Antibodies that bind to thyroid stimulating hormone receptor and uses thereof

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to TSHR, a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, a host cell comprising the same, and related uses. In addition, this invention relates to therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.

Description

Antibodies binding to thyroid stimulating hormone receptor and uses thereof Background Art

[0001] Graves' disease (GD) is a difficult-to-treat autoimmune disease characterized by symptoms such as an enlarged and overactive thyroid gland, an accelerated heart rate, and eye abnormalities. The prominent eyes are also known as Graves' ophthalmopathy (GO) or exophthalmos. The disease is believed to result from a complex interaction of genetic and environmental factors. GO not only severely impacts quality of life but also affects mental and work function. The associated overactivation of thyroid hormone receptors, which stimulates thyroid hormone secretion, increases the risk of mortality. Currently, the main treatments for GO are antithyroid drugs (ATDs), radioactive iodine (RAI), and surgery. However, antithyroid drugs are often ineffective and can cause side effects such as bone marrow suppression and hepatotoxicity. Surgical resection or RAI ablation also carries significant risks, including recurrent neurological damage and hypoparathyroidism after surgery. In addition, glucocorticoid therapy, local orbital radiation therapy, or surgical treatment are also needed to control thyroid function. However, these therapies are symptomatic and do not target the disease pathogenesis. Currently, there is a lack of effective clinical treatment for GO.

[0002] Thyroid-stimulating hormone receptor (TSHR) is mainly expressed on the basolateral surface of thyroid follicular cells, inducing thyroid cell growth, hormone synthesis and hormone secretion. It is also the main autoantigen of autoimmune thyroid diseases, especially GD. As the disease progresses, approximately 25% of GD patients will develop GO.

[0003] Literature has documented that some patients with autoimmune thyroid disease (AITD) develop autoantibodies that react with the TSHR (Rees Smith B, et al 1988. Endocrine Reviews 9:106-121). There are two main types of TSHR autoantibodies (TRAbs): stimulatory and blocking. Thyrotropin-stimulating autoantibodies bind to the TSHR and mimic the effects of TSH, thereby stimulating the thyroid gland to produce high levels of T4 and T3. These autoantibodies are also described as TRAbs with agonist activity (Rees Smith B et al. 2007. Thyroid 17:923-938). When these antibodies are present, the feedback control mechanism of thyroid function is no longer effective, and patients develop clinical symptoms of an overactive thyroid, characterized by excess serum thyroid hormone and its metabolic consequences. This condition is known as Graves' disease (GD). Stimulatory TRAbs may also interact with the TSHR in retroorbital tissue, promoting the development of Graves' ophthalmopathy (GO). Based on current research on the pathogenesis of GO, it is believed that TSHR expression is increased in orbital fibroblasts of GO patients. Activation of this signaling pathway increases the production of hyaluronic acid and adipose tissue in the orbit, leading to elevated intraocular pressure and proptosis, a key cause of GO. Furthermore, TSHR expression is high in the thyroid and testes, while low in other tissues, potentially offering advantages in terms of drug targeting and side effects. Therefore, TSHR is expected to become a potential target for the treatment of GO.

[0004] In addition, the insulin-like growth factor 1 receptor (IGF1R) is considered to be another important role in the pathogenesis of GO. IGF1R blockers can improve GO disease. For example, the marketed drug Teprotumumab can competitively inhibit the signaling pathway downstream of IGF1R receptor activation, especially in improving eye proptosis and reducing inflammation. Its therapeutic efficiency in the treatment of moderate to severe thyroid eye disease (TED) reaches 78%. In orbital fibroblasts of patients with GO, increased expression of TSHR and IGF1R was found, and there is a certain interaction between TSHR and IGF1R. Therefore, the use of direct TSHR blockade (TSAb / antagonist) or combined therapy with antagonism of TSHR and IGF1R receptors may be a way to cure GD or GO disease.

[0005] Currently, there is no drug targeting TSHR for the treatment of GO on the market, and there is a need in this field to develop therapeutic drugs targeting TSHR. Technical Field

[0006] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to the TSHR, pharmaceutical compositions containing the antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, host cells containing the same, and related uses. Furthermore, the invention relates to therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.

[0007] Summary of the Invention

[0008] Through extensive research, the inventors of this application have obtained a murine antibody that specifically binds to the TSHR, exhibiting significant TSHR activity inhibition and cross-binding activity against human, mouse, and monkey TSHRs. Furthermore, based on this murine antibody, the inventors of this application have further obtained a humanized antibody that exhibits TSHR binding and TSHR activity inhibition.

[0009] Based on this, the present application also provides a pharmaceutical composition containing the antibody or its antigen-binding fragment, a nucleic acid molecule encoding the antibody or its antigen-binding fragment and a host cell containing the same, as well as related uses.

[0010] Antibodies or antigen-binding fragments thereof

[0011] mAb001 and its humanized antibodies

[0012] Therefore, in a first aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to TSHR, wherein the antibody or the antigen-binding fragment thereof comprises:

[0013] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 1, 27, 29, and 30; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 5, 28, and 31;

[0014] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0016] The three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 1, 27, 29, and 30; and / or the three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 5, 28, and 31.

[0017] In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering systems. In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat numbering system.

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0019] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 2, VH CDR2 of SEQ ID NO: 3 or 39, and VH CDR3 of SEQ ID NO: 4; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 6, VL CDR2 of SEQ ID NO: 7, and VL CDR3 of SEQ ID NO: 8;

[0020] Wherein, the CDRs are defined by the Kabat numbering system.

[0021] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a murine heavy chain germline sequence, and / or, a light chain framework region of a murine light chain germline sequence.

[0023] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0024] a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 5 or a variant thereof;

[0025] 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 a sequence having 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.

[0026] In certain embodiments, the substitutions are conservative substitutions.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 1 and a VL comprising the sequence shown in SEQ ID NO:5.

[0028] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region comprised in an amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof 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.

[0030] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising a sequence as shown in any one of SEQ ID NOs: 27, 29, 30, or a variant thereof; and / or a light chain variable region (VL) comprising a sequence as shown in SEQ ID NOs: 28 or 31, or a variant thereof;

[0031] 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 a sequence having 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.

[0032] In certain embodiments, the substitutions are conservative substitutions.

[0033] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 27, 29, and 30; and / or a light chain variable region (VL) as shown in SEQ ID NOs: 28 or 31.

[0034] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:27 and a VL comprising the sequence shown in SEQ ID NO:28.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:29 and a VL comprising the sequence shown in SEQ ID NO:28.

[0036] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:30 and a VL comprising the sequence shown in SEQ ID NO:28.

[0037] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:27 and a VL comprising the sequence shown in SEQ ID NO:31.

[0038] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:29 and a VL comprising the sequence shown in SEQ ID NO:31.

[0039] mAb012 and its humanized antibodies

[0040] Therefore, in a second aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to TSHR, wherein the antibody or the antigen-binding fragment thereof comprises:

[0041] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 9, 32, and 34; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in SEQ ID NO: 13 or 33;

[0042] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0043] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0044] The three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 9, 32, and 34; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NOs: 13 or 33.

[0045] In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering systems. In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat numbering system.

[0046] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0047] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 10, VH CDR2 of SEQ ID NO: 11 or 40, and VH CDR3 of SEQ ID NO: 12 or 41; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 14, VL CDR2 of SEQ ID NO: 15, and VL CDR3 of SEQ ID NO: 16;

[0048] Wherein, the CDRs are defined by the Kabat numbering system.

[0049] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0050] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 10, VH CDR2 of SEQ ID NO: 11 or 40, and VH CDR3 of SEQ ID NO: 12; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 14, VL CDR2 of SEQ ID NO: 15, and VL CDR3 of SEQ ID NO: 16;

[0051] Wherein, the CDRs are defined by the Kabat numbering system.

[0052] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0053] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 10, VH CDR2 of SEQ ID NO: 11 or 40, and VH CDR3 of SEQ ID NO: 41; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 14, VL CDR2 of SEQ ID NO: 15, and VL CDR3 of SEQ ID NO: 16;

[0054] Wherein, the CDRs are defined by the Kabat numbering system.

[0055] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0056] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a murine heavy chain germline sequence, and / or, a light chain framework region of a murine light chain germline sequence.

[0057] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0058] a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 13 or a variant thereof;

[0059] 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 a sequence having 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.

[0060] In certain embodiments, the substitutions are conservative substitutions.

[0061] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:9 and a VL comprising the sequence shown in SEQ ID NO:13.

[0062] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0063] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region comprised in an amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof 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.

[0064] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 32 or 34 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 33 or a variant thereof;

[0065] 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 a sequence having 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.

[0066] In certain embodiments, the substitutions are conservative substitutions.

[0067] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) as shown in SEQ ID NO: 32 or 34; and / or, a light chain variable region (VL) as shown in SEQ ID NO: 33.

[0068] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:32 and a VL comprising the sequence shown in SEQ ID NO:33.

[0069] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:34 and a VL comprising the sequence shown in SEQ ID NO:33.

[0070] mAb021 and its humanized antibodies

[0071] Therefore, in a third aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to TSHR, wherein the antibody or the antigen-binding fragment thereof comprises:

[0072] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 17, 35, and 37; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 21, 36, and 38;

[0073] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0074] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0075] The three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 17, 35, and 37; and / or the three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 21, 36, and 38.

[0076] In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering systems. In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat numbering system.

[0077] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0078] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 18, VH CDR2 of SEQ ID NO: 19, and VH CDR3 of SEQ ID NO: 20; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 22, VL CDR2 of SEQ ID NO: 23, and VL CDR3 of SEQ ID NO: 24;

[0079] Wherein, the CDRs are defined by the Kabat numbering system.

[0080] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0081] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a murine heavy chain germline sequence, and / or, a light chain framework region of a murine light chain germline sequence.

[0082] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0083] a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 17 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 21 or a variant thereof;

[0084] 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 a sequence having 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.

[0085] In certain embodiments, the substitutions are conservative substitutions.

[0086] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 17 and a VL comprising the sequence shown in SEQ ID NO: 21.

[0087] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0088] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region comprised in an amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof 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.

[0089] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 35 or 37 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 36 or 38 or a variant thereof;

[0090] 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 a sequence having 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.

[0091] In certain embodiments, the substitutions are conservative substitutions.

[0092] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) as shown in SEQ ID NO: 35 or 37; and / or, a light chain variable region (VL) as shown in SEQ ID NO: 36 or 38.

[0093] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:35 and a VL comprising the sequence shown in SEQ ID NO:36.

[0094] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:37 and a VL comprising the sequence shown in SEQ ID NO:38.

[0095] In certain embodiments, in the first, second, or third aspects, the antibody or antigen-binding fragment thereof further comprises a constant region derived from a mammalian (eg, human or murine) immunoglobulin.

[0096] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof 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 comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ).

[0097] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; and / or,

[0098] The light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 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. The substitutions can be conservative or non-conservative.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a wild-type human immunoglobulin.

[0100] In other embodiments, the antibody or antigen-binding fragment thereof comprises a variant of the heavy chain constant region (CH) of a human immunoglobulin, and the variant of the heavy chain constant region (CH) may have the same or substantially the same properties as the wild-type sequence from which it is derived. In certain embodiments, the variant of the heavy chain constant region (CH) may have one or more conservative substitutions of amino acids compared to the sequence from which it is derived.

[0101] In other embodiments, the antibody or antigen-binding fragment thereof comprises a variant of the heavy chain constant region (CH) of a human immunoglobulin, and the variant of the heavy chain constant region (CH) may comprise one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibody of the invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function, etc. Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue or chemically modifying it, for example, by altering the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby altering the effector function (e.g., reducing or enhancing it). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

[0102] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise variants of the human immunoglobulin heavy chain constant region (CH), which variants may have reduced or abolished effector function compared to the wild-type sequence from which they are derived.

[0103] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise variants of the human immunoglobulin heavy chain constant region (CH), which variants may have enhanced effector function compared to the wild-type sequence from which they are derived.

[0104] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a human immunoglobulin heavy chain constant region (CH) or a variant thereof, which may have the same or substantially the same effector function as the wild-type sequence from which it is derived.

[0105] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a variant of a human immunoglobulin heavy chain constant region (CH) that has: (i) extended half-life, (ii) enhanced pharmacodynamics, and / or, (iii) reduced ADCC activity compared to the wild-type sequence from which it is derived.

[0106] In certain embodiments, the heavy chain constant region (CH) is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0107] In certain embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

[0108] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a murine immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a murine immunoglobulin (e.g., κ or λ).

[0109] In certain embodiments, in the first aspect, the second aspect, or the third aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin IgG4, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ).

[0110] In certain embodiments, the heavy chain constant region derived from human immunoglobulin IgG4 comprises substitution mutations S228P, L235E, M252Y, S254T and / or T256E (e.g., (i) S228P, (ii) L235E, and / or (iii) M252Y, S254T and T256E) compared to the heavy chain constant region of wild-type human immunoglobulin IgG4.

[0111] In certain embodiments, compared with the heavy chain constant region of wild-type human immunoglobulin IgG4, the heavy chain constant region derived from human immunoglobulin IgG4 comprises the substitution mutation S228P; in certain embodiments, the heavy chain constant region derived from human immunoglobulin IgG4 further comprises the substitution mutations M252Y, S254T and T256E; in certain embodiments, the heavy chain constant region derived from human immunoglobulin IgG4 further comprises the substitution mutations L235E, M252Y, S254T and T256E.

[0112] In certain embodiments, the amino acid positions of the substitution mutations are defined by the EU numbering system.

[0113] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in any one of SEQ ID NOs: 25, 42-43, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 26.

[0114] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0115] (1) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 27 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26;

[0116] (2) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 29 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26;

[0117] (3) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 30 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26;

[0118] (4) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 27 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 31 and a CL sequence as shown in SEQ ID NO: 26;

[0119] (5) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 29 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 31 and a CL sequence as shown in SEQ ID NO: 26;

[0120] (6) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 32 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 33 and a CL sequence as shown in SEQ ID NO: 26;

[0121] (7) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 34 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 33 and a CL sequence as shown in SEQ ID NO: 26;

[0122] (8) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 35 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 36 and a CL sequence as shown in SEQ ID NO: 26; or

[0123] (9) A heavy chain comprising a VH sequence shown in SEQ ID NO: 37 and a CH sequence shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence shown in SEQ ID NO: 38 and a CL sequence shown in SEQ ID NO: 26.

[0124] In certain embodiments, the antibody or antigen-binding fragment thereof of the first aspect comprises:

[0125] (1) a heavy chain comprising the sequence shown in SEQ ID NO: 46, and / or a light chain comprising the sequence shown in SEQ ID NO: 51;

[0126] (2) a heavy chain comprising the sequence shown in SEQ ID NO: 47, and / or a light chain comprising the sequence shown in SEQ ID NO: 51; or

[0127] (3) a heavy chain comprising the sequence shown in SEQ ID NO: 48, and / or a light chain comprising the sequence shown in SEQ ID NO: 51.

[0128] In certain embodiments, in the first aspect, the second aspect, or the third aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin IgG1, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ).

[0129] In certain embodiments, the heavy chain constant region derived from human immunoglobulin IgG1 comprises substitution mutations L234A, L235A, M252Y, S254T and / or T256E (e.g., (i) L234A and L235A, and / or (ii) M252Y, S254T and T256E) compared to the heavy chain constant region of wild-type human immunoglobulin IgG1.

[0130] In certain embodiments, the heavy chain constant region derived from human immunoglobulin IgG1 comprises substitution mutations L234A and L235A compared to the heavy chain constant region of wild-type human immunoglobulin IgG1; in certain embodiments, the heavy chain constant region derived from human immunoglobulin IgG1 further comprises substitution mutations M252Y, S254T and T256E.

[0131] In certain embodiments, the amino acid positions of the substitution mutations are defined by the EU numbering system.

[0132] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 44 or 45, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 26.

[0133] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0134] (1) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 27 and a CH sequence as shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26;

[0135] (2) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 29 and a CH sequence as shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26;

[0136] (3) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 30 and a CH sequence as shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26;

[0137] (4) a heavy chain comprising a VH sequence of SEQ ID NO: 27 and a CH sequence of SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence of SEQ ID NO: 31 and a CL sequence of SEQ ID NO: 26;

[0138] (5) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 29 and a CH sequence as shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 31 and a CL sequence as shown in SEQ ID NO: 26;

[0139] (6) a heavy chain comprising a VH sequence of SEQ ID NO: 32 and a CH sequence of SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence of SEQ ID NO: 33 and a CL sequence of SEQ ID NO: 26;

[0140] (7) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 34 and a CH sequence as shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 33 and a CL sequence as shown in SEQ ID NO: 26;

[0141] (8) a heavy chain comprising a VH sequence of SEQ ID NO: 35 and a CH sequence of SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence of SEQ ID NO: 36 and a CL sequence of SEQ ID NO: 26; or

[0142] (9) A heavy chain comprising a VH sequence shown in SEQ ID NO: 37 and a CH sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence shown in SEQ ID NO: 38 and a CL sequence shown in SEQ ID NO: 26.

[0143] In certain embodiments, the antibody or antigen-binding fragment thereof of the first aspect comprises:

[0144] (1) a heavy chain comprising the sequence shown in SEQ ID NO: 49, and / or a light chain comprising the sequence shown in SEQ ID NO: 51; or

[0145] (2) A heavy chain comprising the sequence shown in SEQ ID NO: 50, and / or a light chain comprising the sequence shown in SEQ ID NO: 51.

[0146] In certain embodiments, in the first aspect, the second aspect or the third aspect, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody and single-domain antibody (sdAb); and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or a multispecific antibody.

[0147] In a fourth aspect, the present application also provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, or its heavy chain variable region and / or light chain variable region, or its heavy chain and / or light chain.

[0148] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0149] In a fifth aspect, the present application also provides a vector comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0150] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0151] In a sixth aspect, the present application also provides a host cell comprising the isolated nucleic acid molecule or vector as described above.

[0152] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0153] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0154] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0155] In a seventh aspect, the present application also provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, which comprises culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0156] Therapeutic uses

[0157] In an eighth aspect, the present application also provides a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect.

[0158] In certain embodiments, the bispecific or multispecific molecule specifically binds to the TSHR and additionally specifically binds to one or more other targets.

[0159] In certain embodiments, the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody).

[0160] In certain embodiments, the bispecific or multispecific molecule further comprises at least one second antibody that specifically binds to IGF1R. In certain embodiments, the second antibody has an antagonistic effect on IGF1R signaling.

[0161] In a ninth aspect, the present application further provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

[0162] In certain embodiments, the therapeutic agent is selected from an IGF1R antagonist or inhibitor.

[0163] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0164] In the tenth aspect, the present application also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, the isolated nucleic acid molecule as described in the fourth aspect, the vector as described in the fifth aspect, the host cell as described in the sixth aspect, the bispecific or multispecific molecule as described in the eighth aspect or the immunoconjugate as described in the ninth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0165] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an additional drug for treating an autoimmune thyroid disease (such as Graves' disease or Graves' ophthalmopathy) or thyroid cancer.

[0166] In certain embodiments, the pharmaceutical composition further comprises an IGF1R antagonist or inhibitor, and / or, an additional TSHR antagonist or inhibitor.

[0167] In certain exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0168] In the eleventh aspect, the present application also provides the use of the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, the isolated nucleic acid molecule as described in the fourth aspect, the vector as described in the fifth aspect, the host cell as described in the sixth aspect, the bispecific or multispecific molecule as described in the eighth aspect, the immunoconjugate as described in the ninth aspect or the pharmaceutical composition as described in the tenth aspect for preparing a drug for preventing and / or treating a TSHR-related disease in a subject.

[0169] In certain embodiments, the TSHR-associated disease would benefit from antagonism of TSHR signaling.

[0170] In certain embodiments, the TSHR-associated disease is autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.

[0171] In certain embodiments, the subject is a mammal, such as a human, mouse, or monkey.

[0172] In certain embodiments, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., another drug used to treat autoimmune thyroid disease (such as Graves' disease or Graves' ophthalmopathy) or thyroid cancer), e.g., administered simultaneously or sequentially.

[0173] In certain embodiments, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition is used in combination with an IGF1R antagonist or inhibitor and / or an additional TSHR antagonist or inhibitor, e.g., administered simultaneously or sequentially.

[0174] In the twelfth aspect, the present application also provides a method for preventing and / or treating a TSHR-related disease in a subject, comprising: administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, the isolated nucleic acid molecule as described in the fourth aspect, the vector as described in the fifth aspect, the host cell as described in the sixth aspect, the bispecific or multispecific molecule as described in the eighth aspect, the immunoconjugate as described in the ninth aspect, or the pharmaceutical composition as described in the tenth aspect.

[0175] In certain embodiments, the TSHR-associated disease would benefit from antagonism of TSHR signaling.

[0176] In certain embodiments, the TSHR-associated disease is autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.

[0177] In certain embodiments, the subject is a mammal, such as a human, mouse, or monkey.

[0178] In certain embodiments, the antibody or antigen-binding fragment thereof of the first, second or third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, the host cell of the sixth aspect, the bispecific or multispecific molecule of the eighth aspect, the immunoconjugate of the ninth aspect or the pharmaceutical composition of the tenth aspect can be administered in combination with another pharmaceutically active agent (e.g., another drug for treating autoimmune thyroid disease (e.g., Graves' disease or Graves' ophthalmopathy) or thyroid cancer), for example, simultaneously or sequentially.

[0179] In certain embodiments, the antibody or antigen-binding fragment thereof of the first, second or third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, the host cell of the sixth aspect, the bispecific or multispecific molecule of the eighth aspect, the immunoconjugate of the ninth aspect or the pharmaceutical composition of the tenth aspect can be administered in combination with an IGF1R antagonist or inhibitor and / or an additional TSHR antagonist or inhibitor, e.g., simultaneously or sequentially.

[0180] The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the antibody or antigen-binding fragment thereof of the present invention into an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (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), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0181] The antibody of the present application or its Fab or pharmaceutical composition can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to intended purpose. In certain embodiments, antibody of the present invention or its Fab or pharmaceutical composition are given by intravenous injection or push injection.

[0182] Detection purpose

[0183] In a thirteenth aspect, the present application further provides a conjugate comprising the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, and a detectable label linked to the antibody or antigen-binding fragment thereof.

[0184] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0185] In a fourteenth aspect, the present application further provides a kit comprising the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, or the conjugate as described in the thirteenth aspect.

[0186] In certain embodiments, the kit comprises a detection buffer.

[0187] In certain embodiments, the kit comprises the conjugate of the thirteenth aspect.

[0188] In certain embodiments, the kit comprises the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin.

[0189] In a fifteenth aspect, the present application also provides a method for detecting the presence or level of TSHR in a sample, which comprises using the antibody or antigen-binding fragment thereof as described in the first, second or third aspect or the conjugate as described in the thirteenth aspect.

[0190] In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0191] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0192] In certain embodiments, the method comprises using the conjugate of the thirteenth aspect.

[0193] In certain embodiments, the method comprises using the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide or biotin).

[0194] In certain embodiments, the method comprises: (1) contacting the sample with an antibody, or antigen-binding fragment thereof, or conjugate thereof; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of TSHR or cells expressing TSHR.

[0195] The present application also provides a method for diagnosing a disease associated with TSHR, comprising detecting the presence or level of TSHR in a sample from a subject using the method described in aspect 15. In certain embodiments, when TSHR is present or the level of TSHR is increased compared to a reference level (e.g., compared to a healthy control), it indicates that the subject suffers from a disease associated with TSHR.

[0196] In certain embodiments, the TSHR-associated disease is autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.

[0197] In the sixteenth aspect, the present application also provides the use of the antibody or antigen-binding fragment thereof as described in the first, second or third aspect or the conjugate as described in the thirteenth aspect in the preparation of a detection reagent, wherein the detection reagent is used to detect the presence or level of TSHR in a sample and / or diagnose a disease related to TSHR.

[0198] In certain embodiments, the detection reagent detects the presence or level of TSHR in a sample by the method of aspect 15.

[0199] In certain embodiments, the detection reagent detects the presence or level of TSHR in a sample by the method described in Aspect 15 to diagnose a disease associated with TSHR. In certain embodiments, when TSHR is present or the level of TSHR is increased compared to a reference level (e.g., compared to a healthy control), it indicates that the subject suffers from a disease associated with TSHR.

[0200] In certain embodiments, the TSHR-associated disease is autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.

[0201] In certain embodiments, the sample is a tissue sample from a subject (eg, a mammal, preferably a human, mouse, or monkey).

[0202] Definition of terms

[0203] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0204] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0205] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0206] The term "antibody," as used herein, refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only intact antibodies but also antigen-binding fragments that are capable of specifically binding to a target antigen. "Intact antibodies" are typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing 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 is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The constant domains are not directly involved in antibody-antigen binding but exhibit 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 variable 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 amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The distribution of amino acids among regions or domains can follow the definitions of 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.

[0207] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable region of each of the heavy and light chains 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), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0208] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.

[0209] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0210] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0211] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a 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, which is also referred to as an "antigen-binding portion." See generally, 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 produced by recombinant DNA techniques 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, diabodies, single domain antibodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0212] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0213] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0214] As used herein, the term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0215] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., 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, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants 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 the present 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 be present between the VH and VL of the scFv. In certain embodiments of the invention, scFv can form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the invention, scFv can form a (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0216] As used herein, the term "diabodies" means antibodies whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0217] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody. Single-domain antibodies are also called nanobodies.

[0218] 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 or more (e.g., three or four) different antigens (or epitopes). Bispecific antibodies or multispecific antibodies comprise multiple antigen-binding domains that have binding specificity to different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules. Each antigen-binding domain comprised by a bispecific antibody or multispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or an antigen-binding fragment thereof (e.g., Fv fragment, Fab fragment, F(ab')2 fragment or scFv). In some cases, each antigen-binding domain is connected by a peptide linker.

[0219] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0220] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0221] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). In certain embodiments, the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody can be a mouse antibody with expected 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 framework sequence using methods known in the art. In some cases, the humanized framework sequence may include amino acid mutations that are replaced by corresponding non-human residues. In addition, the humanized antibody may also include residues that are not found in the original donor antibody variable region (e.g., light chain variable region or heavy chain variable region) or human framework sequence to further improve or optimize the performance of the humanized antibody.

[0222] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain 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 in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies in which the heavy chain variable region and light chain variable region of the antibody are derived from a first antibody, and the heavy chain constant region and light chain constant region of the antibody are derived from a second antibody.

[0223] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions 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 molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0224] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0225] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). 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 a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, 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. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0226] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D) indicates. In the present invention, the term "K D ” 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 tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0227] 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 "association 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 K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore. Alternatively, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0228] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), fluorescent proteins (e.g., phycoerythrin (PE)), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa750)), luminescent substances (e.g., chemiluminescent agents, such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin. In certain embodiments, the detectable label as described above can be linked to the antibody or antigen-binding fragment thereof of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0229] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0230] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0231] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by 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 of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). 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, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., 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).

[0232] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present 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.

[0233] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to 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 regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives, and stabilizers. For example, pH regulators 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 that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate 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, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium 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 certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0234] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to expected survival (e.g., survival without treatment).

[0235] As used herein, the term "subject" refers to a mammal, such as a human, mouse, or monkey. In certain embodiments, the subject (e.g., human, mouse, or monkey) suffers from a disease associated with TSHR (e.g., Graves' disease, Graves' ophthalmopathy), or is at risk of suffering from the above-mentioned disease.

[0236] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., Graves' disease, Graves' ophthalmopathy) refers to an amount sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in patients already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use 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 condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0237] Advantageous Effects of the Invention

[0238] The antibodies provided herein have high affinity for the human TSHR and exhibit cross-binding activity against human, mouse, and monkey TSHR. Furthermore, the antibodies described herein have significant inhibitory effects on TSHR activity, inhibiting downstream signaling pathways mediated by the binding of TSHR to activating antibodies by blocking the binding of TSHR to activating antibodies. In vitro functional experiments have demonstrated that the antibodies described herein significantly inhibit the secretion of hyaluronic acid, interleukin-6, and interleukin-8 in primary orbital fibroblasts from patients with GO.

[0239] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention 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 based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0240] Figure 1: Engineered antibodies inhibit TPO production in thyroid cells induced by GD patient serum.

[0241] Sequence information

[0242] A description of the sequences involved in this application is provided in the table below.

[0243] Table 1: Sequence information DETAILED DESCRIPTION

[0244] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit it.

[0245] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods 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., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0246] Example 1: Antibody Preparation

[0247] 1. Preparation of hybridoma antibodies:

[0248] 1.1 After immunizing mice with human TSHR antigen, mice with higher titers were selected for cell fusion and hybridoma preparation.

[0249] 1.2 Prepare myeloma cells. Before fusion, count and test the viability of the cells using a cell counter. Collect SP2 / 0 cells and centrifuge at 400g for 5 minutes at room temperature. Discard the culture medium and resuspend the cell pellet in 20 ml of fusion culture medium.

[0250] 1.3 Collect and culture spleen cells, select mice with strong immunogenic responses for hybridoma cell preparation, and euthanize mice with carbon dioxide according to IACUC-approved methods. Collect pre-fusion plasma, separate and collect serum (FB) as a positive control for hybridoma supernatant screening.

[0251] 1.4 Soak the mouse in 70% alcohol for disinfection, then immediately transfer it to a biosafety cabinet for operation. Collect the spleen and place it in a sterile culture dish. Place the spleen on a filter and repeatedly squeeze the spleen with the plunger of a sterile syringe until there are no obvious clumps. Then rinse the filter with fusion culture medium to obtain a spleen cell suspension and transfer it all to a 50 ml centrifuge tube.

[0252] 1.5 Centrifuge at 400g for 5 minutes and discard the supernatant. Prepare fused cells using electrofusion. Resuspend the spleen cell pellet in 5 ml of red blood cell lysis buffer, then incubate at 4°C for 5 minutes. Terminate the reaction with 50 ml of DEME medium supplemented with 10% FBS. Collect the spleen cell pellet by centrifugation and resuspend the cells in fusion medium. Invert the tube 3-5 times and count the cells. Centrifuge at 400g for 5 minutes, discard the supernatant, retain the cell pellet, and resuspend the cells in 40 ml of fusion medium. Let the spleen cells stand at room temperature for 2-3 minutes and then transfer them to a new 50 ml centrifuge tube. Add SP2 / 0 cells to the spleen cells at a ratio of 4:1 spleen cells to SP2 / 0 cells and make up to 50 ml with fusion medium. Centrifuge the cell mixture, discard the supernatant, and repeatedly tap the bottom of the tube to loosen the cell pellet. For electrofusion, place the cell mixture in an electroporation chamber and perform electroporation according to the optimized protocol.

[0253] After electrofusion, allow the fused cells to rest in the electroporation chamber for an additional 10 minutes. Resuspend the cells in DMEM supplemented with 20% FBS and HAT and plate into 96-well microplates at a volume of 100 μl per well. After 24 hours, add DMEM supplemented with 20% FBS and HAT to the wells of the plate at a volume of 100 μl per well. Transfer all fusion plates back to the CO2 incubator at 37°C, 5% CO2. Monitor cell growth and microbial contamination daily. When hybridoma clones reach a diameter of 1-2 mm (typically 10-14 days after fusion), screen the hybridoma supernatants using Acumen.

[0254] 1.7 Subcloning and expansion culture. Transfer the positive clones to a 24-well plate and culture with expansion culture medium. Collect the hybridoma culture supernatant for FACS detection. Subclone the positive mother clone to obtain a single clone by limiting dilution. Place the subclone 96-well plate in a carbon dioxide incubator, culture for 7 days, and then detect the activity of the screening supernatant. To ensure monoclonality, 1-2 rounds of subcloning are required. Based on the screening results, select the 4 most active monoclones from each subclone plate and transfer them to a 24-well plate for expansion culture.

[0255] 1.8 Cell cryopreservation. Cryopreserve the expanded subcloned cell lines in DMEM supplemented with 20% FBS and 10% DMSO and store them in a liquid nitrogen tank for long-term storage.

[0256] Murine monoclonal antibodies mAb001, mAb012, and mAb021 were prepared by the above method, and their CDR and variable region sequences are shown in Table 2.

[0257] Table 2. Mouse antibody variable region and CDR sequence information

[0258] 2. Humanization of mouse antibodies:

[0259] Humanized antibodies were designed using CDR grafting. The sequence closest to the mouse sequence in the BLAST human antibody was used as the variable region framework, replacing the mouse framework region. The CDRs of the parent antibody were transplanted into the human receptor. All sequences were analyzed for post-translational modifications (PTMs), such as isomerization, deamidation, and glycosylation risks. Appropriate PTM removal mutations were designed to ultimately obtain each humanized light chain and humanized heavy chain for each parent antibody. The humanized light and heavy chain variable regions of each parent antibody were paired with each other for affinity ranking experiments. The heavy and light chain variable regions were then ligated to the human IgG4-S228P heavy chain constant region (SEQ ID NO: 25) and the human kappa light chain constant region (SEQ ID NO: 26), respectively, to obtain the full-length sequences of the humanized antibody heavy and light chains.

[0260] DNA sequences encoding the humanized antibody heavy and light chains were synthesized and inserted into the pcDNA3.4 vector to construct a full-length antibody expression plasmid. The humanized antibody was expressed in Expi293F cell culture, and the supernatant was purified using a Protein A affinity column. The purified antibody buffer was exchanged into PBS using a PD-10 desalting column. The concentration and purity of the purified protein were determined by OD280 and SDS-PAGE, respectively.

[0261] Humanized antibodies of each murine monoclonal antibody were obtained by the above method. The amino acid sequences of the light and heavy chain variable regions of each humanized antibody are shown in SEQ ID NOs: 27-38.

[0262] Example 2: Mouse Antibody Cell Binding Experiment

[0263] Experimental steps:

[0264] The binding strength of the mouse antibodies to 293-human TSHR, 293-mouse TSHR, and 293-monkey TSHR overexpressing cell lines (the cells used were 293 cells overexpressing human, monkey, or mouse TSHR, wherein the amino acid sequences of the human, monkey, and mouse TSHRs are shown in P16473, A0A2K5X226, and P47750 Uniprot, respectively) were detected by flow cytometry (FACS). The experimental method was as follows: cells in the logarithmic growth phase were collected and counted using a cell counter (Countstar; IC1000); the cells were resuspended in cold FACS buffer, which was 500 ml of PBS (HyClone, SH30256.01B) added to 50 ml of FBS (BI; 04-002-1A), and the cell density was adjusted to 5 × 10 6 cells per milliliter; after the cell suspension was allowed to stand at room temperature for 10 to 20 minutes, it was added to a FACS plate (Corning, 3799) at 100 μL per well, centrifuged and the supernatant discarded; the test antibody was diluted in FACS buffer at a starting concentration of 200 nM and diluted 1:3; the diluted antibody was added to the above cells at 100 μL per well and incubated at 4°C for 1 hour; after incubation, the cells were washed three times with FACS buffer (250 μL per well) and centrifuged at 300 g (4°C) for 5 minutes; the secondary antibody anti-mouse IgG-Alexa488 antibody (Invitrogen; A21202) prepared in FACS buffer (1:1k dilution) was added to 100 μL per well and incubated at 4°C in the dark for 1 hour. The cells were then washed again three times with FACS buffer; the cell pellet was resuspended in 100 μL of 4°C FACS buffer, and the fluorescence intensity of each well, i.e., the binding signal, was measured using a flow cytometer (BECKMAN COULTER; B53013).

[0265] Data processing (data processing methods) and experimental data:

[0266] The binding signal values ​​corresponding to each antibody concentration were fitted nonlinearly using GraphPad. The data in Table 3 are the maximum binding signals of the antibodies and the EC50 calculated by curve fitting.

[0267] Table 3 Maximum binding signal and EC50 of each antigen

[0268] Experimental conclusion:

[0269] The selected antibodies all had good binding activity to the 293 cell line overexpressing human TSHR, and all had cross-binding activity to monkey and mouse.

[0270] Example 3: Humanized Antibody Cell Binding Experiment

[0271] Experimental steps:

[0272] The binding of the humanized antibody to 293-human TSHR, 293-mouse TSHR, and 293-monkey TSHR overexpressing cell lines was detected by FACS using the same method as described in Example 2. Anti-human IgG-Alexa 488 antibody (Invitrogen; A-11013) was used as the secondary antibody (1:1k dilution).

[0273] Data processing (data processing methods) and experimental data:

[0274] The binding signal values ​​corresponding to each antibody concentration were subjected to nonlinear fitting using Graphpad. The data in Tables 4 and 5 are the maximum binding signals of the antibodies and the EC50 calculated by curve fitting.

[0275] Table 4 Maximum binding signal and EC50 of each antigen

[0276] Table 5 Maximum binding signal and EC50 of each antigen

[0277] Experimental conclusion:

[0278] The above humanized antibodies have good binding activity to 293-human TSHR cells, and also have the ability to bind to monkey TSHR and mouse TSHR.

[0279] Example 4: Humanized Antibody cAMP Experiment

[0280] Experimental steps:

[0281] Antibody preparation: Antibodies of varying concentrations were prepared in a 384-well plate (3824 Corning) using a Bravo (Agilent) instrument. The preparation solution was DPBS, with a starting concentration of 200 nM and a 1:3 dilution, for a total of 11 points.

[0282] Humanized antibody blocking of agonistic antibody activation of cAMP: Antibody cAMP detection was performed according to the instructions of the cAMP-Gs DYNAMIC Kit (62AM4PEB, Cisbio). The cell line used was the 293-human TSHR cell line; the competing antibodies, Tab01 and Tab02, were agonistic antibodies, used at concentrations of 30 nM and 6.5 nM, respectively. The sample was loaded using a MultiDrop Combi (Thermo) set to low speed; the data was read using an Envision (PerkinElmer) plate reader, detecting signals at wavelengths of 665 nM and 615 nM. A standard curve was constructed using the standard concentration and the signal ratio at 665 nM / 615 nM to determine the relative activity of the antibodies.

[0283] Tab01 is a TSHR agonist antibody, M22, whose sequences are derived from the heavy chain variable region of the antibody sequence numbered No. 1 and the light chain variable region of sequence numbered No. 6 in patent WO 2004 / 050708A2. The heavy and light chain variable regions were ligated to the human IgG1 heavy chain constant region and human lambda light chain constant region, respectively, to obtain the full-length heavy and light chain sequences of Tab01. The sequences of the human IgG1 heavy chain constant region and human lambda light chain constant region were referenced to the positive control, Tab03_hIgG1 heavy chain constant region and light chain constant region sequences.

[0284] Tab02 is a TSHR agonist antibody K1-18, the sequence of which is derived from the heavy chain sequence with sequence number No. 15 and the light chain sequence with sequence number No. 33 in the TSHR antibody patent with patent number US10428153.

[0285] The positive control is Tab03_hIgG1, the sequence of which is derived from the heavy chain sequence No. 51 and the light chain sequence No. 59 in the TSHR antibody patent with patent number US10428153.

[0286] Data processing (data processing methods) and experimental data:

[0287] The ratio of the signals at wavelengths of 665 nM and 615 nM was calculated. This ratio is proportional to the relative level of cAMP and represents the relative activity of the TSHR. A nonlinear regression curve was constructed between the antibody concentration and the corresponding 665 nM / 615 nM ratio to calculate the IC50 and maximum inhibition rate. The results are shown in Tables 6 and 7.

[0288] Table 6 IC50 and maximum inhibition rate of each antibody

[0289] Table 7 IC50 and maximum inhibition rate of each antibody

[0290] Experimental conclusion:

[0291] All antibodies showed significant inhibition of TSHR activity (in the presence of agonistic antibodies Tab01 and Tab02).

[0292] Example 5: Humanized Antibody Hyaluronic Acid (HA) Secretion Experiment

[0293] Experimental steps:

[0294] 1. Obtaining fibroblasts

[0295] The experiment was divided into three groups, and the cell sources were acute GO patients and stable GO patients, respectively. The cell acquisition method was as follows. Tissue specimens from the patient's orbital connective part were obtained, with 2 patients in each group. The specimens were quickly placed in a sterile centrifuge tube containing DMEM / F12 culture medium (containing a mixture of penicillin and streptomycin), and then the centrifuge tube containing the tissue was quickly transferred to the cell culture laboratory; in the clean bench, the tissue in the centrifuge tube was clamped and repeatedly washed with sterile PBS to remove blood stains, and then transferred to a sterile culture dish with DMEM / F12 culture medium (containing a mixture of penicillin and streptomycin), ensuring that the specimen was completely immersed in the culture medium to maintain the tissue moist and active; using micro-ophthalmic scissors and tweezers, the fat tissue and larger blood vessels were carefully removed, and the separated connective tissue was washed 3 times with PBS, placed in a sterile culture dish containing a little DMEM / F12 culture medium, and cut into about 1mm with scissors and tweezers. 3 About 100 pieces of tissue. Rinse the cell culture plate with complete culture medium (DMEM / F12 culture medium plus 20% fetal bovine serum, plus 1% volume fraction of penicillin and streptomycin), and carefully stick the tissue to the cell culture plate after it is slightly dry. Place the cell culture plate with tissue in a 37°C, 5% CO2 cell culture incubator upside down for 30 minutes to allow the tissue to adhere to the wall as soon as possible. After 30 minutes, turn the cell culture plate over and lay it flat, add complete culture medium to just cover the tissue, and culture for several days. After 3 days, change half of the medium, carefully remove floating tissue pieces and residual red blood cells, observe the cells crawling out and add culture medium, use 0.25% trypsin-EDTA to digest every 5 to 7 days, and subculture according to 1:3-1:4.

[0296] 2. Acquisition of serum immunoglobulins (GO-Igs) from GO patients

[0297] Serum samples from 40 GO patients were collected and centrifuged in a 3K15 low-temperature high-speed centrifuge at 4°C, 12,000 rpm / min for 10 min. The samples were filtered through a filter with a 0.45 μm pore size membrane and collected for later use. A 2 mL gravity flow column (29920, Thermo Fisher Scientific) was filled with Thiophillic-Superflow resin (635617, Takara) and precipitated at 4°C overnight. The affinity chromatography gravity flow column was then washed with 10 column volumes of equilibration buffer (50 mM sodium phosphate and 0.5 M sodium sulfate, pH 7.0) for equilibration. The collected serum samples were diluted in sample buffer (50 mM sodium phosphate and 0.55 M sodium sulfate, pH 7.0) at a ratio of 1:10 (volume ratio). The diluted serum samples were added to the affinity chromatography gravity flow column, and the sample flow rate was controlled at 2 ml / min. Unbound proteins were then washed with equilibration buffer (50 mM sodium phosphate and 0.5 M sodium sulfate, pH 7.0). Finally, GO-Igs were eluted with 2-3 column volumes of elution buffer (20 mM sodium phosphate and 20% glycerol, pH 7.0). The eluted products were collected and combined, and the eluted products were placed in Tube-O-Dialyzer dialysis tubes (786-619, G-Biosciences; molecular weight cutoff 50K) and dialyzed in Hanks balanced salt solution (HBSS) containing 10 mM HEPES (pH 7.4) at 4°C overnight. Finally, the eluted products were eluted using Spin-X The dialyzed samples were concentrated using a UF concentrator (CLS431480, Sigma), and the protein concentration of the concentrated product was determined using the Pierce BCA protein assay.

[0298] 3 HA secretion experiment

[0299] 3.1 Cell culture

[0300] Fibroblasts obtained from each group of patients were counted using a Countess 3 automated cell counter (Thermo Fisher Scientific). After adjusting the density, 5,000 or 10,000 cells were seeded per well of a 96-well plate. The 96-well plate was placed in a Forma 3111 water-jacketed CO2 incubator (Thermo Electron) and cultured overnight. All cells adhered to the plate and were starved for 24 hours. The supernatant was removed and the cells were incubated with different concentrations of the test antibody according to the experimental group for 24 hours. GO-Igs were then added and incubated for a further 96 hours. The supernatant was carefully collected and centrifuged at 1,000 × g for 15 minutes using a 3K15 low-temperature high-speed centrifuge. Cell debris was discarded and the supernatant was collected for detection.

[0301] 3.2 HA Detection

[0302] This experiment used a human hyaluronic acid (HA) ELISA kit (ml557801, Shanghai ELISA Biotechnology Co., Ltd.). The specific procedure was as follows: The desired strips were removed from the aluminum foil pouch after equilibration at room temperature for 60 minutes. Standard and sample wells were set up, and 50 μL of the standard at different concentrations (200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, and 6.25 ng / mL) was added to each standard well. 50 μL of the treated supernatant was added to the sample wells; no supernatant was added to the blank wells. The wells were incubated at 37°C for 1 hour. The wells were washed three times with wash buffer. Except for the blank wells, 100 μL of horseradish peroxidase (HRP)-conjugated detection antibody was added to each standard and sample well. The wells were incubated at 37°C for 60 minutes, followed by five washes with wash buffer. 50 μL each of substrates A and B was added to each well and incubated at 37°C in the dark for 15 minutes. 50 μL of stop solution was added to each well, and the OD value of each well was measured at a wavelength of 450 nm using a microplate reader (Tecan Infinite 200 PRO microplate reader, Tecan) within 15 minutes.

[0303] Data processing (data processing methods) and experimental data:

[0304] A standard curve was plotted with the OD value of the measured standard as the horizontal axis and the concentration of the standard as the vertical axis. A linear regression equation was obtained. The OD values ​​of the sample wells were substituted into the equation to calculate the HA concentration in each well. A nonlinear fit of the HA concentration and the antibody concentration was performed using Graphpad Prism 9 to calculate the IC50 concentration. The results are shown in Table 8.

[0305] Table 8 Maximum inhibition rate and IC50 of each antibody

[0306] Experimental conclusion:

[0307] According to the above results, the selected antibodies can inhibit the production of HA in orbital fibroblasts stimulated by GO-Igs, among which cAb01-VH1-DA-VL2, cAb01-VH2-DA-VL2, cAb01-VH3-DA-VL2, cAb01-VH1-DA-VL3, cAb01-VH2-DA-VL3, cAb12-VH1-QG&S-VL3, cAb12-VH3-QG&S-VL3 are effective for It has a good inhibitory effect on cells in the acute phase and stable phase, and the maximum inhibition rate in the stable phase is better than that of the control antibody Tab03_hIgG1; cAb01-VH2-DA-VL3 and cAb12-VH3-QG&S-VL3 IC50 are better than the positive control Tab03_hIgG1; cAb21-VH2-VL2 and cAb21-VH1-VL3 have a good inhibitory effect on cells in the stable phase, and the maximum inhibition rate is better than Tab03_hIgG1.

[0308] Example 6: Humanized Antibody IL6 Secretion Experiment

[0309] Experimental steps:

[0310] 1. Cell culture

[0311] Fibroblasts and GO-Igs were collected according to the method described in Example 5. Cells were counted using a Countess 3 automated cell counter (Thermo Fisher Scientific). After adjusting the density, 5,000 or 10,000 cells were seeded per well of a 96-well plate. The 96-well plate was placed in a Forma 3111 water-jacketed CO2 incubator (Thermo Electron) and cultured overnight. All cells were observed under a microscope to adhere to the wall and then starved for 24 hours. The supernatant was removed and different concentrations of the test antibody were added according to the experimental group and incubated for 24 hours. GO-Igs were then added and incubated for a further 24 hours. The cell supernatant was carefully collected and centrifuged at 1,000 × g for 15 minutes using a 3K15 low-temperature high-speed centrifuge. Cell debris was discarded and the supernatant was collected for detection.

[0312] 2 IL6 detection

[0313] This experiment used a Human IL-6 ELISA kit (CSB-E04638h, Cusabio). The specific procedure was as follows: Before the experiment, the corresponding reagents were incubated at room temperature for 60 minutes. 100 μl of standard and sample were added to each well, with no addition to blank wells. The concentrations of standard were 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.2 pg / ml, 15.6 pg / ml, and 7.8 pg / ml. The plate was sealed and incubated at 37°C for 2 hours. The liquid in each well was discarded, and 100 μl of 1× Biotin-antibody was added to each well and incubated in a 37°C incubator for 1 hour. Wash the plate three times with wash buffer. 100 μl of 1× HRP-avidin was added to each well, and the plate was sealed with a new adhesive strip and incubated at 37°C for 1 hour. After washing five times, 90 μl of TMB solution was added to each well and incubated at 37°C in the dark for 15 minutes. 50 μl of Stop Solution was added to each well, and the plate was gently tapped to ensure thorough mixing. Within 15 min, the OD value of each well was measured at a wavelength of 450 nm using a microplate reader (Tecan Infinite 200 PRO microplate reader, Tecan).

[0314] Data processing (data processing methods) and experimental data:

[0315] A standard curve was plotted with the OD value of the measured standard as the abscissa and the concentration of the standard as the ordinate. A linear regression equation was obtained, and the OD values ​​of the samples were substituted into the equation to calculate the IL6 concentration in the supernatant of each well. Nonlinear fitting of the IL6 concentration and the antibody concentration was performed using Graphpad Prism 9, and the IC50 concentration was calculated. The results are shown in Table 9.

[0316] Table 9 Maximum inhibition rate and IC50 of each antibody

[0317] Experimental conclusion:

[0318] The selected antibodies were able to inhibit GO-Igs-stimulated IL6 production in orbital fibroblasts of patients. All antibodies had good inhibitory effects on cells in the acute and stable phases. Among them, cAb01-VH1-DA-VL2, cAb01-VH1-DA-VL3, cAb01-VH2-DA-VL3, and cAb21-VH2-VL2 had better maximum inhibition rates and IC50 for cells in the acute and stable phases than the control antibody Tab03_hIgG1.

[0319] Example 7: Humanized Antibody IL8 Secretion Experiment

[0320] Experimental steps:

[0321] 1. Cell culture

[0322] Cell culture was performed according to the method described in Example 6, and the supernatant was collected for detection.

[0323] 2 IL8 detection

[0324] In this experiment, the Human IL-8 ELISA kit (D8000C, R&D Company) was used to detect the concentration of IL8 in the supernatant.

[0325] Data processing (data processing methods) and experimental data:

[0326] A standard curve was plotted with the OD value of the standard as the horizontal axis and the concentration of the standard as the vertical axis. A linear regression equation was obtained, and the OD values ​​of the samples were substituted into the equation to calculate the IL8 concentration in the supernatant of each well. A nonlinear fit of the IL8 concentration and the antibody concentration was performed using Graphpad Prism 9 to calculate the IC50 concentration.

[0327] The data analysis results are shown in Table 10.

[0328] Table 10 Maximum inhibition rate and IC50 of each antibody

[0329] Experimental conclusion:

[0330] The selected antibodies were able to inhibit GO-Igs-stimulated IL8 production in orbital fibroblasts of patients. All antibodies had good inhibitory effects on cells in the acute and stable phases, and the maximum inhibition rates were almost all better than the positive control Tab03_hIgG1.

[0331] Example 8: Transformation of humanized antibodies

[0332] In this example, the antibody cAb01-VH2-DA-VL3 (designated hAb01_G4P, whose constant region is of the IgG4 subtype and contains the S228P mutation) was subjected to Fc modification (the modifications included those based on IgG4-S228P and those based on IgG1). The modification aims to extend half-life, increase efficacy, and reduce ADCC. The modified sites are shown in the following table:

[0333] Table 11 hAb01 Fc modification sites

[0334] Example 9: Binding activity of modified antibodies

[0335] The binding of the modified antibody to 293-human TSHR, 293-mouse TSHR, and 293-monkey TSHR overexpressing cell lines was detected by FACS method as described in Example 2. The secondary antibody used was anti-human IgG-Alexa488 antibody (Invitrogen; A-11013) (1:1k dilution).

[0336] Among them, hIgG1 was used as a negative control (purchased from Bio-Index B117901); hIgG4 was used as a negative control (purchased from Bio-Index B107804).

[0337] The test results are as follows:

[0338] Table 12 Maximum binding signal of modified antibodies

[0339] Note: “ / ” indicates a poor fit.

[0340] Experimental conclusion:

[0341] The modified antibody binds well to human TSHR and has cross-binding activity to mouse and monkey.

[0342] Example 10: cAMP activity assay of modified antibodies

[0343] Referring to the cAMP detection method and data processing method described in Example 4, the activity detection results of the modified antibody are as follows:

[0344] Table 13 Maximum inhibition rate and IC50 of cAMP activity of modified antibodies

[0345] Note: “ / ” indicates a poor fit.

[0346] Experimental conclusion:

[0347] In the cAMP activity experiment, all modified antibodies showed the effect of inhibiting the activation of TSHR activity by agonistic antibodies (Tab01 and Tab02), and the inhibitory effect was no weaker than that of the unmodified molecule hAb01_G4P.

[0348] Example 11: ADCC activity of engineered antibodies

[0349] Experimental methods:

[0350] The target cells used in the experiment were 293-human TSHR cells (for the amino acid sequence of the overexpressed human TSHR, see P16473, Uniprot), and the effector cells were human PBMCs (from Miaoshun, P122070903C). The effector PBMCs were resuscitated the day before the experiment and harvested on the day of the experiment. The cell concentration was adjusted to 5 × 10 in experimental buffer (RPMI 1640 + 10% FBS; RPMI Medium 1640, Gibco, A10491-01; FBS, BI, 040021A). 6 / mL, ready for use. Use fluorescent dye (Perkin Elmer, C136-100) to label 1×10 6 Target cells were incubated at 37°C in the dark for 20 minutes, then washed four times with PBS and resuspended in assay buffer. Antibody was diluted in assay buffer starting at 800 nM and serially diluted 1:5 to 11 points for later use. 50 μL of target cells, 100 μL of antibody diluent, and 50 μL of effector cells were added to the assay plate. The following control wells were also set: target cell maximum (50 μL of target cells + 150 μL of assay buffer); target cell autofluorescence (50 μL of target cells + 150 μL of assay buffer); and target and effector cell autofluorescence (50 μL of target cells + 50 μL of effector cells + 100 μL of assay buffer). The assay plate was incubated in a 37°C CO2 incubator for 2 hours. 10 μL of lysis buffer was added to the following control wells: target cell maximum, effector cell maximum, and target and effector cell maximum. Centrifuge the assay plate at 400g for 5 minutes, remove 25 μL of the supernatant and transfer to a flat-bottomed assay plate. Add 200 μL of Europium Solution (Perkin Elmer, C136-100) to each well of the assay plate. Shake the assay plate at 250 rpm for 15 minutes and read in a multifunctional microplate reader.

[0351] The sequences of the light chain variable region and heavy chain variable region of hAb01_G1 are consistent with those of hAb01_G4P, and the constant region subtype is hIgG1 (the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO: 49, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO: 51).

[0352] Data processing (data processing methods) and experimental data:

[0353] Calculation method: Inhibition rate % = (sample value - target cell and effector cell autofluorescence value) / (target cell maximum value - target cell autofluorescence value) × 100%.

[0354] The sample concentration was plotted as the horizontal axis and the inhibition rate as the vertical axis. Nonlinear fitting was performed on the inhibition rate and antibody concentration using Graphpad Prism 9 to calculate the IC50 concentration. The results are shown in Table 14.

[0355] Table 14 Antibody ADCC inhibition rate and IC50

[0356] Note: “None” means no ADCC effect.

[0357] Experimental conclusion:

[0358] Compared with hAb01_G1, hAb01_G1_V4 had weaker ADCC activity. hAb01_G4P, hAb01_G4P_V1, and hAb01_G4P_V2 did not exhibit ADCC activity.

[0359] Example 12: FcRn binding activity of modified antibodies

[0360] Experimental Methods: This experiment used an SA chip (cytiva, BR100531) to capture hFcRn-Avi (Biopsy, FCM-H82W7). Different concentrations of antibody were passed through the chip, and steady-state fitting analysis was performed based on the acquired data. The experimental instrument was a Biacore molecular interaction analyzer (cytiva, Biacore 8K+), and the buffer solution used was HBS-EP Buffer (cytiva, BR100669), pH 6.0. The association time was 240 s, the dissociation time was 240 s, the flow rate was 30 μL / min, and the detection was performed at 25°C. The obtained experimental data were subjected to steady-state fitting analysis using Biacore Insight Evaluation Software 3.0.12. The experimental results are shown in Table 15.

[0361] Table 15 KD values ​​of FcRn of modified antibodies

[0362] Experimental conclusion:

[0363] Comparing antibodies of the same Fc subtype, the affinity of the modified antibody to FcRn was significantly increased, indicating that the modified antibody can prolong its half-life.

[0364] Example 13: In vitro experiments of modified antibodies for GD indications

[0365] Experimental methods:

[0366] 1. Extraction and culture of primary thyroid cells from patients

[0367] Thyroid tissue samples were obtained from normal thyroid tissue of patients undergoing 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 for passage and expansion.

[0368] 2. Thyroid peroxidase (TPO) gene level detection

[0369] The cells from each group were collected and the density was adjusted, and 6×10 4 Cells were cultured overnight. After attachment, starvation cultures were maintained for 24 hours. The supernatant was removed and, according to experimental group, different concentrations of the test antibody and culture medium with or without GD serum (final concentration 1:100, volume ratio) were added. The cells were incubated for an additional 48 hours. Cells were harvested by trypsinization and total RNA was extracted. The reverse transcription reaction system was prepared according to the kit instructions (iScript cDNA, Bio-Rad, 1708891EDU) to obtain cDNA and stored at -70°C.

[0370] Real-time quantitative PCR reaction

[0371] The reaction system was set up on ice according to the reagent instructions (Takara, RR420A) and amplification was performed. Data were processed using the 2-ΔΔCt formula, using the internal reference gene GAPDH as a reference to determine the expression level of the target mRNA. The primers used are shown in Table 16.

[0372] Table 16 - Primers used in real-time quantitative PCR

[0373] Data processing and experimental data:

[0374] The data were analyzed and plotted using Graphpad Prism 9 (Version 9.4.0), and the figures were organized and combined using Adobe Illustrator 2022 (Version 2022). The statistical differences between the groups were tested using one-way ANOVA.

[0375] The experimental results are shown in Figure 1.

[0376] Experimental conclusion:

[0377] All the modified antibodies can inhibit TPO induced by GD patient serum, indicating that the antibodies of the present invention have the potential to treat GD disease.

[0378] Example 14: Experiment on secretion of hyaluronic acid (HA) by modified antibodies

[0379] The experimental method and data processing method are as described in Example 5.

[0380] Data processing and experimental data:

[0381] Note: “ / ” indicates a poor fit.

[0382] Experimental conclusion:

[0383] According to the above results, the modified antibodies can inhibit GO-Igs from stimulating the patient's orbital fibroblasts to produce HA, and have a good inhibitory effect on cells in the acute and stable phases.

[0384] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to TSHR, the antibody or antigen-binding fragment thereof comprising: (a) a VH CDR1 or variant thereof, a VH CDR2 or variant thereof, and a VH CDR3 or variant thereof contained in a heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 1, 27, 29, and 30; and / or a VL CDR1 or variant thereof, a VL CDR2 or variant thereof, and a VL CDR3 or variant thereof contained in a light chain variable region (VL) as shown in any one of SEQ ID NOs: 5, 28, and 31; (b) VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 9, 32, and 34; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in SEQ ID NOs: 13 or 33; or (c) a VH CDR1 or variant thereof, a VH CDR2 or variant thereof, and a VH CDR3 or variant thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 17, 35, and 37; and / or a VL CDR1 or variant thereof, a VL CDR2 or variant thereof, and a VL CDR3 or variant thereof contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 21, 36, and 38; in, The variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) the three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 1, 27, 29, and 30; and / or the three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 5, 28, and 31; (b) the three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 9, 32, and 34; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NOs: 13 or 33; or (c) three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 17, 35, and 37; and / or three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 21, 36, and 38; Preferably, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering systems.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 2, VH CDR2 of SEQ ID NO: 3 or 39, and VH CDR3 of SEQ ID NO:

4. CDR3; and / or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 6, VL CDR2 of SEQ ID NO: 7, and VL CDR3 of SEQ ID NO: 8; (b) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 10, VH CDR2 of SEQ ID NO: 11 or 40, and VH CDR3 of SEQ ID NO: 12 or 41; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 14, VL CDR2 of SEQ ID NO: 15, and VL CDR3 of SEQ ID NO: 16; or, (c) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 18, VH CDR2 of SEQ ID NO: 19, and VH CDR3 of SEQ ID NO: 20; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 22, VL CDR2 of SEQ ID NO: 23, and VL CDR3 of SEQ ID NO: 24; in, The CDRs are defined by the Kabat numbering system.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: (a) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 5 or a variant thereof; (b) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 13 or a variant thereof; or (c) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 17 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 21 or a variant thereof; in, The variant has one or more 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 a sequence having 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 comprises: a VH comprising the sequence shown in SEQ ID NO: 1 and a VL comprising the sequence shown in SEQ ID NO: 5; Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 9 and a VL comprising the sequence shown in SEQ ID NO: 13; Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 17 and a VL comprising the sequence shown in SEQ ID NO:

21.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin; Preferably, the antibody or antigen-binding fragment thereof 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.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein The antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising a sequence as shown in any one of SEQ ID NOs: 27, 29, 30, or a variant thereof; and / or a light chain variable region (VL) comprising a sequence as shown in SEQ ID NOs: 28 or 31, or a variant thereof; (b) a heavy chain variable region (VH) comprising a sequence as shown in SEQ ID NO: 32 or 34 or a variant thereof; and / or a light chain variable region (VL) comprising a sequence as shown in SEQ ID NO: 33 or a variant thereof; or (c) a heavy chain variable region (VH) comprising a sequence as shown in SEQ ID NO: 35 or 37 or a variant thereof; and / or a light chain variable region (VL) comprising a sequence as shown in SEQ ID NO: 36 or 38 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 a sequence having 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 substitution is a conservative substitution; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) comprising a heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 27, 29, and 30; and / or, comprising a light chain variable region (VL) as shown in SEQ ID NOs: 28 or 31; (b) comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 32 or 34; and / or, comprises a light chain variable region (VL) as shown in SEQ ID NO: 33; or, (c) comprising a heavy chain variable region (VH) as shown in SEQ ID NO: 35 or 37; and / or, comprising a light chain variable region (VL) as shown in SEQ ID NO: 36 or 38; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) a VH comprising the sequence shown in SEQ ID NO: 27 and a VL comprising the sequence shown in SEQ ID NO: 28; (2) a VH comprising the sequence shown in SEQ ID NO: 29 and a VL comprising the sequence shown in SEQ ID NO: 28; (3) a VH comprising the sequence shown in SEQ ID NO: 30 and a VL comprising the sequence shown in SEQ ID NO: 28; (4) a VH comprising the sequence shown in SEQ ID NO: 27 and a VL comprising the sequence shown in SEQ ID NO: 31; (5) a VH comprising the sequence shown in SEQ ID NO: 29 and a VL comprising the sequence shown in SEQ ID NO: 31; (6) a VH comprising the sequence shown in SEQ ID NO: 32 and a VL comprising the sequence shown in SEQ ID NO: 33; (7) a VH comprising the sequence shown in SEQ ID NO: 34 and a VL comprising the sequence shown in SEQ ID NO: 33; (8) a VH comprising the sequence shown in SEQ ID NO: 35 and a VL comprising the sequence shown in SEQ ID NO: 36; or (9) VH comprising the sequence shown in SEQ ID NO: 37 and VL comprising the sequence shown in SEQ ID NO:

38.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The antibody or antigen-binding fragment thereof further comprises a constant region derived from a mammalian (e.g., human or mouse) immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof 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 comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ); Preferably, the heavy chain of the antibody or its antigen-binding fragment comprises a heavy chain constant region derived from a murine immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4), and / or the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region derived from a murine immunoglobulin (e.g., κ or λ).

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin IgG4, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ); Preferably, compared with the heavy chain constant region of wild-type human immunoglobulin IgG4, the heavy chain constant region derived from human immunoglobulin IgG4 comprises substitution mutations S228P, L235E, M252Y, S254T and / or T256E (for example, (i) S228P, (ii) L235E, and / or (iii) M252Y, S254T and T256E); Preferably, compared with the heavy chain constant region of wild-type human immunoglobulin IgG4, the heavy chain constant region derived from human immunoglobulin IgG4 comprises the substitution mutation S228P; preferably, the heavy chain constant region derived from human immunoglobulin IgG4 further comprises the substitution mutations M252Y, S254T and T256E; more preferably, the heavy chain constant region derived from human immunoglobulin IgG4 further comprises the substitution mutations L235E, M252Y, S254T and T256E; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in any one of SEQ ID NOs: 25, 42-43, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 26; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 27 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26; (2) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 29 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26; (3) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 30 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26; (4) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 27 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 31 and a CL sequence as shown in SEQ ID NO: 26; (5) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 29 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 31 and a CL sequence as shown in SEQ ID NO: 26; (6) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 32 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 33 and a CL sequence as shown in SEQ ID NO: 26; (7) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 34 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 33 and a CL sequence as shown in SEQ ID NO: 26; (8) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 35 and a CH sequence as shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 36 and a CL sequence as shown in SEQ ID NO: 26; or (9) A heavy chain comprising a VH sequence shown in SEQ ID NO: 37 and a CH sequence shown in any one of SEQ ID NOs: 25, 42-43, and / or a light chain comprising a VL sequence shown in SEQ ID NO: 38 and a CL sequence shown in SEQ ID NO:

26.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin IgG1, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from human immunoglobulin (e.g., κ or λ); Preferably, compared with the heavy chain constant region of wild-type human immunoglobulin IgG1, the heavy chain constant region derived from human immunoglobulin IgG1 comprises the substitution mutations L234A, L235A, M252Y, S254T and / or T256E (for example, (i) L234A and L235A, and / or (ii) M252Y, S254T and T256E); Preferably, compared with the heavy chain constant region of wild-type human immunoglobulin IgG1, the heavy chain constant region derived from human immunoglobulin IgG1 comprises substitution mutations L234A and L235A; preferably, the heavy chain constant region derived from human immunoglobulin IgG1 further comprises substitution mutations M252Y, S254T and T256E; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 44 or 45, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 26; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 27 and a CH sequence as shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26; (2) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 29 and a CH sequence as shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26; (3) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 30 and a CH sequence as shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 28 and a CL sequence as shown in SEQ ID NO: 26; (4) a heavy chain comprising a VH sequence of SEQ ID NO: 27 and a CH sequence of SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence of SEQ ID NO: 31 and a CL sequence of SEQ ID NO: 26; (5) a heavy chain comprising a VH sequence as shown in SEQ ID NO: 29 and a CH sequence as shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence as shown in SEQ ID NO: 31 and a CL sequence as shown in SEQ ID NO: 26; (6) a heavy chain comprising a VH sequence of SEQ ID NO: 32 and a CH sequence of SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence of SEQ ID NO: 33 and a CL sequence of SEQ ID NO: 26; (7) a VH comprising the sequence shown in SEQ ID NO: 34 and the sequence shown in SEQ ID NO: 44 or 45 A heavy chain of CH, and / or a light chain comprising a VL having a sequence as shown in SEQ ID NO: 33 and a CL having a sequence as shown in SEQ ID NO: 26; (8) a heavy chain comprising a VH sequence of SEQ ID NO: 35 and a CH sequence of SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence of SEQ ID NO: 36 and a CL sequence of SEQ ID NO: 26; or (9) A heavy chain comprising a VH sequence shown in SEQ ID NO: 37 and a CH sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL sequence shown in SEQ ID NO: 38 and a CL sequence shown in SEQ ID NO:

26.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody and single-domain antibody (sdAb); and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or a multispecific antibody.

10. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the heavy chain variable region and / or light chain variable region thereof, or the heavy chain and / or light chain thereof.

11. A vector comprising the isolated nucleic acid molecule according to claim 10; preferably, the vector is a cloning vector or an expression vector.

12. A host cell comprising the isolated nucleic acid molecule of claim 10 or the vector of claim 11.

13. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising culturing the host cell according to claim 12 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell.

14. A bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; Preferably, the bispecific or multispecific molecule specifically binds to the TSHR and additionally specifically binds to one or more other targets; Preferably, the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (e.g., a second antibody); Preferably, the bispecific or multispecific molecule further comprises at least one second antibody that specifically binds to IGF1R; preferably, the second antibody has an antagonistic effect on IGF1R signaling.

15. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a therapeutic agent linked to the antibody or antigen-binding fragment thereof; Preferably, the therapeutic agent is selected from IGF1R antagonists or inhibitors; Preferably, the immunoconjugate is an antibody-drug conjugate (ADC).

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the isolated nucleic acid molecule according to claim 10, the vector according to claim 11, the host cell according to claim 12, the bispecific or multispecific molecule according to claim 14, or the immunoconjugate according to claim 15, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises another pharmaceutically active agent, such as another drug for treating autoimmune thyroid disease (such as Graves' disease or Graves' ophthalmopathy) or thyroid cancer; Preferably, the pharmaceutical composition further comprises an IGF1R antagonist or inhibitor, and / or, an additional TSHR antagonist or inhibitor.

17. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the isolated nucleic acid molecule of claim 10, the vector of claim 11, the host cell of claim 12, the bispecific or multispecific molecule of claim 14, the immunoconjugate of claim 15, or the pharmaceutical composition of claim 16 for the preparation of a medicament for preventing and / or treating a TSHR-related disease in a subject; Preferably, the TSHR-related disease will benefit from antagonism of TSHR signaling; Preferably, the TSHR-related disease is autoimmune thyroid disease (such as Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof; Preferably, the subject is a mammal, such as a human, mouse or monkey; Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., another drug used to treat autoimmune thyroid disease (such as Graves' disease or Graves' ophthalmopathy) or thyroid cancer); Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is used in combination with an IGF1R antagonist or inhibitor and / or an additional TSHR antagonist or inhibitor.

18. A method for preventing and / or treating a TSHR-related disease in a subject, comprising: administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the isolated nucleic acid molecule of claim 10, the vector of claim 11, the host cell of claim 12, the bispecific or multispecific molecule of claim 14, the immunoconjugate of claim 15, or the pharmaceutical composition of claim 16; Preferably, the TSHR-related disease will benefit from antagonism of TSHR signaling; Preferably, the TSHR-related disease is autoimmune thyroid disease (such as Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof; Preferably, the subject is a mammal, such as a human, mouse or monkey; Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., another drug used to treat autoimmune thyroid disease (such as Graves' disease or Graves' ophthalmopathy) or thyroid cancer); Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is used in combination with an IGF1R antagonist or inhibitor and / or an additional TSHR antagonist or inhibitor.

19. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and a detectable label linked to the antibody or antigen-binding fragment thereof; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

20. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 19.

21. A method for detecting the presence or level of TSHR in a sample, comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 19; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g. ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the method comprises using the conjugate of claim 19; Preferably, the method comprises using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin); Preferably, the method comprises: (1) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 19; (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex, wherein the formation of the immune complex indicates the presence of TSHR or cells expressing TSHR; Preferably, the method is a method for diagnosing a disease associated with TSHR, wherein the sample is a sample from a subject; Preferably, when TSHR is present or the level of TSHR is increased compared to a reference level (eg compared to a healthy control), it indicates that the subject suffers from a disease associated with TSHR; Preferably, the TSHR-related disease is autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.

22. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 19 in the preparation of a detection reagent for detecting the presence or level of TSHR in a sample and / or diagnosing a disease associated with TSHR; Preferably, the detection reagent is used to detect the presence or level of TSHR in a sample or to diagnose a disease associated with TSHR by the method of claim 21; Preferably, the TSHR-related disease is autoimmune thyroid disease (such as Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof; Preferably, the sample is a tissue sample from a subject (eg, a mammal, preferably a human, mouse or monkey).

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