WO2008066483A1 - Utilisation de nalp5 dans des méthodes de diagnostic et de traitement de troubles associés à la parathyroïde - Google Patents

Utilisation de nalp5 dans des méthodes de diagnostic et de traitement de troubles associés à la parathyroïde Download PDF

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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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nalp5
protein
parathyroid
cells
cancer
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PCT/SE2007/050905
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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/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/635—Parathyroid 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

La présente invention concerne l'utilisation de NALP5 dans des méthodes utiles dans le diagnostic et le traitement de troubles associés à la parathyroïde tels que l'hypoparathyroïdie auto-immune, un cancer originaire d'un tissu exprimant PTHrP et une maladie de la parathyroïde ou des ovaires impliquant des cellules produisant PTHrP. L'invention concerne également des kits pour mettre en œuvre ces procédés.
PCT/SE2007/050905 2006-11-27 2007-11-27 Utilisation de nalp5 dans des méthodes de diagnostic et de traitement de troubles associés à la parathyroïde WO2008066483A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9005982B2 (en) 2010-06-11 2015-04-14 The Regents Of The University Of California Biomarkers associated with autoimmune diseases of the lung
WO2016168711A1 (fr) * 2015-04-17 2016-10-20 The Regents Of The University Of California Procédés de détection d'une agglutination et compositions destinées à être utilisées dans la pratique de ceux-ci

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WO2002032955A1 (fr) * 2000-10-18 2002-04-25 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services. Gene humain indispensable a la fertilite
WO2002061049A2 (fr) * 2001-01-31 2002-08-08 Millennium Pharmaceuticals, Inc. Nouvelles molecules de la famille des proteines pyrin/nbs/lrr et leurs utilisations
US20030125282A1 (en) * 2001-08-10 2003-07-03 Schering Ag Human mater proteins
US20040053292A1 (en) * 2000-11-15 2004-03-18 Jurg Tschopp Proteins and dna sequences underlying these proteins used for treating inflammations
WO2004108891A2 (fr) * 2003-06-05 2004-12-16 Wyeth Methodes de criblage d'inhibiteurs de l'apoptose
WO2007128884A1 (fr) * 2006-05-09 2007-11-15 Oy Jurilab Ltd Gènes et marqueurs atypiques dans le diabète de type 2 et l'obésité

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WO2002032955A1 (fr) * 2000-10-18 2002-04-25 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services. Gene humain indispensable a la fertilite
US20040053292A1 (en) * 2000-11-15 2004-03-18 Jurg Tschopp Proteins and dna sequences underlying these proteins used for treating inflammations
WO2002061049A2 (fr) * 2001-01-31 2002-08-08 Millennium Pharmaceuticals, Inc. Nouvelles molecules de la famille des proteines pyrin/nbs/lrr et leurs utilisations
US20030125282A1 (en) * 2001-08-10 2003-07-03 Schering Ag Human mater proteins
WO2004108891A2 (fr) * 2003-06-05 2004-12-16 Wyeth Methodes de criblage d'inhibiteurs de l'apoptose
WO2007128884A1 (fr) * 2006-05-09 2007-11-15 Oy Jurilab Ltd Gènes et marqueurs atypiques dans le diabète de type 2 et l'obésité

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9005982B2 (en) 2010-06-11 2015-04-14 The Regents Of The University Of California Biomarkers associated with autoimmune diseases of the lung
WO2016168711A1 (fr) * 2015-04-17 2016-10-20 The Regents Of The University Of California Procédés de détection d'une agglutination et compositions destinées à être utilisées dans la pratique de ceux-ci
US11149296B2 (en) 2015-04-17 2021-10-19 The Regents Of The University Of California Methods for detecting agglutination and compositions for use in practicing the same
US12054768B2 (en) 2015-04-17 2024-08-06 The Regents Of The University Of California Methods for detecting agglutination and compositions for use in practicing the same

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