WO2014177719A1

WO2014177719A1 - Methods for the diagnosis or prognosis of rheumatic inflammatory diseases

Info

Publication number: WO2014177719A1
Application number: PCT/EP2014/059115
Authority: WO
Inventors: Abdel-Majid Khatib; Géraldine SIEGFRIED; Martine Cohen-Solal
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université Paris Diderot - Paris 7; Universite de Bordeaux; Assistance Publique-Hôpitaux De Paris (Aphp)
Priority date: 2013-05-03
Filing date: 2014-05-05
Publication date: 2014-11-06

Abstract

The invention relates to methods and kits for diagnosing or prognosing rheumatic inflammatory diseases (such as rheumatoid arthritis) as well as for predicting or monitoring the responsiveness of a patient to a treatment against said diseases (such as TNF inhibitors). Methods of the invention comprise a step of measuring in a body fluid sample obtained from said patient (such as a blood sample): a) the expression level of Furin, b) the activity level of Furin, and/or c) the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of said Furin substrate.

Description

METHODS FOR THE DIAGNOSIS OR PROGNOSIS

OF RHEUMATIC INFLAMMATORY DISEASES

FIELD OF THE INVENTION:

The invention provides methods and kits for diagnosing or prognosing rheumatic inflammatory diseases (such as rheumatoid arthritis) as well as for predicting or monitoring the responsiveness of a patient to a treatment against said diseases (such as TNF inhibitors). BACKGROUND OF THE INVENTION:

Rheumatic inflammatory diseases encompass a group of diseases that affect the musculo-skeletal and connective tissues of the body. These diseases are characterized by chronic inflammation that often leads to permanent tissue damage, deformity, atrophy and disability. Amongst them, Rheumatoid Arthritis (RA) is a chronic, systemic, inflammatory disease that affects the synovial membranes of multiple joints. RA considered an acquired autoimmune disease, and genetic factors appear to play a role in its development. In most cases of RA, the subject has remissions and exacerbations of the symptoms. Rarely does the disease resolve completely, although at times the symptoms might temporarily remit.

Although RA has been extensively studied, the etiology and pathogenesis of the disease remain incompletely understood. It should be further noted that irreversible joint destruction can be prevented by intervention at the early stages of the disease. Therefore, it results that early diagnosis of RA is important. However, definitive diagnosis of RA can be difficult. Immunologic tests that can be performed for the diagnosis of RA include, in particular, measurement of the levels of rheumatoid factor (RF), and anti-cyclic citrullinated peptide (anti-CCP) antibodies. Serological testing for RF is complicated by moderate sensitivity and specificity, and high rates of positivity in other chronic inflammatory and infectious diseases. Anti-CCP antibody testing is particularly useful in the diagnosis of RA, with high specificity, positivity early in the disease process, and ability to identify patients who are likely to have severe disease and irreversible damage. However, a negative result in anti-CCP antibody testing does not exclude RA.

Therefore, there is a great need for new biological markers of rheumatic inflammatory diseases. In particular, biomarkers that would allow reliable diagnosis from the early steps of the disease as well as assessment of the severity of the disease and permit early intervention to potentially prevent pain, joint destruction and long-term disability, are highly desirable. Recently, it was shown that Furin expression and activity were high in the synovial pannus (synovial tissue biopsy) in RA patients and mice with collagen- induced arthritis (CIA) and systemic administration of Furin or its derivatives such as fragments thereof are useful in treating autoimmune disease including RA as disclosed in (Lin et al, 2012).

SUMMARY OF THE INVENTION:

The inventors have now shown that Furin is unexpectedly detected in serum obtained from patients affected with rheumatic inflammatory diseases, including rheumatoid arthritis

(RA) as well as systemic lupus erythematosus (SLE), Reiter's arthritis and Sjogren's syndrome, whereas Furin is not detected in serum from health subjects or osteoarthritic patients (none inflammatory disease).

It results that measuring expression level of Furin in a body fluid sample such a blood sample (e.g. serum sample) may be useful as biomarkers for different aims (diagnosis, prognosis and/or monitoring the responsiveness to a treatment).

Alternatively, the inventors have also shown that the processing level of a Furin substrate involved in said rheumatic inflammatory diseases (such as B-cell activating factor

BAFF) may also be useful as biomarkers for the same aims.

DETAILED DESCRIPTION OF THE INVENTION:

In a first aspect, the invention relates to a method for determining whether a patient has, or is at risk of having or developing a rheumatic inflammatory disease, said method comprising a step of measuring in a biological sample obtained from said patient:

a) the expression level of Furin, and/or

b) the activity level of Furin, and/or

c) the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of said Furin substrate.

In a second aspect, the invention relates to a method for assessing the severity or predicting the outcome of a rheumatic inflammatory disease in a patient, said method comprising a step of measuring in a biological sample obtained from said patient:

a) the expression level of Furin,

b) the activity level of Furin, and/or

c) the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of said Furin substrate. In a third aspect, the invention relates to a method for predicting or monitoring the responsiveness of a patient to a treatment against a rheumatic inflammatory disease, said method comprising a step of measuring in a biological sample obtained from said patient: a) the expression level of Furin, and/or

b) the activity level of Furin, and/or

c) the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of said Furin substrate. In a fourth aspect, the invention provides to a kit suitable for carrying out the method as defined above, comprising means for measuring the expression level of Furin and/or the activity level of Furin and/or the expression level of a Furin substrate.

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

As used herein, "measuring" encompasses detecting or quantifying. Indeed, an expression or activity level can be qualitative or quantitative. Thus, a determination of whether a polynucleotide or polypeptide is present or absent (e.g., detectable or undetectable) constitutes determining its expression level in various embodiments while in other embodiments, a quantitative level is determined. A single measurement can provide information about the level of expression, activity, or both. Thus, evaluating the level of expression or activity of a protein includes evaluating one or more parameters or features that provide information about the level of expression of the protein, the activity of the protein, or both. The term "expression or activity" is not intended to indicate that measurements of these parameters are mutually exclusive. As used herein, "detecting" means determining if at least Furin is present or not in a biological sample and "quantifying" means determining the amount of Furin in a biological sample.

As used herein, the term "Furin" (also known as PCSK3, paired basic amino acid cleaving enzyme or PACE) has its general meaning in the art and refers to an enzyme which belongs to the subtilisin-like proprotein convertases (PCs) family. Similar to many other proteinases, Furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto -inhibitory prodomain. Thus, the term may include naturally occurring profurin and variants thereof including the mature (processed) form of the protein. The naturally occurring profurin protein has an aminoacid sequence as shown in GenBank Accession number NP 002560 and is encoded by the nucleic acid sequence provided in the GenBank database under accession number NM 002569.

As used herein, the term "Furin substrate" refers to a precursor protein (unprocessed form) which is proteolytic processed (mature form) by Furin into its functionally active form through cleavage at the general motif (K/R)-(X)n-(K/R) as above-mentioned and more particularly at the C-terminus of the consensus sequence RXXR, where X is any amino acid. Typically, an example of a Furin substrate is B-cell activating factor (BAFF).

As used herein, the term "B-cell activating factor" (BAFF) refers to a Furin substrate cytokine. Indeed, the transmembrane form can be cleaved from the membrane, generating a soluble protein fragment. A naturally occurring soluble form of BAFF exists, in which proteolytic cleavage occurs between amino acids R133 and A134 in human BAFF resulting in a water-soluble biologically active C-terminal portion of BAFF. Thus, the term may include naturally occurring BAFF and variants thereof including the soluble (processed also called mature) form of the protein. The naturally occurring full-length human BAFF protein has an aminoacid sequence as shown in GenBank Accession number AF116456.

As used herein, the term "rheumatic inflammatory diseases" refers to a family of diseases that often affect the joints and connective tissue with inflammation, thus encompass rheumatoid arthritis (RA), ankylosing spondylitis (AS), scleroderma, systemic lupus erythematosus (SLE), Sjogren's syndrome, Reiter's syndrome, Arthritis related to chronic inflammatory bowel disease (IBD) or Crohn's disease, Polymyositis and Dermatomyositis.

As used herein, the term "patient" refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like), that can be afflicted with a rheumatic inflammatory disease (such as RA), but may or may not have the disease.

As used herein, the term "biological sample" refers to a biological sample obtained for the purpose of in vitro evaluation. In the methods of the invention, the biological sample may comprise any body fluid obtained from a patient. Typical biological samples to be used in the method according to the invention are blood samples (e.g. whole blood sample, serum sample, or plasma sample) or synovial fluid samples. A "control reference level" may be determined, for example, by measuring the expression level of Furin nucleic acid or encoded polypeptide, or the activity level of Furin, in a corresponding biological sample obtained from one or more control subject(s) (e.g., not suffering from a rheumatic inflammatory disease or known not to be susceptible to such a disease). The control reference level can be a threshold value or a range.

When such a control reference level is used, a higher or increased level measured in a biological sample (i.e. test sample obtained from the patient) is indicative for example that said patient has, or is at risk of having or developing a rheumatic inflammatory disease. Alternatively, the control reference level may be obtained from a patient having an established rheumatic inflammatory disease. The control reference level may be established based upon comparative measurements between apparently healthy subjects and patients with established rheumatic inflammatory disease.

As used herein, a "higher" or "increased" level refers to a level of expression or activity in a sample (i.e. test sample obtained from the patient) which is at least 20% higher, in an embodiment at least 30% higher, in a further embodiment at least 40% higher; in a further embodiment at least 50% higher, in a further embodiment at least 100% higher (i.e. 2- fold), in a further embodiment at least 200% higher (i.e. 3-fold), in a further embodiment at least 300% higher (i.e. 4-fold), relative to the control reference level (e.g., biological sample obtained from one or more control healthy subject(s)).

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to rheumatic inflammatory disease (such as for instance RA), and can mean a patient's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a patient compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion.

The term "biomarker", as used herein, refers generally to a molecule, i.e., a gene (or nucleic acid encoding said gene), protein, the expression of which in a biological sample from a patient can be measured by standard methods in the art (as well as those disclosed herein), and is predictive or denotes a condition of the patient from which it was obtained.

Diagnostic methods:

A first aspect of the present invention relates to a method for determining whether a patient has, or is at risk of having or developing a rheumatic inflammatory disease, said method comprising a step of measuring in a biological sample obtained from said patient: a) the expression level of Furin, and/or

b) the activity level of Furin, and/or

In one embodiment, the methods of the invention further comprise an additional step consisting of comparing the measured expression level of Furin, and/or activity level of Furin, and/or the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of said Furin substrate with a control reference level wherein a difference between said measured expression level(s) and/or activity and said control reference level and/or activity is indicative whether said subject has or is at risk of having or developing a rheumatic inflammatory disease.

Typically, a higher or increased expression level of Furin, and/or activity level of Furin, and/or the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of said Furin substrate measured in the biological sample obtained from the patient in comparison with a control reference level and/or activity indicates that said patient has, or is at risk of having or developing a rheumatic inflammatory disease.

In one embodiment, the expression level(s) of Furin mature form and/or Furin unprocessed form is (are) measured. In a particular embodiment, the ratio of expression levels of Furin mature form to Furin unprocessed form is measured.

The method according to the invention comprises determining the concentrations of at least mature form of Furin and unprocessed form of Furin in said biological sample and/or the corresponding concentration ratios [Mature form]/[Unprocessed form].

In another embodiment, the expression level of a Furin substrate, such as a Furin substrate involved in a rheumatic inflammatory disease, is measured.

In one embodiment, the expression level(s) of mature form of a Furin substrate and/or unprocessed form of said Furin substrate is (are) measured.

In a particular embodiment, the ratio of expression levels of a mature form of Furin substrate to an unprocessed form of said Furin substrate is measured. The method according to the invention comprises determining the concentrations of at least mature form of a Furin substrate and unprocessed form of said Furin substrate in said biological sample and/or the corresponding concentration ratios [Mature form]/[Unprocessed form]. Since the diagnostic method may be based on the ratio of expression level(s) of biomarkers, it is not necessary, according to a specific embodiment of the method, to determine the absolute expression level(s) of each bio marker. Determining their relative expression level(s) from one to another may be sufficient.

Once the (relative) expression level of the both forms of the Furin substrate of interest have been determined, a patient is determined as having or being at risk of having or developing a rheumatic inflammatory disease, when the ratio of the expression levels of the forms of the biomarkers in said biological sample is such that:

[Mature form]/[Unprocessed form] of Furin determined for the patient to be tested > [Mature form]/[Unprocessed form] of Furin determined for a healthy subject (used a control reference ratio), or

[Mature form]/[Unprocessed form] of a Furin substrate determined for the patient to be tested > [Mature form]/[Unprocessed form] of said Furin substrate determined for a healthy subject (used a control reference ratio). In one particular embodiment, the Furin substrate involved in a rheumatic inflammatory disease is B-cell activating factor (BAFF).

Accordingly, in one embodiment, the expression level of BAFF is measured.

In another embodiment, the processed form (soluble form) of BAFF is measured.

In a particular embodiment, the ratio [Processed form of BAFF]/[Full-length BAFF] is calculated and compared to [Processed form of BAFF]/[Full-length BAFF] control subject.

When the ratio found in the patient is superior to ratio found in control subject, this indicates that the subject has, or is at risk of having or developing a rheumatic inflammatory disease.

In another particular embodiment, the biological sample is a blood sample (e.g. whole blood sample, serum sample, or plasma sample) or a synovial fluid sample.

In a preferred embodiment, the biological sample is a serum sample.

In another particular embodiment, the rheumatic inflammatory disease is selected from the group consisting rheumatoid arthritis (RA), ankylosing spondylitis, scleroderma, systemic lupus erythematosus (SLE) and Sjogren's syndrome.

In a preferred embodiment, the rheumatic inflammatory disease is RA.

In another preferred embodiment, the rheumatic inflammatory disease is SLE. In another preferred embodiment, the rheumatic inflammatory disease is Sjogren's syndrome. In still another preferred embodiment, the rheumatic inflammatory disease is Reiter's arthritis.

In still another embodiment, one or several other biomarkers associated to rheumatic inflammatory disease are further measured used in combination with the above identified biomarkers (e.g. Furin) in the diagnosis methods of the invention. These biomarkers are selected from the group consisting of rheumatoid factors (RF), anti-cyclic citrullinated peptide (anti-CCP) antibodies and C reactive protein (CRP).

Measurement of expression levels of biomarkers of the invention:

Expression levels may in general be measured by either measuring mRNA from the cells and/or measuring expression products, such as polypeptides and proteins. Expression of the transcripts and/or proteins encoded by the nucleic acids described herein may be measured by any of a variety of known methods in the art. Determination of the expression level of the biomarkers genes by mRNAs measurement Methods for determining the quantity of mR A are well known in the art. For example the nucleic acid contained in the biological samples (e.g., peripheral blood mononuclear cells (PBMC) isolated from a blood sample obtained from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.

Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical.

In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate). In a particular embodiment, the methods of the invention comprise the steps of providing total R As extracted from a biological sample such a blood sample and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi-quantitative RT-PCR.

Determination of the expression level of the biomarkers by measuring gene proteins

Once the biological sample from the patient is prepared, the expression level of Furin or a Furin substrate such as BAFF may be measured by any known method in the art. Methods for determining the level of a bio marker protein in a fluid sample, such as blood sample or a synovial fluid sample are well known in the art.

In a particular embodiment, such methods comprise contacting the biological sample with a binding partner capable of selectively interacting with Furin or a Furin substrate present in the biological sample. In one embodiment, the binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal. In another embodiment, the binding partner may be an aptamer. As used herein, the term "antibody" refers to a protein capable of specifically binding an antigen, typically and preferably by binding an epitope or antigenic determinant or said antigen. The term "antibody" also includes recombinant proteins comprising the binding domains, as well as variants and fragments of antibodies. Examples of fragments of antibodies include Fv, Fab, Fab', F(ab')2, dsFv, scFv, sc(Fv)2, diabodies and multispecific antibodies formed from antibody fragments.

The binding partner may be generally an antibody that may be polyclonal or monoclonal, preferably monoclonal. Polyclonal antibodies directed against Furin or a Furin substrate can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against Furin or a Furin substrate can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler et al. Nature. 1975;256(5517):495-7; the human B-cell hybridoma technique (Cote et al Proc Natl Acad Sci U S A. 1983;80(7):2026-30); and the EBV-hybridoma technique (Cole et al, 1985, In Monoclonal Antibodies and Cancer Therapy (Alan Liss, Inc.) pp. 77-96). Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce anti-CGA, single chain antibodies. Antibodies useful in practicing the present invention also include anti-Furin or Furin substrate fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to Furin or a Furin substrate. For example, phage display of antibodies may be used. In such a method, single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e. g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay.

In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk et al. (1990) Science, 249, 505-510. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena (1999) Clin Chem. 45(9): 1628-50. Peptide aptamers consist of conformational constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al. (1996). Nature, 380, 548-50).

The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled", with regard to the antibody or aptamer, is intended to encompass direct labeling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or lndocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88.

The aforementioned assays generally involve the coating of the binding partner (ie. antibody or aptamer) in a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

In another embodiment of the invention, the measurement of the biomarkers in the biological sample may be achieved by a cytometric bead array system wherein the antibodies that bind to the biomarkers are coated directly or indirectly on beads.

For example, the level of a bio marker protein such as Furin or a Furin substrate may be measured by using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme- labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against Furin or a Furin substrate. A biological sample containing or suspected of containing Furin or a Furin substrate is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody- antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art. Measuring the level of a bio marker protein such as Furin or a Furin substrate (with or without immunoassay-based methods) may also include separation of the proteins: centrifugation based on the protein's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the protein's affinity for the particular solid-phase that is use. Once separated, Furin or a Furin substrate may be identified based on the known "separation profile" e. g., retention time, for that protein and measured using standard techniques.

Alternatively, the separated proteins may be detected and measured by, for example, a mass spectrometer.

Measurement of activity level of the biomarkers of the invention:

Activity levels of Furin may in general be measured by any of a variety of known methods in the art such as for instance enzymatic digestion assay carrying out fluorogenic substrate. Those skilled in the art can easily determine activity levels of Furin using routine experimentation.

For example, activity levels of Furin may be measured by assessing ability to digest the universal PC substrate, the fluorogenic peptide pERTK -AMC, as previously described in Scamuffa et al. 2007.

Typically, the activity assay may be carried out in 96 well plates, and includes the use of a fluorogenic substrate, namely pERTKR-AMC (AMC is amino-methyl-coumarin). The substrate and the biological sample to be tested (e.g. a blood sample) are placed in the wells, and depending on the units of enzyme present, the AMC moiety will be cleaved at a certain rate, such a pmoles/sec. The resultant free AMC is now fluorescent and can be detected with a spectrofluorometer.

Another aspect of the invention relates to the use of serum expression level of Furin or activity level of Furin as a biomarker.

Another aspect of the invention relates to the use of serum expression level of Furin or activity level of Furin as a biomarker of rheumatic inflammatory disease.

In one embodiment, said rheumatic inflammatory disease is selected from the group consisting rheumatoid arthritis (RA), Reiter's arthritis, systemic lupus erythematosus (SLE) and Sjogren's syndrome.

Prognostic methods:

A second aspect of the present invention relates to a method for assessing the severity or predicting the outcome of a rheumatic inflammatory disease in a patient, said method comprising a step of measuring in a biological sample obtained from said patient: a) the expression level of Furin, and/or

b) the activity level of Furin, and/or

Measurement of expression or activity levels of biomarkers of the invention may be carried out by the methods previously described.

Further, in some embodiments, multiple determinations of the expression and/or activity level(s) of biomarker(s) of the invention over time can be made to facilitate prognosing. A temporal change in the expression and/or activity level(s) of biomarker(s) can be used to predict a clinical outcome and/or monitor the progression of a rheumatic inflammatory disease.

In such an embodiment for example, one might expect to see a decrease in the level of expression level of Furin in a biological sample over time in patient with a good prognosis. The methods of diagnosing or prognosing a rheumatic inflammatory disease of the present invention are useful for classifying or stratifying the patients into subgroups having different phenotypes enables a better characterization of said patients and therefore a better selection of a treatment depending on the subgroup to which the patient belongs.

Methods for Predicting or Monitoring Responsiveness:

Accordingly a third aspect of the present invention thus relates to a method for predicting or monitoring the responsiveness of a patient to a treatment against a rheumatic inflammatory disease, said method comprising a step of measuring in a biological sample obtained from said patient: a) the expression level of Furin, and/or

b) the activity level of Furin, and/or

Further, in some embodiments, multiple determinations of the expression and/or activity level(s) of biomarker(s) of the invention over time can be made to facilitate monitoring. A temporal change in the expression and/or activity level(s) of biomarker(s) can be used to monitor efficacy of appropriate therapies directed against a rheumatic inflammatory disease.

In such an embodiment for example, one might expect to see a decrease in the level of expression level of Furin (and potentially at least one additional biomarker) in a biological sample over time during the course of effective therapy.

In one embodiment, the invention relates to a method for monitoring the responsiveness of a patient to a treatment against a rheumatic inflammatory disease, said method comprising the folio wings steps of:

(i) providing a body fluid sample obtained from said patient;

(ii) measuring in said body fluid sample the activity level of Furin; (iii) comparing the measured activity level of Furin with a control reference level, wherein a level higher than the control reference level is indicative that the patient is resistant to said treatment. In a particular embodiment, the body fluid sample is a serum sample and the treatment against a rheumatic inflammatory disease treatment with a DMARD selected from the group consisting of methothrexate, an anti-TNF-a monoclonal antibody, an anti-CD20 monoclonal antibody, an anti-IL6 monoclonal antibody or a CTLA4-Ig immunoadhesin. The term "predicting the responsiveness" to a treatment against a rheumatic inflammatory disease, as used herein, refers to an ability to assess the likelihood that treatment of a patient with an agent useful against a rheumatic inflammatory disease will or will not be more clinically effective (e.g., provide an increased measurable benefit to) in the patient. Patients having for instance an increased level expression level of Furin can then be selected for treatment with a therapeutically active agent useful against a rheumatic inflammatory disease. The ability to assess the likelihood that treatment will or will not be more clinically effective typically is exercised before treatment with agent useful against a rheumatic inflammatory disease is initiated. However, it is also possible that the ability to assess the likelihood that treatment will or will not be clinically effective can be exercised after treatment has begun to aid in optimizing treatment protocols.

As used herein, the term "a treatment against a rheumatic inflammatory disease" refers to therapeutically active agents such as non-steroidal anti-inflammatory agents, corticosteroids, and disease modifying anti-rheumatic drugs useful for treating rheumatic inflammatory disease. Such disease modifying anti-rheumatic drugs (DMARDs) include but are not limited to tumor necrosis factor (TNF) inhibitors such as etanercept (Enbrel®) or an anti-TNF-a monoclonal antibodies (e.g. infliximab (Remicade®), adalimumab (Humira®), certolizumab pegol (Cimzia®) and golimumab (Simponi®)), IL-1 receptor antagonists (ILlra) such as anakinra (Kineret®), B cell depleting agents such as an anti-CD20 monoclonal antibody (e.g. Rituximab (Rituxan®)), an anti-IL-6 monoclonal antibodies (e.g. Tocilizumab, Roactemra®), T-cell costimulatory blockers such as a CTLA4-Ig immunoadhesin (e.g. abatacept (Orencia®)) as well as other drugs such as for instance methotrexate, sulfasalazine, leflunomide, antimalarials, gold salts, d-penicillamine, cyclosporin A, cyclophosphamide and azathioprine. The foregoing therapeutically active agents are listed by way of example and are not meant to be limiting. Other therapeutically active agents which are currently available or that may be developed in the future are equally applicable. In one embodiment, the treatment against inflammatory rheumatic disease is a tumor necrosis factor-alpha (TNF-a) antagonist.

In one particular embodiment, the TNF-a antagonist is an anti-TNF-a monoclonal antibody selected from the group consisting of infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), and golimumab (Simponi).

In one embodiment, the treatment against inflammatory rheumatic disease is an anti- CD20 monoclonal antibody (e.g. Rituximab (Rituxan®)).

In one embodiment, the treatment against a rheumatic inflammatory disease is an anti- IL6 monoclonal antibody (e.g. Tocilizumab, Roactemra®).

In one embodiment, the treatment against a rheumatic inflammatory disease is a CTLA4-Ig immunoadhesin (e.g. abatacept (Orencia®)).

Another aspect of the invention relates to the use of serum expression level of Furin or activity level of Furin as a biomarker of resistance to a treatment with a DMARD.

In one embodiment, said DMARD is selected from the group consisting of methothrexate, an anti-TNF-a monoclonal antibody, an anti-CD20 monoclonal antibody, an anti-IL6 monoclonal antibody or a CTLA4-Ig immunoadhesin.

Another aspect of the invention relates to the use of serum expression level of Furin or activity level of Furin as a biomarker of the responsiveness of a patient to a treatment with biotherapy useful against a rheumatic inflammatory disease.

As used herein, the term "biotherapy" (or biological therapy) refers to the therapeutic use of biological materials. Examples of biotherapy include therapeutic monoclonal antibodies and immunoadhesins (also referred as soluble receptors).

In one embodiment, said biotherapy useful against a rheumatic inflammatory disease is selected from the group consisting of methothrexate, an anti-TNF-α monoclonal antibody, an anti-CD20 monoclonal antibody, an anti-IL6 monoclonal antibody or a CTLA4-Ig immunoadhesin. Kits according to the invention and uses thereof:

The invention is further directed to a kit suitable for carrying out the methods according to the invention. Such a kit may comprise means for measuring the expression level of Furin and/or the activity level of Furin and/or the expression level of a Furin substrate in a biological sample such as a blood sample or a synovial fluid sample.

Typically, said kit comprises as separate components at least one antibody (or aptamer) that binds to Furin and/or at least one antibody that binds to a Furin substrate (e.g. BAFF) and/or at least one fluorogenic peptide (e.g. pERTKR-AMC). Suitable antibodies are similarly identified here above.

In one embodiment, the kit comprises as separate components at least one antibody or aptamer that binds to Furin and at least one fluorogenic peptide (e.g. pERTKR-AMC).

In one embodiment, the kit comprises as separate components at least one antibody or aptamer that binds to Furin, at least one antibody or aptamer that binds to BAFF and at least one fluorogenic peptide (e.g. pERTKR-AMC).

In one embodiment, the antibodies may be coated to a solid support. Suitable examples of solid support are identified here above.

In another embodiment, one or more of the antibodies may be labelled. Suitable examples of labels are similarly identified here above.

The kit may also contain optional additional components for performing the method of the invention. Such optional components are for example containers, mixers, buffers, instructions for assay performance, labels, supports, and reagents necessary to couple the antibody to the support or label.

The invention also relates to the use of a kit comprising as separate components at least one antibody or aptamer that binds to Furin and/or at least one antibody or aptamer that binds to B-cell activating factor (BAFF) and/or at least one fluorogenic peptide (e.g. pERTKR-AMC) in a method of the invention as described above.

In one embodiment, said kit comprises as separate components at least one antibody or aptamer that binds to Furin and at least one fluorogenic peptide (e.g. pERTKR-AMC).

In one embodiment, said kit further comprises at least one antibody or aptamer that binds to BAFF. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

EXAMPLE 1: Furin expression in synovial biopsies as a diagnostic/prognostic marker for rheumatic inflammatory diseases.

Material & Methods: To assess the expression of Furin in humans, synovial tissues were harvested from patients whom rheumatoid arthritis was diagnosed on the basis of joint erosions. Control synovial was obtained from patients with post-traumatic knee pain undergoing arthroscopy. Furin expression was investigated using immunohistochemistry. Biopsies sections were incubated in citrate buffer pH 6.5 at 90°C and treated with 1 mg/ml hyaluronidase for 30 minutes. Vector kit was used according to the manufacturer protocol (Vector, Abcys, France). The Furin antibodies were used a 1 :500 dilution (Santa Cruz, France). Sections were sequentially incubated with secondary biotinylated antibody, with biotin-avidin amplification system and finally with diaminobenzidine (DAB) substrate. The sections were then counterstained with toluidine blue.

Total RNA was subjected to complementary DNA (cDNA) synthesis using the Superscript First-Strand cDNA Synthesis system (Invitrogen). The mRNA synthesis was performed by real-time PCR using the StepOnePlus Real-Time PCR System and Power SYBR Green PCR Master Mix (Applied Biosystems). During the reaction, forty cycles of PCR were performed at 94°C for 15 seconds and at 60°C for 1 minute. Transcription of hypoxanthine guanine phosphoribosyltransferase was evaluated in each sample as an endogenous control.

Results: Immunohistochemistry analysis of Furin expression in the synovial pannus of patients with RA and in controls revealed dramatic levels of Furin in both the cells and extracellular matrix of the synovial pannus in patients with RA. In contrast, Furin was not detected in human control synovium (Figure 1).

Tissues were removed and total RNA was extracted and analyzed by RT-PCR using specific primers for Furin. Results are shown in the bar graph and are expressed as the ratio of Furin mRNA expression relative to the control which was assigned a value of 1. EXAMPLE 2: Furin level in serum and synovial fluid as a diagnostic/prognostic marker for rheumatic inflammatory diseases.

Material & Methods: The measurement of Furin was performed by the use of ELISA kit (R&D Systems, catalog number: DY1503) in serum and synovial fluid derived from healthy individuals and patients. 100 μΐ of frozen samples or standards (included in kit) were added to 96 well plates. To each well the detection antibody was added and incubated for 2h at room temperature. Streptavidin-HRP solution was then added to each well and plates were incubated for 20 min at room temperature prior addition of the substrate solution. Following the arrest of the reaction by the addition to each well of the stop solution, the optical density of each well was determined by the use of a microplate reader set to 450 nm. Following the creation of a standard curve, the concentration of Furin in the samples was evaluated.

Results: Normally, Furin cycles between the plasma membrane, endosomes and the trans-Golgi network (TGN) and processes substrates within. Using Elisa assay we demonstrated that while Furin is not detectable in serums derived from healthy donors, in serums of patients with Rheumatoid arthritis, Lupus, Sjogren's syndrome and Reiter's arthritis this convertase was present and quantifiable (Figure 2). Analysis of Furin levels in the serum of patients with osteoarthritis, chondrocalcinosis and psoriatic arthritis revealed that Furin is not detectable (Table 1).

The concentration range of Furin in the serums of rheumatoid arthritis patients was 1460-4570 pg/ml, in serums of Lupus patients was 1756-3570 pg/ml, in Sjogren's syndrome patients 890-2460 pg/ml and in Reiter's arthritis patients was 1000-4802 pg/ml.

Analysis of Furin levels in RA and Sjogren's syndrome patients following immunoprecipitation of IgM, IgA and IgG rheumatoid factors (RF) revealed that RF doesn't interfere during the Furin Elisa assay.

Similarly, analysis of Furin levels in synovial fluid using Elisa assay we demonstrated that while Furin is not detectable in synovial fluid derived from patients with osteoarthritis, chondrocalcinosis and psoriatic arthritis (Table 1), in synovial fluids of patients with Rheumatoid arthritis and Reiter's arthritis this convertase was present and quantifiable (Figure 3). The concentration range of Furin in the synovial fluid of patients with rheumatoid arthritis was 800-16543 pg/ml and in Reiter's arthritis patients was 100-4353 pg/ml. EXAMPLE 3: Serum Furin activity level as a diagnostic/prognostic marker for rheumatic inflammatory diseases. Material & Methods: Furin activity in samples was assessed by evaluating their ability to digest the universal PCs substrate, the fluorogenic peptide pERTKR-MCA. 5-10 μΐ Samples derived from healthy donors or patients with rheumatic inflammatory diseases, were incubated with pERTKR-MCA (100 μΜ) during various time periods (0-200 min ) in the presence of 25 mM Tris, (pH 7.4), 25 raM methyi-ethane-sulfonic acid, and 2.5 mM CaCl₂, at 37°C and the fluoromctric measurements were performed using a spectrofluorometer (FLUOstar OPTIMA).

Results: Analysis of Furin-like activity using an enzymatic digestion assay, revealed the presence of high Furin-like activity in the serum of RA and Sjogren's syndrome patients as compared to PCs activity found in healthy donors (Figure 4). In the presence of the PC activity inhibitor, Dec-RVKR-CMK (CMK), this activity was completely inhibited at all the time periods examined (0-200 min) and in all the analyzed patient's serums.

EXAMPLE 4: Serum BAFF levels, a proprotein convertases substrate, as a diagnostic/prognostic marker for rheumatic inflammatory diseases.

Material & Methods: This method linked the increased levels of Furin and its substrate BAFF known to be involved in rheumatic inflammatory diseases. The method consists of the evaluation of the levels of BAFF and the degree of its maturation in serum and synovial fluid. The measurement of BAFF was performed by the use of ELISA kit (R&D Systems, catalog number: DBLYS0) in serum and synovial fluid derived from healthy donors and patients. The evaluation of BAFF maturation levels is performed on samples derived from healthy donors or patients with rheumatic inflammatory diseases by the use of immunoblotting or biosynthetic labeling.

Results: Analysis of the Furin substrate BAFF levels, revealed its correlated up- regulated secretion in the serum of RA, Lupus and Sjogren's syndrome patients (Figure 5). The analysis of BAFF maturation in serum and synovial fluid allow the quantification of the ratio: mature BAFF/precursor unprocesssed BAFF in healthy donors and patients. EXAMPLE 5: Serum Furin activity level as a biomarker of response to biotherapy. Material & Methods:

Patients:

1) The first group of patients was patients with inflammatory rheumatic diseases. They were experiencing bone pain, clinical inflammation or progression of the disease despite disease-modifying antirheumatic drugs (DMARD), mostly methotrexate.

2) The second group of patients consisted in patients with inflammatory rheumatic diseases treated with biotherapy for at least 3-6 months. These patients are reassessed as a failure of complete response to biotherapy. Blood samples were collected in 2 groups of patients at the time of their one-day stay hospitalisation, stored at -20°C in aliquot for the measurement of Furin activity. Furin activity was measured in the serum by evaluating its ability to digest the universal PC substrate, the fluorogenic peptide pERTKR-MCA, as previously described in Example 3. Serum were incubated with pERTKR-MCA (100 μιηοΙ/L) during the indicated time periods in the presence of 25 mmol/L Tris, 25 mmol/L methyl-ethane-sulfonic acid, and 2.5 mmol/L CaC12, pH 7.4 at 37° C and the fluorimetric measurements were performed using a spectrofluorometer (FLUOstar OPTIMA; BMG Labtech). The general PC-inhibitor decanoyl-RVKR- cholro methyl ketone was obtained from Calbiochem and recombinant Furin from Sigma. Results:

They were 10 patients without biotherapy (7 women, 3 men, mean age 45 years) and 10 patients with biotherapy (6 women, 4 men, and mean age 50 years) as described below in Table A. Erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) were slightly increased in both groups as a result of a bias of recruitment. However, Furin activity was lower in patients with biotherapy than in patients that did not take any biotherapy.

Table A: Patients with inflammatory rheumatic diseases treated with DMARD or with biotherapies (anti-monoclonal antibodies): Age S Biotherapy VS CRP Turin activity

33 F 0 21,09

53 F 0 16,91

57 H 0 26 5 39,98

57 F 0 28,97

51 H 0 16 14 17,34

39 F 0 15 9 20,4

22 F 0 32 7 36,14

47 F 0 20,45

32 H 0 12 12 26,88

55 F 0 37 22 51,53

Mean 23 11,5 27,969

45 H Humira 32 2 12,1

79 F Enbrel 34 16 22,01

34 H Remicade 4 4 23,17

50 H Remicade 74 75 20,23

45 F Remicade 14 7 20,29

45 H Remicade 11 9 19,43

55 H Remicade 71 31 17,04

72 F Roactemra 2 1

37 F Rituximab 13,69

38 F Orencia 38 35 15,52

53 H Tocilizumab 2 2 17,14

52 F Tocilizumab 4 2

Mean 26 16,73 18,062

These data suggest that serum Furin activity is a biomarker useful for predicting and/or monitoring the responsiveness of a patient to a treatment against a rheumatic inflammatory disease, especially a biomarker of resistance to a treatment with (DMARDs), such as methotrexate or a biotherapy (e.g. TNF-a inhibitors such as anti-TNF-a monoclonal antibodies, B cell depleting agents such as anti-CD20 monoclonal antibodies, anti-IL-6 monoclonal antibodies, and a CTLA4-Ig immunoadhesin) independently of ESR and CRP.

FIGURES:

Figure 1: Synovial tissue was obtained from patients with rheumatoid arthritis and from control patients. Furin is highly expressed in human synovial pannus, but absent in synovial tissue in control patients as assessed by qRT-PCR and immunostaining (X20). Figure 2: Using Elisa kit Furin levels were assessed in serums derived from healthy individuels (20 subjects), in serums of patients with Rheumatoid arthritis (60 subjects), Lupus (15 subjects), Sjogren's syndrome (95 subjects) and reiter's arthritis (3 subjects). This convertase was present and quantifiable in these rheumatic inflammatory diseases and absent in healthy subjects (control). Serum samples were used without any dilution. Data are the median of the indicated value range.

Figure 3: The Furin levels were assessed by Elisa kit in liquid synovial derived from patients with osteoarthritis (10 subjects), patients with Rheumatoid arthritis (60 subjects) and reiter's arthritis (3 subjects). This convertase was present and quantifiable in the rheumatic inflammatory diseases Rheumatoid arthritis and reiter's arthritis (3 subjects) and absent in the none inflammatory disease osteoarthritis (10 subjects). Samples were used without any dilution. Data are the median of the indicated value range.

Figures 4: Furin activity in serum samples was assessed by the measurement of the digestion of the universal PCs substrate, the fluorogenic peptide pERTKR-MCA. 5-10 μΐ Samples derived from healthy donors or patients with Rheumatoid arthritis (20 subjects) or Sjogren's syndrome (20 subjects) were incubated with pERTKR-MCA (100 μΜ) during various time periods ( 0-200 min ) in the presence of 25 mM Tris, (pH 7.4), 25 mM methyl- ethane-sulfonic acid, and 2.5 mM CaCl₂, at 37°C and the fluorometric measurements were performed using a spectrofluorometer.

Figure 5: Using Elisa kit Baff levels were assessed in serums derived from healthy individuels (20 subjects), in serums of patients with Rheumatoid arthritis (30 subjects), Lupus (15 subjects) and Sjogren's syndrome (25 subjects). Baff levels are higher in these rheumatic inflammatory diseases as compared to healthy subjects (control). Serum samples were used without any dilution. Data are the median of the indicated value range. Table 1: Furin expression in rheumatic inflammatory diseases:

Serum Synovial fluid

Healthy donors - X Rheumatoid Arthritis + +

Lupus + X

Sjogre's syndrom + X

Osteoarthritis - -

Chondrocalcinosis - -

Psoriatic arthritis - -

Reiter's arthritis + +

Furin levels analysis in the serum and synovial fluid of healthy individuals (20 subjects) and patients with osteoarthritis (10 subjects), chondrocalcinosis (2 subjects) and psoriatic arthritis (2 subjects) revealed that furin is not detectable as compared to patients with Rheumatoid arthritis (60 subjects), Lupus (15 subjects), Sjogren's syndrome (95 subjects) and reiter's arthritis (3 subjects). - Absence , + : presence, X: not available

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Lin H, Ah Kioon MD, Lalou C, Larghero J, Launay JM, Khatib AM, Cohen-Solal M; Protective role of systemic furin in immune response-induced arthritis; Arthritis Rheum. 2012 Sep;64(9):2878-86.

Scamuffa N, Siegfried G, Bontemps Y, Ma L, Basak A, et al. (2008) Selective inhibition of proprotein convert ases represses the metastatic potential, of human colorectal, tumor cells. J Clin. Invest 1 18: 352-363

Scamuffa N, Basak A, Lalou C, Wargnier A, Marcinkiewicz J, Siegfried G, Chretien

M, Calvo F, Seidah NG, Khatib AM. Regulation of prohepcidin processing and activity by the subtilisin-like proprotein convertases Furin, PC5, PACE4 and PC7. Gut. 2008 Nov;57(l l): 1573-82.

Claims

CLAIMS:

A method for determining whether a patient has, or is at risk of having or developing a rheumatic inflammatory disease, said method comprising a step of measuring in a body fluid sample obtained from said patient: a) the expression level of Furin, and/or b) the activity level of Furin, and/or c) the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of said Furin substrate.

A method for assessing the severity or predicting the outcome of a rheumatic inflammatory disease in a patient, said method comprising a step of measuring in a body fluid sample obtained from said patient: a) the expression level of Furin, b) the activity level of Furin, and/or c) the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of said Furin substrate.

A method for predicting or monitoring the responsiveness of a patient to a treatment against a rheumatic inflammatory disease, said method comprising a step of measuring in a body fluid sample obtained from said patient: a) the expression level of Furin, and/or b) the activity level of Furin, and/or c) the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of said Furin substrate.

The method according to claims 1 to 3, wherein said method comprising a step of comparing the measured expression level of Furin, and/or activity level of Furin, and/or the ratio of expression levels of mature form of a Furin substrate to an unprocessed form of a Furin substrate with a control reference level.

5. The method according to claims 1 to 4, wherein said Furin substrate is B-cell activating factor (BAFF).

6. The method according to any one claims 1 to 5, wherein said body fluid sample is a blood sample (e.g. a serum sample) or a synovial fluid sample.

7. The method according to any one claims 1 to 6, wherein said rheumatic inflammatory disease is selected from the group consisting rheumatoid arthritis (RA), ankylosing spondylitis, scleroderma, Reiter's arthritis, systemic lupus erythematosus (SLE) and Sjogren's syndrome.

8. The method according to claim 7, wherein the rheumatic inflammatory disease is RA.

9. The method according to claim 7, wherein the rheumatic inflammatory disease is SLE.

10. The method according to claim 7, wherein the rheumatic inflammatory disease is Sjogren's syndrome.

11. The method according to claim 7, wherein the rheumatic inflammatory disease is

Reiter's arthritis.

12. The method according to any one claims 3 to 11, wherein the treatment against a rheumatic inflammatory disease is a tumor necrosis factor-alpha (TNF-a) antagonist.

13. The method according to claim 12, wherein the TNF-a antagonist is an anti-TNF-a monoclonal antibody selected from the group consisting of infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), and golimumab (Simponi).

14. The method according to any one claims 3 to 11, wherein the treatment against a rheumatic inflammatory disease is an anti-CD20 monoclonal antibody.

15. The method according to any one claims 3 to 11, wherein the treatment against a rheumatic inflammatory disease is an anti-IL6 monoclonal antibody.

16. The method according to any one claims 3 to 11, wherein the treatment against a rheumatic inflammatory disease is a CTLA4-Ig immunoadhesin. 17. A kit suitable for carrying out the method according to anyone claims 1 to 16, comprising means for measuring the expression level of Furin and/or the activity level of Furin and/or the expression level of a Furin substrate.

18. The kit according to claim 17, comprising as separate components at least one antibody or aptamer that binds to Furin and/or at least one antibody or aptamer that binds to B-cell activating factor (BAFF) and/or at least one fluorogenic peptide (e.g. pERTK -AMC).

19. The kit according to claim 18, comprising as separate components at least one antibody or aptamer that binds to Furin and at least one fluorogenic peptide (e.g. pERTKR-AMC).

20. The kit according to claim 19, further comprising at least one antibody or aptamer that binds to BAFF.

21. Use of a kit comprising as separate components at least one antibody or aptamer that binds to Furin and/or at least one antibody or aptamer that binds to B-cell activating factor (BAFF) and/or at least one fluorogenic peptide (e.g. pERTKR-AMC) in a method according to any one claims 1 to 16.

22. The use according to claim 21, wherein said kit comprises as separate components at least one antibody or aptamer that binds to Furin and at least one fluorogenic peptide (e.g. pERTKR-AMC).

23. Use of serum expression level of Furin or activity level of Furin as a biomarker.

24. Use of serum expression level of Furin or activity level of Furin as a biomarker of rheumatic inflammatory disease.

25. The use according to claim 24, wherein said rheumatic inflammatory disease is selected from the group consisting rheumatoid arthritis (RA), Reiter's arthritis, systemic lupus erythematosus (SLE) and Sjogren's syndrome.

26. Use of serum expression level of Furin or activity level of Furin as a biomarker resistance to a treatment with a disease modifying anti-rheumatic drug (DMARD). 27. The use according to claim 26, wherein said DMARD is selected from the group consisting of methothrexate, an anti-TNF-a monoclonal antibody, an anti-CD20 monoclonal antibody, an anti-IL6 monoclonal antibody or a CTLA4-Ig immunoadhesin.