WO2014122275A1 - Procédé permettant le diagnostic et la surveillance de maladies auto-immunes - Google Patents

Procédé permettant le diagnostic et la surveillance de maladies auto-immunes Download PDF

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WO2014122275A1
WO2014122275A1 PCT/EP2014/052458 EP2014052458W WO2014122275A1 WO 2014122275 A1 WO2014122275 A1 WO 2014122275A1 EP 2014052458 W EP2014052458 W EP 2014052458W WO 2014122275 A1 WO2014122275 A1 WO 2014122275A1
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bankl
blk
plcg2
complex
lymphocytes
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PCT/EP2014/052458
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English (en)
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Marta ALARCÓN RIQUELME
Casimiro CASTILLEJO LÓPEZ
Manuel José BERNAL QUIROS
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Fundación Pública Andaluza Progreso Y Salud
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

  • 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.
  • autoimmune disorders are characterized by the production of autoantibodies against self antigens.
  • systemic lupus erythematous SLE
  • SLE systemic lupus erythematous
  • 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.
  • SLE Systemic lupus Erythematosus
  • GWAS genome wide association studies
  • 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
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • the invention refers to an in vitro method for monitoring the progression of an autoimmune disease in a patient which comprises:
  • 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.
  • the invention relates to an in vitro method for classifying a patient suffering from an autoimmune disease which comprises:
  • 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.
  • the invention in another aspect, 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.
  • BANKl ankyrin repeats
  • PLCg2 Phospho lipase C gamma 2
  • 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.
  • 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.
  • the invention relates to compositions a comprising an inactive BLK variant according to the invention or an inactive BANKl variant according to the invention.
  • 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.
  • the invention refers to a protein complex comprising BANKl and PLCg2.
  • 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.
  • IPs Immunoprecipitates
  • 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.
  • 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).
  • 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.
  • BANKl ankyrin repeats
  • PLCg2 Phospholipase C gamma 2
  • B cell scaffold protein with ankyrin repeats 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).
  • PLCg2 Phospholipase C gamma 2
  • PLCg2 Phospholipase C gamma 2
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • PLA in situ proximity ligation assay
  • 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.
  • PLA probes species-specific secondary antibodies
  • 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.
  • 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.
  • 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.
  • proteins which physically interact with each other e.g.
  • 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.
  • 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.
  • 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
  • GFP Green florescent protein or GFP, yellow florescent protein or YFP, red fluorescent protein or RFP
  • 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.
  • 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.
  • lymphocytes white blood cells
  • 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).
  • BCR B cell receptor
  • 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.
  • a blood sample is used.
  • the term blood sample 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.
  • 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.
  • the first method of the invention is carried out in a
  • 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.
  • 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.
  • 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.
  • lymphocytes B are cultured in vitro under appropriate conditions.
  • 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.
  • 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).
  • BCRs B lymphocytes receptors
  • the antigen 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.
  • B 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.
  • B lymphocytes may be activated in culture medium (e.g.
  • RPMI 1640 culture medium supplemented with amino acids and vitamins
  • FBS heat inactivated fetal bovine serum
  • FCS fetal calf serum
  • 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.
  • 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).
  • BANK1 B cell scaffold protein and ankyrin repeats
  • PLCg2 Phospholipase C gamma 2
  • the invention relates to an in vitro method for diagnosing an autoimmune disease in a patient which comprises:
  • diagnosis 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).
  • 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.
  • 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.
  • 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, fibromyal
  • said autoimmune disease is Systemic Lupus Erythematosus (SLE).
  • SLE Systemic Lupus Erythematosus
  • patient 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.
  • the patient is a human patient.
  • the diagnostic method of the invention comprises activating the
  • B lymphocytes in a sample isolated from said patient containing B lymphocytes.
  • 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.
  • 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.
  • 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.
  • the B lymphocytes are activated by contacting the sample containing said B lymphocytes with anti-IgM antibodies.
  • 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.
  • determining the time-dependent formation of a complex refers to the measurement of the formation of the protein complex (i.e. between BANKl and PLCg2) during certain period of time.
  • 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.
  • 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.
  • interaction 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.
  • an "interaction" is typically exhibited by the binding between the interacting domains or entities.
  • 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.
  • the first protein e.g. BANK1
  • the second protein e.g.
  • 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.
  • the first protein e.g. BANK1
  • the protein complex e.g. BANKl-PLCg2 complex
  • 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.
  • kinetic parameter 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 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).
  • 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.
  • kinetic parameter as used herein, also refers to how long it takes to form a given or desired concentration of BANKl -PLCg2 complex.
  • 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.
  • said desired time is about less than 1 min
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the kinetic parameter which will be determined is the concentration of BANKl-PLCg2 complex.
  • said kinetic parameter is determined by means of PLA or Co-IP.
  • 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.
  • concentration of the complex refers to the concentration wherein the concentration of the complex is highest throughout the time course of the biding reaction.
  • 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.
  • 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.
  • 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
  • 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.
  • the invention contemplates said method for predicting the effectiveness of a treatment administered in a patient suffering from an autoimmune disease.
  • 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:
  • a deviation value of said at least one kinetic parameter with respect to said reference value is indicative of that the treatment is ineffective.
  • 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.
  • the prediction although preferred to be, need not to be correct for 100% of the subjects to be evaluated.
  • the term 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.
  • 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 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.
  • the autoimmune disease is systemic lupus erythematosus (SLE).
  • treatment 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
  • immunosuppressants are substances that decrease the immune response. They may be either exogenous, as immunosuppressive drugs, or endogenous.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • BANK1 "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.
  • 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.
  • 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.
  • the kinetic parameter is the concentration of BANKl -PLCg2 complex.
  • said kinetic parameter is carried out by means of PLA or Co-IP.
  • 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.
  • 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
  • concentration of BANKl -PLCg2 complex formed at the time point wherein said concentration is highest e.g. the concentration of BANKl -PLCg2 complex formed at the time point wherein said concentration is highest
  • 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:
  • 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.
  • 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;
  • all 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.
  • 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.
  • the first sample is taken prior to the subject receiving treatment, e.g. immunosuppressant, and the second sample is taken after treatment.
  • 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.
  • 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.
  • the autoimmune disease is systemic lupus erythematosus (SLE).
  • 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.
  • the first step of the monitoring method of the invention comprises the activation of the B lymphocytes in a sample isolated from a patient.
  • said activation is carried out in a population of isolated lymphocytes.
  • 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.
  • 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.
  • the kinetic parameter is the concentration of BANK- PLCg2 complex.
  • said kinetic parameter is determined by means of PLA or Co-IP.
  • 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.
  • 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.
  • 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:
  • 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.
  • classifying a patient 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).
  • 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.
  • the autoimmune disease is systemic lupus erythematosus (SLE).
  • 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.
  • 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.
  • said activation is carried out in a population of isolated lymphocytes.
  • said activation is carried out with anti-IgM antibody under conditions suitable for the stimulation of the B lymphocytes in said sample.
  • 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.
  • 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.
  • BANK1 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the reference sample is a sample containing B cells of the patient which has not been treated so as to stimulate the B cells.
  • device 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.
  • the kinetic parameter is the concentration of BANK- PLCg2 complex.
  • said kinetic parameter is determined by means of PLA or Co-IP.
  • 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.
  • 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.
  • 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.
  • a patient classified into the first group is susceptible of being suffering an autoimmune disease.
  • 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.
  • 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.
  • a patient classified in the first group would be susceptible to be monitored according to the monitoring method of the invention.
  • 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.
  • a patient classified into the second group is unlikely of suffering an autoimmune disease.
  • 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.
  • 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.
  • a patient classified in the second group would be susceptible to be monitored according to the monitoring method of the invention.
  • 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.
  • 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.
  • the invention in another aspect, 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 inhibitory agent 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.
  • B Lymphocyte Kinase 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.
  • 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 (March 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 the levels of the BLK mRNA e.g. RT-PCR, Northern blot and the like
  • methods for determining the levels of the BLK polypeptide ELISA, RIA, Western blot, and the like
  • BLK activity which can be used for determining inhibitors acting directly on the kinase activity of BLK
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the oligodeoxyribo nucleotide regions derived from the translation start site 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.
  • the oligonucleotide can be conjugated to another molecule, for example, a peptide, a carrier agent, a hybridization-triggered cleavage agent, etc.
  • the BLK inhibitory agent is a BLK-specific interfering RNA (iRNA).
  • iRNA 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.
  • the iRNA is a shRNA" or "small hairpin RNA".
  • 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.
  • the sense strand can precede the nucleotide loop structure and the antisense strand can follow.
  • 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.
  • 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.
  • 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).
  • 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:
  • RNA chain conjugates of the RNA chain with a functional reagent, such as a fluorophore
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • the BLK inhibitor is a BLK inhibitory antibody.
  • 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.
  • 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.
  • active variant 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.
  • 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).
  • the lysine residue is replaced by a hydrophobic residue and, more preferably, by a leucine residue.
  • 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).
  • the agent used in the therapeutics methods according to the invention is a BANKl inhibitory agent.
  • 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).
  • 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 .
  • RNA antisense oligonucleotide
  • ribozyme inhibitor of the BLK inhibitors
  • the BANKl inhibitor is an inactive BANKl variant.
  • 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.
  • inactive variant 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.
  • 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).
  • the tyrosine at position 484 is replaced by an hydrophobic residue.
  • the variant is a Y484F.
  • 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).
  • the tyrosine at position 488 is replaced by an hydrophobic residue.
  • the variant is a Y488F.
  • 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).
  • the tyrosine at positions 484 and 488 are replaced by hydrophobic residues.
  • the variant is a Y484F and Y488F mutant.
  • 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.
  • the inhibitory agents of the invention are used in the treatment of SLE.
  • 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.
  • 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.
  • 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).
  • the lysine residue is replaced by a hydrophobic residue and, more preferably, by a leucine residue.
  • 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).
  • the inactive BLK variant contains an additional palmitoylation site (e.g. by including an amino acid substitution at the position 3).
  • 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.
  • 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).
  • the tyrosine at position 484 is replaced by an hydrophobic residue.
  • the variant is a Y484F.
  • 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).
  • the tyrosine at position 488 is replaced by an hydrophobic residue.
  • the variant is a Y488F.
  • 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).
  • the tyrosine at positions 484 and 488 are replaced by hydrophobic residues.
  • the variant is a Y484F and Y488F mutant.
  • compositions of the invention are provided.
  • the invention in another aspect, relates to a composition comprising a BLK inhibitory agent of the invention or a BANK1 inhibitory agent of the invention.
  • 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.
  • 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.
  • 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.
  • the invention in another aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a BLK inhibitory agent of the invention or a BANK1 inhibitory agent of the invention, and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the complex comprising BANKl and PLCg2 contains a phosphorylated BANKl .
  • the process of phosphorylation refers to the addition of a phosphate (P(V 3 ) group to a protein (e.g. BANKl) or other organic molecule.
  • a protein e.g. BANKl
  • Phosphorylation may occur at one or more serine or tyrosine sites within the BANKl sequence.
  • 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 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.
  • Daudi and embryonic kidney HEK293T cells were each maintained in RPMI
  • HEK293T cells 1640 medium and Dulbecco's modified Eagle's medium containing Glutamax (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen).
  • HEK293T cells (3* 10 6 ) 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.
  • mice 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
  • mouse anti- BLK H00000640-M02 Abnova, Heidelberg, Germany
  • chicken anti-GAPDH SAB3500247 Sigma
  • anti-rabbit and anti-mouse-HRP Zamed,, San Francisco, CA, USA
  • anti-Chicken IgY-HRP Sigma
  • 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.
  • DPBS Dulbecco's phosphate-buffered saline
  • 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.
  • Blk shRNA Lentiviral Particles catalog no. sc-3
  • 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.
  • PKA In situ proximity ligation assay
  • 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.
  • 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 .
  • 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.
  • the PLA signals are mostly localized close to the nucleus, resulting in a merged image with yellow spots, shown by arrowheads.
  • 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).
  • 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.
  • 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
  • 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.
  • 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).
  • EXAMPLE 5 The BANKl-PLCg2 interaction is dependent on the proline rich motif and the phosphorylation of specific tyrosine residues on BANK1
  • 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.
  • 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 .
  • 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).
  • PLCg2 phospholipase C-gamma 2
  • 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.
  • 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 .

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Abstract

La présente invention concerne un procédé permettant le diagnostic et la prédiction de la réponse à un traitement chez un patient souffrant d'une maladie auto-immune. L'invention concerne aussi un procédé permettant la surveillance de la progression d'une maladie auto-immune et un procédé permettant l'affectation d'un patient souffrant d'une maladie auto-immune. La présente invention concerne des agents à des fins d'utilisation dans le traitement d'une maladie auto-immune, des compositions comportant lesdits agents et des compositions pharmaceutiques de ceux-ci.
PCT/EP2014/052458 2013-02-08 2014-02-07 Procédé permettant le diagnostic et la surveillance de maladies auto-immunes WO2014122275A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN108359686A (zh) * 2018-02-24 2018-08-03 武汉伊艾博科技有限公司 融合标签NusA在大肠杆菌表达PLCG2蛋白中的应用
WO2018231322A1 (fr) * 2017-06-13 2018-12-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Classification de troubles des lymphocytes b et sensibilité à des inhibiteurs

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009810A1 (fr) 1987-06-11 1988-12-15 Synthetic Genetics Nouveaux conjugues d'acide nucleique amphiphile
WO1989010134A1 (fr) 1988-04-25 1989-11-02 The Regents Of The University Of California Peptides chimeriques d'administration de neuropeptides a travers la barriere vasculaire du cerveau
US5176996A (en) 1988-12-20 1993-01-05 Baylor College Of Medicine Method for making synthetic oligonucleotides which bind specifically to target sites on duplex DNA molecules, by forming a colinear triplex, the synthetic oligonucleotides and methods of use
US5256775A (en) 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
WO2004035811A2 (fr) 2002-10-14 2004-04-29 University Court Of The University Of Dundee Dosage de modulation de kinases
WO2005059168A1 (fr) * 2003-12-12 2005-06-30 Sirenade Pharmaceuticals Ag Procedes d'identification, de selection et de caracterisation de composes qui modulent l'activite d'une kinase de la famille src
WO2009068481A1 (fr) * 2007-11-26 2009-06-04 Merck Serono S.A. Nouveau variant d'épissage du gène bank1
WO2010128193A1 (fr) * 2009-05-05 2010-11-11 Consejo Superior De Investigaciones Científicas (Csic) Méthode de diagnostic, de pronostic et/ou de traitement du lupus érythémateux systémique
US20110201016A1 (en) * 2010-02-12 2011-08-18 Mediscov Inc. Method for detecting modulator for regulating activity of cellular component in cell

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009810A1 (fr) 1987-06-11 1988-12-15 Synthetic Genetics Nouveaux conjugues d'acide nucleique amphiphile
WO1989010134A1 (fr) 1988-04-25 1989-11-02 The Regents Of The University Of California Peptides chimeriques d'administration de neuropeptides a travers la barriere vasculaire du cerveau
US5176996A (en) 1988-12-20 1993-01-05 Baylor College Of Medicine Method for making synthetic oligonucleotides which bind specifically to target sites on duplex DNA molecules, by forming a colinear triplex, the synthetic oligonucleotides and methods of use
US5256775A (en) 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
WO2004035811A2 (fr) 2002-10-14 2004-04-29 University Court Of The University Of Dundee Dosage de modulation de kinases
WO2005059168A1 (fr) * 2003-12-12 2005-06-30 Sirenade Pharmaceuticals Ag Procedes d'identification, de selection et de caracterisation de composes qui modulent l'activite d'une kinase de la famille src
WO2009068481A1 (fr) * 2007-11-26 2009-06-04 Merck Serono S.A. Nouveau variant d'épissage du gène bank1
WO2010128193A1 (fr) * 2009-05-05 2010-11-11 Consejo Superior De Investigaciones Científicas (Csic) Méthode de diagnostic, de pronostic et/ou de traitement du lupus érythémateux systémique
US20110201016A1 (en) * 2010-02-12 2011-08-18 Mediscov Inc. Method for detecting modulator for regulating activity of cellular component in cell

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
"Goodman and Gilman's The Pharmaceutical Basis of Therapeutics", 1990, PERGAMON PRESS
"Remington's Pharmaceutical Science", 1985, MACK PUBLISHING CO.
"Uniprot database", accession no. 51451
"UniProt database", accession no. P 060405.4
BERNAL-QUIROS MANUEL ET AL: "BANK1 and BLK Act through Phospholipase C Gamma 2 in B-Cell Signaling", PLOS ONE, vol. 8, no. 3, March 2013 (2013-03-01), XP002722689, ISSN: 1932-6203 *
CASTILLEJO-LOPEZ CASIMIRO ET AL: "Genetic and physical interaction of the B-cell systemic lupus erythematosus-associated genes BANK1 and BLK", ANNALS OF THE RHEUMATIC DISEASES, vol. 71, no. 1, January 2012 (2012-01-01), pages 136 - 142, XP002722685 *
CASTILLEJO-LOPEZ ET AL., ANN RHEUM DIS, vol. 71, 2012, pages 136 - 142
CHANG NAN-HUA ET AL: "Altered Response to B Cell Receptor (BCR) Crosslinking in SLE: Correlation with Genetic Risk Variants Predicted to Impact BCR Signaling.", ARTHRITIS & RHEUMATISM, vol. 64, no. 10, Suppl. S, October 2012 (2012-10-01), & ANNUAL SCIENTIFIC MEETING OF THE AMERICAN-COLLEGE-OF-RHEUMATOLOGY (ACR) AND ASSOCIATION-OF-RHEUMATOL; WASHINGTON, DC, USA; NOVEMBER 09 -14, 2012, pages S290, XP002722686 *
DATABASE UniParc [Online] 21 February 2007 (2007-02-21), XP002722687, retrieved from EBI Database accession no. UPI0000F60381 *
DATABASE UniProt [Online] 16 May 2012 (2012-05-16), "SubName: Full=Uncharacterized protein;", XP002722688, retrieved from EBI accession no. UNIPROT:H9GQL2 Database accession no. H9GQL2 *
JULIANNE J. SANDO: "Protein Kinase C Protocols", 2003, HUMANA PRESS
LEMAITRE ET AL., PROC. NATL. ACAD. SCI., vol. 84, 1987, pages 648 - 652
LETSINGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 6553 - 6556
SCHAER ET AL., BLOOD, vol. 107, 2006, pages 373 - 380
STEIN ET AL., CANCER RES, vol. 48, 1988, pages 2659 - 2668
VAN DER KROL ET AL., BIOTECHNIQUES, vol. 6, 1988, pages 958 - 976
ZON, PHARM. RES., vol. 5, 1988, pages 539 - 549

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018231322A1 (fr) * 2017-06-13 2018-12-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Classification de troubles des lymphocytes b et sensibilité à des inhibiteurs
CN108359686A (zh) * 2018-02-24 2018-08-03 武汉伊艾博科技有限公司 融合标签NusA在大肠杆菌表达PLCG2蛋白中的应用

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