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WO2008066483A1 - Use of nalp5 in methods for diagnosis and therapy of parathyroid related disorders - Google Patents

Use of nalp5 in methods for diagnosis and therapy of parathyroid related disorders Download PDF

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Publication number
WO2008066483A1
WO2008066483A1 PCT/SE2007/050905 SE2007050905W WO2008066483A1 WO 2008066483 A1 WO2008066483 A1 WO 2008066483A1 SE 2007050905 W SE2007050905 W SE 2007050905W WO 2008066483 A1 WO2008066483 A1 WO 2008066483A1
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Prior art keywords
nalp5
protein
parathyroid
cells
cancer
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PCT/SE2007/050905
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French (fr)
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Mohammad Alimohammadi
Olle KÄMPE
Åsa HALLGREN
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Mohammad Alimohammadi
Kaempe Olle
Hallgren Aasa
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Publication of WO2008066483A1 publication Critical patent/WO2008066483A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00Drugs for disorders of the endocrine system
    • A61P5/18Drugs for disorders of the endocrine system of the parathyroid hormones
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564Immunoassay; 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57415Specifically defined cancers of breast
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/05Animals comprising random inserted nucleic acids (transgenic)
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00Animals characterised by species
    • A01K2227/10Mammal
    • A01K2227/105Murine
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575Hormones
    • G01N2333/635Parathyroid hormone (parathormone); Parathyroid hormone-related peptides

Definitions

  • NALP5 in methods for diagnosis and therapy of parathyroid related disorders
  • the present invention relates to the field of methods useful in diagnosis and therapy, and kits for performing such methods. More particularly, it relates to the novel use of NALP 5 in such methods.
  • Parathyroid glands are small glands of the endocrine system. In response to changes in the plasma calcium concentration, the parathyroid glands secrete parathyroid hormone (PTH). PTH is the most important endocrine regulator of calcium and phosphorus concentration in extracellular fluid. PTH increases the plasma calcium concentration by enhancing the absorption of calcium from the intestines, increasing the mobilization of calcium from bone and decreasing the excretion of calcium from kidneys 1 . Since an adequate plasma calcium concentration is essential for many of the physiological processes in the human body, disorders affecting the parathyroid glands may lead to severe disability and even death.
  • PTH parathyroid hormone
  • disorders in parathyroid glands may be caused by decreasing parathyroid function (autoimmune disorders, parathyroid infarction or iatrogenic insult on parathyroid glands) or gain of function (cancers, tumours, adenomas, secondary hyperfunction, or tertiary hyperfunction) .
  • NALP5 Human NACHT, leucine rich repeat and PYD containing 5 (NALP5) also named and known as maternal antigen that embryos require (MATER) protein homolog was originally identified as an oocyte specific antigen in mice 2 5 .
  • MATER maternal antigen that embryos require
  • the invention relates to a method for providing support for diagnosis of autoimmune hypoparathyroidism comprising the steps
  • the autoimmune hypoparathyroidism is autoimmune polyendocrine syndrome type 1.
  • One embodiment of this aspect of the invention also relates to a kit comprising a binding reagent binding specifically to antibodies binding specifically to NALP5 or autoreactive T-cells against NALP5 and means for detecting said antibodies or T-cells bound to said binding reagent.
  • the binding reagent is a NALP5 antigen.
  • the invention relates to a method for providing support for diagnosis of cancer originating from tissue expressing PTHrP comprising the steps
  • the cancer is cancer in the parathyroid glands, breast cancer, prostate cancer, myeloma, lung cancer, ovarian cancer or colon cancer.
  • Said method may comprise the steps
  • One embodiment of this aspect of the invention relates to a kit comprising a binding reagent binding specifically to NALP5 protein and means for detecting NALP5 bound to said binding reagent.
  • the kit comprises a binding reagent binding specifically to NALP5 mRNA or NALP5 DNA and means for detecting NALP5 mRNA or DNA bound to said binding reagent.
  • the invention relates to a method for identifying a test compound as a candidate drug, comprising the steps of
  • the invention relates to a method for identifying a test compound as a candidate drug, comprising the steps of
  • the invention relates to a genetically modified cell of an animal species comprising a heterologous DNA molecule encoding the NALP5 protein, and/or having the native gene encoding the NALP5 protein inactivated.
  • the animal species may be a mammal, such as human, non-human primates, and rodents.
  • the invention relates to a complete transgenic animal comprising the genetically modified cell.
  • the invention relates to fusion protein comprising a NALP5 binding domain and a domain capable of providing an analytically detectable signal.
  • the invention relates to pharmaceutical preparations comprising an antibody, or fragment thereof, binding specifically to NALP5, a DNA construct encoding NALP5 or an oligonucleotide capable of binding to a NALP5 mRNA and inhibiting translation thereof, respectively, and optionally pharmaceutically acceptable carriers, adjuvants, diluents and excipients, and to methods of treatment of a patient in need thereof comprising the administration of such pharmaceutical preparations.
  • the invention also relates to a method for treatment of parathyroid or ovarian disease or disorders involving PTHrP producing cells comprising adoptive transfer of T-cells reactive against NALP5 to patients in need thereof.
  • FIG. 1 Protein domains of human NALP5: Pyrin domain, PYD, NACHT
  • NTPase domain NACHT and the Leucine rich repeats LRR
  • Figure 2 A) Autoantibody titers against human NALP5 in sera from APS I patients, patients with other autoimmune disorders and healthy blood donors. B) Autoantibody titers against NALP5 in sera from Aire deficient mice and wild type mice. The figure demonstrates that APS I patients and Aire deficient mice have elevated levels of autoantibodies against NALP5.
  • FIG. 1 Expression pattern/ profile of NALP5 mRNA in adult human tissues. The figure demonstrated the predominant expression of NALP5 in parathyroid glands.
  • FIG. 4 Expression of human NALP5 homologues in different tissues. Left part: Phylogram tree designed in Clustal W illustrating homologies. Right part: mRNA expression in human multiple tissue panel. The figure demonstrates that NALP5 is the only member of NALP-protein -family that is expressed in parathyroid glands.
  • Figure 5 Confirmation /verification of antiserum produced against human and bovine NALP5.
  • FIG. 1 Immunohistochemistry on paraffin embedded parathyroid sections.
  • the expression pattern of NALP 5 is identical in human and cattle.
  • Figure 7 Immunofluorescence on cryosections of bovine parathyroid glands. All sera used in dilution 1 :400 A) background staining, B-D) using sera from three different APS I patients with hypoparathyroidism, E-G) sera from three APS I patients without hypoparathyroidism, H-J) sera from three different healthy blood donors in dilution 1 :400, K) NALP5 antiserum, L) NALP5 antiserum IOOX magnification to analyse subcellular localization. The figure demonstrates the cytoplasmic localization of NALP5 and also presence of autoantibodies against parathyroid cells in sera from APS I patients.
  • FIG. 8 Immunoprecipitation (IP) of NALP5.
  • B Sequential IP of the in vitro transcription and translated 35 S-methionine radiolabeled human parathyroid NALP5 clone.
  • First immunoprecipitation step (Lane 1) no antibody, (lane 2) NALP5 antiserum, (lanes 3-5) Sera from patients with reactivity to NALP5, (lanes 6-8) sera from patient without reactivity to NALP5, (lanes 9-1 1) sera from healthy control blood donors.
  • Second IP step NALP5 antiserum for all lanes.
  • First IP step (lane 1) no antibody, (lane 2) serum from APS I patient with reactivity against NALP5, (Iane3) serum from APS I patient without reactivity against NALP5, (lane 4) serum from healthy blood donor.
  • Second IP step NALP5 antiserum for all lanes.
  • Thymic NALP5 expression (a) Thymic epithelial cells were isolated from embryonic mice as CD45- EpCAM + cells at the indicated days of gestation (E) and analysed for the expression of NALP5. Values have been normalized to FoxNl expression, (b) Thymic epithelial cells expressing high and intermediate levels of MHC class II molecules on their cell surface and thymocytes were isolated from thymic tissue of adult wild type (wt) and Aire-deficient mice (ko) and subsequently analysed for the expression of NALP 5 and Aire. The expression levels for either of these two genes was compared to wt values which was set as 1.0. N. D. denominates not detected. The figure demonstrates the underlying molecular mechanism leading to the loss of tolerance to NALP5.
  • FIGs 10-15 Immunohistochemistry on breast tissue using antiserum against NALP5. Typical regions that get stained are indicated with arrow/ s.
  • Figure 10-1 1 immunohistochemistry on normal healthy breast tissue, the ducts / ductal cells are stained.
  • Figure 12-15 immunohistochemistry on breast different breast cancer specimens. In 3 of 4 specimens, the cancer cells express NALP5 which appears as positive staining pattern in immunohistochemistry. In general, tumours with ductal ( Figures 12 and 14-15) origin display increased expression of NALP5 whereas lobular tumours ( Figure 13) express normal levels of NALP5.
  • the staining pattern of parathyroid chief cells is shown. The parathyroid chief cells get stained.
  • the NALP5 antiserum is used in staining of parathyroid cancer specimens. As shown in the figure, a larger area is stained, indicating increased expression of NALP5 in parathyroid tumours.
  • immunohistochemistry is performed on healthy ovarian tissue and it is shown that the oocytes in the primary ovarian follicles are stained.
  • the immunohistochemistry is performed on ovarian tumour/ cancer. Larger areas of the specimen get stained which indicate the increased expression of NALP5 in these tumours.
  • FIG. 20 The role of NALP5 in calcium sensing and calcium signalling.
  • Parathyroid cells were cultured for six hours in different extracellular calcium concentrations. After six hours, cells were harvested and expression level of mRNA for NALP5, GAPDH and PTH was determined using quantitative PCR. The figure demonstrates that the parathyroid cells respond to increased extracellular calcium concentration by increasing their expression of NALP5. This is an evidence for NALP5s role in calcium sensing and signalling.
  • An antibody or binding reagent binding specifically to a target should be construed as an antibody or binding reagent that binds to the target with high affinity and show no or very low binding to other molecules.
  • NALP5 antigen should be construed as any molecule binding native autoantibodies towards NALP5 with essentially the same or higher binding affinity and specificity as native NALP5.
  • NALP5 antigens include, but are not restricted to, native NALP5 and fragments thereof with retained epitopes binding to autoantibodies.
  • NALP5 protein and NALP5 related compounds and methods such as: NALP5 DNA, NALP5 mRNA, NALP5 protein, isolated naturally or artificially produced antibodies directed against NALP5, detection methods for measurement of NALP5 mRNA, NALP5 protein or antibodies against NALP5 DNA or NALP5 protein in diagnosis and treatment of autoimmune disorders, genetic disorders, parathyroid adenomas, tumour and cancer disorders and development of drugs or treatment methods including immune therapy.
  • NALP5 is important for calcium homeostasis and parathyroid function.
  • NALP5 is also expressed in tissues and cells expressing parathyroid hormone related protein (PTHrP) .
  • PTHrP parathyroid hormone related protein
  • PTHrP is a protein secreted by different cells, especially some cancer cells leading to humeral hypercalcaemia of malignancy.
  • PTHrP is produced commonly in breast cancer, prostate cancer and myeloma, but also other tumours such as lung cancers, ovarian cancers and colon cancers can express PTHrP 6 9 . Consequently NALP5 is expressed in the mentioned tumour/ cancer tissues and NALP5 and the mentioned NALP5 compounds can be used for diagnosis and treatment of these disorders.
  • autoimmune polyendocrine syndrome type 1 and other forms of autoimmune hypoparathyroidism harbour autoantibodies against human NALP5 as well as the murine and bovine orthologoues.
  • the first aspect of the invention is the assessment of autoantibodies and autoreactive T cells against NALP5 used as tool for diagnosis of autoimmune hypoparathyroidism and marker for onset of autoimmunity in patients, domestic animals and animal models.
  • Detection of autoantibodies can be performed using a NALP5 antigen or active fragments thereof, e.g. recombinant produced in a suitable host system, e.g. in bacteria or eukaryotic cells, or translated NALP5 antigen using in vitro transcription and translation systems.
  • the antigen may be labelled either by a marker, facilitating the detection thereof, e.g. by radioactivity 10 12 or fluorescent dye 13 , just to mention two of the most common techniques.
  • NALP5 antigen may be immobilized on a surface or being in soluble form.
  • Detection of an autoantibody-NALP5 antigen complex may be performed using instruments detecting radioactivity or fluorescence. Detection may also be performed using instruments that can distinguish the autoantibody-NALP5 antigen complex by sensing the association/ dissociation of the complex by changes in electrical or optical properties of the NALP5 -autoantibody complex or the surface on which the complex is immobilized on.
  • An example of a technique which doesn't require specific labelling is surface plasmon resonance (SPR) 14 > 15 .
  • SPR surface plasmon resonance
  • NALP5 -autoantibody complex may also be detected using methods based on proximity-ligation 16 18 .
  • Autoreactive T cells may be detected using a NALP5 antigen or MHC Class I or II restricted epitopes of NALP5 as T cell stimulator followed by detection of activity markers of T cells such as intracellular or extracellular cytokines (e.g. gamma interferon or IL2) or surface expression of CD69, CD98, CD401igand, CD30, CD 134 or OX40.
  • cytokines e.g. gamma interferon or IL2
  • CD69, CD98, CD401igand e.g. gamma interferon or IL2
  • NALP5 is increased parathyroid adenomas and parathyroid cancers.
  • NALP5 mRNA and protein expression has also been found to be increased in organs and tumours with PTHrP expression such as breast cancers, prostate cancers, myeloma, lung cancers, ovarian cancers, colon cancers.
  • the second aspect of the invention is thus the detection of increased levels of NALP5 mRNA or NALP5 protein in tissue, cell, blood, plasma, serum or other bodily fluids and used as diagnostic marker for cancers and tumours with origin in the parathyroid glands or any other tissues/ cells where PTHrP is expressed.
  • tissue or cells are breast cancers, prostate cancers, myeloma, lung cancers, ovarian cancers, colon cancers.
  • Detection and quantification of plasma levels of NALP5 may be performed using ELISA (enzyme linked immunoassay) based on either monoclonal or polyclonal antibodies, or fragments thereof, against any orthologue of NALP5, RIA (radio immunoassay) (either direct or indirect technique) based on either monoclonal or polyclonal NALP5 specific antibodies or fragments, and antibody based proximity ligation were the NALP5 specific antibodies (either monoclonal or polyclonal) can be conjugated to DNA probes followed by subsequent PCR quantification.
  • This invention also relates to the detection and quantification of NALP5 mRNA and/ or protein expression in tumour biopsies used in cancer diagnostic 20 ' 21 .
  • the detection and quantification of NALP5 mRNA may be performed with any kind of PCR based technique or real time PCR.
  • the detection/ quantification of the NALP5 protein may be performed using immunohistochemistry and/ or immunofluorescence and /or proximity ligation based on monoclonal or polyclonal NALP5 specific antibodies or active fragments thereof 16 " 18 > 22 > 23 and (Alimohammadi et al. Accepted November 1 (2007) for publication in The New England Journal of Medicine) .
  • kits for diagnosis of parathyroid-related diseases e.g. cancers and tumours.
  • the kit comprises a binding reagent binding specifically to NALP5 protein, NALP5 DNA or NALP5 mRNA, e.g. nucleic acid probes or an antibody or a fragment thereof.
  • the reagent may be provided in solution or optionally the kit comprises a liquid in which the reagent is solubilised prior to measurement.
  • the reagent may also be provided immobilized to a solid phase, such as a micro titre plate, beads, a nitrocellulose membrane, a silicon wafer or any other solid phase used in the art.
  • the kit further comprises a detection reagent and means for detecting any complex formed between the binding reagent, detection reagent and NALP5 protein, mRNA or DNA.
  • the binding reagent and detection reagent may be provided in the same molecule, e.g. as a fluorescently labelled nucleic acid probe, or separately, e.g. as a biotinylated binding reagent and a streptavidin- conjugated detection reagent.
  • Detection and quantification of plasma levels of NALP5 may be performed using ELISA (enzyme linked immunoassay) based on either monoclonal or polyclonal antibodies, or active fragments thereof, against any orthologue of NALP5, RIA (radio immunoassay) (either direct or indirect technique) based on either monoclonal or polyclonal NALP5 specific antibodies or fragments, and antibody based proximity ligation 16 18 ' 22 > 23 were the NALP5 specific antibodies (either monoclonal or polyclonal) can be conjugated to DNA probes followed by subsequent PCR quantification.
  • This invention also relates to the detection and quantification of NALP5 mRNA and/ or protein expression in tumour biopsies used in cancer diagnostic.
  • the detection and quantification of NALP5 mRNA may be performed with any kind of PCR based technique or real time PCR.
  • the detection/ quantification of the NALP5 protein may be performed using immunohistochemistry and/ or immunofluorescence and/ or proximity ligation 16 18 ' 22 > 23 based on monoclonal or polyclonal NALP5 specific antibodies or active fragments thereof.
  • NALP5 is predominantly expressed in parathyroid glands and has an essential role in the parathyroid physiology and other tissues where PTHrP is expressed.
  • NALP5 is involved in calcium signalling and calcium regulation (Figure 20).
  • Another aspect of the invention is thus the use of NALP5 as drug target and target for treatment of diseases in parathyroid glands such as hyperparathyroidism (primary, secondary as well as tertiary hypoparathyroidism) and in other normal or diseased organs and tissues where PTHrP is expressed. This is exemplified in tumours or cancers that express PTHrP which cause disruption in calcium homeostasis in these patients.
  • calcimimetic compounds such as phenylalkylamine calcimimetics have been introduced as treatment for hyperparathyroidism.
  • the drugs based on calcimimetic bind to the calcium sensing receptor and inhibit the secretion of PTH.
  • NALP5 as an alternative drug target for treatment of hyperparathyroidism. Drugs binding to NALP5 and regulating its function could be used for regulation of PTH synthesis and secretion.
  • a further aspect of the invention is methods for identification of components capable of binding NALP5 and methods for screening drugs to identify compounds which interact with and bind to NALP5.
  • the binding protein or an active fragment thereof may be in isolated form, in solution, or in immobilized form or may be genetically engineered to be expressed on the surface of recombinant host cells such as in phage display system or as fusion protein. Alternatively, whole cells or cell fractions comprising NALP5 may be employed in screening protocols. Regardless of the form of the binding protein, a plurality of compounds are contacted with the binding protein under conditions sufficient to form a compound / binding protein complex and compound capable of forming, enhancing or interfering with said complexes are detected 24 .
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to NALP5 binding protein-like sequences.
  • Still another aspect of the invention is the use of an antisense oligonucleotide (RNAi/ siRNA) having a sequence capable of binding with mRNAs encoding NALP5 so as to prevent the translation of NALP5 mRNA in parathyroid and other organs expressing PTHrP 25 .
  • RNAi/ siRNA antisense oligonucleotide
  • a further aspect of the invention is the use of monoclonal or polyclonal antibodies constructed against NALP5 in therapeutical approaches on parathyroid disease such as parathyroid cancer or hyperparathyroidism or other disease in which parathyroid hormones are involved (e.g. osteoporosis, calcifylaxis or calcium/ phosphate dependent heart disease) 26 .
  • parathyroid disease such as parathyroid cancer or hyperparathyroidism or other disease in which parathyroid hormones are involved (e.g. osteoporosis, calcifylaxis or calcium/ phosphate dependent heart disease) 26 .
  • parathyroid disease such as parathyroid cancer or hyperparathyroidism or other disease in which parathyroid hormones are involved (e.g. osteoporosis, calcifylaxis or calcium/ phosphate dependent heart disease) 26 .
  • Such diseases are considered as NALP5 -related diseases in the context of the present invention.
  • These antibodies can be conjugated with different compounds that can affect the genes or living cells such
  • This invention also provides methods for adoptive transfer of T-cells reactive against NALP5 to patients with parathyroid or ovarian disease or disorders involving PTHrP producing cells such as breast cancers 30 ' 31 .
  • T-cells used for adoptive T-cell transfer may be primed against NALP5 DNA, NALP5 protein or fragments thereof by incubation with NALP5 whole protein or T-cell restricted epitopes 32 .
  • DNA constructs containing NALP5 DNA 33 . 34 or encoding NALP5 protein 35 or fragments thereof can be used to vaccinate patients in order to prevent or treat cancer or autoimmune disorders.
  • a further aspect of the invention is transgenic non-human animals comprising or a nucleic acid molecule encoding NALP5, or having the NALP5 gene knocked- out, and methods for use of transgenic animals as models for differential binding protein expression, mutations and immune responses and evaluation as well as in ligand and drug screens 36 .
  • Such a method comprises comparing the results from measurements on a first animal with results from measurements on a second animal, the two animals having a difference in the genome leading to different NALP5 expression.
  • Another aspect of the invention is fusion proteins comprising a NALP5 binding domain and a binding protein /ligand binding indicator domain capable of providing an analytically detectable signal and the use thereof in methods of screening drugs by forming, enhancing or interfering with the detectable signal 37 .
  • fusion proteins comprising a NALP5 binding domain and a binding protein /ligand binding indicator domain capable of providing an analytically detectable signal and the use thereof in methods of screening drugs by forming, enhancing or interfering with the detectable signal 37 .
  • the invention is further exemplified with the following example, which essentially corresponds to a manuscript accepted for publication (Alimohammadi et al, accepted November 1 st 2007 for publication in The New England Journal of Medicine) .
  • This example primarily demonstrate the role of NALP5 in calcium regulation.
  • Figures 10-19 show immunohistochemistry experiments in which tissue specimens have been immunostained by NALP5 -specific antiserum. These figures demonstrate the increased expression of NALP5 mRNA and protein in tissue specimens from human parathyroid tumour, human breast cancer and human ovarian cancer. .
  • an evidential figure demonstrating the response of parathyroid cells to changes in extracellular calcium by increasing the expression of NALP5 is also provided (Figure 20), this figure address the role of NALP5 in calcium sensing and calcium signalling.
  • Example 1 Identification ofNALP5 as a major parathyroid antigen in autoimmune polyendocrine syndrome type 1- a first link between the human disease and the Aire deficient murine model
  • Autoimmune polyendocrine syndrome type 1 is a multi-organ autoimmune disorder caused by mutations in the autoimmune regulator gene. Hypoparathyroidism is one of the cardinal symptoms of autoimmune polyendocrine syndrome type 1 , but up until today, no autoantigen has been identified in the parathyroid glands. A human parathyroid cDNA library, was immunoscreened and NALP5 identified as a major autoantigen. This protein is predominantly expressed in parathyroid chief cells and the presence of autoantibodies to NALP5 correlates with hypoparathyroidism in autoimmune polyendocrinesyndrome type 1. Moreover NALP5 is also recognised by sera from Aire deficient mouse, representing probably the first autoantigen identified in this murine model for APS 1.
  • Autoimmune poly endocrine syndrome type I (APS I) (OMIM240300) is an autosomal recessive disorder caused by mutations in a single gene named autoimmune regulator (AIRE) 38 .
  • Aire is located on chromosome 22q23.3 and encodes a 54 kDa protein expressed in medullary epithelial cells of thymic stroma 39 .
  • AIRE autoimmune regulator
  • endocrine organs such as adrenals, ovaries, pancreas and parathyroid glands are attacked by the immune system and it has been shown that patients harbour autoantibodies directed against key enzymes in these organs 43-45 ⁇ h e presence of autoantibodies against 21 -hydroxylase 46 > 47 , a key enzyme in steroid synthesis only expressed in the adrenal cortex, has been shown to predict the development of autoimmune adrenal insufficiency 48 .
  • Hypoparathyroidism is usually the first symptom of APS I affecting 81% of the patients 49 > 50 .
  • Parathyroid glands secrete parathyroid hormone (PTH) which is essential for the calcium homeostasis, crucial for several cellular functions.
  • PTH parathyroid hormone
  • Untreated hypoparathyroidism leads to hypocalcaemia and is a life threatening condition 51 . It is believed that infants with APS I may die in a hypocalcaemic tetany caused by hypoparathyroidism prior to diagnosis. Although several organ-specific autoantigens have been identified in APS I, none of them has been associated with the parathyroid glands. The Calcium sensing receptor (CaSR) was suggested to be the parathyroid specific antigen in APS I and other acquired hypoparathyroidism 52 , however, these data have not been reproducible in two different reports 50 > 53 . The identification of a parathyroid specific autoantigen would be of significance not only for an improved serological diagnosis of the disease, but also for increased understanding of the underlying pathophysiology and perhaps also of the physiology of the parathyroid glands.
  • CaSR Calcium sensing receptor
  • a murine model for APS I was established in 2002 54 . Until today five other Aire deficient murine models have been constructed 39 > 55 . In all of the constructs, the mice mimic the same mutation in the Aire gene (Finn major PR257X) as the majority of the APS I patients. Aire deficient mice suffer from several features such as infertility and lymphocyte infiltrates in several organs 39 > 55 . The genetic background of the mouse strain used for the construction the knock out model can influence the autoimmune symptoms of these mice 55 . However, in comparison with the APS I patients, the murine phenotype is remarkably mild. Identification of a common B cell autoantigen in APS I patients and the murine model would open for a variety of cellular experiments.
  • NALP5 is a member of NACHT, leucine rich repeat and PYD containing proteins (NALPs) which are a large subfamily of the CATERPILLER protein family 56 and consist of 14 members named NALPl -14.
  • NALPs are characterized by an N- terminal PYD domain suggested to be involved in protein-protein interactions, a central NACHT domain with a potential NTPase activity 57 , a NAD domain with unknown function and C-terminal Leucine rich repeats (LRRs) suggested to be involved in molecular pattern sensing and protein-protein interaction 56 > 58 .
  • NALP5 also named maternal antigen that embryos require (MATER) has been studied in mice and cattle, and recently its expression has been reported to be specifically restricted to oocytes 2 5 . Embryos of NALP5 -/- mice stay in developmental arrest at two-cell stage and die suggesting NALP5s crucial role in oogenesis through an essential role in cell viability.
  • NALP5 also known as MATER
  • MATER is the common B cell autoantigen in human APS I and its murine model, the Aire deficient mouse.
  • a cDNA expression library was constructed by use of the ZAP Express vector system (Stratagene, La Jolla, CA, USA and immunoscreened with sera from APS-I patients with hypoparathyroidism. Positive clones were isolated and sequenced.
  • the diagnosis of APS-I was based on the presence of two of the three major clinical manifestations including hypoparathyroidism, primary adrenal failure and mucocutanous candidiasis. The majority of the included APS-I patients (83 of 87) were also demonstrated to have typical mutations in the AIRE gene. The following diagnostic criteria were used:
  • Mucocutanous candidiasis candidal infections in the oral mucosa, skin or nails for more than 3 months.
  • hypoparathyroidism Subnormal plasma calcium concentration ( ⁇ 2.15 mmol/L) and supranormal plasma phosphate concentration together with low normal or low PTH concentrations, and normal renal function.
  • Addison's disease subnormal serum Cortisol together with elevated plasma ACTH concentrations, or failure to reach s-cortisol of 550 nmol/L at 30 or 60 min in an ACTH stimulation test.
  • the majority of the patients diagnosed with Addison's disease also displayed specific 21 -hydroxylase autoantibodies. All of the control subjects were normocalcemic.
  • Autoantibodies against the calcium sensing receptor (CaSR) were sought using as previously described methodology but could not be detected in the serum of any of the APS- 1 patients included in this study. 50 Mice
  • Wild-type and Aire-deficient C57/B16 mice were either analyzed as adult animals (at 2.5 - 10 months of age) or at defined gestational ages (E) whereby the day of plug defined was scored as EO.5.
  • a full length cDNA clone for human NALP5 (cat#SC306608 obtained from Origene Technologies Inc. Rockville, MD) was used for coupled in vitro transcription/ translation and 35S-methionine labeling using the TnT system (Promega, Madison, WI, USA) according to the manufacturer's protocol.
  • the 35S-radiolabelled recombinant NALP5 was directly immunoprecipitated with patient or control sera in 96-well filtration plates (Millipore). Serum from each individual was analyzed in a double blinded format in duplicates and 20 000 cpm of 35S-NALP5 was used for immunoprecipitation in each well.
  • NALP5 autoantibodies were prepared using 35 S-labelled NALP5 protein (150 000 cpm) using 2.5 ⁇ l of patient or control serum. Antibody-antigen complexes were captured by protein-A Sepharose beads and subsequently removed by centrifugation. The remaining supernatant was then subjected to a second immunoprecipitation step but now using NALP5 -specific rabbit antiserum. The final immunoprecipitate was analyzed on an SDS-PAGE followed by autoradiography.
  • the slides were then incubated with the NALP5 antiserum (dilution 1 :4000) or patient sera (dilution 1 :8000) overnight at +4°C followed by washing and incubation with biotinylated secondary goat anti human or rabbit immunoglobulin antibody (Vector laboratories, Burlingame, CA 94010) for 30 minutes. Peroxidase conjugation was performed using the VECTASTAIN ® ABC system (PK-6100 Vector laboratories, Burlingame, CA 94010) and the staining reaction used ChemMateTM DAKO EnvisionTM Detection kit (Dakocytoformation, Glostrup, Denmark).
  • the specificity of the immunostaining was tested in control slides by omission of the primary antibody, by using pre-immunization serum from the rabbit in which the antiserum was raised, and by blocking the primary antibody through preincubation with the peptide used for the immunization in concentrations of 10-100 nmol/ml.
  • Bovine parathyroid glands were collected immediately after slaughter. Fat and connective tissue was removed, and the glands were minced with scissors. Cell suspensions were prepared by digestion in 1 mg/ml collagenase (Sigma, St Louis, Missouri, USA), 0.05 mg/ml DNase I, 1.5% bovine serum albumin and 1.25 mM Ca 2+ . After digestion in a shaking incubator for 120 min, the suspensions were filtered through nylon mesh (125 ⁇ m) and exposed to 1 mM EGTA in 25 mM HEPES buffer (pH 7.4) containing 142 mM NaCl and 6.7 mM KCl.
  • Debris and dead cells were removed by centrifugation through 25 and 75% standard isotonic Percoll (GE Healthcare) .
  • Cell viability as determined by the Trypan blue exclusion test, exceeded 95%.
  • Cells were cultured for 4 h in DMEM/ Ham's F- 12, 1 mM total calcium, 5% fetal bovine serum, 15 mM HEPES, 100 U/ ml penicillin, 100 ⁇ g/ml streptomycin, 5 ⁇ g/ml insulin, 2 mM glutamine and 1% non-essential amino acids.
  • Freshly isolated cells (10 7 ) were precultured for 30 min in methionine-free RPMI 1640.
  • the medium was changed to methionine-free RPMI 1640 containing 5% dialyzed fetal bovine serum and 0.5 mCi 35 S methionine (GE Healthcare) followed by incubation for 6h.
  • the medium was removed and cells were washed 2 times with ice cold RPMI 1640 medium and 6 times with ice cold PBS. Cells were lysed on ice in a 2OmM Tris-HCl buffer (pH 7.4) containing 0.15 NaCl, 1% Triton X-100, 1 mM phenylmethyl sulfonylfluoride and 1% Trasylol.
  • Insoluble material was removed by centrifugation at 100 000 x g for 20 min at +4°C. Immunoprecipitation was performed with different control sera, patient sera and rabbit antisera for 6h at +4°C and the bound immune complexes were captured by Fast Flow Protein A Sepharose followed by SDS-PAGE analysis.
  • Fisher's exact test was used to compare the frequencies of reactivity to NALP5 with the major clinical manifestations in APS-I patients.
  • the protein encoded by the isolated NALP5 clone was 35 S radio-labelled by in vitro transcription and translation. As the clone lacked the first 170 amino acids the translation started at the following ATG encoding the methionine located at the amino acid position 194. This could be verified by SDS-PAGE analysis which indicated that the translation product had a molecular weight of 1 14 kDa (fig 8b, lane 1) in contrast to the expected 134 kDa for the full length human NALP5.
  • NALP5 mRNA Tissue expression of human NALP5 mRNA was analyzed using real time quantitative PCR carried out on a normalized human multiple tissue cDNA panel (the amplified region is shown in figure 1 E) .
  • NALP5 mRNA was predominantly expressed in parathyroid tissue ( Figure 3) .
  • NALP5 mRNA was also found to be expressed in ovaries as previously described in mice 2 5 .
  • expression of other members in the NALP family with highest rate of homology to NALP5 was checked by a semi-quantitative PCR carried out on the same tissue cDNA panel as used for the real time quantitative PCR.
  • primers for NALP4, NALP5, NALP6, NALP9, NALP 10, NALP 1 1 and NALP 13 only NALP5 mRNA was expressed in parathyroid tissue ( Figure 4) .
  • NALP5 as an autoantigen in the parathyroid glands
  • NALP5 is translated in the parathyroid glands
  • rabbit antiserum was raised by immunization with a NALP5 specific human peptide (aa 897-910), a region with high homology rate to bovine NALP5.
  • the specificity of the antibody was tested in western blot on lysate from bovine parathyroid and human parathyroid adenoma lysate.
  • the antiserum recognized a band of expected molecular weight for human NALP5 as well as bovine NALP5 in both western blot analysis and immunoprecipitation of a 35 S labelled parathyroid cell lysate ( Figure 5A, B).
  • NALP5 expression in the thymus is Aire-dependent
  • NALP5 is a major parathyroid autoantigen in APS I. Hypoparathyroidism is one of the three cardinal symptoms of APS I occurring in up to 80% of the patients 49 > 50 .
  • NALP 5 is a member of the Caterpillar or NACHT-LRR family of proteins, thought to be involved in intracellular recognition of pathogens using a pattern recognition mechanism similar to the TOLL like receptors 59 . Mutations in NALP-molecules are known to cause a number of inflammatory conditions 60 .
  • NALP5 is predominantly expressed in parathyroid tissue, namely in the parathyroid hormone (PTH) producing chief cells and to a lesser extent in the gonads, whereas no expression was found in immune cells so far tested.
  • PTH parathyroid hormone
  • NALP3 may recognise small intracellular molecules such as uric acid 61 .
  • the NALP5 expression increased with increasing calcium levels, thus completely opposite to the effect on PTH ( Figure 20). It is thus plausible to suggest an important role of NALP5 in calcium homeostatis.
  • NALP5 has previously been described as an oocyte specific autoantigen in day 3 neonatal thymectomized mice (d3tx) 2 5 , a well known animal model for organ specific autoimmunity including autoimmune ovarian disease (AOD) 62 .
  • AOD is similar to the human autoimmune gonadal insufficiency - one of the well known manifestations of APS I, affecting 48 % of APS I patients 49 . This may imply NALP5 is also an ovarian autoantigen in APS I patients, and statistical analysis indicates in fact a significant correlation of NALP5 autoantibodies to both hypoparathyroidism and autoimmune ovarian insufficiency (Table 1).
  • hypoparathyroidism in APS I patients is the only disease component with a distinct gender difference appearing more prevalently in females (98%) than males (71%) 53 .
  • Our findings may give an explanation to this intriguing discrepancy.
  • NALP5 in parathyroid chief cells is a new and remarkable finding.
  • NALP5 has been shown to be important in early embryogenesis were the embryos of NALP5 deficient females do not develop further than the two-cell stage 4 .
  • NALP5 Using electron microscopy murine NALP5 was localized to cytoplasm, mitochondria, and the nuclear pore 3 .
  • NALP5 has, however, no signals for either nuclear or mitochondrial localisation and this has been interpreted that other molecules interacting with NALP5 mediate its transport 3 .
  • cytoplasmic protein expression of bovine NALP5 in parathyroid cells using laser scanning confocal microscopy (figure 7L) in concordance with previous reports.
  • the cytoplasmic localisation and the presence of a C-terminal leucine rich repeat and an ATP/ GTP binding domain are strong indicators for NALP5 being involved in protein-protein interaction and cellular signalling.
  • the most important known function of parathyroid chief cells is to sense extracellular calcium concentration and secrete adequate amount of PTH.
  • Murine NALP5 has been shown to be essential for embryogenesis extending the two-cell stage 5 . This developmental stage corresponds to the time when the intracellular signalling must transit to intercellular signalling so that cellular migration can occur correctly and calcium signalling has been shown to be of importance at several stages during embryonic development 63 .
  • the calcium dependent expression of NALP5 mRNA in bovine parathyroid cells cultured in different calcium concentrations shown here may imply a key role in calcium signalling.
  • mice survive and have few anomalities apart from female sterility.
  • Gcm2 glial cell missing 2 transcription factor deficient mice without parathyroid glands 64 can maintain calcium homeostatis, the calcium regulation in mice may be different and perhaps relying more on rPTH than in humans.
  • mice could have another source of parathyroid hormone secretion than the parathyroid glands.
  • Auxiliary production of PTH has been illustrated in mice with deficiency in Glial Cells Missing 2 (Gcm2) transcription factor which causes genetic ablation of the parathyroid glands 64
  • NALP5 has previously been identified as a murine autoantigen in d3tx mice, we investigated whether Aire deficient mice displayed autoantibodies against murine NALP5. High titer autoantibodies was found in 10/44 (22%) Aire- deficient mice and represents to the best of our knowledge the first well characterised autoantigen in the murine model of APS I.
  • Aire deficient mice have decreased fertility (unpublished observation) which could be explained by existence of NALP5 antibodies directed against oocytes. Aire deficient mice have normal serum calcium levels (not shown) and this could raise the question of why hypocalcaemia or symptoms thereof has not been observed in Aire deficient despite existence of NALP5 autoantibodies in 22% of the animals. The importance of the day 3 neonatal time window has been particularly observed/ studied/ since mice do not develop autoimmunity if the thymectomy is performed day 0 or day 7 after birth 65 .
  • Aire is a protein expressed in thymus with structural and functional properties suggestive of a transcription factor 55 and is suggested to control the transcription of several genes controlling the expression of a variety of self- antigen in thymus, important for the clonal deletion of autoreactive T cells 39, 42. Mutations in Aire result in development of autoimmune features in human patients as well as Aire deficient mice. In this study we find serological similarities between the d3tx mouse model and Aire deficient mouse model and APS I patients.
  • NALP5 as a major parathyroid autoantigen in APS I.
  • murine NALP5 orthologue as the first shared autoantigen in APS I patients and Aire deficient mice.
  • the expression pattern of NALP5 during development in Aire deficient and wild type mice supports the previous hypothesis that promiscuous expression of organ-specific autoantigens in thymus at specific developmental stage is critical in order to prevent autoimmunity.
  • NALP3 forms an IL-I beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity 2004;20(3):319-25. 61. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 2006;440(7081):237-

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Abstract

The present invention relates to the use of NALP5 in methods useful in diagnosis and therapy of parathyroid related disorders such as autoimmune hypoparathyroidism, cancer originating from tissue expressing PTHrP and parathyroid or ovarian diseases involving PTHrP producing cells. The invention also relates to kits for performing such methods.

Description

Use of NALP5 in methods for diagnosis and therapy of parathyroid related disorders
Field of the invention
The present invention relates to the field of methods useful in diagnosis and therapy, and kits for performing such methods. More particularly, it relates to the novel use of NALP 5 in such methods.
Background
Parathyroid glands are small glands of the endocrine system. In response to changes in the plasma calcium concentration, the parathyroid glands secrete parathyroid hormone (PTH). PTH is the most important endocrine regulator of calcium and phosphorus concentration in extracellular fluid. PTH increases the plasma calcium concentration by enhancing the absorption of calcium from the intestines, increasing the mobilization of calcium from bone and decreasing the excretion of calcium from kidneys1. Since an adequate plasma calcium concentration is essential for many of the physiological processes in the human body, disorders affecting the parathyroid glands may lead to severe disability and even death.
Disorders in parathyroid glands may be caused by decreasing parathyroid function (autoimmune disorders, parathyroid infarction or iatrogenic insult on parathyroid glands) or gain of function (cancers, tumours, adenomas, secondary hyperfunction, or tertiary hyperfunction) .
There is a need in the art for novel diagnostic methods and treatment approaches for parathyroid related disorders. The present invention disclose such methods and approaches based on the understanding of the parathyroid molecular mechanism. Summary of the invention
Human NACHT, leucine rich repeat and PYD containing 5 (NALP5) also named and known as maternal antigen that embryos require (MATER) protein homolog was originally identified as an oocyte specific antigen in mice 2 5. We have surprisingly discovered the predominant expression of NALP5 in the human parathyroid glands which is a basis for the novel diagnostic and therapeutic approaches of the present invention.
In a first aspect, the invention relates to a method for providing support for diagnosis of autoimmune hypoparathyroidism comprising the steps
- detecting the presence of antibodies binding specifically to NALP5 and/or the presence of autoreactive T-cells against NALP5, in a body fluid sample from a subject wherein the presence of such antibodies and/or autoreactive T-cells is indicative of autoimmune hypoparathyroidism.
In one embodiment of this aspect, the autoimmune hypoparathyroidism is autoimmune polyendocrine syndrome type 1.
One embodiment of this aspect of the invention also relates to a kit comprising a binding reagent binding specifically to antibodies binding specifically to NALP5 or autoreactive T-cells against NALP5 and means for detecting said antibodies or T-cells bound to said binding reagent.
Preferably, the binding reagent is a NALP5 antigen.
In a second aspect, the invention relates to a method for providing support for diagnosis of cancer originating from tissue expressing PTHrP comprising the steps
- measuring the plasma level of NALP5 protein in a subject, and/or
- measuring the expression level of the NALP5 gene in a cell or tissue sample from a subject wherein an increased such level, as compared with healthy controls, is indicative of a cancer originating from tissue expressing PTHrP.
In one embodiment of this aspect, the cancer is cancer in the parathyroid glands, breast cancer, prostate cancer, myeloma, lung cancer, ovarian cancer or colon cancer.
In a further embodiment of this aspect of the invention, it is possible to distinguish between breast cancer of ductal origin and breast cancer of lobular origin. Said method may comprise the steps
- measuring the expression level of NALP5 in a tissue sample from a breast cancer tumour;
- correlating said expression level to the expression levels in healthy controls wherein an increased expression level indicates a breast cancer tumour with ductal origin.
One embodiment of this aspect of the invention relates to a kit comprising a binding reagent binding specifically to NALP5 protein and means for detecting NALP5 bound to said binding reagent.
Preferably, the kit comprises a binding reagent binding specifically to NALP5 mRNA or NALP5 DNA and means for detecting NALP5 mRNA or DNA bound to said binding reagent.
In a further aspect, the invention relates to a method for identifying a test compound as a candidate drug, comprising the steps of
(a) providing a host cell comprising a polynucleotide expressing a NALP5 protein;
(b) contacting the cell with said test compound; and
(c) analysing if said test compound modulates expression of the NALP5 protein. In a further aspect, the invention relates to a method for identifying a test compound as a candidate drug, comprising the steps of
(a) providing a NALP5 protein;
(b) contacting the NALP5 protein with said test compound; and
(c) analysing if said test compound binds to and/ or modulates the activity of the NALP5 protein;
(d) analysing if said test compound inhibits synthesis or secretion of PTH, PTHrP and/ or calcium sensing receptor.
In a further aspect, the invention relates to a genetically modified cell of an animal species comprising a heterologous DNA molecule encoding the NALP5 protein, and/or having the native gene encoding the NALP5 protein inactivated.
The animal species may be a mammal, such as human, non-human primates, and rodents.
In one embodiment of this aspect, the invention relates to a complete transgenic animal comprising the genetically modified cell.
In a further aspect, the invention relates to fusion protein comprising a NALP5 binding domain and a domain capable of providing an analytically detectable signal.
In further aspects, the invention relates to pharmaceutical preparations comprising an antibody, or fragment thereof, binding specifically to NALP5, a DNA construct encoding NALP5 or an oligonucleotide capable of binding to a NALP5 mRNA and inhibiting translation thereof, respectively, and optionally pharmaceutically acceptable carriers, adjuvants, diluents and excipients, and to methods of treatment of a patient in need thereof comprising the administration of such pharmaceutical preparations. The invention also relates to a method for treatment of parathyroid or ovarian disease or disorders involving PTHrP producing cells comprising adoptive transfer of T-cells reactive against NALP5 to patients in need thereof.
Brief description of the drawings
Figure 1. A) Protein domains of human NALP5: Pyrin domain, PYD, NACHT
NTPase domain, NACHT and the Leucine rich repeats LRR B) cDNA sequence of human NALP5 indicating its 14 exons, every other exon in dark grey C) cDNA sequence of the NALP5 clone isolated from the parathyroid cDNA library, D) region used for immunization and production of antiserum, E) region used for expression studies in real time PCR, F) conservation rate of NALP5 between different species.
Figure 2. A) Autoantibody titers against human NALP5 in sera from APS I patients, patients with other autoimmune disorders and healthy blood donors. B) Autoantibody titers against NALP5 in sera from Aire deficient mice and wild type mice. The figure demonstrates that APS I patients and Aire deficient mice have elevated levels of autoantibodies against NALP5.
Figure 3. Expression pattern/ profile of NALP5 mRNA in adult human tissues. The figure demonstrated the predominant expression of NALP5 in parathyroid glands.
Figure 4. Expression of human NALP5 homologues in different tissues. Left part: Phylogram tree designed in Clustal W illustrating homologies. Right part: mRNA expression in human multiple tissue panel. The figure demonstrates that NALP5 is the only member of NALP-protein -family that is expressed in parathyroid glands.
Figure 5. Confirmation /verification of antiserum produced against human and bovine NALP5. A) Western blot analysis on lysate from human parathyroid cells, lane 1 antiserum dilution 1 :400, lane 2 antiserum in dilution 1 :800, lane 3 antiserum in dilution 1 : 1600, lane 4 antiserum in dilution 1 : 1600 preincubated with 20nmol of the peptide used for immunization. B) Immunoprecipitation of bovine 35S radiolabeled parathyroid cell lysate with NALP5 antiserum. C) Immunohistochemistry on bovine ovaries using NALP5 antiserum in dilution 1 :2000 (left image) and NALP5 antiserum in dilution 1 :2000 preincubated with the peptide used for immunization.
Figure 6. Immunohistochemistry on paraffin embedded parathyroid sections. A) background, B) Preimmunization serum in dilution 1 :800, C) NALP5 rabbit antiserum in dilution 1 : 1600 D) NALP5 antiserum in dilution 1 : 1600 preincubated with 20nmol of the peptide used for immunization. The figure demonstrated the expression of NALP 5 protein in parathyroid tissue. The expression pattern of NALP 5 is identical in human and cattle.
Figure 7. Immunofluorescence on cryosections of bovine parathyroid glands. All sera used in dilution 1 :400 A) background staining, B-D) using sera from three different APS I patients with hypoparathyroidism, E-G) sera from three APS I patients without hypoparathyroidism, H-J) sera from three different healthy blood donors in dilution 1 :400, K) NALP5 antiserum, L) NALP5 antiserum IOOX magnification to analyse subcellular localization. The figure demonstrates the cytoplasmic localization of NALP5 and also presence of autoantibodies against parathyroid cells in sera from APS I patients.
Figure 8. Immunoprecipitation (IP) of NALP5. A) Immunoprcipitation of in vitro transcription and translated 35S radiolabeled human parathyroid NALP5 clone with (lane 1) NALP5 antiserum, (lanes 2-3) sera from APS I patients with hypoparathyroidism and reactivity against NALP5, (lanes 4-5) sera from APS I patients without reactivity to NALP5, and (lanes 6-7) control blood donor sera. B) Sequential IP of the in vitro transcription and translated 35S-methionine radiolabeled human parathyroid NALP5 clone. First immunoprecipitation step: (Lane 1) no antibody, (lane 2) NALP5 antiserum, (lanes 3-5) Sera from patients with reactivity to NALP5, (lanes 6-8) sera from patient without reactivity to NALP5, (lanes 9-1 1) sera from healthy control blood donors. Second IP step: NALP5 antiserum for all lanes. C) Sequential immunoprecipitation Of 35S- methionine labelled bovine parathyroid cell lysate. First IP step: (lane 1) no antibody, (lane 2) serum from APS I patient with reactivity against NALP5, (Iane3) serum from APS I patient without reactivity against NALP5, (lane 4) serum from healthy blood donor. Second IP step: NALP5 antiserum for all lanes.
Figure 9. Thymic NALP5 expression . (a) Thymic epithelial cells were isolated from embryonic mice as CD45- EpCAM+ cells at the indicated days of gestation (E) and analysed for the expression of NALP5. Values have been normalized to FoxNl expression, (b) Thymic epithelial cells expressing high and intermediate levels of MHC class II molecules on their cell surface and thymocytes were isolated from thymic tissue of adult wild type (wt) and Aire-deficient mice (ko) and subsequently analysed for the expression of NALP 5 and Aire. The expression levels for either of these two genes was compared to wt values which was set as 1.0. N. D. denominates not detected. The figure demonstrates the underlying molecular mechanism leading to the loss of tolerance to NALP5.
Figures 10-15. Immunohistochemistry on breast tissue using antiserum against NALP5. Typical regions that get stained are indicated with arrow/ s. Figure 10-1 1 immunohistochemistry on normal healthy breast tissue, the ducts / ductal cells are stained. Figure 12-15 immunohistochemistry on breast different breast cancer specimens. In 3 of 4 specimens, the cancer cells express NALP5 which appears as positive staining pattern in immunohistochemistry. In general, tumours with ductal (Figures 12 and 14-15) origin display increased expression of NALP5 whereas lobular tumours (Figure 13) express normal levels of NALP5.
Figures 16, 17. Immunohistochemistry on parathyroid tissue using antiserum against NALP5. In Figure 16, the staining pattern of parathyroid chief cells is shown. The parathyroid chief cells get stained. In Figure 17, the NALP5 antiserum is used in staining of parathyroid cancer specimens. As shown in the figure, a larger area is stained, indicating increased expression of NALP5 in parathyroid tumours. Figures 18-19. Immunohistochemistry on ovarian tissue using NALP5 antiserum. In Figure 18, immunohistochemistry is performed on healthy ovarian tissue and it is shown that the oocytes in the primary ovarian follicles are stained. In figure 19, the immunohistochemistry is performed on ovarian tumour/ cancer. Larger areas of the specimen get stained which indicate the increased expression of NALP5 in these tumours.
Figure 20. The role of NALP5 in calcium sensing and calcium signalling. Parathyroid cells were cultured for six hours in different extracellular calcium concentrations. After six hours, cells were harvested and expression level of mRNA for NALP5, GAPDH and PTH was determined using quantitative PCR. The figure demonstrates that the parathyroid cells respond to increased extracellular calcium concentration by increasing their expression of NALP5. This is an evidence for NALP5s role in calcium sensing and signalling.
Definitions
An antibody or binding reagent binding specifically to a target should be construed as an antibody or binding reagent that binds to the target with high affinity and show no or very low binding to other molecules.
A NALP5 antigen should be construed as any molecule binding native autoantibodies towards NALP5 with essentially the same or higher binding affinity and specificity as native NALP5. NALP5 antigens include, but are not restricted to, native NALP5 and fragments thereof with retained epitopes binding to autoantibodies.
Detailed description of the invention
Disclosed herein is the novel use of NALP5 protein and NALP5 related compounds and methods such as: NALP5 DNA, NALP5 mRNA, NALP5 protein, isolated naturally or artificially produced antibodies directed against NALP5, detection methods for measurement of NALP5 mRNA, NALP5 protein or antibodies against NALP5 DNA or NALP5 protein in diagnosis and treatment of autoimmune disorders, genetic disorders, parathyroid adenomas, tumour and cancer disorders and development of drugs or treatment methods including immune therapy.
Disclosed herein is the novel finding of the predominant expression of the protein NALP5 in human and animal (particularly bovine, equine, murine, feline and canine) parathyroid glands. NALP5 is important for calcium homeostasis and parathyroid function. NALP5 is also expressed in tissues and cells expressing parathyroid hormone related protein (PTHrP) . PTHrP is a protein secreted by different cells, especially some cancer cells leading to humeral hypercalcaemia of malignancy. PTHrP is produced commonly in breast cancer, prostate cancer and myeloma, but also other tumours such as lung cancers, ovarian cancers and colon cancers can express PTHrP6 9. Consequently NALP5 is expressed in the mentioned tumour/ cancer tissues and NALP5 and the mentioned NALP5 compounds can be used for diagnosis and treatment of these disorders.
Diagnosis of autoimmune disorders
Patients with autoimmune polyendocrine syndrome type 1 and other forms of autoimmune hypoparathyroidism harbour autoantibodies against human NALP5 as well as the murine and bovine orthologoues.
The first aspect of the invention is the assessment of autoantibodies and autoreactive T cells against NALP5 used as tool for diagnosis of autoimmune hypoparathyroidism and marker for onset of autoimmunity in patients, domestic animals and animal models. Detection of autoantibodies can be performed using a NALP5 antigen or active fragments thereof, e.g. recombinant produced in a suitable host system, e.g. in bacteria or eukaryotic cells, or translated NALP5 antigen using in vitro transcription and translation systems. The antigen may be labelled either by a marker, facilitating the detection thereof, e.g. by radioactivity10 12 or fluorescent dye13, just to mention two of the most common techniques. Such labelling can be performed either during the synthesis of the antigen as described above or by modifications after the synthesis. The NALP5 antigen, optionally labelled, may be immobilized on a surface or being in soluble form. Detection of an autoantibody-NALP5 antigen complex may be performed using instruments detecting radioactivity or fluorescence. Detection may also be performed using instruments that can distinguish the autoantibody-NALP5 antigen complex by sensing the association/ dissociation of the complex by changes in electrical or optical properties of the NALP5 -autoantibody complex or the surface on which the complex is immobilized on. An example of a technique which doesn't require specific labelling is surface plasmon resonance (SPR) 14> 15. NALP5 -autoantibody complex may also be detected using methods based on proximity-ligation16 18.
Autoreactive T cells may be detected using a NALP5 antigen or MHC Class I or II restricted epitopes of NALP5 as T cell stimulator followed by detection of activity markers of T cells such as intracellular or extracellular cytokines (e.g. gamma interferon or IL2) or surface expression of CD69, CD98, CD401igand, CD30, CD 134 or OX40. Autoreactive T cells may also be detected using MHC class I or class II tetramers or pentamers or hexamers loaded with 7-20mer peptides from
Diagnosis of tumour and cancer disorders
Expression of NALP5 is increased parathyroid adenomas and parathyroid cancers. NALP5 mRNA and protein expression has also been found to be increased in organs and tumours with PTHrP expression such as breast cancers, prostate cancers, myeloma, lung cancers, ovarian cancers, colon cancers.
The second aspect of the invention is thus the detection of increased levels of NALP5 mRNA or NALP5 protein in tissue, cell, blood, plasma, serum or other bodily fluids and used as diagnostic marker for cancers and tumours with origin in the parathyroid glands or any other tissues/ cells where PTHrP is expressed. A few examples of such tissues or cells are breast cancers, prostate cancers, myeloma, lung cancers, ovarian cancers, colon cancers. Detection and quantification of plasma levels of NALP5 may be performed using ELISA (enzyme linked immunoassay) based on either monoclonal or polyclonal antibodies, or fragments thereof, against any orthologue of NALP5, RIA (radio immunoassay) (either direct or indirect technique) based on either monoclonal or polyclonal NALP5 specific antibodies or fragments, and antibody based proximity ligation were the NALP5 specific antibodies (either monoclonal or polyclonal) can be conjugated to DNA probes followed by subsequent PCR quantification. This invention also relates to the detection and quantification of NALP5 mRNA and/ or protein expression in tumour biopsies used in cancer diagnostic20 ' 21. The detection and quantification of NALP5 mRNA may be performed with any kind of PCR based technique or real time PCR. The detection/ quantification of the NALP5 protein may be performed using immunohistochemistry and/ or immunofluorescence and /or proximity ligation based on monoclonal or polyclonal NALP5 specific antibodies or active fragments thereof 16"18> 22> 23 and (Alimohammadi et al. Accepted November 1 (2007) for publication in The New England Journal of Medicine) .
Diagnosis of parathyroid and calcium related disorders
Another aspect of the invention is a kit for diagnosis of parathyroid-related diseases, e.g. cancers and tumours. The kit comprises a binding reagent binding specifically to NALP5 protein, NALP5 DNA or NALP5 mRNA, e.g. nucleic acid probes or an antibody or a fragment thereof. The reagent may be provided in solution or optionally the kit comprises a liquid in which the reagent is solubilised prior to measurement. The reagent may also be provided immobilized to a solid phase, such as a micro titre plate, beads, a nitrocellulose membrane, a silicon wafer or any other solid phase used in the art.
The kit further comprises a detection reagent and means for detecting any complex formed between the binding reagent, detection reagent and NALP5 protein, mRNA or DNA. The binding reagent and detection reagent may be provided in the same molecule, e.g. as a fluorescently labelled nucleic acid probe, or separately, e.g. as a biotinylated binding reagent and a streptavidin- conjugated detection reagent. Detection and quantification of plasma levels of NALP5 may be performed using ELISA (enzyme linked immunoassay) based on either monoclonal or polyclonal antibodies, or active fragments thereof, against any orthologue of NALP5, RIA (radio immunoassay) (either direct or indirect technique) based on either monoclonal or polyclonal NALP5 specific antibodies or fragments, and antibody based proximity ligation16 18 ' 22> 23 were the NALP5 specific antibodies (either monoclonal or polyclonal) can be conjugated to DNA probes followed by subsequent PCR quantification. This invention also relates to the detection and quantification of NALP5 mRNA and/ or protein expression in tumour biopsies used in cancer diagnostic. The detection and quantification of NALP5 mRNA may be performed with any kind of PCR based technique or real time PCR. The detection/ quantification of the NALP5 protein may be performed using immunohistochemistry and/ or immunofluorescence and/ or proximity ligation16 18 ' 22> 23 based on monoclonal or polyclonal NALP5 specific antibodies or active fragments thereof.
Use of NALP5 as drug target
Since NALP5 is predominantly expressed in parathyroid glands and has an essential role in the parathyroid physiology and other tissues where PTHrP is expressed. We have also demonstrated that NALP5 is involved in calcium signalling and calcium regulation (Figure 20). Another aspect of the invention is thus the use of NALP5 as drug target and target for treatment of diseases in parathyroid glands such as hyperparathyroidism (primary, secondary as well as tertiary hypoparathyroidism) and in other normal or diseased organs and tissues where PTHrP is expressed. This is exemplified in tumours or cancers that express PTHrP which cause disruption in calcium homeostasis in these patients.
All subtypes of hyperparathyroidism are today difficult to treat and require surgery for treatment. Recently, calcimimetic compounds such as phenylalkylamine calcimimetics have been introduced as treatment for hyperparathyroidism. The drugs based on calcimimetic bind to the calcium sensing receptor and inhibit the secretion of PTH. We suggest NALP5 as an alternative drug target for treatment of hyperparathyroidism. Drugs binding to NALP5 and regulating its function could be used for regulation of PTH synthesis and secretion.
A further aspect of the invention is methods for identification of components capable of binding NALP5 and methods for screening drugs to identify compounds which interact with and bind to NALP5. The binding protein or an active fragment thereof may be in isolated form, in solution, or in immobilized form or may be genetically engineered to be expressed on the surface of recombinant host cells such as in phage display system or as fusion protein. Alternatively, whole cells or cell fractions comprising NALP5 may be employed in screening protocols. Regardless of the form of the binding protein, a plurality of compounds are contacted with the binding protein under conditions sufficient to form a compound / binding protein complex and compound capable of forming, enhancing or interfering with said complexes are detected24.
Another aspect of the invention is the use of nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to NALP5 binding protein-like sequences.
Still another aspect of the invention is the use of an antisense oligonucleotide (RNAi/ siRNA) having a sequence capable of binding with mRNAs encoding NALP5 so as to prevent the translation of NALP5 mRNA in parathyroid and other organs expressing PTHrP25.
A further aspect of the invention is the use of monoclonal or polyclonal antibodies constructed against NALP5 in therapeutical approaches on parathyroid disease such as parathyroid cancer or hyperparathyroidism or other disease in which parathyroid hormones are involved (e.g. osteoporosis, calcifylaxis or calcium/ phosphate dependent heart disease)26. Such diseases are considered as NALP5 -related diseases in the context of the present invention. These antibodies can be conjugated with different compounds that can affect the genes or living cells such cytostatic or radioactive agents or other bioactive drugs27 29. These antibodies may be humanized or otherwise adapted for use in therapy.
This invention also provides methods for adoptive transfer of T-cells reactive against NALP5 to patients with parathyroid or ovarian disease or disorders involving PTHrP producing cells such as breast cancers30 ' 31. T-cells used for adoptive T-cell transfer may be primed against NALP5 DNA, NALP5 protein or fragments thereof by incubation with NALP5 whole protein or T-cell restricted epitopes32.
DNA constructs containing NALP5 DNA33.34 or encoding NALP5 protein35 or fragments thereof can be used to vaccinate patients in order to prevent or treat cancer or autoimmune disorders.
Use of NALP5 in research and development
A further aspect of the invention is transgenic non-human animals comprising or a nucleic acid molecule encoding NALP5, or having the NALP5 gene knocked- out, and methods for use of transgenic animals as models for differential binding protein expression, mutations and immune responses and evaluation as well as in ligand and drug screens36. Such a method comprises comparing the results from measurements on a first animal with results from measurements on a second animal, the two animals having a difference in the genome leading to different NALP5 expression.
Another aspect of the invention is fusion proteins comprising a NALP5 binding domain and a binding protein /ligand binding indicator domain capable of providing an analytically detectable signal and the use thereof in methods of screening drugs by forming, enhancing or interfering with the detectable signal37. Example
The invention is further exemplified with the following example, which essentially corresponds to a manuscript accepted for publication (Alimohammadi et al, accepted November 1st 2007 for publication in The New England Journal of Medicine) . This example primarily demonstrate the role of NALP5 in calcium regulation. In addition to the example, Figures 10-19 show immunohistochemistry experiments in which tissue specimens have been immunostained by NALP5 -specific antiserum. These figures demonstrate the increased expression of NALP5 mRNA and protein in tissue specimens from human parathyroid tumour, human breast cancer and human ovarian cancer. . Moreover, an evidential figure demonstrating the response of parathyroid cells to changes in extracellular calcium by increasing the expression of NALP5 is also provided (Figure 20), this figure address the role of NALP5 in calcium sensing and calcium signalling.
When understanding and working the present invention the person skilled in the art may also make use of state of the art knowledge and standard technologies not described in detail in the present specification. Such technologies are i.a. disclosed in Current Protocols in Molecular Biology Print ISSN: 1934-3639, Current Protocols in Immunology 2007, John Wiley and Sons, Inc., Sambrook et al. "Molecular cloning: A laboratory manual", 3rd ed. 2001 ; Ausubel et al. "Short protocols in molecular biology", 5th ed. 1995; "Methods in enzymology", Academic Press, Inc.; MacPherson, Hames and Taylor (eds.). "PCR 2: A practical approach", 1995; "Harlow and Lane (eds.) "Antibodies, a laboratory manual" 1988; Freshney (ed.) "Culture of animal cells", 4th ed. 2000; Hogan et al. "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory, 1994; or later editions of these books.
The example and figures are provided for further understanding of the invention and should not be considered as limiting the scope of the invention, which is that indicated by the appended claims. Example 1: Identification ofNALP5 as a major parathyroid antigen in autoimmune polyendocrine syndrome type 1- a first link between the human disease and the Aire deficient murine model
Abstract
Autoimmune polyendocrine syndrome type 1 (APS I) is a multi-organ autoimmune disorder caused by mutations in the autoimmune regulator gene. Hypoparathyroidism is one of the cardinal symptoms of autoimmune polyendocrine syndrome type 1 , but up until today, no autoantigen has been identified in the parathyroid glands. A human parathyroid cDNA library, was immunoscreened and NALP5 identified as a major autoantigen. This protein is predominantly expressed in parathyroid chief cells and the presence of autoantibodies to NALP5 correlates with hypoparathyroidism in autoimmune polyendocrinesyndrome type 1. Moreover NALP5 is also recognised by sera from Aire deficient mouse, representing probably the first autoantigen identified in this murine model for APS 1. Studies on thymi from Aire deficient mice and wild type mice confirms that NALP5 is expressed in medullary epithelial cells of normal murine thymus but absent in thymi from Aire deficient mice, supporting previous speculations about the function of Aire in negative selection, and explaining the underlying molecular mechanism for loss of tolerance against NALP5.
Introduction
Autoimmune poly endocrine syndrome type I (APS I) (OMIM240300) is an autosomal recessive disorder caused by mutations in a single gene named autoimmune regulator (AIRE) 38. Aire is located on chromosome 22q23.3 and encodes a 54 kDa protein expressed in medullary epithelial cells of thymic stroma 39. AIRE's molecular function in regulation of transcription and its involvement in both central and peripheral tolerance has been illustrated recently 39-42. Despite this, the molecular pathophysiology of APS I is not completely understood. The disease starts in early childhood and patients develop multi organ-specific autoimmunity. Typically, endocrine organs such as adrenals, ovaries, pancreas and parathyroid glands are attacked by the immune system and it has been shown that patients harbour autoantibodies directed against key enzymes in these organs 43-45 τhe presence of autoantibodies against 21 -hydroxylase 46> 47, a key enzyme in steroid synthesis only expressed in the adrenal cortex, has been shown to predict the development of autoimmune adrenal insufficiency 48. Hypoparathyroidism is usually the first symptom of APS I affecting 81% of the patients 49> 50. Parathyroid glands secrete parathyroid hormone (PTH) which is essential for the calcium homeostasis, crucial for several cellular functions. Untreated hypoparathyroidism leads to hypocalcaemia and is a life threatening condition 51. It is believed that infants with APS I may die in a hypocalcaemic tetany caused by hypoparathyroidism prior to diagnosis. Although several organ-specific autoantigens have been identified in APS I, none of them has been associated with the parathyroid glands. The Calcium sensing receptor (CaSR) was suggested to be the parathyroid specific antigen in APS I and other acquired hypoparathyroidism 52, however, these data have not been reproducible in two different reports 50> 53. The identification of a parathyroid specific autoantigen would be of significance not only for an improved serological diagnosis of the disease, but also for increased understanding of the underlying pathophysiology and perhaps also of the physiology of the parathyroid glands.
A murine model for APS I was established in 2002 54. Until today five other Aire deficient murine models have been constructed 39> 55. In all of the constructs, the mice mimic the same mutation in the Aire gene (Finn major PR257X) as the majority of the APS I patients. Aire deficient mice suffer from several features such as infertility and lymphocyte infiltrates in several organs 39> 55. The genetic background of the mouse strain used for the construction the knock out model can influence the autoimmune symptoms of these mice 55. However, in comparison with the APS I patients, the murine phenotype is remarkably mild. Identification of a common B cell autoantigen in APS I patients and the murine model would open for a variety of cellular experiments. Despite extensive attempts (Pontynen 2006), Aire deficient mice do no not react with any murine orthologues to the many well characterised human autoantigens 50. In fact, no well characterised murine autoantigen has been identified up until now. NALP5 is a member of NACHT, leucine rich repeat and PYD containing proteins (NALPs) which are a large subfamily of the CATERPILLER protein family 56 and consist of 14 members named NALPl -14. NALPs are characterized by an N- terminal PYD domain suggested to be involved in protein-protein interactions, a central NACHT domain with a potential NTPase activity 57, a NAD domain with unknown function and C-terminal Leucine rich repeats (LRRs) suggested to be involved in molecular pattern sensing and protein-protein interaction 56> 58. Among NALPs, NALP5 also named maternal antigen that embryos require (MATER) has been studied in mice and cattle, and recently its expression has been reported to be specifically restricted to oocytes 2 5. Embryos of NALP5 -/- mice stay in developmental arrest at two-cell stage and die suggesting NALP5s crucial role in oogenesis through an essential role in cell viability.
In order to identify a parathyroid specific antigen we constructed and immunoscreened a human parathyroid cDNA library with sera from APS I patients and identified NALP5 as a novel major parathyroid autoantigen. In this paper we also report that NALP5, also known as MATER, is the common B cell autoantigen in human APS I and its murine model, the Aire deficient mouse.
Materials and Methods
Construction and Screening of cDNA Library
Total RNA was isolated from normal human parathyroid tissue removed during thyroid surgery. A cDNA expression library was constructed by use of the ZAP Express vector system (Stratagene, La Jolla, CA, USA and immunoscreened with sera from APS-I patients with hypoparathyroidism. Positive clones were isolated and sequenced.
Patients and Sera
Serum samples were obtained and analyzed from 11 Swedish, 18 Norwegian, and 58 Finnish APS-I patients who were members of more than 50 independent kindreds. Control sera were included from patients with either isolated hypoparathyroidism (n=20), Graves' disease (n=20), Addison's disease (n=20), type 1 diabetes (n=20), or Sjogren's syndrome (n=20) and from healthy blood donors (n= 193). The diagnosis of APS-I was based on the presence of two of the three major clinical manifestations including hypoparathyroidism, primary adrenal failure and mucocutanous candidiasis. The majority of the included APS-I patients (83 of 87) were also demonstrated to have typical mutations in the AIRE gene. The following diagnostic criteria were used:
Mucocutanous candidiasis: candidal infections in the oral mucosa, skin or nails for more than 3 months.
Hypoparathyroidism: Subnormal plasma calcium concentration (<2.15 mmol/L) and supranormal plasma phosphate concentration together with low normal or low PTH concentrations, and normal renal function.
Addison's disease: subnormal serum Cortisol together with elevated plasma ACTH concentrations, or failure to reach s-cortisol of 550 nmol/L at 30 or 60 min in an ACTH stimulation test. The majority of the patients diagnosed with Addison's disease also displayed specific 21 -hydroxylase autoantibodies. All of the control subjects were normocalcemic. Autoantibodies against the calcium sensing receptor (CaSR) were sought using as previously described methodology but could not be detected in the serum of any of the APS- 1 patients included in this study.50 Mice
Wild-type and Aire-deficient C57/B16 mice, were either analyzed as adult animals (at 2.5 - 10 months of age) or at defined gestational ages (E) whereby the day of plug defined was scored as EO.5.
Generation of 35S-radiolabelled NALP5 and Radioimmunoassay for NALP5 Autoantibodies
A full length cDNA clone for human NALP5 (cat#SC306608 obtained from Origene Technologies Inc. Rockville, MD) was used for coupled in vitro transcription/ translation and 35S-methionine labeling using the TnT system (Promega, Madison, WI, USA) according to the manufacturer's protocol. The 35S-radiolabelled recombinant NALP5 was directly immunoprecipitated with patient or control sera in 96-well filtration plates (Millipore). Serum from each individual was analyzed in a double blinded format in duplicates and 20 000 cpm of 35S-NALP5 was used for immunoprecipitation in each well. The immunoprecipitated radioactivity was evaluated in a liquid scintillation counter (Wallac 1450 MicroBeta). Sera from the APS-I patient used in the immunoscreen recognizing NALP5, and a healthy blood donor were used as positive and negative standard controls, respectively. For each analyzed serum sample, a NALP5 autoantibody index defined as (cpm in the unknown sample- cpm in the negative standard)/ (cpm in the positive standard- cpm in the negative standard)* 100 was calculated. Upper level of normal was defined as the mean results obtained for the healthy blood donors (n= 193) +3 SD and is indicated as a dashed line in the scattergram. The intra-assay and inter-assay coefficient of variations were 8.5 % and 12 %, respectively.
Sequential Immunoprecipitation
To confirm the specificity of the NALP5 autoantibodies in patient sera, sequential immunoprecipitation was performed. First, recombinant 35S-labelled NALP5 protein (150 000 cpm) was precipitated using 2.5 μl of patient or control serum. Antibody-antigen complexes were captured by protein-A Sepharose beads and subsequently removed by centrifugation. The remaining supernatant was then subjected to a second immunoprecipitation step but now using NALP5 -specific rabbit antiserum. The final immunoprecipitate was analyzed on an SDS-PAGE followed by autoradiography.
PCR Expression Analysis
Normalized cDNA from human multiple tissue panel (BD Bio Sciences, Palo Alto, CA) and from human parathyroid gland were used as templates for quantitative PCR. Details on primers and PCR conditions are provided in the Supplemental Appendix. Samples were analyzed in triplicate.
Generation of Antiserum to NALP5 and Immunoblot Analysis An antiserum was raised against human NALP5 by immunization of rabbits with a keyhole limpet hemocyanin conjugated peptide (KLH- CKSLSLAGNKVTDQG). The chosen sequence (aa 897-910) displays a high homology to bovine NALP5. The antiserum obtained was affinity purified on a peptide column. The specificity of the serum was verified by immunoblot with human and bovine parathyroid cells in the absence or presence of the peptide used for immunization.
Immunofluorescence and Laser-scanning Confocal Microscopic Analysis Cryosections (6 μm) of bovine parathyroid tissue were air dried, blocked and incubated with serum (dilution 1 :500) from APS-I patients displaying NALP5 reactivity. Sera from healthy blood donors and from APS- 1 patients without NALP5 autoantibodies as well as an affinity purified rabbit anti NALP5 rabbit serum were used as negative and positive controls, respectively. The slides were incubated with FITC conjugated secondary antibodies (dilution 1 :200) for 30 min and then analyzed on a Zeiss LSM 510 confocal microscope. To verify the specificity of APS-I patient sera for NALP5, absorption studies were performed using sera from 3 patients with NALP5 -specific autoantibodies and positive staining pattern in immunofluorescence. Sera from these patients were diluted (1 :500) to a final volume of 500 μl and incubated over night with either 15 μl PBS (as a negative control), 15 μl TnT derived recombinant 35S-radiolabelled recombinant NALP5 («450 000 cpm), 15 μl TnT derived recombinant 35S- radiolabelled NALP3 («470 000 cpm) or 15 μl TnT derived recombinant luciferase (~1 000 000 cpm). After over night absorption, the sera were tested using immunofluorescence on bovine parathyroid tissue as described above.
Immunohistochemistry
Human parathyroid tissue specimen removed during thyroid surgery, bovine parathyroid gland and bovine ovaries (obtained from the slaughterhouse) were fixed in PBS with 4% paraformaldehyde over night followed by paraffin embedding. Four μm sections were deparaffinized and microwave treated in citrate buffer as standard protocol. Non-specific bindings were blocked by preexposure of the sections for 30 minutes with PBS containing 10% normal goat serum. The slides were then incubated with the NALP5 antiserum (dilution 1 :4000) or patient sera (dilution 1 :8000) overnight at +4°C followed by washing and incubation with biotinylated secondary goat anti human or rabbit immunoglobulin antibody (Vector laboratories, Burlingame, CA 94010) for 30 minutes. Peroxidase conjugation was performed using the VECTASTAIN® ABC system (PK-6100 Vector laboratories, Burlingame, CA 94010) and the staining reaction used ChemMate™ DAKO Envision™ Detection kit (Dakocytoformation, Glostrup, Denmark). The specificity of the immunostaining was tested in control slides by omission of the primary antibody, by using pre-immunization serum from the rabbit in which the antiserum was raised, and by blocking the primary antibody through preincubation with the peptide used for the immunization in concentrations of 10-100 nmol/ml.
Parathyroid cell culture and metabolic 35S radiolabelling of parathyroid cells Bovine parathyroid glands were collected immediately after slaughter. Fat and connective tissue was removed, and the glands were minced with scissors. Cell suspensions were prepared by digestion in 1 mg/ml collagenase (Sigma, St Louis, Missouri, USA), 0.05 mg/ml DNase I, 1.5% bovine serum albumin and 1.25 mM Ca2+. After digestion in a shaking incubator for 120 min, the suspensions were filtered through nylon mesh (125 μm) and exposed to 1 mM EGTA in 25 mM HEPES buffer (pH 7.4) containing 142 mM NaCl and 6.7 mM KCl. Debris and dead cells were removed by centrifugation through 25 and 75% standard isotonic Percoll (GE Healthcare) . Cell viability, as determined by the Trypan blue exclusion test, exceeded 95%. Cells were cultured for 4 h in DMEM/ Ham's F- 12, 1 mM total calcium, 5% fetal bovine serum, 15 mM HEPES, 100 U/ ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin, 2 mM glutamine and 1% non-essential amino acids. Freshly isolated cells (107) were precultured for 30 min in methionine-free RPMI 1640. The medium was changed to methionine-free RPMI 1640 containing 5% dialyzed fetal bovine serum and 0.5 mCi 35S methionine (GE Healthcare) followed by incubation for 6h. The medium was removed and cells were washed 2 times with ice cold RPMI 1640 medium and 6 times with ice cold PBS. Cells were lysed on ice in a 2OmM Tris-HCl buffer (pH 7.4) containing 0.15 NaCl, 1% Triton X-100, 1 mM phenylmethyl sulfonylfluoride and 1% Trasylol. Insoluble material was removed by centrifugation at 100 000 x g for 20 min at +4°C. Immunoprecipitation was performed with different control sera, patient sera and rabbit antisera for 6h at +4°C and the bound immune complexes were captured by Fast Flow Protein A Sepharose followed by SDS-PAGE analysis.
Statistical Analysis
Fisher's exact test was used to compare the frequencies of reactivity to NALP5 with the major clinical manifestations in APS-I patients.
Ethics approval
All contents of this study were approved by the local ethics committese and the local animal ethics committees.
Results
Screening and immunoprecipitation of NALP 5
Screening of the normal human parathyroid cDNA library with sera from APS I patienst with hypoparathyroidism resulted in 14 independent clones. A nucleotide-nucleotide BLAST search at NCBI's human genome database revealed that two of these independent clones were encoding human NALP5. These two clones were fully sequenced and comparison with the published sequence of human NALP5 (Genebank accession number AYl 54460) indicated that both the isolated clones encoded exon 5-14 of the NALP5 cDNA (aa 170- 1 180) . Except lacking of the first 4 exons no other variations or mutations could be found (Figure 1). For verification of the screening results, the protein encoded by the isolated NALP5 clone was 35S radio-labelled by in vitro transcription and translation. As the clone lacked the first 170 amino acids the translation started at the following ATG encoding the methionine located at the amino acid position 194. This could be verified by SDS-PAGE analysis which indicated that the translation product had a molecular weight of 1 14 kDa (fig 8b, lane 1) in contrast to the expected 134 kDa for the full length human NALP5.
Autoantibodies against NALP5 were present in 36 of 87 (41%) sera from APS I patients. No control sera displayed reactivity against NALP5 (Figure 2A).
A strong correlation between the presence of autoantibodies against NALP5 and hypoparathyroidism in APS I patients was found (Table 1). In fact no sera from patients without hypoparathyroidism displayed NALP5 reactivity. Interestingly, a statistically significant correlation with gonad failure could be seen (Table 1).
Clinical disorder Number with Number with antibodies to P disorder/ total NALP5/total
With Without disorder disorder
Hypoparathyroidism 73/87 (84%) 36/73 (49%) 0/ 14 (0%) <0.05
Hypogonadism 28/87 (32%) 19/28 (68%) 17/59 (29%) <0.05
Addison's disease 69/87 (79%) 29/69 (42%) 7/ 18 (39%) 0.81
Insulin-dependent 1 1 /87 (13%) 2/ 1 1 (18%) 34/76 (45%) 0.097 diabetes mellitus
Vitiligo 17/87 (20%) 7/ 17 (41%) 29/70 (41%) 0.99
Alopecia 30/87 (34%) 1 1 /30 (37%) 25/57 (44%) 0.52
Hepatitis 15/87 (17%) 8/ 15 (53%) 28/72 (39%) 0.31
Malabsorption 22/87 (25%) 10/22 (45%) 26/65 (40%) 0.66
Pernicious anemia 14/87 (16%) 5/ 14 (36%) 31 /73 (42%) 0.64 Table 1
Association of clinical components in APS I to presence of antibodies against human parathyroid NALP5 clone. Fisher's two-tailed exact test was used to compare data.
Tissue distήbution ofNALP5mRNA
Tissue expression of human NALP5 mRNA was analyzed using real time quantitative PCR carried out on a normalized human multiple tissue cDNA panel (the amplified region is shown in figure 1 E) . NALP5 mRNA was predominantly expressed in parathyroid tissue (Figure 3) . NALP5 mRNA was also found to be expressed in ovaries as previously described in mice 2 5. To exclude a ubiquitous expression of the NALP family in the parathyroid glands, expression of other members in the NALP family with highest rate of homology to NALP5 was checked by a semi-quantitative PCR carried out on the same tissue cDNA panel as used for the real time quantitative PCR. Using primers for NALP4, NALP5, NALP6, NALP9, NALP 10, NALP 1 1 and NALP 13, only NALP5 mRNA was expressed in parathyroid tissue (Figure 4) .
NALP5 as an autoantigen in the parathyroid glands
To investigate whether NALP5 is translated in the parathyroid glands, rabbit antiserum was raised by immunization with a NALP5 specific human peptide (aa 897-910), a region with high homology rate to bovine NALP5. The specificity of the antibody was tested in western blot on lysate from bovine parathyroid and human parathyroid adenoma lysate. The antiserum recognized a band of expected molecular weight for human NALP5 as well as bovine NALP5 in both western blot analysis and immunoprecipitation of a 35S labelled parathyroid cell lysate (Figure 5A, B). As a control, immunohistochemistry was performed on formalin fixed and paraffin embedded sections of bovine ovaries (Figure 5C) and immunostaining was seen in the oocytes confirming previously published data2- 5. Based on the morphological picture, immunohistochemistry on formalin fixed and paraffin embedded bovine parathyroid tissues showed a specific cytoplasmatic staining of the chief cells (Figure 6) . The oxiphilic cells and stromal cells were not stained. Immunofluorescence on 6μm cryosections of bovine parathyroid tissue was performed (Figure 7) . The NALP5 antiserum and 3 of 4 APS I sera recognizing NALP5 gave raise to fluorescence (Fig. 7B-E) while no immunofluorescence could be detected when sera from patients not recognising NALP5 Fig 7F-H) or healthy blood donors (Fig. 7I-J) were used. Using the rabbit anti aa897-910 NALP5antiserum (Fig. 7 K) the subcellular localisation of NALP could be determined. . In concordance with previous reports 2 5 and prediction algorithms for subcellular localisation (Softberry ProtComp Version 6.1) a cytoplasmic localisation of NALP5 (Fig. 7L) was seen.
NALP5 expression in the thymus is Aire-dependent
Since immunological tolerance to some tissue-restricted antigens is affected by Aire-dependent, promiscuous gene expression in thymic epithelial cells, we next determined in the mouse thymus the temporal and spatial expression pattern of NALP5. Using RT-PCR, NALP5 expression was detected in epithelial cells of the embryonic thymus as early as day 13.5 of gestation (E 13.5) and coincided with the presence of Aire-specific transcripts (Fig. 9A).-NALP5 transcripts were also noticed in the developing parathyroid gland but were only consistently detected there at and beyond E 16.5 (data not shown). Within the postnatal thymus, NALP5 expression occurred in phenotypically mature thymic epithelial cells and was there most prominently detected in the subpopulation of MHC class IIhl§h epithelial cells which encompass medullary epithelial cells (Fig. 9B and data not shown). In Aire-deficient mice, NALP5 transcription was almost absent, revealing its dependency on Aire expression. (Fig. 9B).
Immunoprecipitation
The presence of autoantibodies directed against NALP5 clone was also verified using SDS-PAGE (Figure 8A) with in vitro transcribed and translated 35S NALP5 was used. The rabbit NALP5 antiserum, and sera from APS I patients and healthy blood donors were used (Figure 8 a) .
In order to further obtain proof that the APS I sera recognised NALP5, sequential immunoprecipitations were performed (Figure 8B). 35S-methionine labelled NALP5 was first incubated with a patient serum and a control serum, respectively, followed by subsequent immunoprecipitation using the rabbit peptide antiserum. This demonstrates that the vast majority of the NALP5 was removed by pre-precipitation with sera from NALP5 recognizing patients (lanes 3-5) but not with sera from patients who do not recognize NALP5 (lanes 6-8) or control blood donor sera (lanes 9-1 1). As a positive control for the assay no antibody was added in the first precipitation step (lanel) and as a negative control, the rabbit anti aa897-910 was added already in the first precipitation step (lane 2).
Finally, to verify the expression of NALP5 protein in parathyroid cells from bovine parathyroid cells were radiolabeled with 35S methionine and lysed for immunoprecipitation (Figure 8C). The lysate was used for sequential precipitation as described for figure 8B. As control the lysate was precipitated directly with the anti aa 897-910 rabbit antiserum and a band with the expected molecular weight of 124 kDa for bovine NALP5 could be seen. Preincubation with serum from a patient recognizing NALP5 could remove this band (Iane2), and when serum from a patient not recognizing NALP5 (Iane3) or serum from a blood donor (lane 4) wereused the NALP5 band remained.
NALP5 autoantibodies as a consequence of Aire deficiency
To correlate the detection of NALP5 -specific autoantibodies with a lack in Aire expression, we then compared sera from Aire-deficient and wild type mice for presence of NALP5 -specific autoantibodies. Ten of 44 mice (22%) of the samples revealed high titres of autoantibodies against murine NALP5, whereas such reactivity could not be detected in sera from wild type mice (Fig. 2B).
Calcium dependent expression ofNALP5:
To investigate NALP5s involvement in calcium homeostasis, freshly prepared human parathyroid adenoma cells and bovine normal parathyroid cells were prepared as described above (Methods, section: Parathyroid cell culture) and cultured in different physiological levels of extracellular calcium concentration and the mRNA expression of NALP5, parathyroid hormone (PTH) and calcium sensing receptor (CaSR) was measured by quantitative real time PCR. It is noteworthy that an increase in the extracellular calcium concentration increased mRNA expression of NALP5 whereas the expression of PTH mRNA (Figure 20).
Discussion
Using immunoscreening of a human parathyroid cDNA library, we have identified NALP5 as a major parathyroid autoantigen in APS I. Hypoparathyroidism is one of the three cardinal symptoms of APS I occurring in up to 80% of the patients 49> 50. NALP 5 is a member of the Caterpillar or NACHT-LRR family of proteins, thought to be involved in intracellular recognition of pathogens using a pattern recognition mechanism similar to the TOLL like receptors 59. Mutations in NALP-molecules are known to cause a number of inflammatory conditions 60. As shown in this paper, NALP5 is predominantly expressed in parathyroid tissue, namely in the parathyroid hormone (PTH) producing chief cells and to a lesser extent in the gonads, whereas no expression was found in immune cells so far tested. Interestingly, it has also been shown that a related molecule, NALP3 may recognise small intracellular molecules such as uric acid 61. When parathyroid cells were subjected to different calcium concentrations, the NALP5 expression increased with increasing calcium levels, thus completely opposite to the effect on PTH (Figure 20). It is thus tempting to suggest an important role of NALP5 in calcium homeostatis.
NALP5 has previously been described as an oocyte specific autoantigen in day 3 neonatal thymectomized mice (d3tx) 2 5, a well known animal model for organ specific autoimmunity including autoimmune ovarian disease (AOD) 62. AOD is similar to the human autoimmune gonadal insufficiency - one of the well known manifestations of APS I, affecting 48 % of APS I patients 49. This may imply NALP5 is also an ovarian autoantigen in APS I patients, and statistical analysis indicates in fact a significant correlation of NALP5 autoantibodies to both hypoparathyroidism and autoimmune ovarian insufficiency (Table 1).
Interestingly, it has been reported that hypoparathyroidism in APS I patients is the only disease component with a distinct gender difference appearing more prevalently in females (98%) than males (71%) 53. Our findings may give an explanation to this intriguing discrepancy.
Expression of NALP5 in parathyroid chief cells is a new and remarkable finding. In oocytes NALP5 has been shown to be important in early embryogenesis were the embryos of NALP5 deficient females do not develop further than the two-cell stage 4. Using electron microscopy murine NALP5 was localized to cytoplasm, mitochondria, and the nuclear pore 3. NALP5 has, however, no signals for either nuclear or mitochondrial localisation and this has been interpreted that other molecules interacting with NALP5 mediate its transport 3. In this study we observe cytoplasmic protein expression of bovine NALP5 in parathyroid cells using laser scanning confocal microscopy (figure 7L) in concordance with previous reports. The cytoplasmic localisation and the presence of a C-terminal leucine rich repeat and an ATP/ GTP binding domain are strong indicators for NALP5 being involved in protein-protein interaction and cellular signalling. The most important known function of parathyroid chief cells is to sense extracellular calcium concentration and secrete adequate amount of PTH. Murine NALP5 has been shown to be essential for embryogenesis extending the two-cell stage 5.This developmental stage corresponds to the time when the intracellular signalling must transit to intercellular signalling so that cellular migration can occur correctly and calcium signalling has been shown to be of importance at several stages during embryonic development 63. The calcium dependent expression of NALP5 mRNA in bovine parathyroid cells cultured in different calcium concentrations shown here may imply a key role in calcium signalling. On the other hand NALP5 -deficient mice survive and have few anomalities apart from female sterility. However, as glial cell missing 2 (Gcm2) transcription factor deficient mice without parathyroid glands 64 can maintain calcium homeostatis, the calcium regulation in mice may be different and perhaps relying more on rPTH than in humans.
An explanation for this is that mice could have another source of parathyroid hormone secretion than the parathyroid glands. Auxiliary production of PTH has been illustrated in mice with deficiency in Glial Cells Missing 2 (Gcm2) transcription factor which causes genetic ablation of the parathyroid glands 64
NALP5 has previously been identified as a murine autoantigen in d3tx mice, we investigated whether Aire deficient mice displayed autoantibodies against murine NALP5. High titer autoantibodies was found in 10/44 (22%) Aire- deficient mice and represents to the best of our knowledge the first well characterised autoantigen in the murine model of APS I.
It has been observed that Aire deficient mice have decreased fertility (unpublished observation) which could be explained by existence of NALP5 antibodies directed against oocytes. Aire deficient mice have normal serum calcium levels (not shown) and this could raise the question of why hypocalcaemia or symptoms thereof has not been observed in Aire deficient despite existence of NALP5 autoantibodies in 22% of the animals. The importance of the day 3 neonatal time window has been particularly observed/ studied/ since mice do not develop autoimmunity if the thymectomy is performed day 0 or day 7 after birth 65. The underlying mechanism behind the induction of AOD by d3tx has been discussed extensively and it has been shown that the induction of autoimmunity can be suppressed by adoptive transfer of normal CD4+ spleen cells to the d3tx mice 66> 67. Moreover, studies of the d3tx murine model have led to the discovery of CD4+ CD25+ regulatory T cells 68. A logic explanation for the development of AOD caused by d3tx would be that CD4+ CD25+ regulatory T cells are depleted during third day of life which in turn increases the activity of autorective T cells. The evidence supporting this theory is, however, not completely convincing and more complex causes has been suggested 69. Aire is a protein expressed in thymus with structural and functional properties suggestive of a transcription factor 55 and is suggested to control the transcription of several genes controlling the expression of a variety of self- antigen in thymus, important for the clonal deletion of autoreactive T cells 39, 42. Mutations in Aire result in development of autoimmune features in human patients as well as Aire deficient mice. In this study we find serological similarities between the d3tx mouse model and Aire deficient mouse model and APS I patients.
The similarity in serological reactivity between the Aire deficient and d3tx mice is interesting and implies a critical role for the presentation of NALP5 in the thymus and at day 3. When this is abrogated by either thymectomi or by lack of NALP5 -expression at this stage in Aire deficient mice, autoimmunity may ensue.
In conclusion, we have identified NALP5 as a major parathyroid autoantigen in APS I. Furthermore, we have identified the murine NALP5 orthologue as the first shared autoantigen in APS I patients and Aire deficient mice. The expression pattern of NALP5 during development in Aire deficient and wild type mice supports the previous hypothesis that promiscuous expression of organ-specific autoantigens in thymus at specific developmental stage is critical in order to prevent autoimmunity. References
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Claims

1. Method for providing support for diagnosis of autoimmune hypoparathyroidism comprising the steps
- detecting the presence of antibodies binding specifically to a NALP5 antigen and/or the presence of autoreactive T-cells against a NALP5 antigen, in a body fluid sample from a subject wherein the presence of such antibodies and/or autoreactive T-cells is indicative of autoimmune hypoparathyroidism in said subject.
2. Method according to claim 1 , wherein the autoimmune hypoparathyroidism is autoimmune polyendocrine syndrome type 1.
3. Kit for use in the method according to claim 1 or 2, comprising a binding reagent binding specifically to antibodies specific for NALP5 protein or to autoreactive T-cells against NALP5 and means for detecting said antibodies or T- cells bound to said binding reagent.
4. Kit according to claim 3, wherein the binding reagent is a NALP5 antigen.
5. Method for providing support for diagnosis of cancer originating from tissue expressing PTHrP comprising the steps
- measuring the plasma level of NALP5 protein in a sample from a subject, and/ or
- measuring the expression level of the NALP5 gene in a cell or tissue sample from a subject wherein an increased such level, as compared with healthy controls, is indicative of a cancer originating from tissue expressing PTHrP.
6. Method according to claim 5, wherein the cancer is cancer in the parathyroid glands, breast cancer, prostate cancer, myeloma, lung cancer, ovarian cancer or colon cancer.
7. Method for distinguishing between breast cancer tumours with ductal origin and lobular breast cancer tumours, comprising the steps
- measuring the expression level of NALP5 in a tissue sample from a breast cancer tumour;
- correlating said expression level to the expression levels in healthy controls wherein an increased expression level, as compared to healthy controls, indicates a breast cancer tumour with ductal origin.
8. Kit for use in the method according to claim 5, 6 or 7, comprising a binding reagent binding specifically to NALP5 protein and means for detecting NALP5 bound to said binding reagent.
9. Kit for use in the method according to claim 5, 6 or 7, comprising a binding reagent binding specifically to NALP5 mRNA or NALP5 DNA and means for detecting NALP5 mRNA or DNA bound to said binding reagent.
10. Method for identifying a test compound as a candidate drug, comprising the steps of
(a) providing a host cell comprising a polynucleotide expressing a NALP5 protein;
(b) contacting the cell with said test compound; and
(c) analysing if said test compound modulates expression of the NALP5 protein.
1 1. Method for identifying a test compound as a candidate drug, comprising the steps of
(a) providing a NALP5 protein;
(b) contacting the NALP5 protein with said test compound;
(c) analysing if said test compound binds to and/ or modulates the activity of the NALP5 protein;
(d) analysing if said test compound inhibits synthesis or secretion of PTH, PTHrP and/ or calcium sensing receptor.
12. Pharmaceutical composition comprising an antibody, or fragment thereof, binding specifically to NALP5, and optionally pharmaceutically acceptable carriers, adjuvants, diluents and excipients.
13. Pharmaceutical composition comprising a DNA construct encoding NALP5, and optionally pharmaceutically acceptable carriers, adjuvants, diluents and excipients.
14. Genetically modified cell of an animal species comprising a heterologous DNA molecule encoding the NALP5 protein, and/ or having the gene encoding the native NALP5 protein inactivated.
15. Genetically modified cell according to claim 14, wherein the animal species is a mammal.
16. Genetically modified cell according to any of claim 14-15, wherein the animal species is selected from the group consisting of human, non-human primates, and rodents.
17. Genetically modified non-human animal, comprising a cell according to any of claims 14-16.
18. Fusion protein comprising a NALP5 binding domain and a domain capable of providing an analytically detectable signal.
19. Pharmaceutical preparation comprising an oligonucleotide capable of binding to a NALP5 mRNA and inhibiting translation thereof, and optionally pharmaceutically acceptable carriers, adjuvants, diluents and excipients.
20. Method for treatment of a NALP5 related disease comprising administering a pharmaceutical preparation according to claim 12, 13 or 19 to a patient in need thereof.
21. Method for treatment of parathyroid or ovarian disease or disorders involving PTHrP producing cells comprising adoptive transfer of T-cells reactive against NALP5 to patients in need thereof.
PCT/SE2007/050905 2006-11-27 2007-11-27 Use of nalp5 in methods for diagnosis and therapy of parathyroid related disorders WO2008066483A1 (en)

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