each well and incubated for 45 minutes at room temperature with gentle shaking. The solution was discarded and the wells were washed as noted above. Next, ΙΟΟμΙ of TMB One-Step Substrate Reagent was added to each well and incubated for 30 minutes at room temperature in the dark with gentle shaking. Finally, 50μ1 of Stop Solution was added to each well. The results were read at 450 immediately.

The assessment of high sensitivity C reactive protein (CRP) was done using conventional methods. Many kits, assays and devices for assessing CRP levels are commercially available, for example by Orion Diagnostica, Eurolyser or Genway Biotech Inc. to name just a few.

Results:

To examine the potential involvement of Eotaxin 2 in FMS, the levels of Eotaxin 2 were assessed in the serum of FMS patients as compared to healthy subjects. The results indicated an increase of 40% in Eotaxin 2 (pv<0.0006) in patients with FMS as shown in figure 1.

Without wishing to be bound by theory, it appears that eotaxin 2 may have an important role in FMS. The levels of high sensitivity C reactive protein (CRP), which is a general indicator of inflammation, were also assessed and found not to be correlated to the levels of eotaxin 2. This could indicate a unique role for eotaxin 2 as a standalone biomarker in FMS, unrelated to the progression of an inflammatory process.