WO2011015381A1 - Method for prognosis of pulmonary arterial hypertension by detecting anti-par1-antibodies - Google Patents

Method for prognosis of pulmonary arterial hypertension by detecting anti-par1-antibodies Download PDF

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Publication number
WO2011015381A1
WO2011015381A1 PCT/EP2010/004937 EP2010004937W WO2011015381A1 WO 2011015381 A1 WO2011015381 A1 WO 2011015381A1 EP 2010004937 W EP2010004937 W EP 2010004937W WO 2011015381 A1 WO2011015381 A1 WO 2011015381A1
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WIPO (PCT)
Prior art keywords
parl
receptor
disease
hypertension
patient
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PCT/EP2010/004937
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French (fr)
Inventor
Harald Heidecke
Kai Schulze-Forster
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Celltrend Gmbh
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Publication of WO2011015381A1 publication Critical patent/WO2011015381A1/en

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • 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
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host 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
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/321—Arterial hypertension
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
    • 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

Definitions

  • the present invention is in the filed of biology, chemistry more in particular immunology as well as diagnostics and therapeutics, more in particular in the field of diagnosis of pulmonary arterial hypertension, scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension or sickle-cell disease.
  • SLE systemic lupus erythematosus
  • Systemic sclerosis is a systemic form of scleroderma, a chronic autoimmune disease characterized by a hardening in the skin or other tissues or organs. Systemic sclerosis often has a fatal outcome, as a result of heart, kidney, lung or intestinal damage. Systemic sclerosis is usually associated with generalized fibrosis and particularly fibrosis of the skin. Systemic sclerosis is associated with the blood coagulation cascade and increased levels of coagulation factors. Systemic sclerosis can cause painful ulcers on the fingers or toes which are known as digital ulcers. Also common in systemic sclerosis is calcinosis, i.e. deposition of calcium in lumps under the skin, particularly seen near the elbows, knees or other joints.
  • system sclerosis can cause musculoskeletal, gastrointestinal, renal and other complications.
  • systemic sclerosis is associated with pulmonary hypertension, particularly pulmonary arterial hypertension.
  • Pulmonary hypertension relates to the increase in blood pressure in the lung vasculature (i.e. the pulmonary artery, pulmonary vein, or pulmonary capillaries) and results in shortness of breath, dizziness, fainting, and other symptoms.
  • Other conditions which can be associated with pulmonary arterial hypertension are for example systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • SLE systemic lupus erythematosus
  • graft rejection fibrosis
  • cancer CAD
  • cardiomyopathy malignant hypertension
  • portal hypertension portal hypertension
  • sickle-cell disease a systemic lupus erythematosus
  • the present invention addresses the need for a prognostic tool for the diagnosis of pulmonary arterial hypertension in patients suffering from a primary disease that may be associated with pulmonary arterial hypertension.
  • the present invention addresses the need for a prognostic tool for the prediction of pulmonary arterial hypertension in patients suffering from a primary disease that may be associated with pulmonary arterial hypertension.
  • the present invention relates to a method for diagnosis of a disease wherein, presence or absence of an anti-PARl -receptor antibody is determined in a sample from a patient to be diagnosed and wherein the presence of an anti-PARl -receptor antibody is indicative for the disease.
  • PARl- receptor relates to the protease-activated receptor 1.
  • the disease is selected from the group of scleroderma (particularly systemic sclerosis), preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • the present invention relates to a method for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension, comprising the steps of:
  • the presence of anti-PARl receptor antibodies is indicative for an increased risk of the patient for contracting pulmonary arterial hypertension.
  • the primary disease or condition is preferably selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • the invention relates to an immunoassay.
  • the immunoassay is a luciferase assay and/or an ELISA.
  • the diseases may be diagnosed by detecting the presence or absence of an anti-PARl antibody in a sample from a patient to be diagnosed.
  • the inventors have further found that the presence of high levels of anti-PARl antibodies in the blood of patients suffering from a primary disease or condition such as scleroderma, particularly systemic sclerosis, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease is indicative for a risk of such patients for contracting pulmonary arterial hypertension.
  • a primary disease or condition such as scleroderma, particularly systemic sclerosis, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease is indicative for a risk of such patients for contracting pulmonary arterial hypertension.
  • PARl and “PARl -receptor” equally relate to the protease-activated receptor 1.
  • PAR Protease-activated receptors
  • GPCR 7-trans-membrane-helix G protein-coupled receptor
  • PARl Four different types of PAR receptors have been identified, designated PARl, PAR2, PAR3 and PAR4. PARl and PAR2 are the best studied among the PAR-type receptors and share a sequence homology of 30 %.
  • PAR receptors are activated by the action of serine proteases such as thrombin (P AR 1 , 3 and 4) and trypsin (PAR 2).
  • PAR 1 and PAR2 are both activated by Factor Xa
  • PAR2 is also activated by proteinase III.
  • proteases cleave the N-terminus of the receptor, which in turn acts as a tethered ligand. In the cleaved state, part of the receptor itself acts as the agonist, causing a physiological response.
  • Most of the PAR-type receptors act through the actions of G-proteins, Raf/Ras activation and calcium signaling to cause cellular actions. Inactivation of PAR receptors is, inter alia, mediated by elastases and proteases.
  • PARl -deficient mouse embryos were shown to have a 50% intrauterine lethality. In the surviving knockout mice, an increased deposition of extracellular matrix components in the tissues, infiltration of lymphocytes in the lungs and the occurrence of membranoproliferative glomerulonephritis have been observed. PAR2-deficient mice have shown to exhibit a disturbed leukocyte migration and suffer from nephritis, arthritis and pneumonia.
  • Malignant neoplasia relates to malignant neoplasia.
  • malignant neoplasia include solid and haematological tumors.
  • Solid tumors are exemplified by tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (e.g. thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva.
  • endocrine glands e.g. thyroid and adrenal cortex
  • esophagus e.g. endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx
  • mesothelioma ovary
  • Malignant neoplasia include inherited cancers exemplified by Retinoblastoma and Wilms tumor.
  • malignant neoplasia include primary tumors in said organs and corresponding secondary tumors in distant organs ("tumor metastases").
  • Hematological tumors are exemplified by aggressive and indolent forms of leukemia and lymphoma, namely non-Hodgkins disease, chronic and acute myeloid leukemia (CML / AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma and T-cell lymphoma.
  • kidney cancer particularly renal cell carcinoma (RCC) which is also referred to as hypernephroma.
  • the present invention relates to a method for diagnosis of a disease wherein, presence or absence of an anti-PARl -receptor antibody is determined in a sample from a patient to be diagnosed and wherein the presence of an anti-PARl -receptor antibody is indicative for the disease.
  • the disease may preferably be selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • SLE systemic lupus erythematosus
  • graft rejection in the context of the present invention is preferably renal transplant rejection.
  • the methods according to the present invention may also be used for monitoring the progression of said diseases or for monitoring a therapy against said diseases.
  • the present invention relates to a method for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension, comprising the steps of:
  • anti-PARl receptor antibodies is indicative for an increased risk of the patient for contracting pulmonary arterial hypertension.
  • the primary disease or condition is preferably selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • the primary disease or condition more preferably is scleroderma, preeclampsia, systemic lupus erythematosus (SLE) or renal transplant rejection. It is particularly preferred that the primary disease or condition is scleroderma.
  • the "PARl -receptor” may be present in its natural cellular environment and can be used together with the material associated with the receptor in its natural state as well as in isolated form with respect to its primary, secondary and tertiary structures, the PARl -receptor is well known to those skilled in the art.
  • the isolated receptor should account for at least 0.5%, preferably at least 5% more preferably at least 25%, and in a particular preferred embodiment at least 50%.
  • the receptor is preferably used in isolated form, i.e. essentially free of other proteins, lipids, carbohydrates or other substances naturally associated with the receptor. "Essentially free of means that the receptor is at least 75%, preferably at least 85%, more preferably at least 95% and especially preferably at least 99% free of other proteins, lipids, carbohydrates or other substances naturally associated with the receptor.
  • the naturally occurring receptor as well as all modifications, mutants or derivatives of the PARl-receptor can be used.
  • a PARl- receptor produced by means of recombinant techniques which receptor includes amino acid modifications, such as inversions, deletions, insertions, additions etc. can be used according to the invention provided that this part of the essential function of the PARl-receptor is present, namely the capability of binding antibodies.
  • the PARl-receptor being used may also comprise exceptional amino acids and/or modifications of such as alkylation, oxidation, thiol-modification, denaturation, oligomerization and the like.
  • the receptor can also be synthesized by chemical means.
  • the PARl-receptor particularly can be a protein and/or peptide or a fusion protein, which in addition to other proteins, peptides or fragments thereof, includes the PARl-receptor as a whole or in part.
  • analogous properties can be determined by those skilled in the art.
  • polypeptides or peptides have 50-60%, 70% or 80%, preferably 90%, more preferably 95%, and most preferably 98% homology to peptides identified as PARl-receptor, and said homology can be determined, e.g. by means of Smith- Waterman homology search algorithm, using the MPFRCH program (Oxford Molecular), for example.
  • graft in the meaning of the invention is an organ or tissue which has been transplanted or is to be transplanted.
  • grafts can also be particular implants comprised of materials or components incorporated in a body for a limited period of time or for life in order to assume specific substitute functions.
  • such grafts can be made of inorganic matter coated with organic substances such as cartilage or bone cells.
  • graft rejection is to be understood to be induction of an immune reaction to the graft in the recipient.
  • An immune reaction in the recipient is a specific protective or defense reaction of the body against the antigens of the transplant.
  • Graft rejection in the context of the present invention is preferably a renal transplant rejection.
  • the term "peptide" of an PARl -receptor used in the present invention comprises also molecules differing from the original sequence by deletion(s), insertion(s), substitution(s) and/or other modifications well known in the prior art and/or comprising a fragment of the original amino acid molecule, the PARl -receptor still exhibiting the properties mentioned above.
  • Such a peptide has preferably at least a length of 12 amino acid residues, more preferably a length of at least 20 amino acid residues. Also included are allele variants and modifications. Methods of producing the above changes in the amino acid sequence are well known to those skilled in the art and have been described in the standard textbooks of molecular biology, e.g. Sambrook et al, supra. Those skilled in the art will also be able to determine whether a PARl -receptor, thus, modified still has the properties mentioned above. The amino acid sequence of two isoforms of the PARl receptor is given below and in the attached SEQ ID NO:1 and SEQ ID NO:2.
  • Residues 42 to 425 in SEQ ID NO: 1 and SEQ ID NO:2 relate to the amino acid sequence of the mature receptor, while residues 1 to 26 relate to a signal peptide and residues 27 to 41 are removed from the receptor proprotein upon activation.
  • the receptor may be glycosylated in vivo.
  • all of the above illustrated modifications of the PARl -receptor will be referred to as "functionally analogous peptides or proteins" in brief.
  • sample in the meaning of the invention can be all biological tissues and fluids such as blood, lymph, serum, plasma, urine, cerebral fluid.
  • the sample is collected from the patient and subjected to the diagnosis according to the invention.
  • the antibody can also be modified (e.g. oligomeric, reduced, oxidized and labeled antibodies).
  • anti-PARl -receptor antibody as used herein comprises both intact molecules and also anti-PARl -receptor antibody fragments such as Fab, F(ab') 2 and Fv capable of binding specific epitope determinance of the PARl- receptor, hi these fragments the anti-PARl -receptor antibody(ies) capability of selectively binding its antigen or receptor is retained in part, the fragments being defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be generated by cleavage of a whole antibody using the enzyme papaine, thereby obtaining an intact light chain and part of a heavy chain; (2) the Fab fragment of an antibody molecule can be produced by treatment of a whole antibody with pepsin and subsequent reduction, thereby obtaining an intact light chain and part of a heavy chain, two Fab fragments per antibody molecule are obtained; (3) F(ab') 2 the fragment of the antibody which can be obtained by treatment of a whole antibody with the enzyme peps
  • epitope represents any antigen determinant on the PARl -receptor.
  • Epitope determinance normally consists of chemically active surface groups of molecules such as amino acids or sugar-side chains and normally has specific features of the free dimensional structure as well as specific chart properties.
  • the anti-PARl -receptor antibody binds specifically to the PARl -receptor or in doing so shows specific immuno reactivity when the anti-PARl -receptor antibody assumes its function in a binding reaction in the presence of a heterogeneous population of PARl -receptors or fragments thereof, thereby allowing a conclusion whether the PARl -receptor or another biological structure is present.
  • the above-mentioned anti-PARl - receptor antibodies will preferably bind to a specific portion of the PARl -receptor, while no significant binding to other proteins present in the sample will take place.
  • Patients in the meaning of the invention are understood to be all persons or animals, particularly mammals.
  • any sample collected from cells, tissues, organs, organisms or the like can be a sample of a patient to be diagnosed.
  • the patient according to the invention is a human.
  • the patient is a human suspected to have a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • SLE systemic lupus erythematosus
  • an “immune reaction” in the meaning of the invention is a specific interaction between the PARl-receptor or peptides or proteins of analogous function and anti-PARl -receptor antibodies.
  • the immune reaction can be detected using various immunoassays.
  • Immunoassays in the meaning of the invention are assays utilizing the specific interaction between the PARl-receptor and peptides or proteins of analogous function and the anti-PARl- receptor antibodies, in order to detect the presence or determine the concentration of the anti- PARl -receptor antibodies.
  • a detection in quantification of the anti- PARl -receptor antibodies can be performed with the aid of said peptides or proteins of analogous function, e.g. by immuno precipitation or immuno blotting.
  • the determination of the presence or the absence of an anti- PARl -receptor antibody is done by detecting one or more of the antibodies selected from the group of IgA-antibody, IgG-antibody and IgM-antibody.
  • Human antibodies can be divided into five classes of immunoglobulins.
  • Class A immunoglobulin (IgA) exists in form which is dissolved in blood as well as in secretory variant.
  • IgA comprises two basic classes.
  • Secretory IgA consist of two immunoglobulin basic molecules, together with a J-chain and a secretory component. More, specifically IgA molecules can prevalent in body secretions.
  • Class IgG immunoglobulins represent the major part among the immunoglobulins.
  • the antibodies of the secondary immune response taking place upon contact of the immune system of a particular antigen largely belong to the IgG class.
  • the anti- PARl -receptor antibody is selected from the group of IgGl, IgG2, IgG3 and IgG4.
  • Immunoassays in the meaning of the invention are assays utilizing the specific interaction between the PARl -receptor and peptides or proteins of analogous function and the anti- PARl- receptor antibody, in order to detect the presence or determine the concentration of the anti- PARl -receptor antibodies.
  • the detection and quantification of the anti- PARl- receptor antibody can be performed with the aid of said peptides or proteins an analogous function, e.g. by immunoprecipitation or immunoblotting.
  • immunoassays in the meaning of the invention can be subdivided into the following steps: (1) anti- PARl -receptor antibody/ PARl -receptor reaction, (2) if required separation of the anti- PARl -receptor antibody complex from other components of the reaction mixture especially from non-bound anti- PARl- receptor antibodies an PARl -receptor and (3) measuring the response.
  • anti- PARl- receptor antibody/ PARl-receptor reaction various configurations of passable, e.g. (a) precipitation of one reaction with an access of the other or (b) competition between known quantities of anti- PARl-receptor antibody or PARl-receptor and the material to be investigated.
  • an assay for anti- PARl-receptor antibodies can be performed by a) using excess PARl -receptors/pep tides or proteins of analogous function or b) competition between a labeled anti- PARl-receptor antibody of known amount and non- labeled antibody of unknown amount for a defined quantity of PARl-receptor or peptides of proteins of analogous function.
  • the PARl-receptor can be immobilized on a solid support to allow separation of the anti-P ARl- receptor antibody/P ARl -receptor complex.
  • the solid support material can be nitrocellulose, polyvinylchloride or polystyrene, e.g. the well of a microtiter plate.
  • the immunoassay may take place in a microfluidic environment.
  • To measure the anti-P ARl -receptor antibody/P ARl -receptor interaction it is possible to use labeled anti-P ARl -receptor antibodies, labeled PARl -receptors or secondary reagents, for example.
  • the PARl-receptor can be labeled radioactively or with enzymes or with fluorescent compounds, for example. Irrespective of the label that is used, the response of the anti-PARl receptor antibody/P ARl -receptor interaction can be enhanced by utilizing the affinity of the proteins avidine or streptavidine for biotin.
  • the immunoassays used according to the invention can be: (1) immunoassays using radioactive label: (a) radioimmunoassay with competitive binding (RIA) and (b) immunoradiometric assay (IRMA); (2) immunoassays using an enzyme label: (a) enzyme immunoassays (EIA) and (b) enzyme-linked immunosorbenassys (ELISA); (3) immunoassays using a combination of radioisotope and enzyme labels (ultrasensitive enzyme radio immunoassay) (USERIA).
  • radioactive label (a) radioimmunoassay with competitive binding (RIA) and (b) immunoradiometric assay (IRMA)
  • IRMA immunoradiometric assay
  • EIA enzyme immunoassays
  • ELISA enzyme-linked immunosorbenassys
  • USERIA ultrasensitive enzyme radio immunoassay
  • the immunoassay according to the invention may be a fluorescent immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western Blot, a competitive immunoassay, a non-competitive immunoassay, a homogenous immunoassay, a heterogenous immunoassay, a reporter-assay, e.g. a luciferase assay.
  • the immunoassay is an ELISA.
  • the invention also relates to the use of an PARl -receptor peptide for the diagnosis of a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • the invention relates to the use of an PARl -receptor peptide for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension.
  • the preferred primary diseases and conditions are scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • the invention relates to a research and/or diagnostic kit for the diagnosis of a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease, wherein the kit comprises an PARl -receptor peptide or a functional analogue thereof.
  • a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease
  • the kit comprises an PARl -receptor peptide or a functional analogue thereof.
  • the invention relates in another aspect to a research and/or diagnostic kit for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension, wherein the kit comprises an PARl -receptor peptide or a functional analogue thereof.
  • the preferred primary diseases and conditions are scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • the immunological test kit comprises the PARl -receptor or a functional analog thereof or peptides or proteins of analogous function per se.
  • the test kit of the invention comprises at least one complete PARl -receptor or functionally analogous peptides or proteins of said receptor, optionally bound to a solid phase.
  • the test kit may also comprise buffers, specific conjugate together with an enzyme, wash solution, substrate solution to detect the immune reaction and/or a quenching solution. Using these substances a person skilled in the art will be able to perform, e.g. an ELISA to detect the anti- PARl -receptor antibodies.
  • the buffers, specific conjugate plus enzyme, wash solution, substrate solution to detect immune reaction and quenching solution are well known to those skilled in the art.
  • the test comprise a freeze-dried PARl -receptor or peptides or proteins of PARl -receptor analogous function and to add the buffers and other solutions immediately prior to testing the biological material.
  • the test kit with the PARl -receptor or its functionally analogous peptides of proteins bound to a solid phase.
  • the specific conjugate, wash solution, substrate solution and quenching solution which can be components of the test kit, have to be added according to a mode well known to those skilled in the art.
  • the test kit is a test strip comprising the PARl -receptor or its functionally analogous peptides or proteins immobilized on a solid phase.
  • the test strip can be immersed in serum or other patient samples and incubated.
  • a specific biochemical reaction on the test strip after formation of the PARl-receptor/anti- PARl-recepetor antibody complex a specific color reaction can be triggered by means of which the anti- PARl -receptor antibody can be detected.
  • the test system of the invention permits quantification of the anti- PARl -receptor antibodies directly in a sample, e.g. in plasma or serum of patients.
  • the detection method according to the invention is time saving and cost effected. Large amounts of the samples can be tested and, owing to the low amount of the equipment required, routine laboratories can be used.
  • the invention also relates to an inhibitor of an anti- PARl -receptor antibody and an inhibitor of an PARl -receptor for the production of a medicament.
  • inhibitor refers to an agent that binds to the receptor but does not provoke the normal biological response.
  • an inhibitor may be any molecule which, when bound to an PARl -receptor, decreases the activity of or reduces the expression levels of the PARl-receptor
  • the medicament is for the treatment or prevention of pulmonary arterial hypertension.
  • the invention also relates to inhibitors of the anti- PARl-receptor antibody such as antibodies and anti-sense molecules.
  • the present invention relates to the use of an inhibitor of an anti-PARl -receptor antibody or an inhibitor of an PARl-receptor for the production of a medicament for the treatment or prevention of pulmonary arterial hypertension.
  • the invention relates to the use of an inhibitor of an anti-PARl -receptor antibody or an inhibitor of an PARl-receptor for the production of a medicament for the treatment or prevention of pulmonary arterial hypertension, wherein the inhibitor of the PARl- receptor is selected from the group of a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
  • the invention pertains to a method for the removal of anti-PARl -receptor antibodies from isolated blood, (i) wherein in a first step the presence or absence of an anti- PARl -receptor antibody is determined in a blood sample from a patient to be diagnosed, wherein the patient is thought to have a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease, and (ii) wherein, upon determining the presence of an anti-PARl -receptor antibody the isolated blood of the patient is subjected to a plasmapheresis.
  • a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hyper
  • the invention also relates to a method for the removal of anti-PARl -receptor antibodies from isolated blood, (i) wherein in a first step the presence or absence of an anti-PARl -receptor antibody is determined in a blood sample from a patient to be diagnosed, wherein the patient is thought to have pulmonary arterial hypertension or thought to be at risk to contract pulmonary arterial hypertension, and (ii) wherein, upon determining the presence of an anti-PARl -receptor antibody the isolated blood of the patient is subjected to a plasmapheresis.
  • the invention also relates to the use of plasmapheresis for the isolation of an anti- PARl -receptor antibody from blood.
  • the present invention also relates to an antibody specifically binding to PARl.
  • the antibody is a human antibody.
  • antibody in the context of the present invention comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called “single- chain-antibodies” (Bird R. E. et al (1988) Science 242:423-6), chimeric, humanized, in particular CDR-grafted antibodies, and dia- or tetrabodies (Holliger P. et al (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-8). Also comprised are immunoglobulin like proteins that are selected through techniques including, for example, phage display to specifically bind to the molecule of interest contained in a sample.
  • the term "specific binding” refers to antibodies raised against the molecule of interest or a fragment thereof.
  • An antibody is considered to be specific, if its affinity towards the molecule of interest or the aforementioned fragment thereof is at least 50- fold higher, preferably 100- fold higher, more preferably at least 1000- fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to make antibodies and to select antibodies with a given specificity.
  • each assay result obtained may be compared to a "normal" value, or a value indicating a particular disease or outcome.
  • a particular diagnosis/prognosis may depend upon the comparison of each assay result to such a value, which may be referred to as a diagnostic or prognostic "threshold".
  • assays for one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicator(s) in the assay.
  • an assay can be designed so that a positive signal only occurs above a particular threshold concentration of interest, and below which concentration the assay provides no signal above background.
  • ROC curves Receiver Operating Characteristic curves
  • a threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal.
  • the area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition.
  • a threshold is selected to provide a ROC curve area of greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9.
  • the term "about” in this context refers to +/- 5% of a given measurement.
  • the horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives.
  • the vertical axis of the curve represents sensitivity, which increases with the rate of true positives.
  • the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained.
  • the area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition.
  • the area under the ROC curve can be used to determine the effectiveness of the test.
  • a positive likelihood ratio, negative likelihood ratio, odds ratio, or hazard ratio is used as a measure of a test's ability to predict risk or diagnose a disease.
  • a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased” and “control” groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group.
  • a value of 1 indicates that a negative result is equally likely among subjects in both the "diseased” and “control” groups; a value greater than 1 indicates that a negative result is more likely in the test group; and a value less than 1 indicates that a negative result is more likely in the control group.
  • a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and “control” groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group.
  • a value of 1 indicates that the relative risk of an endpoint (e.g., death) is equal in both the "diseased" and “control” groups; a value greater than 1 indicates that the risk is greater in the diseased group; and a value less than 1 indicates that the risk is greater in the control group.
  • associating a diagnostic or prognostic indicator, with a diagnosis or with a prognostic risk of a future clinical outcome is a statistical analysis.
  • a marker level of greater than X may signal that a patient is more likely to suffer from an adverse outcome than patients with a level less than or equal to X, as determined by a level of statistical significance.
  • a change in marker concentration from baseline levels may be reflective of patient prognosis, and the degree of change in marker level may be related to the severity of adverse events.
  • Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value.
  • Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.
  • EXAMPLES We measured the anti-PARl autoantibody by a sandwich ELISA (CellTrend GmbH Luckenwalde, Germany). The microtiter 96-well polystyrene plates were coated with extracts from Chinese hamster ovary cells overexpressing the human PARl. To maintain the conformational epitopes of the receptor, 1 mM calcium chloride was added to every buffer. Duplicate samples of a 1:100 serum dilution were incubated at 4°C for 2 hours. After washing steps, plates were incubated for 60 minutes with a 1:20.000 dilution of horseradish-peroxidase- labeled goat anti-human IgG (Jackson, USA) used for detection. In order to obtain a standard curve, plates were incubated with test sera from an anti-PARl autoantibody positive index patient. The ELISA was validated according to the FDA's "Guidance for industry: Bioanalytical method validation".
  • Figure 1 ROC analysis for the diagnosis of scleroderma based on PARl auto-antibody detection.
  • the control group are normal (healthy) subjects plus patients suffering from rheumatoid arthritis (RA).
  • RA rheumatoid arthritis
  • Figure 2 ROC analysis for the diagnosis of scleroderma based on PARl auto-antibody detection.
  • the control group are normal (healthy) subjects only.

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Abstract

The invention relates to a method for diagnosis of a disease, wherein presence or absence of an anti-PAR1 antibody is determined in a sample from a patient to be diagnosed. The disease according to the invention is in particular selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease. The invention further relates to the use of an inhibitor of an anti-PAR1 antibody or an inhibitor of a PAR1 for the production of a medicament as well as a method for removing PAR1 antibodies from isolated blood by means of plasmapheresis.

Description

METHOD FOR PROGNOSIS OF PULMONARY ARTERIAL HYPERTENSION BY
DETECTING ANTI-PARl ANTIBODIES
FIELD OF THE INVENTION
The present invention is in the filed of biology, chemistry more in particular immunology as well as diagnostics and therapeutics, more in particular in the field of diagnosis of pulmonary arterial hypertension, scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension or sickle-cell disease. BACKGROUND OF THE INVENTION
Systemic sclerosis is a systemic form of scleroderma, a chronic autoimmune disease characterized by a hardening in the skin or other tissues or organs. Systemic sclerosis often has a fatal outcome, as a result of heart, kidney, lung or intestinal damage. Systemic sclerosis is usually associated with generalized fibrosis and particularly fibrosis of the skin. Systemic sclerosis is associated with the blood coagulation cascade and increased levels of coagulation factors. Systemic sclerosis can cause painful ulcers on the fingers or toes which are known as digital ulcers. Also common in systemic sclerosis is calcinosis, i.e. deposition of calcium in lumps under the skin, particularly seen near the elbows, knees or other joints. In addition, system sclerosis can cause musculoskeletal, gastrointestinal, renal and other complications. In some patients, systemic sclerosis is associated with pulmonary hypertension, particularly pulmonary arterial hypertension. Pulmonary hypertension relates to the increase in blood pressure in the lung vasculature (i.e. the pulmonary artery, pulmonary vein, or pulmonary capillaries) and results in shortness of breath, dizziness, fainting, and other symptoms. Other conditions which can be associated with pulmonary arterial hypertension are for example systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease. Patients suffering from pulmonary arterial hypertension have a very poor outcome. No therapy for the treatment of pulmonary arterial hypertension is currently available.
The present invention addresses the need for a prognostic tool for the diagnosis of pulmonary arterial hypertension in patients suffering from a primary disease that may be associated with pulmonary arterial hypertension.
Furthermore, the present invention addresses the need for a prognostic tool for the prediction of pulmonary arterial hypertension in patients suffering from a primary disease that may be associated with pulmonary arterial hypertension.
SUMMARY OF THE INVENTION
The present invention relates to a method for diagnosis of a disease wherein, presence or absence of an anti-PARl -receptor antibody is determined in a sample from a patient to be diagnosed and wherein the presence of an anti-PARl -receptor antibody is indicative for the disease. "PARl- receptor" relates to the protease-activated receptor 1.
Preferably, the disease is selected from the group of scleroderma (particularly systemic sclerosis), preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
In a further aspect the present invention relates to a method for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension, comprising the steps of:
a. providing a sample from said patient, and
b. determining the presence or absence of anti-PARl receptor antibodies in said sample,
wherein the presence of anti-PARl receptor antibodies is indicative for an increased risk of the patient for contracting pulmonary arterial hypertension. The inventors have found that the presence of anti-PARl receptor antibodies in the blood a patients suffering from certain primary diseases is associated with the risk of such patients for contracting pulmonary arterial hypertension. The primary disease or condition is preferably selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease. hi one embodiment of the invention the invention relates to an immunoassay. There are numerous ways of performing an immunoassay in a preferred embodiment of the invention, the immunoassay is a luciferase assay and/or an ELISA.
DETAILED DESCRIPTION OF THE INVENTION The inventors have found that the diseases may be diagnosed by detecting the presence or absence of an anti-PARl antibody in a sample from a patient to be diagnosed. The inventors have further found that the presence of high levels of anti-PARl antibodies in the blood of patients suffering from a primary disease or condition such as scleroderma, particularly systemic sclerosis, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease is indicative for a risk of such patients for contracting pulmonary arterial hypertension.
In the context of the present invention the terms "PARl" and "PARl -receptor" equally relate to the protease-activated receptor 1.
Protease-activated receptors (PAR) are involved in a number of essential biological processes such as blood clotting, regulation of vascular tone and vascular permeability, motility of the gastrointestinal tract, perception of pain, inflammatory response (including arthritis), angiogenesis, muscle growth and bone cell differentiation and proliferation. PAR are members of the 7-trans-membrane-helix G protein-coupled receptor (GPCR) super family and are activated by cleavage of part of their extracellular domain. They are expressed throughout the body. Particularly high expression occurs in platelets, but also on endothelial cells, myocytes and neurons. Expression of PAR on cells is influenced by the presence of cytokines such as TNFα.
Four different types of PAR receptors have been identified, designated PARl, PAR2, PAR3 and PAR4. PARl and PAR2 are the best studied among the PAR-type receptors and share a sequence homology of 30 %.
PAR receptors are activated by the action of serine proteases such as thrombin (P AR 1 , 3 and 4) and trypsin (PAR 2). PAR 1 and PAR2 are both activated by Factor Xa, PAR2 is also activated by proteinase III. These proteases cleave the N-terminus of the receptor, which in turn acts as a tethered ligand. In the cleaved state, part of the receptor itself acts as the agonist, causing a physiological response. Most of the PAR-type receptors act through the actions of G-proteins, Raf/Ras activation and calcium signaling to cause cellular actions. Inactivation of PAR receptors is, inter alia, mediated by elastases and proteases.
A number of agonist and antagonist of PAR have been developed, however, their effects on activation and inhibition of the receptors is highly dependent on the type of cells carrying the respective receptor, the surrounding environment of these cells and ligand concentration. PARl -deficient mouse embryos were shown to have a 50% intrauterine lethality. In the surviving knockout mice, an increased deposition of extracellular matrix components in the tissues, infiltration of lymphocytes in the lungs and the occurrence of membranoproliferative glomerulonephritis have been observed. PAR2-deficient mice have shown to exhibit a disturbed leukocyte migration and suffer from nephritis, arthritis and pneumonia.
"Cancer" herein relates to malignant neoplasia. Examples of malignant neoplasia include solid and haematological tumors. Solid tumors are exemplified by tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (e.g. thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva. Malignant neoplasia include inherited cancers exemplified by Retinoblastoma and Wilms tumor. In addition, malignant neoplasia include primary tumors in said organs and corresponding secondary tumors in distant organs ("tumor metastases"). Hematological tumors are exemplified by aggressive and indolent forms of leukemia and lymphoma, namely non-Hodgkins disease, chronic and acute myeloid leukemia (CML / AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, cancers of unknown primary site as well as AIDS related malignancies. In a particular embodiment of the present invention, the cancer is kidney cancer, particularly renal cell carcinoma (RCC) which is also referred to as hypernephroma.
Thus, the present invention relates to a method for diagnosis of a disease wherein, presence or absence of an anti-PARl -receptor antibody is determined in a sample from a patient to be diagnosed and wherein the presence of an anti-PARl -receptor antibody is indicative for the disease.
The disease may preferably be selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease. Preferably the disease is scleroderma. Graft rejection in the context of the present invention is preferably renal transplant rejection.
The methods according to the present invention may also be used for monitoring the progression of said diseases or for monitoring a therapy against said diseases.
Furthermore, the present invention relates to a method for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension, comprising the steps of:
a. providing a sample from said patient, and
b. determining the presence or absence of anti-PARl receptor antibodies in said sample,
wherein the presence of anti-PARl receptor antibodies is indicative for an increased risk of the patient for contracting pulmonary arterial hypertension.
The primary disease or condition is preferably selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease. The primary disease or condition more preferably is scleroderma, preeclampsia, systemic lupus erythematosus (SLE) or renal transplant rejection. It is particularly preferred that the primary disease or condition is scleroderma.
It has been possible to demonstrate that there is a relationship between the presence of said anti- PARl -receptor antibody and the risk of such patients for contracting pulmonary arterial hypertension
The "PARl -receptor" may be present in its natural cellular environment and can be used together with the material associated with the receptor in its natural state as well as in isolated form with respect to its primary, secondary and tertiary structures, the PARl -receptor is well known to those skilled in the art. Based on the weight of the whole receptor in the preparation to be used according to the invention, the isolated receptor should account for at least 0.5%, preferably at least 5% more preferably at least 25%, and in a particular preferred embodiment at least 50%. The receptor is preferably used in isolated form, i.e. essentially free of other proteins, lipids, carbohydrates or other substances naturally associated with the receptor. "Essentially free of means that the receptor is at least 75%, preferably at least 85%, more preferably at least 95% and especially preferably at least 99% free of other proteins, lipids, carbohydrates or other substances naturally associated with the receptor.
In connection with the present invention, the naturally occurring receptor as well as all modifications, mutants or derivatives of the PARl-receptor can be used. Similarly, a PARl- receptor produced by means of recombinant techniques, which receptor includes amino acid modifications, such as inversions, deletions, insertions, additions etc. can be used according to the invention provided that this part of the essential function of the PARl-receptor is present, namely the capability of binding antibodies. The PARl-receptor being used may also comprise exceptional amino acids and/or modifications of such as alkylation, oxidation, thiol-modification, denaturation, oligomerization and the like. The receptor can also be synthesized by chemical means. According to the invention the PARl-receptor particularly can be a protein and/or peptide or a fusion protein, which in addition to other proteins, peptides or fragments thereof, includes the PARl-receptor as a whole or in part. Using conventional methods, peptides or polypeptides of the PARl-receptor which have functionally analogs, analogous properties can be determined by those skilled in the art. For example such polypeptides or peptides have 50-60%, 70% or 80%, preferably 90%, more preferably 95%, and most preferably 98% homology to peptides identified as PARl-receptor, and said homology can be determined, e.g. by means of Smith- Waterman homology search algorithm, using the MPFRCH program (Oxford Molecular), for example.
In connection with the present invention, a number of general terms will be used as follows: "graft" in the meaning of the invention is an organ or tissue which has been transplanted or is to be transplanted. In the meaning of the invention, however, grafts can also be particular implants comprised of materials or components incorporated in a body for a limited period of time or for life in order to assume specific substitute functions. For example, such grafts can be made of inorganic matter coated with organic substances such as cartilage or bone cells.
According to the invention "graft rejection" is to be understood to be induction of an immune reaction to the graft in the recipient. An immune reaction in the recipient is a specific protective or defense reaction of the body against the antigens of the transplant. Graft rejection in the context of the present invention is preferably a renal transplant rejection. The term "peptide" of an PARl -receptor used in the present invention, comprises also molecules differing from the original sequence by deletion(s), insertion(s), substitution(s) and/or other modifications well known in the prior art and/or comprising a fragment of the original amino acid molecule, the PARl -receptor still exhibiting the properties mentioned above. Such a peptide has preferably at least a length of 12 amino acid residues, more preferably a length of at least 20 amino acid residues. Also included are allele variants and modifications. Methods of producing the above changes in the amino acid sequence are well known to those skilled in the art and have been described in the standard textbooks of molecular biology, e.g. Sambrook et al, supra. Those skilled in the art will also be able to determine whether a PARl -receptor, thus, modified still has the properties mentioned above. The amino acid sequence of two isoforms of the PARl receptor is given below and in the attached SEQ ID NO:1 and SEQ ID NO:2. Residues 42 to 425 in SEQ ID NO: 1 and SEQ ID NO:2 relate to the amino acid sequence of the mature receptor, while residues 1 to 26 relate to a signal peptide and residues 27 to 41 are removed from the receptor proprotein upon activation. The receptor may be glycosylated in vivo. In the present specification all of the above illustrated modifications of the PARl -receptor will be referred to as "functionally analogous peptides or proteins" in brief.
Amino acid sequence of the human PARl receptor (isoform 1) [SEQ ID NO : 1 ] :
1 MGPRRLLLVA ACFSLCGPLL SARTRARRPE SKATNATLDP RSFLLRNPND
51 KYEPFWEDEE KNESGLTEYR LVSINKSSPL QKQLPAFISE DASGYLTSSW
101 LTLFVPSVYT GVFWSLPLN IMAIWFILK MKVKKPAWY MLHLATADVL
151 FVSVLPFKIS YYFSGSDWQF GSELCRFVTA AFYCNMYASI LLMTVISIDR
201 FLAWYPMQS LSWRTLGRAS FTCLAIWALA IAGWPLLLK EQTIQVPGLN
251 ITTCHDVLNE TLLEGYYAYY FSAFSAVFFF VPLIISTVCY VSIIRCLSSS
301 AVANRSKKSR ALFLSAAVFC IFIICFGPTN VLLIVHYSFL SHTSTTEAAY
351 FAYLLCvcvs SISCCIDPLI YYYASSECQR YVYSILCCKE SSDPSSYNSS
401 GQLMASKMDT CSSNLNNSIY KKLLT Amino acid sequence of the human PARl receptor (isoform 2) [SEQ ID NO : 2 ] : 1 MGPRRLLLVA ACFSLCGPLL SARTRARRPE SKATNATLDP RSFLLRNPND
51 KYEPFWEDEE KNESGLTEYR LVSINKSSPL QKQLPAFISE DASGYLTSSW
101 LTLFVPSVYT GVFWSLPLN IMAIWFILK MKVKKPAWY MLHLATADVL
151 FVSVLPFKIS YYFSGSDWQF GSELCRFVTA AFYCNMYASI LLMTVISIDR
201 FLAWYPMQS LSWRTLGRAS FTCLAIWALA IAGWPLLLK EQTIQVPGLN
251 ITTCHDVLNE TLLEGYYAYY FSAFSAVFFF VPLIISTVCY VSIIRCLSSS
301 AVANRSKKSR ALFLSAAVFC IFIICFGPTN VLLIAHYSFL SHTSTTEAAY
351 FAYLLCVCVS SISCCIDPLI YYYASSECQR YVYSILCCKE SSDPSSYNSS
401 GQLMASKMDT CSSNLNNSIY KKLLT
"Sample" in the meaning of the invention can be all biological tissues and fluids such as blood, lymph, serum, plasma, urine, cerebral fluid. The sample is collected from the patient and subjected to the diagnosis according to the invention. The "anti-PARl -receptor antibody" in the meaning of the invention, which is to be detected, binds the PARl -receptor in a specific fashion. The antibody can also be modified (e.g. oligomeric, reduced, oxidized and labeled antibodies). The term anti-PARl -receptor antibody as used herein comprises both intact molecules and also anti-PARl -receptor antibody fragments such as Fab, F(ab')2 and Fv capable of binding specific epitope determinance of the PARl- receptor, hi these fragments the anti-PARl -receptor antibody(ies) capability of selectively binding its antigen or receptor is retained in part, the fragments being defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be generated by cleavage of a whole antibody using the enzyme papaine, thereby obtaining an intact light chain and part of a heavy chain; (2) the Fab fragment of an antibody molecule can be produced by treatment of a whole antibody with pepsin and subsequent reduction, thereby obtaining an intact light chain and part of a heavy chain, two Fab fragments per antibody molecule are obtained; (3) F(ab')2 the fragment of the antibody which can be obtained by treatment of a whole antibody with the enzyme pepsin without subsequent reduction, F(ab')2 is a dimer comprised of two Fab fragments held together by two disulfate bonds; (4) Fv defined as fragment modified by genetic engineering which includes the variable region of the light chain and the variable region of the heavy chain is expressed in the form of two chains; and (5) single- chain antibody (SCA) defined as a molecule modified by genetic engineering, which includes the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker to perform a genetically fused single-chain molecule. The term "epitope" as used in the present invention represents any antigen determinant on the PARl -receptor. Epitope determinance normally consists of chemically active surface groups of molecules such as amino acids or sugar-side chains and normally has specific features of the free dimensional structure as well as specific chart properties. The anti-PARl -receptor antibody binds specifically to the PARl -receptor or in doing so shows specific immuno reactivity when the anti-PARl -receptor antibody assumes its function in a binding reaction in the presence of a heterogeneous population of PARl -receptors or fragments thereof, thereby allowing a conclusion whether the PARl -receptor or another biological structure is present. Under the present conditions of an immunoassay, the above-mentioned anti-PARl - receptor antibodies will preferably bind to a specific portion of the PARl -receptor, while no significant binding to other proteins present in the sample will take place.
"Patients" in the meaning of the invention are understood to be all persons or animals, particularly mammals. In the meaning of the invention, any sample collected from cells, tissues, organs, organisms or the like can be a sample of a patient to be diagnosed. In a preferred embodiment the patient according to the invention is a human. In a further preferred embodiment of the invention the patient is a human suspected to have a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
An "immune reaction" in the meaning of the invention is a specific interaction between the PARl-receptor or peptides or proteins of analogous function and anti-PARl -receptor antibodies. The immune reaction can be detected using various immunoassays. "Immunoassays" in the meaning of the invention are assays utilizing the specific interaction between the PARl-receptor and peptides or proteins of analogous function and the anti-PARl- receptor antibodies, in order to detect the presence or determine the concentration of the anti- PARl -receptor antibodies. For example, a detection in quantification of the anti- PARl -receptor antibodies can be performed with the aid of said peptides or proteins of analogous function, e.g. by immuno precipitation or immuno blotting.
In a preferred embodiment of the invention the determination of the presence or the absence of an anti- PARl -receptor antibody is done by detecting one or more of the antibodies selected from the group of IgA-antibody, IgG-antibody and IgM-antibody. Human antibodies can be divided into five classes of immunoglobulins. Class A immunoglobulin (IgA) exists in form which is dissolved in blood as well as in secretory variant. IgA comprises two basic classes. Secretory IgA consist of two immunoglobulin basic molecules, together with a J-chain and a secretory component. More, specifically IgA molecules can prevalent in body secretions.
Class IgG immunoglobulins represent the major part among the immunoglobulins. The antibodies of the secondary immune response taking place upon contact of the immune system of a particular antigen largely belong to the IgG class. In a particularly preferred embodiment of the invention, the anti- PARl -receptor antibody is selected from the group of IgGl, IgG2, IgG3 and IgG4.
"Immunoassays" in the meaning of the invention are assays utilizing the specific interaction between the PARl -receptor and peptides or proteins of analogous function and the anti- PARl- receptor antibody, in order to detect the presence or determine the concentration of the anti- PARl -receptor antibodies. For example, the detection and quantification of the anti- PARl- receptor antibody can be performed with the aid of said peptides or proteins an analogous function, e.g. by immunoprecipitation or immunoblotting. For example, immunoassays in the meaning of the invention can be subdivided into the following steps: (1) anti- PARl -receptor antibody/ PARl -receptor reaction, (2) if required separation of the anti- PARl -receptor antibody complex from other components of the reaction mixture especially from non-bound anti- PARl- receptor antibodies an PARl -receptor and (3) measuring the response. As for the anti- PARl- receptor antibody/ PARl-receptor reaction various configurations of passable, e.g. (a) precipitation of one reaction with an access of the other or (b) competition between known quantities of anti- PARl-receptor antibody or PARl-receptor and the material to be investigated.
For example, an assay for anti- PARl-receptor antibodies can be performed by a) using excess PARl -receptors/pep tides or proteins of analogous function or b) competition between a labeled anti- PARl-receptor antibody of known amount and non- labeled antibody of unknown amount for a defined quantity of PARl-receptor or peptides of proteins of analogous function.
The PARl-receptor can be immobilized on a solid support to allow separation of the anti-P ARl- receptor antibody/P ARl -receptor complex. For example, the solid support material can be nitrocellulose, polyvinylchloride or polystyrene, e.g. the well of a microtiter plate. The immunoassay may take place in a microfluidic environment. To measure the anti-P ARl -receptor antibody/P ARl -receptor interaction, it is possible to use labeled anti-P ARl -receptor antibodies, labeled PARl -receptors or secondary reagents, for example. The PARl-receptor can be labeled radioactively or with enzymes or with fluorescent compounds, for example. Irrespective of the label that is used, the response of the anti-PARl receptor antibody/P ARl -receptor interaction can be enhanced by utilizing the affinity of the proteins avidine or streptavidine for biotin. The immunoassays used according to the invention can be: (1) immunoassays using radioactive label: (a) radioimmunoassay with competitive binding (RIA) and (b) immunoradiometric assay (IRMA); (2) immunoassays using an enzyme label: (a) enzyme immunoassays (EIA) and (b) enzyme-linked immunosorbenassys (ELISA); (3) immunoassays using a combination of radioisotope and enzyme labels (ultrasensitive enzyme radio immunoassay) (USERIA). Further, the immunoassay according to the invention may be a fluorescent immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western Blot, a competitive immunoassay, a non-competitive immunoassay, a homogenous immunoassay, a heterogenous immunoassay, a reporter-assay, e.g. a luciferase assay. In a preferred embodiment of the invention the immunoassay is an ELISA. The invention also relates to the use of an PARl -receptor peptide for the diagnosis of a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
Furthermore, the invention relates to the use of an PARl -receptor peptide for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension. The preferred primary diseases and conditions are scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
In one aspect, the invention relates to a research and/or diagnostic kit for the diagnosis of a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease, wherein the kit comprises an PARl -receptor peptide or a functional analogue thereof. The invention relates in another aspect to a research and/or diagnostic kit for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension, wherein the kit comprises an PARl -receptor peptide or a functional analogue thereof. The preferred primary diseases and conditions are scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
The immunological test kit according to the invention comprises the PARl -receptor or a functional analog thereof or peptides or proteins of analogous function per se. The test kit of the invention comprises at least one complete PARl -receptor or functionally analogous peptides or proteins of said receptor, optionally bound to a solid phase. Furthermore, the test kit may also comprise buffers, specific conjugate together with an enzyme, wash solution, substrate solution to detect the immune reaction and/or a quenching solution. Using these substances a person skilled in the art will be able to perform, e.g. an ELISA to detect the anti- PARl -receptor antibodies. The buffers, specific conjugate plus enzyme, wash solution, substrate solution to detect immune reaction and quenching solution are well known to those skilled in the art. For example, it would be sufficient to have the test comprise a freeze-dried PARl -receptor or peptides or proteins of PARl -receptor analogous function and to add the buffers and other solutions immediately prior to testing the biological material. However, it is also possible to provide the test kit with the PARl -receptor or its functionally analogous peptides of proteins bound to a solid phase. To detect the anti- PARl -receptor antibodies the specific conjugate, wash solution, substrate solution and quenching solution, which can be components of the test kit, have to be added according to a mode well known to those skilled in the art. In another advantageous embodiment of the invention, it is envisioned that the test kit is a test strip comprising the PARl -receptor or its functionally analogous peptides or proteins immobilized on a solid phase. For example, the test strip can be immersed in serum or other patient samples and incubated. Using a specific biochemical reaction on the test strip after formation of the PARl-receptor/anti- PARl-recepetor antibody complex, a specific color reaction can be triggered by means of which the anti- PARl -receptor antibody can be detected.
The test system of the invention permits quantification of the anti- PARl -receptor antibodies directly in a sample, e.g. in plasma or serum of patients. The detection method according to the invention is time saving and cost effected. Large amounts of the samples can be tested and, owing to the low amount of the equipment required, routine laboratories can be used.
The invention also relates to an inhibitor of an anti- PARl -receptor antibody and an inhibitor of an PARl -receptor for the production of a medicament. The term "inhibitor" refers to an agent that binds to the receptor but does not provoke the normal biological response. For example, an inhibitor may be any molecule which, when bound to an PARl -receptor, decreases the activity of or reduces the expression levels of the PARl-receptor In a preferred embodiment the medicament is for the treatment or prevention of pulmonary arterial hypertension.
The invention also relates to inhibitors of the anti- PARl-receptor antibody such as antibodies and anti-sense molecules.
In another aspect the present invention relates to the use of an inhibitor of an anti-PARl -receptor antibody or an inhibitor of an PARl-receptor for the production of a medicament for the treatment or prevention of pulmonary arterial hypertension. In yet another aspect the invention relates to the use of an inhibitor of an anti-PARl -receptor antibody or an inhibitor of an PARl-receptor for the production of a medicament for the treatment or prevention of pulmonary arterial hypertension, wherein the inhibitor of the PARl- receptor is selected from the group of a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
In yet another aspect the invention pertains to a method for the removal of anti-PARl -receptor antibodies from isolated blood, (i) wherein in a first step the presence or absence of an anti- PARl -receptor antibody is determined in a blood sample from a patient to be diagnosed, wherein the patient is thought to have a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease, and (ii) wherein, upon determining the presence of an anti-PARl -receptor antibody the isolated blood of the patient is subjected to a plasmapheresis. The invention also relates to a method for the removal of anti-PARl -receptor antibodies from isolated blood, (i) wherein in a first step the presence or absence of an anti-PARl -receptor antibody is determined in a blood sample from a patient to be diagnosed, wherein the patient is thought to have pulmonary arterial hypertension or thought to be at risk to contract pulmonary arterial hypertension, and (ii) wherein, upon determining the presence of an anti-PARl -receptor antibody the isolated blood of the patient is subjected to a plasmapheresis.
The invention also relates to the use of plasmapheresis for the isolation of an anti- PARl -receptor antibody from blood.
The present invention also relates to an antibody specifically binding to PARl. Preferably, the antibody is a human antibody.
The term "antibody" in the context of the present invention comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called "single- chain-antibodies" (Bird R. E. et al (1988) Science 242:423-6), chimeric, humanized, in particular CDR-grafted antibodies, and dia- or tetrabodies (Holliger P. et al (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-8). Also comprised are immunoglobulin like proteins that are selected through techniques including, for example, phage display to specifically bind to the molecule of interest contained in a sample. In this context the term "specific binding" refers to antibodies raised against the molecule of interest or a fragment thereof. An antibody is considered to be specific, if its affinity towards the molecule of interest or the aforementioned fragment thereof is at least 50- fold higher, preferably 100- fold higher, more preferably at least 1000- fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to make antibodies and to select antibodies with a given specificity.
The levels of the autoantibodies as obtained by the methods or the use of the methods according to the present invention may be analyzed in a number of fashions well known to a person skilled in the art. For example, each assay result obtained may be compared to a "normal" value, or a value indicating a particular disease or outcome. A particular diagnosis/prognosis may depend upon the comparison of each assay result to such a value, which may be referred to as a diagnostic or prognostic "threshold". In certain embodiments, assays for one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicator(s) in the assay. For example, an assay can be designed so that a positive signal only occurs above a particular threshold concentration of interest, and below which concentration the assay provides no signal above background.
The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test, they also depend on the definition of what constitutes an abnormal result. In practice, Receiver Operating Characteristic curves (ROC curves), are typically calculated by plotting the value of a variable versus its relative frequency in "normal" (i.e. apparently healthy) and "disease" populations. For any particular marker, a distribution of marker levels for subjects with and without a disease will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition. ROC curves can be used even when test results don't necessarily give an accurate number. As long as one can rank results, one can create a ROC curve. For example, results of a test on "disease" samples might be ranked according to degree (e.g. l=low, 2=normal, and 3=high). This ranking can be correlated to results in the "normal" population, and a ROC curve created. These methods are well known in the art. See, e.g., Hartley et al. 1982. Radiology 143: 29-36. Preferably, a threshold is selected to provide a ROC curve area of greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. The term "about" in this context refers to +/- 5% of a given measurement.
The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cut-off selected, the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test. In other embodiments, a positive likelihood ratio, negative likelihood ratio, odds ratio, or hazard ratio is used as a measure of a test's ability to predict risk or diagnose a disease. In the case of a positive likelihood ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group. In the case of a negative likelihood ratio, a value of 1 indicates that a negative result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the test group; and a value less than 1 indicates that a negative result is more likely in the control group.
In the case of an odds ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group.
In the case of a hazard ratio, a value of 1 indicates that the relative risk of an endpoint (e.g., death) is equal in both the "diseased" and "control" groups; a value greater than 1 indicates that the risk is greater in the diseased group; and a value less than 1 indicates that the risk is greater in the control group.
The skilled artisan will understand that associating a diagnostic or prognostic indicator, with a diagnosis or with a prognostic risk of a future clinical outcome is a statistical analysis. For example, a marker level of greater than X may signal that a patient is more likely to suffer from an adverse outcome than patients with a level less than or equal to X, as determined by a level of statistical significance. Additionally, a change in marker concentration from baseline levels may be reflective of patient prognosis, and the degree of change in marker level may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.
EXAMPLES We measured the anti-PARl autoantibody by a sandwich ELISA (CellTrend GmbH Luckenwalde, Germany). The microtiter 96-well polystyrene plates were coated with extracts from Chinese hamster ovary cells overexpressing the human PARl. To maintain the conformational epitopes of the receptor, 1 mM calcium chloride was added to every buffer. Duplicate samples of a 1:100 serum dilution were incubated at 4°C for 2 hours. After washing steps, plates were incubated for 60 minutes with a 1:20.000 dilution of horseradish-peroxidase- labeled goat anti-human IgG (Jackson, USA) used for detection. In order to obtain a standard curve, plates were incubated with test sera from an anti-PARl autoantibody positive index patient. The ELISA was validated according to the FDA's "Guidance for industry: Bioanalytical method validation".
FIGURE CAPTIONS
Figure 1 : ROC analysis for the diagnosis of scleroderma based on PARl auto-antibody detection. The control group are normal (healthy) subjects plus patients suffering from rheumatoid arthritis (RA).
Figure 2: ROC analysis for the diagnosis of scleroderma based on PARl auto-antibody detection. The control group are normal (healthy) subjects only.

Claims

Claims
1. Method for diagnosis of a disease wherein presence or absence of an anti-PARl -receptor antibody is determined in a sample from a patient to be diagnosed and wherein the presence of an anti-PARl -receptor antibody is indicative for the disease.
2. Method according to claim 1, wherein the disease is selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
3. A method for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension, comprising the steps of:
a. providing a sample from said patient, and
b. determining the presence or absence of anti-PARl receptor antibodies in said sample,
wherein the presence of anti-PARl receptor antibodies is indicative for an increased risk of the patient for contracting pulmonary arterial hypertension.
4. Method according to claim 3, wherein the primary disease or condition is selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
5. Method according to claims 1 to 4, wherein the determination of presence or absence of an PARl antibody is done by detecting one or more of the antibodies selected from the group of IgA-antibody, IgG-antibody and IgM-antibody.
6. Method according to claim 6, wherein one or more of the antibodies is selected from the group of IgGl, IgG2, IgG3 and IgG4.
7. Method according to claims 1 to 6, wherein the immunoassay is selected from the group of immunoprecipitation, enzyme immunoassay (EIA)), radioimmunoassay (RIA) or fluorescent immunoassay, a chemiluminescent assay, an agglutination assay, nephelometric assay, turbidimetric assay, a Western Blot, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay and a reporter assay such as a luciferase assay.
8. Method according to claim 7, wherein the immunoassay is an ELISA.
9. Use of an PARl -receptor peptide for the diagnosis of a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
10. Use of an PARl -receptor peptide for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension.
11. Research and/or diagnostic kit for the diagnosis of a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease, wherein the kit comprises an PARl -receptor peptide or a functional analogue thereof.
12. Research and/or diagnostic kit for predicting the risk of a patient suffering from a primary disease or condition for contracting pulmonary arterial hypertension or for identifying a patient suffering from a primary disease or condition having an enhanced risk for contracting pulmonary arterial hypertension, wherein the kit comprises an PARl -receptor peptide or a functional analogue thereof.
13. Use of an inhibitor of an anti-PARl -receptor antibody or an inhibitor of an PARl- receptor for the production of a medicament for the treatment or prevention of pulmonary arterial hypertension, wherein the inhibitor of the PARl -receptor is selected from the group of a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease.
14. Use of an inhibitor of an anti-PARl -receptor antibody or an inhibitor of an PARl- receptor for the production of a medicament for the treatment or prevention of pulmonary arterial hypertension.
15. Method for the removal of anti-PARl -receptor antibodies from isolated blood
a. wherein in a first step the presence or absence of an anti-PARl -receptor antibody is determined in a blood sample from a patient to be diagnosed, wherein the patient is thought to have a disease selected from the group of scleroderma, preeclampsia, systemic lupus erythematosus (SLE), graft rejection, fibrosis, cancer, cardiomyopathy, malignant hypertension, portal hypertension and sickle-cell disease, b. wherein, upon determining the presence of an anti-PARl -receptor antibody the isolated blood of the patient is subjected to a plasmapheresis.
16. Method for the removal of anti-PARl -receptor antibodies from isolated blood
a. wherein in a first step the presence or absence of an anti-PARl -receptor antibody is determined in a blood sample from a patient to be diagnosed, wherein the patient is thought to have pulmonary arterial hypertension or thought to be at risk to contract pulmonary arterial hypertension,
b. wherein, upon determining the presence of an anti-PARl -receptor antibody the isolated blood of the patient is subjected to a plasmapheresis.
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