Methods for treating autoimmune thyroid disease

Abstract

The present invention provides methods of treating autoimmune thyroid disease using an antibody, or antigen-binding fragment thereof, capable of specifically binding to TSHR.

Description

[0001] This application claims the benefit of Chinese application No. 202411253713.9, filed on September 6, 2024, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biomedicine, in particular, the present application relates to a method for treating autoimmune thyroid disease using an antibody or antigen-binding fragment thereof that specifically binds to TSHR. BACKGROUND

[0003] The thyrotropin receptor (TSHR) is mainly present on the lateral surface of the follicular basal cells of the thyroid gland, testis, and orbital fibroblasts of patients with thyroid-associated ophthalmopathy. When combined with the pituitary gland-secreted thyrotropin (TSH), it can stimulate the formation and release of thyroxine (T4) and triiodothyronine (T3). The release of TSH is controlled by the levels of T4, T3 in the circulation and the hypothalamus-secreted thyrotropin-releasing hormone (TRH), which in turn controls the stimulation of the thyroid gland and the levels of thyroid hormones in the serum (Szkudlinski M W, et al, 2002 supra).

[0004] Graves' disease (also known as diffuse toxic goiter, Graves' disease, GD for short) is an autoimmune thyroid disease (AITD) that can cause hyperthyroidism, i.e., the thyroid gland secretes too much thyroid hormone (T3, T4), the main clinical manifestations are hypermetabolic syndrome, diffuse goiter, eye signs, skin lesions, and thyroid acropathy. The clinical symptoms of AITD patients include the production of autoantibodies that bind to TSHR (Rees Smith B, et al 1988. Endocrine Reviews 9: 106-121), i.e., TRAb. There are mainly two types of TRAb: one is stimulating type, and the other is inhibitory type. When stimulating antibodies are present, the feedback control mechanism of thyroid function is no longer effective, and patients develop clinical symptoms of thyroid hyperactivity, characterized by excessive thyroid hormones (T3, T4) in the serum, which is referred to as GD. Tab01 is a stimulating human monoclonal antibody isolated from GD patient lymphocytes, which is described in detail in WO2004 / 050708A2. In AITD patients, the frequency of inhibitory TRAb is lower than that of stimulating autoantibodies. Inhibitory autoantibodies bind to TSHR, preventing TSH from binding to the receptor, but have no ability to stimulate TSHR activity. Therefore, the formation and secretion of thyroid hormones (T4 and T3) are greatly reduced, and such TRAb patients can exhibit clinical symptoms of hypothyroidism.

[0005] According to the above characteristics of the TSHR target, there is currently an antibody drug for the GD indication in clinical phase I study. The drug is TSHR antibody drug K1-70, which is a TSHR inhibitory antibody isolated from the lymphocytes of a hypothyroid patient, and competitively binds to TSHR. The results of the clinical phase I study of K1-70 in treating GD patients show that after a single intramuscular injection of 25 mg or a single intravenous injection of 50 mg or 150 mg of K1-70, the levels of free T3, free T4 and TSH progress to the hypothyroid range (Jadwiga Furmaniak, et al. Clinical Endocrinology. 2022; 96: 878-887.). SUMMARY

[0006] The present application provides a method for treating autoimmune thyroid disease by administering a TSHR inhibitory antibody, wherein the TSHR inhibitory antibody can effectively inhibit or block the activating activity of stimulating TSHR antibodies (TRAb), can effectively inhibit the production of thyroid peroxidase (TPO), and has been proven to be able to effectively inhibit the secretion of thyroid hormones (T3 and / or T4) in an acute GD model mouse, thereby being able to be effectively used for the treatment of autoimmune thyroid disease. Moreover, unlike existing GD treatment antibodies, the TSHR inhibitory antibody provided by the present application does not cause hypothyroidism (hypothyroidism) and / or sub-hypothyroidism (subclinical hypothyroidism), thereby not requiring combined administration with a drug for treating hypothyroidism or sub-hypothyroidism. In addition, the TSHR inhibitory antibody provided by the present application also has a significantly prolonged in vivo half-life, thereby being able to be administered to a subject at a lower administration frequency and / or at a lower administration dose.

[0007] Therefore, in a first aspect, the present application provides a method for preventing and / or treating autoimmune thyroid disease, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof capable of specifically binding to TSHR;

[0008] wherein the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises: a VH CDR1, a VH CDR2 and a VH CDR3 contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 7; and / or a VL CDR1, a VL CDR2 and a VL CDR3 contained in a light chain variable region (VL) as shown in SEQ ID NO: 8;

[0009] wherein the method does not comprise administering to the subject a drug and / or therapy for preventing and / or treating hypothyroidism and / or subclinical hypothyroidism.

[0010] Based on the disclosure of this application, those skilled in the art will readily understand that the methods provided in this aspect do not include administering drugs and / or therapies for the prevention and / or treatment of hypothyroidism to the subject, or, the methods provided in this aspect do not include administering drugs and / or therapies for the prevention and / or treatment of subclinical hypothyroidism to the subject, or, the methods provided in this aspect do not include administering drugs and / or therapies for the prevention and / or treatment of hypothyroidism to the subject, nor do they include administering drugs and / or therapies for the prevention and / or treatment of subclinical hypothyroidism to the subject.

[0011] In some implementations, the method of this aspect does not include administering drugs and / or therapies for the prevention and / or treatment of hypothyroidism to the subject.

[0012] In some implementations, the CDRs are defined by the Kabat, IMGT, Chothia, or AbM numbering systems.

[0013] For example, the antibody or its antigen-binding fragment that specifically binds to TSHR is not administered concurrently with or successively with drugs or therapies for the prevention and / or treatment of hypothyroidism and / or subclinical hypothyroidism.

[0014] In some embodiments, the medication for preventing and / or treating hypothyroidism and / or subclinical hypothyroidism includes levothyroxine tablets (Euthyrox), and / or the therapy for preventing and / or treating hypothyroidism and / or subclinical hypothyroidism includes administering levothyroxine tablets (Euthyrox) to the subject.

[0015] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is the only pharmaceutically active agent administered to the subject for the prevention and / or treatment of the autoimmune thyroid disease.

[0016] In some implementations, the subject contains an autoantibody (e.g., a stimulatory TRAb) capable of activating TSHR.

[0017] In some implementations, the subject contains an amount of autoantibodies (e.g., stimulatory TRAb) that can activate TSHR, exceeding a reference level (e.g., healthy control level).

[0018] In some embodiments, the autoimmune thyroid disease is selected from: Graves' disease, Graves' ophthalmopathy, thyroid-associated ophthalmopathy, pretibial myxedema, Hashimoto's thyroiditis, thyroid cancer, and any combination thereof.

[0019] In some implementations, the autoimmune thyroid disease is Graves' disease.

[0020] In some implementations, the autoimmune thyroid disease is thyroid-associated ophthalmopathy.

[0021] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises:

[0022] (a) Heavy chain variable regions (VH) comprising VH CDR1, VH CDR2, and VH CDR3 as shown in SEQ ID NOs:1-3, respectively; and / or,

[0023] (b) Light chain variable regions (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in SEQ ID NOs:4-6 respectively.

[0024] In some implementations, the CDRs are defined by the Kabat numbering system.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a framework region sequence derived from human immunoglobulin.

[0026] In some embodiments, the antibody or antigen-binding fragment capable of specifically binding to TSHR comprises a framework region contained in the amino acid sequence encoded by a human germline antibody gene. In some embodiments, the antibody or antigen-binding fragment capable of specifically binding to TSHR comprises: a heavy chain framework region of a human heavy chain germline sequence, and / or, a light chain framework region of a human light chain germline sequence.

[0027] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises:

[0028] (1) Heavy chain variable region (VH), which contains the sequence shown in SEQ ID NO: 7 or a variant thereof; and / or,

[0029] (2) Light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 8 or a variant thereof;

[0030] The variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence, or has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the substitutions are conservative substitutions.

[0031] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain variable region (VH) as shown in SEQ ID NO:7 and a light chain variable region (VL) as shown in SEQ ID NO:8.

[0032] In some embodiments, the antibody or its antigen-binding fragment that is capable of specifically binding to TSHR further comprises a constant region derived from mammalian (e.g., human) immunoglobulins.

[0033] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain constant region derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4), and / or, the light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a light chain constant region derived from human immunoglobulins (e.g., a light chain constant region of the κ or λ chain).

[0034] Those skilled in the art will readily understand that the heavy chain constant region derived from human immunoglobulins includes both heavy chain constant regions directly derived from human immunoglobulins and heavy chain constant regions obtained by modifying or altering heavy chain constant regions derived from human immunoglobulins (e.g., heavy chain constant region variants); the light chain constant region derived from human immunoglobulins includes both light chain constant regions directly derived from human immunoglobulins and light chain constant regions obtained by modifying or altering light chain constant regions derived from human immunoglobulins (e.g., light chain constant region variants).

[0035] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain constant region of human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or,

[0036] The light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a light chain constant region of human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids). The substitutions may be conserved or non-conserved.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises the heavy chain constant region of wild-type human immunoglobulin.

[0038] In other embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a variant of the heavy chain constant region of a human immunoglobulin, the variant of which may have the same or substantially the same characteristics as its derived wild-type sequence. In some embodiments, the variant of the heavy chain constant region may have one or more conserved substitutions of amino acids compared to its derived sequence.

[0039] In other embodiments, the antibody or antigen-binding fragment capable of specifically binding TSHR comprises a variant of the heavy chain constant region of a human immunoglobulin. This variant may contain one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibody or antigen-binding fragment capable of specifically binding TSHR: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function. Functional alterations can be produced by replacing at least one amino acid residue in the constant region of the antibody or antigen-binding fragment capable of specifically binding TSHR with a different residue or by chemical modification. For example, altering the antibody's affinity for effector ligands (such as FcγR, FcRn, or complement C1q) can alter (e.g., decrease or enhance) effector functions (e.g., the Fc region of the antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, FcRn binding, etc.).

[0040] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a variant of the constant region of the human immunoglobulin heavy chain, which may have reduced or eliminated effector functions compared to the wild-type sequence from which it is derived (e.g., the Fc region of the antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, FcRn binding, etc.).

[0041] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR contains a variant of the constant region of the human immunoglobulin heavy chain, which may have enhanced effector functions compared to the wild-type sequence from which it is derived (e.g., the Fc region of the antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, FcRn binding, etc.).

[0042] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR comprises a constant region of human immunoglobulin heavy chain or a variant thereof, which may have the same or substantially the same effector functions as the wild-type sequence from which it is derived (e.g., the Fc region of the antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, FcRn binding, etc.).

[0043] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain constant region derived from human immunoglobulin IgG4, and / or, the light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a light chain constant region derived from human immunoglobulin (e.g., a light chain constant region of the κ or λ chain).

[0044] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain constant region derived from human immunoglobulin IgG4, and / or, the light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a light chain constant region derived from the human immunoglobulin κ chain.

[0045] In some embodiments, the heavy chain constant region is selected from the heavy chain constant region of wild-type human immunoglobulin IgG4 and its variants. In some embodiments, the heavy chain constant region variants have altered effector functions compared to the heavy chain constant region of wild-type human immunoglobulin IgG4.

[0046] In some embodiments, the heavy chain constant region variant has enhanced FcRn binding activity compared to the heavy chain constant region of wild-type human immunoglobulin IgG4.

[0047] In some embodiments, the heavy chain constant region variant contains the substitution mutation S228P compared to the heavy chain constant region of wild-type human immunoglobulin IgG4. In some embodiments, the amino acid position of the substitution mutation is defined by the EU numbering system.

[0048] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR includes a heavy chain constant region (CH) as shown in SEQ ID NO:9, and / or, the light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR includes a light chain constant region (CL) as shown in SEQ ID NO:10.

[0049] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises:

[0050] A heavy chain comprising the sequence shown in SEQ ID NO:11, and / or a light chain comprising the sequence shown in SEQ ID NO:12.

[0051] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises murine antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, or multispecific antibodies.

[0052] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody, and sdAb.

[0053] In some implementations, administration of the antibody or its antigen-binding fragment that specifically binds to TSHR does not cause hypothyroidism or subclinical hypothyroidism in the subject.

[0054] In some implementations, the method avoids one or more side effects caused by the administration of TSHR inhibitors.

[0055] In some embodiments, the TSHR inhibitor is a TSHR-inhibiting antibody or antigen-binding fragment thereof other than the antibodies or antigen-binding fragments thereof that are capable of specifically binding to TSHR as defined above.

[0056] In some implementations, one or more side effects include causing the subject to develop hypothyroidism or subclinical hypothyroidism.

[0057] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is administered once every 4-24 weeks (e.g., every 4-20 weeks, every 4-16 weeks, every 4-12 weeks, every 4-8 weeks, every 8-24 weeks, every 8-20 weeks, every 8-16 weeks, every 8-12 weeks, every 12-24 weeks, every 12-20 weeks, every 12-16 weeks).

[0058] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is administered once every 4-24 weeks (e.g., every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, every 16 weeks, every 17 weeks, every 18 weeks, every 19 weeks, every 20 weeks, every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks).

[0059] In some embodiments, the antibody or its antigen-binding fragment that is capable of specifically binding to TSHR is administered once every 12 weeks (e.g., every 3 months).

[0060] In some implementations, the antibody that specifically binds to TSHR or its antigen-binding fragment is administered every 4-12 weeks before the subject achieves 40%-70% improvement in symptoms of the autoimmune thyroid disease, or within 0-24 weeks of administration.

[0061] In some implementations, the antibody or antigen-binding fragment that specifically binds to TSHR is administered every 12-24 weeks after the subject has achieved a 40%-70% improvement in symptoms of the autoimmune thyroid disease, or after 24 weeks of administration of the antibody or antigen-binding fragment that specifically binds to TSHR.

[0062] In some embodiments, the single-dose administration of the antibody or its antigen-binding fragment capable of specifically binding to TSHR is 0.1-20 mg / kg (e.g., 0.1-16 mg / kg, 0.1-12 mg / kg, 0.1-10 mg / kg, 0.1-7.5 mg / kg, 0.1-5 mg / kg, 0.1-3 mg / kg, 0.1-1 mg / kg, 0.5-20 mg / kg, 0.5-16 mg / kg, 0.5-12 mg / kg, 0.5-10 mg / kg). g / kg, 0.5-7.5mg / kg, 0.5-5mg / kg, 0.5-3mg / kg, 0.5-1mg / kg, 1-20mg / kg, 1-16mg / kg, 1-12mg / kg, 1-10mg / kg , 1-7.5mg / kg, 1-5mg / kg, 1-3mg / kg, 3-20mg / kg, 3-16mg / kg, 3-12mg / kg, 3-10mg / kg, 3-7.5mg / kg, 3-5mg / kg).

[0063] In some embodiments, the single dose of the antibody or its antigen-binding fragment that specifically binds to TSHR is 0.1-12 mg / kg.

[0064] In some embodiments, the single dose of the antibody or antigen-binding fragment specifically binding to TSHR is 0.1-12 mg / kg, prior to a 40%-70% improvement in the subject's symptoms of the autoimmune thyroid disease, or within 0-24 weeks of administration of the antibody or antigen-binding fragment specifically binding to TSHR.

[0065] In some embodiments, after the subject achieves a 40%-70% improvement in symptoms of the autoimmune thyroid disease, or after 24 weeks of administration of the antibody or antigen-binding fragment thereof that specifically binds to TSHR, the single dose of the antibody or antigen-binding fragment thereof that specifically binds to TSHR is 0.1-7.5 mg / kg.

[0066] In some embodiments, the total dose of the antibody or its antigen-binding fragment that specifically binds to TSHR is administered periodically (e.g., annually, semi-annually, or every 3 months) at a dose of 0.4-48 mg / kg.

[0067] In some embodiments, the total annual dose of the antibody or its antigen-binding fragment that specifically binds to TSHR is 0.4-48 mg / kg.

[0068] In some implementations, the total amount of the antibody or its antigen-binding fragment that specifically binds to TSHR administered every six months is 0.2-24 mg / kg.

[0069] In some embodiments, the total amount of the antibody or its antigen-binding fragment that specifically binds to TSHR is administered every 3 months in the form of 0.1-12 mg / kg.

[0070] As used herein, the phrase "40%-70% improvement in symptoms of autoimmune thyroid disease" describes the degree of improvement in symptoms of autoimmune thyroid disease in subjects that can be observed or detected. Those skilled in the art will readily understand that the degree of improvement in symptoms of autoimmune thyroid disease can be assessed based on a variety of physiological or clinical indicators (e.g., thyroid function indicators (T3 levels, T4 levels), autoantibody levels (e.g., anti-TSHR antibodies, anti-Tg antibodies, anti-TPO antibodies), symptom rating scales, etc.).

[0071] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is administered for a period selected from 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 40 weeks, 80 weeks, 120 weeks, 150 weeks, 200 weeks, 250 weeks, 300 weeks or longer.

[0072] The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR can be administered by any suitable method known in the art.

[0073] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is administered to the subject via a route selected from:

[0074] Intravenous, intra-arterial, transdermal, sublingual, oral, intraperitoneal, intramuscular, rectal, intranasal, inhalation, intraocular, subcutaneous, intra-fat, intra-articular, intrathecal, and any combination thereof.

[0075] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is administered to the subject via intravenous or subcutaneous injection.

[0076] In some implementations, the subject is selected from mammals.

[0077] In some implementations, the subject is a human being.

[0078] In some embodiments, the antibody or antigen-binding fragment of which is capable of specifically binding to TSHR may be administered in combination with other pharmaceutically active agents (e.g., other drugs for treating autoimmune thyroid diseases such as Graves' disease or thyroid-associated ophthalmopathy), for example, simultaneously or sequentially.

[0079] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR may be administered in combination with an IGF1R antagonist or inhibitor and / or another TSHR antagonist or inhibitor, for example, simultaneously or sequentially.

[0080] In another aspect, this application provides the use of an antibody or antigen-binding fragment thereof capable of specifically binding to TSHR as defined in the first aspect in the preparation of a medicament for the prevention and / or treatment of autoimmune thyroid diseases.

[0081] In some implementations, the drug is used to prevent and / or treat autoimmune thyroid disease in subjects via the method of the first aspect.

[0082] In some embodiments, the drug preparation is in the form of a kit containing instructions describing the method of the first aspect.

[0083] In some embodiments, the drug is prepared as a kit containing a dosing unit (e.g., a single-dose dosing unit, a daily dosing unit, a weekly dosing unit, a monthly dosing unit, a dosing unit every two months, a dosing unit every three months, or a dosing unit every six months) of the antibody capable of specifically binding to TSHR.

[0084] In some implementations, the subject contains an autoantibody (e.g., a stimulatory TRAb) capable of activating TSHR.

[0085] In some implementations, the subject contains an amount of autoantibodies (e.g., stimulatory TRAb) that can activate TSHR, exceeding a reference level (e.g., healthy control level).

[0086] In some embodiments, the autoimmune thyroid disease is selected from: Graves' disease, Graves' ophthalmopathy, thyroid-associated ophthalmopathy, pretibial myxedema, Hashimoto's thyroiditis, thyroid cancer, and any combination thereof.

[0087] In some implementations, the autoimmune thyroid disease is Graves' disease.

[0088] In some implementations, the autoimmune thyroid disease is thyroid-associated ophthalmopathy.

[0089] In another aspect, this application also provides the use of antibodies or antigen-binding fragments thereof capable of specifically binding to TSHR as defined in the first aspect in the preparation of regimens for the prevention and / or treatment of autoimmune thyroid diseases.

[0090] In some implementations, the protocol includes a method of administering the first aspect to the subject to whom it is thus required.

[0091] In some implementations, administration of the regimen does not cause hypothyroidism or subclinical hypothyroidism in the subject.

[0092] In some implementations, the subject contains an autoantibody (e.g., a stimulatory TRAb) capable of activating TSHR.

[0093] In some implementations, the subject contains an amount of autoantibodies (e.g., stimulatory TRAb) that can activate TSHR, exceeding a reference level (e.g., healthy control level).

[0094] In some embodiments, the autoimmune thyroid disease is selected from: Graves' disease, Graves' ophthalmopathy, thyroid-associated ophthalmopathy, pretibial myxedema, Hashimoto's thyroiditis, thyroid cancer, and any combination thereof.

[0095] In some implementations, the autoimmune thyroid disease is Graves' disease.

[0096] In some implementations, the autoimmune thyroid disease is thyroid-associated ophthalmopathy.

[0097] In a second aspect, this application provides a method for preventing and / or treating autoimmune thyroid disease, comprising administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof capable of specifically binding to TSHR.

[0098] The antibody or its antigen-binding fragment that can specifically bind to TSHR includes: VH CDR1, VH CDR2 and VH CDR3 contained in the heavy chain variable region (VH) as shown in SEQ ID NO:7; and / or, VL CDR1, VL CDR2 and VL CDR3 contained in the light chain variable region (VL) as shown in SEQ ID NO:8;

[0099] The antibody or its antigen-binding fragment that specifically binds to TSHR is administered once every 4-24 weeks (e.g., every 4-20 weeks, every 4-16 weeks, every 4-12 weeks, every 4-8 weeks, every 8-24 weeks, every 8-20 weeks, every 8-16 weeks, every 8-12 weeks, every 12-24 weeks, every 12-20 weeks, every 12-16 weeks).

[0100] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is administered once every 4-24 weeks (e.g., every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, every 16 weeks, every 17 weeks, every 18 weeks, every 19 weeks, every 20 weeks, every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks).

[0101] In some embodiments, the antibody or its antigen-binding fragment that is capable of specifically binding to TSHR is administered once every 12 weeks (e.g., every 3 months).

[0102] In some implementations, the CDRs are defined by the Kabat, IMGT, Chothia, or AbM numbering systems.

[0103] In some embodiments, the method does not include administering to the subject drugs and / or therapies for the prevention and / or treatment of hypothyroidism and / or subclinical hypothyroidism. For example, the antibody capable of specifically binding to TSHR or its antigen-binding fragment is not administered concurrently with, or subsequently with, drugs or therapies for the prevention and / or treatment of hypothyroidism and / or subclinical hypothyroidism.

[0104] Based on the disclosure of this application, those skilled in the art will readily understand that the methods provided in this aspect do not include administering drugs and / or therapies for the prevention and / or treatment of hypothyroidism to the subject, or, the methods provided in this aspect do not include administering drugs and / or therapies for the prevention and / or treatment of subclinical hypothyroidism to the subject, or, the methods provided in this aspect do not include administering drugs and / or therapies for the prevention and / or treatment of hypothyroidism to the subject, nor do they include administering drugs and / or therapies for the prevention and / or treatment of subclinical hypothyroidism to the subject.

[0105] In some implementations, the method of this aspect does not include administering drugs and / or therapies for the prevention and / or treatment of hypothyroidism to the subject.

[0106] In some embodiments, the medication for preventing and / or treating hypothyroidism and / or subclinical hypothyroidism includes levothyroxine tablets (Euthyrox), and / or the therapy for preventing and / or treating hypothyroidism and / or subclinical hypothyroidism includes administering levothyroxine tablets (Euthyrox) to the subject.

[0107] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is the only pharmaceutically active agent administered to the subject for the prevention and / or treatment of the autoimmune thyroid disease.

[0108] In some implementations, the subject contains an autoantibody (e.g., a stimulatory TRAb) capable of activating TSHR.

[0109] In some implementations, the subject contains an amount of autoantibodies (e.g., stimulatory TRAb) that can activate TSHR, exceeding a reference level (e.g., healthy control level).

[0110] In some embodiments, the autoimmune thyroid disease is selected from: Graves' disease, Graves' ophthalmopathy, thyroid-associated ophthalmopathy, pretibial myxedema, Hashimoto's thyroiditis, thyroid cancer, and any combination thereof.

[0111] In some implementations, the autoimmune thyroid disease is Graves' disease.

[0112] In some implementations, the autoimmune thyroid disease is thyroid-associated ophthalmopathy.

[0113] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises:

[0114] (a) Heavy chain variable regions (VH) comprising VH CDR1, VH CDR2, and VH CDR3 as shown in SEQ ID NOs:1-3, respectively; and / or,

[0115] (b) Light chain variable regions (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in SEQ ID NOs:4-6 respectively.

[0116] In some implementations, the CDRs are defined by the Kabat numbering system.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a framework region sequence derived from human immunoglobulin.

[0118] In some embodiments, the antibody or antigen-binding fragment capable of specifically binding to TSHR comprises a framework region contained in the amino acid sequence encoded by a human germline antibody gene. In some embodiments, the antibody or antigen-binding fragment capable of specifically binding to TSHR comprises: a heavy chain framework region of a human heavy chain germline sequence, and / or, a light chain framework region of a human light chain germline sequence.

[0119] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises:

[0120] (1) Heavy chain variable region (VH), which contains the sequence shown in SEQ ID NO: 7 or a variant thereof; and / or,

[0121] (2) Light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 8 or a variant thereof;

[0122] The variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence, or has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the substitutions are conservative substitutions.

[0123] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain variable region (VH) as shown in SEQ ID NO:7 and a light chain variable region (VL) as shown in SEQ ID NO:8.

[0124] In some embodiments, the antibody or its antigen-binding fragment that is capable of specifically binding to TSHR further comprises a constant region derived from mammalian (e.g., human) immunoglobulins.

[0125] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain constant region derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4), and / or, the light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a light chain constant region derived from human immunoglobulins (e.g., a light chain constant region of the κ or λ chain).

[0126] Those skilled in the art will readily understand that the heavy chain constant region derived from human immunoglobulins includes both heavy chain constant regions directly derived from human immunoglobulins and heavy chain constant regions obtained by modifying or altering heavy chain constant regions derived from human immunoglobulins (e.g., heavy chain constant region variants); the light chain constant region derived from human immunoglobulins includes both light chain constant regions directly derived from human immunoglobulins and light chain constant regions obtained by modifying or altering light chain constant regions derived from human immunoglobulins (e.g., light chain constant region variants).

[0127] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain constant region of human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or,

[0128] The light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a light chain constant region of human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids). The substitutions may be conserved or non-conserved.

[0129] In some embodiments, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises the heavy chain constant region of wild-type human immunoglobulin.

[0130] In other embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a variant of the heavy chain constant region of a human immunoglobulin, the variant of which may have the same or substantially the same characteristics as its derived wild-type sequence. In some embodiments, the variant of the heavy chain constant region may have one or more conserved substitutions of amino acids compared to its derived sequence.

[0131] In other embodiments, the antibody or antigen-binding fragment capable of specifically binding TSHR comprises a variant of the heavy chain constant region of a human immunoglobulin. This variant may contain one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibody or antigen-binding fragment capable of specifically binding TSHR: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function. Functional alterations can be produced by replacing at least one amino acid residue in the constant region of the antibody or antigen-binding fragment capable of specifically binding TSHR with a different residue or by chemical modification. For example, altering the antibody's affinity for effector ligands (such as FcγR, FcRn, or complement C1q) can alter (e.g., decrease or enhance) effector functions (e.g., the Fc region of the antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, FcRn binding, etc.).

[0132] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a variant of the constant region of the human immunoglobulin heavy chain, which may have reduced or eliminated effector functions compared to the wild-type sequence from which it is derived (e.g., the Fc region of the antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, FcRn binding, etc.).

[0133] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR contains a variant of the constant region of the human immunoglobulin heavy chain, which may have enhanced effector functions compared to the wild-type sequence from which it is derived (e.g., the Fc region of the antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, FcRn binding, etc.).

[0134] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR comprises a constant region of human immunoglobulin heavy chain or a variant thereof, which may have the same or substantially the same effector functions as the wild-type sequence from which it is derived (e.g., the Fc region of the antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, FcRn binding, etc.).

[0135] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain constant region derived from human immunoglobulin IgG4, and / or, the light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a light chain constant region derived from human immunoglobulin (e.g., a light chain constant region of the κ or λ chain).

[0136] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a heavy chain constant region derived from human immunoglobulin IgG4, and / or, the light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises a light chain constant region derived from the human immunoglobulin κ chain.

[0137] In some embodiments, the heavy chain constant region is selected from the heavy chain constant region of wild-type human immunoglobulin IgG4 and its variants. In some embodiments, the heavy chain constant region variants have altered effector functions compared to the heavy chain constant region of wild-type human immunoglobulin IgG4.

[0138] In some embodiments, the heavy chain constant region variant has enhanced FcRn binding activity compared to the heavy chain constant region of wild-type human immunoglobulin IgG4.

[0139] In some embodiments, the heavy chain constant region variant contains the substitution mutation S228P compared to the heavy chain constant region of wild-type human immunoglobulin IgG4. In some embodiments, the amino acid position of the substitution mutation is defined by the EU numbering system.

[0140] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR includes a heavy chain constant region (CH) as shown in SEQ ID NO:9, and / or, the light chain of the antibody or its antigen-binding fragment capable of specifically binding to TSHR includes a light chain constant region (CL) as shown in SEQ ID NO:10.

[0141] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises:

[0142] A heavy chain comprising the sequence shown in SEQ ID NO:11, and / or a light chain comprising the sequence shown in SEQ ID NO:12.

[0143] In some embodiments, the antibody or its antigen-binding fragment capable of specifically binding to TSHR comprises murine antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, or multispecific antibodies.

[0144] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody, and sdAb.

[0145] In some implementations, administration of the antibody or its antigen-binding fragment that specifically binds to TSHR does not cause hypothyroidism or subclinical hypothyroidism in the subject.

[0146] In some implementations, the method avoids one or more side effects caused by the administration of TSHR inhibitors.

[0147] In some embodiments, the TSHR inhibitor is a TSHR-inhibiting antibody or antigen-binding fragment thereof other than the antibodies or antigen-binding fragments thereof that are capable of specifically binding to TSHR as defined above.

[0148] In some implementations, one or more side effects include causing the subject to develop hypothyroidism or subclinical hypothyroidism.

[0149] In some implementations, the antibody that specifically binds to TSHR or its antigen-binding fragment is administered every 4-12 weeks before the subject achieves 40%-70% improvement in symptoms of the autoimmune thyroid disease, or within 0-24 weeks of administration.

[0150] In some implementations, the antibody or antigen-binding fragment that specifically binds to TSHR is administered every 12-24 weeks after the subject has achieved a 40%-70% improvement in symptoms of the autoimmune thyroid disease, or after 24 weeks of administration of the antibody or antigen-binding fragment that specifically binds to TSHR.

[0151] In some embodiments, the single-dose administration of the antibody or its antigen-binding fragment capable of specifically binding to TSHR is 0.1-20 mg / kg (e.g., 0.1-16 mg / kg, 0.1-12 mg / kg, 0.1-10 mg / kg, 0.1-7.5 mg / kg, 0.1-5 mg / kg, 0.1-3 mg / kg, 0.1-1 mg / kg, 0.5-20 mg / kg, 0.5-16 mg / kg, 0.5-12 mg / kg, 0.5-10 mg / kg). g / kg, 0.5-7.5mg / kg, 0.5-5mg / kg, 0.5-3mg / kg, 0.5-1mg / kg, 1-20mg / kg, 1-16mg / kg, 1-12mg / kg, 1-10mg / kg , 1-7.5mg / kg, 1-5mg / kg, 1-3mg / kg, 3-20mg / kg, 3-16mg / kg, 3-12mg / kg, 3-10mg / kg, 3-7.5mg / kg, 3-5mg / kg).

[0152] In some embodiments, the single dose of the antibody or its antigen-binding fragment that specifically binds to TSHR is 0.1-12 mg / kg.

[0153] In some embodiments, the single dose of the antibody or antigen-binding fragment specifically binding to TSHR is 0.1-12 mg / kg, prior to a 40%-70% improvement in the subject's symptoms of the autoimmune thyroid disease, or within 0-24 weeks of administration of the antibody or antigen-binding fragment specifically binding to TSHR.

[0154] In some embodiments, after the subject achieves a 40%-70% improvement in symptoms of the autoimmune thyroid disease, or after 24 weeks of administration of the antibody or antigen-binding fragment thereof that specifically binds to TSHR, the single dose of the antibody or antigen-binding fragment thereof that specifically binds to TSHR is 0.1-7.5 mg / kg.

[0155] In some embodiments, the total dose of the antibody or its antigen-binding fragment that specifically binds to TSHR is administered periodically (e.g., annually, semi-annually, or every 3 months) at a dose of 0.4-48 mg / kg.

[0156] In some embodiments, the total annual dose of the antibody or its antigen-binding fragment that specifically binds to TSHR is 0.4-48 mg / kg.

[0157] In some implementations, the total amount of the antibody or its antigen-binding fragment that specifically binds to TSHR administered every six months is 0.2-24 mg / kg.

[0158] In some embodiments, the total amount of the antibody or its antigen-binding fragment that specifically binds to TSHR is administered every 3 months in the form of 0.1-12 mg / kg.

[0159] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is administered for a period selected from 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 40 weeks, 80 weeks, 120 weeks, 150 weeks, 200 weeks, 250 weeks, 300 weeks or longer.

[0160] The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR can be administered by any suitable method known in the art.

[0161] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is administered to the subject via a route selected from:

[0162] Intravenous, intra-arterial, transdermal, sublingual, oral, intraperitoneal, intramuscular, rectal, intranasal, inhalation, intraocular, subcutaneous, intra-fat, intra-articular, intrathecal, and any combination thereof.

[0163] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR is administered to the subject via intravenous or subcutaneous injection.

[0164] In some implementations, the subject is selected from mammals.

[0165] In some implementations, the subject is a human being.

[0166] In some embodiments, the antibody or antigen-binding fragment of which is capable of specifically binding to TSHR may be administered in combination with other pharmaceutically active agents (e.g., other drugs for treating autoimmune thyroid diseases such as Graves' disease or thyroid-associated ophthalmopathy), for example, simultaneously or sequentially.

[0167] In some embodiments, the antibody or its antigen-binding fragment that specifically binds to TSHR may be administered in combination with an IGF1R antagonist or inhibitor and / or another TSHR antagonist or inhibitor, for example, simultaneously or sequentially.

[0168] In another aspect, this application provides the use of an antibody or antigen-binding fragment thereof capable of specifically binding to TSHR as defined in the second aspect in the preparation of a medicament for the prevention and / or treatment of autoimmune thyroid diseases.

[0169] In some implementations, the drug is used to prevent and / or treat autoimmune thyroid disease in subjects via a second aspect of the method.

[0170] In some embodiments, the drug preparation is in the form of a kit containing instructions describing the method of the second aspect.

[0171] In some embodiments, the drug is prepared as a kit containing a dosing unit (e.g., a single-dose dosing unit, a daily dosing unit, a weekly dosing unit, a monthly dosing unit, a dosing unit every two months, a dosing unit every three months, or a dosing unit every six months) of the antibody capable of specifically binding to TSHR.

[0172] In some implementations, the subject contains an autoantibody (e.g., a stimulatory TRAb) capable of activating TSHR.

[0173] In some implementations, the subject contains an amount of autoantibodies (e.g., stimulatory TRAb) that can activate TSHR, exceeding a reference level (e.g., healthy control level).

[0174] In some embodiments, the autoimmune thyroid disease is selected from: Graves' disease, Graves' ophthalmopathy, thyroid-associated ophthalmopathy, pretibial myxedema, Hashimoto's thyroiditis, thyroid cancer, and any combination thereof.

[0175] In some implementations, the autoimmune thyroid disease is Graves' disease.

[0176] In some implementations, the autoimmune thyroid disease is thyroid-associated ophthalmopathy.

[0177] In another aspect, this application also provides the use of antibodies or antigen-binding fragments thereof capable of specifically binding to TSHR as defined in the second aspect in the preparation of regimens for the prevention and / or treatment of autoimmune thyroid diseases.

[0178] In some implementations, the protocol includes a method of administering the second aspect to the subject who requires it.

[0179] In some implementations, administration of the regimen does not cause hypothyroidism or subclinical hypothyroidism in the subject.

[0180] In some implementations, the subject contains an autoantibody (e.g., a stimulatory TRAb) capable of activating TSHR.

[0181] In some implementations, the subject contains an amount of autoantibodies (e.g., stimulatory TRAb) that can activate TSHR, exceeding a reference level (e.g., healthy control level).

[0182] In some embodiments, the autoimmune thyroid disease is selected from: Graves' disease, Graves' ophthalmopathy, thyroid-associated ophthalmopathy, pretibial myxedema, Hashimoto's thyroiditis, thyroid cancer, and any combination thereof.

[0183] In some implementations, the autoimmune thyroid disease is Graves' disease.

[0184] In some implementations, the autoimmune thyroid disease is thyroid-associated ophthalmopathy.

[0185] The antibodies or antigen-binding fragments of the present invention capable of specifically binding to TSHR can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The antibodies or antigen-binding fragments of the present invention capable of specifically binding to TSHR should be sterile and stable under manufacturing and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the necessary dose of the antibody or antigen-binding fragment of the present invention capable of specifically binding to TSHR into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0186] Terminology Definition

[0187] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0188] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0189] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0190] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen via at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context explicitly indicates otherwise, it includes not only the complete antibody but also the antigen-binding fragment capable of specifically binding to a target antigen. A "complete antibody" typically consists of two pairs of polypeptide chains, each pair comprising one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0191] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272), or the IMGT numbering system (Lefranc et al.). The definitions in Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003 are provided. For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0192] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat, Chothia, AbM, or IMGT numbering systems.

[0193] As used herein, the term “frame region” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0194] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0195] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). The term "multispecific antibody" refers to an antibody that has binding specificity to at least two (e.g., three or four) different antigens (or epitopes). Bispecific or multispecific antibodies comprise multiple antigen-binding domains that have binding specificity to different antigens (or epitopes), thereby enabling them to bind to at least two different binding sites and / or target molecules. The individual antigen-binding domains comprised in a bispecific or multispecific antibody may be independently selected from full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., Fv fragments, Fab fragments, F(ab')2 fragments, or scFv). In some cases, the individual antigen-binding domains are linked by peptide linkers.

[0196] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). In some embodiments, the CDR region of the humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies typically retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, etc. In this application, the donor antibody may be a murine antibody with the intended properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare a humanized antibody, the CDR region of the donor antibody can be inserted into a human frame sequence using methods known in the art. In some cases, the human frame sequence may contain amino acid mutations substituted by corresponding non-human residues. Furthermore, the humanized antibody may also contain residues not found in either the variable region of the initial donor antibody (e.g., the light chain variable region or the heavy chain variable region) or the human frame sequence, to further improve or optimize the performance of the humanized antibody.

[0197] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In some embodiments, the term "chimeric antibody" may include an antibody in which the variable regions of the heavy chain and light chain are derived from a first antibody, while the constant regions of the heavy chain and light chain are derived from a second antibody.

[0198] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety”. See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, diabody, single-domain antibody, and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0199] As used herein, the term "Fd" refers to an antibody fragment consisting of VH and CH1 domains; the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region; and the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0200] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0201] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some embodiments of the present invention, scFv can form di-scFv, which refers to two or more individual scFvs linked together to form an antibody. In some embodiments of the present invention, scFv can form (scFv)2, which refers to two or more individual scFvs linked together in parallel to form an antibody.

[0202] As used herein, the term “biantibody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0203] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0204] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0205] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0206] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of that interaction. In this invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0207] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the “binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of KD, kon, and kdis can be measured using any effective method. In some embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). Alternatively, bioluminescent interferometry or Kinexa can be used to measure the dissociation constant.

[0208] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length.

[0209] The determination of percentage identity between two sequences can also be achieved using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as an improvement upon that in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are integrated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0210] As used herein, the term "variant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide containing an amino acid sequence altered by the introduction of amino acid residue substitutions, deletions, or additions. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, polypeptides can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, variants have similar, identical, or improved functions to the polypeptide or peptide from which they are derived.

[0211] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0212] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0213] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0214] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (e.g., survival without treatment).

[0215] As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject (e.g., a human) has an autoimmune thyroid disease (e.g., Graves' disease, thyroid-associated ophthalmopathy), or is at risk of having such a disease.

[0216] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., autoimmune thyroid disease) means an amount sufficient to prevent, stop, or delay the onset of said disease; an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0217] Beneficial effects of the invention

[0218] The TSHR inhibitory antibody provided in this application can effectively inhibit or block the activation activity of stimulating TSHR antibody (TRAb), and can effectively inhibit the production of thyroid catalase (TPO). It has been shown to effectively inhibit the secretion of thyroid hormone (T4) in acute GD model mice, thus making it effective for the treatment of autoimmune thyroid diseases. Furthermore, unlike existing GD therapeutic antibodies, the TSHR inhibitory antibody provided in this application does not cause hypothyroidism or subacute hypothyroidism, thus eliminating the need for combination therapy with drugs for treating hypothyroidism or subacute hypothyroidism. In addition, the TSHR inhibitory antibody provided in this application has a significantly prolonged in vivo half-life, expected to achieve a human dosing interval of once every 3 months.

[0219] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0220] Figure 1 Results of the assay of the activity of anti-TSHR antibody in inhibiting T4 production in acute GD model animals.

[0221] Figure 2 Results of T4 content detection of anti-TSHR antibody in normal monkeys.

[0222] Figure 3 Results of T3 content detection of anti-TSHR antibody in normal monkeys.

[0223] Figure 4 Results of TSH content detection in normal monkeys using anti-TSHR antibodies.

[0224] Figure 5 Pharmacokinetic results of anti-TSHR antibody in mice.

[0225] Figure 6 Pharmacokinetic results of anti-TSHR antibody in cynomolgus monkeys.

[0226] Sequence information

[0227] The descriptions of the sequences involved in this application are provided in the table below.

[0228] Table 1: Sequence Information

[0229]

[0230]

[0231] Detailed Implementation

[0232] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0233] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0234] The anti-TSHR monoclonal antibody hAb01_G4p used in the embodiments of this application is derived from patent application WO 2024 / 046384A1, the entire contents of which are incorporated herein by reference.

[0235] The VH CDRs and VL CDRs (defined by the Kabat numbering system) contained in the anti-TSHR monoclonal antibody hAb01_G4p are shown in SEQ ID NOs:1-6; its heavy chain variable region and light chain variable region are shown in SEQ ID NO:7 and 8, respectively; its full-length heavy chain and full-length light chain are shown in SEQ ID NO:11 and 12, respectively.

[0236] Example 1: Anti-TSHR antibody inhibits the activity of primary thyroid cells in producing TPO.

[0237] Experimental methods:

[0238] 1. Extraction and culture of primary thyroid cells from patients

[0239] Thyroid tissue samples were obtained from normal thyroid tissue of patients who underwent total thyroidectomy for thyroid cancer. The tissue was resuspended in PBS solution containing 3 mg / mL type IV collagenase (Life Technologies) to obtain dispersed cells, which were then passaged and amplified in DMEM medium containing 10% PBS.

[0240] 2. TPO testing

[0241] Cells from each group were collected, and after adjusting the density, 6 × 10⁶ cells were seeded into each well of a 24-well plate. 4 Cells were cultured overnight. After adhesion, they were starved for 24 hours, the supernatant was discarded, and different concentrations of the test antibody and culture medium containing or without GD patient serum (final concentration 1:100) were added according to the experimental groups. Cells were incubated for another 48 hours, and cells were collected using trypsin to extract total RNA samples. The reverse transcription reaction system was prepared according to the instructions of the kit (iScript cDNA, Bio-Rad, 1708891EDU) to obtain cDNA, which was stored at -70℃.

[0242] 3. Real-time quantitative PCR reaction

[0243] The reaction system was prepared on an ice box according to the reagent (Takara, RR420A) instructions, and the cDNA obtained in the previous step was amplified. The data were processed using the 2-ΔΔCt formula, and the expression level of the target mRNA was obtained with the internal reference gene GAPDH as a reference. The primers used are shown in Table 2.

[0244] Table 2: Primers used in real-time quantitative PCR

[0245]

[0246] Data processing and experimental data:

[0247] Data were analyzed and plotted using Graphpad Prism 9 (Version 9.4.0), and compiled using Adobe Illustrator 2022 (Version 2022). One-way ANOVA was used to analyze statistical differences between groups.

[0248]

[0249] The positive control antibody Tab03 is a TSHR inhibitory antibody, consistent with the variable region sequence of K1-70 in patent US9073992B2. The heavy chain variable region sequence originates from sequence number No. 51 in patent US9073992B2, and the light chain variable region sequence originates from sequence number No. 69 in patent US9073992B2. The heavy chain constant region and light chain constant region are the hIgG1 heavy chain constant region (SEQ ID NO ID: 17) and the Lambda light chain constant region (SEQ ID NO ID: 18), respectively. The negative control antibodies hIgG1 (BIOINTRON, B117901) and hIgG4 (BIOINTRON, B107804) are unrelated negative control antibodies. The negative control group (Control) is a control group that does not contain serum from GD patients or the test antibody.

[0250] The experimental results are shown in Table 3.

[0251] Table 3: Activity of anti-TSHR antibody in inhibiting TPO production by primary thyroid cells

[0252]

[0253]

[0254] Experimental conclusions: Negative control molecules hIgG1 and hIgG4 had no therapeutic effect on thyroid cells induced by serum from GD patients; sample molecules Tab03 and hAb01_G4p significantly inhibited TPO mRNA production in primary thyroid cells stimulated by serum from GD patients in a dose-dependent manner, and at the same dose, hAb01_G4p was more effective than Tab03 in inhibiting TPO mRNA production in primary thyroid cells stimulated by serum from GD patients.

[0255] Example 2: Anti-TSHR antibody inhibits the activity of T4 production in acute GD model animals.

[0256] Modeling principle: Tab01 is an antibody isolated from patients with Graves' disease and hyperthyroidism, possessing thyroid-stimulating activity. In mice, administration of Tab01 rapidly induces an increase in the thyroid hormone T4.

[0257] Experimental Methods: 7-8 week old female Balb / C mice (source: Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were used in the experiment. The modeling method was the Tab01 antibody stimulation method: From Day 4 before modeling, all animals were fed 3,3′,5-triiodo-L-thyroxine (T3, final concentration 3 μg / ml) through free access to water, with fresh T3-prepared drinking water changed every 2 days until the experimental endpoint. On the day of modeling (Day 0), the modeling reagent Tab01 antibody (0.5 μg / mouse, intramuscular injection) was administered for stimulation; within 10 minutes after Tab01 antibody injection, the experimental animals were treated with a single subcutaneous (SC) dose based on body weight. The model control group (Model) consisted of mice treated with both T3 and Tab01, while the negative control group consisted of mice treated with only T3. The experimental endpoint was defined as 24 hours after administration of the test substance. Blood was collected at the end of the experiment, serum was separated, and T4 levels in mouse serum samples were detected using a fully automated biochemical and immunoassay system (Roche cobas 8000).

[0258] Tab01 is a TSHR-stimulated antibody (M22). The heavy chain variable region and light chain variable region sequences are derived from the antibody heavy chain variable region with sequence number No.1 and the light chain variable region with sequence number No.6 in patent WO2004 / 050708A2. The heavy chain constant region and light chain constant region are the hIgG1 heavy chain constant region (SEQ ID NOID: 17) and the Lambda light chain constant region (SEQ ID NO ID: 18), respectively.

[0259] Data processing and experimental data:

[0260] T4 was described using mean ± standard error (XSEM). T4 was first tested for homogeneity of variance using the LEVENE test. If variances were unequal (P ≤ 0.05), the Kruskal-Wallis H rank-sum test (KW method) was used for statistical analysis. If the Kruskal-Wallis H rank-sum test showed statistical significance (P ≤ 0.05), the Mann-Whitney U test (MW method) was used to compare differences between groups. This experiment compared the differences between the model control group and the negative control group, as well as the differences between the hAb01_G4p dose group and the model control group.

[0261] All tests were two-tailed tests, with α = 0.05. Statistical analysis was performed using Stata / IC 15.0 for Windows. P ≤ 0.05 indicated a statistically significant difference; P ≤ 0.01 indicated a highly significant difference; and P ≤ 0.001 indicated an extremely significant difference.

[0262] Experimental results are as follows Figure 1 As shown.

[0263] Experimental conclusion: In Tab01-induced GD thyroid model mice, hAb01_G4p (0.2-7.5 mg / kg) showed dose-dependent T4 inhibitory activity, indicating that hAb01_G4p can block the activity of Tab01, making it a potential GD therapeutic drug that blocks the activation activity of autoantibodies.

[0264] Example 3: Performance of thyroid function in normal mice using anti-TSHR antibody

[0265] Experimental Methods: ICR mice (source: Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiment, with an equal number of males and females. The mice were administered medication subcutaneously. The dosage of hAb01_G4p was 0 (negative control), 20, 60, and 200 mg / kg, and the dosage of Tab03 was 60 mg / kg. Administration was once every two weeks. The experimental endpoint was day 28 of the administration period. Blood was collected at the endpoint, serum was separated, and T3 and T4 were detected using a competitive assay (using a cobas 8000 instrument), while TSH was detected using an ELISA kit (Abnova brand).

[0266] Data processing:

[0267] The quantitative indicators T3, T4, and TSH are described using the mean.

[0268] The experimental results are shown in Table 4.

[0269] Table 4: T3, T4, and TSH content in each group

[0270]

[0271] Experimental conclusions: The Tab03 group showed a significant increase in TSH and a decrease in T3 and T4, indicating hypothyroidism or subclinical hypothyroidism in the animals. The hAb01_G4p group showed no significant changes in T3, T4, or TSH, and the animals did not exhibit hypothyroidism or subclinical hypothyroidism.

[0272] Example 4: Performance of anti-TSHR antibody on thyroid function in normal monkeys

[0273] Experimental Methods: Cynomolgus monkeys (source: Guangxi Nanning Tongling Biotechnology Co., Ltd.), aged 3-5 years, all female, were used in the experiment. Three monkeys were in the hAb01_G4p group and two in the Tab03 group. The administration route was a single subcutaneous dose. The dosages for Tab03 and hAb01_G4p were 15 and 30 mg / kg, respectively. Blood samples were collected before administration and at 24h, 72h, 120h, 168h, 240h, and 336h after administration. Serum was separated, and T3 and T4 were detected using a competitive assay (using a cobas 8000 instrument), while TSH was detected by ELISA (using a FineTest kit).

[0274] Data processing:

[0275] The quantitative indicators T3, T4, and TSH are described using individual data.

[0276] According to historical statistical data provided by Chengdu Huaxi Haiqi Pharmaceutical Technology Co., Ltd., the background range of T4 in normal monkeys is (42.69~109.68 nmol / L), and the background range of T3 is (1.63~4.01 nmol / L).

[0277] Experimental results are as follows Figures 2-4 As shown.

[0278] Experimental conclusion:

[0279] In the Tab03 group, T3 and T4 were significantly decreased, and TSH was significantly increased, indicating hypothyroidism in the animals.

[0280] No significant changes were observed in T3, T4, and TSH in the hAb01_G4p group; all values ​​remained within the background range, and the animals did not exhibit hypothyroidism.

[0281] Example 5: Pharmacokinetic Study of Anti-TSHR Antibody in Mice

[0282] Experimental methods:

[0283] Thirty-two 7-8 week old ICR mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were divided into four groups of eight mice each, with half males and half females. In the hAb01_G4p intravenous injection group, hAb01_G4p was administered as a single intravenous injection of 3 mg / kg at 5 mL / kg. Blood samples were collected before administration and at 10 minutes, 24, 48, 72, 96, 168, 240, 336, 504, 672, 840, 1008, 1176, and 1344 hours after administration. In the hAb01_G4p subcutaneous injection low, medium, and high dose groups, hAb01_G4p was administered as a single subcutaneous injection of 1, 3, and 12 mg / kg at 5 mL / kg, respectively. Blood samples were collected before administration and at 6, 24, 48, 72, 96, 168, 240, 336, 504, 672, 840, 1008, 1176, and 1344 hours after administration. Serum drug concentrations were determined by ELISA, with a standard curve range of 78.1–5000 ng / mL and a limit of quantitation of 78.1 ng / mL. Pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2. Additionally, anti-drug antibodies (ADAs) in serum were detected before administration and at 15, 29, 43, and 57 days post-administration using Bridging-ELISA.

[0284] The administration method and dosage are shown in Table 5.

[0285] Table 5: Administration Method and Dosage

[0286]

[0287] Experimental results are as follows Figure 5 As shown in Table 6.

[0288]

[0289] Experimental conclusion:

[0290] hAb01_G4p was administered subcutaneously at doses of 1, 3, and 12 mg / kg. Serum drug concentrations increased with increasing dosage, reaching peak levels between 24.0 and 96.0 hours post-administration. The average serum drug concentration in ICR mice of each group was [not specified]. 1 / 2 The mean AUC of serum hAb01_G4p in each group was 341, 358, and 336 hours, respectively, within the dose range of 1–12 mg / kg. last Average AUC 0-∞ Average C max The increase was basically proportional to the increase in dosage, and there were no significant gender differences in the main pharmacokinetic parameters within each group.

[0291] After a single intravenous injection of 3 mg / kg hAb01_G4p, the average serum drug concentration (t) was... 1 / 2The duration of treatment was 327 hours. There were no significant differences in the main pharmacokinetic parameters between genders. The bioavailability of subcutaneous injection at this dose was 98.6%.

[0292] Example 6: Pharmacokinetic Study of Anti-TSHR Antibody in Monkeys

[0293] Experimental methods:

[0294] Twenty-four crab-eating macaques aged 3 to 6 years (purchased from Hainan Jingang Biotechnology Co., Ltd.) were divided into four groups of six, with half males and half females. In the hAb01_G4p intravenous injection group, hAb01_G4p was administered as a single intravenous injection of 0.12 mL / kg, with a dose of 3 mg / kg. Blood samples were collected before administration and at 10 minutes, 8, 24, 48, 72, 96, 120, 168, 240, 336, 504, 672, 840, 1008, 1176, and 1344 hours after administration. In the hAb01_G4p subcutaneous injection low, medium, and high dose groups, hAb01_G4p was administered as a single subcutaneous injection of 0.12 mL / kg, with doses of 1, 3, and 12 mg / kg, respectively. Blood samples were collected before administration and at 2, 8, 24, 48, 72, 96, 120, 168, 240, 336, 504, 672, 840, 1008, 1176, and 1344 hours after administration. Serum drug concentrations were determined using a validated ELISA method, with a standard curve range of 78.1–5000 ng / mL and a limit of quantitation of 78.1 ng / mL. Pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2. Additionally, serum anti-drug antibodies (ADAs) were detected using a validated Bridging-ELISA method before administration and at 14, 28, 42, and 56 days post-administration.

[0295] The administration method and dosage are shown in Table 7.

[0296] Table 7: Administration Method and Dosage

[0297]

[0298] Experimental results are as follows Figure 6 As shown in Table 8.

[0299]

[0300] Experimental conclusion:

[0301] hAb01_G4p was administered subcutaneously at doses of 1, 3, and 12 mg / kg. Serum drug concentrations increased with increasing dosage, reaching peak levels between 24.0 and 96.0 hours post-administration. The average serum drug concentration (t) in each group of cynomolgus monkeys was [not specified]. 1 / 2The mean AUC of serum hAb01_G4p in each group was 407, 457, and 479 h, respectively, within the dose range of 1–12 mg / kg. last Average AUC 0-∞ Average C max The increase was basically proportional to the increase in dosage, and there were no significant gender differences in the main pharmacokinetic parameters within each group.

[0302] After a single intravenous injection of 3 mg / kg hAb01_G4p, the average serum drug concentration (t) was... 1 / 2 The duration of treatment was 462 hours, and there were no significant gender differences in the main pharmacokinetic parameters. The bioavailability of subcutaneous injection at this dose was 89.6%.

[0303] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method of preventing and / or treating an autoimmune thyroid disease, comprising administering to a subject in need thereof an effective amount of an antibody or an antigen-binding fragment thereof capable of specifically binding to TSHR; wherein the antibody or the antigen-binding fragment thereof capable of specifically binding to TSHR comprises: a VH CDR1, a VH CDR2, and a VH CDR3 contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 7; and / or, a VL CDR1, a VL CDR2, and a VL CDR3 contained in a light chain variable region (VL) as set forth in SEQ ID NO: 8; wherein the method does not comprise administering to the subject a drug and / or a therapy for preventing and / or treating hypothyroidism and / or subclinical hypothyroidism.

2. The method of claim 1, wherein, the drug for preventing and / or treating hypothyroidism and / or subclinical hypothyroidism comprises levothyroxine tablets (Euthyrox), and / or, the therapy for preventing and / or treating hypothyroidism and / or subclinical hypothyroidism comprises administering to the subject levothyroxine tablets (Euthyrox).

3. The method of claim 1, wherein, the method does not comprise administering to the subject a drug and / or a therapy for preventing and / or treating hypothyroidism.

4. The method of any one of claims 1-3, wherein, the antibody or the antigen-binding fragment thereof capable of specifically binding to TSHR is the only pharmaceutically active agent administered to the subject for preventing and / or treating the autoimmune thyroid disease.

5. The method of any one of claims 1-4, wherein, the subject has in his / her body an autoantibody capable of activating TSHR.

6. The method of any one of claims 1-5, wherein, the autoimmune thyroid disease is selected from the group consisting of Graves' disease, Graves' ophthalmopathy, thyroid-associated ophthalmopathy, pretibial myxedema, Hashimoto's thyroiditis, thyroid cancer, and any combination thereof.

7. The method of any one of claims 1-6, wherein, the antibody or the antigen-binding fragment thereof capable of specifically binding to TSHR comprises: (a) a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in SEQ ID NOs: 1-3, respectively; and / or, (b) a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in SEQ ID NOs: 4-6, respectively.

8. The method of any one of claims 1-7, wherein, the antibody or the antigen-binding fragment thereof capable of specifically binding to TSHR comprises: (1) a heavy chain variable region (VH) comprising a sequence as set forth in SEQ ID NO: 7 or a variant thereof; and / or, (2) a light chain variable region (VL) comprising a sequence as set forth in SEQ ID NO: 8 or a variant thereof; wherein the variant has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, or has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%; preferably, the substitutions are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain variable region (VH) comprising the sequence as set forth in SEQ ID NO: 7 and a light chain variable region (VL) comprising the sequence as set forth in SEQ ID NO:

8.

9. The method of any one of claims 1-8, wherein, The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR further comprises a constant region derived from a mammalian (e.g., human) immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4), and / or the light chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a light chain constant region derived from a human immunoglobulin (e.g., a light chain constant region of a kappa or lambda chain).

10. The method of any one of claims 1-9, wherein, The heavy chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain constant region derived from a human immunoglobulin IgG4, and / or the light chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a light chain constant region derived from a human immunoglobulin (e.g., a light chain constant region of a kappa or lambda chain); Preferably, the heavy chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain constant region derived from a human immunoglobulin IgG4, and / or the light chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a light chain constant region derived from a human immunoglobulin kappa chain; Preferably, the heavy chain constant region is selected from the heavy chain constant region of wild-type human immunoglobulin IgG4 and a variant thereof; Preferably, the heavy chain constant region variant comprises a substitution mutation S228P as compared to the heavy chain constant region of wild-type human immunoglobulin IgG4; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 9, and / or the light chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a light chain constant region (CL) as set forth in SEQ ID NO:

10.

11. The method of any one of claims 1-10, wherein, The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises: a heavy chain comprising the sequence as set forth in SEQ ID NO: 11, and / or a light chain comprising the sequence as set forth in SEQ ID NO:

12.

12. The method of any one of claims 1-11, wherein, The administration of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR does not result in hypothyroidism or subclinical hypothyroidism in the subject.

13. The method of any one of claims 1-12, wherein, The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered once every 4-24 weeks (e.g., once every 4-20 weeks, once every 4-16 weeks, once every 4-12 weeks, once every 4-8 weeks, once every 8-24 weeks, once every 8-20 weeks, once every 8-16 weeks, once every 8-12 weeks, once every 12-24 weeks, once every 12-20 weeks, once every 12-16 weeks); Preferably, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered once every 12 weeks (e.g., once every 3 months).

14. The method of any one of claims 1-13, wherein, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered at a single administration dose of 0.1-20 mg / kg (e.g., 0.1-16 mg / kg, 0.1-12 mg / kg, 0.1-10 mg / kg, 0.1-7.5 mg / kg, 0.1-5 mg / kg, 0.1-3 mg / kg, 0.1-1 mg / kg, 0.5-20 mg / kg, 0.5-16 mg / kg, 0.5-12 mg / kg, 0.5-10 mg / kg, 0.5-7.5 mg / kg, 0.5-5 mg / kg, 0.5-3 mg / kg, 0.5-1 mg / kg, 1-20 mg / kg, 1-16 mg / kg, 1-12 mg / kg, 1-10 mg / kg, 1-7.5 mg / kg, 1-5 mg / kg, 1-3 mg / kg, 3-20 mg / kg, 3-16 mg / kg, 3-12 mg / kg, 3-10 mg / kg, 3-7.5 mg / kg, 3-5 mg / kg); Preferably, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered at a single administration dose of 0.1-12 mg / kg.

15. The method of any one of claims 1-14, wherein, The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered for a period of time selected from the group consisting of 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 40 weeks, 80 weeks, 120 weeks, 150 weeks, 200 weeks, 250 weeks, 300 weeks or more.

16. The method of any one of claims 1-15, wherein, The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered to the subject via a route selected from the group consisting of: intravenous, intra-arterial, transdermal, sublingual, oral, intraperitoneal, intramuscular, rectal, intranasal, inhalation, intraocular, subcutaneous, intralipid, intra-articular, intrathecal, and any combination thereof.

17. The method of any one of claims 1-16, wherein, The subject is selected from the group consisting of mammals; Preferably, the subject is a human.

18. A method of preventing and / or treating autoimmune thyroid disease, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof capable of specifically binding to TSHR; wherein The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises: a VH CDR1, a VH CDR2, and a VH CDR3 contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 7; and / or a VL CDR1, a VL CDR2, and a VL CDR3 contained in a light chain variable region (VL) as set forth in SEQ ID NO: 8; wherein the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered once every 4-24 weeks (e.g., once every 4-20 weeks, once every 4-16 weeks, once every 4-12 weeks, once every 4-8 weeks, once every 8-24 weeks, once every 8-20 weeks, once every 8-16 weeks, once every 8-12 weeks, once every 12-24 weeks, once every 12-20 weeks, once every 12-16 weeks).

19. The method of claim 18, wherein the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered once every 12 weeks (e.g., once every 3 months).

20. The method of claim 18 or 19, which does not comprise administering to the subject a drug and / or a therapy that prevents and / or treats hypothyroidism and / or subclinical hypothyroidism.

21. The method of any one of claims 18-20, which does not comprise administering to the subject a drug and / or a therapy that prevents and / or treats hypothyroidism.

22. The method of any one of claims 18-21, wherein, the drug that prevents and / or treats hypothyroidism or subclinical hypothyroidism comprises levothyroxine tablets (Euthyrox), and / or the therapy that prevents and / or treats hypothyroidism or subclinical hypothyroidism comprises administering to the subject levothyroxine tablets (Euthyrox).

23. The method of any one of claims 18-22, wherein, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is the only pharmaceutically active agent administered to the subject to prevent and / or treat the autoimmune thyroid disease.

24. The method of any one of claims 18-23, wherein, the subject has in his / her body autoantibodies capable of activating TSHR.

25. The method of claims 18-24, wherein, the autoimmune thyroid disease is selected from the group consisting of: Graves' disease, Graves' ophthalmopathy, thyroid-associated ophthalmopathy, pretibial myxedema, Hashimoto's thyroiditis, thyroid cancer, and any combination thereof.

26. The method of any one of claims 18-25, wherein, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises: (a) a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, as set forth in SEQ ID NOs: 1-3, respectively; and / or, (b) a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, as set forth in SEQ ID NOs: 4-6, respectively.

27. The method of any one of claims 18-26, wherein, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises: (1) a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof; and / or, (2) a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof; wherein the variant has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) as compared to the sequence from which it was derived, or has a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions; preferably, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain variable region (VH) comprising SEQ ID NO: 7 and a light chain variable region (VL) comprising SEQ ID NO:

8.

28. The method of any one of claims 18-27, wherein, the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR further comprises a constant region derived from a mammalian (e.g., human) immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain constant region derived from a human immunoglobulin (e.g. IgG1, IgG2, IgG3 or IgG4), and / or the light chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a light chain constant region derived from a human immunoglobulin (e.g. a light chain constant region of a kappa or lambda chain).

29. The method of any one of claims 18-28, wherein, Preferably, the heavy chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain constant region derived from a human immunoglobulin IgG4, and / or the light chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a light chain constant region derived from a human immunoglobulin (e.g. a light chain constant region of a kappa or lambda chain). Preferably, the heavy chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain constant region derived from a human immunoglobulin IgG4, and / or the light chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a light chain constant region derived from a human immunoglobulin kappa chain. Preferably, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of wild-type human immunoglobulin IgG4 and variants thereof. Preferably, the heavy chain constant region variant comprises the substitution mutation S228P compared to the heavy chain constant region of wild-type human immunoglobulin IgG4. Preferably, the heavy chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 9, and / or the light chain of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises a light chain constant region (CL) as set forth in SEQ ID NO:

10.

30. The method of any one of claims 18-29, wherein, The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR comprises: a heavy chain comprising a sequence as set forth in SEQ ID NO: 11, and / or a light chain comprising a sequence as set forth in SEQ ID NO:

12.

31. The method of any one of claims 18-30, wherein, The administration of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR does not result in hypothyroidism or subclinical hypothyroidism in the subject.

32. The method of any one of claims 18-31, wherein, The single administration dose of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is 0.1-20 mg / kg (e.g., 0.1-16 mg / kg, 0.1-12 mg / kg, 0.1-10 mg / kg, 0.1-7.5 mg / kg, 0.1-5 mg / kg, 0.1-3 mg / kg, 0.1-1 mg / kg, 0.5-20 mg / kg, 0.5-16 mg / kg, 0.5-12 mg / kg, 0.5-10 mg / kg, 0.5-7.5 mg / kg, 0.5-5 mg / kg, 0.5-3 mg / kg, 0.5-1 mg / kg, 1-20 mg / kg, 1-16 mg / kg, 1-12 mg / kg, 1-10 mg / kg, 1-7.5 mg / kg, 1-5 mg / kg, 1-3 mg / kg, 3-20 mg / kg, 3-16 mg / kg, 3-12 mg / kg, 3-10 mg / kg, 3-7.5 mg / kg, 3-5 mg / kg); Preferably, the single administration dose of the antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is 0.1-12 mg / kg.

33. The method of any one of claims 18-32, wherein, The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered for a period selected from the group consisting of 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 40 weeks, 80 weeks, 120 weeks, 150 weeks, 200 weeks, 250 weeks, 300 weeks or more.

34. The method of any one of claims 18-33, wherein, The antibody or antigen-binding fragment thereof capable of specifically binding to TSHR is administered to the subject by a route selected from the group consisting of: intravenous, intra-arterial, transdermal, sublingual, oral, intraperitoneal, intramuscular, rectal, intranasal, inhalation, intraocular, subcutaneous, intralipid, intra-articular, intrathecal, and any combination thereof.

35. The method of any one of claims 18-34, wherein, The subject is selected from the group consisting of mammals; Preferably, the subject is a human.

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