JP5345556B2 - Human monoclonal antibody against thyrotropin receptor acting as an antagonist - Google Patents

Description

  The present invention relates to an antibody that reacts with a thyrotropin (TSH) receptor (TSHR), and is not particularly limited, but particularly relates to an antibody that can bind to TSHR and inhibit stimulation of TSHR by TSH or TSHR stimulating antibodies.

Thyrotropin, thyroid stimulating hormone (TSH), is a pituitary hormone that regulates thyroid function through TSHR (Szkudlinski MW, Fremont V, Ronin C, Weintraub BD 2002 Thyroid-stimulating hormone and TSHR structure-function relationships.
Physiological Reviews 82: 473-502). TSHR is a G protein-coupled receptor and consists of three domains: a leucine rich domain (LRD), a cleavage domain (CD), and a transmembrane domain (TMD) (Nunez Miguel R, Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Blundell TL, Rees Smith B, Furmaniak J 2004 Analysis of the thyrotropin receptor-thyrotropin interaction by comparative modeling. Thyroid 14: 991-1011). Binding of TSH to TSHR causes a receptor signal that leads to stimulation of the formation and release of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). The mechanism of negative feedback related to circulating T4 and T3 concentrations controls the release of TSH from the pituitary gland (and the thyrotropin-releasing hormone secreted from the hypothalamus), which in turn causes thyroid stimulation and serum To control the concentration of thyroid hormones.

  It is well documented in the art that certain autoimmune thyroid disease (AITD) patients have autoantibodies that react with TSHR (Rees Smith B, McLachlan SM, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocrine Reviews 9: 106-121). In most cases, these autoantibodies stimulate the thyroid to produce high concentrations of T4 and T3 by binding to TSHR and mimicking the action of TSH. These autoantibodies are described as thyroid stimulating autoantibodies or TSHR autoantibodies (TRAb) having stimulating activity or TSH agonist activity. The physiological feedback mechanism of thyroid function control described above is not effective in the presence of such thyroid-stimulating autoantibodies and in patients with signs of hyperthyroidism or thyroid poisoning (excess thyroid hormone in serum) . This disease is known as Graves' disease. In some patients, stimulatory TRAbs are involved in the interaction of posterior orbital tissue with TSHR and contribute to the signs of Graves' eye disease. Details of a human monoclonal autoantibody (TSHR1, a hMAb) that acts as a potent thyroid stimulator are described in patent application WO 2004 / 050708A2.

On the other hand, in patients with AITD, autoantibodies bind to TSHR and block TSH binding to the receptor, but do not have the ability to stimulate TSHR. This type of autoantibody is known as a TRAb with blocking or TSH antagonist activity, and patients with blocking TRAb in their serum can have inactive thyroid symptoms (hypothyroidism) ( Rees Smith B, McLachlan SM, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocrine Reviews 9: 106-121). In particular, the presence of TRAb with blocking activity in the serum of pregnant women may cross the placenta and block fetal thyroid TSHR, causing severe neonatal hypothyroidism and severe developmental consequences. Furthermore, TRAbs with blocking activity are found in the breast milk of affected mothers, which may further contribute to infant clinical hypothyroidism. To date, human monoclonal autoantibodies against TSHR with TSH antagonist activity are not available. Thus, how this type of autoantibody interacts with TSHR and how its interaction with TSHR is compared with stimulating autoantibodies (eg M22) and with TSH and TSHR. Detailed research on this is limited.

  Human chorionic gonadotropin is a hormone with a mild thyroid stimulating effect produced during pregnancy.

Characterizing the properties of TRAbs by stimulatory or blocking activity is critical in studies aimed at improving the diagnosis and management of diseases associated with autoimmune responses to TSHR. The invention described in patent application WO 2004/050708 A2 provides details on the nature of human monoclonal autoantibodies with potent stimulatory activity and their interaction with TSHR. Further, patent application WO 2006 / 016121A discloses a mutant TSHR formulation comprising at least one point mutation, which comprises a patient serum stimulating TSHR autoantibody, a patient serum blocking TSHR autoantibody, and Used for differential screening and identification of TSH in screened patient fluid samples. Patent application WO 2004/050708 A2 also describes a mouse monoclonal antibody (9D33) with TSHR blocking activity. 9D33 binds to TSHR with high affinity (2 × ^{10 10} L / mol), is an effective antagonist of TSH, the hMAb TSHR1 (M22), and patient serum TRAb with stimulating or blocking activity (patent application international publication) 2004 / 050708A2 and Sanders J,
Allen F, Jeffreys J, Bolton J, Richards T, Depraetere H, Nakatake N, Evans M, Kiddie A, Premawardhana LD, Chirgadze DY, Miguel RN, Blundell TL, Furmaniak J, Rees Smith B 2005 Characteristics of a monoclonal antibody to the thyrotropin receptor that acts as a powerful thyroid-stimulating autoantibody antagonist. Thyroid 15: 672-682). Although this mouse monoclonal antibody 9D33 exhibits at least some of the characteristics of patient serum TRAb with blocking activity, it is a mouse antibody made by immunizing experimental animals with TSHR, and autoimmunity to TSHR in humans. It cannot truly represent TSHR autoantibodies produced in the reaction process. As a mouse monoclonal antibody, 9D33 needs to be humanized for in vivo applications in humans. This can be disadvantageous given the cost and complexity involved in the humanization process.

  The present invention derives from the production and characterization of human monoclonal autoantibodies (5C9) against TSHR, which are effective antagonists of TSH and stimulating TRAbs in patient sera. 5C9 was isolated from peripheral lymphocytes of patients with hypothyroidism and high concentrations of TSHR autoantibodies. The lymphocytes were immortalized by Epstein-Barr virus (EBV) infection, and positive clones were fused with a mouse / human cell line to generate stable clones. IgG was purified from the clonal culture supernatant, and the ability of 5C9 IgG to bind to TSHR and the effect on TSHR activity were evaluated. In particular, the ability of 5C9 to inhibit TSH binding to TSHR and to inhibit cyclic AMP-stimulating activity of TSH was examined. In addition, the ability of 5C9 to inhibit the binding of stimulating or blocking patient serum TRAbs to TSHR and their biological activity was also evaluated. In addition, the use of 5C9 in assays for TSHR antibodies, TSH, and related compounds was also investigated. The 5C9 heavy chain (HC) and light chain (LC) variable region (V region) gene sequences were sequenced to identify the complementarity determining region (CDR).

  According to a first aspect of the present invention there is provided an isolated human antibody against TSHR that is an antagonist of TSH.

  According to a second aspect of the invention, there is provided an isolated humanized antibody against TSHR that is an antagonist of TSH.

  An antibody according to either the first or second aspect of the invention is an “antibody according to the invention”.

  The antibody according to the invention may be an antagonist of a thyroid stimulating antibody.

  The antibodies according to the invention may have the TSH antagonist properties of patient serum TSHR autoantibodies that are TSH antagonists.

  The antibodies according to the invention may be antagonists of TSH and thyroid stimulating antibodies.

  The antibodies according to the invention may have the antagonist properties of patient serum TSHR autoantibodies which are antagonists of thyroid stimulating antibodies.

  An antibody according to the present invention may be an inhibitor of binding to TSHR or a part thereof by an antibody having stimulatory activity on TSH, M22, or TSHR or an antibody having blocking activity. A portion of TSHR may include LRD or a substantial portion thereof. Preferably, the antibody blocks such binding.

The antibody according to the invention may be a monoclonal or recombinant antibody or may comprise or consist of a fragment thereof that is an antagonist of TSH. The antibodies according to the invention, V _H containing one or more amino acid sequences having substantial homology to one or more of the CDR or their CDR, is selected from the CDR1, CDR2 or CDR3, it is shown in Figure 2 An area may be included. In addition or alternatively, the antibody according to the present invention may be one or more CDRs selected from the CDR1, CDR2 or CDR3 shown in FIG. 3, or one or more amino acid sequences having substantial homology to these CDRs. V _L region including

The antibody according to the invention may have a binding affinity of about 10 ¹⁰ L / mol for human full-length TSHR. The antibody according to the invention preferably has a binding affinity of about 10 ⁹ L / mol for human full-length TSHR.

  The present invention helps those skilled in the art to understand the immune mechanisms that drive the generation and production of stimulatory and blocking TSHR autoantibodies. Furthermore, the present invention helps those skilled in the art to understand the molecular differences between TSHR autoantibodies having thyroid stimulating activity and those having blocking activity. Furthermore, the medical treatment and pharmacological compositions of the present invention provide a new treatment for thyroid related diseases.

  A preferred antibody according to the invention is 5C9. 5C9 was unexpectedly found to inhibit the constitutive activity of the thyroid stimulating hormone receptor, ie the production of cyclic AMP in the test system in the absence of thyroid stimulating hormone or M22. This is particularly advantageous for the treatment of thyroid cancer cells that have remained or metastasized in the thyroid gland, especially for preventing or delaying regrowth where these cells can grow more rapidly as a result of constitutive activity of the thyroid stimulating hormone receptor. It is.

As used herein, the terms “antibody” and similar terms such as “antibodies” are used according to circumstances, such as monoclonal and polyclonal antibodies, single chain antibodies, multispecific antibodies, etc. Binding moieties that include immunoglobulin-based binding moieties and that may be replaced by those skilled in the art to such immunoglobulin-based binding moieties, such as domain antibodies, bispecific antibodies, IgGΔC _H 2, F (ab ′) ₂ , Fab, scFv, V _L , V _H , dsFv, Minibody, Triabody, Tetrabody, (scFv) ₂ , scFv-Fc, F (ab ′ ₃ ) (Holliger P, Prospero T, Winter G 1993 “Diabodies: small bivalent and bispecific antibody fragments” Proc Natl Acad Sci USA 90: 6444-6448.) (Carter PJ 2006 “Potent antibody the rapeutics by design "Nat Rev Immunol 6: 343-357).

  The term “TSHR” applies to full-length human thyroid stimulating hormone receptor having the amino acid sequence shown in FIG. 4, or a variant or fragment thereof having high homology to the thyroid stimulating hormone receptor. . Preferably such variants and fragments have 70 to 99.9% homology with the amino acid sequence shown in FIG.

According to another aspect of the invention,
a) a nucleotide sequence encoding an antibody according to the first aspect of the invention;
b) the nucleotide sequence shown in FIG. 2 or 3 encoding the amino acid sequence of the antibody V _H domain, antibody _VL domain, or CDR shown in FIG. 2 or 3; or c) the nucleotide sequence of a) or b) Nucleotides comprising a nucleotide sequence encoding an antibody having high homology and binding to TSHR with an affinity of at least about 10 ⁹ L / mol are provided.

  According to another aspect of the present invention, there is provided a vector comprising a nucleotide according to the above aspect of the present invention.

  The vector may be a plasmid, virus or fragment thereof. Many different types of vectors are known to those skilled in the art.

  According to another aspect of the invention there is provided an isolated cell comprising an antibody according to the invention; a nucleotide or / vector. The isolated cell may express an antibody according to the invention. Preferably, the isolated cell secretes the antibody according to the invention. Preferably, the isolated cell according to the present invention is derived from a stable hetero-hybridoma cell line.

  According to a further aspect of the invention there is provided a composition comprising a defined concentration of TSHR autoantibodies and comprising an antibody according to the invention. Such a composition may comprise a defined concentration of TSHR autoantibodies with TSH antagonist activity and comprises an antibody according to the invention.

  Alternatively, a composition according to this aspect of the invention may comprise a defined concentration of a TSHR autoantibody that is an antagonist of a thyroid stimulating antibody and comprises an antibody according to the invention. The composition may comprise a defined concentration of a TSHR autoantibody with TSH antagonist activity that is an antagonist of a thyroid stimulating antibody and comprises an antibody according to the invention.

  According to another aspect of the present invention there is provided a pharmaceutical composition comprising an antibody according to the present invention together with a pharmacologically acceptable carrier for administration to a mammalian subject for the treatment of thyroid related diseases. The thyroid-related disease may be selected from hyperthyroidism, Graves eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, anterior cervical myxedema, thyroid cancer, and thyroiditis.

  The pharmaceutical composition according to the invention may be suitable for human administration. Preferably, the pharmaceutical composition according to the invention has no significant adverse effect on the immune system of the subject.

  The pharmaceutical composition according to the invention may comprise a further thyroid stimulating hormone receptor antagonist. A suitable additional thyroid stimulating hormone receptor antagonist is 9D33 disclosed in WO 2004/050708.

  Various formats are contemplated for the pharmaceutical composition according to the invention. The pharmaceutical composition according to the invention for use in the treatment of thyroid related diseases may be in an injectable form. The pharmaceutical composition according to the invention for use in the treatment of anterior tibial myxedema is preferably in a topical form. The pharmaceutical composition according to the invention for use in the treatment of Graves eye disease is preferably in the form of eye drops.

  The pharmaceutical composition of the invention comprises any antibody according to the invention of the invention together with any pharmacologically acceptable carrier, adjuvant, or vehicle. Pharmacologically acceptable carriers, adjuvants, and vehicles that may be used in the pharmaceutical compositions of the present invention include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, phosphate Buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, Zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol And wool fat but not limited to.

  The pharmaceutical compositions of the invention may be administered orally, parenterally by inhalation spray, topically by eye drops, rectally, nasally, buccally, vaginally or via an implantable reservoir. Preferably, it is oral administration or administration by injection. The term “parenteral” as used herein refers to subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intracapsular, intralesional, and intracranial injection or infusion methods. including.

  The pharmaceutical composition may be in the form of a sterile injectable preparation, such as an aqueous or oily sterile injectable suspension. This suspension may be formulated according to methods known in the art using suitable dispersing or wetting agents (eg, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally administrable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are mannitol, water, Ringer's solution and physiological saline. Furthermore, conventionally, sterile fixed oils have been used as solvents or suspending agents. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid and glyceride derivatives thereof are useful in the preparation of injectable solutions as natural pharmacologically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated form. is there. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.

  The pharmaceutical compositions of the present invention may be administered orally by any orally usable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. A lubricant such as magnesium stearate is also typically added. Useful diluents for oral administration in capsule form include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents may be added.

The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of the present invention with a suitable non-irritating excipient. Because this excipient is solid at room temperature but liquid at rectal temperature, it can be dissolved in the rectum to release the active ingredient. Such materials include, but are not limited to cocoa butter, beeswax and polyethylene glycols.

  Topical administration of the pharmaceutical composition of the invention is particularly useful when the desired treatment involves a site or organ that is readily available by local administration. For topical administration to the skin, the pharmaceutical composition must be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, mineral oil, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active ingredient suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical composition of the invention may also be administered topically to the lower intestinal tract by rectal suppository formulation or a suitable enema formulation. Topical transdermal patches are also included in the present invention.

  The pharmaceutical composition of the invention may be administered by nasal aerosol or inhalation. Such compositions are prepared by methods known in the art of pharmaceutical formulation and include benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or It may be prepared as a physiological saline solution using other solubilizers or dispersants known in the art.

  According to a further aspect of the present invention there is provided a method for producing an antibody according to the present invention comprising culturing one or more isolated cells according to the present invention such that the cells express the antibody. Preferably, the antibody is secreted by the cell.

  According to a further aspect of the present invention there is provided a method of treating a thyroid related disease in a mammalian subject or cells derived from said subject, said method comprising contacting said subject or said cell with an antibody according to the present invention. including.

  According to another aspect of the present invention, there is provided a method of inhibiting thyroid stimulating autoantibodies that stimulate TSHR in the thyroid gland of a mammalian subject, said method comprising contacting said subject with an antibody according to the present invention. . Preferably, binding of thyroid stimulating autoantibodies to TSHR is blocked.

  In accordance with another aspect of the present invention, there is provided a method of inhibiting thyroid stimulating autoantibodies that bind to extrathyroid gland TSHR of a mammalian subject, said method comprising contacting said subject with an antibody according to the present invention. The extrathyroid gland TSHR may be present in posterior orbital tissue and / or anterior tibial tissue of the subject. The antibodies according to the invention preferably block TSHR autoantibodies that bind to extrathyroid gland TSHR when used in the method.

  According to another aspect of the present invention, there is provided a method of treating thyroid cancer that is within or has metastasized within a thyroid gland, in a thyroid cell within or within a subject, said method comprising intracellular homeostasis. In order to inhibit target thyroid stimulating hormone receptor activity, the method comprises contacting cancerous cells with an antibody according to the present invention. Preferably, regrowth of thyroid cancer cells is prevented or delayed.

Also provided is a method of treating hyperthyroidism due to constitutive thyroid activity in a subject or in a thyroid cell derived from the subject, the method for inhibiting hyperthyroidism due to constitutive thyroid activity. Contacting a subject or thyroid cell with an antibody according to the invention.

  The subject to be treated in the various methods of the present invention is preferably a human.

  According to another aspect of the present invention there is provided the use of an antibody according to the present invention in the treatment of thyroid related diseases. Alternatively, there is provided the use of an antibody according to the invention in the preparation of a medicament for the treatment of thyroid related diseases.

  There is also provided an antibody according to the invention for use in medical therapy. In particular, there is provided the use of an antibody according to the invention for use in the treatment of thyroid related diseases. The thyroid-related disease may be selected from hyperthyroidism, Graves eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, anterior cervical myxedema, thyroid cancer, and thyroiditis.

  According to another aspect of the invention, there is provided a method for characterizing a TSHR antibody comprising determining binding of a tested TSHR antibody to a polypeptide having a TSHR-related amino acid sequence, wherein the method Relates to process steps involving the use of antibodies according to the invention. Preferably, the method comprises determining the effect of the antibody according to the invention in binding of the TSHR antibody to the polypeptide. Said polypeptide having a TSHR-related amino acid sequence preferably comprises full-length human TSHR.

  According to another aspect of the present invention, there is provided a method for characterizing TSH and related molecules comprising determining the binding of the TSH or related molecule being tested to a polypeptide having a TSHR-related amino acid sequence. Where the method involves a method step comprising the use of an antibody according to the invention.

  The above-described TSHR antibodies, or methods for characterizing TSH and related methods may be by ELISA format.

  According to another aspect of the present invention, there is provided a method for determining a TSHR amino acid involved in binding of a TSHR autoantibody acting as an antagonist, said method comprising the first TSHR-related amino acid sequence to which an antibody according to the present invention binds. Providing a polypeptide having, modifying at least one amino acid in the TSHR-related amino acid sequence, and determining the effect of such modification on antibody binding.

  A method for modifying an antibody according to the invention comprises modifying at least one amino acid of the antibody and determining the effect of such modification in binding to a TSHR-related sequence. Modified TSHR antibodies are preferably selected that have enhanced affinity for TSHR.

  According to another aspect of the invention, there is provided a method for identifying a molecule that inhibits a thyroid stimulating antibody that binds to TSHR, the method providing at least one antibody according to the invention as a reference. including. The molecule to be tested is preferably selected to block thyroid stimulating antibodies that bind to TSHR.

  Also provided are methods for identifying molecules that inhibit thyroid blocking antibodies that bind to TSHR, the methods comprising providing at least one antibody according to the invention as a reference. Preferably, molecules are selected that block thyroid blocking antibodies that bind to TSHR.

  The antibodies and methods according to the invention will now be described by way of example only and with reference to the accompanying FIGS.

Effect of serum from Graves' disease patients (n = 40) and healthy blood donors (n = 10) on binding of ¹²⁵ I-5C9 IgG, ¹²⁵ I-TSH, or ¹²⁵ I-M22 to TSHR-coated tubes A series of three graphs illustrating the comparison. a. ¹²⁵ I-5C9 IgG versus ¹²⁵ I-TSH binding Effect of serum from Graves' disease patients (n = 40) and healthy blood donors (n = 10) on binding of ¹²⁵ I-5C9 IgG, ¹²⁵ I-TSH, or ¹²⁵ I-M22 to TSHR-coated tubes A series of three graphs illustrating the comparison. b. ¹²⁵ I-5C9 IgG vs. ¹²⁵ I-M22 IgG binding Effect of serum from Graves' disease patients (n = 40) and healthy blood donors (n = 10) on binding of ¹²⁵ I-5C9 IgG, ¹²⁵ I-TSH, or ¹²⁵ I-M22 to TSHR-coated tubes A series of three graphs illustrating the comparison. c. ¹²⁵ I-TSH vs. ¹²⁵ I-M22 IgG binding The sequence of the variable region sequence of 5C9 heavy chain (HC) is shown. a. The oligonucleotide sequence of 5C9HC is shown in an unannotated and annotated format. In the annotated format, sequences used for PCR primers are underlined, individual complementarity determining regions (CDRs) are boxed, and constant regions are shown in bold. The sequence of the variable region sequence of 5C9 heavy chain (HC) is shown. b. The amino acid sequence of 5C9HC from the oligonucleotide sequence is shown in an unannotated and annotated format. The sequence of the variable region sequence of 5C9 light chain (LC) is shown. a. The oligonucleotide sequence of 5C9LC is shown in an unannotated and annotated format (as in FIG. 2). The sequence of the variable region sequence of 5C9 light chain (LC) is shown. b. The amino acid sequence of 5C9LC from the oligonucleotide sequence is shown in an unannotated and annotated format. The consensus amino acid sequence of human TSHR (Accession number P16473, http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62298994) is illustrated.

Method Lymphocyte Separation and Cloning of Human Monoclonal TSHR Autoantibody 5C9 Monoclonal autoantibody 5C9 was usually isolated using the method described in WO 2004 / 050708A2. Lymphocytes were first isolated from blood samples collected from patients with postpartum hypothyroidism and high levels of TRAb (with approval from the local ethics committee). The lymphocytes are infected with Epstein-Barr virus (EBV) (European Collection of Cell Cultures-ECACC; Porton Down, SP4 OJG5, UK) and, as described in WO 2004/050708 A2, mouse macrophage feeder Cultured on the layer. Immortal lymphocytes secreting TSHR autoantibodies were fused with K6H6 / B5 (ECACC), a mouse / human hybrid cell line, and cloned four times by limiting dilution to obtain a single colony. The presence of TSHR autoantibodies in the cell culture supernatant at various stages of cloning was detected by inhibition of binding of ¹²⁵ I-labeled TSH to TSHR (WO 2004/050708 A2). Single clones producing TSHR autoantibodies were grown and culture supernatants were collected for autoantibody purification.

Purification and labeling of 5C9 IgG preparations 5C9 IgG was obtained from Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Kiddie A, Brereton K, Premawardhana LD, Chirgadze DY, Nunez Miguel R, Blundell TL, Furmaniak J, Rees Smith B. 2004 Characteristics of a human monoclonal autoantibody
Purified from the culture supernatant using protein A affinity chromatography with MabSelect ™ (GE Healthcare, UK) as described in Thyroid 2004 14: 560-570 Was evaluated by SDS polyacrylamide gel electrophoresis (PAGE).

  The heavy chain isotype of 5C9 was determined using a radiation diffusion assay (The Binding Site; Birmingham, B296AT, UK), and the light chain isotype was determined by anti-human kappa chain and anti-human lambda chain specific mouse monoclonal antibodies (Sigma- Determined by Western blotting using Aldrich Company Ltd (Pool, UK).

10 mg / mL 5C9 IgG in 20 mmol / L sodium acetate (pH 4.5) was shaken for 4 and a half hours at room temperature with immobilized pepsin prepared according to the manufacturer's instructions (Perbio Science UK Ltd, Cramlington, UK). Incubated with aging. The immobilized pepsin was then removed by centrifugation (1000 × g, 5 minutes at room temperature) and the supernatant dialyzed overnight at 4 ° C. against 300 mmol / L NaCl, 10 mmol / L Tris-HCl, pH 7.5. . The dialyzed mixture containing 5C9F (ab ′) ₂ and a small amount of intact IgG was separated using a Sephacryl S-300 High Resolution Matrix (GE Healthcare, Chalfont St. Giles, UK). The 5C9F (ab ′) ₂ formulation purified by this method did not contain intact IgG as judged by SDS-PAGE and HPLC gel filtration analysis.

Further, F (ab ′) ₂ was reduced at 37 ° C. for 1 hour using L-cysteine having a final concentration of 100 mmol / L. The reaction was stopped at room temperature for 30 minutes with iodoacetamide at a final concentration of 50 mmol / L. F (ab ′) was purified using a Sephacryl S-300 column as described above. The F (ab ′) formulation purified by this method did not contain F (ab ′) ₂ as judged by SDS-PAGE and HPLC gel filtration analysis.

  In addition, 5C9 IgG was treated with Mercury Papain (Sigma, UK) at an enzyme / protein ratio of 1: 100, dialyzed into 50 mmol / L NaCl, Tris-HCl, pH 9.0, and intact IgG from the Fab formulation. Alternatively, an anion exchange Sepharose (Q-Sepharose Fast flow obtained from GE Healthcare) column was passed through to separate Fc. Analysis by SDS-PAGE and gel filtration (Sephacryl S-300 column; above) showed that no intact IgG was detected in the Fab formulation.

5C9 IgG is Sanders J, Oda Y, Roberts S, Kiddie A, Richards T, Bolton J, McGrath V, Walters S, Jaskolski D, Furmaniak J, Rees Smith B 1999. The interaction of TSH receptor autoantibodies with ¹²⁵ I-labelled TSH Journal of Clinical Endocrinology and Metabolism. 1999 84: 3797-3802. Labeled with ¹²⁵ I or labeled with biotin hydrazide (Perbio Science, Cramlington, UK).

Inhibition of ¹²⁵ I-TSH or ¹²⁵ I-M22 or ¹²⁵ I-5C9 binding to TSHR Binding inhibition assays were performed using TSHR-coated tubes according to the description of WO 2004 / 050708A2. In this assay, 100 μL of test sample (MAb formulation, patient serum or unlabeled TSH) and 50 μL of starting buffer (RSR Ltd) are incubated for 2 hours at room temperature with gentle shaking in a tube coated with TSHR. did. After aspiration, the tube was washed, 100 μL of ¹²⁵ I-labeled protein (5 × 10 ⁴ cpm) was added, and incubated at room temperature for 1 hour with shaking. Tubes were then aspirated and washed and counted in a gamma counter.

コントロール物質は種々の実験結果に示すように、健常血液ドナー血清のプール、または個々の健常血液ドナー血清、またはその他の物質であった。 Inhibition of the binding of the labeled protein was calculated as follows.

The control substance was a pool of healthy blood donor sera, or individual healthy blood donor sera, or other substances, as shown in various experimental results.

Scatchard analysis of 5C9 IgG binding to TSHR Unlabeled 5C9 IgG and 50 μL ¹²⁵ I in 50 μL assay buffer (50 mmol / L NaCl, 10 mmol / L Tris, pH 7.8, and 1% Triton X-100) Labeled 5C9 IgG (30,000 cpm in assay buffer) is incubated for 2 hours at room temperature with shaking in TSHR coated tubes (maximum binding occurred under these conditions), aspirated and 1 mL Was washed twice with the assay buffer and counted in a gamma counter. To determine the binding constant, the binding concentration / free concentration was plotted against the concentration of bound IgG (Scatchard G 1949 The attraction of proteins for small molecules and ions. Annals of the New York Academy of Sciences 51: 660-672). .

Analysis of cyclic AMP production stimulation The ability of 5C9 IgG and other preparations to stimulate cyclic AMP production in Chinese hamster ovary (CHO) cells into which human TSHR was introduced was tested according to the description in WO 2004 / 050708A2. After adaptation of CHO cells expressing about 5 × 10 ⁴ or about 5 × 10 ⁵ TSHR per cell in 96-well plates to 3 × 10 ⁴ cells per well and DMEM without fetal calf serum (Invitrogen Ltd, Paisley, UK) Add test sample (TSH, IgG or patient serum) (1 g / L glucose, 20 mmol / L HEPES, 222 mmol / L sucrose, 15 g / L bovine serum albumin, and 0.5 mmol / L 3 isobutyl) 1-methylxanthine pH 7.4, diluted in cyclic AMP assay buffer, a Hanks buffered salt solution without NaCl, 100 μL) and incubated at 37 ° C. for 1 hour. After removal of the test solution, the cells were lysed and the cyclic AMP concentration in the lysate was assayed by one of the following two methods: 1) obtained from GE Healthcare (Chalfont St. Giles, UK) Use of Biotrak enzyme immunoassay system or 2) Use of Direct Cyclic AMP Correlate-EIA kits obtained from Assay Designs; Cambridge Bioscience (UK). Results were expressed as cyclic AMP in cell lysate (200 μL) in pmol / mL or in fmol per cell well.

Measurement of Antagonist (Blocking) Activity In CHO cells expressing TSHR, 5C9 IgG and other formulations were tested for porcine (p) TSH, native human (h) TSH, and recombinant human (rh) TSH, MAbM22, and patient serum TRAb. The ability to inhibit stimulatory activity was evaluated. This will stimulate the stimulatory effect of TSH, M22, or TRAb 5C9 IgG (or other tested formulations)
By comparing in the absence and presence of. The assay consists of adding 50 μL of 5C9 (or other tested formulation) diluted in cyclic AMP assay buffer to cell wells, followed by 50 μL of TSH or M22 or patient serum (in cyclic AMP assay buffer). Was performed as described above, except that the appropriate dilution) was added, and incubated and tested in the same manner as the stimulation assay described above.

In addition to 5C9, sera from patients with other MAbs and blocking TRAbs were tested in this assay.

Variable region gene analysis 5C9 heavy chain and light chain variable region genes are 1x10 secreting 5C9 IgG to produce mRNA for RT-PCR (reverse transcriptase PCR) reaction according to the description of WO 2004 / 050708A2. Determined using total RNA prepared from ⁷ heterohybridoma cells. Sense and antisense strand oligonucleotide primers specific for IgG1HC and kappa LC were designed using the Medical Research Council V-base (http://vbase.mrc-cpe.cam.ac.uk/) and Invitrogen (Paisley, PA4 9RF, UK). The RT reaction was performed at 50 ° C. for 15 minutes, followed by 40 cycles of PCR at 94 ° C. for 15 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 30 seconds. The DNA product was cloned into pUC18 and sequenced by the Sanger-Coulson method (Sanger
F, Nicklen S, Coulson AR 1977 DNA sequencing with chain terminating inhibitors.
Proceedings of the National Academy of Sciences of USA 74: 5463-5467). The sequence of the V region was compared with the sequence of an available human Ig gene using Ig blast (http://www.ncbi.nlm.nih.gov/igblast/).

Analysis of the effect of amino acid mutations in the human TSHR sequence on 5C9 activity The method used to introduce specific mutations in the TSHR sequence is described in patent application WO 2006 / 016121A. In addition, transfection of mutant TSHR constructs into CHO cells using the Flp-In system is also described in WO 2006 / 016121A.

  Flp-In-CHO cells expressing either wild type or mutant TSHR are seeded in 96-well plates as described above to test the ability of 5C9 formulations to block the stimulatory activity of TSH, M22, or patient serum TRAbs. used.

5C9-based assay for TSHR antibody, TSH and related molecules 2.55 mL of 5C9 IgG is dialyzed into 100 mmol / L sodium phosphate buffer pH 8.5 and the EZ molecular ratio of 1/10 to EZ -Link NHS-LC-Biotin (Perbio) was reacted. Test serum samples (75 μL) were incubated at room temperature for 2 hours with shaking (500 shakes per minute) in TSHR coated ELISA plate wells (RSR Ltd). After removing and washing the test sample, biotin-labeled 5C9 IgG (2 ng in 100 μL) was added and incubation continued at room temperature for 25 minutes without shaking. Wells were emptied, washed and 100 μL streptavidin-peroxidase (10 ng in 100 μL; RSR Ltd) was added and incubated at room temperature for 20 minutes without shaking. The wells were then washed three times, tetramethylbenzidine (TMB; 100 μL; RSR Ltd), a substrate for peroxidase, was added and incubated in the dark at room temperature for 30 minutes without shaking. Thereafter, 50 μL of 0.5 mol / L H 2 SO 4 was added to stop the reaction, and the absorbance of each plate well was read at 450 nm using an ELISA plate reader. Inhibition of 5C9IgG-biotin binding was calculated as follows.

Results Isolation and Cloning of 5C9 Secreting Cell Line Lymphocytes (27 × 10 ⁶ ) from 20 mL of patient blood were infected with EBV and seeded with ¹ × 10 ⁶ cells per well on a mouse macrophage feeder layer in a 48-well plate. It was. On day 13 after EBV infection, the plate well supernatant was monitored for inhibition of ¹²⁵ I-TSH binding. Cells from positive wells were expanded and fused with the K6H6 / B5 hybridoma cell line and seeded in 96 well plates. One clone that stably produced an antibody having inhibitory activity of ¹²⁵ I-TSH binding was obtained and recloned four times. The monoclonal antibody purified from hetero-hybridoma culture supernatant and named 5C9 is a subclass of IgG1 with a kappa light chain.

Binding and blocking activity of 5C9 IgG to TSHR Table 1 shows the ability of 5C9 IgG at various concentrations to inhibit the binding of labeled TSH or labeled M22 or labeled 5C9 itself to TSHR. As shown in Table 1, 12% inhibition of ¹²⁵ I-TSH binding was observed with a minimal amount of 0.005 μg / mL of 5C9 IgG, which was dose dependent up to 84% inhibition with 100 μg / mL of 5C9. Increased. This can be compared to the inhibition of ¹²⁵ I-TSH binding by donor serum IgG, ie a dose-dependent increase from 13% inhibition at 0.05 mg / mL to 94% inhibition at 1 mg / mL. In the case of donor plasma, 16% inhibition of ¹²⁵ I-TSH binding was observed at 1: 160 dilution of healthy blood donor pool serum, and 95% inhibition was observed at 1:10 dilution.

5C9 IgG also had an effect on the binding of ¹²⁵ I-M22 IgG to TSHR-coated tubes (Table 1). With 0.01 μg / mL 5C9 IgG, 9% inhibition of ¹²⁵ I-M22 IgG binding was observed, and increasing concentrations resulted in a dose-dependent increase in inhibition up to 85% at 100 μg / mL It was done. Donor serum IgG was effective at 0.01 mg / mL causing 9% inhibition, and this effect increased in a dose-dependent manner to 89% inhibition at 1 mg / mL. Donor serum plasma showed 13% inhibition of ¹²⁵ I-M22 IgG binding at a dilution of 1: 320 and 91% inhibition at a dilution of 1:10.

Unlabeled 5C9 IgG dose-dependently inhibits ¹²⁵ I-5C9 binding to TSHR-coated tubes (from 11% inhibition at 0.005 μg / mL to 88% inhibition at 100 μg / mL) (Table 1). -2) ¹²⁵ I-5C9 binding was also inhibited by donor serum IgG (15% inhibition at 0.05 mg / mL and 91% inhibition at 1 mg / mL), as well as by dilution of donor plasma (1 : 10% inhibition at 320 dilution and 92% inhibition at 1:10 dilution).

Tables 2a-c show the blocking ability of 5C9 IgG upon stimulation of cyclic AMP via TSH and M22 in CHO cells expressing TSHR. Porcine TSH (3 ng / mL) strongly stimulated cyclic AMP production (19,020 ± 2154 fmol / cell well; mean ± SD; n = 3) (Table 2a). In the presence of 0.1 μg / mL 5C9 IgG, the stimulatory activity of porcine TSH is reduced to 11874 ± 4214 fmol / cell well (mean ± SD; n = 3) and only 2 in the presence of 1 μg / mL 5C9. 208 ± 32
Since 9 fmol / cell of cyclic AMP was produced, this inhibitory effect was dependent on 5C9 concentration (Table 2a). Lymphocyte donor sera also had a potent inhibitory effect on TSH-mediated cyclic AMP stimulation in CHO-TSHR cells. As shown in Table 2a, cyclic AMP production is present in the presence of 1:10 dilution of donor serum (total serum IgG concentration at this dilution is 1.43 mg / mL) compared to 19000 fmol / cell well in the absence of serum. Below, there was an inhibition that decreased to about 6000 fmol / cell well. This effect was consistent with the effect of approximately 0.37 μg / mL of purified 5C9 IgG (calculated from the dilution curves of the effects of various concentrations of 5C9 IgG shown in Table 2a). This indicates that purified 5C9 IgG is about 3900 times more active than donor serum IgG in terms of its ability to block the ability of TSH to stimulate cyclic AMP production.

Fragments of 5C9, such as 5C9F (ab ′) ₂ and 5C9 Fab, were also effective inhibitors of TSH stimulation. In particular, the TSH stimulation inhibitory activities of 5C9IgG, 5C9F (ab ′) ₂ and 5C9Fab at 100 μg / mL were equivalent (Table 2b). All three formulations at 10 μg / mL, namely 5C9IgG, 5C9F (ab ′) ₂ , and 5C9Fab, were also potent inhibitors of TSH stimulating activity, while 5C9IgG was more potent than 5C9F (ab ′) ₂ or 5C9Fab It was considered more effective (Table 2b).

  M22 Fab (3 ng / mL) is a potent stimulator of cyclic AMP (9,432 ± 822 fmol / cell well) (Table 2c). The stimulatory effect of M22 Fab in the presence of 5C9 was inhibited in a dose-dependent manner, and the cyclic AMP concentration in the presence of 0.1 μg / mL 5C9 IgG was reduced to 1,298 ± 134 fmol / cell well. Complete inhibition of M22 stimulation was caused by 100 μg / mL 5C9 (Table 2c).

Scatchard analysis showed that the binding constant of ¹²⁵ I-labeled 5C9 binding to TSHR was 4 × ^{10 10} L / mol.

Inhibition of ¹²⁵ I-5C9 IgG binding to TSHR by serum TRAb The ability of serum TRAb to inhibit ¹²⁵ I-5C9 IgG binding to tubes coated with TSHR is shown in Table 3 and FIG. The effect of the same serum TRAb on ¹²⁵ I-TSH binding and ¹²⁵ I-M22 IgG binding was also shown for comparison.

¹²⁵ I-5C9 IgG binding to TSHR was not clearly inhibited by sera from 10 different healthy blood donors (N1-N10, Table 3) (range of inhibition was 3.4-18.9%). ). ^{In 125} I-TSH and ¹²⁵ I-M22 inhibition assays (Table 3), sera from 40 Graves disease patients (G1-G40, Table 3) all positive for TSHR autoantibodies were TSHR-coated tubes. Inhibition of ¹²⁵ I-5C9 to a greater degree of inhibition than serum from healthy blood donors (inhibition range 22.0-85.2%) (Table 3). The ability of patient serum TRAb to inhibit ¹²⁵ I-5C9 IgG, ¹²⁵ I-TSH, or ¹²⁵ I-M22 IgG binding to TSHR was determined by the Pearson correlation coefficient r = 0.95 ( ¹²⁵ I-5C9 IgG vs ¹²⁵ I-TSH; FIG. 1a ) And r = 0.95 ( ¹²⁵ I-5C9 IgG vs ¹²⁵ I-M22 IgG; FIG. 1 b). FIG. 1c shows a comparison of inhibition of ¹²⁵ I-TSH and ¹²⁵ I-M22 IgG by the same serum (Pearson correlation coefficient r = 0.99).

  These experiments indicate that 5C9 IgG binding to TSHR is effectively inhibited by serum TRAb, and that the inhibitory effect of serum TRAb on 5C9 IgG binding is similar to their inhibitory effect on TSH or M22 binding. ing.

Table 4 shows inhibition of ¹²⁵ I-5C9 IgG binding by various dilutions of patient serum TRAb with TSH blocking activity (B1-B5) and patient serum TRAb with strong thyroid stimulating activity (S1, S2, S4). . ¹²⁵ I-5C9 IgG binding was inhibited by serum B1-B5 as well as by serum S1, S2, and S4 in a dose-dependent manner. The blocking and stimulating sera also inhibited ¹²⁵ I-TSH and ¹²⁵ I-M22 IgG binding in a dose-dependent manner, and the percentage of inhibition for all three labeled ligands was comparable at the same dilution of serum. (Tables 4a and b).

  These results indicate that TSHR autoantibodies with both stimulatory and blocking activities inhibit 5C9 binding to TSHR.

Inhibition of ¹²⁵ I-5C9 IgG binding to TSHR by mouse MAbs having TSH binding inhibitory activity
Of various mouse TSHRMAb with ¹²⁵ I-TSH binding inhibition activity, and test the inhibitory capacity of ¹²⁵ I-5C9 binding to TSHR, it was compared to the effects of ¹²⁵ I-M22IgG binding (Table 5). As shown in Table 5, although all MAb with ability to inhibit ¹²⁵ I-TSH and ¹²⁵ I-M22IgG binding was also inhibited ¹²⁵ I-5C9 binding, if some of the MAb, ¹²⁵ I-5C9 and ¹²⁵ The inhibitory effect on I-M22 binding was weaker than that on ¹²⁵ I-TSH binding.

  These experiments suggest that there is considerable overlap between the binding site for 5C9 on TSHR and the binding site for murine TSHRMAb that has the ability to inhibit TSH binding.

Effect of 5C9 IgG on Stimulation of Cyclic AMP Production by Patient Serum in CHO Cells Expressing TSHR As shown in Table 2, 5C9 IgG blocks stimulation of TSH or M22 on cyclic AMP concentration in CHO cells expressing TSHR. We were able to. In a series of different experiments, the effect of 5C9 IgG on the stimulating activity of patient serum TRAb was tested. The results are shown in Table 6a. Serum T1-T9 and T11-T18 stimulate cyclic AMP production in CHO-TSHR cells, and incubation with control MAb IgG (2G4 specific for human thyroid peroxidase) has no effect on their stimulatory activity It was. However, in the presence of 5C9 IgG (50 μL of 200 μg / mL), the stimulatory activity of all sera tested was clearly reduced (Table 6a).

  The dose response effects of six different sera (T1, T6, T3, T19, T20, T21) are shown in Tables 6b-g. In these experiments, concentrations of 5C9 IgG ranging from 0.1 μg / mL to 100 μg / mL cause a dose-dependent decrease in serum stimulating activity, and the effect of 5C9 IgG is blocked in all tested sera except serum T3. The effect was comparable to that of the mouse monoclonal antibody 9D33 (described in WO 2004 / 050708A2) against TSHR having activity. In the case of T3 serum (Tables 6a and 6d), approximately 50% inhibition of cyclic AMP production was observed in the presence of 100 μg / mL 5C9 IgG, while 100 μg / mL 9D33 IgG resulted in almost complete inhibition. This suggested that there may be some minor differences between the epitopes recognized by 5C9 and 9D33.

Effect of 5C9 IgG on basic (ie, unstimulated) cyclic AMP production in CHO cells expressing TSHR Similar to the inhibition of the stimulatory activity of TSH and TSHR antibodies, 5C9 is in the absence of these thyroid stimulators. The amount of cyclic AMP produced was inhibited. In particular, Table 6b shows 1207 ± produced in the presence of 100 μg / mL control monoclonal IgG (2G4).
It shows that the cyclic AMP in 123 fmol / cell well was reduced to 301 ± 38 fmol / cell well in the presence of 100 μg / mL 5C9 IgG. The effect of 9D33 IgG was less than the production of 721 ± 183 fmol / cell well in the presence of 100 μg / mL 9D33 IgG. Similar results were obtained in separate experiments shown in Tables 6d, 6e, 6f and 6g. This indicates that 5C9 IgG has a significant effect on the basic or constitutive activity of TSHR.

Effect of 5C9 IgG in Stimulating Production of Cyclic AMP Intracellular CHO Expressing TSHR Containing Amino Acid Mutation TSHR Single Amino Acid Mutation in the Blocking Ability of 5C9 to Cyclic AMP Stimulating Activity of Porcine TSH in CHO-TSHR Cells The effects are shown in Table 7. In particular, cyclic AMP in CHO cells expressing TSHR in which the following residues: Lys58, Ile60, Arg80, Tyr82, Thr104, Arg109, Lys129, Phe134, Asp151, Lys183, Gln235, Arg255, Trp258, Ser281 were mutated to alanine The effect of 5C9 on production stimulation was examined. Furthermore, in the case of TSHR residues: Arg80Asp, Asp151Arg, Lys183Asp, Arg255Asp, the effect of change of charge mutation was examined. Here, according to conventional notation, the substituted amino acid residue and its position in the primary sequence polypeptide are shown before the substituted amino acid residue. Previous studies have shown that Asp160Lys, a change of charge mutation in TSHR, caused loss of reactivity of TSHR to TSH, but the response to M22 was not affected. (Patent Application International Publication No. WO2006 / 016121A). Therefore, the effect of Asp160Lys mutation of TSHR on 5C9 biological activity was investigated by using M22 as a cyclic AMP stimulator in CHO-TSHR cells (Table 71).

  Of all TSHR mutations studied, only three mutations were found to affect the ability of 5C9 to act as an antagonist. The mutation of Lys129 to Ala (Table 7h) resulted in a complete loss of 5C9 IgG's ability to block TSH stimulation of cyclic AMP production.

  The TSHR mutation Lys183Ala also caused a partial decrease in the blocking activity of 5C9 IgG. During the test, 28% inhibition of TSH stimulation was observed with the TSHR Lys183Ala mutation at 1 μg / mL, compared to 84% inhibition with wild-type TSHR (Table 7m). Even in 100 μg / mL 5C9 IgG, only partial inhibition (43%) of TSH stimulation was detected in the experiment using the Lys183Ala mutation of TSHR, but in the experiment using the wild type receptor, TSH stimulation was performed at the same concentration. A complete block of activity (93%) was observed (Table 7m). When positively charged Lys183 was mutated to negatively charged aspartic acid, the effect on 5C9 biological activity was similar to that observed by the Lys183Ala mutation (Table 7m). This suggests that Lys183 is important for 5C9 biological activity. In the case of Asp151Ala mutation, a slight decrease in 5C9 IgG blocking activity; 49% inhibition was observed at 1 μg / mL compared to 88% blocking by wild type (Table 7j). However, in the presence of 100 μg / mL 5C9 IgG, this activity was comparable to that with wild-type TSHR. When negatively charged Asp151 was mutated to positively charged arginine, no significant decrease in 5C9 IgG blocking activity was observed (Table 7k).

The effect of TSHR mutation on 5C9 activity can be compared to the effect of TSHR mutation on 9D33 activity. As described in patent application WO 2006 / 016121A, mutations in Lys58, Arg80, Tyr82, Arg109, Lys129 and Phe134 in TSHR had an effect on 9D33 activity. However, none of these mutations except Lys129 also had an effect on 5C9 activity. Further, Lys129, which is a mutation that affects M22 activity (Arg80, Tyr82, Glu107, Arg109, Lys129, Phe130, Lys183, Tyr185, Arg255, and Trp258) as described in Patent Application Publication No. 2006 / 016121A is excluded. None of these mutations affected 5C9 activity. Furthermore, the Lys183 mutation had a partial effect on 5C9 activity and M22 activity, but no effect on 9D33 activity.

  These results indicate that the TSHR residues important for interaction with thyroid stimulating human autoantibody M22, mouse blocking antibody 9D33, and human blocking autoantibody 5C9 are different. Thus, the combination of 5C9 with other TSHR antibodies with antagonist activity (eg 9D33) provides a particularly effective means for inhibiting the stimulation activity of patient serum TRAb, other stimulants and / or constitutive activity of TSHR. obtain.

5C9 variable region sequence Sequence analysis of the gene encoding 5C9 showed that the HC V region gene was derived from the VH3-53 family, the D gene was derived from the D2-2 family, and the J gene was derived from the JH4 family. In the case of LC, the V region gene was derived from the O12 family, and the J region gene was derived from the germline of JK2. The nucleotide and amino acid sequences of HC are shown in FIGS. 2a and 2b, respectively, and the nucleotide and amino acid sequences of LC are shown in FIGS. 3a and 3b, respectively.

  Compared with germline sequences, somatic mutations in HC gene sequences, in particular one silent mutation in FWR1, two substitution mutations in CDR2, one silent mutation and one substitution mutation in CDR3 And there is one silent mutation in FWR4. However, the HC V region sequence is characterized by two insertions, one of 6 base pairs in length between the V and D genes and one of 15 base pairs in length between the D and J genes. . Thus, CDR1 of HC is 5 amino acids in length, CDR2 is 16 amino acids in length, and CDR3 is 18 amino acids in length (FIG. 2b).

  In the LC sequence, one silent mutation in FWR1, one substitution mutation in CDR1, one substitution mutation in CDR3, and one insertion of 6 base pairs in length between V and J genes Exists. The CDR1 of LC consists of 11 amino acids, CDR2 consists of 7 amino acids, and CDR3 consists of 10 amino acids (FIG. 3b).

Example 5C9 Based Assay for TSH or TRAb Detection An example of an ELISA based on 5C9 IgG-biotin binding to TSHR coated plate wells to detect TSHR autoantibodies is shown in Table 8. In this assay, all samples positive for inhibition of TSH-biotin binding were also positive for 5C9 IgG-biotin binding. Furthermore, absorbance signals, percent inhibition, and assembly unit / L values were comparable between TSH-biotin and 5C9-biotin assays (Table 8).

Effect of 5C9 IgG on the stimulating activity of mouse thyroid-stimulating monoclonal antibody (mouse TSHRMAb; TSMAb) on CHO cells expressing TSHR As shown in Table 9, 5C9 IgG is a protein of all five TSMAbs tested (1, 2, It was possible to block the stimulation of cyclic AMP production by 4,5 and 7). For example, the concentration of cyclic AMP stimulated by TSMAb1 (Table 9) was 18.94 ± 7.4 pmol / mL but only 1.24 ± in the presence of 100 μg / mL 5C9 IgG.
0.07 pmol / mL cyclic AMP was produced. This can be compared to 16.5 ± 1.1 pmol / mL, the concentration of cyclic AMP in the presence of 100 μg / mL control MAb2G4 (Table 9).

  The cyclic AMP concentrations shown in Table 9 below and similarly in Tables 10-15 are expressed in pmol / mL. That is, the amount of cyclic AMP per cell well is pmol / mL ÷ 5 (representing 200 μL of sample from each assayed well).

Effect of 5C9 IgG on the stimulating activity of native and recombinant human TSH on CHO cells expressing TSHR The ability of 5C9 IgG to block cyclic AMP stimulation by porcine TSH is shown in Table 2 and Table 10. In addition, 5C9 IgG is against cyclic AMP stimulation of both natural human TSH (NIBSC reference preparation 81/565 from National Institute for Biological Standards and Control, South Mimms, Potters Bar EN6 3QG UK) and recombinant TSH (NIBSC reference preparation 94/674). Inhibitory ability was shown (Table 10). In particular, stimulation of cyclic AMP production with either 100 ng / mL recombinant or native human TSH requires 0.1-1.0 μg / mL 5C9 IgG to obtain complete inhibition of cyclic AMP production. The circulating TSH concentration in the donor serum at the time of blood collection for 5C9 separation is 160 mU / L (about 32 ng / mL), and the results obtained (Table 2a and Table 10) show that this concentration of circulating TSH is in serum. It can be completely blocked by the presence of 32-320 ng / mL of 5C9 IgG. The concentration of TSHR autoantibodies in the donor serum was determined by inhibiting ¹²⁵ I-M22 binding to TSHR (Nakatake N, Sanders J, Richards T, Burne P, Barrett C, Dal Pra C, Presotto F, Betterle C, Furmaniak J, Rees Smith
B700 Estimation of serum TSH receptor autoantibody concentration and affinity. (As described in Thyroid 16: 1077-1084) was estimated to be 1700 ng / mL (120 ng / mg). This is several times higher than the 5C9 concentration required to block thyroid stimulation by TSH.

Effect of 5C9IgG on basic (ie unstimulated) cyclic AMP activity in CHO cells expressing TSHR with activating mutations S281I, I568T and A623I 5C9IgG is a thyroid activator (ie TSH or TSHR antibody) In the absence of), the amount of cyclic AMP produced in CHO cells expressing TSHR with activating mutations could be reduced. As shown in Table 11a, the basic cyclic AMP concentration in CHO cells expressing TSHR with activating mutation S281I is 9.90 ± 1.51 pmol / mL in the absence of 5C9. Decreased to 4.17 ± 0.60 pmol / mL in the presence of 0.01 μg / mL 5C9 IgG and decreased to 3.44 ± 0.63 pmol / mL in the presence of 1 μg / mL 5C9 IgG. Blocking mouse TSHRMAb9D33 had as little effect as control MAb2G4 (Table 11a).

Similar results were obtained with the TSHR-activating mutation I568T, which shows a basic cyclic AMP concentration of 21.39 ± 5.31 pmol / mL (Table 11b). This is reduced to 5.29 ± 0.75 pmol / mL by the addition of 1 μg / mL 5C9 IgG, and 20.52 ± 0.95 pmol / mL and 21.65 ± 1.2 when 2G4IgG and 5C9IgG are added. Compared to 99 pmol / mL respectively. In the case of A623I, the third TSHR activating mutation studied, with a basic cyclic AMP concentration of 36.89 pmol / mL, the addition of 1 μg / mL 5C9 IgG resulted in a cyclic AMP concentration of 16.43 ±. Decreased to 1.27 pmol / mL, which is a slight effect (28.96 ± 2.29 pmol / mL) with 1 μg / ml control IgG 2G4 or 1 μg / mL 9D33 IgG (40.09 ± 7.73 pmol / mL) mL) (Table 11c).

  These results indicate that 5C9 is different from mouse blocking MAb 9D33, even though TSHR activating mutations are present in different parts of TSHR (ie, S281I is in the extracellular domain and I568T is in the transmembrane domain). In the second extracellular loop, and A623I is in the third intracellular loop of the transmembrane domain), indicating a significant effect on cyclic AMP production associated with these mutations.

Comparison of the effects of 5C9 and mouse TSHR blocking monoclonal antibody 9D33 and a mixture of these two antibodies on stimulation of TSH-mediated cyclic AMP production in CHO cells expressing wild type TSHR as shown in the previous experiment and in Table 12 Thus, human TSHR blocking MAb5C9 and mouse TSHR blocking MAb9D33 at a concentration of at least 1 μg / mL have the ability to block TSH's TSHR cyclic AMP-stimulating activity in CHO-TSHR cells. As shown in Table 1 Experiment 1-5, the effects of 9D33 IgG and 5C9 IgG on the stimulation of cyclic AMP via TSH were additive. The same additive effect was observed when using two different concentrations of TSH (3 ng / mL and 0.3 ng / mL) for stimulation (Table 12; Experiments 1-3 and Experiments 4 and 5) ).

Comparison of the effects of 5C9 and mouse TSHR blocking monoclonal antibody 9D33 and mixtures of these two antibodies on stimulation of M22-mediated cyclic AMP production in CHO cells expressing wild type TSHR As previously indicated, 5C9 And 9D33 can also inhibit the stimulation of cyclic AMP via M22 Fab in CHO-TSHR cells. The effects of 9D33 IgG and 5C9 IgG on cyclic AMP stimulation through M22 were additive (Table 13, Experiments 1-4). The same additive effect was observed when using two different concentrations of M22 Fab (3 ng / mL and 0.3 ng / mL) for stimulation (Table 13; each of Experiments 1 and 2, and Experiments 3 and 4). Experiment).

  The additive effects of 5C9IgG and 9D33IgG were similar for stimulation of cyclic AMP production via both TSH and M22 (Tables 12 and 13).

Effect of 5C9 on basal (ie unstimulated) cyclic AMP activity in CHO cells that express a large number of wild-type TSHR per cell CHO cell lines expressing about 5 × 10 ⁵ receptors per cell have been shown in previous experiments (eg, A high concentration of basic (ie unstimulated) cyclic AMP was shown compared to the standard CHO cell line used in Tables 9-13) (expressing about 5 × 10 ⁴ TSHR per cell). That is, each concentration is 47.1 ± 11.7 pmol / mL compared to about 1.0 pmol / mL. The effect of 5C9IgG and 9D33IgG on the basal activity of wild type TSHR was evaluated using cell lines that expressed multiple receptors per cell. Incubation with 9D33 IgG and a negative control anti-GAD antibody (5B3) resulted in 0-5.3% inhibition of basal cyclic AMP activity (Table 14; Experiment 1). This indicates that mouse blocking MAb9D33 or control MAb has no effect on basal cyclic AMP production in CHO cells expressing wild type TSHR. However, in the case of 5C9 IgG, a clear inhibition of basal cyclic AMP activity was observed (Table 14; experiment 2). That is, concentrations of 0.1 μg / mL and 10 μg / mL caused 45.7% and 74.6% inhibition, respectively. In addition, 5C9 Fab and 5C9F (ab ′) were also effective inhibitors of basal cyclic AMP activity in CHO cells expressing multiple TSHRs per cell (Table 14, Experiment 3). For example, 1 μg / mL and 100 μg / mL 5C9 Fab show 39% and 61% inhibition of basal cyclic AMP production, respectively, compared to 48% inhibition by 100 μg / mL 5C9F (ab ′). (Table 14, Experiment 3).

Effect of patient serum TSHR autoantibodies with antagonist (ie blocking) activity on basal (ie unstimulated) cyclic AMP activity of CHO cells expressing TSHR with activating mutation I568T Cyclic AMP by TSHRI 568T cells Basic cyclic AMP production (20.5 ± 8.7 pmol / mL) in the presence of assay buffer is normal pooled serum (NPS) from healthy blood donors tested or 3 different healthy individuals It was essentially unaffected by the addition of blood donor serum (N1-N3) at 1/10 and 1/50 dilutions. Basic cyclic AMP production in the presence of NPS and N1-N3 serum showed 0-14% inhibition compared to basic cyclic AMP production in the presence of cyclic AMP assay buffer. (Table 15). However, in the presence of 4 different sera with high concentrations of blocking TRAb (B2-B5), 23-89% inhibition of basal cyclic AMP production was observed (Table 15). In the presence of 5C9 IgG (1 μg / mL), 83% inhibition of the basic cyclic AMP activity of TSHRI568T was observed. The dose-dependent effects of the two blocking sera (B3 and B4) on the basic cyclic AMP production of CHO cells expressing TSHR with the I568T mutation are also shown in Table 15.

  These results indicate that 5C9 has the TSHR blocking activity properties of patient blocking TSHR autoantibodies, particularly with respect to the inhibition of basic cyclic AMP production in the TSHR activating mutation I568T.

Effect of patient serum TSHR autoantibodies with antagonist activity on basal (ie unstimulated) cyclic AMP activity of CHO cells expressing TSHR with activating mutation S281I in the presence of cyclic AMP assay buffer by TSHRS281I cells Basic cyclic AMP production at 11.2 ± 2.0 pmol / mL, and incubation with healthy blood donor pool serum or individual healthy blood donor serum (1/10 or 1/50 dilution) has no effect Not shown (Table 16). On the other hand, in the presence of 4 different sera with high concentrations of blocking TRAb (B2-B5), 31-56% inhibition of basic cyclic AMP production was observed (Table 16). In experiments with TSHRS281I, 1 μg / mL 5C9 IgG inhibited basal cyclic AMP activity by 71%.

Effect of patient serum TSHR autoantibodies with antagonist activity on basal (ie unstimulated) cyclic AMP activity of CHO cells expressing TSHR with activating mutation A623I In the case of TSHRA623I cells, cyclic AMP assay buffer Basic cyclic AMP production in the presence was 43.5 ± 11.2 pmol / mL and was essentially unaffected by incubation with healthy blood donor pool sera or individual sera. (Table 17). Incubation with 4 different sera with high concentrations of blocking TRAb (B2-B5) inhibited -1% to 56% of cyclic AMP in these experiments (Table 17). This can be compared to 49% inhibition by 1 μg / mL 5C9 IgG in the same experiment.

Effect of patient serum TSHR autoantibodies with antagonist activity on basal (ie unstimulated) cyclic AMP activity of CHO cells expressing about 5 × 10 ⁵ wild-type TSHR per cell A relatively large number of wild-type TSHR per cell The basic cyclic AMP production of CHO cells expressing 28.1 ± 0.7 pmol / mL in this series of experiments. When the cells are incubated with 1/10 dilution of healthy blood donor pool serum or individual sera (N1-N3), the basic cyclic AMP concentration in the presence of cyclic AMP assay buffer is 99% to 146%. The range was 93% to 137% at 1/50 dilution. Of the four sera tested with blocking TSHR autoantibodies, one sera (B2) had no effect on basal cyclic AMP production (Table 18). In the case of two sera (B3 and B5), the cyclic AMP concentration was increased compared to the concentration observed in the presence of cyclic AMP assay buffer (Table 18). It is quite possible that sera B3 and B5 contain a mixture of TSHR autoantibodies with stimulatory and blocking activity. On the other hand, serum B4 had a clear inhibitory effect on basic cyclic AMP production at 1/10 and 1/50 dilutions. That is, the basic cyclic AMP concentration was 31% and 61%, respectively, compared to the concentration in the presence of cyclic AMP buffer (Table 18). This can be compared with the concentration in the presence of 1 μg / mL 5C9 IgG being 33% compared to the concentration in the presence of cyclic AMP assay buffer (Table 18).

  In general, 5C9 IgG has a similar effect on basal cyclic AMP production in CHO cells transfected with TSHR with activating mutations on the effects observed with sera from patients positive for wild-type TSHR or blocking TSHR autoantibodies. Indicates. However, the effects with individual patient sera vary in the case of various mutations (Table 19). In the case of wild type TSHR, some sera show a stimulating effect. This is probably due to the presence of TSHR stimulating autoantibodies as well as blocking autoantibodies (Table 19).

Effect of 5C9 on stimulation of cyclic AMP production in CHO cells expressing TSHR containing amino acid mutations The effect of 5C9 on stimulation of cyclic AMP production in CHO cells expressing TSHR having amino acid mutations is Covering the inclusion of mutations: Asp43, Glu61, His105, Glu107, Phe130, Glu178, Tyr185, Asp203, Tyr206, Lys209, Asp232, Lys250, Glu251, Thr257, Arg274, Asp276 (summarized in Tables 20a-p and Table 21).

  Mutations of TSHR amino acids Asp43, Glu61, His105, Glu107, Tyr185, Asp232 and Thr275 to alanine had no effect on the ability of 5C9 IgG to inhibit cyclic AMP production by TSH stimulation. The ability of 5C9 to inhibit TSH-stimulated cyclic AMP production was reduced by mutation of TSHR to Phe130, Glu178, Asp203, Tyr206, Lys250, Glu251 and Asp276 to alanine. In the case of two mutations, Lys209Ala and Asp274Ala, the ability of 5C9 IgG to inhibit TSH-mediated cyclic AMP production was increased.

  In summary (Tables 7, 20, and 21), all 10 TSHR residues of Lys129, Phe130, Asp151, Glu178, Lys183, Asp203, Tyr206, Lys250, Glu251 and Asp276 are directed against cyclic AMP stimulation by 5C9 TSH. Inhibitory capacity was reduced compared to wild type TSHR. Mutations of TSHR Lys129 and Asp203 showed the greatest effect and caused complete inhibition of 5C9 activity.

Effect of blocking serum B2-B5 on stimulation of cyclic AMP production via TSH in CHO cells expressing wild type TSHR by comparison with Asp203Ala mutant TSHR The blocking effect of 1 μg / mL 5C9 in wild type TSHR is ( 92% inhibition of cyclic AMP stimulation induced by TSH), reduced to 4% in Asp203Ala mutant TSHR (Table 22).

  The activity of blocking serum B4 was not affected by the Asp203Ala mutation in TSHR, but in blocking serum B2 and B3, the Asp203Ala mutation in TSHR showed cyclic AMP stimulation induced by TSH compared to wild-type TSHR. There was a slight decrease in percent inhibition.

  In the case of single serum B5, a significant decrease was observed in the percent inhibition of cyclic AMP stimulation induced by TSH; ie, 69% inhibition with wild-type TSHR versus 30% inhibition with mutant TSHR. there were.

  Although the effect of TSHR Asp203Ala mutation on 5C9 activity was greater than that on blocking serum activity, the blocking activity of 3/4 sera tested was affected to varying degrees. This may indicate that the blocking TSHR autoantibodies and 5C9 binding sites overlap, but there are some differences in the actual TSHR amino acids that come into contact with various sera.

Summary and Conclusions The above experiments demonstrate that antibodies according to the invention such as 5C9 can block the stimulating activity of various thyroid stimulants including human and mouse TSHR stimulating antibodies, natural human and animal TSH, and recombinant human TSH. To do. In addition, two different blocking antibodies, human MAb5C9 and mouse MAb9D33, when tested individually, have the ability to block cyclic AMP stimulation via TSH or M22 in CHO cells expressing TSHR when mixed. It is demonstrated to show an additive blocking effect on TSH or M22 stimulation.

  Antibodies according to the present invention, such as 5C9, have a novel effect on the basic (ie unstimulated) cyclic AMP activity of TSHR. These effects have been studied by experiments using TSHR-introduced CHO cells with a relatively high concentration of basic cyclic AMP. That is, the blocking effect on the basic cyclic AMP activity of the antibody according to the present invention such as 5C9 has been confirmed. In addition, some sera with TSHR autoantibodies with blocking (antagonist) activity have been shown to have the ability to block basal cyclic AMP activity in these TSHR-introduced cells. This experiment also demonstrates the blocking effect of antibodies according to the invention such as 5C9 and serum TSHR blocking autoantibodies on the basic cyclic AMP activity associated with activated TSHR mutations.

  These results highlight that 5C9 is a human MAb that exhibits the properties of blocking TSHR autoantibodies, ie, typical of patient serum TSHR autoantibodies associated with thyroid autoimmune disease.

  The described experiment also allowed the identification of several TSHR amino acids important for the blocking activity of the antibodies according to the invention.

In general, this result shows that antibodies according to the invention, such as 5C9, are similar in TSHR binding activity and TSHR function similar to TSHR blocking autoantibodies found in various sera from patients with thyroid autoimmune disease. It exhibits the biological effects of Thus, antibodies according to the invention, such as 5C9, which have the properties and biological activity of blocking TSHR autoantibodies in serum, can be applied to inactivate TSHR in various pathologies. These pathologies include activation of TSHR via TSH, activation of TSHR via thyroid stimulating TSHR autoantibodies, basic (unstimulated, constitutive) TSHR activity, and TSHR due to activated TSHR mutations. Activation. Therefore, the antibody according to the present invention such as 5C9 can be used for the pathological condition caused by the activation of the above-mentioned TSHR; Applicable to the management and control of hyperthyroidism, thyroid cancer, and thyroid cancer metastasis.

ＴＳＨＲをコートしたチューブへの標識ラベルの平均結合％
¹²⁵Ｉ−ＴＳＨを用いた実験において１４％
¹²⁵Ｉ−Ｍ２２ＩｇＧを用いた実験において２１％
および、５Ｃ９ＩｇＧを用いた実験において２０％

ＨＢＤプール＝健常人血液ドナープール血清
２Ｇ４＝コントロールのＩｇＧ（ヒト甲状腺ペルオキシダーゼに対するヒトＭＡｂ）

Average% binding of labeled label to TSHR coated tube
14% in experiments with ¹²⁵ I-TSH
21% in experiments with ¹²⁵ I-M22 IgG
And 20% in experiments with 5C9 IgG

HBD pool = healthy blood donor pool serum 2G4 = control IgG (human MAb against human thyroid peroxidase)

¹１回の決定
^a ドナー血清は示されたようにサイクリックＡＭＰ検定緩衝液で希釈した
^b 比濁法により決定された非希釈血清のＩｇＧ濃度は１４．３ｍｇ/ｍＬであった
５Ｃ９ＩｇＧおよびＴＳＨはサイクリックＡＭＰアッセイ緩衝液で希釈した。

¹ One decision
^a Donor serum diluted in cyclic AMP assay buffer as indicated
^b IgG concentration of undiluted serum determined by nephelometry was 14.3 mg / mL 5C9 IgG and TSH were diluted with cyclic AMP assay buffer.

¹２組のサンプルの平均
２Ｇ４＝コントロールのＩｇＧ（ヒト甲状腺ペルオキシダーゼに対するヒトＭＡｂ）
抗体およびＴＳＨ標本はサイクリックＡＭＰアッセイ緩衝液で希釈した。

¹ Average 2G4 of duplicate samples = control IgG (human MAb against human thyroid peroxidase)
Antibody and TSH specimens were diluted with cyclic AMP assay buffer.

¹２組のサンプルの平均
９Ｄ３３は、ＴＳＨＲが発現するＣＨＯ細胞において、ＴＳＨおよびＴＲＡｂが介在するサイクリックＡＭＰ生産を遮断するマウス抗体である。
抗体およびＴＳＨ標本はサイクリックＡＭＰアッセイ緩衝液で希釈した。

¹ Average 9D33 of the two sets of samples are mouse antibodies that block cyclic AMP production mediated by TSH and TRAb in CHO cells expressing TSHR.
Antibody and TSH specimens were diluted with cyclic AMP assay buffer.

Ｎ１−１０＝健常人血液ドナーからの血清
Ｇ１−Ｇ４０＝グレーブス病にかかった経験のある患者からの血清
結果はほぼ一致する二重測定の平均である。

ここで、Ａ＝テスト血清存在下での結合；Ｂ＝健常人血液ドナーからの血清ＨＢＤプールのプールの存在下での結合。
ＨＢＤプール存在下での¹²⁵Ｉ−５Ｃ９は２０％の結合率であり、ＨＢＤプール存在下での¹²⁵Ｉ−ＴＳＨは１２％の結合率であり、ＨＢＤプール存在下での¹²⁵Ｉ−Ｍ２２は２０％の結合率であった。

N1-10 = serum from healthy blood donors G1-G40 = serum results from patients with experience with Graves' disease are the average of two matched duplicates.

Where A = binding in the presence of test serum; B = binding in the presence of a pool of serum HBD pools from healthy blood donors.
¹²⁵ I-5C9 in the presence of the HBD pool is 20% binding, ¹²⁵ I-TSH in the presence of the HBD pool is 12% binding, and ¹²⁵ I-M22 in the presence of the HBD pool is 20%. % Binding rate.

Ｂ１−Ｂ５はアンタゴニスト（遮断）活性を伴う高いレベルのＴＲＡｂを有する患者血清である。
Ｂ３は５Ｃ９に対するリンパ球ドナーからの血清である
Ｓ１、Ｓ２、およびＳ４はアンタゴニスト（遮断）活性を伴う高いレベルのＴＲＡｂを有する患者血清である。
アッセイキャリブレーター４０Ｕ／Ｌ、８Ｕ／Ｌ、２Ｕ／Ｌ、および１Ｕ／Ｌ、は、Ｍ２２ ＩｇＧを健常人血液ドナー血清（ＨＢＤプール）で希釈し、ＮＩＢＳＣ９０／６７２のＵ／Ｌ活性を、標識したＴＳＨがＴＳＨＲでコートしたチューブへ結合することの阻害率を用いて測定したものである。

ここで、Ａ＝テストサンプル；Ｂ＝ＨＢＤプール
ＮＴ＝テストしない
１／５、１／１０等はＨＢＤプールにおけるテスト血清の希釈率を示し、ニートは希釈無し、を示す。
ＨＢＤプールの存在下、¹²⁵Ｉで標識したＭ２２ＩｇＧ、５Ｃ９ＩｇＧ、ＴＳＨの、それぞれ約２０％、１７％、１２％がコートしたチューブへ結合した。

B1-B5 is patient serum with high levels of TRAb with antagonist (blocking) activity.
B3 is sera from lymphocyte donors for 5C9, S1, S2, and S4 are patient sera with high levels of TRAb with antagonist (blocking) activity.
Assay calibrators 40 U / L, 8 U / L, 2 U / L, and 1 U / L were prepared by diluting M22 IgG with healthy blood donor serum (HBD pool) and labeling the T / L activity of NIBSC 90/672 with labeled TSH. Is measured using the inhibition rate of binding to a tube coated with TSHR.

Here, A = test sample; B = HBD pool NT = not tested 1/5, 1/10 etc. indicate the dilution rate of the test serum in the HBD pool, and neat indicates no dilution.
In the presence of the HBD pool, ¹²⁵ I-labeled M22IgG, 5C9IgG, and TSH were bound to tubes coated with approximately 20%, 17%, and 12%, respectively.

抗体は健常人血液ドナー血清のプールで希釈した（ＨＢＤプール）。

ここで、Ａ＝テストサンプル存在下での結合率（％）；Ｂ＝ＨＢＤプール存在下での結合率（％）
¹甲状腺刺激活性を有するマウスＴＳＨＲ ＭＡｂ
²ＴＳＨおよびＴＲＡｂ両方が仲介するサイクリックＡＭＰ生産の刺激を遮断するマウスＴＳＨＲ ＭＡｂ
³甲状腺ペルオキシダーゼＭＡｂに対するヒトＭＡｂ（ネガティブコントロール）
⁴ＴＳＨ遮断活性を有するマウスＴＳＨＲ ＭＡｂ（ＴＳＨＲアミノ酸３８１−３８５により形成されるエピトープを認識する）
⁵ＴＳＨ遮断活性を有するマウスＴＳＨＲ ＭＡｂ（ＴＳＨＲアミノ酸３６−４２により形成されるエピトープを認識する）
⁶ＴＳＨ遮断活性を有するマウスＴＳＨＲ ＭＡｂ（ＴＳＨＲアミノ酸２４６−２６０により形成されるエピトープを認識する）
⁷マウスＴｇＭＡｂ（ネガティブコントロール）
ＨＢＤプールの存在下、¹²⁵Ｉで標識したＭ２２ＩｇＧ、５Ｃ９ＩｇＧ、ＴＳＨの、それぞれ約１３％、２４％、１５％がコートしたチューブへ結合する。

The antibody was diluted in a pool of healthy human blood donor sera (HBD pool).

Here, A = binding rate in the presence of test sample (%); B = binding rate in the presence of HBD pool (%)
¹ Mouse TSHR MAb with thyroid stimulating activity
² Mouse TSHR MAb that blocks stimulation of cyclic AMP production mediated by both TSH and TRAb
³ Human MAb against thyroid peroxidase MAb (negative control)
⁴ Mouse TSHR MAb with TSH blocking activity (recognizes the epitope formed by TSHR amino acids 381-385)
⁵ Mouse TSHR MAb with TSH blocking activity (recognizes epitope formed by TSHR amino acids 36-42)
⁶ Mouse TSHR MAb with TSH blocking activity (recognizes epitope formed by amino acids 246-260 of TSHR)
⁷ mouse TgMAb (negative control)
In the presence of the HBD pool, ¹²⁵ I-labeled M22IgG, 5C9IgG, and TSH bind to approximately 13%, 24%, and 15% coated tubes, respectively.

ｕｄ＝検出されず
¹＝二重測定
ＨＢＤプールは健常人血液ドナー血清のプールである；これらの実験では、１：１０に希釈したサイクリックＡＭＰアッセイ緩衝液を使用した。
Ｔ１−Ｔ９およびＴ１１−Ｔ１８はＴＳＨＲを発現するＣＨＯ細胞においてサイクリックＡＭＰ産生を刺激する血清である。Ｔ１―Ｔ９およびＴ１１−Ｔ１８はサイクリックＡＭＰアッセイ緩衝液で１：１０に希釈しテストした。
２Ｇ４は甲状腺ペルオキシダーゼに対するヒトモノクローナル抗体である（ネガティブコントロール）。
２Ｇ４ ＩｇＧおよび５Ｃ９ ＩｇＧは１００μｇ/ｍＬでテストした。

ud = not detected
¹ = Dual measurement HBD pool is a pool of healthy blood donor sera; in these experiments, cyclic AMP assay buffer diluted 1:10 was used.
T1-T9 and T11-T18 are sera that stimulate cyclic AMP production in CHO cells expressing TSHR. T1-T9 and T11-T18 were tested diluted 1:10 in cyclic AMP assay buffer.
2G4 is a human monoclonal antibody against thyroid peroxidase (negative control).
2G4 IgG and 5C9 IgG were tested at 100 μg / mL.

Ｔ１は、ＴＳＨＲを発現するＣＨＯ細胞におけるサイクリックＡＭＰ産生を刺激する、患者の血清サンプルである；これらの実験ではサイクリックＡＭＰアッセイ緩衝液で１：１０に希釈したものを用いた。
２Ｇ４は甲状腺ペルオキシダーゼに対するヒトモノクローナル抗体である（ネガティブコントロール）。
９Ｄ３３は、ＴＳＨＲを発現するＣＨＯ細胞における、ＴＳＨおよびＴＲＡｂによるサイクリックＡＭＰ産生を遮断するマウス抗体である（表２を参照）。

T1 is a patient serum sample that stimulates cyclic AMP production in CHO cells expressing TSHR; these experiments were diluted 1:10 in cyclic AMP assay buffer.
2G4 is a human monoclonal antibody against thyroid peroxidase (negative control).
9D33 is a mouse antibody that blocks cyclic AMP production by TSH and TRAb in CHO cells expressing TSHR (see Table 2).

¹一回の測定
説明注釈については表６ａおよび６ｂを参照

¹ for a single measurement Description Comments see Table 6a and 6b

説明注釈については表６ａおよび６ｂを参照

See Tables 6a and 6b for explanatory notes

¹二重サンプルの平均
²１回の測定
説明注釈については表６ａおよび６ｂを参照

Average of ¹ double sample
² See Tables 6a and 6b for remarks on single measurements

¹二重サンプルの平均
ＮＴ＝テストせず
説明注釈については表６ａおよび６ｂを参照

Average of ¹ duplicate sample = not tested, see Tables 6a and 6b for explanatory notes

説明注釈については表６ａおよび６ｂを参照

See Tables 6a and 6b for explanatory notes

¹二重測定の平均値
ｐＴＳＨ濃度＝３ｎｇ／ｍＬ
全ての希釈液はサイクリックＡＭＰアッセイ緩衝液
２Ｇ４は甲状腺ペルオキシダーゼに対するヒトモノクローナル抗体である（５Ｃ９に対するネガティブコントロール）

¹ Average value of double measurement pTSH concentration = 3 ng / mL
All dilutions are cyclic AMP assay buffer 2G4 is a human monoclonal antibody against thyroid peroxidase (negative control for 5C9)

説明注釈については表７ａを参照

See Table 7a for explanatory notes

説明注釈については表７ａを参照

See Table 7a for explanatory notes

説明注釈については表７ａを参照

See Table 7a for explanatory notes

説明注釈については表７ａを参照

See Table 7a for explanatory notes

説明注釈については表７ａを参照

See Table 7a for explanatory notes

説明注釈については表７ａを参照

See Table 7a for explanatory notes

説明注釈については表７ａを参照

See Table 7a for explanatory notes

説明注釈については表７ａを参照

See Table 7a for explanatory notes

説明注釈については表７ａを参照

See Table 7a for explanatory notes

説明注釈については表７ａを参照

See Table 7a for explanatory notes

Ｍ２２濃度＝３ｎｇ／ｍＬ
説明注釈については表７ａを参照

M22 concentration = 3 ng / mL
See Table 7a for explanatory notes

説明注釈については表７ａを参照。

See Table 7a for explanatory notes.

説明注釈については表７ａを参照。

See Table 7a for explanatory notes.

説明注釈については表７ａを参照。

See Table 7a for explanatory notes.

説明注釈については表７ａを参照。

See Table 7a for explanatory notes.

説明注釈については表７ａを参照。

See Table 7a for explanatory notes.

説明注釈については表７ａを参照。

See Table 7a for explanatory notes.

説明注釈については表７ａを参照。

See Table 7a for explanatory notes.

アッセイキャリブレーター４０Ｕ／Ｌ、８Ｕ／Ｌ、２Ｕ／Ｌ、および１Ｕ／ＬはＭ２２ ＩｇＧを健常人血液ドナー血清（ＨＢＤプール）で希釈したものであり、ＮＩＢＳＣ ９０／６７２のＵ／Ｌ活性により、標識されたＴＳＨのＴＳＨＲをコートしたチューブへの結合の阻害を測定した。アッセイのネガティブコントロールはＨＢＤプールである。

Assay calibrators 40 U / L, 8 U / L, 2 U / L, and 1 U / L are M22 IgG diluted with healthy blood donor serum (HBD pool) and labeled by the U / L activity of NIBSC 90/672 Inhibition of binding of the prepared TSH to TSHR-coated tubes was measured. The negative control for the assay is the HBD pool.

^aサイクリックＡＭＰアッセイ緩衝液による希釈液
^b２Ｇ４はヒト甲状腺ペルオキシダーゼモノクローナル自己抗体コントロールである。

^a Dilution with cyclic AMP assay buffer
^b 2G4 is a human thyroid peroxidase monoclonal autoantibody control.

^a２Ｇ４はヒト甲状腺ペルオキシダーゼモノクローナル抗体コントロールである。

^a 2G4 is a human thyroid peroxidase monoclonal antibody control.

^a２回の測定

^a 2 measurements

コントロールのＣＨＯ細胞では、ＴＳＨＲが発現されず、サイクリックＡＭＰアッセイ緩衝液の存在下での基礎的なサイクリックＡＭＰ産生は１．０２±０．０６ｐｍｏｌ／ｍＬ（平均±ＳＤ；ｎ＝３）であり、３ｎｇ／ｍＬ ＴＳＨの存在下では０．７４±０．３２ｐｍｏｌ／ｍＬ（平均±ＳＤ；ｎ＝３）であった。
−ｖe＝ネガティブ、すなわちサイクリックＡＭＰ産生の阻害が無い。
^b５Ｂ３はグルタミン酸デカルボキシラーゼ（ＧＡＤ）ヒトモロクローナル抗体である（５Ｃ９に対するネガティブコントロール）。

コントロールのＣＨＯ細胞では、ＴＳＨＲが発現されず、サイクリックＡＭＰアッセイ緩衝液の存在下での基礎的なサイクリックＡＭＰ産生は０．０３ｐｍｏｌ／ｍＬ（ｎ＝２）であり、３ｎｇ／ｍＬ ＴＳＨの存在下では０．４０±０．０９ｐｍｏｌ／ｍＬ（平均±ＳＤ；ｎ＝３）であった。
−ｖe＝ネガティブ、すなわちサイクリックＡＭＰ産生の阻害が無い。
^b５Ｂ３はグルタミン酸デカルボキシラーゼ（ＧＡＤ）ヒトモロクローナル抗体である（５Ｃ９に対するネガティブコントロール）。

コントロールのＣＨＯ細胞では、ＴＳＨＲが発現されず、サイクリックＡＭＰアッセイ緩衝液の存在下での基礎的なサイクリックＡＭＰ産生は１．０３ｐｍｏｌ／ｍＬ（平均±ＳＤ；ｎ＝２）であり、３ｎｇ／ｍＬ ＴＳＨの存在下では０．０４±０．０９ｐｍｏｌ／ｍＬ（平均±ＳＤ；ｎ＝３）であった。
−ｖe＝ネガティブ、すなわち還元サイクリックＡＭＰ産生の阻害が無い。
Ｆ（ａｂ‘） Ｆ（ａｂ‘）₂の還元により準備される；詳細な説明の記載を参照

In control CHO cells, TSHR is not expressed and basal cyclic AMP production in the presence of cyclic AMP assay buffer is 1.02 ± 0.06 pmol / mL (mean ± SD; n = 3) Yes, in the presence of 3 ng / mL TSH, 0.74 ± 0.32 pmol / mL (mean ± SD; n = 3).
-Ve = negative, ie no inhibition of cyclic AMP production.
^b 5B3 is glutamate decarboxylase (GAD) human moloclonal antibody (negative control for 5C9).

In control CHO cells, TSHR is not expressed, basal cyclic AMP production in the presence of cyclic AMP assay buffer is 0.03 pmol / mL (n = 2) and the presence of 3 ng / mL TSH Below was 0.40 ± 0.09 pmol / mL (mean ± SD; n = 3).
-Ve = negative, ie no inhibition of cyclic AMP production.
^b 5B3 is glutamate decarboxylase (GAD) human moloclonal antibody (negative control for 5C9).

In control CHO cells, TSHR is not expressed, basal cyclic AMP production in the presence of cyclic AMP assay buffer is 1.03 pmol / mL (mean ± SD; n = 2), 3 ng / 0.04 ± 0.09 pmol / mL (mean ± SD; n = 3) in the presence of mL TSH.
-Ve = negative, ie no inhibition of reduced cyclic AMP production.
F (ab ′) prepared by reduction of F (ab ′) ₂ ; see description in detailed description

−ｖe＝ネガティブ、すなわちサイクリックＡＭＰ産生の阻害が無い。
ＨＢＤプール＝健常人血液ドナー血清のプール
Ｎ１−Ｎ３＝個々の健常人血液ドナー
全ての血清はサイクリックＡＭＰアッセイ緩衝液で希釈された。

-Ve = negative, ie no inhibition of cyclic AMP production.
HBD pool = pool of healthy blood donor sera N1-N3 = sera of all individual healthy blood donors were diluted in cyclic AMP assay buffer.

説明注釈については表１５を参照。
１μｇ／ｍＬの５Ｃ９ ＩｇＧは、ＴＳＨＲ Ｓ２８１Ｉを用いた実験における基本的なサイクリックＡＭＰ活性の阻害の７１％を引き起こした。

See Table 15 for explanatory notes.
1 μg / mL 5C9 IgG caused 71% inhibition of basal cyclic AMP activity in experiments with TSHR S281I.

説明注釈については表１５を参照。
１μｇ／ｍＬの５Ｃ９ ＩｇＧは、ＴＳＨＲ Ａ６２３Ｉを用いた実験における基本的なサイクリックＡＭＰ活性の４９％阻害を引き起こした。

See Table 15 for explanatory notes.
1 μg / mL 5C9 IgG caused 49% inhibition of basal cyclic AMP activity in experiments with TSHR A623I.

１μｇ／ｍＬの５Ｃ９ ＩｇＧの存在下、基本的なサイクリックＡＭＰレベルはサイクリックＡＭＰアッセイ緩衝液存在下でのレベルに比べ３３％に減少した。

In the presence of 1 μg / mL 5C9 IgG, basal cyclic AMP levels were reduced to 33% compared to levels in the presence of cyclic AMP assay buffer.

ＨＢＤプール＝健常人血液ドナー血清のプール。
ＨＢＤプールおよびＢ２〜Ｂ５の血清はサイクリックＡＭＰアッセイ緩衝液中で１：１０に希釈した。
５Ｃ９ ＩｇＧはサイクリックＡＭＰアッセイ緩衝液中１μｇ／ｍＬの濃度で用いた。
Ｂ２−Ｂ５は、野生型ＴＳＨＲを発現するＣＨＯ細胞中での、ＴＳＨおよびＭ２２の両方によるサイクリックＡＭＰ産生刺激を遮断した。

HBD pool = pool of healthy blood donor serum.
HBD pool and B2-B5 sera were diluted 1:10 in cyclic AMP assay buffer.
5C9 IgG was used at a concentration of 1 μg / mL in cyclic AMP assay buffer.
B2-B5 blocked cyclic AMP production stimulation by both TSH and M22 in CHO cells expressing wild type TSHR.

Average of ¹ double measurement
² Single measured pTSH concentration = 3 ng / mL.
All dilutions were made with cyclic AMP assay buffer.
^a 5B3 is a human monoclonal antibody against glutamate decarboxylase (GAD) (negative control for 5C9).

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

ＵＤ＝アッセイ測定限界以下。
説明注釈については表２０aを参照。

UD = below assay measurement limit.
See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

説明注釈については表２０aを参照。

説明注釈については表２０aを参照。

See Table 20a for explanatory notes.

See Table 20a for explanatory notes.

使用したｐＴＳＨ濃度＝３ｎｇ／ｍＬ
ＴＳＨＲの比較した効果は野生型とともに観察された活性のパーセンテージで以下のように表される：−＋＋＋＋＋＝野生型活性の１００％；＋＋＋＋＝野生型活性の８０％以上１００％未満；＋＋＋＝野生型活性の６０％以上８０％未満；＋＋＝野生型活性の４０％以上６０％未満；＋＝野生型活性の２０％以上４０％未満、０＝野生型の２０％未満、および野生型と比較して増加した活性：＞１００％は＋＋＋＋＋＋＋。ＮＴ＝テストせず。^aこの実験のためのサイクリックＡＭＰ刺激は、ＴＳＨの応答を欠失したＭ２２を用いた（詳しくは明細書を参照）。

PTSH concentration used = 3 ng / mL
The compared effect of TSHR is expressed as a percentage of activity observed with wild type: − ++++++ = 100% of wild type activity; +++++ = 80% to less than 100% of wild type activity; +++ = wild 60% or more and less than 80% of type activity; ++ = 40% or more and less than 60% of wild type activity; + = 20% or more and less than 40% of wild type activity, 0 = less than 20% of wild type, and compared with wild type Increased activity:> 100% +++++++. NT = not tested. ^a Cyclic AMP stimulation for this experiment used M22 lacking the TSH response (see specification for details).

^aサイクリックＡＭＰアッセイ緩衝液での希釈
^bＴＳＨの関与するサイクリックＡＭＰ刺激の阻害率％

¹二重サンプルの平均
^c最終濃度３ｎｇ／ｍＬで使用されたｐＴＳＨ
ＨＢＤプール＝健常人ドナー血清のプール

^a Dilution with cyclic AMP assay buffer
^b % inhibition of cyclic AMP stimulation involving TSH

Average of ¹ double sample
^c pTSH used at a final concentration of 3 ng / mL
HBD pool = pool of healthy donor serum

Claims (47)

An isolated human monoclonal or recombinant antibody against thyroid stimulating hormone receptor (TSHR) comprising:
The antibody is
An antagonist of thyrotropin (TSH),
Has a binding affinity of at least 10 ⁹ L / mol for human full-length TSHR;
a) SNYMS (CDR1, SEQ ID NO: 1);
b) VTYSGGSTSYADSVKG (CDR2, SEQ ID NO: 2); and c) GGRYCSSISCYARSGCDY (CDR3, SEQ ID NO: 3)
A V _H region containing
a) RASQSISSYLN (CDR1, SEQ ID NO: 4);
b) AASSLQS (CDR2, SEQ ID NO: 5); and c) QQSYSSPSTT (CDR3, SEQ ID NO: 6)
_VL region including
The antibody is
SEQ ID NO: 7
t
And the amino acid sequence encoded by
SEQ ID NO: 8
gccatccagatgacccagtctccttcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtt
tgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagttccccctccaccacttttggccaggggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccg t
An antibody comprising the amino acid sequence encoded by. 2. The antibody of claim 1 having a binding affinity of at least 10 < ¹⁰ > L / mol for human full-length TSHR.   The antibody according to claim 1 or 2, which inhibits constitutive activity of TSHR.   The antibody according to any one of claims 1 to 3, which is an antagonist of a thyroid stimulating antibody.   5. The antibody according to any one of claims 1 to 4, which has the TSH antagonist property of a patient serum TSHR autoantibody that is an antagonist of TSH.   6. The antibody according to claim 4 or 5, which is an antagonist of TSH and an antagonist of thyroid stimulating antibody.   The antibody according to any one of claims 1 to 6, which has antagonist properties of a patient serum TSHR autoantibody that is an antagonist of a thyroid stimulating antibody.   The antibody according to any one of claims 1 to 7, which is an antibody having a stimulating activity on TSH, M22 or TSHR, or an inhibitor of binding to TSHR or a part thereof by an antibody having a blocking activity.   9. The antibody of claim 8, wherein a portion of the TSHR comprises a TSHR leucine rich domain (LRD) or a substantial portion thereof.   The antibody according to claim 8 or 9, which blocks the binding.   11. A nucleotide comprising a nucleotide sequence encoding the antibody of any one of claims 1-10.   A vector comprising the nucleotide according to claim 11.   An isolated cell comprising the antibody according to any one of claims 1 to 10.   The isolated cell which expresses the antibody of any one of Claims 1-10.   The isolated cell which secretes the antibody of any one of Claims 1-10.   16. The isolated cell according to claim 13, 14, or 15, which is derived from a stable hetero-hybridoma cell line.   A composition comprising a TSHR autoantibody at a defined concentration, comprising the antibody of any one of claims 1-10.   A composition comprising a defined concentration of a TSHR autoantibody having TSH antagonist activity, comprising the antibody of any one of claims 1-10.   A composition comprising a TSHR autoantibody that is an antagonist of a thyroid stimulating antibody at a defined concentration, comprising the antibody of any one of claims 1-10.   A composition comprising a TSHR autoantibody having a defined concentration of a TSH antagonist activity and an antagonist of a thyroid stimulating antibody, comprising the antibody of any one of claims 1-10.   A pharmaceutical composition comprising the antibody according to any one of claims 1 to 10 together with a pharmacologically acceptable carrier for administration to a mammalian subject for the treatment of a thyroid-related disease.   22. The thyroid-related disease is selected from hyperthyroidism, Graves eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, anterior cervical myxedema, thyroid cancer, and thyroiditis. A pharmaceutical composition according to 1.   23. A pharmaceutical composition according to claim 21 or 22, suitable for human administration.   24. A pharmaceutical composition according to claim 21, 22 or 23, wherein the antibody has no significant adverse effect on the immune system of the subject.   25. A pharmaceutical composition according to any one of claims 21 to 24, wherein the composition comprises one or more additional thyroid stimulating hormone receptor antagonists.   26. The pharmaceutical composition according to claim 25, wherein said further thyroid stimulating hormone receptor antagonist is 9D33.   27. A pharmaceutical composition according to any one of claims 21 to 26 for use in the treatment of thyroid related diseases, which is in an injectable form.   27. A pharmaceutical composition according to any one of claims 21 to 26 for use in the treatment of anterior tibial myxedema which is in a topical form.   27. A pharmaceutical composition according to any one of claims 21 to 26 for use in the treatment of Graves eye disease, which is in the form of eye drops.   A method for producing the antibody according to any one of claims 1 to 10, wherein the cell is expressed by culturing the cell according to any one of claims 13 to 16. Including methods.   32. The method of claim 30, wherein the antibody is secreted by the cell.   Use of an antibody according to any one of claims 1 to 10 in the preparation of a medicament for the treatment of thyroid related diseases.   11. An antibody according to any one of claims 1 to 10 for use in medical therapy. 11. An antibody according to any one of claims 1 to 10 for use in the treatment of thyroid related diseases.   35. The thyroid-related disease is selected from hyperthyroidism, Graves eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, anterior cervical myxedema, thyroid cancer, and thyroiditis. The antibody according to 1.   11. A method for characterizing a TSHR antibody, comprising determining binding of a tested TSHR antibody to a polypeptide having a TSHR-related amino acid sequence, wherein the antibody of any one of claims 1-10. A method involved in a method step including use.   37. The method of claim 36, comprising determining the effect of the antibody of any one of claims 1-10 on binding of a TSHR antibody to the polypeptide.   38. The method of claim 37, wherein the TSHR-related polypeptide comprises full length human TSHR.   11. A method for characterizing TSH and related molecules comprising determining the binding of a TSH to be tested or a related molecule to a polypeptide having a TSHR-related amino acid sequence. A method relating to a method step comprising the use of the antibody according to Item.   40. The method of claim 39, wherein the method is in an ELISA format.   A method for determining a TSHR amino acid involved in binding of a TSHR autoantibody that acts as an antagonist, comprising: a polypeptide having a first TSHR-related amino acid sequence to which the antibody of any one of claims 1 to 10 binds. Providing, modifying at least one amino acid in the TSHR-related amino acid sequence, and determining the effect of such modification in antibody binding.   11. A method for modifying an antibody according to any one of claims 1 to 10, wherein at least one amino acid of the antibody is modified, and the effect of such modification on a TSHR-related sequence. A method comprising determining in binding.   43. The method of claim 42, wherein modified TSHR antibodies are selected that have enhanced affinity for TSHR.   11. A method for identifying a molecule that inhibits a thyroid stimulating antibody that binds to TSHR, comprising providing at least one antibody of any one of claims 1 to 10 as a reference.   45. The method of claim 44, wherein a molecule is selected that blocks a thyroid stimulating antibody that binds to TSHR.   11. A method for identifying a molecule that inhibits a thyroid blocking antibody that binds to TSHR, comprising providing at least one antibody of any one of claims 1 to 10 as a reference.   47. The method of claim 46, wherein a molecule that blocks a thyroid blocking antibody that binds to TSHR is selected.

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