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WO2000000590A1 - Compositions a base de recepteur de la thyrotropine humaine et leur utilisation - Google Patents

Compositions a base de recepteur de la thyrotropine humaine et leur utilisation Download PDF

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WO2000000590A1
WO2000000590A1 PCT/US1999/014640 US9914640W WO0000590A1 WO 2000000590 A1 WO2000000590 A1 WO 2000000590A1 US 9914640 W US9914640 W US 9914640W WO 0000590 A1 WO0000590 A1 WO 0000590A1
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tshr
cells
tsh
endocrinol
autoantibodies
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PCT/US1999/014640
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English (en)
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Basil Rapoport
Sandra M. Mclachlan
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Basil Rapoport
Mclachlan Sandra M
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Priority to AU47255/99A priority Critical patent/AU4725599A/en
Publication of WO2000000590A1 publication Critical patent/WO2000000590A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

  • the present invention relates generally to the fields of cell physiology, endocrinology and immunology. More particularly, the present invention relates to human thyrotropin receptor compositions and to diagnostic and therapeutic methods of using them.
  • TSHR thyrotropin receptor
  • TSHR expressed in mammalian cells are well-recognized by autoantibodies in TBI assays involving intact cells (Harfst, et al, Molec. Cell Endocrinol, 83:117-123 (1992); Filetti, et al, /. Clin. Endocrinol Metab., 72:1096-1101 (1991)), cell particulate fractions (Matsuba, et al, /. Biochem., 118:265-270 (1995); Libert, et al,
  • TSHR is of pivotal importance in the regulation of thyroid gland physiological function and is also the direct cause of hyperthyroidism when targeted by autoantibodies in Graves' disease (Rees Smith, et al, Endocr. Rev.,
  • Graves' disease is a very common ( ⁇ 1% prevalence)(Vanderpump, et al, Clin. Endocrinol, 43:55-68 (1995)), organ-specific autoimmune disease, affecting only humans.
  • type I a less common organ-specific disease, there is no spontaneous animal model for Graves' disease.
  • one specific antigen is unequivocally and directly involved in the pathogenesis of Graves' disease, namely the thyrotropin receptor (TSHR).
  • TSHR thyrotropin receptor
  • autoantibodies to the TSHR activate the receptor, leading to thyroid overactivity and thyrotoxicosis (Rees Smith, et al, Endocr. Rev., 5 9:106-121 (1988)).
  • the interaction between autoantibodies and the TSHR is, therefore, of interest from the theoretical, diagnostic and therapeutic points of view.
  • TSHR autoantibodies like TSH, predominantly recognize discontinuous, highly conformational epitopes (Nagayama, et al, Proc. Natl. Acad. Sci. USA, 88:902-905 (1991); Nagayama, et al, /. Clin. Invest, 88:336-340 (1990); Tahara, et al, Biochem. Biophys. Res. Comm., 179:70-77 (1991)).
  • TSHR expressed on the surface of mammalian cells are conformationally intact and are unquestionably recognized by autoantibodies in patients' sera (Wadsworth, et al, Science, 249:1423-1425 (1990); Ludgate, et al, Molec. Cell Endocrinol, 73:R13-R18 (1990); Filetti, et al, /. Clin. Endocrinol Metab., 72:1096-1101 (1991)).
  • large numbers of TSHR- expressing mammalian cells can be produced in fermentors (Matsuba, et al, /.
  • a number of factors may contribute to the difficulty in detecting TSHR autoantibody binding by direct means, including: i) low TSHR concentration, ii) requirement for conformational integrity of the antigen, iii) the high background observed with polyclonal antibodies in human sera and, iv) low autoantibody titer.
  • Autoantibodies to thyroid peroxidase may be present at very high concentrations (Beever, et al, Clin. Chem., 35:1949-1954 (1989)).
  • An early study suggested that TSHR autoantibodies are present at very high concentrations in serum (2-3% of total IgG).
  • TSHR A two subunit form of TSHR has been recognized for many years.
  • covalent crosslinking of radiolabeled TSH to thyrocyte membranes revealed a ligand-binding glycoprotein A subunit linked by disulfide bonds to a membrane- spanning B subunit (Buckland, et al, FEBS Letters, 145:245-249 (1982)).
  • the TSHR is encoded by a single mRNA species (Parmentier, et al, Science, 246:1620-1622 (1989); Nagayama, et al, Biochem. Biophys. Res. Comm., 165:1184-1190
  • the A and B subunits must be formed by intramolecular cleavage.
  • an uncleaved single chain TSHR is also present on the surface of cultured thyroid cells (Furmaniak, et al, FEBS Letters, 215:316-322 (1987)) and transfected mammalian cells (Russo, et al, Mol. Endocrinol, 5:1607-1612 (1991)).
  • the functional importance of TSHR cleavage is presently an enigma.
  • TSH can activate chimeric TSH-LH/CG receptors that do not cleave into two subunits (Chazenbalk, et al, Biochem. Biophys. Res. Comm., 225:479-484 (1996)). Identification of the cleavage site in the TSHR ectodomain would be important for elucidating the structure-function relationship of the TSHR subunits.
  • TSHR and lutropin/chorionic gonadotropin receptor are closely related members of the G protein-coupled receptor family with glycoprotein hormone ligands. Both the TSHR and LH/CGR have large, heavily glycosylated ectodomains with leucine-rich repeats and are encoded by numerous exons (Gross, et al, Biochem. Biophys. Res. Comm., 177:679 (1991); Tsai-Morris, et al, /. Biol Chem., 266:11355 (1991); Koo, et al, Endocrinology, 128:2297 (1991)).
  • the two subunit TSHR involves a ligand binding, glycosylated A subunit and a membrane-associated B subunit linked by disulfide bonds (Buckland, et al, FEBS Letters, 145:245 (1982)). Because the TSHR is encoded by a single mRNA species (Parmentier, et al, Science, 246:1620 (1989);
  • the A and B subunits must be formed by intramolecular cleavage, a process believed to involve a matrix mellatoprotease (Couet, et al, /.
  • Panel A Specific 125 I-TSH binding to CHO cell monolayers expressing the human TSHR on their surface.
  • 4 kb TSHR cells and TSHR-0 cells were cultured in 24 well and
  • Panel B TBI assay using 4 kb TSHR and TSHR-0 cells cultured in 24 well and 96 well plates, respectively. The assay was performed with the same Graves' patient serum at a saturating concentration of 125 I-bTSH (10 6 cpm/ml), as described in Example 1. Data shown in both panels are the mean of closely agreeing values from duplicate dishes of cells.
  • FIG. 2 Comparison of radiolabeled human and bovine TSH in a TBI assay. Data are shown for 12 sera assayed using TSHR-0 cells (-150,000 TSHR per cell) cultured in a 96 well microtiter plate. TBI assay was as described in Example 1. Fifty ml of either 125 I-bTSH or 125 I-hTSH (5 x 10 4 cpm; 10 6 cpm/ml) was added to each well. Values shown are the mean of closely agreeing triplicate wells for each tracer.
  • Tracer TSH binding in the presence of normal serum was: 125 I-bTSH, 12,396 cpm (mean of 12,044, 12,323, 12821 cpm); hTSH, 2442 cpm (mean of 2509, 2362, 2454 cpm).
  • FIG. 3 Scraping and homogenizing TSHR-10,000 cells reduces the yield of TSHR extracted with detergent.
  • Cells were scraped from 50 confluent 10 cm dishes (5 x 10 s cells) and pelleted. After extraction with buffer containing 1% Triton X-100 (Example 1), aliquots (50 ml) derived from the indicated number of cells were substituted for the same volume of porcine TSHR normally used in the kit.
  • cells were "directly" extracted with buffer containing 1% Triton X-100 without removing the cells from the culture dishes (Example 1). Three ml of detergent-containing buffer were added to a 10 cm diameter culture dish (10 7 cells).
  • FIG. 4 Comparison of solubilized porcine and human TSHR in a TSH binding inhibition assay.
  • TBI activity was determined in 30 sera sent to a clinical laboratory for known or suspected Graves' disease. Sera were tested with a commercial kit using porcine TSHR. In addition, the same sera were assayed with the same reagents with the exception that solubilized human TSHR was substituted for porcine TSHR (Example 1). The cut-off point for positivity, as defined in the kit (TBI >15%), is indicated. The arrow indicated two sera that have detectable TBI activity with the human TSHR, but not with the porcine TSHR.
  • FIG. 5 Correlation between thyroid stimulating immunoglobulin (TSI) activity and TSH binding inhibitory (TBI) activity determined with solubilized human (panel A) or porcine (panel B) TSHR.
  • Figure 6 Panel A: Immunoprecipitation of precursor-labeled TSH receptors in TSHR-10,000 cells.
  • Panel B Specificity of mAb for the TSHR. Immunoprecipitations were performed after an overnight chase using both TSHR-10,000 cells and untransfected CHO cells. In addition, precursor-labeled TSHR-10,000 cells were incubated with a mAb to TPO. Immunoprecipitated proteins (not enzymatically deglycosylated) were applied to a 7.5% polyacrylamide gel. Note the non-specificity of the 48 kDa band.
  • Figure 7 Immunoblots of the TSHR overexpressed in TSHR-10,000 cells.
  • Panel B Specificity of the T3-495 and T3-365 mAb, as determined by their lack of interaction with untransfected cells.
  • Panel C Confirmation of the size of the deglycosylated A subunit on immunoblotting with mAb AlO (epitope including TSHR amino acid residues 22-35). Similar data were obtained with another mAb to the TSHR A subunit, mAb All (data not shown).
  • Panel D Specificity of mAb AlO for the TSHR as determined by its lack of recognition of untransfected CHO cells, as well as by the lack of detection of the TSHR by a mAb to a non-relevant antigen (TPO).
  • TPO non-relevant antigen
  • FIG. 8 Schematic representation of the TSH receptor subunits. The diagram is not drawn to scale and is a modification of our previous version (Chazenbalk et al, /. Biol. Chem., 269:32209-32213 (1994)) based on the new information in the present study.
  • the amino terminal two-thirds of the TSHR ectodomain contains 9 leucine rich repeats, each with an ⁇ -helix and ⁇ -sheet, and is based on the three-dimensional structure of ribonuclease A inhibitor (Kobe et al, Nature, 366:751-756 (1993)). A more detailed model of this region has been performed by Kajava et al, Structure, 3:867-877 (1995).
  • the mass of the A subunit polypeptide backbone is 35 kDa, hence the estimated cleavage site #1 at approximately amino acid residue 330 (Nagayama et al, Biochem. Biophys. Res. Comm., 165:1184-1190 (1989)).
  • the primary non-glycosylated B subunit is -42 kDa, thereby placing the second cleavage site at about residue 380. Cleavage at these two sites would release a putative C peptide of -50 amino acid residues.
  • the thick line between residues 317-366 represents a 50 amino acid "insertion" that is unique to the TSHR relative to the other glycoprotein hormone receptors 5 (Nagayama et al, Biochem. Biophys. Res.
  • TSHRmyc contains a c-myc epitope substituted for residues 338-
  • Panel A Cell proteins were labeled with 35 S- methionine and 35 S-cysteine (one hr pulse; overnight chase) followed by immunoprecipitation with either mAb AlO (A subunit) or mAb 9E10 (c-myc epitope). Precipitated samples were left untreated (Con) or were treated with endoglycosidase H (Endo H) or N-glycosidase F (Endo F)(Example 2). The
  • glycosylated A subunit 20 products were subjected to polyacrylamide gel electrophoresis (10%) under reducing conditions.
  • immunoprecipitation by mAb AlO of TSHR-10,000 cells (approximately 1/20 the amount of the TSHRmyc material) is shown in the extreme right hand lane.
  • the glycosylated A subunit is a diffuse band overlapping with a sharper, non-glycosylated band at - 62 kDa.
  • Panel C Specificity of the deglycosylated, 35 kDa A subunit band recognized by mAb AlO, as determined by immunoprecipitation of untransfected HEK cells, as well as of TSHRmyc cells with a non-relevant mAb
  • Figure 10 Comparison of t25 I-TSH cross-linking to TSHR on the surface of intact TSHRmyc and TSHR-0 cells. Both cell types express similar number of
  • TSHR ( - 10 5 and - 1.5 x 10 5 per cell, respectively).
  • Radiolabeled TSH crosslinking, PAGE (10%) under reducing conditions and autoradiography (18 hr) were as described in Example 2.
  • the ligand, 125 I-TSH itself contains two subunits linked by disulfide bonds. Under reducing conditions, only one of these subunits ( ⁇ 14 kDa) remains covalently linked to the TSHR. Therefore, the size of the TSHR can be deduced by subtracting this mass from the complex.
  • Figure 11 Additional evidence for the existence of two cleavage sites in the TSHR.
  • the ectodomain of the TSHR is shown divided into 5 arbitrary domains (A through E) that were used in creating chimeric TSH-LH/CG receptor molecules (Nagayama et al, Proc. Natl. Acad. Sci. USA, 88:902-905 (1991)). Three chimeric receptors that are relevant to the present study are indicated, as well as a TSHR mutant in which amino acid residues 317-366 are deleted (Wadsworth et al, Science, 249:1423-1425 (1990)). These 50 amino acids are not present in the LH/CG receptor. Therefore, when domain D of the TSHR is replaced with the corresponding segment of the LH/CG receptor, residues 317-360 are missing. The site of the c-myc epitope in TSHRmyc is shown relative to the other segments.
  • FIG. 12 Immunoprecipitation of TSHR protein in whole CHO cells.
  • Cell proteins were precursor-labeled with 35 S-methionine and 35 S-cysteine (one hour pulse and 8 hour chase in the experiment shown) followed by immunoprecipitation under native conditions using mouse monoclonal antibodies to the TSHR (A9 + AlO; Dr. Paul Banga, London, U.K.).
  • the following clonal CHO cell lines were used:- CHO - untransfected cells; TSHR-WT - cell transfected with the 4 kb TSHR cDNA containing both 5'- and 3'-untranslated regions
  • the arrow indicates the 100 kDa TSHR holoreceptor form previously observed by Misrahi et al, Eur. J. Biochem., 222:711- 719 (1994)) that was quantitated by densitometry.
  • FIG. 13 Crosslinking of 125 I-TSH to TSHR on the surface of intact cells.
  • the cells used are described in the legend to Fig. 12.
  • Radiolabeled TSH crosslinking, PAGE (7.5%) under reducing conditions and autoradiography (20 hr) were as described in Example 3.
  • Equal amounts of cell membrane protein were applied to each lane.
  • Much longer periods of autoradiography at least 10 days) were required for visualization of the TSHR in the TSHR-WT cells stably transfected with the 4 kb TSHR cDNA (Nagayama et al, Biochem. Biophys. Res. Comm., 165:1184-1190 (1989)). Similar results were obtained in a second experiment.
  • FIG. 14 Cyclic cAMP levels in stably-transfected CHO cell lines expressing different numbers of TSHR.
  • Cells were incubated in hypotonic medium (Fig. 14A) or in isotonic medium (Fig. 14B), as described in Example 3. These media were either deficient in, or were supplemented with, 1 mU/ml TSH (10 9 M). This concentration is supramaximal in cells exposed to hypotonic medium (Kasagi et al, /. Clin. Endocrinol.
  • Figure 15 Binding of 125 I-TSH to CHO cells expressing the TSHR in the presence of increasing concentrations of unlabeled TSH.
  • the stably-transfected cells lines are described in the legend to Fig. 12. Intact cells in monolayer were incubated in medium containing TSH, as described in Example 3. The brackets indicate the mean + S.E. of values obtained in triplicate dishes of cells.
  • the TSHR-800 and TSHR-10,000 cells were diluted 1:50 with untransfected CHO cells. Because the maximum binding differed for each cell line, the values are expressed as a % of maximum TSH binding in the abence of unlabeled TSH.
  • FIG. 16 TSH binding to cells expressing the TSHR by 125 I-TSH in the absence of unlabeled TSH. Cells in 96-well plates were incubated in 125 I-TSH concentrations up to 1.25 x 10 6 cpm/well (2.5 x 10 7 cpm/well), as described in Example 3. The data shown are the means + S.E. of values obtained in triplicate dishes of cells. The data are representative of three separate experiments.
  • Figure 17 Schematic representation of three TSHR ectodomain variants truncated at their carboxyl-termini.
  • the serpentine transmembrane and cytoplasmic portions of the holoreceptor (764 amino acid residues including signal peptide) are not shown.
  • Six histidine residues (6H) followed by a stop codon are inserted after the indicated TSHR residues. Insertion of a stop codon at residue 418 has previously been shown to generate an ectodomain containing predominantly high mannose carbohydrate that is largely retained within the cell and is not recognized by TSHR autoantibodies in Graves' patients sera (Rapoport et al, /. Clin. Endocrinol Metab., 81:2525-2533 (1996)). As shown in the present study, progressive carboxyl-terminal truncations of the TSHR ectodomain lead to a high level of secretion of material with mature, complex carbohydrate that can completely neutralize TSHR autoantibody activity.
  • Figure 18 Relative secretion into the culture medium of TSHR ectodomain variants.
  • CHO cells stably expressing TSHR ectodomain variants truncated at amino acid residues 261, 289 and 309 were precursor labeled for 1 hr followed by a chase of 16 hr (Example 4).
  • TSHR in medium (M) and cells (C) was then immunoprecipitated with a murine mAb (AlO) to amino acid residues 22-35 (Nicholson et al, /. Mol Endocrinol, 16:159-170 (1996)).
  • TSHR in medium was also recovered using Ni-NTA resin that binds to the 6 histidine residues inserted at the C-termini of the ectodomain variants.
  • Ni-NTA is not an effective method for identifying precursor-labeled TSHR in cells because of its interaction with a large number of CHO cell proteins. Autoradiography in the experiment shown was for 12 hr.
  • Figure 19 Recognition of TSHR ectodomain variants by TSHR autoantibodies in Graves' disease serum.
  • the assay involves the ability of autoantibodies to compete for 125 I-TSH binding to TSHR in solubilized porcine thyroid membranes (Shewring et al, Clin. Endocrinol, 17:409-417 (1982))(Example 4).
  • Left panel In the absence of conditioned medium from CHO cells, serum from a Graves' patient, unlike serum from a normal individual, reduces 12a I-TSH binding by - 60%.
  • Right panel Conditioned medium from a non-relevant cell culture secreting thyroid peroxidase (TPO) has no effect on TSH binding inhibitory (TBI) activity. In contrast, conditioned medium from TSHR-261 and
  • TSHR-289 cell cultures (unlike TSHR-309) nearly completely reverses the TBI activity. Bars indicate the mean + range of duplicate determinations. See Fig. 25 for data on 18 Graves' sera using TSHR-261 after partial purification from conditioned medium.
  • Figure 20 Lectin specificity for TSHR-261 ectodomain variant.
  • Conditioned medium from CHO cell cultures expressing TSHR-261 was adsorbed with Sepharose linked to the indicated lectins (Example 4). Both unadsorbed (“Flow-through”) and material recovered from the beads (“Eluate”) was spotted (neat or after dilution) on nitrocellulose filters and probed with mAb AlO to the amino terminus of the TSHR.
  • Figure 21 Immunoblots of TSHR-261 enriched from conditioned medium using lectins. Material obtained from equivalent volumes of the same medium using Bandeiraea simplificifolia, Concanavalin A or Wheat germ agglutinin was either left untreated (-) or was subjected to endoglycosidase H (Endo H) or endoglycosidase F (Endo F) digestion (Example 4). The samples were electrophoresed on a 10% polyacrylamide gel. Proteins were transferred to PVDF membrane and probed with murine mAb AlO (Example 4).
  • FIG 22 Immunoblot of TSHR ectodomain variants.
  • TSHR-261, TSHR- 289 and TSHR-309 were affinity-enriched from conditioned medium using Concanavalin A (Example 4). Material was either left untreated (-) or was subjected to endoglycosidase H (Endo H) or endoglycosidase F (Endo F) digestion (Example 4). The samples were electrophoresed on a 10% polyacrylamide gel. Proteins were transferred to PVDF membrane and probed with murine mAb AlO to amino acid residues 22-35 (Nicholson, et al, /. Mol. Endocrinol, 16:159-170 (1996)) using the ECL system (Example 4).
  • Figure 23 Direct visualization and quantitation of TSHR-261. Left panel:
  • Serum from a Graves' patient with moderate TSH binding inhibitory (TBI) activity was assayed using the commercial autoantibody kit (Example 4) in the presence of increasing concentrations of partially purified TSHR-261.
  • Serum from a normal individual does not inhibit 125 I-TSH binding to solubilized porcine thyroid TSHR (hatched bar).
  • TSH binding is reduced to - 40% of maximum.
  • Incubation volume in the assay is 0.2 ml. Bars indicate the mean + range of duplicate determinations.
  • FIG. 25 Neutralization of TSHR autoantibodies by TSHR-261 partially purified from conditioned medium.
  • FIG. 26 Flow cytometric analysis of IgG-class TSHR autoantibody binding to CHO cells expressing different numbers of TSHR on their surface.
  • TSHR-WT cells are stably transfected with the 4 kb TSH cDNA (Nagayama et al, Biochem. Biophys. Res. Comm., 165:1184-1190 (1989)).
  • TSHR-0 cells contain the 2.3 kb translated region of the TSHR cDNA (Kakinuma, et al, Endocrinology, 137:2664- 2669 (1996)).
  • TSHR-800 and TSHR-10,000 cells the transgenome has been amplified and TSHR expression has been increased to - 10 6 and - 1.9 x 10 6 per cell, respectively, as shown in Example 3.
  • Cells were incubated with serum (1:10) from a normal individual (open histogram) and from a patient with Graves' disease (BB1) containing high levels of TSHR autoantibodies as measured by the TSH binding inhibition assay (shaded histogram). Fluorescence was developed as described in Example 5.
  • FIG. 27 How cytometric analysis of TSHR-10,000 cells using mouse monoclonal and rabbit polyclonal antisera to the TSHR.
  • TSHR-10,000 cells were incubated with mouse monoclonal antibodies A9 (l:100)(panel A) and AlO (l:100)(panel B), as well as with rabbit serum R8 (l:60)(panel C)(shaded histograms).
  • Controls sera open histograms
  • Figure 28 Effect of adsorption of sera with untransfected CHO cells on the specificity of the autoantibody fluorescence signal on flow cytometry with TSHR-expressing cells. Representative examples are shown of sera from two individuals, 7H and BB1 (shaded histograms), each with very high TBI values (81.7% and 100% inhibition, respectively). Sera (1:10 dilution) were either not preadsorbed (upper panels) or preadsorbed on untransfected CHO cells (lower panels) prior to incubation with TSHR-10,000 cells (Example 5). Included as a negative control is serum from a normal individual without TSHR autoantibodies detectable by the TBI assay (open histogram).
  • Figure 29 Adsorption of TSHR autoantibodies using cells expressing the recombinant TSHR on their surface.
  • Sera BB1, 10H, 3H and 10M that generated a fluorescent signal on FACS analysis with TSHR-10,000 cells (Table 1) were preadsorbed (0.5 hours at room temperature, three times) on TSHR-10,000 cells prior to analysis using the same cells (open histograms). Huorescence generated by the same sera after preadsorption on untransfected CHO cells is shown by the shaded histograms.
  • TAI TSH binding inhibition
  • Figure 30 Relative titers of TPO and TSHR autoantibodies in the BB1 serum, as determined by flow cytometry on CHO cells expressing either the TSHR or TPO on their surface.
  • TSHR-10,000 cells as described in Example 3
  • CHO-TPO cells previously described as C4C cells (Kaufman et al, Molec. Cell EndorcinoL, 78:107-114 (1991))
  • C4C cells previously described as C4C cells
  • Serum BB1 serum from a normal individual without TSHR or TPO autoantibodies detectable by clinical assay (open histograms) were tested at dilutions between 1:10 and 1:1000.
  • FIG. 31 The ectodomain of the TSHR is shown divided into 5 arbitrary domains (A through E) that were used in creating chimeric TSH-LH/CG receptor molecules (Nagayama et al, Proc. Natl. Acad. Sci. USA, 88:902 (1991)). Three chimeric receptors that are relevant to the present study are indicated. Note that amino acid residues 317-366 are not present in the LH/CG receptor. Therefore, when domain D of the TSHR is replaced with the corresponding segment of the LH/CG receptor, residues 317-360 are missing.
  • Figure 32 A. Amino acid substitutions introduced in the region of putative cleavage site 1 in the TSHR. Mutations were made in the D domain of chimeric receptor TSH-LHR-5 (Fig. 31). The dashed line for the LH/CG receptor indicates that this region is unique to the TSHR and is absent in the LH/CG receptor.
  • the ligand 125 I-TSH, binds to the uncleaved TSH holoreceptor or to the ligand-binding A subunit in the cleaved TSHR, indicating the presence of both cleaved and uncleaved TSHR on the cell surface.
  • the mass of the hormone ligand complex includes one subunit of the ligand, which itself contains two subunits linked by disulfide bonds. Under reducing conditions, only one ligand subunit ( ⁇ 14 kDa) remains covalently linked to the TSHR.
  • Figure 33 A. Amino acid substitutions introduced in the region of putative cleavage site 2 in the TSHR. Mutations were made in the E domain of chimeric receptor TSH-LHR-4 (Fig. 31). The mutations shown in bold prevent cleavage, as determined by TSH cross-linking. In the El, E2 and E3 mutations,
  • Figure 34 The presence of the GQE 2 7 . 269 NET mutation does not prevent cleavage of the wild-type TSHR.
  • Cross-linked 125 I-TSH-TSHR products were reduced and subjected to PAGE (10%) and autoradiography.
  • the present invention is directed to novel human thyrotropin compositions which are useful in diagnostic and therapeutic methods for the diagnosis and treatment of autoimmune diseases, particularly Graves'
  • the present invention is directed broadly to methods for the therapeutic treatment of autoimmune disease, and particularly, Graves' disease, involving administration of the therapeutic compositions described herein and such variants thereof as will be appreciated by those of skill. Accordingly, in one aspect, the invention is directed to a method for the treatment of Graves' disease comprising administering to a patient suffering from Graves' disease an effective amount of a composition comprising one or more of the substantially isolated human thyrotropin compositions of the invention, or a variant thereof, alone or in conjunction with one or more other active ingredients.
  • Such compositions useful according to the methods of the invention are preferably selected for use according to the invention by means of one or more assay methods disclosed herein or known to those of skill.
  • compositions useful in the methods of the invention are preferably substantially isolated human thyrotropin receptor, which may be recombinantly produced according to the invention, or variants or mutants thereof, capable of modulating Graves' disease in a patient, and may be selected, as described herein, by means of appropriate assays. TSH compositions so selected may be administered by methods known to those of skill in order to achieve the desired therapeutic result.
  • compositions of the invention may be desirable, for example, to increase or decrease the half life of the resulting peptide in the bloodstream or tissue.
  • compositions useful according to the methods of the invention by introducing therein alterations, such as are known in the art, which may include the use of synthetic or non-traditional amino acid residues, side chains, non-amide bonds as in peptides, and the like, which may act as blocking groups to protect the peptide against degradation.
  • alterations such as are known in the art, which may include the use of synthetic or non-traditional amino acid residues, side chains, non-amide bonds as in peptides, and the like, which may act as blocking groups to protect the peptide against degradation.
  • compositions useful according to the invention may be isolated from natural sources and purified. However, it is preferred to synthesize the compositions by means well known in the art. Preferred is solid phase synthesis, although any suitable method of synthesis may be employed.
  • LH/CG receptor ectodomain at a position (amino acid residue 294) similar to the TSHR variants generates a non-secreted protein (Koo, et al, Endocrinology, 134:126 (1994)).
  • the LH/CG receptor truncated at residue 329 or further downstream is secreted to a limited extent (VuHai-LuuThi, et al, Biochem., 31:8377-8383 (1992); Tsai-Morris, et al, /. Biol. Chem., 265:19385-19388 (1990)).
  • TSHR mRNA Alternately-spliced truncated forms of TSHR mRNA have been detected in thyroid tissue (Graves, et al, Biochem. Biop hys. Res. Comm., 187:1135-1143 (1992); Takeshita, et al, Biochem. Biophys. Res. Comm., 188:1214-1219 (1992)), however whether these transcripts are actually expressed and, if so, secreted by thyrocytes is unknown.
  • TSHR-261 The lectin specificity of TSHR-261 is consistent, in part, with previous data on the extraction of TSH holoreceptor activity from detergent-solubilized thyroid membranes.
  • B. simplificifolia was effective for bovine, but not for human, TSHR (Kress, et al, Endocrinology, 118:974-979 (1986)).
  • TSHR TSHR
  • the bovine TSH holoreceptor was also bound well by Wheat germ agglutinin and was irreversibly bound by Concanavalin A (Kress, et al, Endocrinology, 118:974-979 (1986)).
  • TSHR-289 shares many of the advantages of TSHR-261, and may be more stable than TSHR-261, which loses activity over several hours at room temperature. Thus, TSHR-289 may be preferred according to the present invention although TSHR-261 is somewhat more effectively secreted. Also within the scope of the present invention are other truncations between 261 and 309, which may be utilized by those of skill without undue experimentation who have read and understood the teachings of the present invention.
  • TSHR-261 Autoantibody-reactivity of TSHR-261 is reminiscent of the observation nearly three decades ago that freezing and thawing thyroid tissue releases a water soluble factor (LATS absorbing activity; LAA) that neutralizes TSHR autoantibodies (Dirmikis, et al, /. Endocrinology, 58:577-590 (1973); Smith, /. Endocrinology, 46:45-54 (1970)).
  • LAA (like TSHR-261) was estimated to be - 50 kDa (Dirmikis, et al, /. Endocrinology, 58:577-590 (1973)).
  • the TSHR ectodomain variant TSHR-261 despite its potent autoantibody neutralizing activity, does not bind TSH.
  • the TSH binding site on the TSHR is discontinuous and involves multiple segments throughout the entire ectodomain, including segments downstream of residue 261 (Nagayama, et al, Biochem. Biophys. Res. Comm., 173:1150-1156 (1990); Nagayama, et al, Proc. Natl. Acad. Sci. USA, 88:902-905 (1991)).
  • the TSHR autoantibody epitope(s) may, therefore, be more limited than the TSH binding site. Support for this notion is provided by data from most (Rapoport, et al, /. Clin.
  • TSHR-261 contains the dominant portion of a discontinuous epitope, sufficient to neutralize most TSHR reactivity in Graves' patients sera.
  • TSHR-261 is highly potent in interacting with TSHR autoantibodies.
  • Antigenically active TSHR will provide a major impetus for future studies on the diagnosis, pathogenesis and immunotherapy of Graves' disease. Those of skill will appreciate that when treating populations of cells, such as thyroidal tissue sells, a therapeutic effect may be observed by any number of known clinical endpoints.
  • the compounds of the present invention also have
  • the compounds of the invention may be used in conjunction with other agents.
  • Mechanisms of drug resistance are described, for example, in Remington's Pharmaceutical Sciences, 18th Edition, supra.
  • the quantities of active ingredient necessary for effective therapy will depend on many different factors, including means of administration, target site, physiological state of the patient, and other medicaments administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the active ingredients. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, for example, Goodman and Gilman's The Pharmacological Basis of Therapeutics. 7th Ed., MacMillan Publishing Co,, New York (1985), and Remington's Pharmaceutical Sciences 18th Ed., Mack Publishing Co., Easton, Perm (1990). Methods for administration are discussed therein, including oral, intravenous, intraperitoneal, intramuscular, transdermal, nasal, iontophoretic administration, and the like.
  • a treatment regimen will be selected which will achieve a sufficient concentration of the composition(s) to achieve the desired therapeutic effect in the target cells.
  • Those of skill will be able to determine an effective dose, which will vary depending upon the manner and mode of administration, in order to achieve effective concentrations.
  • compositions of the present invention are inhalation of dry-powder formulations comprising the composition(s).
  • the powder In order to allow for absorption of the active components through the alveoli into the bloodstream, the powder must be very fine; on the order of 1-5 micron particles.
  • the highly disbursable powder is delivered via an inhaler which generates an aerosol cloud containing the bolus of drug at the top of the inhalation chamber.
  • compositions of the present invention will lend themselves to injection into the bloodstream of a patient.
  • the half life of the active compositions so administered may be manipulated for best therapeutic effect by employing known drug technologies.
  • One example of such technologies is known as DEPOFOAM phospholipid spheres (Depo Tech Corp., San Diego, CA), which gradually release the active component(s) over a period of days to weeks. This allows for a constant level of systemic concentration with lower initial drug levels and injection frequency.
  • compositions of the present invention may be employed to accommodate several different routes of drug delivery.
  • One example of such technologies is the TECHNOSPHERE powder (Pharmaceutical Discovery Corp., Elmsford, NY), which reliably forms two micron diameter spheres under conditions preserving the structural and functional integrity of the active peptide component.
  • the pH-sensitive spheres when injected into the blood, dissolve and release the active component, which is rapidly absorbed. Fine powders such as these are suitable for pulmonary, oral, intravenous and intraperitoneal administration.
  • the site of administration and cells will be selected by one of ordinary skill in the art based upon an understanding of the particular disorder being treated.
  • the dosage, dosage frequency, and length of course of treatment can be determined and optimized by one of ordinary skill in the art depending upon the particular disorder being treated.
  • the particular mode of administration can also be readily selected by one of ordinary skill in the art and can include, for example, oral, intravenous, subcutaneous, intramuscular, etc. Principles of pharmaceutical dosage and drug delivery are known and are described, for example, in Ansel, H. C. and Popovich, N. G., Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Ed, Lea & Febiger, Pub., Philadelphia, PA (1990).
  • liposomes to specifically deliver the agents of the invention.
  • Such liposomes can be produced so that they contain additional bioactive compounds and the like such as drugs, radioisotopes, lectins and toxins, which would act at the target site.
  • Nucleic acid compositions encoding the compositions and variants thereof useful according to the invention will generally be in RNA or DNA forms, mixed polymeric forms, or any synthetic nucleotide structure capable of binding in a base-specific manner to a complementary strand of nucleic acid.
  • Such a nucleic acid embodiment is typically derived from genomic DNA or cDNA, prepared by synthesis, or derived from combinations thereof.
  • the DNA compositions generally include the complete coding region encoding the compositions, or fragments thereof.
  • composition of the invention and is intended to include “fragments,” “variants,” “analogs,” “homologs,” or “chemical derivatives” possessing such activity or characteristic.
  • Functional equivalents of a peptide according to the invention may not share an identical amino acid sequence, and conservative or non- conservative amino acid substitutions of conventional or unconventional amino acids are possible.
  • references herein to "conservative" amino acid substitution is intended to mean the interchangeability of amino acid residues having similar side chains.
  • glycine, alanine, valine, leucine and isoleucine make up a group of amino acids having aliphatic side chains; serine and threonine are amino acids having aliphatic-hydroxyl side chains; asparagine and glutamine are amino acids having amide-containing side chains; phenylalanine, tyrosine and tryptophan are amino acids having aromatic side chains; lysine, arginine and histidine are amino acids having basic side chains; and cysteine and methionine are amino acids having sulfur-containing side chains.
  • Preferred conservative substitution groups include asparagine-glutamine, alanine-valine, lysine-arginine, phenylalanine-tyrosine and valine-leucine-isoleucine.
  • mutant refers to a peptide having an amino acid sequence which differs from that of a known peptide or protein by at least one amino acid. Mutants may have the same biological and immunological activity as the known protein. However, the biological or immunological activity of mutants may differ or be lacking. For example, a mutant may lack the biological activity which characterizes a TSHR of the invention, but may be useful as an eptitopic determinant for raising antibodies or for the detection or purification of antibodies thereagainst, or as an agonist (competitive or non-competitive),
  • Suitable labels for use in assays according to the invention include a detectable label such as an enzyme, radioactive isotope, fluorescent compound, chemiluminescent compound, or bioluminescent compound. Those of ordinary skill in the art will know of other suitable labels or will be able to ascertain such using routine experimentation. Furthermore, the binding of these labels to the peptides is accomplished using standard techniques known in the art.
  • TSHR further isolation, purification and sequencing of TSHR according to the invention may be accomplished by standard biochemical methods such as, for example, those described in Cantor, C, ed., Protein purification: Principles and Practice, Springer Verlag, Heidelberg, Publisher (1982); Hancock, W., ed., New Methods in Peptide Mapping for the Characterization of Proteins, CRC Press, Boca Raton, Florida, Publisher (1996), following the teachings of the present invention.
  • Peptidomimetic agents are of use in the therapeutic treatment of disease. Such peptidomimetics are also provided by the present invention, and can act as drugs for the modulation of autoimmune disease, such as Graves' disease. Peptidomimetics are commonly understood in the pharmaceutical industry to include non-peptide drugs having properties analogous to those of those of the mimicked peptide. The principles and practices of peptidomimetic design are known in the art and are described, for example, in Fauchere J., Adv. Drug Res. 15: 29 (1986); and Evans, et al, ⁇ . Med. Chem. 30: 1229 (1987). Peptidomimetics which bear structural similarity to
  • therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • such peptidomimetics have one or more peptide linkages optionally replaced by a linkage which may convert desirable properties such as resistance to chemical breakdown in vivo.
  • Peptidomimetics may exhibit enhanced pharmacological properties (biological half life, absorption rates, etc.), different specificity, increased stability, production economies, lessened antigenicity and the like which makes their use as therapeutics particularly desirable.
  • compositions according to the invention may vary depending upon a number of factors.
  • a given protein may be obtained as an acidic or basic salt, or in neutral form, since ionizable carboxyl and amino groups are found in the molecule.
  • any form of peptide comprising human TSHR which retains the therapeutic or diagnostic activity of the compositions of the present invention is intended to be within the scope of the invention.
  • compositions of the present invention may be produced by recombinant DNA techniques known in the art.
  • nucleotide sequences encoding human TSHR of the invention may be inserted into a suitable DNA vector, such as a plasmid, and the vector used to transform a suitable host.
  • the recombinant human TSHR is produced in the host by expression.
  • the transformed host may be a prokaryotic or eukaryotic cell.
  • Preferred nucleotide sequences for this purpose encoding a human TSHR are disclosed herein.
  • Synthetic polynucleotide sequences may be constructed by known chemical synthetic methods for the synthesis of oligonucleotides. Such synthetic methods are described, for example, in Blackburn, G.M. and Gait, M.J., Eds., Nucleic Acids in Chemistry and Biology, IRL Press, Oxford, England (1990), and it will be evident that commercially available oligonucleotide synthesizers also may be used according to the manufacturer's instructions. One such manufacturer is Applied Bio Systems.
  • PCR Polymerase chain reaction
  • primers based on the nucleotide sequence data disclosed herein may be used to amplify DNA fragments from mRNA pools, cDNA clone libraries or genomic DNA.
  • PCR nucleotide amplification methods are known in the art and are described, for example, in Erlich, H.A., Ed., PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, New York (1989); U.S. Patent No. 4,683,202; U.S. Patent No. 4,800,159; and U.S. Patent No. 4,683,195.
  • nucleotide deletions, additions and substitutions may be incorporated into the polynucleotides of the invention as will be recognized by those of skill, who will also recognize that variation in the nucleotide sequence encoding human TSHR may occur as a result of, for example, allelic polymorphisms, minor sequencing errors, and the like.
  • the polynucleotides encoding the peptides of the invention may include short oligonucleotides which are useful, for example, as hybridization probes and PCR primers.
  • the polynucleotide sequences of the invention also may comprise a portion of a larger polynucleotide and, through polynucleotide linkage, they may be fused, in frame, with one or more polynucleotide sequences encoding different proteins.
  • the expressed protein may comprise a fusion protein.
  • the polynucleotide sequences of the invention may be used in the PCR method to detect the presence of mRNA encoding autoantibodies in the diagnosis of disease or in forensic analysis.
  • sequence of amino acid residues in a protein or peptide is designated herein either through the use of their commonly employed three-letter designations or by their single-letter designations. A listing of these three-letter and one-letter designations may be found in textbooks such as Lehninger, A., Biochemistry, 2d Ed, Worth Publishers, New York, New York (1975). When the amino acid sequence is listed horizontally, the amino terminus is intended to be on the left end whereas the carboxy terminus is intended to be at the right end.
  • the residues of amino acids in a peptide may be separated by hyphens. Such hyphens are intended solely to facilitate the presentation of a sequence.
  • Suitable agents for use according to the invention include human TSHR and mimetics, fragments, functional equivalents and /or hybrids or mutants thereof, as well as mutants, and vectors containing cDNA encoding any of the foregoing. Agents can be administered alone or in combination with and/or concurrently with other suitable drugs and /or courses of therapy.
  • the agents of the present invention are suitable for the treatment of autoimmune diseases which are characterized by inappropriate cell death.
  • Autoimmune diseases are disorders caused by an immune response directed against self antigens. Such diseases are characterized by the presence of circulating autoantibodies or cell-mediated immunity against autoantigens in conjunction with inflammatory lesions caused by immunologically competent cells or immune complexes in tissues containing the autoantigens.
  • diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis, and Graves' disease.
  • the human TSHR peptides, mimetics, agents and the like disclosed herein, as well as vectors comprising nucleotide sequences encoding them or their corresponding antisense sequences, and hosts comprising such vectors, may be used in the manufacture of medicaments for the treatment of diseases including autoimmune diseases.
  • the present invention describes improved assays for the detection of TSHR autoantibodies. Surprisingly and unexpectedly, it has been found that mammalian cells expressing more TSHR on their surface provide a less sensitive TBI assay when this assay is performed with intact cells in monolayer culture.
  • TBI thyroid peroxidase
  • An optimum TBI assay according to the present invention should utilize a small amount of receptor and a very effective ligand, as is the case with the affinity-purified bovine 125 I-TSH in the procedure developed by Rees-Smith (Shewring, et al, Clin. Endocrinol, 17:409-417 (1982); Rees Smith, et al, Methods in Enzymology, 74:405-420 (1981)).
  • a TBI assay using cell monolayers is at a serious practical disadvantage relative to assays utilizing solubilized TSHR.
  • the present invention demonstrates this low recovery of effective TSHR from resuspended cells and indicates that the direct extraction of TSHR from cell monolayers can overcome the evident fragility of this very difficult receptor.
  • a cell line such as TSHR-10,000, that expresses very high levels of TSHR, can provide TSHR suitable for direct use in a TBI assay according to the invention without further purification or concentration, and is preferred on that basis.
  • TSHR species may be important in bioassays for stimulatory autoantibodies (Murakami, et al, Eur. ⁇ . Endocrinol, 133:80-86 (1995); Vitti, et al, /. Clin. Endo. Metab., 76:499-503 (1993); Endo, et al, Biochem. Biophys. Res. Comm., 186:1391-1396 (1992)).
  • human rather than
  • TSHR would be advantageous in a TBI assay has not heretofore been established. This factor was considered during the original development of the
  • porcine TSHR became the standard in TBI assays. It is worth noting that most sera in this study had relatively high TBI values, making discrimination at the very important low end of the assay difficult to discern.
  • TSH ligand
  • the present invention also provides evidence for two cleavage sites in the TSHR ectodomain. This evidence must be viewed in the context of previous data that (in retrospect) support this notion.
  • TSH crosslinking studies with certain chimeric TSH-LH/CG receptors.
  • D domain a domain of TSHR residues 261-360
  • E domain a domain of TSHR residues 363-418
  • the smaller than expected sum of the sizes of the deglycosylated TSHR A subunit and the non-glycosylated B subunit support the concept of two cleavage sites in the TSHR, with the loss of an intervening portion of the polypeptide chain. While we recognize that size estimations cannot be absolutely precise, they are sufficiently reproducible among different laboratories, using different methodologies for TSHR detection, to suggest that a piece of the TSHR has been lost during intramolecular cleavage. We observed the deglycosylated A subunit and the B subunit to be -35 kDa and -42 kDa, respectively. Others have reported A and B subunits of similar sizes (Loosfelt, et al, Proc. Natl. Acad.
  • a third line of evidence suggesting two cleavage sites in the TSHR ectodomain is the present observation of the selective loss of a strategically situated c-myc epitope in the two subunit, but not in the single chain, form of the TSHR.
  • This phenomenon There are three possible explanations for this phenomenon: (i) loss of a peptide fragment containing the c-myc epitope during intramolecular cleavage at two different sites, (ii) cleavage at a single cleavage site within the c-myc epitope leading to loss of antibody recognition, (iii) a combination of these two events (two cleavage sites, one of these within the c-myc epitope).
  • Persistent cleavage in such a highly mutated region would, therefore, indicate lack of amino acid sequence specificity for a TSHR cleavage site. For all these reasons, there are likely to be two cleavage sites in the TSHR ectodomain, the more upstream of which may, or may not, be with the region of the c-myc epitope.
  • the c-myc epitope lies within a 50 amino acid segment (residues 317-366) that we observed to be unique to the TSHR when compared to other glycoprotein hormone receptors (Nagayama, et al, Biochem. Biophys. Res. Comm., 165:1184-1190 (1989)). Although the precise boundaries of this 50 amino acid "insertion" are uncertain (because of low homology among the receptors in adjacent regions), this TSHR segment has been the subject of intense study.
  • residues 317-366 led us to speculate that it was a projection on the exterior of the TSHR molecule, perhaps important in ligand specificity (Wadsworth, et al, Science, 249:1423-1425 (1990)). Surprisingly, however, its deletion had no effect on TSH binding or on TSH-mediated signal transduction (Wadsworth, et al, Science, 249:1423-1425 (1990)).
  • the deduced superficial topography of TSHR residues 317-366 was also the reason for selection of this region for c-myc epitope tagging. Other investigators have used synthetic peptides corresponding to portions of this region for generating antisera to the TSHR.
  • the present invention achieves high level of expression of the human TSHR in mammalian cells.
  • This level of expression 10-12-fold higher than previously attained in stably-transfected mammalian cells (Costagliola, et al, /. Clin. Endocrinol. Metab., 75:1540-1544 (1992); Matsuba, et al, /.
  • TSHR- 10,000 cells will facilitate study of subunit structure, carbohydrate composition, interaction with its physiological ligand, TSH, as well as intracellular trafficking.
  • the TSHR-10,000 cells are also of potential value in providing antigen for the study of TSHR autoantibodies.
  • these uses are the development of new assays for TSHR autoantibodies, flow cytometric analysis of TSHR autoantibodies in patients' sera and the isolation of human monoclonal autoantibodies from immunoglobulin gene combinatorial libraries derived from patients' B cells.
  • the high basal cAMP levels in these cells reduces their sensitivity and makes them suboptimal for the bioassay of TSHR functional activity.
  • TSHR-10,000 cells provide insight into the function of the TSHR, at least as expressed in CHO cells.
  • the data on stably-transfected cell lines strongly support transient transfection data (Van Sande, et al, Eur. J. Biochem., 229:338-343 (1995)) that the TSHR maintains a moderate level of activity in the absence of ligand.
  • Such spontaneous activity was first observed with the ⁇ lB- adrenergic receptor (Kjelsberg, et al, /. Biol. Chem., 267:1430-1433 (1992)).
  • thyroid overactivity could result from an increased number of TSHR expressed on the thyrocytes of a particular individual consequent to mutations in the mRNA untranslated region in the same way as mutations in the mRNA coding region can increase the increased constitutive activity of an unchanged number of receptors (Duprez, et al, Nature Genet, 7:396-401 (1994); Parma, et al, Mol. Endocrinol, 9:725-733 (1995)).
  • a second interesting feature of the TSHR overexpressed in the TSHR- 10,000 cells is the ability of TSH to bind to single chain TSHR expressed on the cell surface.
  • the proportion of single chain and two-subunit TSHR on the surface of CHO cells is independent of the number of receptors expressed.
  • the stronger signal with the TSHR-10,000 cells makes the phenomenon much more clear and supports our previous evidence using chimeric TSH-LH/CG receptors
  • TSHR aggregate or "patch" in the fluid plasma membrane, especially at high density. Conformational changes in the TSHR itself or steric hindrance to TSH binding could also lead to an apparent reduction in affinity, as could adducts between holoreceptors and TSHR B subunits. Excess B subunits have been observed previously in cells expressing the TSHR (Couet, et al, /. Biol.
  • TSHR affinity could also, perhaps, occur if a greater proportion of TSHR were coupled to G proteins.
  • TSHR expressed at high density will require caution in extrapolating structural information on these receptors to the TSHR present in thyrocytes. Nevertheless, it is feasible that TSHR negative cooperativity could represent another type of autoregulation, so typical of the thyroid, associated with maintaining stable thyroid function.
  • the present invention facilitated by the availability of CHO cells overexpressing the human TSHR (Example 3), also describes and demonstrates that IgG class autoantibodies to the TSHR can be detected directly by flow cytometry.
  • an unequivocal signal by flow cytometry was obtained with only 4 of 21 TBI positive sera, including some selected on the basis of their high TSHR autoantibody activity. Indeed, 11 of these sera had high TBI levels (>50% inhibition).
  • TSHR autoantibodies all 20 sera with TPO autoantibodies detected by ,2S I-TPO binding gave strongly positive signals on flow cytometry using TPO-expressing CHO cells.
  • TPO autoantibodies are known to be of similar affinity for their antigen (McLachlan, et al, Clin. Exp. Immunol, 101:200-206 (1995)). Finally, a 1:1000 dilution of a potent serum for TSHR autoantibodies nearly eliminated its signal on flow cytometry, whereas at the same dilution, TPO autoantibodies in this serum continued to produce a very strong signal.
  • TSHR autoantibodies by flow cytometry in a serum (10M) with only moderate TBI activity, provides support for the concept that, in addition to autoantibodies to the TSH binding site, some TSHR autoantibodies recognize epitopes outside of this region.
  • the present invention also demonstrates and describes that the evolutionary divergence of the TSHR into a receptor that cleaves into two subunits is unique and enigmatic. Unlike thrombin (Vu, et al, Cell, 64:1057 (1991)), TSH does not cleave its receptor; two subunit TSHR are present in transfected cells cultured in the absence of TSH (Misrahi, et al, Eur. ⁇ . Biochem., 222:711 (1994); Koo, et al, Endocrinology, 128:2297 (1991)). TSH binds to both cleaved and uncleaved forms of the TSHR.
  • TSH action does not require a cleaved receptor (Russo, et al, Endocrinology, 130:2135 (1992); Chazenbalk, et al, Biochem. Biophys. Res. Comm., 225:479 (1996)).
  • TSHR receptor cleavage including the release of a small polypeptide between cleavage sites 1 and 2, may be related to the very common occurrence of disease-causing autoantibodies, a phenomenon rarely, if ever, encountered with other members of the G protein-coupled receptor family.
  • Data obtained from thirty three new TSHR variants stably expressed in
  • CHO cells (i) provide proof for the existence of two cleavage sites in the TSHR, (ii) identify three amino acid residues in the TSHR that, when substituted with the homologous residues of the LH/CG receptor, prevent cleavage at the second, downstream cleavage site (site 2) and, (iii) reveal that cleavage or non-cleavage at this second site is related to N-linked glycosylation.
  • site 2 downstream cleavage site
  • site 2 downstream cleavage site 2
  • site 2 downstream cleavage site 2
  • cleavage or non-cleavage at this second site is related to N-linked glycosylation.
  • the presence or absence of glycosylation represents a novel mechanism by which two closely-related receptors have evolved into having a difference in subunit structure. Greater understanding of its structural features will also contribute to a better understanding of why the TSHR is a pivotal autoantigen in human autoimmune disease.
  • Example 1 describes TSH binding inhibition assays employing recombinant human TSHR compositions.
  • Example 2 presents the surprising and novel finding for G protein- coupled receptors that contrary to the prevailing concept of one cleavage site in the TSHR, there are, in fact, two such sites.
  • the TSHR like insulin, may release a C peptide during intramolecular cleavage into two subunits.
  • Example 3 describes high level expression of TSHR in CHO cells, as well as high constitutive activity of the TSHR in the absence of ligand, and the binding of TSH to the single subunit, uncleaved TSHR. Moreover, high level expression is associated with apparent negative co-operativity among the
  • TSHR in terms of their affinity for ligand.
  • Example 4 demonstrates that carboxyl-terminal truncation of the human TSHR ectodomain generates a secreted protein with complex carbohydrate that neutralizes autoantibodies in Graves' patients' sera.
  • Antigenically active TSHR is useful for the diagnosis, pathogenesis and immunotherapy of Graves' disease.
  • Example 5 presents data providing the strongest support for the notion that TSHR autoantibodies in the sera of patients with autoimmune thryoid disease are present at much lower levels than are TPO autoantibodies. This finding has important implications for the diagnostic detection of TSHR autoantibodies and for understanding the pathogenesis of Graves' disease.
  • Example 6 identifies the amino acid residues related to the putative cleavage sites of TSHR.
  • TBI Thyrotropin Receptor
  • TSHR-0 cells expressing only the coding region of the TSHR cDNA, were renamed from the previously cumbersome pTSHR-5'3'TR-NEO-ECE (Kakinuma, et al, Endocrinology, 137:2664-2669 (1996)).
  • the transgenome in the TSHR-10,000 cells was amplified by adaptation of these cells to 10,000 nM methotrexate (Chazenbalk, et al, Endocrinology, 137:4586-4591 (1996)).
  • all cell lines (cloned by limiting dilution in selection medium) were grown in Ham's F-12 medium supplemented with 10% fetal calf serum and standard antibiotics. Cells were cultured to confluence in either 10 cm diameter, 24 well cluster or 96 well microtiter culture dishes, as described in the text. Table 1: Characteristics of cell lines expressing the human TSH receptor
  • TSH binding inhibition TBD assay using intact cells: Highly purified bovine TSH (N.I.H.) or recombinant human TSH (Sigma, St.
  • Solubilized TSH receptor preparation Receptors were prepared from TSHR-10,000 cells in two procedures: (i) Cells removed from the culture dishes: Fifty confluent 10 cm diameter dishes of cells (10 7 cells per dish) were rinsed once with PBS and cells were resuspended by scraping into buffer A (10 mM Tris, pH 7.5, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin and 2 ⁇ g/ml pepstatin A; Sigma)(3 ml/dish).
  • buffer A (10 mM Tris, pH 7.5, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin and 2 ⁇ g/ml pepstatin A; Sigma)(3 ml/dish).
  • the 500 - 20,000 x g particulate fraction was processed according to the protocol of Rees Smith et al. (Shewring, et al, Clin. Endocrinol, 17:409-417 (1982); Rees Smith, et al, Methods in Enzymology, 74:405-420 (1981)).
  • the final extraction was with 5 ml of 10 mM Tris, pH 7.5, 50 mM NaCl and 1% Triton X-100.
  • TSH binding inhibition TAI1 assay using solubilized TSHR: Sera were assayed using TSHR antibody (TRAb) kits purchased from Kronus, San
  • Thyroid stimulating immunoglobulin CTSI Thyroid stimulating immunoglobulin CTSI assay: TSHR-0 cells, grown to confluence in 96 well culture plates, were assayed as previously described for human thyroid cells (Hinds, et al, /. Clin. Endocrinol. Metab., 52:1204-1210 (1981); Rapoport, et al, /. Clin. Endocrinol. Metab., 58:332-338 (1984)). This procedure employs the modification of using hypotonic medium (Kasagi, et al, /. Clin. Endocrinol Metab., 54:108-114 (1982)).
  • IgG was precipitated with PEG (see above) and resuspended in the hypotonic medium supplemented with 10 mM Hepes, pH 7.4, 1 mM 3-isobutyl 1-methylxanthine and 0.3% BSA. Cells were incubated in this medium (0.1 ml) for 2 hr at 37°C. Cyclic AMP in the medium, diluted in 50 mM Na acetate, pH 6.2 and acetylated (Rapoport, et al, /. Clin. Endocrinol.
  • TSI activity was expressed as a percentage of the cyclic AMP value in the test serum relative to cAMP measured after concurrent incubation with serum from normal individuals.
  • the TBI assays performed were of two main types (Table 2) involving either intact CHO cells or solubilized TSHR.
  • TSH binding inhibition (TBI) assays performed.
  • TSH binding inhibition (TBI) assay using cells in monolayer TSH binding inhibition (TBI) assay using cells in monolayer:
  • TBI values % inhibition of 125 I-TSH binding
  • % inhibition of 125 I-TSH binding were 79%, 91% and 83% with the commercial assay versus 10%, 13% and 57% with the CHO cells, respectively.
  • TSHR-0 cells (150,000 TSHR per cell) (Kakinuma, et al, Endocrinology, 137:2664-2669 (1996)) cultured in microtiter (0.36 cm 2 ) wells provide only - 2-fold more receptors per well than the "4kb" TSHR cell line (16,000 TSHR per cell)(Nagayama, et al, Biochem. Biophys. Res. Comm.,
  • hTSH in a TBI assay is presently limited because this species of TSH is a less effective ligand than bTSH (Pierce, et al, Ann. Rev. Biochem., 50:465 ⁇ 95 (1981)). Indeed, in the experiment shown, maximal 125 I-TSH binding was 5-fold lower with hTSH than with bTSH (4.9% vs. 24.8%).
  • TSH binding inhibition assay using detergent-extracted recombinant human TSHR As mentioned above, intact CHO cells expressing large numbers of receptors on their surface cannot be used for TBI assays. However, we wished to determine whether such cells would be a good source of recombinant TSHR in a soluble receptor assay. For this purpose, we used cells (TSHR-10,000), that express very large numbers ( ⁇ 1.9 x 10 6 ) of TSHR on their surface (Chazenbalk, et al, Endocrinology, 137:4586-4591 (1996)).
  • TBI values obtained with the solubilized porcine TSHR are known to correlate only weakly with thyroid stimulating activity determined in a bioassay (TSI) involving activation of the human TSHR (Filetti, et al, /. Clin. Endocrinol. Metab., 72:1096-1101 (1991); Murakami, et al, Eur. J. Endocrinol, 133:80-86 (1995); Vitti, et al, /. Clin. Endo. Metab., 76:499-503 (1993)).
  • TBI bioassay
  • TSI activity was available from 28 of the 30 samples depicted in Fig. 4 to permit determination of TSI activity using CHO cells stably transfected with the human TSHR.
  • TSHR like insulin, may release a C peptide during intramolecular cleavage.
  • TSHR-10,000 (Chazenbalk, et al, Endocrinology, 137:4586-4591 (1996)) is a Chinese hamster ovary (CHO) cell line overexpressing the human TSHR ( ⁇ 2 x 10 6 receptors per cell). Overexpression was attained using a dihydrofolate reductase minigene to amplify the stably- transfected TSHR cDNA transgenome.
  • TSHR-0 are CHO cells expressing the same TSHR cDNA, but without transgenome amplification (-1.5 x lO 3 receptors per cell)(Chazenbalk, et al, Endocrinology, 137:4586-4591 (1996); Kakinuma, et al, Endocrinology, 137:2664-2669 (1996)).
  • TSHRmyc are 293 human embryonal kidney (HEK) cells stably expressing the unamplified gene for an epitope-tagged human TSHR (Tanaka, et al, Biochem. Biophys. Res. Comm., 228:21-28 (1996)).
  • Epitope-tagging was achieved by replacing TSHR amino acids 338 to 349 with the human c-myc peptide EEQKLISEEDLL.
  • Cells were propagated in Ham's F-12 medium (CHO cells) or Dulbecco's modified Eagle's medium (DMEM)(293 HEK cells), supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), gentamicin (50 ⁇ g/ml) and amphotericin B (2.5 ⁇ g/ml).
  • Immunoprecipitation of precursor-labeled TSHR Cells near confluence in 100 mm diameter culture dishes were rinsed with phosphate-buffered saline (PBS) and pre-incubated (0.5 h, twice) in DME-H21 methionine- and cysteine- free medium containing 5% heat-inactivated FCS. The cells were then pulsed (1 h at 37 C) in 5 ml fresh medium supplemented with - 0.5 mCi of 33 S- methionine/cysteine (> 1000 Ci/mmole, DuPont NEN, Wilmington DE).
  • PBS phosphate-buffered saline
  • the cells were pelleted (5 min, 100 x g), washed twice with PBS and resuspended in buffer A containing 1% Triton X-100. After 90 min at 4°C with occasional vortexing, the mixture was centrifuged for 45 min at 100,000 x g and the supernatant was diluted 1:4 in immunoprecipitation buffer (20 mM Hepes pH 7.2, 300 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% Nonidet-P40, 2 mM EDTA).
  • immunoprecipitation buffer (20 mM Hepes pH 7.2, 300 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% Nonidet-P40, 2 mM EDTA).
  • solubilized cell proteins were precleared for 1 h at 4°C with - 150 ⁇ g normal mouse serum IgG prebound to 25 ⁇ l packed and washed protein A-agarose (Sigma). The protein A was removed by centrifugation (3 min at 10,000 X g) in a microcentrifuge. Mouse monoclonal antibodies (mAb) were then added, as described in the text. AlO and All (kind gifts of Dr Paul Banga, London, U.K.) both recognize TSHR amino acid residues 22-35 (Nicholson, et al, /. Mol. Endocrinol, 16:159-170 (1996)) and were used at a final dilution of 1:1000.
  • mAb monoclonal antibodies
  • the anti-myc mAb 9E10 (obtained from ATCC) was used at a final dilution of 1:500.
  • T3-365, a mAb to the TSHR B subunit (kindly provided by Drs. E. Milgrom and H. Loosfelt, Le Kremlin- Bicetre, France) and a mAb to human thyroid peroxidase (kindly provided by Dr. Scott Hutchison, Nichols Institute, San Juan Capistrano, CA, USA) were used at a dilution of 1:1000. After 3 h at 4°C, 25 ⁇ l packed and washed protein A-agarose was added and the tubes were tumbled for 1 h at 4°C.
  • the protein A was recovered by centrifugation for 3 min at 10,000 X g (4°C), washed 5 times with 1 ml immunoprecipitation buffer, and then once with 10 mM Tris, pH 7.4, 2 mM EDTA and 0.5% sodium dodecyl sulfate (SDS). Finally the pellet was resuspended in Laemmli sample buffer (Laemmli, Nature, 227:680-685 (1970)) with 0.7 M ⁇ -mercaptoethanol (30 min at 50°C) and electrophoresed on 7.5 % or 10% SDS-polyacrylamide gels (BioRad, Hercules, CA). Prestained molecular weight markers (BioRad) were included in parallel lanes. We precalibrated these markers against more accurate unstained markers to obtain the molecular weights indicated in the text. Radiolabeled proteins were visualized by autoradiography on Kodak XAR-5 X-ray film (Eastman Kodak, Rochester, NY).
  • Immunoblots of TSHR proteins Stably transfected TSHR-10,000 cells (in two 100 mm diameter dishes) were resuspended by incubation in Ca ++ - and Mg + ree PBS with 0.5 mM EDTA. The cells were pelleted (5 min., 100 x g, 4°C), resuspended in 1.5 ml of 10 mM Tris-HCl, pH 7.4, containing the protease inhibitors described above and homogenized with a Polytron homogenizer (Brinkman Instruments, Westbury, CT). After centrifugation for 10 min at 500 x g (4°C), the supernatant was recentrifuged for 20 min at 10,000 x g (4°C).
  • the pellet was resuspended in 0.1 ml of the same buffer, after which Laemmli buffer with 0.7 M ⁇ -mercaptoethanol was added (30 min at 50°C) and the sample electrophoresed on 10% SDS-polyacrylamide gels. Prestained molecular weight markers are described above. Proteins were transferred to ProBlott membranes (Applied Biosystems, Foster City, CA), which were processed as described previously (Rapoport, et al, /. Clin.
  • Membranes were incubated overnight (4°C) with mAb AlO or All to the A subunit, or with mAb to the B subunit; T3-495 (TSH-R1; Transbio, Boulogne, France) or T3-365 (final dilutions of 1:1000). After rinsing, the membranes were incubated for 1 hr at room temperature with alkaline phosphatase conjugated goat anti-mouse immunoglobulin G (1:400 dilution)(Cappel, Durham, NC).
  • the signal was developed with nitroblue tetrazolium and 5-bromo, 4-chloro, 3-indolyl phosphate in lOOmM Tris-HCl buffer, pH 9.5, containing 100 mM NaCl and 5 mM MgCl 2 .
  • Enzymatic deglycosylation of TSHR protein The Protein A/IgG/TSHR complex or the 10,000 x g crude membrane fraction (see above) were incubated (10 min., 100°C) in denaturing buffer containing 0.5% SDS, 1% ⁇ - mercaptoethanol. Enzymatic deglycosylation was performed according to the protocol of the manufacturer (N.E. Biolabs, Beverly MA). N-glycosidase F digestion (100 U for 2 h at 37°C) was in 50 mM Na phosphate, pH 7.5, 1% NP- 40. Endoglycosidase H digestion (50 U for 2 h at 37°C) was in 50 mM Na citrate, pH 5.5. Samples were then subjected to SDS-PAGE, as described above. Covalent cross-linking of radiolabeled TSH: Highly purified bovine
  • TSH (5 ⁇ g, 30 U/mg protein) was radiolabeled with 125 I to a specific activity of - 80 ⁇ Ci/ ⁇ g protein using the Bolton-Hunter reagent (4400 Ci/mmol; DuPont- NEN) according to the protocol of the manufacturer, followed by Sephadex G- 100 chromatography (Goldfine, et al, Endocrinology, 95:1228-1233 (1974)).
  • Confluent 100 mm diameter dishes of TSHR-expressing cells were incubated for 2h at 37 C with 5 ⁇ Ci 125 I-TSH in 5 ml modified Hank's buffer (without NaCl), supplemented with 280 mM sucrose and 0.25% BSA (binding buffer).
  • Unbound 125 I-TSH was removed by rinsing the cells three times with ice-cold binding buffer.
  • Disuccinimidyl suberate (DSS; 1 mM; Sigma) in 10 mM Na phosphate buffer, pH 7.4, containing the protease inhibitors described above was then added for 20 min at room temperature.
  • the cross-linking reaction was terminated by the addition of 20 mM ammonium acetate (final concentration).
  • the cells were rinsed twice with PBS and scraped into 10 mM Tris, pH 7.5, containing the same protease inhibitors. Cells were homogenized using a Polytron homogenizer and centrifuged for 5 min at 4°C
  • Immunodetection of the TSHR in TSHR-10,000 cells Immunoprecipitation studies of precursor-labeled TSHR-10,000 cells were performed with mAb AlO (Nicholson, et al, /. Mol. Endocrinol, 16:159-170 (1996)) to TSHR amino acid residues 22-35 at the amino terminus of the A subunit. Multiple forms of the TSHR were observed under reducing conditions after chase periods of 3 hr and 16 (Fig. 6A).
  • Two forms of single subunit (uncleaved) TSHR were:- (i) -115 kDa in size (complex carbohydrate resistant to endoglycosidase H) and, (ii) -100 kDa in size (immature, high mannose carbohydrate sensitive to endoglycosidase H)(Fig. 6).
  • N-glycosidase F digestion removed both forms of carbohydrate, exposing a -84 kDa polypeptide backbone. Cleaved (two subunit) TSHR was also present.
  • the extracellular A subunit was visible as a broad -62 kDa band with complex carbohydrate, which upon deglycosylation became a more focused 35 kDa band.
  • the largely transmembrane B subunit could not be visualized in these immunoprecipitation experiments (Fig. 6A), presumably because of its detachment from the antibody-bound A subunit and consequent loss during the very stringent washing procedure.
  • the specificity of mAb for the TSHR was evident in control experiments using untransfected CHO cells and with a mAb to thyroid peroxidase (TPO)(Fig. 6B).
  • mAb T3-495 and T3-365 was confirmed on immunoblotting with untransfected CHO cells (Fig. 7B). As in the immunoprecipitation experiments, the size of the A subunit could clearly be determined by immunoblotting with mAb AlO to the TSHR amino terminus.
  • the -62 kDa mature A subunit contained complex carbohydrate (endoglycosidase H resistant)(Fig. 7C). Most important, deglycosylation with N-glycosidase F confirmed a -35 kDa A subunit backbone. Lesser fragments of -39 kDa and -42 kDa were also evident.
  • the specificity of mAb AlO to the A subunit was confirmed on immunoblots with untransfected CHO cells and on immunoblotting of TSHR- 10,000 cells with a mAb to TPO (Fig. 7D).
  • Conundrum of a missing piece of the TSHR The human TSHR, without its 21 amino acid residue signal peptide, has a predicted polypeptide backbone of 84.5 kDa (743 amino acid residues). However, from the immunoprecipitation and immunoblot studies shown above, the sum of the enzymatically deglycosylated A subunit (35 kDa) and the primary B subunit fragment (-42 kDa) was only -77 kDa. A 35 kDa polypeptide backbone for the TSHR A subunit would place the cleavage site in the region of amino acid residue 330, taking into account the absence of the signal peptide.
  • a -42 kDa size for the non-glycosylated B subunit would be consistent with a holoreceptor cleavage site at about residue 380. It, therefore, appeared that a "C peptide" fragment in the vicinity of residues 330 - 380 could be missing from the cleaved TSHR ectodomain (Fig. 8). This deduction is inconsistent with the prevailing concept of a single cleavage site in the TSHR ectodomain.
  • TSHR subunits in TSHRmyc cells In order to explore the possibility of two cleavage sites in the TSHR, we used TSHRmyc cells that express a receptor with a 12 amino acid human c-myc epitope in place of residues 338- 349. This epitope lies within the segment of the TSHR predicted to be missing if the two cleavage site hypothesis is correct, namely residues - 330-380 (Fig. 8). Detection by a mAb to c-myc of only the single subunit forms of the TSHR, and not the cleaved A subunit, would support the concept that a portion of the ectodomain is lost during intramolecular cleavage. Cleavage within the c-myc epitope could also lead to loss of this epitope. The two cleavage site hypothesis would also predict detection in TSHRmyc cells of the
  • the TSHRmyc cells do not contain an amplified transgenome and express fewer receptors (-100,000 per cell)(Tanaka, et al, Biochem. Biophys. Res.
  • the 35 kDa deglycosylated A subunit band detected by mAb AlO in the TSHRmyc and TSHR-10,000 cells was not an artifact because no such band was detected by immunoprecipitation with mAb 10 of precursor-labeled, untransfected HEK cells, nor did a non-relevant mAb (to TPO) detect this band in TSHRmyc cells (Fig. 9C).
  • TSHRmyc cells as in a cell line expressing similar numbers of wild-type TSHR (TSHR-0) (Kakinuma, et al, Endocrinology, 137:2664-2669 (1996)).
  • EXAMPLE 3 EVIDENCE FOR NEGATIVE COOPERATIVITY
  • ECD A 2.7 kb TSHR cDNA containing the coding region and 0.4 kb of the 3' untranslated region was excised with Sal I and Xba I from pTSHR-5'TR- NEO-ECE (Kakinuma, et al, Endocrinology, (In Press) (1995)). This fragment was restricted with Hinc II to release the 2.3 kb TSHR coding region, which was then inserted into the Xba I (blunted) and Sal I sites in pSV2-DHFR-ECE- TPO (Kaufman, et al, Molec. Cell. Endocrinol, 78:107-114 (1991)) following removal of its Sal I-Xba I insert.
  • the new plasmid termed pSV2-DHFR-ECE- TSHR, was transfected by the calcium phosphate precipitation method (Chen, et al, Mol. Cell. Biol, 7:2745-2752 (1987)) into dihydrofolate reductase (dhfr)-deficient CHO cells (CHO-DG44; kindly provided by Dr. Robert
  • Stably transfected cells were selected in thymidine-, guanine-, and hypoxanthine-free Ham's F-12 medium supplemented with 10% dialyzed fetal calf serum, penicillin (100 U/ml), gentamicin (50 ⁇ g/ml) and amphotericin B (2.5 ⁇ g/ml). After limiting dilution, 14 different clones were tested for TSHR expression by measurement of intracellular cAMP after TSH stimulation (see below). The clone with the greatest response was selected for transgenome amplification.
  • Methotrexate (MTX) was added to the selective cell culture medium at an initial 5 concentration of 20 nM and surviving cells were expanded. The methotrexate concentration was sequentially increased until a final concentration of 10,000 nM (10 ⁇ M) was reached. Cells were recloned by limiting dilution at 800 nM and 10,000 nM methotrexate.
  • aprotinin (1 ⁇ g/ml), and pepstatin A (2 ⁇ g/ml) (all from Sigma, St. Louis, MO)(buffer A).
  • the cells were pelleted (5 min, 100 x g), washed twice with PBS and resuspended in buffer A containing 1% Triton X-100. After 90 min at 4C with occasional vortexing, the mixture was centrifuged for 45 min at 100,000 x g and the supernatant was diluted 1:4 in immunoprecipitation buffer (20 mM Hepes pH 7.2, 300 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% Nonidet-P40 and 2 mM EDTA).
  • solubilized cell proteins were precleared by incubation for 1 h at 4C with 150 ⁇ g normal mouse serum IgG prebound to 25 ⁇ l packed and washed protein A-agarose (Sigma). After removal of the protein A by centrifugation (3 min at 10,000 X g) in a microcentrifuge, mouse monoclonal antibodies to the TSHR (mixture of A9 and AlO, a kind gift of Dr Paul Banga, London, U.K., final dilution of 1:1000) were added. A9 and AlO recognize TSHR regions between amino acids 147-229 and 22-35, respectively (Nicholson, et al, /. Mol Endocrinol, 16:159-170 (1996)).
  • the cells were rinsed twice with PBS and scraped into 10 mM Tris, pH 7.5, containing the same protease inhibitors.
  • Cells were homogenized using a Polytron homogenizer (Brinkmann Instruments, Westbury CT) and centrifuged for 5 min at 4 C (500 x g). The supernatant was centrifuged (15 min, 10,000 x g, 4C) and the pellet was resuspended in 50 ⁇ l 10 mM Tris, pH 7.5. Protein concentrations were determined by the method of Bradford (Bradford, Anal Biochem., 72:248-254 (1976)).
  • TSH binding CHO cells stably transfected with TSHR cDNA were grown to confluence in 96-well culture plates. Cells were then incubated for 2 h at 37°C in 50 ⁇ l binding buffer (see above) containing approximately 50,000 cpm of 125 I-TSH in the presence or absence of increasing concentrations of 5 unlabeled bovine TSH (Sigma).
  • the cells were rapidly rinsed three times with binding buffer (4°C), solubilized with 0.1 ml NaOH and radioactivity measured in a gamma-counter.
  • binding buffer 4°C
  • solubilized with 0.1 ml NaOH radioactivity measured in a gamma-counter.
  • Non- 10 specific 12S I-binding to untransfected CHO cells was subtracted from total counts bound to provide specific counts bound. These values were ⁇ 10% of total counts bound at the highest tracer concentration used (2.5 x 10 7 cpm/ml).
  • TSH stimulation of intracellular cAMP Transfected CHO cells, grown to confluence in 24-well or 96-well culture plates, were incubated for 2 h at 15 37°C in either hypotonic medium (Kasagi, et al, /. Clin. Endocrinol. Metab., 54:108-114 (1982); Rapoport, et al, Metabolism, 31:1159-1167 (1982)) or in Ham's F-12 medium containing 1% bovine serum albumin, 1 mM isobutyl methylxanthine, with or without added bovine TSH (Sigma). Cyclic AMP was measured directly in the hypotonic medium.
  • Cyclic AMP was measured by radioimmunoassay using cAMP, 2'-0-succinyl 125 I-iodotyrosine methyl ester (Dupont NEN) and a rabbit anti-cAMP antibody from Calbiochem, San Diego CA.
  • Quantisation by immunoprecipitation of TSHR protein expression in whole CHO cells The process of stable transfection and progressive amplification of the TSHR holoreceptor/ dhfr-minigene complex in the genome of CHO cells took approximately one year. During this period, TSHR expression in the selected clone was confirmed intermittently by 125 I-TSH binding. After completion of the amplification process, we assessed the level of TSHR expression within whole cells by precursor 35 S-methionine and 35 S- cysteine labeling followed by immunoprecipitation under native conditions using mouse monoclonal antibodies to the TSHR (A9 + A10)(Nicholson, et al, /. Mol. Endocrinol, 16:159-170 (1996)).
  • FIG. 12 we used clonal cell lines stably transfected with (i) 4 kb TSHR cDNA containing both 5'- and 3'-untranslated regions (wild-type TSHR) (Nagayama, et al, Biochem. Biophys. Res.
  • the upper band represents a single chain holoreceptor (Russo, et al, Mol. Endocrinol, 5:1607-1612 (1991); Russo, et al, Endocrinology, 130:2135-2138 (1992)) with an apparent mass of - 115 kDa and the lower band a dissociated A subunit (Buckland, et al, FEBS Letters, 5 145:245-249 (1982)) of - 60 kDa.
  • TSHR Function of the overexpressed TSHR in the absence of ligand: Basal cAMP levels were assessed in stably-transfected cell lines expressing different numbers of TSHR. In cells cultured in hypotonic medium (Kasagi, et al, /. Clin. Endocrinol. Metab., 54:108-114 (1982); Rapoport, et al, Metabolism,
  • TSHR-0 cells Kd - 5 x 10 "10 M) (Kakinuma, et al, Endocrinology, (In Press)
  • Plasmid constructs We generated three plasmids for expression in mammalian cells of limited TSHR ectodomain truncations (Fig. 17): (i) TSHR- 261: Plasmid TSHR-5TR-NEO-ECE (Kakinuma, et al, Endocrinology, 137:2664-2669 (1996)) contains an Afl II site at codon 260 and an Xba I site in the vector at the 3 end of the insert. The Afl Il-Xba I fragment was excised and replaced with a cassette coding for 6 histidine residues (6H) followed by 2 stop codons.
  • TSHR-289 A cDNA fragment including the Afl II site at codon 260 continuing to codon 289 followed by an Spe I site was generated by PCR using Pfu DNA polymerase (Stratagene, San Diego, CA).
  • TSHR-309 Construction used the identical strategy to that of TSHR-289 except that the Afl II - Spe cDNA fragment generated by PCR extended up to codon 309. After confirmation of the nucleotide sequences of the relevant areas, the
  • TSHR-261, TSHR-289 and TSHR-309 cDNAs were excised with Sal I and Xba I and transferred to the vector pSV2-ECE-dhfr (Kaufman, et al, Molec. Cell. Endocrinol, 78:107-114 (1991)).
  • TSHR ectodomain variants Cell lines, stably transfected with the above TSHR ectodomain cDNA variants were established in CHO dhfr-cells (CHO-DG44; kindly provided by Dr. Robert Schimke, Stanford University, Palo Alto, CA), using procedures described previously (Rapoport,
  • Transgenome amplification was achieved by progressive adaptation to growth in methotrexate (final concentration 10 ⁇ M)(Rapoport, et al, /. Clin. Endocrinol.
  • TSHR ectodomain variants in medium and in cells CHO cells to be tested for TSHR ectodomain variant expression were metabolically
  • Radiolabeled proteins were visualized by autoradiography on Kodak XAR-5 X-ray film (Eastman Kodak, Rochester, NY). TSHR secreted into the medium was also detected by means of their 6H tag using Ni-NTA resin (QIAGEN, Inc, Chatsworth, CA) according to the procedure reported previously (Rapoport, et al, /. Clin. Endocrinol Metab., 81:2525-2533 (1996)).
  • TSHR autoantibody kits were purchased from Kronus, San Clemente, CA.
  • the principal of this assay is the ability of autoantibodies to compete for 125 I-TSH binding to TSHR solubilized from porcine thyroid glands ("TSH binding inhibition" or TBI assay) (Shewring, et al, Clin. Endocrinol, 17:409-417 (1982)).
  • solubilized TSHR 50 ml
  • solubilized TSHR 50 ml
  • Buffer containing 125 I-TSH is then added (2 h at room temperature).
  • Solubilized TSHR complexed with TSH is precipitated by polyethylene glycol.
  • Antibody activity is measured as % inhibition of 125 I-TSH binding relative to a standard serum from a normal individual without autoantibodies.
  • We modified this assay by preincubating (30 min at room temperature) serum from Graves' patients (25 ml) with conditioned medium from cells expressing TSHR ectodomain variants (25 ml). Solubilized TSHR (50 ml) was then added to the serum/medium mixture (50 ml).
  • serum from normal individuals and conditioned medium from CHO cells secreting a truncated form of thyroid peroxidase Kerman, et al, Molec. Cell.
  • Lectin adsorption Binding of TSHR in conditioned medium to different lectins was determined for three Sepharose-linked lectins: Wheat germ agglutinin (WGA), Bandeiraea simplificifolia and Concanavalin A (Con A)(Pharmacia, Piscataway, NJ). Medium (40 ml) was slowly stirred for 2 hr at room temperature with 0.4 ml of Sepharose-lectin.
  • TBS 150 mM NaCl
  • BSA bovine serum albumin
  • TSHR-261 partial purification Conditioned medium was harvested from CHO cells expressing TSHR-261 cultured in non-selective F12 medium containing 10% fetal calf serum, antibiotics and 5 mM Na butyrate (Dorner, et al, /. Biol. Chem., 264:20602-20607 (1989)). Medium (2 liters) was applied to a
  • TSHR-261 recovery was monitored by bioassay (TBI neutralization; see above).
  • TBI neutralization The N-terminal amino acid sequence of the deglycosylated TSHR-261, cut out from a PVDF membrane, was determined by the Protein Structure Laboratory, University of California at
  • TSHR ectodomain variants CHO-DG44 cells were stably transfected with plasmids coding for TSHR ectodomain variants truncated at amino acid residues 261, 289 and 309 (Fig. 17). Individual clones were obtained by limiting dilution and transgenome amplification was performed by progressive adaptation to growth in methotrexate (final concentration 10 mM). One clone of each TSHR ectodomain variant, selected for high level of TSHR expression, was expanded and used for further studies.
  • TSHR-261 The ectodomain variant with the greatest degree of C-terminal truncation (TSHR-261) was entirely secreted into the medium, as detected by immunoprecipitation after an overnight chase, with no receptor remaining in the cells (Fig. 18).
  • TSHR-289 truncated to a lesser extent was secreted to an intermediate degree.
  • the receptor remaining within the cells was present in multiple forms, the dominant band having a molecular weight lower than the secreted form.
  • TSHR-309 the least truncated ectodomain, secretion into the medium was relatively inefficient. Thus, proportionately less receptor was secreted than remained within the cells, the latter primarily in lower molecular weight form.
  • TSH binding inhibition (TBI) assay to test whether conditioned medium from cultured cells expressing TSHR-261, TSHR-289 and TSHR-309 could neutralize autoantibody activity in a Graves' patient's serum.
  • THI TSH binding inhibition
  • TSHR ectodomain variants Adsorption of TSHR ectodomain variants to lectins: Although the Ni- NTA resin was effective in purifying radiolabeled TSHR secreted by CHO cells into tissue culture medium (Fig. 18), we were unable to purify unlabeled TSHR protein from medium using this approach. The Ni-NTA bound to many unlabeled proteins despite attempts to minimize non-specific interactions with imidazole and adsorption at lower pH (data not shown). We, therefore, attempted partial purification of TSHR ectodomain variants from conditioned medium using lectins. TSHR-261 in conditioned medium bound poorly to wheat germ agglutinin and Bandeiraea simplicifolia (Fig. 20). Almost all of this material remained in the "flow-through" and minimal amounts could be recovered by elution with specific sugar. In contrast, concanavalin A (Con A) was effective in extracting TSHR-261 from the medium.
  • Con A concanaval
  • Con A-enriched TSHR-261 showed that, like the material immunoprecipitated from conditioned medium with mAb AlO (Fig. 18), the secreted receptor was endoglycosidase H resistant and endoglycosidase F sensitive (complex carbohydrate)(Fig. 21). Indeed, this pattern was similar to the smaller amounts of TSHR-261 that could be recovered from conditioned medium using wheat germ agglutinin (Fig. 21). Most important, the Con-A enriched material was highly active in neutralizing autoantibody TBI activity in patients' sera (data not shown for these experiments; see data below on TBI activity in more extensive studies using multiple Graves' sera; Fig. 24).
  • TSHR-289 and TSHR-309 were also extracted from culture medium using Con A. Immunoblotting indicated that TSHR-289 and TSHR-309, like TSHR-261, contained only mature, complex carbohydrate (Fig. 22). Remarkably, TSHR-261 contains - 20 kDa of N-linked glycosylation, 40% of its mass. The apparent molecular weights of the deglycosylated proteins ( ⁇ 30, 32 and 34 kDa for TSHR-261, TSHR-289 and TSHR-309, respectively) were slightly ( ⁇ 2 kDa) greater than predicted from their known amino acid sequences (including 6 H tags).
  • TSHR-261 Autoantibody neutralization by partially purified TSHR-261: In the preceding studies, the secreted TSHR variants could be detected qualitatively by immunoprecipitation, immunoblotting or by autoantibody neutralization. However, it was important to determine quantitatively the amount of receptor that was interacting with TSHR autoantibodies in patients' sera. No TSHR standards are available for this purpose. Indeed, the TSHR of mammalian cell origin has never been purified sufficiently for direct visualization on a polyacrylamide gel. We selected TSHR-261 for further study because, of the three ectodomain variants, it was secreted to the greatest extent (Fig. 18) and because its "bioactivity" in terms of autoantibody recognition appeared equal to that of TSHR-289 (Fig. 19).
  • Con A chromatography provided an initial purification of - 100-fold. Subsequently, and in contrast to its use as an initial capture stem, Ni-chelate chromatography was quite effective in generating sufficient TSHR-261 for direct visualization and quantisation by Coomassie blue staining (Fig. 23, left panel). Enzymatic deglycosylation confirmed the immunoblot evidence for a -30 kDa polypeptide backbone with -20 kDa of complex carbohydrate (Fig. 23, right panel) and also provided the best means to quantitate the amount of receptor recovered.
  • TSHR-261 In 3 separate preparations from 2 liters of conditioned medium, recovery of TSHR-261 (corrected for a 40% glycan component) was 0.3 - 0.4 mg/ liter. Amino acid sequencing of the 30 kDa deglycosylated band confirmed the amino terminus of the TSHR (Met, Gly, X, Ser, Ser, Pro, Pro; X represents a Cys residue). Further purification of TSHR-261 (Sephacryl S-200) was associated with a major loss in activity (data not shown). However, semi- purified TSHR-261 (20-40% estimated purity)(Fig. 23) was quite stable at -80°C.
  • TSHR-261 Partially-purified TSHR-261 was highly potent in neutralizing TSHR autoantibody TBI activity in patients' sera.
  • TBI TSH binding inhibitory
  • 30 ng of TSHR-261 per tube (0.15 mg/ml) completely neutralized autoantibody activity (Fig. 24). Therefore, in studies on further sera, we used 50 ng of TSHR-261 per tube (0.25 mg/ml). This concentration of TSHR-261 neutralized all or most TSH binding inhibitory activity in the 18 Graves' sera examined (Fig. 25).
  • TSHR-10,000 Chinese hamster ovary (CHO) cells express high numbers of mature TSHR on their surface in vivo (described in Example 3).
  • TPO thyroid peroxidase
  • Serum BB1 Serum BB1 kindly provided by Dr. Stephanie Lee, New England Medical Center Hospitals, Boston, was from a patient with Graves' disease who subsequently became hypothyroid with the development of blocking antibodies.
  • Thirty sera were generously provided to us by Mr. Juan Tercero, Corning-Nichols Institute, San Juan Capistrano, California. These sera were submitted without clinical information for the assay of autoantibodies to the TSHR (see below), presumably for suspicion of Graves' disease.
  • Ten of the 30 sera were selected for the absence of TSHR autoantibodies, 10 for the presence of moderate TSH binding inhibitory (TBI) activity and a further 10 for their high (>50%) TBI activities.
  • TBI moderate TSH binding inhibitory
  • TPO and TSHR Construction of plasmids and Chinese hamster ovary (CHO) stable transfections for overexpression of the human TSHR and TPO have been described elsewhere (Example 3; Kaufman et al, Molec. Cell. Endocrinol, 78:107-114 (1991)). These cells, as well as untransfected CHO cells (DG44; kindly provided by Dr. Robert Schimke, Stanford University, Palo Alto, CA) were propagated in Ham's F-12 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), gentamicin (50 ⁇ g/ml) and amphotericin B (2.5 ⁇ g/ml).
  • DG44 untransfected CHO cells
  • FACS Flow cytometric analysis
  • untransfected CHO cells were resuspended in 0.18 ml of buffer A (phosphate-buffered saline, 10 mM Hepes, pH 7.4, 0.05% Na azide and 2% fetal calf serum heat-inactivated at 56°C for 30 min).
  • Sera (20 ⁇ l) were added (final concentration of 1:10, or as specified in the text) and gently mixed for 60 min at 4°C. After removal of the cells by centrifugation, the sera were divided in two 0.1 ml aliquots. One aliquot was added to CHO cells expressing either the TSHR or TPO, the other aliquot to untransfected CHO cells.
  • Dickinson FACScan-CELLQuest system Three parameters (forward scatter, FSC, 90° side scatter, SSC, and FL1 detector) were use for the analysis. All assays included cells treated with second antibody alone and serum from normal individuals. All sera were analyzed by flow cytometry at least twice.
  • TSHR-10,000 cells using mouse monoclonal antibodies (A9 and A10)(l:100 final concentration)(Nicholson, et al, /. Mol. Endocrinol, 16:159-170 (1996)) and rabbit antiserum (1:60 final concentration) (R8) (Vlase, et al, Endocrinology, 136:4415-4423 (1995)) to the TSHR (all kindly provided by Dr.
  • Flow cytometric analysis of CHO cells expressing the TSHR We have previously been unable to detect the TSHR stably expressed on the surface of CHO cells using Graves' sera. Recently, however, two reagents became available to us; CHO cells expressing very high numbers of TSHR (nearly 2 x 10 6 ) on their surface (Example 3), and a Graves' serum (BB1) particularly potent in the indirect TSH binding inhibition (TBI) assay. Using this serum, a very small specific signal (relative to control untransfected cells) was detected with our original line (Nagayama, et al, Biochem. Biophys. Res.
  • TSHR-10,000 cells Use of TSHR-10,000 cells to detect TSHR autoantibodies in different sera: In preliminary studies, we observed that some sera, regardless of whether or not they contained TSHR autoantibodies, elicited high fluorescence on flow cytometry with TSHR-10,000 cells. We observed similar high fluorescence when these sera were incubated with untransfected CHO cells not expressing the TSHR (data not shown). Therefore, some sera contained antibodies against unknown antigens on the surface of CHO cells. For this reason, we instituted a preadsorption step, in which sera were preincubated with untransfected CHO cells prior to addition to the TSHR-10,000 cells. Preadsorption was effective in eliminating, or greatly reducing, this nonspecific background.
  • this panel included 10 sera (IL - 10L) without TBI activity (1 - 4.2% inhibition); 10 sera (1M - 10M) with moderately high TBI values (17.3 - 39.4% inhibition); 10 sera (IH - 10H) with high TBI levels (52 - 95.1% inhibition); four normal individuals without autoimmune thyroid disease and four patients with systemic lupus erythematosus with anti-DNA and/or anti-cardiolipin antibodies. None of the sera from normal individuals, individuals with negative TBI values or patients with systemic autoimmunity generated a positive signal on flow cytometry.
  • the TSHR autoantibody titers in the four sera positive for the TSHR on flow cytometry were determined in the TBI assay (Table 2). Dilution of these sera indicated that BBl and 10H had similar, high TSHR autoantibody titers, consistent with their strong fluorescence signals on flow cytometry. The lower TBI titer of serum 3H was also consistent with its relatively low fluorescence signal. Surprisingly, serum 10M, with the lowest TSHR autoantibody titer, generated a strong signal on flow cytometry, raising the possibility of the presence in this serum of "neutral" autoantibodies to the TSHR that do not inhibit TSH binding.
  • TPO autoantibodies detected by flow cytometry In view of the small number of sera that could unequivocally recognize the TSHR by flow cytometry, we studied TPO autoantibodies in the same sera by the same approach using CHO cells overexpressing TPO on their surface (Kaufman, et
  • TPO autoantibodies commonly coexist with TSHR autoantibodies. Indeed, of the 20 TBI positive sera (1-lOM and 1-lOH), 19 bound > 13% 125 I-TPO, well above the upper limit detected in the sera of normal individuals (2.6% binding)(Table 1). In addition, two of the TBI-negative sera (7L and 10L) were also TPO autoantibody positive by this method. Strikingly, all 20 sera with detectable TPO autoantibodies were clearly positive on flow cytometry with CHO-TPO cells.
  • Example 2 suggested that cleavage sites 1 and 2 were in TSHR domains D and E, respectively.
  • Plasmids were stably transfected with Lipofectin (Gibco-BRL, Gaithersburg, MD) into Chinese hamster ovary (CHO) cells cultured in Ham's F-12 medium supplemented with 10% fetal calf serum (FCS) and standard antibiotics.
  • Lipofectin Gibco-BRL, Gaithersburg, MD
  • FCS fetal calf serum
  • this 50 amino acid segment (estimated to be between residues 317-366) is very hydrophilic and can be deleted from the wild-type TSHR without loss of ligand binding and function (Wadsworth, et al, Science, 249:1423 (1990)) and without preventing cleavage into A and B subunits (Russo, et al, Mol
  • the deglycosylated TSHR A subunit is - 35 kDa in size (Example 4;
  • the deglycosylated A subunit would be 33 kDa or less, smaller than observed by multiple investigators (Graves, et al, Endocrinology, 137:3915 (1996); Example 4; Misrahi, et al, Eur. ⁇ . Biochem., 222:711 (1994)); (ii) The specificity of cleavage at site 1 was "relaxed” and, (iii) our hypothesis for two cleavage sites was wrong, despite the strong supporting evidence that we had obtained previously (Example 2).
  • the mutation that abrogates cleavage at site 2 introduces a consensus sequence for an N-linked glycosylation site.
  • a carbohydrate side-chain in this vicinity could, therefore, prevent ectodomain cleavage, for example by steric hindrance of a proteolytic enzyme.
  • the mutagenesis strategy used to investigate cleavage site 2 in the TSHR involved substitution into the TSHR of the corresponding segments of the non-cleaving LH/CG receptor.
  • a motif that would not be a potential glycosylation site (Fig. 33A).
  • TSH-LHR ⁇ -GQE ⁇ . ⁇ AAA did cleave (Fig. 33B).

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Abstract

La présente invention concerne des compositions à base de récepteur de la thyrotropine ainsi que des méthodes d'utilisation de ces compositions. Ces compositions conviennent particulièrement à des utilisations thérapeutiques et diagnostiques.
PCT/US1999/014640 1998-06-30 1999-06-29 Compositions a base de recepteur de la thyrotropine humaine et leur utilisation WO2000000590A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002034042A3 (fr) * 2000-10-26 2003-09-04 Deltagen Inc Souris transgeniques contenant des disruptions geniques du recepteur de l'hormone de stimulation thyroidienne (tch-r)
DE10207135A1 (de) * 2002-02-20 2003-09-11 Euroimmun Gmbh Verfahren und Kit zum Nachweis von spezifischen Antikörpern mittels Immunfluoreszenz
WO2007036511A1 (fr) * 2005-09-26 2007-04-05 Ulrich Loos Procede d'identification d'autoanticorps contre le recepteur de la tsh et nouveaux chimeres du recepteur de la tsh
DE102014011653A1 (de) 2014-06-20 2015-12-24 Ulrich Loos Nachweis von Autoantikörpern gegen den TSH-Rezeptor

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5614363A (en) * 1990-01-25 1997-03-25 New England Medical Center Hospitals, Inc. TSH receptor
US5744348A (en) * 1989-09-08 1998-04-28 New England Medical Center Hospitals, Inc. TSH receptor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5744348A (en) * 1989-09-08 1998-04-28 New England Medical Center Hospitals, Inc. TSH receptor
US5614363A (en) * 1990-01-25 1997-03-25 New England Medical Center Hospitals, Inc. TSH receptor

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002034042A3 (fr) * 2000-10-26 2003-09-04 Deltagen Inc Souris transgeniques contenant des disruptions geniques du recepteur de l'hormone de stimulation thyroidienne (tch-r)
DE10207135A1 (de) * 2002-02-20 2003-09-11 Euroimmun Gmbh Verfahren und Kit zum Nachweis von spezifischen Antikörpern mittels Immunfluoreszenz
WO2007036511A1 (fr) * 2005-09-26 2007-04-05 Ulrich Loos Procede d'identification d'autoanticorps contre le recepteur de la tsh et nouveaux chimeres du recepteur de la tsh
US8999727B2 (en) 2005-09-26 2015-04-07 Ulrich Loos Innovative TSH-R-Ab-kit
DE102014011653A1 (de) 2014-06-20 2015-12-24 Ulrich Loos Nachweis von Autoantikörpern gegen den TSH-Rezeptor
US11592444B2 (en) 2014-06-20 2023-02-28 Ulrich Loos Detection of autoantibodies against the TSH receptor

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