WO2012098256A1 - Auto-anticorps anti-cd146 et leurs utilisations - Google Patents

Auto-anticorps anti-cd146 et leurs utilisations Download PDF

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WO2012098256A1
WO2012098256A1 PCT/EP2012/050971 EP2012050971W WO2012098256A1 WO 2012098256 A1 WO2012098256 A1 WO 2012098256A1 EP 2012050971 W EP2012050971 W EP 2012050971W WO 2012098256 A1 WO2012098256 A1 WO 2012098256A1
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risk
autoimmune disease
pregnancy
antibodies
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PCT/EP2012/050971
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Nathalie Bardin
Marcel Blot-Chabaud
Elise KASPI
Brigitte REY GRANEL
Florence Bretelle
Françoise DIGNAT-GEORGE
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Université De La Méditerranée Aix - Marseille Ii
Assistance Publique - Hôpitaux De Marseille
Institut National De La Santé Et De La Recherche Médicale (Inserm)
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Publication of WO2012098256A1 publication Critical patent/WO2012098256A1/fr

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the invention relates to in vitro or ex vivo methods comprising the determination of the presence and/or the measure of the quantity or level of anti-CD 146 auto-antibodies in a biological sample from a subject for (i) the detection of an autoimmune disease or of a high-risk pregnancy (diagnostic), (ii) the determination of a predisposition to an autoimmune disease or a high-risk pregnancy, (iii) the determination of the prognosis or monitoring of the course of an autoimmune disease or of a high-risk pregnancy (prognostic), or (iv) assessing the efficiency of a treatment of an autoimmune disease or of a disorder associated to a high-risk pregnancy in the subject.
  • Scleroderma, lupus, in particular systemic lupus erythematosus (also herein identified as SLE, systemic lupus or lupus erythematosus), and rheumatoid arthritis are typical examples of vascular disorders-associated autoimmune diseases.
  • Scleroderma is a clinically heterogeneous, systemic disorder which affects the connective tissue of the skin, internal organs and the walls of blood vessels. It is characterized by early alterations of the microvasculature, by disturbances of the immune system and by massive (excessive) deposition of collagen and other matrix substances in the connective tissue ("fibrosis").
  • fibrosis a connective tissue
  • the term “Scleroderma” refers to any known kind of scleroderma and more particularly to "systemic sclerosis” (SSc) also herein identified as "Progressive systemic sclerosis”.
  • Progressive systemic sclerosis is characterized by a deep induration and by the thickening of the layers of the skin and of underlying tissues. It is almost always associated with similar deteriorations concerning the conjunctive tissue of viscera or organs.
  • a particular classification has been proposed by the Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee (Arthritis Rheum 1980; 23: 581-90.) which can be used to accurately diagnose scleroderma and distinguish it from unrelated diseases.
  • This classification includes one major criterion and three minor criteria. According to this classification, the patient should fulfil the major criterion or two of the three minor criteria to be diagnosed as a patient suffering of a "systemic sclerosis” (SSc).
  • SSc systemic sclerosis
  • proximal scleroderma also identified as “proximal diffuse sclerosis” which mainly damages the subject's trunk and is responsible for manifestations such as skin tightness, thickening, and non-pitting induration.
  • the three minor criteria are 1) sclerodactily, 2) digital pitting scars of fingertips or loss of substance of the distal finger pat, and 3) bilateral basilar pulmonary fibrosis.
  • lcSSc - limited systemic scleroderma or limited systemic sclerosis
  • Typical manifestations are for example skin calcification, telangiectasia and nail fold capillaries presenting dilatation without significant giant capillaries.
  • a Raynaud's phenomenon may precede skin manifestations by years.
  • Gastrointestinal organs may further be affected as well as lungs.
  • a pulmonary arterial hypertension may occur in about 8 to 15% of patients and is the most serious complication for this form of scleroderma.
  • SSc This form of SSc was previously referred to as CREST (Calcinosis. Raynaud's, Esophageal dysmotility, Sclerodactyly, Telangiectasia).
  • dcSSc diffuse systemic scleroderma or diffuse systemic sclerosis
  • Typical manifestations are for example interstitial lung disease, renovascular hypertensive crisis, nail fold capillaries presenting dilatation and giant capillaries, gastrointestinal disorders and myocardial disorders.
  • a Raynaud's phenomenon can start within the first year of disease or may appear on the occasion of skin changes.
  • Prognosis is generally good for limited cutaneous scleroderma patients who escape arterial pulmonary complications.
  • Prognosis is worse for diffuse cutaneous disease, particularly in older age, and for males. Death occurs most often from pulmonary and heart complications but also, although less often, from kidney and digestive complications. In diffuse cutaneous disease, 5-year survival is 70% and 10-year survival only 55%.
  • auto-antibodies directed against intracellular antigens are present. These auto-antibodies can induce the expression of adhesion molecules and cause apoptosis of endothelial cells.
  • an individual patient's serum contains only a limited number of self antigens.
  • the particular auto-antibody present is often indicative of clinical expression, disease course and overall severity. In systemic sclerosis, wherein clinical features are highly variable, such information is a valuable aid to the diagnosis and prognosis of an individual patient.
  • Total antinuclear antibodies are present in the serum of 80 to 98% of patients with systemic sclerosis but they are not specific since they are also found in other connectivitis. Some of these autoantibodies are very specific of scleroderma and considered to be mutually exclusive (Haustein, Systemic sclerosis, 2002, Dermatology online journal, volume 8, number 1), like the anti- topoisomerase I (also identified as anti-Scl-70) antibodies (ATA) (Tamby and Al, 2007), more frequently present in the diffuse cutaneous forms of the disease (dcSSc), the anti-centromere antibodies (ACA), generally associated with the limited scleroderma cutaneous forms (lcssC) (Moroi and Al, 1980). ATA and ACA are present in about 50% of SSc indicating that some SSc patients are seronegative for specific markers, leading to the search of other markers.
  • Anti-RNA polymerase III (RNAP III) antibodies are associated with the diffuse scleroderma cutaneous forms and with renal crisis (Bunn and Al, 1998) although less frequent.
  • SSc non-specific auto-antibodies like the anti-ribonucleoprotein antibodies, the anti-cardiolipin antibodies as well as the rheumatoid factor are sometimes detected during SSc.
  • Anti-endothelial cell antibodies AECA
  • AECA Anti-endothelial cell antibodies
  • These autoantibodies are able to induce the expression of molecules of adhesion and to cause the endothelial cells apoptosis in the presence of natural killer cells (Bordron and al., 1998). Little is known about the targets of AECA during scleroderma.
  • Anti-fibroblasts Antibodies were further identified among sclerodermic patients. These antibodies are able to activate fibroblasts, to increase the expression of molecules of adhesion, like the inter-cellular adhesion molecule- 1 (I CAM), and of pro-inflammatory molecules (increase in the levels of ARNm, IL-laIL- ⁇ and IL-6) as well as to increase the collagen synthesis (Chizzolini and al., 2002). It was recently highlighted that the AFA could be fixed by the topoisomerase 1 adsorbed at the surface of fibroblasts (Henault and Al, 2006). In ELISA (Enzyme-linked immunosorbent assay), AFA were found in 30% of the patients with a SSc associated with HTAP (pulmonary hypertension) (Tamby and Al, 2006).
  • the previously mentionned autoantibodies in particular the anti -centromere B (ACA) and anti-topoisomerase I (ATA) antibodies (which are the antibodies mainly analyzed in routine), may be absent, making difficult the diagnosis of SSc.
  • cardiac manifestations are common in SSc, with an estimated clinical prevalence of 15-35% (Kahan et al., "Cardiac complications of systemic sclerosis”; Rheumatology 2009; 48:iii45-iii48 ). Patients who develop myocardial manifestations are known to be at greater risk of clinical deterioration. However in the majority of SSc patients, cardiac manifestations can remain subclinical. Monitoring of myocardial involvement represents an important aspect of the disease management.
  • lupus erythematosus or systemic lupus erythematosus (SLE), discoid lupus erythematosus, drug-induced lupus erythematosus and neonatal lupus erythematosus.
  • SLE systemic lupus erythematosus
  • SLE is the most common and serious form of lupus. SLE affects nine times as many women as men. Almost all people with SLE have joint pain and swelling. Some develop arthritis.
  • One of the common symptoms of lupus erythematosus is sensitivity to sunlight. Photosensitivity's relationship to and influence on the systemic manifestations of lupus remain to be defined. Mechanisms for photosensitivity might notably include: modulation of autoantibody location, cytotoxic effects, apoptosis induction with autoantigens in apoptotic blebs, upregulation of adhesion molecules and cytokines etc.
  • Tests used to diagnose SLE may include antibody tests and/or antinuclear antibody panels.
  • Antibodies usable to diagnose SLE are typically: anti-double strand DNA (anti ds DNA) antibodies, anti-Smith (anti-SM) antibodies and anti-PCNA antibodies. Other antibodies can also be found: antiphospholipid antibodies, anti-SSA antibodies, anti-SSB antibodies, and anti-RNP antibodies.
  • Anti-nucleosome antibodies, anti-Clq antibodies were also in some cases investigated.
  • AECA have been detected in sera of 50-74% of patients with systemic lupus erythematosus (Ihn et al, 1999 - see also Duval et al., Rheumatology 49, 2010, pages 1049-105, who evaluated endothelial dysfunction in systemic lupus with low diseased activity by quantification and characterization of endothelial microparticles (EMps) and AECA.
  • EMps endothelial microparticles
  • Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. The pathology of the disease process often leads to the destruction of articular cartilage and ankylosis of the joints. RA can also produce diffuse inflammation in the lungs, pericardium, pleura, and sclera, and also nodular lesions, most common in subcutaneous tissue under the skin. Although the cause of rheumatoid arthritis is unknown, autoimmunity plays a pivotal role in both its chronicity and progression, and RA is considered as a systemic autoimmune disease.
  • RA rheumatoid factor
  • IgGFc rheumatoid factor
  • ACPAs anti-citrullinated protein antibodies
  • anti-CCP anti-cyclic citrullinated peptide
  • RF RF
  • these tests are positive in only a proportion (67%) of all RA cases.
  • the most common tests for ACPAs are the anti-CCP test and the anti-MCV assay (antibodies against mutated citrullinated Vimentin).
  • Antinuclear antibodies are also frequently found in people with rheumatoid arthritis. Pickl et al.
  • CD146 identified in vivo expression of CD146 on synovial fluid T cells of rheumatoid arthritis patients, but also on CD3+ T cells infiltrating delayed-type hypersensitivity lesions of the skin, and on distinct T leukemia cells (see abstract). They further found CD146 to be released into culture supematants of cell lines and to be present in human serum from healthy individuals (see page 2114, second paragraph). The authors do not refer to autoimmunity, do not suggest looking for CD 146 autoantibodies, and a fortiori establish no correlation between such CD 146 AAA and rheumatoid arthritis. They only conclude with a role of the CD 146 moiety in adhesion, cell migration and/or homing of T lymphocytes.
  • Vasculitis refers to a heterogeneous group of disorders that are characterized by inflammatory destruction of blood vessels. Both arteries and veins are affected. Lymphangitis is considered as a type of vasculitis. Vasculitis is primarily due to leukocyte migration and resultant damages.
  • ESR erythrocyte sedimentation rate
  • CRP C-reactive protein
  • anaemia increased white blood cell count and eosinophilia.
  • Other possible findings are elevated antineutrophil cytoplasmic antibody (ANCA) levels and hematuria.
  • ANCA antineutrophil cytoplasmic antibody
  • Other organ functional tests may be abnormal (specific abnormalities depend on the degree of various organs involvement).
  • vasculitis The definite diagnosis of vasculitis is established after a biopsy of involved organ or tissue, such as skin, sinuses, lung, nerve, and kidney.
  • the biopsy elucidates the pattern of blood vessel inflammation.
  • An alternative to biopsy can be an angiogram (x-ray test of the blood vessels). It can demonstrate characteristic patterns of inflammation in affected blood vessels.
  • the Antiphospholipid syndrome (APS or APLS) or antiphospholipid antibody syndrome or Hughes syndrome is a disorder of coagulation that causes blood clots (thrombosis) in both arteries and veins as well as pregnancy-related complications such as miscarriage, stillbirth, preterm delivery, or severe preeclampsia.
  • the syndrome occurs due to the autoimmune production of antibodies directed against phospholipids of the cell membrane (aPL).
  • primary antiphospholipid syndrome is used when APS occurs in the absence of any other related disease. APS is commonly seen in conjunction with other autoimmune diseases; the term “secondary antiphospholipid syndrome” is used when APS coexists with other diseases such as systemic lupus erythematosus (SLE). In some cases, APS leads to rapid organ failure due to generalized thrombosis and a high risk of death; this is termed “catastrophic antiphospholipid syndrome” (CAPS).
  • Antibodies usable to diagnose APS are typically selected from Lupus anticoagulant antibodies, anticardiolipin antibodies, anti-p2 glycoprotein antibodies, anti-phosphatidylethanolamine antibodies, anti-prothrombin antibodies, anti-annexin V antibodies, and anti-LDLox antibodies, in particular antibodies of the IgG or IgM isotype.
  • vascular disorders-associated obstetric pathologies like preeclampsia (PE) and intrauterine growth retardation (IUGR) are examples of disorders associated to high-risk pregnancies.
  • a high-risk pregnancy is also considered as a pregnancy associated with a high risk that an anomaly or adverse pregnancy outcome such as a fetal loss (FL) or spontaneous abortion, typically spontaneous early abortion or miscarriage (occurring before the 20 th weeks of gestation), spontaneous late abortion (occurring after the 20 th weeks of gestation) or stillbirth; or preterm delivery occurs.
  • FL fetal loss
  • spontaneous abortion typically spontaneous early abortion or miscarriage (occurring before the 20 th weeks of gestation), spontaneous late abortion (occurring after the 20 th weeks of gestation) or stillbirth; or preterm delivery occurs.
  • Thrombotic lesions of the placenta are a common feature in women with recurrent fetal loss or with adverse pregnancy outcomes.
  • the etiology of fetal loss or other associated adverse conditions of the pregnancy is unknown. It is believed that these are associated with abnormal placental vasculature and haemostatic disturbances leading to inadequate maternal fetal circulation
  • Due to the known thrombotic nature of the placental lesions and to the risk associated with both acquired and genetic thrombophilias a cause-effect relationship between thrombophilias and severe obstetric complications has been suggested (Vora, Placental histomorphology in unexplained fetal loss with Thrombophilia, Indian J Med Res 129, February 2009, pp 144-149).
  • Preeclampsia is a medical condition in which hypertension arises in pregnancy in association with significant amounts of protein in the urine.
  • Preeclampsia refers to a set of symptoms rather than any causative factor, and there are many different causes for the condition. It appears likely that there are substances from the placenta that can cause endothelial dysfunction in the maternal blood vessels of susceptible women. While blood pressure elevation is the most visible sign of the disease, it involves generalized damage to the maternal endothelium, kidneys, and liver, with the release of vasoconstrictive factors being secondary to the original damage. Although eclampsia is potentially fatal, preeclampsia is often asymptomatic, and so its detection depends on signs or investigations.
  • Intrauterine growth retardation is most commonly caused by inadequate maternal -fetal circulation, with a resultant decrease in fetal growth. It is generally characterized by a fetal weight of less than 10 percent of the predicted fetal weight for gestational age. It may result in significant fetal morbidity and mortality if not properly diagnosed. In the United States, IUGR is linked to an increase of 6 to 10 times in perinatal mortality. Currently, IUGR is often suspected on the basis of the fundal height measurements. A significant lag in fundal height is a 4 cm or greater difference than expected for gestational age.
  • Spontaneous late abortion is characterized by a fetal loss after 20 weeks or more of gestation. Acquired and genetic thrombotic conditions, both organ and non organ specific, are associated with increased fetal wastage. Currently, there is no test to predict a spontaneous abortion. A simple, reliable and specific test of diagnosing and monitoring anomalies, diseases or disorders such as those described previously or of determining whether a patient is at risk of expressing or developing such an anomaly, disease or disorder is still lacking, and would be of high value to set up quickly therapeutic strategies intended to improve patient's health and/or chances of survival. This is the aim of the present invention.
  • Inventors herein demonstrate, for the first time, the presence of auto-antibodies directed against CD146 (AAACD146) in human biological samples. They further demonstrate that these auto- antibodies are biomarkers abnormally expressed in patients affected by or at risk of developing an autoimmune disease such as those herein described in the background part, and/or in women suffering of or at risk of developing a disorder associated to a high-risk pregnancy, or in women experiencing or at risk of experiencing an anomaly such as those herein described.
  • the present invention in particular allows the detection in a subject of a high risk to experience an embryo implantation failure (IF).
  • IF embryo implantation failure
  • CD 146 also known as MCAM, MUC18, or Mel-CAM
  • MCAM MUC18
  • Mel-CAM is a component of the endothelial junction which belongs to the immunoglobulin superfamily (Bardin N, Anfosso F, Masse IM, Cramer E, Sabatier F, Le Bivic A, Sampol J, Dignat-George F. Identification of CD 146 as a component of the endothelial junction involved in the control of cell-cell cohesion. Blood. 2001; 98:3677-84).
  • CD 146 is involved in the control of cell and tissue architecture, as demonstrated by the regulation of its expression during endothelium monolayer formation, its involvement in the control of paracellular permeability (Bardin N, Anfosso F, Masse JM, Cramer E, Sabatier F, Le Bivic A, Sampol ⁇ Dignat- George F. Identification of CD 146 as a component of the endothelial junction involved in the control of cell-cell cohesion. Blood. 2001; 98:3677-84) and its colocalization with the actin cytoskeleton (Anfosso F, Bardin N, Vivier E, Sabatier F, Sampol J, Dignat-George F.
  • CD 146 soluble form of CD 146
  • inventors Bardin N, Frances V, Combes V, Sampol J, Dignat-George F. CD 146: Biosynthesis and production of a soluble form in human cultured endothelial cells. FEBS Lett. 1998; 421: 12-4).
  • This soluble form of the protein, present in the human serum has been structurally and functionally characterized by inventors in patent application WO 2010/086405 (also identified as EP2216399).
  • AAACD146 anti-CD 146 auto-antibodies
  • IF embryo implantation failure
  • the present invention provides, in an embodiment, an in vitro or ex vivo method for the detection of an autoimmune disease or of a high-risk pregnancy in a subject, or for the determination of the predisposition of a subject to develop an autoimmune disease, to develop a high-risk pregnancy or to experience an implantation failure.
  • the method comprises the determination of the presence of and/or the measure of the quantity (typically expressed as a ratio or as an arbitrary unit) of at least auto antibodies directed against CD 146 (AAACD146) in a biological sample of the subject.
  • AAACD146 in the subject is typically indicative of the presence of an autoimmune disease, of a disorder or anomaly associated to a high-risk that an embryo implantation failure (IF) occurs, or of a high-risk pregnancy, in particular is indicative of the presence of a disorder associated to a high-risk pregnancy or is indicative of a high risk for an anomaly as herein described to occur.
  • IF embryo implantation failure
  • AAACD146 The presence of AAACD146 in the subject can further be indicative of a predisposition or risk to develop such a disease or disorder or experience such an anomaly.
  • the level or amount of AAACD146 in the biological sample of the subject is compared to a control value (typically a cut-off value obtained in a control population), the presence of AAACD146 in a quantity different (below or above) from, typically above, the control value being indicative of the presence of an autoimmune disease, of a high-risk pregnancy (i.e., of a disease or disorder associated to a high-risk pregnancy), or of a disorder or anomaly associated to a high-risk that an embryo implantation failure (IF) occurs; or of a predisposition or risk for the subject to develop an autoimmune disease, or a disease or a disorder associated to a high-risk pregnancy; or of an increased risk that an implantation failure occurs, or that an anomaly (as herein described), associated to a high- risk pregnancy, occurs.
  • a control value typically a cut-off value obtained in a control population
  • the invention provides an in vitro or ex vivo method of determining the prognosis of, or of monitoring the course of, an autoimmune disease or of a high-risk pregnancy, typically of a particular disease, disorder or anomaly associated to a high-risk pregnancy or to a high risk that an implantation failure occurs, in a subject.
  • the method comprises the determination of the presence of and/or the measure of the quantity of at least auto antibodies directed against CD146 (AAACD146) in a biological sample of the subject, at different times, the presence (following the absence) or a variation (increase or decrease), typically an increase, in the quantity of auto antibodies detected during time being indicative of a worsening of the autoimmune disease or of the high-risk pregnancy, i.e., is indicative of a worsening of the disease or disorder associated to the high-risk pregnancy (as herein described) or is indicative of a stabilized or increased risk that an anomaly (as herein described), associated to a high-risk pregnancy or to a high risk of implantation failure, occurs.
  • AAACD146 the measure of the quantity of at least auto antibodies directed against CD146
  • the invention relates to an in vitro or ex vivo method of assessing the efficiency of a treatment of an autoimmune disease, of a disease, disorder or anomaly associated to a high-risk pregnancy, or of a disorder associated to a high-risk that an implantation failure occurs in a subject.
  • the method comprises the determination of the presence of and/or the measure of the quantity of at least autoantibodies directed against CD 146 (AAACD146) in a biological sample of the subject, at different times before, during and/or after the treatment, the absence (following the presence) or a variation (increase or decrease), typically a decrease, in the quantity of auto antibodies during time being indicative of an improvement of the health's subject regarding the autoimmune disease, the disease or disorder associated to the high-risk pregnancy, or the disorder associated to a high-risk that an implantation failure occurs.
  • Such an absence or variation may be indicative of a decreased risk that an anomaly (as herein described), associated to a high-risk pregnancy or associated to a high-risk of embryo implantation failure (IF), occurs
  • AAACD146 auto-antibodies directed against CD 146
  • inventors investigated their presence in a cohort of 140 patients suffering of an autoimmune disease and compared the results with those obtained from healthy subjects using immunoassays (ELISA, anti autoantibody absorption tests, western-blots). The comparison reveals significantly higher level (quantity of antibodies) and prevalence (patient positive for antibody in the autoimmune disease) of AAACD146 in patients suffering of an autoimmune disease than in healthy subjects. They discovered that patients suffering of systemic sclerosis (SSc) exhibited a particularly high level and prevalence of AAACD146.
  • SSc systemic sclerosis
  • AAACD146 as a new and very efficient (highly sensitive and specific) immunological biomarker capable of detecting patients suffering of or at risk of developing an autoimmune disease or a high-risk pregnancy or at risk of experiencing an implantation failure.
  • “Auto antibody directed against CD146” or “Anti-CD 146 auto-antibody” or “AAACD146” refers to a human antibody produced by the immune system of a human subject which is directed against a CD 146 protein, peptide or amino acid molecule.
  • “Human CD 146 protein” or “CD 146” refers to a human protein, peptide or amino acid molecule, mainly present in the membrane of endothelial cells, which is either the "long CD 146 protein” or the “short CD 146 protein”.
  • Human long CD 146 protein or “long CD 146” refers to a human protein, peptide or amino acid molecule having an amino acid sequence corresponding to the following SEQ ID NO: 8 :
  • Human short CD 146 protein or “short CD 146” refers to a human protein, peptide or amino acid molecule having an amino acid sequence corresponding to the following SEQ ID NO: 9 :
  • An example of a human soluble CD 146 protein according to the present invention comprises at least residues 1 to 552 inclusive, preferably at least residues 1 to 557 inclusive, of the ammo acid sequence SEQ ID NO: 8.
  • soluble CD 146 of the invention comprises an amino acid sequence consisting in SEQ ID NO: 1
  • SEQ ID NO: 5 MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLSHVD WFSVHKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLCQGKRPRSQ EYRIQLRVYKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYKNGRPLKEEKNR VHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYRLPSGNHMKESREVTVPVFYPTE KVWLEVEPVGMLKEGDRVEIRCLADGNPPPHFSISKQNPSTREAEEETTNDNGVLVLEPARKE HSGRYECQAWNLDTMISLLSEPQELLVNYVSDVRVSPAAPERQEGSSLTLTCEAESSQDLEFQ WLREETDQVLERGPVLQLHDLKREAGGGYRCVASVPSIPGLNRTQLV
  • SEQ ID NO: 6 MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLSHVD WFSVHKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLCQGKRPRSQ EYRIQLRVYKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVrWYKNGRPLKEEKNR VHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYRLPSGNHMKESREVTVPVFYPTE KVWLEVEPVGMLKEGDRVEIRCLADGNPPPHFSISKQNPSTREAEEETTNDNGVLVLEPARKE HSGRYECQAWNLDTMISLLSEPQELLVNYVSDVRVSPAAPERQEGSSLTLTCEAESSQDLEFQ WLREETDQVLERGPVLQLHDLKREAGGGYRCVASVPSIPGLNRTQ
  • CD 146 protein, peptide or amino acid molecule (long, short or soluble form) herein described, in particular any recombinant CD 146 protein, peptide or amino acid molecule, usable in the context of the present invention, is able to recognize, link or interact with AAACD146.
  • the term "biological sample” includes any biological sample from a subject, in particular a mammalian subject, typically a human being.
  • the biological sample may be a tissue biopsy, for example a skin biopsy, or a biological fluid sample.
  • Typical examples of biological samples usable in the context of the present invention may be selected from a plasma, a blood, a serum, a urinary, a cerebrospinal fluid, and a saliva sample.
  • the biological sample is a plasma, a blood or a serum sample, even more preferably a serum sample.
  • subject refers to any testable subject and typically designates a patient.
  • the subject is a mammal, even more preferably a human being.
  • the invention may be used both for an individual and for an entire population.
  • the subject may be tested whatever his/her age or sex.
  • the subject can be a subject at risk of developing any disease or disorder leading to the expression, in particular to an overexpression, of AAACD146 and/or to vascular damages, for example a disease or disorder associated to a variation of the concentration of soluble CD 146 and/or to a variation of the CD146 membrane expression.
  • the subject can be a subject at risk, or suspected to be at risk, of developing a specific disease, disorder or anomaly as herein described.
  • the patient can be a subject predisposed to (or suspected to be predisposed to) develop a scleroderma, in particular a cardiac complication associated to SSc, and/or a disease, disorder or anomaly independent of pregnancy such as implantation failure (IF) or associated with a high-nsk pregnancy as previously defined (see the background part).
  • a scleroderma in particular a cardiac complication associated to SSc
  • a disease, disorder or anomaly independent of pregnancy such as implantation failure (IF) or associated with a high-nsk pregnancy as previously defined (see the background part).
  • the subject can be asymptomatic, or present early or advanced signs of such a disease, disorder or anomaly.
  • the subject may be selected for example from a pregnant women with a genetic predisposition to a disorder associated to a high-risk pregnancy or to an anomaly (IF for example) as herein described, a multipara pregnant women who suffered, in the context of a previous pregnancy, from at least one such disorder associated to a high- risk pregnancy, or a women with an historic of implantation failure, and a woman positive for a marker selected from the anti-SSA antibody, the anti-phospholipid antibody, and the anti-thyroid antibody.
  • the control population is also a population of pregnant women.
  • the control population is preferably also a population of non pregnant women.
  • the subject may be selected for example from patient presenting at least one symptom of a known autoimmune disease, in particular a scleroderma symptom, typically a symptom selected from a deep induration of the layers of the skin and/or of underlying tissues, a thickening of the layers of the skin and/or of underlying tissues, a non-pitting induration, a deterioration of the conjunctive tissue of an organ, a proximal scleroderma symptom, sclerodactily, digital pitting scars of fingertips, loss of substance of the distal finger pat, a bilateral basilar pulmonary fibrosis, a cutaneous manifestation of lcSSc or dcSSc, a Raynaud's phenomenon, a calcinosis, a gastrointestinal disorder, typically an esophageal dysmotility, a sclerodactily, a telangiectasia, a pulmonary arterial hypertension, a intersti
  • the symptom may further be a symptom of lupus, in particular SLE, a symptom of rheumatoid arthritis, a symptom of vasculitis, or a symptom of the antiphospholipid syndrome as described previously.
  • the subject may further be selected from a subject the serum of whom comprises an auto-antibody selected for example from an anti-endothelial cell antibody (AECA); an antinuclear antibody (ANA), typically an anti-topoisomerase I antibody (ATA), an anti-centromere antibody (ACA), an anti-RNA polymerase III (RNAP III) antibody; an anti-ribonucleoprotein antibody; the rheumatoid factor; an anti-fibroblasts antibody (AFA); and an autoantibody directed against the PDGF receptor; an anti- double strand DNA (anti ds DNA) antibody; an anti-Smith (anti-SM) antibody, an anti-PCNA antibody; an antiphospholipid antibody; an anti-SSA antibody; an anti-SSB antibody; an anti-RNP
  • a particular subject is a subject who is seronegative for ATA and/or ACA.
  • Another particular subject has at least one symptom of a scleroderma, as explained previously, and is seronegative for ATA and/or ACA.
  • Autoimmune disease refers to a disease caused by auto antibodies produced by the body against its own tissues.
  • the autoimmune disease leads to inflammation and tissue damages.
  • autoimmune diseases should not be mixed up with inflammatory diseases such as psoriasis for example.
  • Autoimmune disease according to the present invention is characterized by an abnormal expression (expression versus non expression, over expression or underexpression), typically an overexpression (when compared to a reference value, i.e., the corresponding value in a control population), of AAACD146 as a biomarker. This abnormal expression of AAACD146 is typically responsible for vascular damages.
  • autoimmune diseases include, without limitation, scleroderma (as herein defined), for example systemic sclerosis (SSc); a scleroderma related disorder in particular a heart, lung, kidneys or Esophagus complication, more particularly a cardiac complication such as left heart failure or severe pulmonary arterial hypertension or any other known cardiac complication; lupus, typically systemic lupus erythematosus (SLE); rheumatoid arthritis (RA); anti-phospholipid syndrome (APS); and vasculitis.
  • SSc systemic sclerosis
  • a scleroderma related disorder in particular a heart, lung, kidneys or Esophagus complication, more particularly a cardiac complication such as left heart failure or severe pulmonary arterial hypertension or any other known cardiac complication
  • lupus typically systemic lupus erythematosus (SLE); rheumatoid arthritis (RA); anti-phospholipid syndrome (APS); and va
  • a disorder associated with a high-risk pregnancy is characterized by an abnormal expression (expression versus non expression, over expression or underexpression), typically an overexpression, of AAACD146 as a biomarker.
  • This abnormal expression of AAA CD 146 is typically responsible for vascular damages.
  • Typical examples of such diseases or disorders associated to high-risk pregnancies include, without limitation pre-eclampsia (PE) and intrauterine growth retardation (IUGR).
  • PE pre-eclampsia
  • IUGR intrauterine growth retardation
  • a high-risk pregnancy is also considered as a pregnancy associated with a high risk that an anomaly or adverse pregnancy outcome such as a fetal loss (FL) or spontaneous abortion, typically spontaneous early abortion or miscarriage (occurring before the 20 th weeks of gestation), spontaneous late abortion (occurring after the 20 th weeks of gestation) or stillbirth; or preterm delivery occurs.
  • FL fetal loss
  • spontaneous abortion typically spontaneous early abortion or miscarriage (occurring before the 20 th weeks of gestation), spontaneous late abortion (occurring after the 20 th weeks of gestation) or stillbirth; or preterm delivery occurs.
  • Another anomaly which may be detected with the present invention is a high risk that an implantation failure (IF) occurs.
  • IF implantation failure
  • Diagnostic refers to the detection or identification of a disease or disorder as herein defined, or to the evaluation (dosing, comparison) of the severity or of the progression of such a disease or disorder in a subject as herein defined.
  • a diagnostic method of the invention comprises the determination of the presence and/or the measure of the quantity of AAACD 146 present in a biological sample of a subject, and preferably the comparison of the quantity to a control value. More precisely, the presence (versus absence) of AAACD146, or the presence of AAACD146 in a quantity distinct or different from (below or above), typically above, the control value, in the biological sample of the subject, is indicative of the presence, in the subject, of an autoimmune disease, of a disease, disorder or anomaly associated to a high-risk pregnancy, or of a disease, disorder or anomaly associated to a high risk that an implantation failure occurs.
  • Prediction refers to the assessment of a predisposition of the subject to develop an autoimmune disease or a high-risk pregnancy or to experience an implantation failure (as herein described).
  • prognostic refers to the assessment or monitoring of the progression (course) of a disease or disorder (as herein defined) in a subject (as herein defined), treated or not, typically the prediction of the worsening of such a disease or disorder and associated harmful effects or, on the contrary, the prediction of an improvement of the subj ec s health.
  • a prognostic method of the invention can comprise one or several steps of monitoring, dosing, comparing the measured quantity(ies) or level(s) of AAACD146, etc., at various stages, including early, pre-symptomatic stages, and late stages, in a biological sample or in biological samples from the subject.
  • Prognosis typically includes the assessment (prediction) of the progression of an autoimmune disease or of a disorder associated with high-risk pregnancy, and the characterization of a subject to define the most appropriate treatment.
  • control value or "cut-off value” can refer to a basal value corresponding to the mean of values (measured levels, quantities or concentrations) obtained with the biological samples of a reference population, typically of a population or cohort of healthy subjects, i.e., of subjects who do not suffer from a disease, disorder or anomaly as herein defined.
  • autoimmune disease subjects who do not suffer of an autoimmune disease or subjects who suffer of an autoimmune disease distinct from the autoimmune disease suspected in the tested subject;
  • the control value can also be a statistic or discriminating value, i.e., a value which has been determined by measuring the parameter in both a healthy control population and a population with a known disease or disorder as herein defined.
  • the discriminating value identifies the diseased population with a predetermined specificity and/or a predetermined sensitivity based on an analysis of the relation between the parameter values and the known clinical data of the healthy control population and of the diseased patient population (see the detailed discussion in the examples herein). The discriminating value determined in this manner is valid for the same experimental setup in future individual tests
  • ROC Receiver Operating Characteristic
  • a ROC curve is a graphical plot of the sensitivity (or true positive rate), vs. false positive rate (1 - specificity or 1 - true negative rate), for a binary classifier system. Each point on the ROC plot represents a sensitivity/specificity pair corresponding to a particular decision threshold.
  • the area under the ROC curve is a measure of how well a parameter can distinguish between two diagnostic groups (diseased/normal). In other words, "specificity" is defined as the proportion of positives (i. e.
  • sensitivity is defined as the proportion of negatives (i. e. individuals having a parameter representing the concentration of AAACD146 in body fluid samples different, typically lower, than a predefined diagnostic level) that are correctly identified by the described method.
  • the discriminating value may be expressed as a concentration of the biomarker in the biological sample of the tested subject for a particular specificity and/or sensitivity, or may be a normalized cutoff value expressed as a ratio for a particular specificity and/or sensitivity.
  • the cut-off value can easily be changed by the skilled person.
  • an odd ratio may be calculated.
  • a predictive positive value risk of developing the disease in the presence of AAACD146
  • a negative one risk of developing the disease in the absence of AAA CD 1466
  • a strong odd ratio of 13.5 [1.57 - 116]
  • a predictive positive value of 90% and a negative one of 60% has been calculated in SSc patients with cardiac complications.
  • the control value depends of the pregnancy status. Indeed antibodies levels are lower in pregnant women than in non pregnant women as demonstrated in the experimental part. In order to assess the evolution of a disease or control the efficiency of the treatment, testing a patient and testing the same patient several days, weeks or months later can be of help. In such a situation, the results (measured value(s)) of the second test are compared with the results of the first test.
  • a quantity of antibody "above the control value” or “higher than the control value” may mean a significant statistical increase, for example of at least 2 standard deviations.
  • an in vitro or ex vivo method for the detection of an autoimmune disease or of a high-risk pregnancy in a subject, or for the determination of the predisposition of a subject to develop an autoimmune disease, to develop a high-risk pregnancy or to experience an implantation failure is herein described.
  • the method comprises the determination of the presence of and/or the measure of the level or quantity (typically expressed as a ratio or as an arbitrary unit) of at least auto antibodies directed against CD146 (AAACD146) in a biological sample of the subject.
  • AAACD146 auto antibodies directed against CD146
  • the presence of AAACD146 in the subject, or the presence in a quantity different, typically above a control value, is indicative for the presence of an autoimmune disease, of a disorder or anomaly associated to a high-risk that an embryo implantation failure (IF) occurs, or of a disorder associated to a high-risk pregnancy, or of a predisposition or risk for said subject to develop such a disease or disorder or experience such an anomaly.
  • IF embryo implantation failure
  • the present invention provides an in vitro method for the detection of an autoimmune disease, preferably selected from scleroderma, in particular systemic sclerosis (SSc); lupus; rheumatoid arthritis; anti-phospholipid syndrome (APS) and vasculitis, even more preferably from SSc, lupus, rheumatoid arthritis and anti-phospholipid syndrome, in a subject, or for the determination of the predisposition of a subject to develop an autoimmune disease, to develop a high- risk pregnancy or to experience an implantation failure, comprising the determination of the presence of and/or the measure of the quantity of auto antibodies directed against CD 146 in a biological sample of the subject and comparison to a control value.
  • the autoimmune disease is SSc.
  • a scleroderma related disorder in particular a cardiac complication or disorder
  • a subject suffering of a SSc or for the determination of the predisposition of a subject suffering of a SSc to develop a scleroderma related disorder, comprising the determination of the presence of and/or the measure of the quantity of auto antibodies directed against CD 146 in a biological sample of the subject and comparison to a control value.
  • the presence of antibodies directed against CD 146, or the presence in a quantity different from the control value is indicative for the presence of a disease or disorder associated to a high-risk pregnancy in the tested subject or for a predisposition of said subject to develop such a disease or disorder.
  • a particular in vitro method for the detection of an intrauterine growth retardation (IUGR) in a pregnant subject, or for the determination of the predisposition of a subject to suffer from an IUGR comprises the determination of the presence of and/or the measure of the quantity of auto antibodies directed against CD146 in a biological sample of the subject and comparison to a control value.
  • IUGR intrauterine growth retardation
  • a particular in vitro method for the determination of the predisposition of a subject to suffer from embryo implantation failure comprises the determination of the presence of and/or the measure of the quantity of auto antibodies directed against CD 146 in a biological sample of the subject and comparison to a control value.
  • an in vitro or ex vivo method of determining the prognosis of, or of monitoring the course, in a subject, of, an autoimmune disease, of a high-risk pregnancy or of a disorder associated to a high risk that an implantation failure occurs is herein described.
  • the method comprises the determination of the presence of and/or the measure of the quantity of at least auto antibodies directed against CD146 (AAACD146) in a biological sample of the subject, at different times, the presence (following the absence) or a variation (increase or decrease), typically an increase, in the quantity of auto antibodies detected during time being indicative of a worsening of the autoimmune disease or of the high-risk pregnancy, i.e., is indicative of a worsening of the disease or disorder associated to the high-risk pregnancy or is indicative of a stabilized or increased risk that an anomaly (as herein described), associated to a high-risk pregnancy or to a high risk of implantation failure, occurs.
  • AAACD146 the measure of the quantity of at least auto antibodies directed against CD146
  • an in vitro or ex vivo method of assessing, in a subject, the efficiency of a treatment of an autoimmune disease, of a disease, disorder or anomaly associated to a high-risk pregnancy, or of a disorder associated to a high-risk that an implantation failure occurs is herein provided.
  • This method comprises the determination of the presence of and/or the measure of the quantity of at least auto antibodies directed against CD 146 (AAACD146) in a biological sample of the subject, at different times before, during and/or after the treatment, the absence (following the presence) or a variation (increase or decrease), typically a decrease, in the quantity of auto antibodies during time being indicative of an improvement of the health's subject regarding the autoimmune disease, the disease or disorder associated to the high-risk pregnancy, or the disorder associated to a high-risk that an implantation failure occurs.
  • Such an absence or variation may be indicative of a decreased risk that an anomaly (as herein described), associated to a high-risk pregnancy or associated to a high-risk of embryo implantation failure (IF), occurs.
  • the anti-CD 146 auto-antibody used in the herein described methods of the invention may be used as a biomarker of a particular disease or disorder of interest alone or together with (in combination with) at least one, possibly two or more, distinct known antibody(ies). Detection and/or quantification of AAACD146 may therefore be combined to the detection of the at least one, possibly two or more (possibly each known antibodies for a particular disease or for several diseases), distinct known antibody(ies) using known antigens such as CENP B, Scl-70, RNA polymerase III, Fibrillarine, and PM-Scl.
  • known antigens such as CENP B, Scl-70, RNA polymerase III, Fibrillarine, and PM-Scl.
  • Such a distinct antibody may be selected:
  • AECA anti-endothelial cell antibody
  • AN A antinuclear antibody
  • ATA anti-topoisomerase I antibody
  • ACA anti-centromere antibody
  • RNAP III anti-KNA polymerase III
  • AFA autoantibody directed against the PDGF receptor
  • an anti-endothelial cell antibody AECA
  • an anti-citrullinated protein antibody ACPA
  • anti-CCP anti-cyclic citrullinated peptide
  • an antinuclear antibody ACPA
  • ANCA antineutrophil cytoplasmic antibody
  • a lupus anticoagulant antibody from a lupus anticoagulant antibody, a anticardiolipin antibody, a anti-p2 glycoprotein antibody, an anti-phosphatidylethanolamine antibody, an anti-prothrombin antibody, an anti-annexin V antibody, and an anti-LDLox antibody, in particular an antibody of the IgG or IgM type.
  • the anti-SSA antibody for a disease or disorder associated to a high risk pregnancy, from the anti-SSA antibody and the anti-phospholipid antibody.
  • the distinct antibody is selected from an anti-nuclear antibody, an anti- fibroblast antibody, an anti-phospholipid antibody, and an anti-PDGF receptor antibody.
  • any CD 146 protein, peptide or amino acid molecule as well as any fragment thereof capable of recognizing, linking or interacting with AAACD146 may be used in the context of the present invention for determining the presence or measuring the amount of AAA CD 146 in a biological sample of a subject.
  • the CD146 protein, peptide or amino acid molecule is a CD146 protein, peptide or amino acid molecule (long, short or soluble form) as herein described.
  • the CD 146 protein, peptide or amino acid molecule has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8 and SEQ ID NO: 9.
  • Each of the herein described antigens may be labelled through direct labelling (using a luminescent enzyme, a radioisotope, a fluorochrome, a tag polypeptide sequence such as the c-myc tag, etc.) or through indirect labelling (using for example a labelled antibody or labelled affinity pair reagents such as, but not exclusively, the avidine-biotine pair).
  • the biological sample is typically a fluid sample.
  • Typical examples of biological samples usable in the context of the present invention may be selected from a blood, a serum, a plasma, a urinary, a cerebrospinal fluid, and a saliva sample.
  • the biological sample is a blood or a serum sample.
  • the biological sample is preferably a diluted sample, the dilution being typically of 1/50, 1/100, 1/200 or 1/400.
  • the determination of the presence as well as the measure of the quantities of auto antibodies, in particular of AAACD146 is determined in an immunoassay through a one step method wherein the subject's biological sample is directly contacted with the appropriate antigen or through a method implying a preliminary treatment of the biological sample.
  • the immunoassay can be performed through well known methods of the art: in solid phase or homogeneous phase, in one or two steps, through competitive method, etc.
  • said immunoassay is selected from the group consisting of ELISA, FEIA, western blot, dot blot, bead-based assay, antigen array and Radio Immuno Assay.
  • an antigen In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a colored product.
  • the colored product is fluorescent.
  • the final product is radioactive.
  • Protein detection using the dot blot protocol is similar to western blotting in that both methods allow for the identification and analysis of proteins of interest.
  • Dot blot methodology differs from traditional western blot techniques by not separating protein samples using electrophoresis. Sample proteins are instead spotted onto membranes and hybridized with an antibody probe. Bead-based assay or antigen array are new approaches for investigators to simultaneously measure multiple analytes in biological and environmental samples.
  • Semi-quantitative measurements can be obtained with each of the previously described methods using for example normal controls to normalize the value and then establish a ratio, or using a positive control as a calibrator (expressed in arbitrary units).
  • the detection may be performed on a solid support, for example a microplaque, on which are laid out in a definite and ordered way the antigens corresponding to plurality of antibodies, in addition to AAACD146, to be detected and/or quantified, or solid particles, test tubes, etc.
  • a solid support for example a microplaque, on which are laid out in a definite and ordered way the antigens corresponding to plurality of antibodies, in addition to AAACD146, to be detected and/or quantified, or solid particles, test tubes, etc.
  • kits comprising any one or more of the herein-described products or compositions.
  • the kit comprises at least one CD 146 protein or a fragment thereof as herein described capable of detecting AAA CD 146.
  • the kit also comprises one or more containers filled with one or more of the herein described products.
  • a labelling notice providing instructions for using the products in the context of a method according to the present invention can further be provided.
  • FIG. 3 AAACD146 ratio: comparison between pregnant and non pregnant women
  • FIG. 4 AAACD146 ratio in pathologic pregnant complications
  • FIG. 5 AAACD146 ratio is upregulated in women with implantation failure - panel 1
  • Ratio of anti-CD 146 autoAb in the cohort of auto immune diseases Mann Whitney test P ⁇ 0.0001.
  • Obstetrical APS or SAPL obstetrical primary anti-phospholipid syndrom
  • FIG. 11 AAACD146 ratio is upregulated in women with implantation failure - panel 2
  • FIG. 12 AAACD146 ratio decrease is observed during normal pregnancy but not during abnormal pregnancy.
  • EXAMPLE 1 Identification of AAACD146 as an immunological biomarker of autoimmune diseases
  • Venous blood samples were sent to an immunology laboratory by physicians working in the Public Assistance of Marseilles Hospitals for an immunological investigation (or testing). After serum separation, samples were stored at -80°C until further analysis. Patients were issued from the internal medicine, rheumatology, and dermatology units of the university teaching hospital in Marseilles. 153 sera from patients with autoimmune diseases were collected and retrospectively analyzed: 55 with systemic sclerosis (SSc), 36 with rheumatoid arthritis (RA), 37 with systemic lupus erythematosus (SLE), and 9 patients with vasculitis. As controls, inventors studied the sera of 50 age and sex matched blood donors.
  • SSc systemic sclerosis
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • ANA Total antinuclear antibodies
  • HEp-2 cells Bio-Rad, Hercules, CA, USA
  • Extractible antinuclear antibodies directed against topoisomerase I (Scl70) and centromeric B proteins (CentB) were detected by ELISA using ENA ELIATM test (Phadia GmbH, FREIBURG, Germany) performed on the Phadia Laboratory Systems ImmunoCAP.
  • the recombinant protein was separated by 10 % NuPage SDS-Polyacrylamide gels electrophoresis (Invitrogen; Frederik; Maryland; USA). Gels were transferred onto a nitrocellulose membrane (Invitrogen) using the iBlotTM device (Invitrogen; Frederik; Maryland; USA). Membrane blots were saturated in PBS containing 5% nonfat dry milk, supplemented with 1% of FCS, then with the anti- sera at a 1 :50 dilution.
  • Antibodies directed against CD 146 were detected using a solid phase absorbed rsCD146 (Biocytex, Marseille, France).
  • the protein was coated onto micro-ELISA plates (Nunc, Maxisorb, Roskilde, Denmark), at 10 ⁇ g ml in a bicarbonate buffer at pH 9,6, overnight at +4°C. After being washed with phosphate buffered saline [PBS] containing 0.1% Tween 20, the plates were saturated by incubation for 2 hours at 37°C with PBS containing 10% fetal calf serum [FCS].
  • Plasma samples were tested at a dilution of 1 : 100 in PBS containing 10% FCS, during 1 hour at +4°C.
  • a positive control was performed using anti-CD146 or S-Endo-1 at a dilution of 1:5000 in PBS containing 10% FCS (Biocytex, Marseille, France).
  • Negative controls were performed using the same protocol with uncoated wells. Plates were washed with PBS-Tween 20 0,5%.
  • the binding was detected by the addition of a monoclonal F(ab') 2 donkey anti-human IgG antibodies coupled to horseradish peroxydase (Jackson ImmunoResearch, Baltimore, USA) at 1 :2000 in PBS-10% FCS or goat anti-mouse IgG antibodies (positive control, Invitrogen, Carlsbad, USA) at 1 : 10000 in PBS-10% FCS, both conjugated with horseradish peroxydase, during 1 hour at +4°C.
  • a monoclonal F(ab') 2 donkey anti-human IgG antibodies coupled to horseradish peroxydase Jackson ImmunoResearch, Baltimore, USA
  • goat anti-mouse IgG antibodies positive control, Invitrogen, Carlsbad, USA
  • Sera were selected based on their positivity and on available serum volumes adequate for the following experiments. Two hundred microliters of diluted patient sera (1: 100) were added to the CD146-coated plate and incubated for 1 h at room temperature. After the first absorption, the second absorption was carried out by the same procedure and this absorption procedure was repeated identically up to six times. The absorbance was measured at each time as previously described, and the optical density (OD) measured from uncoated well was removed to the OD measured from the CD 146 corresponding coated well.
  • OD optical density
  • anti-CD146 autoAb Evidence for the presence of auto-antibodies directed against CD146 (anti-CD146 autoAb) in a cohort of patients with autoimmune diseases.
  • Anti-endothelial cell antibodies are a heterogeneous group of antibodies associated with several autoimmune diseases.
  • CD146 a cell adhesion molecule located in the endothelial junction and involved in angiogenesis, constitute, as herein demonstrated, a new antigenic target present on endothelial cells.
  • AAACD146 auto-antibodies directed against CD146
  • SSc systemic sclerosis
  • AAACD146 constitute a relevant marker of heart damage.
  • AAACD146 In the SSc population studied, 9 sera (18%) were negative for ACA and ATA. Among them, 6 (66%) were positive for AAACD146 Moreover no difference was observed for the presence of AAACD146 in limited or diffuses forms of the disease, showing that, conversely to the other conventional markers of SSc, ACA and ATA, the reactivity to CD 146 was not associated with the disease form. Thus, search for AAACD146 can be proposed to patients with both clinical features of SSc and in particular in patients seronegative for the immunological markers of the disease known in the art.
  • AAACD146 are in addition of great help as they can be detected at an early stage of the SSc disease since the earliest manifestation of the disease consists unequivocally of endothelial cell damage.
  • Venous blood samples were sent to an immunology laboratory by physicians working in the Public Assistance of Marseilles Hospitals for an immunological investigation (or testing). After serum separation, samples were stored at -80°C until further analysis. Patients were issued from the obstetrical units of the university teaching hospital in Marseilles.
  • Auto-antibodies directed against CD146 were detected using a solid phase absorbed rsCD 146 (Biocytex, Marseilles, France).
  • the protein was coated onto micro- ELISA plates (Nunc, Maxisorb, Roskilde, Denmark), at 10 ⁇ g/ml in a bicarbonate buffer at pH 9,6, overnight at +4°C. After being washed with phosphate buffered saline [PBS] containing 0.1% Tween 20, the plates were saturated by incubation for 2 hours at 37°C with PBS containing 10% fetal calf serum [FCS] .
  • PBS phosphate buffered saline
  • Plasma samples were tested at a dilution of 1 : 100 in PBS containing 10% FCS, during 1 hour at +4°C.
  • a positive control was performed using anti-CD146 or S-Endo-1 at a dilution of 1:5000 in PBS containing 10% FCS (Biocytex, Marseille, France).
  • Negative controls were performed using the same protocol with uncoated wells. Plates were washed with PBS-Tween 20 0,5%. The binding was detected by the addition of a monoclonal F(ab') 2 donkey anti-human IgG antibodies coupled to horseradish peroxydase (Jackson ImmunoResearch, Baltimore, USA) at 1 :2000 in PBS-10% FCS or goat anti-mouse IgG antibodies (positive control, Invitrogen, Carlsbad, USA) at 1 : 10000 in PBS-10% FCS, both conjugated with horseradish peroxydase, during 1 hour at +4°C.
  • a monoclonal F(ab') 2 donkey anti-human IgG antibodies coupled to horseradish peroxydase Jackson ImmunoResearch, Baltimore, USA
  • goat anti-mouse IgG antibodies positive control, Invitrogen, Carlsbad, USA
  • Ratio corresponded to Axl00/M, where A is the corrected absorbance of serum tested, and M is the mean of corrected absorbance obtained with sera controls.
  • PE pre-eclampsia
  • IUGR intrauterine growth retardation
  • EXAMPLE 3 Contribution of anti-CD146 auto-antibodies to the diagnosis of systemic lupus erythematosus (SLE)
  • EXAMPLE 4 Contribution of anti-CD146 auto-antibodies to the diagnosis of rheumatoid arthritis (RA)
  • EXAMPLE 5 Contribution of anti-CD146 auto-antibodies to the diagnosis of anti-phospholipid syndrom (APS or SAPL)
  • AAACD146 ratio seems to be lower in pregnant women with normal pregnancy (NP) than in non pregnant women with an history of normal pregnancy (hNP) (figure 3).
  • AAACD146 ratio decreases during normal pregnancy. In contrast, this decrease of the AAACD146 ratio is not observed in pregnant women with preeclampsia and intrauterine growth retardation ( Figure 12).

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Abstract

L'invention concerne des méthodes in vitro pour la détection d'une maladie auto-immune ou d'une grossesse à haut risque chez un sujet, ou pour la détermination de la prédisposition d'un sujet de développer une maladie auto-immune, de développer une grossesse à haut risque ou de présenter un défaut d'implantation, comprenant la détermination de la présence d'auto-anticorps anti-CD146 et/ou la mesure de la quantité d'auto-anticorps anti-CD 146 dans un échantillon biologique du sujet et la comparaison à une valeur témoin.
PCT/EP2012/050971 2011-01-21 2012-01-23 Auto-anticorps anti-cd146 et leurs utilisations WO2012098256A1 (fr)

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EP3629024A1 (fr) * 2018-09-26 2020-04-01 Assistance Publique - Hôpitaux de Marseille Cd146 et ses utilisations en tant que biomarqueur et cible thérapeutique dans le diagnostic et le traitement de la fibrose

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