WO2014122275A1

WO2014122275A1 - Method for the diagnosis and monitoring of autoinmune diseases

Info

Publication number: WO2014122275A1
Application number: PCT/EP2014/052458
Authority: WO
Inventors: Marta ALARCÓN RIQUELME; Casimiro CASTILLEJO LÓPEZ; Manuel José BERNAL QUIROS
Original assignee: Fundación Pública Andaluza Progreso Y Salud
Priority date: 2013-02-08
Filing date: 2014-02-07
Publication date: 2014-08-14

Abstract

The present invention relates to a method for diagnosing and predicting the response to a treatment in a subject suffering from an autoimmune disease. The invention also relates to a method for monitoring the progression of an autoimmune disease and to a method for allocating a subject suffering from an autoimmune disease. The present invention relates to agents for use in the treatment of an autoimmune disease, compositions comprising said agents and pharmaceutically compositions thereof.

Description

METHOD FOR THE DIAGNOSIS AND MONITORING OF AUTOINMUNE

DISEASES

FIELD OF THE INVENTION

The present invention relates to the diagnosis and/or prognostic of patients with autoimmune diseases based on determining the interaction between the B cell scaffold protein and ankyrin repeats (BANK1), the Phospho lipase C gamma 2 (PLCg2) and the B lymphoid tyrosine kinase (BLK). The invention also relates to agents aimed at the treatment of autoimmune diseases based on the alteration of interactions between BANK1 , PLCg2 and BLK.

BACKGROUND OF THE INVENTION

Numerous autoimmune disorders are characterized by the production of autoantibodies against self antigens. For example, systemic lupus erythematous (SLE) is an autoimmune disease in which the excess of autoantibodies causes organ damage. The autoantibodies bind to host cells leading to activation of the immune system and they can form immune complexes that deposit in organs such kidney and vascular tissues. Sjogren's syndrome is an autoimmune disease characterized by inflammation in the glands of the body. Other autoimmune disorders sharing similar pathology are IgA nephropathy, psoriasis, rheumatoid arthritis, multiple sclerosis, ankylosing spondylitis, etc

Systemic lupus Erythematosus (SLE) is the prototype autoimmune disease, characterized by B-cell hyperactivity and pathologic production of autoantibodies. SLE is a complex genetic disease in which multiple susceptibility genes and environmental factors interplay in concert toward a disease phenotype. The genetic predisposition to SLE thus represents the integrated effect of enhancing and protective genetic variants. Over the last decades, many susceptibility genes have been identified mainly through genome wide association studies (GWAS). Despite this wealth of genetic information, little is known about the mechanistic contribution of genetic risk variants to the disease. A prevalence of several hundred thousand patients with lupus has been estimated in the United States - it may in fact approach 1 million to 2 million individuals according to the Lupus Foundation of America - and almost the same figures are given in Europe. Diagnosing and monitoring disease activity are both problematic in patients with

SLE. Diagnosis is problemat ic because the spectrum of disease is broad and ranges from subtle or vague symptoms to life threatening multi-organ failure. There are other diseases with multi-system involvement that can be mistaken for systemic lupus, or vice versa. Several criteria have been dev eloped for the ciassificat ion of SLE. The American

College of Rheumatism created a list of 1 1 criteria to help physicians to diagnose lupus.

It is considered an SLE case when a patient displays at least four of the eleven criteria.

The classification is further compromised because determining the presence or absence of the criteria often requires subjetive interpretations. Furthermore, the range of clinical manifestation in SLE can vary in the level of activity and severity from, one patient to another. Thus SLE is often misdiagnosed.

Diagnosing patients with SLE is also carried out by serological tests based on the detection in the serum of ant i-nuclear ant ibodies (ANAs) directed against the autologous components present in the cell nucleus or antibodies to extractable nuclear antigens (ENAs) directed against single stranded DNA (ssDNA) and individual histories. However, in the presence of typical features of SLE, a negative ANA or EN A test does not exclude the diagnosis.

Untreated SLE can be fatal as the disease progress from attacks of skin and joints to internal organs, including lung, heart and kidneys, thus making prognosis particularly crit ical. However monitoring of the SLE progression is complicated because it mainly appears as a series of flare-ups with intervening periods of litt le no disease manifestation.

Thus, it would be advantageous to hav e molecular-based methods for diagnosing patients w ith SLE and prognostic methods for predicting the clinical outcome in lupus as well as molecular-based method that can be used to object ively monitoring the progression of said disease.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention refers to an in vitro method for diagnosing an autoimmune disease or for predicting the effectiveness of a treatment administered in a patient suffering from an autoimmune disease which comprises: (i) activating the B lymphocytes in a sample isolated from said patient containing B lymphocytes,

(ii) determining the time-dependent formation of a complex comprising BANK1 and PLCg2 in the B lymphocytes present in the sample in response to said activation, wherein said determination is carried out until the interaction between said proteins reaches a stationary level;

(iii) comparing the value of at least one kinetic parameter of the formation of said complex with a reference value for said parameter,

wherein a deviation in the value of said at least one kinetic parameter with respect to said reference value is indicative of the presence of the disease or that the treatment is ineffective.

In another aspect, the invention refers to an in vitro method for monitoring the progression of an autoimmune disease in a patient which comprises:

(i) activating the B lymphocytes in a sample isolated from said patient containing B lymphocytes,

(iii) determining the variation of the value of at least one kinetic parameter of the formation of said complex with a reference value for said variation,

wherein a deviation in the value of said at least one kinetic parameter in comparison with the variation of the same parameter at an earlier point of the disease in the patient is indicative that the autoimmune disease shows a bad progression.

In another aspect, the invention relates to an in vitro method for classifying a patient suffering from an autoimmune disease which comprises:

(i) activating the B lymphocytes in a sample isolated from said patient containing B lymphocytes, (ϋ) determining the time-dependent formation of a complex comprising BANKl and PLCg2 in the B lymphocytes present in the sample in response to said activation, wherein said determination is carried out until the level of complex reaches stationary level,

wherein the patient is classified in a first group if a deviation in the value of said at least one kinetic parameter with respect to said reference value is detected or classified in a second group if a deviation value of the value of said at least one kinetic parameter with respect to said reference value is not detected.

In another aspect, the invention relates to an in vitro method for detecting a protein complex between the B cell scaffold protein with ankyrin repeats (BANKl) and Phospho lipase C gamma 2 (PLCg2) in a sample comprising B lymphocytes wherein said B lymphocytes are activated.

In another aspect, the invention relates to an agent selected from the group consisting of a B lymphoid tyrosine kinase (BLK) inhibitor and a BANKl inhibitor for use in the treatment of an autoimmune disease.

In another aspect, the invention relates to an inactive BLK variant selected from the group consisting of a BLK variant carrying a mutation at position 269 with respect to the human BLK, a BLK variant lacking the myristoylation site and a BLK variant containing an additional palmitoylation site.

In another aspect, the invention relates to an inactive BANKl variant which carries a mutation at position 484 and/or a mutation at position 488 with respect to human BANKl isoform 1.

In a further aspect, the invention relates to compositions a comprising an inactive BLK variant according to the invention or an inactive BANKl variant according to the invention. In yet another aspect, the invention relates to pharmaceutical compositions comprising an inactive BLK variant according to the invention or an inactive BANKl variant according to the invention and a pharmaceutical acceptable carrier. In another aspect, the invention refers to a protein complex comprising BANKl and PLCg2.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: A) Time variation of PL A BANKl -PLCg2 interaction upon stimulation with anti-IgM in two human B-cell lines, the Burkitt^'s derived Daudi and the non- Hodgkin^'s lymphoma derived RL. Both cell lines show similar kinetic of interaction. Results are shown as the mean from three independent experiments. Error bars represent the SD from the mean; B) Time course upon anti-IgM stimulation of co- localization between the PLA signal and the nucleus. The graft shows two independent experiments, each point represents the analysis of at least 300 cells. C) Immunoprecipitation of the BANKl -PLCg2 complex in Daudi cells. Anti-PLCg2 immunoprecipitates (above) and total cell lysate (below) were analyzed by immunoblotting with anti-BANKl antibody (BANKl -ET-52). The position of the BANKl protein is indicated by arrows.

Figure 2: A) Schematic representation of the constructs used to study the association between BANKl and PLCg2 in transfected HEK293 cells. The constructs coding for wild-type forms of BLK, LYN, GFP and PLCg2 or the indicated mutated forms were fused to the epitope V5 at the C-termini. BANKl was targeted with the Flag epitope at the N- terminus; B) HEK293 cells were transiently co -transfected with plasmids coding for the wild-type form of BLK, its functionally mutated forms (KL and YF), LYN or GFP in addition to plasmids expressing BANKl and PLCg2. The lysates were immunoprecipitated using anti-PLCg2 antibody (above) and immunoblotted sequencially with anti-BANKl antibody, anti-V5 to detect PLCg2, Srcs kinases and GFP and anti-phosphotyrosine antibody; C) Mutation of lipidation sites of the kinases influence the formation of the BANKl -PLCg2 complex and the overall tyrosine phosphorylation on PLCg2. The blots were interrogates as in B.

Figure 3: A) Immunoblot of extracts derived from human Daudi B-cells showing efficient silencing of endogenous BLK protein. Top, western blot analysis using antibody to BLK; bottom, western blot analysis using antibody to GAPDH as loading control. B) The relative BLK mRNA is reduced to half in the silencing line (shBLK). C) Immunoprecipitates (IPs) of stimulated silenced shBLK cells with anti-IgM, using anti-PLCg2 antibody and interrogated with anti-BANKl to assay BANKl-PLCg2 association. D) Kinetics of the association between BANKl-PLCg2 assayed with in situ Proximity Ligation (PLA) in control and BLK- silenced cell lines.

Figure 4: A) The Dof/BCAP/BANK (DBB) motif (amino acids 199-327), the double ankyrin repeat-like (ANK) motifs (amino acids 339-402) and the presumptive coiled coils (CC) region (amino acids 677-705) are indicated. Tyrosine residues susceptible to be phosphorylated are shown by Y. Residues Y125, Y146, Y161, Y416, Y484 and Y488 that predict the putative SH2 binding sites are indicated as bold Y. The positions of the mutated amino acids are indicated above the BANK1 drawing. B) Alignment of the BANK1 amino acid motifs in different species indicating the mutated residues corresponding to putative SH2 and SH3 binding domains C) Phosphorylation and immunoprecipitation analysis of the wild type and mutated forms of BANK1 co-expressed with the constitutively active form of BLK (BLK- YF) and PLCg2. D) Quantification of BANK1 phosphorylation and immunoprecipitation using anti-PLCg2 antibody. Bands of the western blots were quantified using ImageJ program. E) Co-expression in HEK293 cells of the two iso forms of BANK1 renders equivalent recovery of both iso forms in the anti-PLCg2 immunoprecipitate, indicating that exon 2 does not participated in the binding between BANK1 and PLCg2.

Figure 5: A) Solid growth assay on DO-3 medium of transformants carrying coding fragments of BANK1. B) Summary of the results of the assay.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have found, surprisingly, that the B cell scaffold protein with ankyrin repeats (BANK1) interacts physically with Phospholipase C gamma 2 (PLCg2) protein in B lymphocytes upon activation of the B lymphocytes. The authors of the present invention have also found, surprisingly, that hyperstimulated B lymphocytes show altered levels of said protein complex and that the formation of said complex is modulated by the kinase activity of the B lymphoid tyrosine kinase (BLK). Methods for detecting a complex between BANKl and PLCg2 (First method of the invention)

In a first aspect, the invention relates to an in vitro method for detecting a protein complex between the B cell scaffold protein with ankyrin repeats (BANKl) and Phospholipase C gamma 2 (PLCg2) in a sample comprising B lymphocytes, wherein said B lymphocytes are activated.

The term "B cell scaffold protein with ankyrin repeats (BANKl)" as used herein, includes variants, isoforms and species homologous of human BANKl . It is involved in B-cell receptor induced Ca2+ mobilization from intracellular stores and promotes Lyn mediated phosphorylation of IP3 receptors 1 and 2. The complete cDNA sequence for human BANKl has the Genebank accession number NM 001083907.2 (release of 18 January, 2002). The complete protein sequences for isoforms 1, 2 and 3 of human BANKl are available in the Uniprot database under accession numbers NP_060405.4 (1 l-Nov-2012), NP_001077376 (1 l-Nov-2012) and NP 001120979 (I lNov-2012), respectively.

The term the "Phospholipase C gamma 2 (PLCg2)" as used herein, includes variants, isoforms and species homologous of human PLCg2. Enzymes of the phospholipase C family catalyze the hydrolysis of phospholipids to yield diacylglycerols and water-soluble phosphorylated derivatives of the lipid head groups. A number of these enzymes have specificity for phosphoinositides. Of the phosphoinositide-specific phospholipase C enzymes, C-beta is regulated by heterotrimeric G protein-coupled receptors, while the closely related C-gamma-1 (PLCG1; MIM 172420) and C-gamma-2 enzymes are controlled by receptor tyrosine kinases. The C-gamma-1 and C-gamma-2 enzymes are composed of phospholipase domains that flank regions of homology to noncatalytic domains of the SRC oncogene product, SH2 and SH3. The complete cDNA sequence for human PLCg2 has the Genebank accession number NM 002661.3 (November 17, 2012). The complete protein sequence for human PLCg2 has the Uniprot accession number PI 6885 (August 1, 1990).

The term "protein complex" or "complex" as used herein, is related to a group of two or more associated polypeptide chains. Preferably, said polypeptides interact with each other at the same time and place. Protein complexes are a form of quaternary- structure. Proteins in protein complexes are linked by non-covalent protein-protein interactions, and different complexes have different degrees of stability over time. Details and further embodiments of the complex are explained below under the heading "Complex of the invention". The different embodiments mentioned below are equally applicable to the method according to the present invention.

The detection of protein complex can be carried out by any method known in the art. Said methods are based on the identification of the interacting partners of one target protein. For example, the interacting partners of one protein (i.e. BANK1) can be identified using fusion-based affinity protein purification. Briefly, this technology is based in heterologue expression of the bait protein fused to an appropriate tag. Suitable tags include peptide-based tags. The tag is generally placed at the amino- or the carboxyl-terminus of the bait protein. Various tag polypeptides are well known in the art. Examples include poly-histidine (poly- his) or poly-histidine-glycine (poly-his-gly), the flu HA tag, the c-myc tag, the streptavidin tag or the flag tag. Bait fusion protein is transfected and appropriate expressed in suitable cells. After an appropriate expression period, cells are lysed and the tagged bait together with bound proteins, is isolated using a specifical chemical or biological ligand linked to a solid support. Eluted proteins can be separated by gel-electrophoresis and specifically bound proteins (i.e. proteins absent from the control are identified by mass spectrometry.

In a particular embodiment, the complex between BANK1 and PLCg2 is determined by means of in situ proximity ligation assay (PLA), as shown in the examples of the invention. Briefly, this technology allows the detection and localization of protein targets with single molecule resolution and objectively quantified in unmodified cells and tissues. Two primary antibodies raised in different species recognize antigens of interest. A typical experimental approach starts by seeding cells (a lymphoblastic cell line could be used in this assay) in suitable slides followed by incubation with rabbit anti-human BANK1 antibody together with mouse anti human PLCg2 antibody. The antibodies can be incubated over night at 4°C; however the conditions and the incubation time of the antibodies can be modified according to the antibodies used. After the incubation the slides are incubated with species-specific secondary antibodies, called PLA probes, each with a unique short DNA strand attached to it, bind to the primary antibodies. Thus, mouse minus and rabbit plus PLA probes can be added under conditions suitable for the ligation reaction, for example 37°C and 30 minutes. Only when the PLA probes are in close proximity, the DNA strands interact and can be sealed by a subsequent addition of two other circle- forming DNA oligonucleotides. After joining of the two added DNA hybrid by enzymatic ligation, they are amplified using rolling circle amplification using a polymerase. After the amplification reaction, several hundredfold replication of the DNA circle has occurred and labeled complementary oligonucleotides probes highlight the product. The resulting high concentration of fluorescence in each single-molecule amplification product is easily visible as a distinct bright spot when viewed with a fluorescence microscope. The values of the complex formation between BANKl and PLCg2 can be quantified using appropriate software.

In another particular embodiment, the detection of a protein complex between BANKl and PLCg2 is determined by means of protein co-immunoprecipitation (Co-IP) in which the protein of interest (e.g. BANKl) is isolated with a specific antibody and the molecules (proteins) which interacts with the protein (e.g. PLCg2) are subsequently identified by means of western blot. Co-IP can be carried out on cell extracts or tissue which endogenously expressing the proteins of interest. If desired, expression levels of said proteins can be increased by the exogenous expression thereof in tissues or cells of interest using techniques known in the art such cellular transfection or cellular transduction. Optionally, proteins which physically interact with each other (e.g. BANKl and PLCg2) can be covalently cross-linked using crosslinking agents. The formation of crosslinks between two distinct proteins is a direct and convincing evidence of their close proximity. Crosslinking agents are either homo- or hetero- bifunctional reagents with identical reactive groups, respectively, permitting the establishment of inter- as well as intra-molecular crosslikages. Examples of crosslinking agents include the imidoester crosslinker dimethyl suberimidate, the N- Hydroxysuccinimide ester crosslinker BS3 and formaldehyde. Each of these crosslinker induces nucleophilic attack of the amino group of lysine and consequent covalent binding via the crosslinker. A crude cellular extract is treated with the crossliking reagent and immunoprecipitation with antibodies specific for the target protein (e.g. BANKl or PLCg2) is used to recover the assorted complex containing it. Quantification of the formed complex between BANKl and PLCg2 may be carried out by means of band intensity analysis using appropriate software.

In another preferred embodiment, the protein complex between BANKl and PLCg2 is determined by means of protein colocalization which refers to observation of the spatial overlap between two (or more) different fluorescent labels, each having a separate emission wavelength, to see if the different targets (e.g. BANKl and PLCg2) are located in the same area of the cell or very near to one to another. Fluorescent proteins (e.g. Green florescent protein or GFP, yellow florescent protein or YFP, red fluorescent protein or RFP) can be attached to desired proteins to form a fusion protein, synthesized in cells after transfection of a suitable expression plasmid.

Additional biochemical methods for determining protein complex generally known in the art include but are not limited to, protein affinity chromatography, affinity blotting, pull down, and the like. The binding constant for two interacting proteins, which reflects the strength of quality the interaction can also be determined using methods known in the art.

The complex between BANKl and PLCg2 is detected, preferably in a sample comprising B lymphocytes wherein said B lymphocytes are activated. B cells belong to a group of white blood cells known as lymphocytes, making them vital part of the immune system, specifically the humoral immunity branch of the adaptative immune system. The person skilled in the art can easily distinguish B lymphocytes from other lymphocytes, such T lymphocytes and natural killer cells, by the presence of a protein on the outer surface of the lymphocytes B, known as B cell receptor (BCR). In the context of the present invention, plasma B cells, memory B cells, Bl cells, marginal- zone B cells, and follicular B cells are also included. Examples of suitable samples containing B lymphocytes are peripheral blood, spleen, bone marrow or lymph nodes.

Conveniently a blood sample is used. The term blood sample, as used herein, shall include hematopoietic biological samples such blood, lymph, leukophoresis product, bone marrow and the like. The blood sample is drawn from any site, preferably by venicpunture. Blood samples will usually be from 1 to 100 ml of whole blood and may be treated with anticoagulants, e.g., heparin, EDTA, citrate, acid citrate dextrose or citrate phosphate dextrose as known in the art. The sample may be subjected to treatment such as dilution in buffered medium, concentration, filtration, or other gross treatment that will not involve the destruction of B lymphocytes. The sample may derived from any mammal, including primate, particularly human, murine, equine, bovine, porcine, lagomorpha, canine, feline, etc.

A preparation of nucleated cell may be made from the sample using any acceptable procedure that can separate living nucleated cells from erythrocites. The use of whole blood allows detection of eosinophils in addition to basophil detection. The use of Ficoll-Hypaque density gradients or elutriation is well documented in the literature. Alternatively, the blood cells may be resuspended in a solution which selectively lyses adult erythrocites, e.g. ammonium chloride-potassium, ammonium- oxalate, etc. Treatments may also include removal of cells by various techniques including centrifugation, using Ficoll-Hypaque, panning, affinity separation, using antibodies specific for one or more markers present as surface membrane proteins on the surface of the cells, or other techniques that provide for enrichment of lymphocytes B.

In a preferred embodiment, the first method of the invention is carried out in a

"population of isolated B lymphocytes". Many different approaches may be used for isolation the of B lymphocytes from blood sample. If desired, B lymphocytes can be isolated by means of magnetic beads. Said technique is based on the positive selection of said cells bearing specific Ig-receptors with magnetic beads coated by respective antigens. Optionally, unwanted cells can be targeted for removal with specific antibodies recognizing CD2, CD3, CD14, CD16, CD43, CD56, CD56b, glycophorin A and coated magnetic particles. The labeled cells are separated using a magnet. Alternatively, B lymphocytes may be isolated using cytometric assays to stain with one or more specific markers conjugated florescent dye (such R-phycoerytrin or PE, fluorescein isothiocyanate or FITC, Cy5 etc.) whose expression patterns correlate (not necessarily exclusively) with lymphocyte development (i.e. IgD vs. CD38), the state of proliferation/ cell cycle (e.g. Ki67, CFSE, Hoechst 33342 and pyronin Y), activation state (e.g. CD69 and CD71) predisposition for cell: cell interactions (e.g., CD40, CD25, CD70, CD80/86), and cell survival state (e.g. annexin V and propidium iodide) among others. Thus, fluorescently stained cells are sorted in one or more separate fractions and, then optionally, lymphocytes B are cultured in vitro under appropriate conditions. In a preferred embodiment, the sample contains at least 10%, 20%, 30%>, 40%>, 50%, 75%, 85%, 95%, 100% of B lymphocytes.

The first method of the invention is carried out in a sample comprising B lymphocytes wherein said B lymphocytes in the sample are activated. The term "B lymphocyte activation" or "B lymphocyte stimulation" as used herein, refers to the biochemical changes generated in B lymphocytes after encounter with an antigen. The first activation signal occurs upon antigen binding to B lymphocytes receptors (BCRs). Upon binding BCR, the antigen is internalized by receptor-mediated endocytosis, digested and complexed with MHC II molecules on the B surface. A second activation signal occurs via either a thymus-independent or a thymus-dependent mechanism. Presentation of antigen-class II MHC complex on a B lymphocyte enables it to act as an antigen-presenting cell (APC) to T cells. Preferably, the activation is carried out by contacting the sample with anti-IgM antibodies under conditions suitable for the stimulation of the B lymphocytes of the sample. Alternatively, lymphocytes B can be stimulated using methods well known in the art; non limiting examples of method for stimulating B lymphocytes include culturing said cells with anti-IgD antibodies, anti- CD40 antibodies, interleukin 4 (IL4), arachidonic acid or alpha-interferon (a-INF) in conditions suitable for said stimulation.. If desired, B lymphocytes may be activated in culture medium (e.g. RPMI 1640 culture medium supplemented with amino acids and vitamins) supplemented with heat inactivated fetal bovine serum (FBS) or fetal calf serum (FCS) at 10-40%, by adding appropriate concentration of a human IgM fragment [F(ab')2 human IgM] (e.g. 10 μg/ml) during the suitable time for stimulation. In a preferred embodiment the suitable time for stimulation is about less than 1 min, in another preferred embodiment is about 1 min, in another preferred embodiment the suitable time for stimulation is about 4 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, about 60 min, about 120 min, about 24 hours, about 48 hours or more.

Methods for diagnosing an autoimmune disease in a patient (Second method of the invention)

The invention also provides an in vitro method for diagnosing an autoimmune disease in a patient based in the detection of the protein complex between the B cell scaffold protein and ankyrin repeats (BANK1) and the Phospholipase C gamma 2 (PLCg2).

Thus, in a second aspect the invention relates to an in vitro method for diagnosing an autoimmune disease in a patient which comprises:

(ii) determining the time-dependent formation of a complex comprising BANK1 and PLCg2 in the B lymphocytes present in the sample in response to said activation, wherein said determination is carried out until the interaction between BANK1 and PLCg2 reaches a stationary level;

(iii) comparing the value of at least one kinetic parameter of the formation of said complex with a reference value for said parameter, wherein, a deviation value of said at least one kinetic parameter with respect to said reference value is indicative of the presence of the disease.

The term "diagnosis" as used herein, refers both to the process of attempting to determine and/or identify a possible disease in a subject, i.e. the diagnostic procedure, and to the opinion reached by this process, i.e. the diagnostic opinion. As such, it can also be regarded as an attempt at classification of an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. As the person skilled in the art will understand, such a diagnosis may not be correct for 100% of the subject to diagnose, although preferred it is. The term however requires that can identify a statistically significant proportion of subject suffering from such pathologies (in this case, autoimmune disease). The skilled in the art may determine whether a party is statistically significant using different statistical evaluation tools well known, for example, by determination of confidence intervals, the p-value determination, Student ^'s-test, the Mann- Whitney, etc. (see Dowdy and Wearden, 1983). Preferred confidence intervals are at least, 50%>, at least 60%>, at least 70%>, at least 80%>, at least 90%> or at least 95%. The p-values are preferably, 0.05, 0.025, 0.001 or lower.

The term "autoimmune disease" refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunological reaction of the subject to its own cells, tissues and/or organs. Illustrative, non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospho lipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjogren's syndrome, Good pasture's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, Wegener's granulomatosis, Anti-Glomerular Basement Membrane Disease, Antiphospho lipid Syndrome, Autoimmune Diseases of the Nervous System , Familial Mediterranean Fever, Lambert-Eaton Myasthenic Syndrome, Sympathetic Ophthalmia, polyendocrinopathies, psoriasis, etc.

In a particular embodiment, said autoimmune disease is Systemic Lupus Erythematosus (SLE).

The term "patient" as used herein refers to any recipient of health care services. The patient is most often ill or injured and in need of treatment by a physician, physician assistant, advanced practice registered nurse, veterinarian or other health care provider. In a preferred embodiment the patient is a human patient.

In the first step, the diagnostic method of the invention comprises activating the

B lymphocytes in a sample isolated from said patient containing B lymphocytes. The term "B lymphocyte activation" has been described in detail in the context of the first method of the invention and is equally applicable to the second method of the invention.

In a preferred embodiment the diagnostic method of the invention is carried out in a population of isolated B lymphocytes. Many different approaches may be used for isolation the of B lymphocytes from blood sample. Said methods are described in the context of the first method of the invention and are equally applicable to the diagnostic method of the invention.

In a preferred embodiment said lymphocyte activation is carried out by contacting the sample with anti-IgM antibodies under conditions suitable for the stimulation of B lymphocytes in said sample. Said conditions have been detailed in the context of the method for detecting a complex between BANKl and PLCg2 and are equally applicable to the diagnostic method of the invention. In a preferred embodiment, the B lymphocytes are activated by contacting the sample containing said B lymphocytes with anti-IgM antibodies.

In the second step, the method for diagnosing an autoimmune disease in a patient comprises determining the time-dependent formation of a complex comprising BANKl and PLCg2 in the B lymphocytes present in the sample in response to said activation, wherein said determination is carried out until the interaction of said proteins reaches a stationary level.

The term "determining the time-dependent formation of a complex" as used herein, refers to the measurement of the formation of the protein complex (i.e. between BANKl and PLCg2) during certain period of time. As mentioned previously concerning the first method of the invention, the detection of a protein complex between BANKl and PLCg2 can be done by different approaches known in the art, such as, for example PLA or Co-IP detailed above. If desire, the time-dependent formation of a protein complex between BANKl and PLCg2 can be detected during the suitable time for stimulation, which in a preferred embodiment is about less than 1 min, in another preferred embodiment the suitable time for stimulation is about 1 min, about 4 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, about 60 min, about 120 min, about 24 hours, about 48 hours or more. In a more preferred embodiment the time-dependent formation of a protein complex between BANK1 and PLCg2 is carried out until the interaction between said proteins reaches a stationary level.

The term "interaction" as used herein, means that two domains or independent entities exhibit sufficient physical affinity to each other so as to bring the two interacting domains or entities physically close to each other. An extreme case of interaction is the formation of a chemical bound the results in continual, stable proximity of the two domains. Interactions that are based solely on physical affinities, although usually more dynamic than chemical bonded interactions, can be equally effective at co-localizing independent entities. Examples of physical affinities and chemical bonds include but are not limited to, forces caused by electrical charge differences, hydrophobicity, hydrogen bonds, van der Waals force, ionic force, covalent linkages, and combinations thereof. Although not necessarily, an "interaction" is typically exhibited by the binding between the interacting domains or entities.

The term "stationary level" or "equilibrium phase" as used herein, refers to the state of the interaction between two reactants, (e.g. proteins BANK1 and PLCg2) wherein the rate of association equals the rate of dissociation. Thus, in said phase the protein complex comprising BANK1 and PLCg2 formation has no further tendency to change with time. The formation of the BANKl-PLCg2 complex may be determined by parameters well known in the art, such as the rate of association (k_a (M^_1 s^"1)) which can be measured during the association phase of the interaction between two proteins. In said phase, the first protein (e.g. BANK1) is flowed across the surface of the second protein (e.g. PLCg2) and binding is measured, allowing the determination of the rate of formation of the complex between said proteins (e.g. BANK1 and PLCg2) over the time. There is an associated increase in response units over time as the complex form. If desired, the complex formation can be further measured by means of the determination of the rate of dissociation (kj (s ¹)), which is determined during the dissociation phase of the interaction between two proteins. In said phase, the first protein (e.g. BANK1) is removed of the protein complex (e.g. BANKl-PLCg2 complex) and comes to concentration zero. There is an associated decrease in response units over time as the complex form. According to the second step of the diagnosis method of the invention, the determination of time-dependent formation of a protein complex comprising BANKl and PLCg2 is carried out by any suitable means known in the art, such for example, PLA or Co-IP, until the association rate of the protein complex comprising BANKl and PLCg2 equals the dissociation rate of said protein complex.

The third step of the diagnosis method of the invention comprises comparing the value of at least one kinetic parameter of the formation of the complex between BANKl and PLCg2 with a reference value for said parameter. The term "kinetic parameter" as used herein, refers to any measurable binding kinetic factor that at least partially defines a given molecular interaction (e.g. between BANKl and PLCg2) and can be employed to define its behaviour. Examples of kinetic parameters are well known in the state of the art. Said parameters include but are not limited to, "activation energy (E_a)", which is defined as the minimum energy required to start a chemical reaction, "reaction rate" (or "velocity of reaction") which defines the velocity of molecular interactions, e.g. the velocity of the formation of the complex between BANKl and PLCg2. The rate of a reaction can be expressed as average rate, which refers to changes in molar concentration of either reactants (e.g. proteins BANKl or PLCg2) or products (e.g. complex which comprises BANKl and PLCg2) in unit time. The rate of a reaction can also be determined as instantaneous rate which relates to changes in molar concentration of either reactants (e.g. proteins BANKl or PLCg2) or products (e.g. complex between BANKl and PLCg2) at an instant of time (or in infinitesimally small interval of time). The term "kinetic parameter" as used herein, also refers to the "association rate constant" and also refers to the "dissociation rate constant", terms which have been defined above. The term "kinetic parameter" as used herein, also refers to how long it takes to form a given or desired concentration of BANKl -PLCg2 complex.

The term "kinetic parameter" in the context of the present invention and in a preferred embodiment, also refers to the "concentration of BANKl -PLCg2 complex" at a given or desired time. In a preferred embodiment, said desired time is about less than 1 min, in another preferred embodiment the suitable time for stimulation is about 1 min, about 4 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, about 60 min, about 120 min, about 24 hours, about 48 hours or more.

As used herein, a "reference value" refers to the median or average value of at least one of any kinetic parameter related to BANKl-PLCg2 complex (defined below) measured in a reference sample, wherein the reference sample can be a sample obtained from a subject which is not suffering or has no history of having suffered an autoimmune disease. Alternatively, the reference value can be the median or average value of any kinetic parameter related to BANKl-PLCg2 complex (defined below) obtained from a collection of samples from control individuals (e.g. people with no diagnosis of autoimmune diseases). In another embodiment, the reference sample is a sample containing B cells of the patient which has not been treated so as to stimulate the B cells.

Once this median value is established, said kinetic parameter in samples from patients can be compared with this median value, and thus it will be considered as "deviation value" if said value is included in any of "low" or decreased" value, " or "high" or "increased" value. In a preferred embodiment, an deviation value is an increase of a kinetic parameter of BANKl-PLCg2 complex of at least 1.1 -fold, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared with the reference value is considered as "increased" level of said kinetic parameter. In a preferred embodiment, a deviation value is a decrease of a kinetic parameter of BANKl-PLCg2 complex of at least 0.9-fold, 0.75- fold, 0.2-fold, 0.1 -fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01 -fold, 0.005-fold or even less compared with the reference value is considered as "decreased" level of said kinetic parameter. The term "same level" as used herein, refers to a kinetic parameter of BANK-PLCg2 complex which is substantially unaltered with respect to the reference value. For instance, the kinetic parameter under study is considered to be the same as in the reference sample when the levels differ by no more than 0.1%, no more than 0,2%, no more than 0,3%, no more than 0,4%, no more than 0,5%, no more than 0,6%, no more than 0,7%, no more than 0,8%, no more than 0,9%, no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10% or no more than the percentage value that is the same as the error associated to the experimental method used in the determination.

In a preferred embodiment, the kinetic parameter which will be determined is the concentration of BANKl-PLCg2 complex. In more preferred embodiment, said kinetic parameter is determined by means of PLA or Co-IP.

In a preferred embodiment, the comparison of the concentration of the BANK1- PLCg2 complex with the reference value is determined at the time point wherein the concentration of said complex is highest. The term "highest concentration of the complex", as used herein, refers to the concentration wherein the concentration of the complex is highest throughout the time course of the biding reaction. As mentioned above, activation of the B cells is followed by an increase in the intracellular concentration of the complex. This concentration reaches a maximum level, from where the concentration starts decreasing until the stationary levels are reached again. The comparison is then carried out at the time point wherein the levels are the highest. In a preferred embodiment, the highest value refers to a relative concentration of the complex which is at least 1% higher, at least 5% higher, at least 10 % higher, at least 20% higher, at least 30 % higher, at least 30% higher, at least 40 % higher, at least 50 % higher, at least 60 % higher, at least 70 % higher, at least 80%>, at least 90%> highest or at least 100% or more than the concentration in the sample from which the reference value is obtained. It will be understood that the comparison of the concentration of the complex between the test sample and the sample from where the reference value is obtained has to be carried out at the same time after stimulation of the B cells.

Once at least one kinetic parameter of the formation of a complex comprising BANK1 and PLCg2 (e.g. the concentration of BANKl-PLCg2 complex formed at the time point wherein said concentration is highest) has been measured in a sample from a patient and compared with the reference value, if said kinetic parameter shows a deviation value with respect to said reference value it can be conclude that said subject can be diagnosed as suffering an autoimmune disease.

Methods for predicting the response to a treatment administered in a patient suffering from an autoimmune disease

Alternatively, since the intracellular levels of the BANK1 PLCg2 complex in B cells in response to stimulation of the B cells is an indicative of the presence of an autoimmune disease, it is also possible that the levels of the complex can be used to monitor the progression of the disease, being the progression of the disease associated with altered levels of the BANKl-PLCg2 complex in comparison to patients wherein the disease is stationary. Similarly, in those patients which are being treated and wherein the disease is remitting, the levels of the complex should also reach levels similar to those observed in a reference sample. Thus, in another aspect, the invention contemplates said method for predicting the effectiveness of a treatment administered in a patient suffering from an autoimmune disease. Thus, in a second aspect, the invention relates to an in vitro method (hereinafter third method of the invention or prognostic method of the invention) for predicting effectiveness of a treatment administered in a patient suffering from an autoimmune disease which comprises:

wherein, a deviation value of said at least one kinetic parameter with respect to said reference value is indicative of that the treatment is ineffective.

The terms "predicting the effectiveness of a treatment" and "predicting the response to a treatment" are used herein interchangeably and relate to the likelihood that a patient will have a positive or negative response to said treatment. As will be understood by those skilled in the art, the prediction, although preferred to be, need not to be correct for 100% of the subjects to be evaluated. The term however, requires that a statistically significant portion of subject can be identified as having an increased probability of having a given response to specific treatment. Whether a subject is statistically significant can be determined using the statistical evaluation described above. The skilled in the art may determine whether a party is statistically significant using different statistical evaluation tools well known, for example, by determination of confidence intervals, the p-value determination, Student^'s-test, the Mann- Whitney, etc. (see Dowdy and Wearden, 1983). Preferred confidence intervals are at least, 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The p-values are preferably, 0.05, 0.025, 0.001 or lower.

In a preferred embodiment, the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, Behcet disease, amyotrophic lateral sclerosis, multiple sclerosis and Devic disease, spondyloartheopathy, fibromyalgia, rheumatic fever, Wegener granulomatosis, systemic lupus erythematosus, Antiphospho lipids syndrome or Huges syndrome, polymyositis, dermamyositis, chronic inflammatory demyelinating polyradiculoneuropathy, psoriasis, immune thrombocytopenic purpura , sarcoidosis, chronic fatigue syndrome, Guillain-Barre syndrome, systemic vasculitis or vitiligo. In a still more preferred embodiment, the autoimmune disease is systemic lupus erythematosus (SLE).

The term "treatment" or "to treat" in the context of this specification means administration of a compound or formulation according to the invention to prevent, ameliorate or eliminate the disease or one or more symptoms associated with said disease. "Treatment" also encompasses preventing, ameliorating or eliminating the physiological sequel of the disease

The treatment of autoimmune diseases is done typically with immunosuppressants. These are substances that decrease the immune response. They may be either exogenous, as immunosuppressive drugs, or endogenous. Examples of immunosuppresive drugs are glucocorticoids, which diminishes both B cell clone expansion and antibody synthesis; cytostatic, which affect the proliferation of both T cells and B cells, examples of cytostatic are akylating agents, antimetabolites (for example, folic acid analogues, purine analogues, pyrimidine analogues, protein synthesis inhibitors); antibodies or drugs acting on immunophilins such ciclosporin, tacrolimus or sirolimus. SLE is treated with immunosupression, mainly with cyclophosphamide or corticosteroids.

The term "patient" and preferred embodiment thereof has been defined in the context of the diagnostic method of the invention and equally applies for the prognostic method of the invention. In the first step, the method for predicting the effectiveness of a treatment administered to a patient suffering an autoimmune disease comprises activating the B lymphocytes in a sample isolated form said patient containing B lymphocytes.

In a preferred embodiment the prognostic method of the invention is carried out in a population of isolated lymphocytes. Many different approaches may be used for isolation the of B lymphocytes from blood sample. Said methods are described in the context of the first and the second method of the invention and are equally applicable to the diagnostic method of the invention.

In a preferred embodiment the activation of B lymphocytes in a sample containing B lymphocytes form a patient suffering from an autoimmune disease is carried out by contacting the sample with anti-IgM antibodies under conditions suitable for the stimulation of B lymphocytes in said sample.

In the second step, the method for predicting the effectiveness of a treatment administered to a patient suffering an autoimmune disease comprises determining the time-dependent formation of a complex comprising BANK-1 and PLCg2 in the B lymphocytes present in the sample in response to the activation carried out in the first step of said method, wherein said determination is carried out until the said complex reaches a stationary level.

The terms "BANK1", "PLCg2", "time-dependent formation of a complex", "interaction" and "stationary level" have been detailed in the context of the diagnostic method of the invention and are equally applicable to the third method of the invention.

In the third step, the method for predicting the effectiveness of a treatment administered to a patient suffering an autoimmune disease comprises comparing the value of at least one kinetic parameter of the formation of the BANKl-PLCg2 complex with a reference value for said parameter. The term "kinetic parameter" has been defined in the context of the diagnostic method of the invention and is equally applicable to the third method of the invention.

The term "reference value" as used herein to refer to method for predicting the effectiveness of a treatment administered to a patient suffering an autoimmune disease, refers to the value of at least one kinetic parameter of the formation of a complex comprising BANK1 and PLCg2 in subjects suffering an autoimmune diseases and which are known to respond to a treatment directed to the autoimmune disease. In a preferred embodiment, the kinetic parameter is the concentration of BANKl -PLCg2 complex. In more preferred embodiment said kinetic parameter is carried out by means of PLA or Co-IP.

In a preferred embodiment, the comparison of the concentration of the BANKl - PLCg2 complex with the reference value is determined at the time point wherein the concentration of said complex is highest.

Once at least one kinetic parameter of the formation of a complex comprising BANKl and PLCg2 (e.g. the concentration of BANKl -PLCg2 complex formed at the time point wherein said concentration is highest) has been measured in a sample form a patient suffering an autoimmune disease and compared with the reference value, if said kinetic parameter shows a deviation value with respect to said reference value it can be conclude that said treatment is ineffective. The term "deviation value" applied to the kinetic parameter of the formation of the complex comprising BANKl and PLCg2 has been previously defined.

Methods for monitoring the progression of an autoimmune disease in a patient

Alternatively, since the intracellular levels of the BANKl PLCg2 complex in B cells in response to stimulation of the B cells is an indicative of the presence of an autoimmune disease, it is also possible that in those patients which are being treated and wherein the disease is remitting, the levels of the complex should also reach levels similar to those observed in a reference sample. Thus, in another aspect, the invention relates to an in vitro method (hereinafter, fourth method of the invention) for monitoring the progression of an autoimmune disease in a patient which comprises:

(ϋ) determining the time-dependent formation of a complex comprising BANKl and PLCg2 in the B lymphocytes present in the sample in response to said activation, wherein said determination is carried out until the interaction between said proteins reaches a stationary level; (iii) determining the variation of the value of at least one kinetic parameter of the formation of said complex with a reference value for said variation,

wherein, a deviation value of said at least one kinetic parameter in comparison with the variation of the same parameter at an earlier point of the disease in the patient is indicative that the autoimmune disease shows a bad progression.

The term "monitoring the progression of an autoimmune disease", as used herein, relates to the determination of one or several parameters indicating the progression of the disease in a patient diagnosed with autoimmune disease. Parameters suitable for determining the evolution of a subject diagnosed with autoimmune disease are selected from the group of risk of relapse, disease-free survival and/or overall survival of the subject. As used herein, the expression "risk of relapse" is understood as the probability of a subject developing an autoimmune disease or symptoms of the same again after a disease-free period; "disease-free survival" is understood as the time period after the treatment in which the autoimmune disease is not found; and "overall survival of the subject" is understood as the percentage of subjects who survive, from the time of the diagnosis or treatment, after a defined time period.

According to this inventive aspect, the kinetic parameter (or kinetic parameters) of the formation of the complex comprising BANK1 and PLCg2 determined in the sample from a subject having an autoimmune disease obtained at a first time (first sample) and the kinetic parameter (or kinetic parameters) of the formation of the complex comprising BANK1 and PLCg2 determined in the sample from the subject at a second period of time (second subject sample) are compared allowing the progression of said autoimmune disease in said subject having an autoimmune disease to be monitoring. The second subjects sample can be taken at any time after the first period of time, e.g., one day, one week, one month, two months, three months, 1 year, 2 years, or more after the first subject sample. In a particular embodiment, the first sample is taken prior to the subject receiving treatment, e.g. immunosuppressant, and the second sample is taken after treatment. In another particular embodiment, the first sample is taken after the subject has started/received treatment, e.g. immunosuppressant and the second sample is taken later, at different time periods during a course of treatment. These methods allow for the evaluation of the progression of the autoimmune disease in a selected subject previously diagnosed as suffering from said autoimmune disease. Consequently, if the autoimmune disease has a bad prognosis, a further therapy should be designed to treat said autoimmune disease in said subject. The progression of the autoimmune disease after said new treatment can be easily followed according to the teachings of this invention.

In a preferred embodiment, said autoimmune disease is selected from the group consisting of: rheumatoid arthritis, Behcet disease, amyotrophic lateral sclerosis, multiple sclerosis and Devic disease, spondyloartheopathy, fibromyalgia, rheumatic fever, Wegener granulomatosis, systemic lupus erythematosus, Antiphospho lipids syndrome or Huges syndrome, polymyositis, dermamyositis, chronic inflammatory demyelinating polyradiculoneuropathy, psoriasis, immune thrombocytopenic purpura , sarcoidosis, chronic fatigue syndrome, Guillain-Barre syndrome, systemic vasculitis or vitiligo. In a still more preferred embodiment, the autoimmune disease is systemic lupus erythematosus (SLE).

The term "patient" and preferred embodiment thereof has been defined in the context of the diagnostic and prognostic method of the invention and equally applies for the prognostic method of the invention.

As mentioned previously concerning the diagnostic and prognostic method of the invention, the first step of the monitoring method of the invention comprises the activation of the B lymphocytes in a sample isolated from a patient. In a more preferred embodiment, said activation is carried out in a population of isolated lymphocytes. In another yet preferred embodiment said activation is carried out with anti-IgM antibody under conditions suitable for the stimulation of the B lymphocytes in said sample.

The second step of the monitoring method of the invention comprises determining the time-dependent formation of a complex comprising BANK1 and PLCg2 in the B lymphocytes present in the sample in response to the activation, wherein said determination is carried out until the interaction between said proteins reaches a stationary level. The terms "BANK1", "PLCg2", "time-dependent formation of a complex", "interaction" and "stationary level" have been detailed in the context of the diagnostic and prognostic method of the invention and are equally applicable to the monitoring method of the invention. In the third step, the method for monitoring a progression of an autoimmune disease in a patient comprises comparing the value of at least one kinetic parameter of the formation of the BANKl-PLCg2 complex with a reference value for said parameter. The term "kinetic parameter" has been defined in the context of the diagnostic and prognostic method of the invention and is equally applicable to the forth method of the invention.

In a preferred embodiment, the kinetic parameter is the concentration of BANK- PLCg2 complex. In more preferred embodiment, said kinetic parameter is determined by means of PLA or Co-IP. In another yet more preferred embodiment, the comparison of the concentration of the BANKl-PLCg2 complex with the reference value is determined at the time point wherein the concentration of said complex is highest.

Thus, once the comparison of at least one kinetic parameter of the formation of the complex comprising BANK1 and PLCg2 in the subject samples, at different periods of time (first and second samples) has been determined, it is necessary to identify if there is deviation value of said, at least one, kinetic parameter in the second subject sample in comparison with the same kinetic parameter or parameters in the first subject sample. Thus, a deviation value of said at least one kinetic parameter of the formation of the BANKl-PLCg2 complex in the second subject sample with respect to said at least one kinetic parameter in the first subject sample is indicative that the autoimmune disease in the subject under study is in progression (i.e., it has a bad prognosis); thus, the therapy administered to the subject under study should be changed and a new therapy should be designed to treat the autoimmune disease in said subject. The progression of the autoimmune disease in the subject can be easily followed according to this method.

On the contrary, if no increase or decrease, that is to say if said kinetic parameter has the same value in the second subject sample with respect to the same kinetic parameter in the first subject sample is achieved, then the autoimmune disease in the subject under study is remitting. Methods for classifying patients suffering from an autoimmune disease In another aspect, the invention refers to an in vitro method (hereinafter fifth method of the invention) for classifying a patient suffering from an autoimmune disease which comprises:

(ii) determining the time-dependent formation of a complex comprising BANK1 and PLCg2 in the B lymphocytes present in the sample in response to said activation, wherein said determination is carried out until the level of complex reaches stationary level,

The term "classifying a patient" as used herein, refers to the process of attempting to determine or identify of a patient by means of classification of individual^'s condition into separate and distinct categories (or groups) preferably in one of two or more groups of subjects. The classification of said patient may be done by means of any suitable method which allows the identification of said subject (e.g. by the detection of a clinical symptom or a specific clinical parameter) against the rest of the population or against other specific population group (e.g. people with no diagnosis of an autoimmune disease).

In a preferred embodiment, the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, Behcet disease, amyotrophic lateral sclerosis, multiple sclerosis and Devic disease, spondyloarthropathy, fibromyalgia, rheumatic fever, Wegener granulomatosis, systemic lupus erythematosus, Antiphospho lipids syndrome or Huges syndrome, polymyositis, dermamyositis, chronic inflammatory demyelinating polyradiculoneuropathy, psoriasis, immune thrombocytopenic purpura , sarcoidosis, chronic fatigue syndrome, Guillain-Barre syndrome, systemic vasculitis or vitiligo. In a still more preferred embodiment, the autoimmune disease is systemic lupus erythematosus (SLE).

The term "patient" and preferred embodiment thereof has been defined in the context of the diagnostic, prognostic and monitoring method of the invention and equally applies for the prognostic method of the invention.

As mentioned previously concerning the diagnostic, prognostic and monitoring method of the invention, the first step of the method for allocating a patient suffering from an autoimmune disease of the invention comprises the activation of the B lymphocytes in a sample isolated from a patient. In a more preferred embodiment, said activation is carried out in a population of isolated lymphocytes. In another yet preferred embodiment said activation is carried out with anti-IgM antibody under conditions suitable for the stimulation of the B lymphocytes in said sample.

In a preferred embodiment the method for allocating a patient suffering an autoimmune disease of the invention is carried out in a population of isolated lymphocytes. Many different approaches may be used for isolation the of B lymphocytes from blood sample. Said methods are described in the context of previous methods of the invention and are equally applicable to the method for allocating a patient of the invention.

In the second step, the method for allocating a patient suffering an autoimmune disease comprises determining the time-dependent formation of a complex comprising BANK-1 and PLCg2 in the B lymphocytes present in the sample in response to the activation carried out in the first step of said method, wherein said determination is carried out until the said complex reaches a stationary level.

The terms "BANK1", "PLCg2", "time-dependent formation of a complex", "interaction" and "stationary level" have been detailed in the context of the diagnostic, prognostic and monitoring method of the invention and are equally applicable to the third method of the invention.

In the third step, the method for allocating a patient suffering an autoimmune disease comprises comparing the value of at least one kinetic parameter of the formation of the BANKl-PLCg2 complex with a reference value for said parameter. The term "kinetic parameter" has been defined in the context of the diagnostic method of the invention and is equally applicable to the third method of the invention. As used herein, a "reference value" refers to the median or average value of at least one of any kinetic parameter related to BANKl-PLCg2 complex measured in a reference sample, wherein the reference sample can be a sample obtained from a subject which is not suffering or has no history of having suffered an autoimmune disease. Alternatively, the reference value can be the median or average value of any kinetic parameter related to BANKl-PLCg2 complex (defined below) obtained from a collection of samples from control individuals (e.g. people with no diagnosis of autoimmune diseases). In another embodiment, the reference sample is a sample containing B cells of the patient which has not been treated so as to stimulate the B cells.

The term "deviation value" has been defined for the previously detailed methods of the invention and applies equally for the method for allocating a patient suffering from an autoimmune disease.

Thus, once the comparison of at least one kinetic parameter of the formation of the complex comprising BANK1 and PLCg2 in the subject samples has been determined, a subject can be classified in a first group if a deviation in the value of said at least one kinetic parameter with respect to said reference value is detected or classified in a second group if a deviation value of the value of said at least one kinetic parameter with respect to said reference value is not detected. In the context of the present invention, the first group refers to said group of patients with a deviation value of at least one kinetic parameter of the formation of the BANKl-PLCg2 complex. Thus, a patient classified into the first group is susceptible of being suffering an autoimmune disease. In a preferred embodiment, a patient classified in the first group would be then susceptible of being diagnosed with an autoimmune disease (e.g. diagnosed with SLE) according to the diagnostic method of the invention. In another preferred embodiment, a patient classified in the first group would be then susceptible of being evaluated according to the method for predicting the effectiveness of a treatment of the invention. In another preferred embodiment, a patient classified in the first group would be susceptible to be monitored according to the monitoring method of the invention.

Conversely, the second t group refers to said group of patients without a detectable deviation in the value of at least one kinetic parameter of the formation of the BANKl -PLCg2 complex. Thus, a patient classified into the second group is unlikely of suffering an autoimmune disease. In a preferred embodiment, a patient classified in the second group would not be susceptible of being diagnosed with an autoimmune disease (e.g. diagnosed with SLE) according to the diagnostic method of the invention. In another preferred embodiment, a patient classified in the second group would not be susceptible of being evaluated according to the method for predicting the effectiveness of a treatment of the invention. In another preferred embodiment, a patient classified in the second group would be susceptible to be monitored according to the monitoring method of the invention. Methods for the treatment of autoimmune diseases and personalized therapeutic methods

The authors of the present invention have found that activated B cells show increased concentration of the complex comprising BANKl -PLCg2 and that the interaction between BANKl and PLCg2 to form BANKl -PLCg2 protein complex can be modulated by either BLK or by inactive forms of BANKl which are not longer able to interaction with PLCg2. Thus, in another aspect, the invention relates to an agent selected from the group consisting of a BLK inhibitory agent and a BANKl inhibitory agent for use in the treatment of an autoimmune disease.

In another aspect, the invention relates to the use of an agent selected from the group consisting of a BLK inhibitory agent and a BANKl inhibitory agent for the manufacture of a medicament for the treatment of an autoimmune disease.

In another aspect, the invention relates to a method for the treatment of an autoimmune disease in a subject in need thereof comprising the administration to said subject an agent selected from the group consisting of a BLK inhibitory agent and a BANKl inhibitory agent.

BLK inhibitors The term "BLK inhibitory agent" as used herein, refers to any molecule capable of completely or partially inhibiting the expression of the gene encoding BLK, both by preventing the expression product of said gene form being produced (interrupting the BLK gene transcription and/or blocking the translation of the mRNA coming from the BLK gene expression) or by directly inhibiting the BLK protein activity.

As used herein the term "B Lymphocyte Kinase (BLK)" refers to a kinase of the Src family tyrosine kinases that includes also the related proteins SRC, LYN, FYN, YES, HCK, FGR and LCK and which are characterized by a modular structure composed of a N-terminal region with attachment sites for fatty acid modifications, a unique region, a Src-homology 3 (SH3) domain, a Src-homology 2 (SH2) domain, a tyrosine kinase domain and a C-terminal kinase inhibitory domain. The term BLK includes variants, isoforms and species orthologs of human BLK. BLK kinase activity allows the phosphorylation of BANK1. The complete cDNA sequence for human BLK has the GenBank accession number NM 001715.2 (November, 2012). The complete protein sequence for human BLK can be found in the Uniprot database under accession number P51451 (August 21, 2007).

Methods suitable for determining whether an inhibitor is capable of inhibiting the expression of the gene encoding BLK are well-known in the art and include, without limitation, methods for determining the levels of the BLK mRNA (e.g. RT-PCR, Northern blot and the like), which can be used for the determination of inhibitors acting at the transcriptional level, methods for determining the levels of the BLK polypeptide (ELISA, RIA, Western blot, and the like), which can be used for determination of inhibitors acting at a translational or transcriptional level and methods for determining BLK activity, which can be used for determining inhibitors acting directly on the kinase activity of BLK. Methods for determining whether an inhibitor acts by inhibiting the kinase activity of BLK are known by the person skilled in the art. By way of illustration, the inhibitor can be identified by determining its ability to block the phsophorylation of BANK1 by BLK using the assays shown in the examples of the present invention (see examples 3 and 5). The kinase activity and the inhibition thereof can be detected by any suitable method, for example, by means of radio/chemical/photochemical bond of a phosphate and the subsequent detection of its incorporation into the substrate. Other BLK activation assays may be used for determining whether an agent is a BLK inhibitor, such as "Pull-down" method or an ELISA-based method, where the BLK activation is measured by means of luminescence. ELISA-based techniques consist of incubating the test in plate having a phosphorylation domain of a BLK effector protein. The active form of BLK will bind to said domain and can subsequently be detected using a BLK-specific antibody. Other assays to measure the modulation of kinases are known by the persons skilled in the art such as those described in Julianne J. Sando (Protein Kinase C Protocols. 2003 Humana Press) and WO/2004/035811.

In a particular embodiment, the BLK inhibitory agent is selected from the group consisting of a BLK specific interfering R A, a BLK specific antisense oligonucleotide, a BLK specific ribozyme, a BLK specific inhibitory antibody and an inactive variant of BLK.

In one embodiment, the BLK inhibitory agent is a BLK-specific antisense oligonucleotide. Antisense oligonucleotides usually can bind to their potential target by means of conventional base complementarity, or, for example, in the case of binding to double-stranded DNA, through specific interactions in the major groove of the double helix. The antisense oligonucleotide can be provided, for example, as an expression plasmid which, when transcribed into the cell, produces RNA that is complementary to at least one unique part of the cellular mRNA encoding the protein of interest, e.g. BLK. Alternatively, the antisense construct is an oligonucleotide probe generated ex vivo and which, when introduced into the cell, causes inhibition of gene expression by hybridizing with the mRNA and/or genomic sequences of the target nucleic acid. Those oligonucleotide probes are preferably oligonucleotides which are resistant to endogenous nucleases, for example exonucleases and/or endonucleases, and are therefore stable in vivo. Illustrative nucleic acid molecules for use as antisense oligonucleotides include DNA analogs of phosphoramidate, phosphothionate and methylphosphonate (see, for example, US5176996, US5264564 and US5256775). Additionally, for a review of general approaches for constructing oligomers useful in antisense therapy, see, for example, Van der Krol et ah, BioTechniques 6: 958-976, 1988; and Stein et al, Cancer Res 48: 2659-2668, 1988.

Regarding the antisense oligonucleotide, the oligodeoxyribo nucleotide regions derived from the translation start site, for example, between -10 and +10 of the target gene, are preferred. Antisense approaches involve designing oligonucleotides (either DNA or RNA) complementary to the mRNA encoding the target polypeptide. Antisense oligonucleotides will bind to mRNA transcripts and prevent translation.

Oligonucleotides complementary to either the 5' or 3' untranslated, non- encoding regions of a gene could also be used in an antisense approach to inhibit translation of that mRNA. The oligonucleotides complementary to the 5' untranslated region of the mRNA must include the complement of the AUG start codon. The oligonucleotides complementary to the coding regions of the mRNA are less effective translation inhibitors but could also be used according to the invention. If they are designed to hybridize with the 5' or 3' or coding region of the mRNA, the antisense nucleic acids must be at least 6 nucleotides long and preferably less than about 100, and more preferably less than about 50, 25, 17 or 10 nucleotides long.

The antisense oligonucleotides can be single- stranded or double stranded DNA, RNA or chimeric mixtures or derivatives or modified versions thereof. The oligonucleotide can be modified at the base, in the sugar or in the phosphate backbone, for example, to improve the stability of the molecule, its hybridization capacity, etc. The oligonucleotide can include other groups bound thereto, such as peptides (for example to direct them to host cell receptors) or agents to facilitate transport across the cell membrane (Letsinger et ah, Proc. Natl. Acad. Sci. USA 86: 6553-6556, 1989; Lemaitre et al, Proc. Natl. Acad. Sci. 84: 648-652, 1987; WO88/09810) or the blood-brain barrier (WO89/10134), or intercalating agents (Zon, Pharm. Res. 1988. 5: 539-549). For this purpose, the oligonucleotide can be conjugated to another molecule, for example, a peptide, a carrier agent, a hybridization-triggered cleavage agent, etc.

In another particular embodiment, the BLK inhibitory agent is a BLK-specific interfering RNA (iRNA). The term "iRNA", as used herein, refers to a means of selective post- transcriptional gene silencing by destruction of specific mRNA by molecules that bind and inhibit the processing of mRNA, for example inhibit mRNA translation or result in mRNA degradation.

In a preferred embodiment, the iRNA is a shRNA" or "small hairpin RNA". In one embodiment, these shRNAs are composed of a short, e.g. about 19 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand can precede the nucleotide loop structure and the antisense strand can follow.

In another embodiment, the iR A is a siRNA, which is a nucleic acid that forms a double stranded RNA, which has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is present or expressed in the same cell as the target gene, sEH. The double stranded RNA siRNA can be formed by the complementary strands. In one embodiment, a siRNA refers to a nucleic acid that can form a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 19-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

In a particular embodiment, the siRNAs that can be used in the present invention are substantially homologous to the mRNA of the gene encoding the protein of interest (e.g. BLK) or to the genomic sequence encoding said protein. "Substantially homologous" is understood as having a sequence that is sufficiently complementary or similar to the target mRNA, such that the siRNA is able to cause degradation thereof by RNA interference. The siRNAs suitable for causing said interference include siRNAs formed by RNA and siRNAs containing different chemical modifications such as:

- siRNA in which the bonds between the nucleotides are different from those found in nature, such as phosphorothioate bonds;

- conjugates of the RNA chain with a functional reagent, such as a fluorophore;

- modifications to the ends of the RNA chains, particularly the 3' end by means of modifying hydroxyl at position 2' with different functional groups;

- nucleotides with modified sugars such as O-alkylated moieties at position 2', such as 2'-0-methylribose or 2'-0-fluororibose;

- nucleotides with modified bases such as halogenated bases (for example 5- bromouracil and 5-iodouracil), alkylated bases (for example 7- methylguanosine) .

The siRNAs and shRNAs that can be used in the present invention can be obtained using a series of techniques known by the person skilled in the art. The region of the nucleotide sequence encoding BLK used as a basis for designing the siRNAs is not limiting and can contain a region of the coding sequence (between the start codon and stop codon), or it can alternatively contain sequences of the 5' or 3' untranslated region, preferably between 25 and 50 nucleotides in length and at any position 3' sense position with respect to the start codon. One way to design a siR A involves identifying the AA(N19)TT motifs, where N can be any nucleotide in the sequence encoding the protein of interest, and selecting those having a high G/C content. If the motifs are not found, it is possible to identify the NA (N21) motive, where N can be any nucleotide. In a preferred embodiment, lentiviral particles which coding BLK specific siRNA, are used for the treatment of an autoimmune disease. Transduction of cells with lentiviral particles coding siRNA are well known in the state of the art.

In another particular embodiment, the BLK inhibitory agent is a BLK-specific microRNA (miRNA), which is either a naturally-occurring miRNA or an artificial miRNA. miRNAs are single-stranded RNAs having a length of between 21 and 25 nucleotides, and it has the capacity to regulate the expression of other genes by means of different processes, using the ribo interference pathway to that end.

In another embodiment, the BLK inhibitory agent is a BLK-specific ribozyme. Riboyzmes are RNA molecules designed to catalytically cleave target mRNA transcripts, thereby preventing translation of the mRNAs into the corresponding polypeptide. Suitable ribozymes include hammerhead ribozymes, RNA endoribonucleases, and the like. Ribozymes can be made up of modified oligonucleotides (for example to improve stability, direction, etc.) and they must be distributed in vivo to cells expressing the target gene.

In another particular embodiment, the BLK inhibitor is a BLK inhibitory antibody. As used herein "inhibitor antibody" refers to any antibody that is capable of specifically binding to BLK and inhibiting one or more functions of said protein, preferably the kinase activity. Suitable inhibitory antibodies include antibodies as well as fragments thereof such as Fab, F(ab')2, Fab', single chain Fv fragments (scFv), diabodies and nanobodies. The antibodies can be obtained using any methods known by the person skilled in the art.

In another embodiment, the BLK inhibitory agent is a dominant negative inactive BLK variants. This are variants which, when introduced into a cell, competes with native BLK leading to a decrease in BANK1 phosphorylation and consequently, to a decrease in the amount of BANKl -PLCg2 complex.

The term "inactive variant", as used herein, refers to a modified form of BLK which lacks partially or completely one or more of the activities of the active variant. Inactive variants according to the invention show less than 95%, less than 90%>, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%), less than 30%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% of the activity of the wild-type variant or undetectable activity using the any of the assays mentioned above for detecting the BLK activity.

In one embodiment, the inactive BLK variant contains a mutation at the lysine at position 269 with respect to the sequence of human BLK (UniProt accession number P51451). Preferably, the lysine residue is replaced by a hydrophobic residue and, more preferably, by a leucine residue. In another embodiment, the inactive BLK variant is a variant lacking the myristoylation site (mutation of the Gly residue at position 2, preferably a glycine to valine substitution) or containing an additional palmitoylation site (e.g. by including an amino acid substitution at the position 3).

BANKl inhibitory agent

In another embodiment, the agent used in the therapeutics methods according to the invention is a BANKl inhibitory agent.

The term "BANKl inhibitory agent" as used herein, refers to any molecule capable of completely or partially inhibiting the expression of the gene encoding BANKl, either by preventing the expression product of said gene form being produced (interrupting the BANKl gene transcription and/or blocking the translation of the mRNA coming from the BANKl gene expression) or by directly inhibiting the BANKl protein activity.

The term "BANKl" has been defined above and is equally applicable to the therapeutic methods.

Methods suitable for determining whether an inhibitor is capable of inhibiting the expression of the gene encoding BANKl are well-known in the art and have been described above in the context of the BLK- inhibitory agents. Methods for determining whether an inhibitor acts by inhibiting BANKl can be identified by determining its ability to prevent interaction of BANKl with PLC2g in B cells upon stimulation of the B-cell receptor (see example 5 of the present invention).

In a particular embodiment, the BLK inhibitory agent is selected from the group consisting of a BANKl specific interfering RNA, a BANKl specific antisense oligonucleotide, a BANKl specific ribozyme, a BANKl specific inhibitory antibody and an inactive variant of BANKl .

The terms "iRNA", "antisense oligonucleotide", "ribozyme", "inhibitory antibody" have been described in the context of the BLK inhibitors and are equally applicable to the BANKl inhibitors.

In another embodiment, the BANKl inhibitor is an inactive BANKl variant. As used herein "inactive BANKl variant" refers to a BANKl variant into which a mutation such a deletion, substitution, addition, insertion or the like of amino acids has been introduced, and has reduced or deleted activity comparing to active BANK. l In a preferred embodiment, said activity refers to the BANKl capacity to interact with PLCg2. An inactive variant of BANKl may be a naturally existing or may be a variant into which a mutation has been artificially introduced. Techniques for introducing such mutation are well known in the art.

The term "inactive variant", as used herein, refers to a mutated form of BANKlwhich lacks partially or completely one or more of the activities of the active variant. Inactive variants according to the invention show less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%), less than 30%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% of the activity of the wild-type variant or undetectable activity using the any of the assays mentioned above for detecting the BANKl activity.

In a preferred embodiment, an inactive BANKl variant carries a mutation at the tyrosine at position 484 with respect to the sequence of the human BANKl isoform 1 (Accession number NP 060405.4 in the UniProt database). Preferably the tyrosine at position 484 is replaced by an hydrophobic residue. More preferably, the variant is a Y484F.

In a preferred embodiment, the inactive BANKl variant carries a mutation at the tyrosine at position 488 with respect to the sequence of the human BANK1 isoform 1 (Accession number NP 060405.4 in the UniProt database). Preferably the tyrosine at position 488 is replaced by an hydrophobic residue. More preferably, the variant is a Y488F.

In a preferred embodiment, an inactive BANK1 variant carries mutations at the tyrosines at positions 484 and 488 with respect to the sequence of the human BANK1 isoform 1 (Accession number NP 060405.4 in the UniProt database). Preferably the tyrosine at positions 484 and 488 are replaced by hydrophobic residues. More preferably, the variant is a Y484F and Y488F mutant.

In a preferred embodiment, the agent for use according to the therapeutic method of the invention is used for the treatment of an autoimmune disease is selected from the group consisting of: rheumatoid arthritis, Behcet disease, amyotrophic lateral sclerosis, multiple sclerosis and Devic disease, spondyloartheopathy, fibromyalgia, rheumatic fever, Wegener granulomatosis, systemic lupus erythematosus, Antiphospho lipids syndrome or Huges syndrome, polymyositis, dermamyositis, chronic inflammatory demyelinating polyradiculoneuropathy, psoriasis, immune thrombocytopenic purpura , sarcoidosis, chronic fatigue syndrome, Guillain-Barre syndrome, systemic vasculitis or vitiligo. In a more preferred embodiment, the inhibitory agents of the invention are used in the treatment of SLE.

In another aspect, the invention relates an inhibitory agent selected from the group consisting of a BLK inhibitory agent and a BANK1 inhibitory agent for use in the treatment of a subject suffering an autoimmune disease, wherein said subject is classified in the first group according to the method for classifying patients according to the invention.

Inactive BLK variants

In one embodiment, the inactive BLK variant contains a mutation at the lysine at position 269 with respect to the sequence of human BLK (UniProt accession number P51451). Preferably, the lysine residue is replaced by a hydrophobic residue and, more preferably, by a leucine residue. In another embodiment, the inactive BLK variant is a variant lacking the myristoylation site (mutation of the Gly residue at position 2, preferably a glycine to valine substitution). In another embodiment, the inactive BLK variant contains an additional palmitoylation site (e.g. by including an amino acid substitution at the position 3).

Inactive BANK1 variants

In another embodiment, the invention relates to an inactive BANK1 variant which carries a mutation at position 484 and/or a mutation at position 488 with respect to human BANK1 isoform 1.

In a preferred embodiment, an inactive BANK1 variant carries a mutation at the tyrosine at position 484 with respect to the sequence of the human BANK1 isoform 1 (Accession number NP 060405.4 in the UniProt database). Preferably the tyrosine at position 484 is replaced by an hydrophobic residue. More preferably, the variant is a Y484F.

In a preferred embodiment, the inactive BANK1 variant carries a mutation at the tyrosine at position 488 with respect to the sequence of the human BANK1 isoform 1 (Accession number NP 060405.4 in the UniProt database). Preferably the tyrosine at position 488 is replaced by an hydrophobic residue. More preferably, the variant is a Y488F.

In a preferred embodiment, an inactive BANK1 variant carries mutations at tyrosines at positions 484 and 488 with respect to the sequence of the human BANK1 isoform 1 (Accession number NP 060405.4 in the UniProt database). Preferably the tyrosine at positions 484 and 488 are replaced by hydrophobic residues. More preferably, the variant is a Y484F and Y488F mutant.

Compositions of the invention

In another aspect, the invention relates to a composition comprising a BLK inhibitory agent of the invention or a BANK1 inhibitory agent of the invention.

The terms "BLK inhibitory agent" and "BANK1 inhibitory agent" have been defined in the context of the methods for the treatment of autoimmune diseases and are equally applicable for the compositions of the invention.

In a preferred embodiment the composition of the invention comprises an inactive BLK variant selected form the group consisting of a BLK variant carrying a mutation at position 269 with respect to the human BLK, a BLK variant lacking the myristoylation site and a BLK variant containing an additional palmitoylation site.

In a preferred embodiment, the composition of the invention comprises an inactive BLK variant which carries a mutation at position 484 and/or a mutation at position 488 with respect to human BANK1 isoform 1. Pharmaceutical compositions of the invention

In another aspect, the invention relates to a pharmaceutical composition comprising a BLK inhibitory agent of the invention or a BANK1 inhibitory agent of the invention, and a pharmaceutically acceptable carrier.

In a preferred embodiment the composition of the invention comprises an inactive BLK variant selected form the group consisting of a BLK variant carrying a mutation at position 269 with respect to the human BLK, a BLK variant lacking the myristoylation site and a BLK variant containing an additional palmitoylation site, and a pharmaceutically acceptable carrier.

In a preferred embodiment, the composition of the invention comprises an inactive BLK variant which carries a mutation at position 484 and/or a mutation at position 488 with respect to human BANK1 isoform 1, and a pharmaceutically acceptable carrier.

Appropriate amounts of the inhibitory agents of the invention can be formulated with pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, or European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. The composition, if desired, can also contain minor amounts of pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a prophylactically or therapeutically effective amount of a corticospinal upper motor neuron or a cell population of corticospinal upper motor neurons of the invention preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration. In a preferred embodiment, the pharmaceutical compositions are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

The pharmaceutical composition of the invention may be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as lyophilized preparations, liquids solutions or suspensions, injectable and infusible solutions, etc. The preferred form depends on the intended mode of administration and therapeutic application.

The pharmaceutical composition of the invention can be delivered to a subject by a variety of routes. Exemplary routes include intrastriatal, intracerebroventricular, intrathecal, intraparenchymal (e.g., in the striatum), intranasal, and ocular delivery. The composition can also be delivered systemically, e.g., by intravenous, subcutaneous or intramuscular injection, which is particularly useful for delivery of the conjugates to peripheral neurons. Additionally, it is also possible to administer the conjugates of the invention intranasally which allows systemic administration by a non-aggressive mode of administration. Also, intraventricular administration may also be adequate. A preferred route of delivery is directly to the brain, e.g., into the ventricles or the hypothalamus of the brain, or into the lateral or dorsal areas of the brain. Those of skill in the art are familiar with the principles and procedures discussed in widely known and available sources as Remington's Pharmaceutical Science (17th Ed., Mack Publishing Co., Easton, Pa., 1985) and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics (8th Ed., Pergamon Press, Elmsford, N.Y., 1990).

Protein complex comprising BAN 1 and PLCg2 The authors of the present invention have surprisingly found that PLCg2 can be found in association with BANKl both in vivo and in vitro. Thus, in another aspect, the invention relates to a protein complex comprising BANKl and PLCg2. The terms "BANKl" and "PLCg2" and "protein complex" have been detailed above and are equally applicable to this aspect of the invention. It will be understood that the complex according to the invention may comprise exclusively one or more copies of each BANKl and PLCg2. Alternatively, the complex may comprise further components associated to the complex. In some embodiments, the complex contains the same number of molecules of BANKl and PLCg2, i.e. the stoichiometry is 1 : 1. In further embodiments, the stoichiometry of the BANKl/PLCg2 complex is of 100: 1, 50: 1 , 40: 1, 30: 1, 20: 1, 10: 1, 5: 1, 4: 1, 3:1, 2: 1, 1 : 1, 1;2, 1 :3, 1 :4, 1 :5, 1 :10. 1 :20, 1 :30, 1 :40, 1 :50 or 1 : 100.

In a preferred embodiment, the complex comprising BANKl and PLCg2 contains a phosphorylated BANKl . As the person skilled in the art will understand, the process of phosphorylation refers to the addition of a phosphate (P(V³) group to a protein (e.g. BANKl) or other organic molecule. In those complexes containing more than one molecule of BANKl, in some embodiments all molecules are phosphorylated, in some embodiments all molecules are non-phosphorylated and in other embodiments, some of the molecules are phosphorylated and some molecules are not phosphorylated. Phosphorylation may occur at one or more serine or tyrosine sites within the BANKl sequence. By way of example, BANKl may be phosphorylated at one or more sites selected from the group consisting of serine 66, serine 148, at serine 479, at serine 480, at tyrosine 484, at tyrosine 488, at tyrosine 588, at tyrosine 630, at serine 644 and tyrosine 737, wherein the numbering of the positions is defined according to the human BANKl sequence as defined above.

The invention is detailed below by means of the following examples which are merely illustrative and by no means limiting for the scope of the invention.

EXAMPLES METHODS

Cloning, expression vectors and mutagenesis

Human BANK1 cDNA was PCR-amplified from human peripheral blood mononuclear cells and cloned into the expression vectors pcDNA3.1D/V5-His- TOPO (Invitrogen, Boston, MA, USA) and pires2-EGFP (Clontech, Palo Alto, CA). The coding sequences of BLK, ATG4b and CD 163 were amplified from BJAB cells' cDNA and cloned into pcDNA3.1D/V5-His-TOPO (Invitrogen). Fluorescent fusion proteins were added in frame at the C-terminal using the cloning sites Notl/Xbal. BANK1 and BLK mutants were generated by site-directed mutagenesis. The constitutively active form of BLK (BLK-YF) has a substitution of a tyrosine residue to phenylalanine in the C-terminal regulatory domain (Y501F), the kinase dead form (BLK-KL) was generated by the K269L substitution in the catalytic site. PLCg2 and LYN were amplified from the I.M.A.G.E. full length cDNA clone IRAUp969G0437D and pME-LYN (Yokoyama et el 2002), respectively, and cloned into pcDNA3.1D/V5-His-TOPO (Invitrogen). All clones were confirmed by sequencing.

Cell culture and transfections

Daudi and embryonic kidney HEK293T cells were each maintained in RPMI

1640 medium and Dulbecco's modified Eagle's medium containing Glutamax (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). HEK293T cells (3* 10⁶) were transiently transfected with 20 uL Lipofectamine 2000 (Invitrogen) and 8 ug of each DNA vector following the manufacturer instructions. The analysis of cells was performed 48 hours after transfections.

Antibodies

The antibodies used for immunoprecipitation and western blot were: Mouse anti-V5 (Invitrogen), mouse anti-Phospho-Tyrosine #9411 (Cell signaling, Beverly, MA ), mouse anti-PLCg2 ab89625 (Abeam, Cambridge, UK), rabbit anti-BANKl- ET52, rabbit anti-BANKl HPA037002 (Sigma, St. Louis, Mo, USA), mouse anti- BLK H00000640-M02 (Abnova, Heidelberg, Germany) and chicken anti-GAPDH SAB3500247 (Sigma), anti-rabbit and anti-mouse-HRP (Zymed,, San Francisco, CA, USA) anti-Chicken IgY-HRP (Sigma).

Co-immunoprecipitation and Western blot analysis

For immunoprecipitation, HEK293T transfected cells (10* 10⁶) or Daudi cells

(3* 10⁷) were solubilized in NP-40 lysis buffer containing 1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM Na3V04, 1 mM PMSF and protease and phosphatase inhibitor cocktails (Roche) for 10 minutes on ice and centrifuged at 20000g for 10 minutes at 4°C. An aliquot of each lysate was saved for input analysis and the remaining lysates were immunoprecipitated with 3 ug of anti-PLCG2 (Abeam) previously bound to 50 uL protein G Dynabeads (Invitrogen) for 3 hours at 4°C with rotation. Dynabeads-Ab-Ag complexes were washed 3 times with ice cold Dulbecco's phosphate-buffered saline (DPBS) including proteases and phosphatase inhibitors and eluted in 30 uL elution buffer containing NuPAGE LDS Sample Buffer lx and NuPAGE reducing agent lx (Invitrogen) by heating at 70°C for 10 minutes. Lysates and immunoprecipitates were separated by 4-15% gradient SDS- PAGE gels (BioRad, Barcelona, Spain), transferred to PVDF membranes (Biorad) and detected with the appropriate antibodies on a ECL system. Cell stimulation and silencing

For stimulation, Daudi cells were washed with DPBS and changed to RPMI1640 medium without FBS two hours before addition of the stimulus. Cells were resuspended and stimulated in Opti-MEM I medium (Invitrogen) with 10 ug/mL goat F(ab')2 anti-human IgM (Southernbiotech, Birmingham, Alabama, USA) at 37°C for the indicated times. The cells were transferred to ice to stop the stimulation. Before co-immunoprecipitation, the cells were washed with ice-cold DPBS and lysed with NP-40 lysis buffer. For silencing, Daudi cells were transduced with Blk shRNA Lentiviral Particles (cat no. sc-39227-V) or control scrambled shRNA Lentiviral Particles (cat no. sc- 108080, Santa Cruz Biotechnology Santa Cruz, CA, USA) following the manufacturer instructions.

Microscopy Cells were grown and transfected on Lab-Tek chamber slides coated with poly-D-lysine (Beckton Dickinson, Oxford, UK). Twenty-four hours after transfection cells were fixed at room temperature for 20 minutes with 3.7% paraformaldehyde in a buffer containing PBS with 0.18% Triton-X. Fluorescent fusion proteins were visualized directly after fixation, FX enhancer treatment (Invitrogen) and mounted with Vectashield (Vector Lab. Peterborough, UK) or SlowFade Gold Antifade Reagent (Invitrogen) containing DAPI. Confocal microscopy was performed using a Zeiss 510 Meta confocal scanning microscope with a Zeiss plan-Apochromat 63x oil-immersion objective (Zeiss, Stockholm, Sweden). Dual- or triple-color images were acquired by consecutive scanning with only 1 laser line active per scan to avoid cross-excitation. Image analysis was carried out using ImageJ software.

In situ proximity ligation assay (PLA)

Daudi cells were seeded into an 8-well culture slide coated with polylysine.

Cells were grown for 4 hours and stimulated with 10 ug/mL goat F(ab')2 anti-human IgM (Southernbiotech) diluted in Opti-MEM I medium at 37°C for the indicated times. Stimulation was stopped by fixation of cells with paraformaldehyde solution at 4% final concentration. Slides were incubated for 20 minutes at RT, washed with PBS-Tween 0.05% and permeabilized with methanol: acetone (1 : 1) for 10 minutes at -20°C. After permeabilization, cells were washed twice with PBS-Tween 0.05% and residual liquid dried at RT.

Proximity ligation assay was done with the Duolink II kit according to the manufacturer^'s protocol (Olink Bioscience, Uppsala, Sweden)). Briefly, slides were incubated with blocking solution in a pre-heated humidity chamber for 30 minutes at 37°C. Cells were incubated with rabbit anti-human BANK1 ET52 (Castillejo-Lopez et al, 2012, Ann Rheum Dis 71 : 136-142), alternatively rabbit anti-human BANK1 HPA037002 (Sigma) together with mouse anti-human PLCG2 antibody (ab89625, Abeam) overnight in a humidity chamber at 4°C. After incubation, slides were washed twice and incubated with mouse minus and rabbit plus PLA probes for lh at 37°C. Ligation was carried out for 30 minutes at 37°C and amplification for 100 min at 37°C. Finally, slides were washed, dried at RT in the dark and mounted with SlowFade Gold Antifade Reagent (Invitrogen) containing DAPI for nuclei staining.

The images were taken with a confocal microscope and the quantification was done using the free software BlobFinder (Centre for Image Analysis, Uppsala University, Uppsala, Sweden) (for PC) or alternatively using our own developed plug-in for ImageJ.

Y2H screen

Two independent screens were performed as a service by Hybrigenics S.A. (Paris, France). The first one was done using as a bait the human full-length BANKl (amino acids 1-785), and the second one using a truncate form of BANKl (amino acids 331-785) In both screens, the bait constructs were transformed with a Human leukocyte and mononuclear cell library (Hybrigenics). A total of 6x10⁶ interactions were tested in each screen. EXAMPLE 1: Ectopically expressed BANKl co-localizes with Phospholipase C- gamma 2 (PLCg2)

Co-expression of BANKl and PLCg2 showed perfect co-localization while CD 163 and ATG4b show only partial co- localization with BANKl . BANKl is a cytoplasmic protein that when ectopically expressed shows a variable pattern of expression. BANKl distributes homogeneously through the cytoplasm and under certain circumstances concentrates in punctate structures. Cells showing an evenly distributed cytoplasmic pattern of BANKl do present an equally distributed PLCg2. Likewise, in cells where BANKl showed punctate structures, PLCg2 co-localized with the majority of the dots. The expression of CD 163 shows a cytoplasmic distribution with a reticulate and punctate pattern that partially co-localizes with BANKl . In our experiments, the sub-cellular location of CD 163 is identical to the distribution of the endogenous CD 163 protein (Schaer et al, 2006, Blood 107: 373- 380), suggesting the expression system and the addition of the fluorescent tag did not interfere in the localization of the exogenously expressed proteins. At this point, we concluded that there were no obvious sub-cellular constrains for the interaction between BANKl and PLCg2 and both proteins shared cellular compartments. EXAMPLE 2: BANK1-PLCG2 complex formation is transient and induced by IgM stimulation.

In order to validate the physical interaction between BANK1 and PLCg2 proteins, co-immunoprecipitation with the ectopically expressed fusion proteins was performed. The co-inmunoprecipitation of BANKl-PLCg2 using extracts of HEK293 cells expressing both proteins was not observed.

The detection of interaction between said proteins was then carried out using an in situ Proximity Ligation Assay (PL A). Single interacting protein events are visible as bright dots when viewed with a fluorescence microscope. Next, the kinetic of BANKl-PLCg2 PLA interaction was determined in Daudi and follicular lymphoma derived RL B-cell lines upon IgM stimulation. In non-stimulated cells only a few interactions were detected but still above the noise level obtained in the negative control HEK cells. After stimulation with anti-lgM the interaction of the two proteins increased dramatically indicating that both proteins translocated to close proximity in response to anti-lgM stimulation. Both cells lines showed similar kinetics, increasing the interaction at one minute after stimulation and returning to initial levels after twenty minutes (Fig. 1A).

The stimulation with IgM was tested in order to see if it leads to the translocation of the BANK1-PLCG2 complex away from their perinuclear location. Using co -localization coefficients between the PLA signal and the DAPI staining the localization of the BANK1-PLCG2 interactions were quantified. In non-stimulated cells (0 min), the PLA signals are mostly localized close to the nucleus, resulting in a merged image with yellow spots, shown by arrowheads. After one minute of IgM stimulation the PLA signals lose their perinuclear location appearing as red spots in the merge image. The translocation of the PLA signal was quantified in two independent experiments (Fig. IB). In non-stimulated cells, the few BANK1-PLCG2 interactions are close to the nuclei, they lose the perinuclear localization at one minute of stimulation and at 20 minutes return to a position close to the nuclei.

The dynamics of the interactions upon BCR cross-linking was further addressed using conventional immunoprecipitation methods. Fig. 1C shows that in resting B-cells the interaction between BANK1 and PLCg2 is negligible while an evident immunoprecipitate was obtained upon stimulation. The reverse immunoprecipitation using BANK1 antibody produced similar results (Fig. 1C). Based on these data it can be concluded that BANKl -PLCg2 interaction is transient and inducible upon BCR stimulation.

EXAMPLE 3: The kinase activity and the lipidation of BLK contribute to the BANKl-PLCg2 interaction

To test directly that tyrosine phosphorylation of the adaptor protein enhances the BANKl -PLCG2 interaction, the interacting proteins, BANKl and PLCg2 were coexpresed with the BLK and LYN kinases as well as with mutant forms of BLK (Fig. 2A). Complex formation was observed when BANKl and PLCG2 were co- expressed with the constitutive active form of BLK (YF), (Fig. 2B, lane 3). Co- expression with the wild type (WT) kinases generated a weak but visible precipitate, lanes 4 and 6. The immunoprecipitate was absent when co-transfection was done with the kinase dead form of BLK (Fig. 2B, lane 5) or when using a protein lacking kinase activity (GFP), (Fig. 2B, lane 1). Quantification in various independent experiments showed stronger immunoprecipitates using BLK-WT versus Lyn-WT, suggesting that BLK could be more specific than Lyn in the BANKl -PLCg2 interaction. However, because the expression of Lyn constructs was consistently lower it was not possible to address adequately the contribution of each kinase to the BANKl -PLCg2 interaction; see for example the lysate, IB-v5 (Fig. 2B). Difference in tyrosine phosphorylation of PLCg2 is observed; see lysate, IB-P-Tyr (Fig. 2B). While the expression of BLK-WT phosphorylates PLCg2, the LYN-WT does not (compare lanes 4 and 6 in Figure 4B, IB: P-Tyr). To address if the sub-cellular localization of the kinases could influence the BANKl -PLCG2 interaction, expression vectors with mutated lipidation sites in BAK were constructed (Fig. 2A).

The BANKl -PLCg2 interaction decreased when the constitutive active form of BLK lacked the myristoylation site or when an additional palmitoylation site is added. The last mutation mimics the lipidation pattern of Lyn, which suggests that single myristoylation of the kinase favors the BANKl -PLCg2 interaction (compare lanes 2, 3 and 4 of top panel of Fig. 2C). Accordingly with this result, using the Lyn construct harboring the lipidation pattern of BLK renders a large amount of precipitate (lane 5 of top panel of Figure 4C), Thus both the kinase activity of BLK and its proper lipidation contributed to the specificity of BLK in the BANKl-PLCg2 interaction.

EXAMPLE 4: Silencing of BLK reduces the association between BANK1 and PLCg2

Commercially available lentiviral particles coding for three BLK-specific siRNAs were used to silence the kinase in the human B-cell line Daudi. A substantial reduction of protein and mRNA expression in the silenced cell lines was obtained (Fig. 3A and B).

Silencing of BLK leads to a reduction of the immunoprecipitation between

PLCg2 and BANK1 upon stimulation with IgM (Fig. 3C). The kinetics of the interaction determined by proximity ligation follows the previously observed pattern (Fig. 3D). The PL A signals reached a maximum at one minute after stimulation and decreased to the basal level after 15 minutes. In non- stimulated cells and after 15 minutes of IgM stimulation the differences in PLA interactions were significant between silencing and control cells (P< 0.0001 in t-test). At one minute after stimulation the difference did not reach the significance level (P=0.0561). These results suggest that other kinases are able to compensate for the reduction of BLK during intensive stimulation. They also suggest a role for BLK in the maintenance of a homeostatic modulation of the BANKl-PLCg2 interaction. The depletion of BLK leads to larger fluctuations of the interaction between the two proteins than in the presence of BLK.

EXAMPLE 5: The BANKl-PLCg2 interaction is dependent on the proline rich motif and the phosphorylation of specific tyrosine residues on BANK1

BCR stimulation or the co-expression of an active tyrosine kinase induced the phosphorylation of BANK1 and enhanced its association with PLCg2. This suggests that certain tyrosine residues on BANK1 are important for the interaction. The fact that the prey clones retrieved in our Y2H screen coded for phosphotyrosine-binding domains (SH2) reinforced this notion. The full-length isoform of BANK1 (FL) has thirteen potential tyrosine phosphorylation residues. Two of them are absent in the short isoform (D2) lacking the second exon (Fig. 4A). Because the differential expression of each isoform may have functional relevance to susceptibility to SLE, these two residues were targeted and performed a binding assay. In addition, two adjacent tyrosines (Y484 and Y488) that are predicted to form a strong SH2 binding motif were mutated. Because the prey clones also contain proline rich binding motifs (SH3), the effect of BANKl proline substitutions was analyzed in the binding assay. The mutated sites have a variable degree of conservation on orthologous proteins, thus, the proline P20 is poorly conserved while the sequence surrounding the prolines P611 and P612 is highly conserved (Fig. 4B). The mutated BANKl proteins were expresed in cells transfected with the kinase constitutive active form of BLK (BLK- YF) and PLCg2 (Fig. 4C). The association was measured by immunoprecipitation using the anti-PLCg2 antibody and the level of BANKl tyrosine phosphorylation was estimated with the anti-pan-tyrosine antibody (Fig. 4D). Substitution of Y484 and Y488 to F lead to an overall decrease of tyrosine phosphorylation of BANKl (Fig. 4C, lane 2, first row), the substitution of Y 125 did not influence the BLK- mediated phosphorylation and the substitution of Y 146 only marginally reduced the level of tyrosine phosphorylation, indicating that Y484 and/or Y488 were phosphorylated by the constitutively active form of BLK while Y125 was not. The BANKl association to PLCg2 was significantly reduced in the Y484-488 substitution but not completely abolished, which suggested additional binding sites. The PP513LL seemed to represent such an additional binding site because its mutation leads to a reduction of the association. In this case, the association is independent from the overall tyrosine phosphorylation of BANKl (Figure 5D). We addressed further the interaction of PLCg2 with the natural occurring isoforms of BANKl .

Immunoprecipitation of co-expressed BANKl isoforms (FL and D2) showed as expected that both proteins associated equally to PLCg2 (Fig. 3). Thus, BANKl has two defined domains, one containing exon 2 that binds to type 2 IP3R and a PLCg2 binding domain composed of a phosphotyrosine motif (Y484-488) that probably binds to the SH2 domains of PLCg2 and a proline rich motif (PP513-514) that probably binds to the SH3 domain of PLCg2. The two domains connect the enzyme responsible of the generation of IP3 (PLCg2) and the receptor of this second messenger (IP3R). The signaling cascade is initiated by BCR mediated phosphorylation of BANKl .

EXAMPLE 6: Identification of protein partners interacting with the B -cell scaffold protein with ankyrin repeats (BANKl)

Two independent yeast two-hybrid screens were carried out to identify interacting partners of BANKl . In the first one, we used as bait the full-length form of BANKl (aa 1-785). Due to the high autoactivity of the full-length BANKl construct, a mapping of BANKl domains was performed to identify fragments that do not or mildly autoactivate the transcription of the reporter gene in the yeast two- hybrid system (Figure 6). Once the non-activating domains were identified, an additional screen with the N-terminal truncated form of BANKl (aa 331-785) was also performed.

The screen with the full-length form of BANKl identified 9 clones with good or moderate confidence in the interaction. The interaction found with the Src kinase FYN rendered the results of the screen reliable. The hypothesis preceding the screen was to recover prey clones coding for conserved Src family of kinases because it has been previously shown that BANKl interacts physically in vivo with two related Src kinases, namely LYN and BLK The higher confidence for interaction was however obtained with the phospholipase C-gamma 2 (PLCg2). Two independent clones coding for the regulatory region specific for the PLCg family were recovered. Both clones code for the carboxy terminal SH2 domain (cSH2), the complete SH3 domain and one of clones included the carboxy terminal catalytic Y-core.

The second screen with the truncated form of BANKl (aa 331-785) produced high confidence interactions, which suggests that this fragment of BANKl is, at least partially well folded. The higher scores in this screen were given to the genes G22P1 coding for the Ku70 protein, required for V(D)J recombination, protection of telomeres and originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases and the genes PSAP and Saposin C coding for the saposin precursor and the mature Saposin C form, respectively. In this screen, we identified once again fragments as prey clones coding for the SH2 and SH3 domains of the related Src kinases LYN, FYN and HCK . In addition a single clone coding for a polypeptide from PLCgl was found. The aminoacid sequence is highly homologous to PLCg2 and correspond to aa 647-843 that comprise the cSH2 domain and the complete SH3 domain. Surprisingly, the clone had a 25 aa deletion that removes two tyrosine residues previously implicated in phosphorylation-dependent activation of the lipase, suggesting that this domain is dispensable for the binding to BANKl . With the only exception of the clone A-14 coding the kinase domain of FYN, all the recovered clones belonging to PLCg and Src-kinase families expressed the SH3 and a truncated SH2 domain, which indicates that these motifs are implicated in the interaction with BANKl .

Claims

1. An in vitro method for diagnosing an autoimmune disease or for predicting the effectiveness of a treatment administered in a patient suffering from an autoimmune disease which comprises:

(ii) determining the time-dependent formation of a complex comprising BANK1 and PLCg2 in the B lymphocytes present in the sample in response to said activation, wherein said determination is carried out until the level of complex reaches a stationary level;

2. An in vitro method for monitoring the progression of an autoimmune disease in a patient which comprises:

wherein, a deviation in the value of said at least one kinetic parameter in comparison with the variation of the same parameter at an earlier point of the disease in the patient is indicative that the autoimmune disease shows a bad progression.

An in vitro method for classifying a patient suffering from an autoimmune disease which comprises:

Method according to any of claims 1 to 3 wherein the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, Behcet disease, amyotrophic lateral sclerosis, multiple sclerosis and Devic disease, spondyloartheopathy, fibromyalgia, rheumatic fever, Wegener granulomatosis, systemic lupus erythematosus, Antiphospho lipids syndrome or Huges syndrome, polymyositis, dermamyositis, chronic inflammatory demyelinating polyradiculoneuropathy, psoriasis, immune thrombocytopenic purpura , sarcoidosis, chronic fatigue syndrome, Guillain-Barre syndrome, systemic vasculitis or vitiligo.

Method according to claim 4, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

6. The method according to any of claims 1 to 5 wherein the activation of the B lymphocytes is carried out by contacting the sample with anti-IgM antibodies.

7. The method according to any of claims 1 to 6, wherein the determination of the protein complex between BANK1 and PLCg2 is carried out in a population of isolated B lymphocytes.

8. The method according to any of claims 1 to 7 wherein the kinetic parameter is the concentration of BANKl-PLCg2 complex.

9. The method according to claim 8 wherein the kinetic parameter is the concentration of BANKl-PLCg2 complex at the time point wherein the concentration of the complex is highest.

10. The method according to claims 1 to 9 wherein the detection of BANKl-PLCg2 complex or the determination of the concentration of said complex is determined by means of in situ proximity ligation assay (PL A) or by an immunoprecipitation assay.

11. The method according to any of claims 1 to 10 wherein the patient is a human being.

12. An in vitro method for detecting a protein complex between the B cell scaffold protein with ankyrin repeats (BANK1) and Phospho lipase C gamma 2 (PLCg2) in a sample comprising B lymphocytes wherein said B lymphocytes are activated.

13. The method according to claim 12 wherein the detection of BANKl-PLCg2 complex is carried out by means of in situ proximity ligation assay (PLA) or by an immunoprecipitation assay.

14. An agent selected from the group consisting of a B lymphoid tyrosine kinase (BLK) inhibitor and a BANK1 inhibitor for use in the treatment of an autoimmune disease.

15. The agent for use according to claim 14 wherein the BLK inhibitor is selected from the group consisting of a BLK specific interfering RNA, a BLK specific antisense oligonucleotide, a BLK specific ribozyme, a BLK specific inhibitory antibody and an inactive BLK variant.

16. The agent for use according to claim 15, wherein the inactive BLK variant is selected from the group consisting of a BLK variant carrying a mutation at position 269 with respect to the human BLK, a BLK variant lacking the myristoylation site and a BLK variant containing an additional palmitoylation site.

17. The agent for use according to claim 14 wherein the BANKl inhibitor is selected from the group consisting of a BANKl specific interfering RNA, a BANKl specific antisense oligonucleotide, a BANKl specific ribozyme, a BANKl specific inhibitory antibody and an inactive BANKl variant.

18. The agent for use according to claim 17, wherein the inactive BANKlvariant carries a mutation at position 484 and/or a mutation at position 488 with respect to human BANK liso form 1.

19. The agent for use according to claim 18 wherein the amino acid mutation at position 484 is alanine and/or wherein the amino acid mutation at position 488 is alanine.

20. The agent for use according to any of claims 14 to 19 wherein said subject is a subject classified in the first group according to the of claim 3.

21. The agent for use according to any of claims 14 to 20, wherein the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, Behcet disease, amyotrophic lateral sclerosis, multiple sclerosis and Devic disease, spondyloartheopathy, fibromyalgia, rheumatic fever, Wegener granulomatosis, systemic lupus erythematosus, Antiphospho lipids syndrome or Huges syndrome, polymyositis, dermamyositis, chronic inflammatory demyelinating polyradiculoneuropathy, psoriasis, immune thrombocytopenic purpura, sarcoidosis, chronic fatigue syndrome, Guillain-Barre syndrome, systemic vasculitis or vitiligo.

22. The agent for use according to claim 21 wherein the autoimmune disease is SLE.

23. An inactive BLK variant selected from the group consisting of a BLK variant carrying a mutation at position 269 with respect to the human BLK, a BLK variant lacking the myristoylation site and a BLK variant containing an additional palmitoylation site.

24. An inactive BANKl variant which carries a mutation at position 484 and/or a mutation at position 488 with respect to human BANKl isoform 1.

25. An inactive BANKl variant according to claim 24 wherein the amino acid mutation at position 484 is alanine and/or wherein the amino acid mutation at position 488 is alanine.

26. A composition comprising an agent according to any of claims 23 to 25 or a pharmaceutical composition comprising an agent according to any of claims 23 to 25 and a pharmaceutical acceptable carrier.

27. A protein complex comprising BANKl and PLCg2.

28. A protein complex according to claim 27 wherein BANKl is phosphorylated.