System for detecting igg4-related hypophysitis and / or igg4-related disease

Abstract

The present invention relates to a system for detecting IgG4-related hypophysitis and / or IgG4-related disease in a subject at risk of IgG4-related hypophysitis, comprising an anti-Secretogranin-2 (SCG2) antibody, an anti-Vasopressin-neurophysin2-copeptin (AVP) antibody, and / or an anti-Polyubiquitin-B (UBB) antibody.

Description

Detection system for IgG4-related hypophysitis and / or IgG4-related diseases

[0001] The present invention relates to a detection system for IgG4-related hypophysitis and / or IgG4-related diseases.

[0002] IgG4-related diseases are diseases in which, in addition to high blood IgG4 levels, marked infiltration of lymphocytes and IgG4-positive plasma cells and marked fibrosis result in enlargement, nodules, hypertrophic lesions, etc. being observed in various organs throughout the body simultaneously or metachronously. The cause of IgG4-related diseases remains unclear. The affected organs include multiple organs such as the pancreas, lacrimal gland / salivary gland, bile duct, kidney, lung, retroperitoneum, artery, central nervous system, thyroid gland, liver, digestive tract, prostate, lymph node, skin, breast, etc., and the lesions often involve multiple organs, but single-organ lesions may also occur.

[0003] The etiology of IgG4-related diseases is still mostly unclear, and diagnostic markers targeting autoantigens of IgG4-related diseases have been searched worldwide, and Laminin 511, etc. have been reported in autoimmune pancreatitis, which is one of the IgG4-related diseases (Non-Patent Document 1).

[0004] On the other hand, IgG4-related hypophysitis is a disease in which tumorous lesions are recognized in the hypothalamus-pituitary gland and pituitary hormone hyposecretion is exhibited. Although it presents clinical symptoms similar to those of hypothalamus-pituitary tumors, etc., since the treatment and prognosis are very different, although it is essential to distinguish IgG4-related hypophysitis from hypothalamus-pituitary tumors, etc., there is a problem that simple discrimination is difficult. For a definitive diagnosis, an invasive pituitary biopsy that cuts the head / face bones is necessary. Moreover, even when a pituitary biopsy is performed, it may be a study of a small biopsy tissue piece, and IgG4-positive cell infiltration associated with non-specific inflammation may be recognized, making the diagnosis difficult. Also, although high serum IgG4 levels are known as a marker for IgG4-related hypophysitis, cases that do not show high levels are not rare, and in addition, it is known that there are many discrepancies between blood IgG4 levels and pathological findings. Therefore, there are still many unresolved problems in the diagnosis of IgG4-related hypophysitis.

[0005] The main symptoms of IgG4-related hypophysitis include: 1. Local symptoms due to pituitary tumorous lesions or symptoms due to pituitary hypofunction; 2. Symptoms due to central diabetes insipidus.

[0006] As mentioned above, diagnosing IgG4-related hypophysitis is often difficult. The following are considered as diagnostic and pathological findings for IgG4-related hypophysitis: 1. Decreased baseline and target levels of one or more anterior pituitary hormones in the serum 2. Decreased responsiveness to anterior pituitary hormone secretion stimulation tests 3. Laboratory findings consistent with central diabetes insipidus 4. Diffuse enlargement of the pituitary gland or thickening of the pituitary stalk on imaging studies 5. Increased serum IgG4 concentration 6. IgG4-positive plasma cell infiltration in pituitary biopsy tissue 7. IgG4-positive plasma cell infiltration in lesional tissue of other organs

[0007] IgG4-related hypophysitis presents as anterior pituitary hypofunction or central diabetes insipidus depending on the site of impairment. Pituitary MRI imaging reveals diffuse enlargement of the pituitary gland or thickening of the pituitary stalk, as well as increased serum IgG4 concentration (≥135 mg / dl). Pituitary biopsy tissue shows IgG4-positive plasma cell infiltration, but since IgG4-positive cell infiltration can be secondarily observed in conditions such as Rathke's cleft, craniopharyngioma, malignant lymphoma, and granulomatosis with polyangiitis, careful differential diagnosis is necessary, along with clinical findings such as lesions in other organs and steroid responsiveness. Furthermore, the presence of anti-pituitary antibodies has been reported in IgG4-related hypophysitis, suggesting the involvement of an autoimmune mechanism (Non-Patent Literature 2).

[0008] Laminin 511 is a target antigen in autoimmune pancreatitis. Sci Transl Med. 2018 Aug 8;10(453). Anti-pituitary antibodies against corticotrophs in IgG4-related hypophysitis. Pituitary. 2017 Jun;20(3):301-310.

[0009] Therefore, the problem that the present invention aims to solve is to provide a non-invasive detection system for IgG4-related hypophysitis and / or IgG4-related disease using disease-specific autoantigens in the blood of IgG4-related hypophysitis. Another problem that the present invention aims to solve is to provide a detection marker for IgG4-related hypophysitis and / or IgG4-related disease.

[0010] As a result of diligent research by the inventors, it was discovered that a specific antibody is useful for detecting IgG4-related hypophysitis and / or IgG4-related diseases, thus completing the present invention.

[0011] The present invention is as follows: <1> A detection system for IgG4-related hypophysitis and / or IgG4-related disease in subjects at risk of IgG4-related hypophysitis, comprising an anti-Secretogranin-2 (SCG2) antibody, an anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or an anti-Polyubiquitin-B (UBB) antibody. <2> A detection marker for IgG4-related hypophysitis in subjects at risk of IgG4-related hypophysitis, comprising an anti-Secretogranin-2 (SCG2) antibody, an anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or an anti-Polyubiquitin-B (UBB) antibody. <3> A detection marker for IgG4-related disease in a subject suspected of having IgG4-related hypophysitis, comprising anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody. <4> An auxiliary method for diagnosing IgG4-related hypophysitis and / or IgG4-related disease, comprising the step of detecting anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in a sample derived from the subject, wherein the subject is a subject suspected of having IgG4-related hypophysitis. <5> A method for diagnosing IgG4-related hypophysitis and / or IgG4-related disease, comprising the steps of detecting anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in a sample derived from a subject, and determining that the subject has IgG4-related hypophysitis and / or IgG4-related disease when the presence of anti-SCG2 antibody, anti-AVP antibody and / or anti-UBB antibody is confirmed, wherein the subject is suspected to have IgG4-related hypophysitis (except in cases where the diagnosis is made by a physician). <6> The method according to <4> or <5>, wherein the sample is blood, plasma, serum, cerebrospinal fluid, or urine.

[0012] The present invention may also be described as follows: <A1> Anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody for use in the detection of IgG4-related hypophysitis and / or IgG4-related disease in subjects at risk of IgG4-related hypophysitis. <A2> Anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody for use as a detection marker for IgG4-related hypophysitis in subjects at risk of IgG4-related hypophysitis. <A3> Anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody for use as detection markers for IgG4-related disease in subjects at risk of IgG4-related hypophysitis. <B1> Use of anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in the manufacture of a detection system for IgG4-related hypophysitis and / or IgG4-related disease in subjects at risk of IgG4-related hypophysitis. <B2> Use of anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in the manufacture of detection markers for IgG4-related hypophysitis in subjects at risk of IgG4-related hypophysitis. <B3> Use of anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in the manufacture of detection markers for IgG4-related disease in subjects at risk of IgG4-related hypophysitis.<C1> Use of anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody for the detection of IgG4-related hypophysitis and / or IgG4-related disease in subjects at risk of IgG4-related hypophysitis. <C2> Use of anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody as a detection marker for IgG4-related hypophysitis in subjects at risk of IgG4-related hypophysitis. <C3> Use of anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody as detection markers for IgG4-related disease in subjects at risk of IgG4-related hypophysitis. In this specification, the descriptions in "1. Detection system," "2. Detection marker," "3. Auxiliary method for diagnosis," and "4. Diagnostic method" of the present invention as described herein may apply to A1 to C3 above.

[0013] The results of confirming the expression of human recombinant proteins of autoantigen candidates identified by proteomics analysis using Western blotting with anti-EGFP antibodies are shown. Images were captured using Image Quant 800 at 7.5% gel, exposure time: 1.4 seconds (automatic). While the results shown are for other proteins besides SCG2, AVP, and UBB, SCG2, AVP, and UBB were considered suitable as autoantigen candidates. M represents the molecular weight marker, and NT represents the background. Identification of candidate diagnostic markers for IgG4-associated hypophysitis (IgG4-RH) by Western blotting. The results for anti-SCG2 antibody (Secretogranin-2 (SCG2 gene)) are shown. White arrows indicate detected bands. Identification of candidate diagnostic markers for IgG4-associated hypophysitis (IgG4-RH) by Western blotting. The results for anti-AVP antibody (Vasopressin-neurophysin 2-copeptin (AVP gene)) are shown. White arrows indicate detected bands. Identification of a candidate diagnostic marker for IgG4-associated hypophysitis (IgG4-RH) by Western blotting. The results for anti-UBB antibody (Polyubiquitin-B (UBB gene)) are shown. Antigen-antibody reactions with patient serum were observed for three bands (corresponding to 0, digit 1, 2, or 3) with one, two, or three ubiquitin molecules attached to EGFP (black arrows). EGFP + 1 ubiquitin molecule: 35.5 kD, EGFP + 2 ubiquitin molecules: 44 kD, EGFP + 3 ubiquitin molecules: 52.5 kD. The results of ELISA-based experiments against the anti-AVP antibody (Vasopressin-neurophysin 2-copeptin (AVP gene)) are shown.

[0014] 1. Detection System The first embodiment of the present invention relates to a detection system for IgG4-related hypophysitis and / or IgG4-related diseases in subjects at risk of IgG4-related hypophysitis, comprising an anti-Secretogranin-2 (SCG2) antibody, an anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or an anti-Polyubiquitin-B (UBB) antibody.

[0015] The detection system in the first embodiment includes an antibody that binds to Secretogranin-2 (SCG2), Vasopressin-neurophysin2-copeptin (AVP), or Polyubiquitin-B (UBB).

[0016] Secretogranin-2 (SCG2) is a member of the chromogranin / secretogranin family of neuroendocrine secretory proteins and is suggested to be involved in the packaging and sorting of peptide hormones and neuropeptides into secretory vesicles. Because SCG2 is a molecule involved in secretion, its autoantibodies may be involved in pituitary disorders or abnormalities in pituitary hormone secretion, i.e., in pathological conditions. Secretogranin-2 (SCG2) may have the amino acid sequence shown in SEQ ID NO: 1, or it may have the nucleotide sequence shown in SEQ ID NO: 2, which is the Secretogranin-2 gene. The anti-Secretogranin-2 (SCG2) antibody may recognize a protein having an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 1. In this case, it is preferable that the protein has the function of Secretogranin-2 (SCG2). The anti-Secretogranin-2 (SCG2) antibody may recognize a protein having the amino acid sequence shown in SEQ ID NO: 1. In this case, it is preferable that the protein has the function of Secretogranin-2 (SCG2). Furthermore, the anti-Secretogranin-2 (SCG2) antibody may recognize a protein substantially consisting of the amino acid sequence shown in SEQ ID NO: 1, or it may recognize a protein consisting of the amino acid sequence shown in SEQ ID NO: 1. The anti-Secretogranin-2 (SCG2) antibody may also be an antibody that recognizes a protein translated from a nucleic acid having a base sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the base sequence shown in Sequence ID No. 2. In this case, it is preferable that the protein has the function of Secretogranin-2 (SCG2). The anti-Secretogranin-2 (SCG2) antibody may also be an antibody that recognizes a protein translated from a nucleic acid having the base sequence shown in Sequence ID No. 2.In this case, it is preferable that the protein has the function of Secretogranin-2 (SCG2). Furthermore, the anti-Secretogranin-2 (SCG2) antibody may be an antibody that recognizes a protein translated from a nucleic acid consisting substantially of the base sequence shown in SEQ ID NO: 2, or an antibody that recognizes a protein translated from a nucleic acid consisting of the base sequence shown in SEQ ID NO: 2. In this specification, SEQ ID NOs 2, 4, and 6 represent DNA strands, but may also represent RNA strands, or complementary strands having sequences complementary to SEQ ID NOs 2, 4, or 6, respectively.

[0017] Vasopressin-neurophysisin2-copeptin (AVP) directly exerts an antidiuretic effect on the kidneys, also causes peripheral vasoconstriction, and is suggested to act by binding to vasopressin receptors. Since AVP is an antidiuretic hormone, its autoantibodies may be involved in pituitary disorders such as central diabetes insipidus, or abnormalities in antidiuretic hormone secretion, i.e., in the pathological condition. Vasopressin-neurophysisin2-copeptin (AVP) may have the amino acid sequence shown in SEQ ID NO: 3, or it may have the nucleotide sequence shown in SEQ ID NO: 4, which is the vasopressin-neurophysisin2-copeptin gene. The anti-vasopressin-neurophysisin2-copeptin (AVP) antibody may also be an antibody that recognizes a protein having an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in Sequence ID No. 3. In this case, it is preferable that the protein has the function of vasopressin-neurophysisin2-copeptin (AVP). The anti-vasopressin-neurophysisin2-copeptin (AVP) antibody may also be an antibody that recognizes a protein having the amino acid sequence shown in Sequence ID No. 3. In this case, it is preferable that the protein has the function of Vasopressin-neurophysisin2-copeptin (AVP). Furthermore, the anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody may be an antibody that recognizes a protein substantially consisting of the amino acid sequence shown in SEQ ID NO: 3, or an antibody that recognizes a protein consisting of the amino acid sequence shown in SEQ ID NO: 3. The anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody may also be an antibody that recognizes a protein translated from a nucleic acid having a base sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the base sequence shown in SEQ ID NO: 4.In this case, it is preferable that the protein has the function of Vasopressin-neurophysisin2-copeptin (AVP). The anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody may also be an antibody that recognizes a protein translated from a nucleic acid having a base sequence containing the base sequence shown in Sequence ID No. 4. In this case, it is preferable that the protein has the function of Vasopressin-neurophysisin2-copeptin (AVP). Furthermore, the anti-Vasopressin-neurophysisin2-copeptin (AVP) antibody may also be an antibody that recognizes a protein translated from a nucleic acid having a base sequence substantially consisting of the base sequence shown in Sequence ID No. 4, or an antibody that recognizes a protein translated from a nucleic acid having a base sequence consisting of the base sequence shown in Sequence ID No. 4.

[0018] Polyubiquitin-B (UBB) is a protein necessary for the rapid metabolic turnover of ATP-dependent non-lysosomal intracellular proteins, both abnormal and normal, and it has been suggested that it covalently binds to and labels proteins being degraded. UBB-modified proteins are found in organ damage, including the central nervous system, and anti-ubiquitin antibodies may be useful in evaluating the accumulation of abnormal proteins and in understanding their involvement in pathogenesis. Polyubiquitin-B (UBB) may have the amino acid sequence shown in SEQ ID NO: 5, or it may have the nucleotide sequence shown in SEQ ID NO: 6, which is the Polyubiquitin-B (UBB) gene. The anti-Polyubiquitin-B (UBB) antibody may recognize a protein having an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 5. In this case, it is preferable that the protein has the function of Polyubiquitin-B (UBB). The anti-Polyubiquitin-B (UBB) antibody may recognize a protein having an amino acid sequence shown in SEQ ID NO: 5. In this case, it is preferable that the protein has the function of Polyubiquitin-B (UBB). Furthermore, the anti-Polyubiquitin-B (UBB) antibody may recognize a protein substantially composed of the amino acid sequence shown in SEQ ID NO: 5, or it may recognize a protein consisting of the amino acid sequence shown in SEQ ID NO: 5. The anti-Polyubiquitin-B (UBB) antibody may also be an antibody that recognizes a protein translated from a nucleic acid having a base sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the base sequence shown in Sequence ID No. 6. In this case, it is preferable that the protein has the function of Polyubiquitin-B (UBB). The anti-Polyubiquitin-B (UBB) antibody may also be an antibody that recognizes a protein translated from a nucleic acid having the base sequence shown in Sequence ID No. 6.In this case, it is preferable that the protein has the function of Polyubiquitin-B (UBB). Furthermore, the anti-Polyubiquitin-B (UBB) antibody may be an antibody that recognizes a protein translated from a nucleic acid consisting substantially of the base sequence shown in SEQ ID NO: 6, or an antibody that recognizes a protein translated from a nucleic acid consisting of the base sequence shown in SEQ ID NO: 6.

[0019] The first embodiment is a detection system for IgG4-related hypophysitis and / or IgG4-related diseases in subjects at risk of IgG4-related hypophysitis, using anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody.

[0020] "Subjects suspected of having IgG4-related hypophysitis" to which the detection system applies refer to subjects who have at least one symptom known to be caused by IgG4-related hypophysitis, or subjects who do not have such symptoms but are suspected of having IgG4-related hypophysitis. Subjects may generally be human. "Subjects suspected of having IgG4-related hypophysitis" may also be subjects who need to be differentiated from those who have IgG4-related hypophysitis, and may also be subjects who require a pituitary biopsy to differentiate from those who have IgG4-related hypophysitis.

[0021] In the first embodiment, it is detected whether the subject is IgG4-related hypophysitis and / or an IgG4-related disease. Here, IgG4-related diseases are not particularly limited, but include IgG4-related hypophysitis, IgG4-related hypertrophic pachymeningitis, IgG4-related thyroiditis, IgG4-related dacryoadenitis, IgG4-related eye diseases, IgG4-related sialadenitis, asthma-like symptoms, IgG4-related kidney disease, IgG4-related respiratory diseases, IgG4-related sclerosing cholangitis, IgG4-related prostatitis, IgG4-related retroperitoneal fibrosis, type 1 autoimmune pancreatitis, and IgG4-related lymphadenitis. In the first embodiment, it is preferable that IgG4-related diseases and IgG4-related hypophysitis are understood separately, so it is preferable that IgG4-related diseases exclude IgG4-related hypophysitis. IgG4-related hypophysitis is distinguished from other types of hypophysitis, including lymphocytic hypophysitis, pituitary lesions due to local diseases (germinoma, Rathke's cleft, craniopharyngioma, pituitary adenoma), and pituitary lesions due to systemic diseases (sarcoidosis, Langerhans cell histiocytosis, granulomatosis with polyangiitis, syphilis, tuberculosis, drug-induced hypophysitis, and immune checkpoint inhibitors such as anti-CTLA-4 antibodies and anti-PD-1 antibodies).

[0022] The "detection system" according to the first embodiment is useful for diagnosing IgG4-related hypophysitis and / or IgG4-related disease. This system detects whether a subject suspected of having IgG4-related hypophysitis is suffering from IgG4-related hypophysitis and / or IgG4-related disease by detecting the presence of anti-SCG2 antibodies, anti-AVP antibodies, and / or anti-UBB antibodies.

[0023] The "detection system" detects the presence of anti-SCG2 antibodies, anti-AVP antibodies, and / or anti-UBB antibodies in a sample derived from the subject, but "presence" can be detected as the "amount" or "concentration" of each antibody. The "detection system" can also determine that IgG4-related hypophysitis and / or IgG4-related disease is present if the amount of anti-SCG2 antibodies, anti-AVP antibodies, and / or anti-UBB antibodies present in a sample derived from the subject is higher than a certain predetermined value, or that IgG4-related hypophysitis and / or IgG4-related disease is present simply because anti-SCG2 antibodies, anti-AVP antibodies, and / or anti-UBB antibodies are present in a sample derived from the subject. Alternatively, detection by the "detection system" may be used to indicate the high probability that the subject is suffering from IgG4-related hypophysitis and / or IgG4-related disease.

[0024] The "detection system" may include the following steps: (1) a step of detecting anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in a sample derived from a subject suspected of having IgG4-related hypophysitis; (2) a step of determining that the subject has IgG4-related hypophysitis and / or IgG4-related disease if the presence of anti-SCG2 antibody, anti-AVP antibody and / or anti-UBB antibody is confirmed.

[0025] In step (1), anti-SCG2 antibody, anti-AVP antibody, and / or anti-UBB antibody are detected in the sample. This detection may be performed by measuring the amount of these antibodies present, and it is not necessarily required to quantify the amount of these antibodies in the sample.

[0026] The specimens used in the "detection system" are not particularly limited as long as they originate from subjects suspected of having IgG4-related hypophysitis, but are preferably components derived from subjects for whom the likelihood of developing IgG4-related hypophysitis and / or IgG4-related diseases needs to be predicted. Examples include blood, plasma, serum, cerebrospinal fluid, and urine, with blood, plasma, and serum being more preferred. Conventional treatments may be applied to the specimens. Conventional treatments include adding noncoagulants or stabilizers.

[0027] Since the detection of antibodies in a sample is generally carried out by conventionally known methods, such as contacting the sample with a detection reagent for detecting antibodies present in the sample to generate an immune complex, the "detection system" in the first embodiment may also use a conventionally known method. In the "detection system," the sample and the detection reagent are brought into contact in order to detect anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysin 2-copeptin (AVP) antibody, and / or anti-Polyubiquitin-B (UBB) antibody in the sample. Contact between the sample and the detection reagent is generally carried out by adding the detection reagent to the sample. By adding a detection reagent, the "detection system" generates immune complexes, and by measuring the presence of these immune complexes, it detects anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysin2-copeptin (AVP) antibody, and / or anti-Polyubiquitin-B (UBB) antibody in the sample. The method for detecting immune complexes is not particularly limited and can be carried out by known methods for detecting complexes through antigen-antibody reactions.

[0028] While not limited to these methods, examples of measurement methods for detecting immune complexes include latex agglutination, fluorescence immunoassay (FIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blotting. Preferred measurement methods include FIA ​​and EIA (including ELISA), with ELISA being a more preferred method. These conventional methods enable highly sensitive, rapid, and convenient detection of immune complexes.

[0029] Examples of labeling substances, though not particularly limited, include enzymes such as peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, β-D-galactosidase, glucose oxidase, and glucose-6-phosphate dehydrogenase; fluorescent substances such as fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and europium; chemiluminescent substances such as luminol, isoluminol, and acridinium derivatives; coenzymes such as NAD; biotin; and radioactive substances such as 131I and 125I.

[0030] Next, step (2) may be performed to determine whether the subject has IgG4-related hypophysitis and / or IgG4-related disease. Step (2) may be performed by confirming the presence of anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in the sample via the presence of immune complexes in the sample. If the presence of anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysin2-copeptin (AVP) antibody, and / or anti-Polyubiquitin-B (UBB) antibody is confirmed in the sample, the possibility that the subject (or sample derived from the subject) has IgG4-related hypophysitis and / or IgG4-related disease may be determined. Alternatively, in step (2), the determination may be made based on a comparison between a reference detection value and a detection value from a sample derived from the subject. As the reference detection value, a sample from a healthy individual, a subject suspected of having IgG4-related hypophysitis, a subject suffering from IgG4-related hypophysitis, a subject suspected of having IgG4-related disease, or a subject suffering from IgG4-related disease may be used. The determination may be qualitative or quantitative, and it may be performed automatically / mechanically without the judgment of a person with specialized knowledge such as a physician or laboratory technician.

[0031] Alternatively, step (2) may be performed by comparison with a reference value. For example, if the detected value in the subject is higher than the reference value, it may be determined that "the disease is IgG4-related hypophysitis and / or IgG4-related disease," and if the detected value is lower than the reference value, it may be determined that "the disease is not IgG4-related hypophysitis and / or IgG4-related disease," "there is a low probability of having IgG4-related hypophysitis and / or IgG4-related disease," or "the subject does not have IgG4-related hypophysitis and / or IgG4-related disease."

[0032] 2. Detection Markers A second embodiment of the present invention relates to detection markers for IgG4-related hypophysitis or IgG4-related diseases. Detection markers are important indicators for distinguishing and / or evaluating IgG4-related hypophysitis and / or IgG4-related diseases.

[0033] "Detection marker" means a marker capable of detecting IgG4-related hypophysitis and / or IgG4-related disease in a target specimen, and in the second embodiment, the detection marker includes anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody. The "detection marker" is useful for diagnosing IgG4-related hypophysitis and / or IgG4-related disease. The "detection marker" may also be a kit or tool containing other reagents for diagnosing IgG4-related hypophysitis and / or IgG4-related disease. Alternatively, it may be a detection system containing the detection marker according to the second embodiment.

[0034] The detection marker may be a detection kit or a detection reagent, and the description of a detection marker is understood in this specification to be replaced with "kit" or "reagent." The detection kit and detection reagent may include known reagents used in measurements such as latex agglutination, fluorescence immunoassay (FIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blotting. The detection kit and detection reagent may also include instructions describing the experimental protocol.

[0035] 3. Auxiliary Method for Diagnosis A third embodiment of the present invention relates to an auxiliary method for diagnosing IgG4-related hypophysitis and / or IgG4-related disease. Examples of auxiliary methods include data acquisition methods. The auxiliary method for diagnosis according to the third embodiment is an auxiliary method for diagnosing IgG4-related hypophysitis and / or IgG4-related disease, wherein the subject is a subject suspected of having IgG4-related hypophysitis, and includes the step of detecting anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in a sample derived from the subject.

[0036] The step of detecting anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysin2-copeptin (AVP) antibody, and / or anti-Polyubiquitin-B (UBB) antibody in a sample derived from the target is as described above.

[0037] In an adjunct method for diagnosing IgG4-related hypophysitis and / or IgG4-related disease, data on the detection of anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysin2-copeptin (AVP) antibody, and / or anti-Polyubiquitin-B (UBB) antibody in the specimen may be provided as an adjunct diagnostic tool. The detection data may include the amount of antibody present in the specimen, its presence or absence, or data used by physicians or other medical professionals for diagnosis based on this detection data. This information can provide support or corroboration to the conventional diagnosis of IgG4-related hypophysitis and / or IgG4-related disease. This information is beneficial for determining more appropriate treatment strategies, promoting improved treatment effectiveness and a higher quality of life (QOL) for the patient. On the other hand, the present invention may be used to monitor the disease state in order to prevent the disease from becoming intractable, worsening, or recurring.

[0038] 4. Diagnostic Method A fourth embodiment of the present invention relates to a diagnostic method for IgG4-related hypophysitis and / or IgG4-related diseases. However, the diagnostic method according to the fourth embodiment excludes cases where the diagnosis is made by a physician.

[0039] The diagnostic method according to the fourth embodiment includes the steps of detecting anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in a sample derived from a subject, and determining that the subject has IgG4-related hypophysitis and / or IgG4-related disease when the presence of anti-SCG2 antibody, anti-AVP antibody and / or anti-UBB antibody is confirmed, and is a diagnostic method for IgG4-related hypophysitis and / or IgG4-related disease in which the subject is suspected to have IgG4-related hypophysitis (except in cases where the diagnostic method is performed by a physician). The step of detecting anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysin2-copeptin (AVP) antibody, and / or anti-Polyubiquitin-B (UBB) antibody in a sample derived from the subject is as described above. In the diagnostic method according to the fourth embodiment, the presence or absence of disease in the subject (subject sample) can be determined based on objective indicators such as the amount of anti-SCG2 antibody, anti-AVP antibody, and / or anti-UBB antibody present.

[0040] The anterior pituitary gland was excised from rats, and an anterior pituitary protein extract was obtained, along with IgG from the blood of patients with a confirmed diagnosis of IgG4-related disease (see [1] to [2] below). These were reacted to obtain antigen-autoantibody complexes for each patient, and the antigens were separated and analyzed to determine candidate autoantibody proteins (see [3] below). Furthermore, full-length human proteins were synthesized by genetic recombination based on the amino acid sequences of human proteins corresponding to the proteins determined as candidate autoantibodies. Autoantigens and diagnostic markers (autoantibodies) were identified by Western blotting using these synthesized proteins and serum collected from each patient group.

[0041] [1] Preparation of rat anterior pituitary protein extract The anterior pituitary gland was excised from male SD rats. The excised anterior pituitary tissue was homogenized with lysis buffer (20 mM Tris / HCl, 120 mM NaCl, 1 mM EDTA, 1% SDS, PhosSTOP®, protein inhibitor, pH 7.4), then sonicated, reduced with DTT, and the supernatant obtained from ultracentrifugation at 43,000 rpm for 1 hour was used as the anterior pituitary protein extract.

[0042] [2] IgG was isolated and purified from the blood of three patients (#AH28, #1014, #AH134) with IgG4-related hypophysitis (hypophysitis observed, no other organ lesions) confirmed by pituitary biopsy, three patients (#AH115, #1276, #AH38) with IgG4-related disease (hypophysitis and other organ lesions observed), and three healthy individuals using the IgG Purification Kit (Cosmo Bio).

[0043] [3] Immunoprecipitation, mass spectrometry, and data analysis. Purified IgG and rat anterior pituitary protein extract were mixed, Protein A beads were added, and the mixture was inverted and mixed at 4°C for 16 hours. After immunoprecipitation, the antigen was eluted from the immunoprecipitate by washing and addition of trypsin. The eluted antigen was reduced and alkylated, then digested with trypsin (in-solution digestion) and analyzed using an LC-MS / MS system (Orbitrap Fusion). Based on the SwissProt database, etc., the MS data obtained using Mascot (Matrix Science Ltd.) software was analyzed to identify candidate self-antigen proteins.

[0044] The candidate proteins obtained by creating human recombinant proteins were subcloned into an expression vector. Subsequently, they were introduced into HEK293 cells to create human recombinant proteins. The expression vectors were created by VectorBuilder. Using Lipofectamine 3000 reagent (Thermo Fisher Scientific), each vector was transfected into HEK293 cells cultured in a 12-well plate according to the manufacturer's instructions. After 24 hours, the transfected HEK293 cells were collected. The expression of each recombinant protein was confirmed by Western blotting using an anti-EGFP antibody (Invitrogen). The results are shown in Figure 1.

[0045] The antigen-antibody reaction between the self-antigen candidates identified by proteomic analysis of the detection marker candidates and patient sera was confirmed by Western blotting. The recombinant proteins were separated by SDS-PAGE, and the antigen-antibody reaction was analyzed by Western blotting using patient and healthy sera as primary antibodies and HRP-labeled anti-human IgG antibody as the secondary antibody. As a result, the anti-SCG2 antibody was observed in 3 IgG4-RH patients, suggesting the possibility of a novel detection marker for IgG4-RH (Figure 2). The detected bands are indicated by white arrows. The anti-AVP antibody was observed in 2 IgG4-RH patients, suggesting the possibility of a novel detection marker (Figure 3). The detected bands are indicated by white arrows. The anti-UBB antibody was observed in 3 IgG4-RH patients, suggesting the possibility of a novel detection marker (Figure 4). The detected bands are indicated by white arrows (corresponding to the numbers 1, 2, or 3 in 0 digits).

[0046] Subsequently, it was also confirmed by ELISA that autoantibodies in patient sera could be detected. [1] Preparation of antigen-coated ELISA plate Using the antigen dissolved in cold PBS, an amount of 0.5 μg / well was added to each well of the ELISA plate to immobilize the antigen consisting of the amino acid sequence set forth in SEQ ID NO: 3. After standing overnight at 4°C, the antigen aqueous solution was discarded, and the blocking buffer (50 mM NaHCO ３[2] Add 100 μL / well of the patient serum to an ELISA plate and leave it standing overnight at 4°C. Then discard the blocking buffer and store it with the desiccant at 4°C until use. [2] Add the reaction buffer (containing BSA, 2-CAA, Block Ace, and PBS) to the ELISA serum to prepare the patient serum diluent. Add 100 μL / well of the patient serum diluent (1:101 dilution) to the antigen-immobilized plate and shake at room temperature for 2 hours to allow the reaction to occur. Then discard the patient serum diluent and wash the ELISA plate with the wash buffer. Next, add 100 μL / well of the secondary antibody solution Anti-Human IgG (γ-chain)-HRP to the ELISA plate and shake at room temperature for 1 hour. Discard the secondary antibody solution and wash the ELISA plate with the wash buffer. Next, 100 μL / well of the 4°C reaction substrate solution was added to the ELISA plate and left to stand at room temperature for 15 minutes. After stopping the reaction by adding 100 μL / well of the reaction stop solution (1 M sulfuric acid), the wavelength at 450 nm (secondary wavelength 620 nm) was measured. [3] Results Serum from IgG4-related hypophysitis patients reacted with AVP protein by ELISA, and the presence of anti-AVP antibodies in the patients' serum was confirmed (Figure 5, n=3). Therefore, it was shown that autoantibodies detected in serum by Western blotting can be similarly detected by ELISA.

[0047]

[0048] Vasopressin-neurophysin 2-copeptin (AVP) Amino acid sequence (SEQ ID NO: 3) MPDTMLPACFLGLLAFSSACYFQNCPRGGKRAMSDLERQCLPCCGPGGKGRCFGPSICC ADELGCFVGTAEALRCQEE NYLPSPCQS GQKACGSGGRCA AFGVCCNDE S CVTEPECR EGFHRRARASDRSNATQLDGPGAGALLLR LVQLAGAPE PFEP AQPDAY Vasopressin-neurophysin 2-copeptin (AVP) Gene sequence (SEQ ID NO: 4) ATGCC TGACAC CATGCT GCCCG CTTCCT CGGCC TACTGG CCTTC TCCTC CCGCG CTACTT CCAGA ACTGCC CAGAG GGGC GGCA AGAG GGCC ATGTCC GACCT GGAGC TGAGA CAGTG CCTCC CCTGC GGGCC CCGGG GGCA AAGGC CGCCT GCTTC GGGC CCA GCA TCTGCT GCGCG GACGA GCTGG GCTGC TTCGT GGGC ACGGC TGAGG CGCTG CGCCT GCCAG GAGGA AACTAC CTGCC GTCGC CCTGC CAGTC CGGCC AGAAG GGCG TGCGG GAGCG GGGG GCCG CTGCG CCGCC TTCGG CGTTT GCTGA AACGA CGAGAG CTGCG TGACC GAGCC CAGAG TGCCG CGAGG GGCT TTCAC CGGCC GCGCC CCGCG CCAGC GACCG GGAGC AACGC CACTG CAGCT GGACG GGCCG GGCC GGGC CTTCG CTGCT GCGGG CTGGT GCAGCT GGCC GGGGG CGCCC GAGCC CTTCE GCCCC CAGCC CCGAC GCTTA CTGA

[0049] P]uuouuuth-B(UBB) Amino acid sequence (SEQ ID NO: 5) MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQRLIAAIN IQKESTLHLVLRLRGGQIKVKTLTGKTITLEVE@DTIENVKKIQDKEGIPPPQQRIA KIQDKEGIPPDQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGC POLIIAAGLBOOOOOOOOOOOOOOOOOOOOOOOOOOO - - ΑΚΟΑΡΑΑΚΑΡΟΣΣΑΡΑAAGGAGTCGGACCGCACCGAACCGCCCGGAGCGJACGCAGACTTCGGAAAA - - - ΑΚΑΚΟΣATCGAAAATGTGAAGACAAGATCCAGATAAAAGGCACAAAAA -

Claims

1. A detection system for IgG4-related hypophysitis and / or IgG4-related disease in subjects at risk of IgG4-related hypophysitis, comprising an anti-Secretogranin-2 (SCG2) antibody, an anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody, and / or an anti-Polyubiquitin-B (UBB) antibody.

2. A detection marker for IgG4-related hypophysitis in subjects at risk of IgG4-related hypophysitis, comprising anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody.

3. A detection marker for IgG4-related disease in subjects at risk of IgG4-related hypophysitis, comprising anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody, and / or anti-Polyubiquitin-B (UBB) antibody.

4. An auxiliary method for diagnosing IgG4-related hypophysitis and / or IgG4-related disease, comprising the step of detecting anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in a sample derived from a subject, wherein the subject is suspected of having IgG4-related hypophysitis.

5. A method for diagnosing IgG4-related hypophysitis and / or IgG4-related disease, comprising the steps of detecting anti-Secretogranin-2 (SCG2) antibody, anti-Vasopressin-neurophysiin 2-copeptin (AVP) antibody and / or anti-Polyubiquitin-B (UBB) antibody in a sample derived from a subject, and determining that the subject has IgG4-related hypophysitis and / or IgG4-related disease when the presence of anti-SCG2 antibody, anti-AVP antibody and / or anti-UBB antibody is confirmed, wherein the subject is suspected of having IgG4-related hypophysitis (except in cases where the diagnosis is made by a physician).

6. The method according to claim 4 or 5, wherein the specimen is blood, plasma, serum, cerebrospinal fluid, or urine.

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