TREATMENT OF LUPUS

FIELD OF THE INVENTION:

The present invention relates to EphB2 polypeptides and uses thereof for the diagnosis and treatment of lupus.

BACKGROUND OF THE INVENTION:

Lupus is a chronic autoimmune disease in which the immune system malfunctions cannot distinguish between foreign invaders and healthy tissue. Antibodies are produced against the body's healthy cells and tissues, causing inflammation, pain and damage in various parts of the body.

There are several types of lupus. The most common type is systemic lupus erythematosus (SLE). SLE is a connective tissue autoimmune disease, where vasculopathy is one of the most typical symptoms (Cruz D, Lupus 1998). Vascular involvement is frequent in SLE patients and represents the most frequent cause of death in established disease.

Symptoms of SLE can be mild or severe. It can affect the skin, joints, kidneys, brain, and other organs. SLE is much more common in women than men. It may occur at any age, but appears most often in people between the ages of 10 and 50. African Americans and Asians are affected more often than people from other ethnies. Symptoms vary from person to person. The disease is discontinuous in activity with flare and remission. Almost everyone with SLE has joint pain and swelling. Some develop arthritis. The joints of the fingers, hands, wrists, and knees are often affected. There is presently no cure for SLE. It is mainly treated with immunosuppressive drugs such as cyclophosphamide or anti-inflammatory drugs as corticosteroids. SLE can be lethal, the leading cause of death is from cardiovascular disease due to accelerated atherosclerosis. Another type of lupus is discoid lupus erythematosus (DLE) which is a chronic skin disorder in which a red, raised rash appears on the face, scalp or elsewhere on the body. Subacute cutaneous lupus erythematosus refers to skin lesions that appear on parts of the body exposed to sun. The lesions do not cause scarring.

In some cases, lupus may be induced by some medications: drug-induced lupus such as some antiseizure, high blood pressure, thyroid medications, antibiotics, antifungals and oral contraceptive pills. The symptoms usually completely disappear once the drug is stopped.

Neonatal lupus is a rare disease that can occur in newborn babies of women with SLE, Sjogren's syndrome, or with no disease at all. Most babies of mothers with SLE are healthy. Eph receptors are the largest family of receptor tyrosine kinases, consisting of EphA (A1-A8, A10) and EphB (B1-B4, B6), which are distinguished by their extracellular domain sequence homologies (Pasquale, E. B. (2010) Nat. Rev. Cancer). Their ligands, known as ephrins, are also divided into two classes, ephrin A (A1-A5) and ephrin B (B1-B3). The interactions of Eph receptors and ephrins can signal bidirectionally into Eph-expressing cells (forward signaling) and ephrin-expressing cells (reverse signaling) (Kullander, K (2002) Nat. Rev. Mol. Cell Biol). Eph-ephrin interactions are involved in neural development, angiogenesis, vasculogenesis, and control monocyte and T cell migration in inflammatory processes.

Angiogenesis is the formation of new capillaries from pre-existing vasculature, whereas vasculogenesis is the term used for the formation of new blood vessels when there are no preexisting ones. Both play a critical role in the pathogenesis of several inflammatory autoimmune diseases such as rheumatoid arthritis (RA), spondyloarthropathies, psoriasis, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and atherosclerosis (Chapter 7 Vasculitis and Vasculopathy in Rheumatic Diseases Mislav Radic and Josipa Radic and Steyers CM Int. J. Mol. Sci. 2014).

EphB2, a receptor for both ephrinBl and ephrinB2 (Kullander, K (2002) Nat. Rev. Mol. Cell Biol), could be an actor, direct or indirect, in angiogenesis. Indeed a few studies have recently shown in the field of cancer that blocking ephrin B2 hampers Vascular Endothelial Growth Factor receptor 2 (VEGFR-2) internalization and angiogenesis signaling (Sawamiphak S. Nature 2010; Abengozar, Blood 2012). Moreover EphB2 expression is up-regulated in macrophages and binding of ephrinB-ligands (on endothelial cells) to EphB2 stimulates their motility and release of pro-inflammatory cytokines (Liu et al. 2014) Thrombosis and Haemostasis). Pro -inflammatory cytokines are capable of stimulating endothelial cell migration and/or proliferation and thus contribute to the formation of new capillaries (Jackson JR 1997 FASEB J).

It takes on average 4 years to obtain a correct diagnosis for lupus, in part due to the range and complexity of symptoms and the necessity to discount other possible causes. In the case of lupus, new methods and composition are needed to diagnosis lupus. The American College of Rheumatologists has established eleven criteria to assist in the diagnosis of lupus for the inclusion of patients in clinical trials and developed the SLE Disease Activity Index (SLEDAI) to assess lupus activity. Some of these criteria are very specific for lupus but have poor sensitivity, but none of these tests provides a definitive diagnosis and so the results of multiple differing tests must be integrated to enable a clinical judgement by an expert. Thus there is a need to identify strong and non-invasive methods to diagnose lupus.

SUMMARY OF THE INVENTION:

The present invention relates to EphB2 polypeptides and uses thereof for the diagnosis and treatment of lupus. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Using Invitrogen protein arrays (9483 human proteins), the inventors have previously identified, in a pilot study, 6 autoantigens specific for systemic sclerosis (SSc). Among them, the kinase domain of EphB2 (ephrin B2 receptor), a crucial regulator of vascular assembling is particularly interesting in the context of SSc vasculopathy. Nevertheless, inventors previously experienced that patients with Systemic Lupus Erythematosus (SLE) have many autoantibodies which could cross-react with SSc-autoantigens. Moreover, both SLE and SSc are characterized by vascular inflammation, altered angiogenesis, and increased cardiovascular morbidity and mortality. Furthermore, a case of disease Acute Necrotizing Encephalopathy (ANE) complicated with SLE, described anti-EphB2 antibodies (recognizing the extracellular part of the receptor). Thus the inventors investigated the presence of anti-EphB2 (kinase domain) autoantibodies by ELISA on 336 sera from 63 patients with SLE, 107 with SSc, 81 with other rheumatic diseases (RD), including Rheumatoid Arthritis (RA), Psoriatic Arthritis (PsoA) and Ankylosing Spondylitis (AS) and 85 healthy controls (HC). To further decipher which part of EphB2 was recognized by patient's autoantibodies, they screened thirty- four 15-mer peptides encompassing the kinase domain of EphB2. The kinase domain of EphB2 is significantly more often recognized by sera from patients with SLE than by sera from patients with SSc or with other RD or HC. Epitope mapping revealed a 15-mer peptide (P7) to be specifically recognized by sera from patients with SLE compared to all controls. Moreover, higher anti-P7 antibody titers correlated with higher SLE Disease Activity Indexes (SLEDAI, Spearman's correlation p=0.02). Polypeptide EphB2 and its derivatives

Accordingly, a first object of the invention relates to a polypeptide comprising a sequence having at least 90% of identity with the sequence ranging from the amino acid at position 666 to the amino acid at position 681 in SEQ ID NO: 1. SEQ ID NO: l; EphB2 (Homo Sapiens)

MALR LGAALLLLPLLAAVEETLMDSTTATAELGWMVHPPSGWEEVSGYDE NMNTIRTYQVCNVFESSQ NWLRTKFIRR GAHRIHVEMKFSVRDCSSIPSVPGSCKE TFNL Y Y YE ADFD S ATKTFPN WMENP WVKVDTI AADE SF S Q VDLGGRVMKINTE VRS FGPVSRSGFYLAFQDYGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVAARG SCIANAEEVDVPIKLYCNGDGEWLVPIGRCMCKAGFEAVENGTVCRGCPSGTFKANQ GDEACTHCPINSRTTSEGATNCVCRNGYYRADLDPLDMPCTTIPSAPQAVISSVNETS LMLEWTPPRDSGGREDLVYNIICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYISD LLAHTQYTFEIQAVNGVTDQSPFSPQFASVNITTNQAAPSAVSIMHQVSRTVDSITLS WSQPDQPNGVILDYELQYYEKELSEYNATAIKSPTNTVTVQGLKAGAIYVFQVRART VAGYGRYSGKMYFQTMTEAEYQTSIQEKLPLIIGSSAAGLVFLIAVVVIAIVCNRRGF ERADSEYTDKLQHYTSGHMTPGMKIYIDPFTYEDPNEAVREFAKEIDISCVKIEQVIGA GEFGEVCSGHLKLPGKREIFVAI TL SGYTE QRRDFLSEASIMGOFDHPNVIHLEG VVTKSTPVMIITEFMENGSLDSFLRQNDGQFTVIQLVGMLRGIAAGMKYLADMNYV HRDLAARNILVNSNLVCKVSDFGLSRFLEDDTSDPTYTS ALGGKIPIRWTAPEAIQYR KFTSASDVWSYGIVMWEVMSYGERPYWDMTNQDVINAIEQDYRLPPPMDCPSALH QLMLDCWQKDRNHRPKFGQIVNTLDKMIRNPNSLKAMAPLSSGINLPLLDRTIPDYT SFNTVDEWLEAIKMGQYKESFANAGFTSFDVVSQMMMEDILRVGVTLAGHQK ILN SIQVMRAQMNQIQSVEGQPLARRPRATGRTKRCQPRDVTK TCNSNDGKK GMGK K TDPGRGREIQGIFFKEDSHKESNDCSCGG.

As used herein, the term "polypeptide" has its general meaning in the art and refers to amino acid sequences of a variety of lengths, either in their neutral (uncharged) forms or as salts, and either unmodified or modified by glycosylation, side chain oxidation, or phosphorylation, or citrullination. In some embodiments, the amino acid sequence is a fu.ll- length native protein. In other embodiments, the amino acid sequence is a smaller fragment of the full-length protein. The expressions "polypeptides and its derivatives" refer to the amino acid sequence which may be modified by additional substituents attached to the amino acid side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversion of the chains such as oxidation of sulfhydryl groups. Thus, the term "polypeptide" is intended to include the amino acid sequence of the full- length native protein, or a fragment thereof, subject to those modifications that do not significantly change its specific properties. In particular, the term "protein" encompasses protein isoforms, i.e., variants that are encoded by the same gene, but that differ in their pi or MW, or both. Such isoforms can differ in their amino acid sequence (e.g., as a result of alternative splicing or limited proteolysis), or in the alternative, may arise from differential post- translational modification (e.g., glycosylation, acylation, phosphorylation).

As used herein, the term "amino acid" refers to natural or unnatural amino acids in their D and L stereoisomers for chiral amino acids. It is understood to refer to both amino acids and the corresponding amino acid residues, such as are present, for example, in peptidyl structure. Natural and unnatural amino acids are well known in the art. Common natural amino acids include, without limitation, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Uncommon and unnatural amino acids include, without limitation, allyl glycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphtylalanine (2-Nal), ornithine (Orn) and pentafluorophenylalanine. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first amino acid sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol, 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al. Nuc. Acids Res., 16: 10881-10890, 1988; Huang et al, Comp. Appls Biosci., 8: 155-165, 1992; and Pearson et al, Meth. Mol. Biol, 24:307-31, 1994). Altschul et al, Nat. Genet., 6: 119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CABIOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al, J. Mol. Biol, 215:403-410, 1990; Gish. & States, Nature Genet., 3 :266-272, 1993; Madden et al. Meth. EnzymoL, 266: 131-141, 1996; Altschul et al, Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997. In some embodiments, the polypeptide of the invention comprises 15; 16; 17; 18; 19;

20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44;

45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69;

70; 71; 72; 73; 74; 75; 76; 77 ; 78; 79; 80; 81 ; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94;

95; 96; 97; 98; 99; 100; 101; 102; 103; 104; 105; 106; 107; 108; 109; 110; 111 ; 112; 113; 114; 115; 116; 117; 118; 119; 120; 121; 122; 123; 124; 125; 126; 127; 128; 129; 130; 131; 132; 133;

134; 135; 136; 137; 138; 139; 140; 141; 142; 143; 144; 145; 146; 147; 148; 149; 150; 151; 152;

153; 154; 155; 156; 157; 158; 159; 160; 161; 162; 163; 164; 165; 166; 167; 168; 169; 170; 171 ;

172; 173; 174; 175; 176; 177; 178; 179; 180; 181; 182; 183; 184; 185; 186; 187; 188; 189; 190;

191; 192; 193; 194; 195; 196; 197; 198; 199; or 200 amino acids. In some embodiments, the polypeptide of the invention comprises less than 150 amino acids, preferably less than 100 amino acids, more preferably less than 50 amino acids or even more preferably less than 25 amino acids.

In some embodiments, the polypeptide of the present invention is fused to at least one heterologous polypeptide to form a fusion protein. In some embodiments, the polypeptide of the present invention is fused either directly or via a spacer at its C-terminal end to the N- terminal end of the heterologous polypeptide, or at its N-terminal end to the C-terminal end of the heterologous polypeptide. As used herein, the term "directly" means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the polypeptide of the present invention is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide. In other words, in this embodiment, the last amino acid of the C- terminal end of said polypeptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said polypeptide is directly linked by a covalent bond to the last amino acid of the C- terminal end of said heterologous polypeptide. As used herein, the term "spacer" refers to a sequence of at least one amino acid that links the polypeptide of the invention to the heterologous polypeptide. Such a spacer may be useful to prevent steric hindrances. Typically a spacer comprises 2, 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; or 20 amino acids.

The polypeptide of the present invention is produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. For instance, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptide, by standard techniques for production of amino acid sequences. For instance, the polypeptide can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Bio systems, Foster City, California) and following the manufacturer's instructions. Alternatively, the polypeptide of the present invention can be synthesized by recombinant DNA techniques as is now well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired polypeptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

Method for diagnosing lupus

A further object of the present invention is a method for diagnosing lupus in a subject, said method comprising the step of detecting the presence of anti- EphB2 autoantibodies in a biological sample obtained from the subject wherein the presence of said autoantibodies indicates that the subject suffers from lupus.

In some embodiments, the method of the present invention comprises detecting in the biological sample the presence of autoantibodies having specificity for the polypeptide of the present invention wherein the presence of said autoantibodies indicates that the subject suffers from lupus.

The term "lupus" has its general meaning in the art and refers to a chronic inflammatory disease that is caused by the autoimmunity. Patients with lupus have in their blood unusual antibodies that are targeted against their own body tissues. The term "lupus" refers, without limitation, to systemic lupus erythematosus or SLE, discoid lupus erythematosus, drug-induced lupus erythematosus or DLE, neonatal lupus erythematosus. The term "subject" refers to a subject that presents one or more symptoms indicative of lupus (e.g. joint pain, stiffness and swelling), or that is screened for lupus (e.g., during a physical examination). Alternatively or additionally, a subject suspected of having lupus may have one or more risk factors (e.g., age, sex, family history, smoking, etc). The term encompasses subjects that have not been tested for lupus as well as subjects that have received an initial diagnosis.

In some embodiments, the method of the invention is suitable for diagnosing systemic lupus erythematosus (SLE) in a subject. In some embodiments, the method of the invention is suitable for diagnosing discoid lupus erythematosus in a subject. In some embodiments, the method of the invention is suitable for diagnosing drug-induced lupus in a subject. In some embodiments, the method of the invention is suitable for diagnosing neonatal lupus in a subject.

The term "autoantibody", as used herein, has general meaning in the art, and refers to an antibody that is produced by the immune system of a subject and that is directed against subject's own proteins. Autoantibodies may attack the body's own cells, tissues, and/or organs, causing inflammation and damage.

The term "biological sample" has its general meaning. A biological sample is generally obtained from a subject. Frequently, a sample will be a "clinical sample", i.e., a sample derived from a patient. Such samples include, but are not limited to, bodily fluids which may or may not contain cells, e.g., blood (e.g., whole blood, serum or plasma), synovial fluid, saliva, tissue or fine needle biopsy samples, and archival samples with known diagnosis, treatment and/or outcome history. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. The term "biological sample" also encompasses any material derived by processing a biological sample. Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample, or proteins extracted from the sample. Processing of a biological sample may involve one or more of: filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. In some embodiments of the invention, the biological sample is a blood sample i.e. a whole blood, a serum sample or a plasma sample obtained from a subject.

In some embodiments, the method of the present invention comprises the steps of i) contacting the biological sample with the polypeptide of the invention for a time and under conditions allowing polypeptide-antibody complexes to form between the polypeptide and the anti-EphB2 autoantibodies present in the biological sample and ii) detecting any polypeptide- antibody complex formed at step i) wherein the detection of said polypeptide-antibody complex indicates that the subject suffers from lupus. In some embodiments, the method of the present invention comprises a step of i) quantifying the level of anti-EphB2 autoantibodies present in the biological sample obtained from the subject ii) comparing the level determined at step i) with a predetermined reference value, and iii) concluding that the subject suffers from lupus when the level determined at step i) is higher than the predetermined reference value.

Typically, the predetermined reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of level of autoantibodies in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the levels of autoantibodies in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured levels of autoantibodies in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1- specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

The predetermined reference value can also be relative to a number or value derived from population studies, including without limitation, subjects of the same or similar age range, subjects in the same or similar ethnic group, and subjects having the same severity of lupus. Such predetermined reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices. In some embodiments, the predetermined reference values are derived from the level of autoantibodies in a control sample derived from one or more subjects who do not suffer from lupus. Furthermore, retrospective measurement of the level of autoantibodies in properly banked historical subject samples may be used in establishing these predetermined reference values.

In some embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found. For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. For example, a subject may be assessed by comparing values obtained by measuring the level of autoantibodies, where values greater than 5 reveal that the subject suffers from a lupus and values less than 5 reveal that the subject does not suffer from a lupus. In some embodiments, a subject may be assessed by comparing values obtained by measuring the level of autoantibodies and comparing the values on a scale, where values above the range of 4-6 indicate that the subject suffers from a lupus and values below the range of 4-6 indicate that the subject does not suffer from a lupus, with values falling within the range of 4-6 indicate that further explorations are needed to conclude whether the subject suffers or not from a lupus.

According to the invention, the detection and/or quantification of anti-EphB2 autoantibodies in a biological sample is achieved by any method well known in the art. Typically, the methods involve detection or quantification of a polypeptide-antibody complex formed between the polypeptide of the present invention and an autoantibody present in the biological sample tested. In the practice of the invention, detection of such a complex may be performed by any suitable method (see, for example, E. Harlow and A. Lane, "Antibodies: A Laboratories Manual", 1988, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY). In some embodiments, the polypeptide used in the diagnosis method is immobilized on a solid carrier or support.

For example, detection or quantification of a polypeptide-antibody complex may be performed using an immunoassay. A wide range of immunoassay techniques is available, including radioimmunoassay, enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), and immunofluorescence immunoprecipitation. Immunoassays are well known in the art. Methods for carrying out such assays as well as practical applications and procedures are summarized in textbooks. Examples of such textbooks include P. Tijssen, In: Practice and theory of enzyme immunoassays, eds. R.H. Burdon and v. P.H. Knippenberg, Elsevier, Amsterdam (1990), pp. 221-278 and various volumes of Methods in Enzymology, Eds. S.P. Colowick et al, Academic Press, dealing with immunological detection methods, especially volumes 70, 73, 74, 84, 92 and 121. Immunoassays may be competitive or noncompetitive.

For example, any of a number of variations of the sandwich assay technique may be used to perform an immunoassay. Briefly, in a typical sandwich assay applied to the detection of, for example, anti-EphB2 autoantibodies according to the present invention, an unlabeled polypeptide of the present invention is immobilized on a solid surface (as described above) and the biological sample to be tested is brought into contact with the bound polypeptide for a time and under conditions allowing formation of a polypeptide-antibody complex. Following incubation, an antibody that is labeled with a detectable moiety and that specifically recognizes antibodies from the species tested (e.g., an anti- human IgG for human subjects) is added and incubated under conditions allowing the formation of a ternary complex between any polypeptide-bound autoantibody and the labeled antibody. Any unbound material is washed away, and the presence of any anti- EphB2 autoantibody in the sample is determined by observation/detection of the signal directly or indirectly produced by the detectable moiety. Variations on this assay include an assay, in which both the biological sample and the labeled antibody are added simultaneously to the immobilized polypeptide. The second antibody (i.e., the antibody added in a sandwich assay as described above) may be labeled with any detectable moiety, i.e., any entity which, by its chemical nature, provides an analytically identifiable signal allowing detection of the ternary complex, and consequently detection of the polypeptide- autoantibody complex. Detection may be either qualitative or quantitative. Methods for labeling biological molecules such as antibodies are well-known in the art (see, for example, "Affinity Techniques. Enzyme Purification: Part B", Methods in EnzymoL, 1974, Vol. 34, W.B. Jakoby and M. Wilneck (Eds.), Academic Press: New York, NY; and M. Wilchek and E.A. Bayer, Anal. Biochem., 1988, 171 : 1-32). The most commonly used detectable moieties in immunoassays are enzymes and fluorophores. In the case of an enzyme immunoassay (EIA or ELISA), an enzyme such as horseradish perodixase, glucose oxidase, beta-galactosidase, alkaline phosphatase, and the like, is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. The substrates to be used with the specific enzymes are generally chosen for the production of a detectable color change, upon hydrolysis of the corresponding enzyme. In the case of immunofluorescence, the second antibody is chemically coupled to a fluorescent moiety without alteration of its binding capacity. After binding of the fluorescently labeled antibody to the polypeptide-antibody complex and removal of any unbound material, the fluorescent signal generated by the fluorescent moiety is detected, and optionally quantified. Alternatively, the second antibody may be labeled with a radioisotope, a chemiluminescent moiety, or a bioluminescent moiety.

In some embodiments, the detection and quantification is performed with a protein array wherein the polypeptide of the present invention is immobilized into said array.

A used herein the term "protein array" has its general meaning in the art and refers to a substrate surface on which different proteins or polypeptides are immobilized, in an ordered manner, at discrete spots on the substrate. Protein arrays may be used to identify protein/protein interactions (e.g., antigen/antibody interactions), to identify the substrates of enzymes, or to identify the targets of biologically active small molecules. The term "microarray" specifically refers to an array that is miniaturized so as to require microscopic examination for visual evaluation.

Arrays can be made on any suitable support, whether in one part or several. In some embodiments the solid support can be any material that is an insoluble matrix and can have a rigid or semi-rigid surface. In some embodiments, the support is a membrane, such as nitrocellulose, nylon, and the like, among other membrane materials suitable to act as supports for applying proteins to make arrays. Such membrane supports may be free standing or may be themselves supported, such as nitrocellulose membrane material on a glass slide. In some embodiments supports may be glass, such as glass slides, silicon, including the surfaces of elements in integrated circuits and MEMs devices, and plastics, including plastic plates, such as microtiter plates, including, for instance, 96-well, 384-well microtiter plates, as well as those of other capacities. Exemplary solid supports include, but are not limited to, substrates such as nitrocellulose (e.g., in membrane on a glass slide or in microtiter well form); polyvinylchloride (e.g., sheets, on glass or in microtiter wells); polystyrene latex (e.g., bead, on glass or in microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, etc. Particular supports include plates, pellets, disks, capillaries, hollow fibers, needles, pins, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N- N -bis-acryloylethylenediamine, and glass particles coated with a hydrophobic polymer.

Arrays can be made in a wide variety of formats, sizes, modularity and can be made with a wide variety of positions, proteins, features, feature sizes, feature spaces, feature occupancy, controls, alignment markers and references among others.

The polypeptide of the present invention is attached to the solid support by any conventional method in the art. Typically, the solid is conjugated to an avidin moiety that can create an avidin-biotin complex with the biotinylated polypeptides of the present invention. The term "biotinylated" as used herein, refer to any covalent or non-covalent adduct of bio tin with other moieties such as the polypeptide of the present invention. As used herein the term "avidin" comprises the native egg-white glycoprotein avidin, as well as any derivatives, analogs and other non-native forms of avidin that can specifically bind to biotin moieties. In some embodiments, the avidin moiety can comprise deglycosylated forms of avidin, bacterial streptavidins produced by selected strains of Streptomyces, e.g., Streptomyces avidinii, to truncated streptavidins, and to recombinant avidin and streptavidin as well as to derivatives of native, deglycosylated and recombinant avidin and of native, recombinant and truncated streptavidin, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyl and N-succinyl avidin, and the commercial products ExtrAvidin®, Captavidin®, Neutravidin® and Neutralite Avidin®. All forms of avidin-type molecules, including both native and recombinant avidin and streptavidin as well as derivatized molecules, e.g. nonglycosylated avidins, N-acyl avidins and truncated streptavidins, are encompassed within the terms "avidin" and "avidin moiety". As used herein, the term "biotin-avidin complex" and its variants refers to a specific linkage formed between a biotin moiety and an avidin moiety. Typically, a biotin moiety can bind with high affinity to an avidin moiety, with a dissociation constant Kd typically in the order of 10-14 to 10-15 mol/L. Typically, such binding occurs via non-covalent interactions. In some embodiments, the antigen and the antibody is monobiotinylated, i.e., is conjugated to only on biotin moiety.

Kit for diagnosing lupus

A further object of the invention is a kit comprising:

i) a polypeptide of the invention and/or ii) a protein array as provided herein and

iii) at least one reagent for the detection of a polypeptide-antibody complex formed between the polypeptide included in the kit and an autoantibody present in a biological sample.

In some embodiment, the polypeptide of the invention which is included in the kit may or may not be immobilized on the substrate surface (e.g., beads, array, and the like). Thus, in some embodiments, a kit of the invention includes a protein array for diagnosing lupus as provided herein. Alternatively, a substrate surface may be included in the kit of the invention for immobilization of the polypeptide of the invention.

In some embodiments, the kit comprises instructions for using its components for the diagnosis of lupus, in a subject according to the method of the invention. Instructions for using the kit according to methods of the invention may comprise instructions for processing the biological sample obtained from the subject and/or for performing the test, and/or instructions for interpreting the results. A kit may also contain a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products.

Method for treating angiogenesis and/or vasculogenesis impairment

A further object of the present invention relates to a method of treating angiogenesis and/or vasculogenesis impairment in a subject suffering from lupus in need thereof comprising a step of administrating to said subject a therapeutically effective amount of the polypeptide of the invention.

In the context of the invention, the term "treatment" or "treat" as used herein, refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

A "therapeutically effective amount" is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a patient is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder.

The term "vasculopathy" has its general meaning refers to damage to a blood vessel by a disease process affecting the opening (lumen) or walls of the vessel. In the context of the invention, vasculopathy refers to vascular lesions in lupus, commonly known as the lupus vasculopathy; a typical lupus vasculitis with inflammatory and vascular wall necrosis and a thrombus in the lumen of affected artery occurs less often. Lupus vasculopathy is usually seen in cutaneous vessels, in renal glomeruli, coronary and brain vessels, the brain, lung alveoli and less often in the gastrointestinal tract (Chapter 7 Vasculitis and Vasculopathy in Rheumatic Diseases Mislav Radic and Josipa Radic).

The term "angiogenesis" has its general meaning in the art and refers to the development of new blood vessels from pre-existing vessels. Angiogenesis plays a fundamental role in embryonic development, tissue and wound repair, resolution of inflammation, and onset of neoplasia. In some embodiments the polypeptide of the invention interferes with EphB2 signaling pathway and stimulates angiogenesis and/or vasculogenesis in a subject suffering from lupus. Thus, the polypeptide of the present invention reverse the disease process and restore the normal angiogenesis and/or vasculogenesis function in a subject suffering from lupus.

The term "interfere" means that the polypeptide of the invention blockades or inhibits the interaction between the EphB2 and autoantibodies present in the subject.

In some embodiments, the present invention relates to a pharmaceutical composition comprising the polypeptide of the invention. The polypeptide of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

In the pharmaceutical compositions of the present invention, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The polypeptide of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a 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 vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The polypeptide according to this invention, may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

FIGURES:

Figure 1: Detection of autoantibodies for EphB2 kinase domain in patients and controls. Sera from 63 patients with Systemic Lupus Erythematosus (SLE), 107 patients with scleroderma (SSc), 81 controls with other Rheumatic Diseases (RD) and 85 healthy controls (HC) were tested at dilution 1/100 and defined as positive when AOD >0. The kinase domain of EphB2 protein was coated at a concentration of 0^g/ well. Under our ELISA conditions, EphB2 is significantly recognized by sera from patients with systemic sclerosis (SSc) and patients with Systemic Lupus Erythematosus (SLE), when compared to both sera from healthy controls (HC) and sera from patients with other rheumatic diseases (RD). All p values (P) are calculated after Bonferroni correction. PI : patients with SSc or SLE compared to HC, P2: compared to RD, P3: compared to both HC and RD.

Figure 2: Anti-EphB2 antibody titers in patients with SLE with or without anti- dsDNA antibodies. Titers of autoantibodies for EphB2 kinase domain were analyzed in two groups of patients with Systemic Lupus Erythematosus (SLE). Twenty-eight patients with SLE were negative for anti-double strand DNA antibodies (dsDNA) and 32 with anti-dsDNA antibodies. Sera were tested at dilution 1/100 and defined as positive when ΔΟϋ >0 (on or above the dotted black line). EphB2 was tested at a concentration of 0.2μ§/ well. Black bars represent medians with interquartile ranges.

Figure 3: Autoantibodies against peptide 7 (P7 from EphB2 protein) analyzed in patients with scleroderma (SSc), lupus (SLE), Rheumatoid Arthritis (RA) and healthy controls (HC). Sera reactivity against the peptide 7 (P7 at 10μg/well) is given by Absorbance (Abs) values for all patients and controls. Sera were tested at dilution 1/100 and defined as positive when Abs>0 (on or above the dotted black line). Black bars represent medians with interquartile ranges. Percentage of individuals positive for anti-P7 antibodies are indicated in the upper part of the graph. One data point for SLE is outside the Y axis limit (-0,41) and not represented here but counted for statistics. P value is calculated using Mann Whitney test by comparing patients with SLE to all controls (SSc, RA and HC, n=157).

Figure 4: Correlation between anti-P7 antibody titers and SLEDAI in patients with SLE. Disease activity is indicated by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) for 48 patients with SLE. Spearman's correlation, p (two-tailed) <0.02.

Figure 5: ROC curve analysis with comparison of the ELISA for THEX1 and EphB2 in SSc and SLE. Here are compared diagnostic abilities of EphB2 and THEX1 for SSc and SLE. Controls in both cases are all other individuals, which means patients with SSc were also included among controls when SLE is regarded as the tested disease and inversely patients with SLE were included among controls when SSc is regarded as the tested disease. Areas under the curve (AUC) for THEX1 and EphB2 in SLE are respectively 0,80 and 0,74. AUC for THEXland EphB2 in SSc are respectively 0,59 and 0,58.

Figure 6: ROC curve analysis for peptide 7 from EphB2 protein (P7). Here are compared diagnostic abilities of P7 for SSc and SLE. Controls in both cases are all other individuals, which means patients with SSc were included among controls when SLE is regarded as the tested disease and inversely patients with SLE were included among controls when SSc is regarded as the tested disease. EphB2 assays SSc ^a SLE RA PsA AS HC

N=107 ^b N=63 N=41 N=27 N=13 N=99

Mean age, [range] 53 [23-78] 39 [15-84] 65 [37-90] 56 [38- 59 [39- 60 [45- 85] 84] 77]

Sex : female % 82% 84% 76% 59% 38% 100%

Positive for ACA 21% dsDNA 53.3% ACPA NA ^d NA NA autoantibodies ⁰ , % ATA 34% (N=60) 100%

ACA/ATA ^ncg 45%

(N=106)

Mean age at 47[12-75] UKN ^C 44 [22-73] 38 [15- 42 [24- NA diagnosis, [range] 73] 67]

Clinical subtype, % LcSSc, In remission, NA NA NA NA

51% 62%

DcSSc, Active disease,

49% 38% (N=60)

Caucasian, % 79% 95% (N=59) U N UKN UKN 98%

Diseases: SSc, systemic sclerosis ; SLE, systemic lupus erythematosus ; RA, rheumatoid arthritis ;

PsoA, psoriatic arthritis ; AS, ankylosing spondylitis ; HC, healthy controls; ^b Number of individuals considered for calculation is indicated for each column otherwise indicated between brackets when clinical data are missing; Autoantibodies: ACA, anti-centromere antibodies ; ATA, anti-topoisomerase antibodies ; ACA ATA ^neg , without ACA and ATA ; dsDNA, anti-double strand DNA antibodies ; ACPA, anti-citrullinated protein antibodies ; ^d NA, not applicable, ^e UKN, unknown.

Table 1: Participants' characteristics for EphB2 ELISA assays. EXAMPLE

Material & Methods:

Patient and healthy control clinical characteristics (see Table 1)

Patients with Systemic sclerosis (SSc) were recruited at Claude Huriez Hospital, Lille; Nord and La Conception Hospitals, Marseille; St Louis and St Antoine Hospitals, Paris. They all fulfilled the criteria of LeRoy for SSc Patients and healthy control characteristic are presented in the table 1.

Patients with SLE were recruited at Hopitaux Universitaires Strasbourg; La Conception Hospital, Marseille and CHU Bretonneau, Tours. The 63 patients with SLE tested fulfilled the American College of Rheumatology revised criteria for SLE [13] as updated in 1997. We had obtained Disease Activity Indexes (SLEDAI) for 60 of them.

Patients with RA, PsA and AS were recruited in the Rheumatology Unit of St Marguerite Hospital in Marseille. All patients with RA satisfied the 2010 revised criteria of the American College of Rheumatology and were all positive for Anti-Citrullinated Protein Antibodies (ACPA). Patients with Psoriatic Arthritis (PsA) fulfilled the ClASsification of Psoriatic ARthritis (CASPAR) criteria and all 20 patients with Ankylosing Spondylitis (AS) fulfilled the Assessment of Spondylo Arthritis international Society classification criteria.

Healthy controls were recruited at the Centre d'Examen de Sante de l'Assurance Maladie (CESAM), Marseille, France. They had no symptom or familial history of autoimmune disorder.

Ethics statements

All participants signed informed consent according to the Declaration of Helsinki [18]. The study is registered at the INSERM under the Biomedical Research Protocol number RBM- 04-10 and received the approval of the "Comite de Potection des Personnes de Marseille Π" or as a collection registered under the number DC-2008-327.

Detection of anti-EphB2 autoantibodies by ELISA

ProtoArrays allowed the identification of autoantibodies against six proteins. Among them, EphB2 was specifically recognized by patients with SSc. EphB2 protein was purchased from Invitrogen and was identical to the one coated on protoarrays. Contrary to its denomination, it coPlates (Nunc, Kamstrupvej, Denmark) were coated 8 hours at 4°C with 0.2 μg of EphB2 per well diluted in PBS. Then, plates were blocked with PBS 2% BSA overnight. After blocking solution removal, sera samples diluted at 1 : 100 in PBS 1%BSA were added. After 2 hours of incubation at room temperature, plates were washed 3 times (2 minute) with PBS 0.1% Tween 20 and then peroxidase-conjugated anti-human IgG (Sigma Aldrich, St Quentin-Fallavier, France) was added for half an hour, then washed 2 times (2 minute) with PBS 0.1% Tween 20 and one more time with only PBS, before being revealed with tetramethylbenzidine (TMB) liquid substrate system (Sigma-Aldrich, St Louis, MO, USA). After 30 minutes, optical density (OD) was read at 405 nm on a PowerWave XS microplate spectrophotometer (Biotek, Colmar, France). For each individual, background OD was obtained by adding sera on duplicated wells without tested protein. Positive sera were defined by an OD value superior or equal to twice the background OD (positive AOD= 0 or more).

Epitope mapping on EphB2 protein: peptide ELISA

In order to determine which part of the protein was recognized by autoantibodies, we performed an epitope mapping for EphB2. A total of thirty- four 15-mer peptides encompassing residues from EphB2 (locus NM_004442.3) overlapping on 7-8 amino acids were synthesized using the solid-phase system, and then purified (Polypeptide Laboratories, Strasbourg, France). Plates were coated overnight with 10 μg/well peptides diluted in PBS, pH 7.4. Plates were blocked and washed similarly to the ELISA plates presented above. Sera, diluted to 1 : 100 in PBS 1% BSA, were incubated for 2 h. Positive wells were defined as above.

Statistical analysis

To determine whether EphB2 was significantly better recognized by autoantibodies from patients with SSc or patients with SLE rather than healthy controls and/or patients with other rheumatic diseases, p values were calculated using the χ ² test.

For AOD comparisons between groups, p values were evaluated using Mann Whitney test (Graphpad Prism 6). Correlation between anti-EphB2 (or P7) antibody titers and severity of the disease (SLEDAI) for SLE was assessed by Spearman's rank test.

Results

The kinase domain of EphB2 is specifically recognized by patients with SLE

The kinase domain of EphB2, abbreviated EphB2 below, is significantly recognized by 56% of sera from patients with SLE compared to 34% of patients with SSc, 16% of patients with other rheumatic diseases (patients with RA, with PsoA and with AS) and 13% of healthy controls (see p values Figure 1). A significant difference is also observed in anti-EphB2 antibody titers between SLE patients and other controls (p<0.0001 , data not shown). Interestingly, EphB2 was recognized by 54% of patients with SLE without anti-dsDNA- autoantibodies (N=28, Figure 2). Epitope mapping of EphB2: Peptide P7 is specifically recognized by sera from patients with SLE.

To further decipher which part of the protein was recognized by patient's autoantibodies, we first screened the thirty- four 15-mer EphB2 peptides with sera samples from 1 1 patients with SSc, 5 patients with SLE and 4 healthy controls. This first screening eliminated 31 of the 34 peptides because of absence of reactivity or non-specificity.

Three peptides were further tested: Peptide #1 with a slight specificity for Lupus, although recognized by 1/1 1 patients with SSc; Peptide # 7 which was recognized by 4/5 patients with SLE but none of the patients with SSc or controls and Peptide # 22 which was recognized by 4/1 1 patients with SSc but none of the patients with SLE or controls. When tested on larger number of patients, P#l was not specific for Lupus or SSc, as it was recognized by 1/23 SSc, 1/23 SLE, 2/23 RA and 2/23 HC and was not further investigated. Peptide # 22, which seemed promising for SSc at first screening, did not remain specific when tested on 56 patients with SLE, 56 with SSc, 46 with other RD and 54 controls.

Only Peptide #7 (Phe-Leu-Ser-Glu-Ala-Ser-Ile-Met-Gly-Gln-Phe-Asp-His-Pro-Asn -

NH2) was specifically recognized by 35.4% of patients with SLE compared to 5.4% of patients with SSc, 6.5% of patients with RA and 3.6% of healthy controls (p<10 ^~7 ) with titers significantly higher in patients with SLE than any other subjects (p<0.0001, Figure 3).

P7 was also recognized by 30% of patients with SLE without anti-dsDNA-autoantibodies (data not shown).

Anti-P7 antibody titers correlate with higher SLE Disease Activity Indexes (SLEDAl)

Active disease was defined as having a SLEDAl strictly superior to 4 (>4). We had SLEDAl information for the 48 patients with SLE who had been tested for the presence of anti- peptide P7 antibodies. We showed that higher anti-P7 antibody titers correlated with higher disease indexes and could be a marker of disease activity, although the correlation was marginally significant (Spearman's rank test, p<0.02, Figure 4). Conclusion:

The 15-mer peptide (P7) is specifically recognized by sera from patients with SLE compared to all controls. Moreover, higher anti-P7 antibody titers correlates with higher SLE Disease Activity Indexes (SLEDAl, Spearman's correlation p=0.02). EphB2 (receptor of ephrinB2) is a candidate involve in the mechanisms triggering vasculopathy and angiogenesis impairment. Ephrin-B2 (ligand) interacts through PDZ domain with Vascular Endothelial Growth Factor Receptor (VEGFR)-2 on endothelial cells Indeed, Acker-Palmer's group and others have recently shown that Ephrin B2 (ligand of EphB2 or EphB4) is required for VEGFR internalization and signaling and is necessary for angiogenesis and lymphangiogenesis. Accordingly the polypeptide based on the P7 peptide could thus be suitable to reverse the disease process and restore normal angiogenesis and/or vasculogenesis function in patients suffering from lupus.

Inventors have obtained more results which confirm that the anti- EphB2 autoantibodies can be used to diagnose lupus. This validation is shown in the following Tables 2 and 3. They observe in their study that patients with SLE positive for anti-EphB2 AAb had more often experienced cardiovascular events than patients negative for this AAb (P=0.015, two-sided Fisher's exact test, Table 2), which argues in favor of a role for Anti-EphB2 Ab in vasculopathy. Otherwise patients with SLE were not significantly different in gender, ethnicity, whether they were positive or negative for anti-EphB2 (Table 2).

Among patients with SSc they could observe that men with SSc had a tendency to have more often anti-EphB2 AAbs than women with SSc, although without reaching significance (Table 3, P= 0.053). Otherwise patients with SSc were not significantly different in age at diagnosis, had similar disease duration and did not differ in ethnicity or clinical subtypes (diffuse or limited SSc), whether they were positive or negative for anti-EphB2 AAb. Patients had similar organ involvement at the time of their blood draw whether they were positive or negative for anti-EphB2 AAb except for joint involvement which was marginally more frequent for patients negative for EphB2 AAb (P=0.04). Treatments were similar in any groups (data not shown). Nevertheless, when patients were classified according to classical SSc- specific AAb status (ATApos, ACApos or ATA/ACAneg), those without classical SSc- AAb (ACA/ATAneg) were the one who had more often anti-EphB2 AAb (44%) and with the highest Absorbance levels (PO.0001).

To evaluate diagnostic ability of EphB2 for SLE, plots of sensitivity versus 1-Specifity or Receiver Operating Characteristic (ROC) curves have been realized by comparing results in each disease to all other subjects. Results are represented Figure 5 and include also results from another protein (THEX1). The presence of anti-EphB2 AAb is a good diagnostic tool for SLE even when patients with SSc are also considered as controls with healthy controls and controls with RD (areas under the curve (AUC) of 0.80 and 0.74 respectively). On the opposite, and expected from results above, EphB2 is not diagnostic tools for SSc when among controls patients with SLE are also included (Figure 5).

Peptide 7 was recognized by 35.4% of patients with SLE (n=48, P<10 ^"7 ) compared to 5% of all other individuals (N=157), including 5.4% of patients with SSc (n=56), 6.5% of patients with RA (n=46) and 3.6% of healthy controls (n=55). Sera samples from patients with SLE had titers significantly higher than any other sera (P<0.0001, Figure 3). Roc curve for P7 showed an AUC of 0.73 (Figure 6). Anti-P7 AAb detection assay allowed a sensitivity of 35% and a specificity of 95% for SLE diagnosis (Figure 6). Anti-EphB2 AAb

n=63 ^a

sera samples positive negative

from patients with SLE (n=35) (n=28)

Gender female % 80 89

(n) (n=25)

Disease in remission % 57 68

activity active % 43 32

Ethnic Caucasian % 91 100

diversity asian % 2 0

african % 6 0

AutoAb dsDNA % 54 48 (n=25)

status ^b Sm % 29 (n=14) 20 (n=15)

RNP % 21 n=33j 19 (n=26j

SSA % 56 fn=76) 50 (n=14)

SSB % 40 (n=15) 23 (n=73j

(n=31) (n=27j

Organ skin % 23 29

involvement joint % 36 19

kidney % 19 33

cardiovascular % 26* 0

hematology % 23 19

brain % 0 0 a Number of individuals considered for calculation is indicated ahead of each column, otherwise indicated between brackets (when clinical data are not available for all individuals). b AutoAb status: Autoantibody status: dsDNA, anti-double strand DNA antibodies, Sm: anti-Smith antibodies, RNP: anti-ribonucleoprotein antibodies, SSA, anti-SSA antibodies, SSB, anti-SSB antibodies.

*p value <0.05 with two-sided Fisher's exact test. Table 2- Clinical and serological characteristics of patients with SLE, positive or negative for anti-EphB2 antibodies.

Anti-EphB2 AAb

n=107 ^a

sera samples positive negative

from patients with SSc (n=36) (n=71)

Gender female % 72* 87

Mean age at diagnosis (years) 45 46

Disease duration (years) 7.1 6.5

Ethnic Caucasian % 83 80

diversity asian % 14 7

african % 0 14

Disease's Lc-SSc % 53 51

subtype" Dc-SSc % 47 49

Organ lung % 73 (n=15) 67 (n=39)

involvement cardio-vascular % 33 (n=12) 36 (n=31)

joint % 42 (n=12) 76** (n=25)

a Number of individuals considered for calculation is indicated ahead of each column, otherwise indicated between brackets (when clinical data are not available for all individuals) .b Patients with SSc are divided into 2 clinical subtypes: limited cutaneous and diffuse cutaneous SSc, respectively, Lc-SSc and Dc-SSc.

*p value = 0.053 (χ ² test).

** p value = 0.04 (χ ² test).

Table 3- Clinical and serological characteristics of patients with SSc either positive or negative for anti-EphB2 antibodies.

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